Dispersed-phase composition for producing peptide-containing microspheres, method for producing peptide-containing microspheres using same, and peptide-containing microspheres

Abstract

The present invention relates to a dispersed-phase composition for producing peptide-containing microspheres, a method for producing peptide-containing microspheres using the dispersed-phase composition, and peptide-containing microspheres.

Description

Composition of a dispersed phase for manufacturing peptide-containing microspheres, a method for manufacturing peptide-containing microspheres using the same, and peptide-containing microspheres

[0001] The present invention relates to a dispersed phase composition for producing peptide-containing microspheres and a method for producing peptide-containing microspheres using the dispersed phase composition.

[0002] In addition, the present invention relates to peptide-containing microspheres.

[0003]

[0004] Conventional techniques for manufacturing drug-loaded microspheres include the solvent evaporation method, spray drying, membrane emulsification, and microfluidic method. In manufacturing microspheres using the above techniques, depending on the solubility of the drug and the type of polymer used, formulations in which the outermost solution is water during the manufacturing process, such as Oil in Water (O / W), Water in Oil in Water (W / O / W), Solid in Oil in Water (S / O / W), and Solid in Water (S / W), can be manufactured, as well as formulations in which the outermost solution is an organic solvent that is immiscible with water, such as Water in Oil (W / O), Solid in Oil (S / O), and Solid in Water in Oil (S / W / O).

[0005] In selecting the form of the aforementioned drug-loading microsphere formulation, the most commonly used polyester polymers in pharmaceuticals, such as PLGA, PLA, and PCL, can be used as the drug release substrate. Since the above polyester polymers are well soluble in organic solvents that are immiscible with water (especially dichloromethane), water-soluble drugs have mainly been developed into W / O / W formulations, or research and development have been conducted into O / W formulations in which a solvent capable of dissolving the drug and being miscible with the organic solvent in which the polymer is dissolved is selected and utilized as a co-solvent.

[0006] As explained above, even in the case of microparticle formulations utilizing mainly water-soluble peptide drugs, they have been developed as W / O / W formulations or O / W formulations using an organic solvent in which the peptide is dissolved as a co-solvent, or S / O / W formulations obtained by solid-dispersing the peptide in a solvent in which a polymer is dissolved into nano or micro sizes.

[0007] Among conventional methods for manufacturing microspheres, membrane emulsification and microfluidic methods have the advantage of being able to produce uniform microspheres. Among these, microfluidic methods are well known as a technology that can produce droplets very stably and can significantly increase the encapsulation rate when encapsulating peptide drugs.

[0008] Meanwhile, GLP-1 agonist peptide drugs, such as semaglutide, are peptides that structurally have glycine and arginine at the C-terminal end and are well known as water-soluble drugs that can be used for adult diseases such as diabetes, obesity, and non-alcoholic fatty liver disease. However, in the case of microparticle formulations containing 9% or more of semaglutide, the initial dissolution rate increased rapidly, which was a disadvantage of the prior art (KR 10-2022-0077146) that made it difficult to use in the human body.

[0009] In addition, when a final W / O / W formulation is prepared using a general W / O type dispersion phase consisting only of peptides and polyester polymers as a primary emulsion to manufacture microspheres containing peptides with glycine and arginine at the C-terminus, such as semaglutide, there was a limitation in that drug release from the microspheres was not smoothly achieved due to strong interactions with polyester polymers such as PLGA and PLA caused by arginine within the peptide chemical structure.

[0010] In addition, the dispersed phase for producing microspheres has high viscosity due to the interaction between arginine and polyester polymer within the chemical structure of the peptide, making it unsuitable for producing peptide-containing microspheres by microfluidic methods. Furthermore, the colloidal stability of the primary emulsion in the W / O form is low, causing phase separation after a certain period of time, making it difficult to produce uniform microspheres in the W / O / W form by microfluidic methods for a long time.

[0011] Therefore, in order to uniformly produce large quantities of microspheres containing peptides with glycine and arginine at the C-terminus when using microfluidic methods, there remain issues regarding 1) the reduction of fluid viscosity to ensure smooth fluid flow inside the microfluidic tube, and 2) the stability of the dispersed phase flowing inside the microfluidic tube.

[0012] Against this backdrop, the inventors conducted diligent research to solve the problems of manufacturing peptide-containing microspheres as described above. As a result, they confirmed the possibility of extending the administration interval from one month to several months when using a microfluidic method, and confirmed that when using a dispersed phase composition containing arginine, the interaction between arginine and polyester polymers within the peptide chemical structure is competitively blocked during the manufacturing process, allowing the peptide drug to be smoothly released from the microspheres.

[0013] Furthermore, the inventors confirmed that when a peptide drug is used together with hydroxypropyl-beta-cyclodextrin (HP-β-CD), hydroxypropyl-beta-cyclodextrin (HP-β-CD) influences the structural environment of the peptide, which tends to alleviate non-specific interactions with polyester polymers, thereby improving the stability and release characteristics of the peptide.

[0014] In addition, when the above-mentioned dispersed phase composition was left standing for a certain period of time and then microspheres were prepared using a microfluidic method, it was confirmed that the viscosity of the dispersed phase decreased without phase separation, making it suitable for the production of uniform microspheres. This phase separation inhibition effect is believed to be the result of the dispersed phase remaining stable as arginine contained in the dispersed phase mitigates electrostatic interactions with polyester polymers, and hydroxypropyl-beta-cyclodextrin (HP-β-CD) interacts with some hydrophobic sites of the peptide to improve interfacial stability.

[0015]

[0016] The present invention was completed by confirming that it is possible to manufacture uniform microspheres without phase separation while reducing the viscosity of the dispersed phase by utilizing the interaction effect of arginine and / or hydroxypropyl-beta-cyclodextrin (HP-β-CD).

[0017]

[0018] One objective of the present invention is to provide a dispersed composition for producing peptide-containing microspheres.

[0019] Another objective of the present invention is to provide a method for manufacturing peptide-containing microspheres using the above-mentioned dispersed phase composition.

[0020] Another objective of the present invention is to provide microspheres comprising a peptide, arginine, and a polyester-based polymer.

[0021] Another objective of the present invention is to provide a use of a dispersed phase composition for producing peptide-containing microspheres.

[0022]

[0023] The composition of the dispersed phase for preparing peptide-containing microspheres according to the present invention effectively inhibits physicochemical interactions between the peptide and the polyester polymer, thereby enabling the microspheres prepared using the dispersed phase to smoothly elute the peptide.

[0024] In addition, the method for manufacturing peptide microspheres using the composition of the dispersed phase for manufacturing peptide-containing microspheres according to the present invention is a method for manufacturing particles of uniform size containing a high content of peptide of 10% or more, and since the viscosity of the dispersed phase can be lowered while increasing the stability of the dispersed phase during the manufacturing process so that phase separation does not occur, it has the effect of facilitating the manufacturing of microspheres using a microfluidic method.

[0025] In addition, the peptide-containing microspheres of the present invention are particles of uniform size containing 10% or more of peptide, and have high potential for industrial application due to the low dosage and have therapeutic effects for obesity, diabetes, or non-alcoholic fatty liver disease.

[0026]

[0027] Figure 1 is a figure showing a comparison of the dispersed phases of Example 1 and Comparative Example 1 according to the addition of arginine.

[0028] Figure 2 is a figure showing the decrease in viscosity over time of the dispersed phase (Example 1) with added arginine.

[0029] Figure 3 is a figure showing scanning electron microscope images of microspheres of Example 2 and Comparative Example 2 according to the settling time after preparation of the dispersed phase.

[0030] Figure 4 is a scanning electron microscope image of microspheres of Example 2, Comparative Example 3, and Comparative Example 4 with the addition of arginine to the dispersed phase.

[0031] Figure 5 is a scanning electron microscope image of microspheres of Examples 2, 3, and 4 according to the type of polymer.

[0032] Figure 6 is a figure showing scanning electron microscope images of microspheres of Example 5, Example 6 and Comparative Example 5 according to the concentration of hydroxypropyl-beta-cyclodextrin (HP-β-CD) when the polymer composition is lactic acid:glycolic acid = 50:50.

[0033] Figure 7 is a figure showing scanning electron microscope images of microspheres of Example 7, Example 8 and Comparative Example 6 according to the concentration of hydroxypropyl-beta-cyclodextrin (HP-β-CD) when the polymer composition is lactic acid:glycolic acid = 75:25.

[0034]

[0035] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0036] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. In addition, such equivalents are intended to be included in the present invention.

[0037]

[0038] According to one embodiment of the present invention for achieving the above objective,

[0039] i) a primary water phase containing peptides and arginine; and

[0040] ii) Provides a composition of a dispersed phase for manufacturing peptide-containing microspheres comprising a secondary oil phase comprising a polyester-based polymer and an organic solvent.

[0041] In the present invention, the primary aqueous phase may additionally comprise hydroxypropyl-beta-cyclodextrin (HP-β-CD).

[0042] The term "dispersed phase" in the present invention may be used interchangeably with "composition of the dispersed phase" or "dispersed phase composition," and may refer to a solution containing a drug and a polymer among the solutions used to produce microspheres by microfluidic methods. In one example, the dispersed phase in the present invention may include a peptide having glycine and arginine at the C-terminus as a drug, a polyester polymer, and arginine as a polymer, and in another example, the dispersed phase may include a peptide having glycine and arginine at the C-terminus as a drug, a polyester polymer, arginine, and hydroxypropyl-beta-cyclodextrin (HP-β-CD) as polymers, but is not limited thereto. In addition, the dispersed phase may refer to an emulsion in which a peptide, a polyester polymer, and arginine are dissolved or dispersed in the present invention, or may refer to an emulsion in which a peptide, a polyester polymer, arginine, and hydroxypropyl-beta-cyclodextrin are dissolved or dispersed.

[0043] The term "peptide" of the present invention may mean a peptide having glycine and arginine at the C-terminus, and, for example, may be a peptide having glycine and arginine repeatedly at the C-terminus, but is not limited thereto. The above peptide comprises 10 to 40%, 10 to 38%, 10 to 36%, 10 to 34%, 10 to 32%, 10 to 30%, 10 to 28%, 10 to 26%, 10 to 25%, 11 to 40%, 11 to 38%, 11 to 36%, 11 to 34%, 11 to 32%, 11 to 30%, 11 to 28%, 11 to 26%, 11 to 25%, 12 to 40%, 12 to 38%, 12 to 36%, 12 to 34%, 12 to 32%, 12 to 30%, 12 to 28%, 12 to 26%, 12 to 25%, 13 to 40%, 13 to 38%, 13 to 36%, 13 to 34%, 13 to 32%, 13 to 30%, 13 to 28%, 13 to 26%, 13 to 25%, 14 to 40%, 14 to 38%, 14 to 36%, 14 to 34%, 14 to 32%, 14 to 30%, 14 to 28%, 14 to 26%, 14 to 25%, 15 to 40%, 15 to 38%, 15 to 36%, 15 to 34%, 15 to 32%, or corresponding to 15 to 30% It may be included in the dispersion in an amount (w / w%) corresponding to, for example, 15 to 28%, specifically 15 to 26%, more specifically 15 to 25%.

[0044] In addition, the above peptide comprises 10 to 40%, 10 to 38%, 10 to 36%, 10 to 34%, 10 to 32%, 10 to 30%, 10 to 28%, 10 to 26%, 10 to 25%, 11 to 40%, 11 to 38%, 11 to 36%, 11 to 34%, 11 to 32%, 11 to 30%, 11 to 28%, 11 to 26%, 11 to 25%, 12 to 40%, 12 to 38%, 12 to 36%, 12 to 34%, 12 to 32%, 12 to 30%, 12 to 28%, 12 to 26%, 12 to 25%, 13 to 40%, 13 to 38%, 13 to 36%, 13 to 34%, 13 to 32%, 13 to 30%, 13 to 28%, 13 to 26%, 13 to 25%, 14 to 40%, 14 to 38%, 14 to 36%, 14 to 34%, 14 to 32%, 14 to 30%, 14 to 28%, 14 to 26%, 14 to 25%, 15 to 40%, 15 to 38%, 15 to 36%, 15 to 34%, 15 to It may be included in the dispersion phase in an amount corresponding to 32% or 15 to 30% (w / w%), for example, 15 to 28%, specifically 15 to 26%, more specifically 15 to 25% (w / w%).

[0045] In the present invention, the peptide may refer to a peptide drug contained within a microsphere, or a drug.

[0046] According to one embodiment of the present invention, if the amount of peptide included in the dispersion phase is less than 10% of the total weight of the peptide, polyester polymer, and arginine, or if the amount of peptide included in the dispersion phase is less than 10% of the total weight of the peptide, polyester polymer, arginine, and hydroxypropyl-beta-cyclodextrin, the drug content in the microspheres prepared with the dispersion phase of the present invention is too low to be utilized as a long-acting formulation for more than one month, and if the amount of peptide included in the dispersion phase exceeds 40% of the total weight of the peptide, polyester polymer, and arginine, or if the amount of peptide included in the dispersion phase exceeds 40% of the total weight of the peptide, polyester polymer, arginine, and hydroxypropyl-beta-cyclodextrin, it is difficult to control the initial release of the drug as a number of pores are formed on the surface of the final microspheres prepared with the dispersion phase of the present invention.

[0047] The polyester polymer included in the dispersion phase is a polymer that is biodegradable and can gradually biodegrade after injection to release the peptide encapsulated therein. Examples include polylactic-co-glycolide copolymer, polylactic-co-glycolic acid, polylactide, polylactic acid, polycaprolactone, etc., but are not limited thereto.

[0048] The above polyester-based polymer is present in an amount of 5 to 25%, 5 to 23%, 5 to 21%, 5 to 19%, 5 to 17%, 5 to 15%, 6 to 25%, 6 to 23%, 6 to 21%, 6 to 19%, 6 to 17%, 6 to 15%, 7 to 25%, 7 to 23%, 7 to 21%, 7 to 19%, 7 to 17%, 7 to 15%, 8 to 25%, 8 to 23%, 8 to 21%, 8 to 19%, 8 to 17%, 8 to 15%, 9 to 25%, 9 to 23%, 9 to 21%, 9 to It may be included in a weight (w / v%) of 19%, 9 to 17%, 9 to 15%, 10 to 25%, 10 to 23%, or 10 to 21%, for example, 10 to 19%, specifically 10 to 17%, more specifically 10 to 15% by weight (w / v%), but is not limited thereto.

[0049] According to one embodiment of the present invention, if the concentration of the polyester-based polymer dissolved in the secondary oil phase organic solvent is less than 5%, the yield of microspheres produced using the dispersion phase of the present invention is very low, making it difficult to use industrially, and if it exceeds 25%, the viscosity of the final dispersion phase is too high, making it difficult to produce by the microfluidic method.

[0050] The arginine (Arginine, Arg, R) included in the above dispersion phase refers to a polymer that necessarily includes arginine, a pharmaceutically acceptable salt thereof, an arginine derivative, or polyarginine, and must be 2 to 25 times the molar ratio of the peptide based on a single molecule of arginine. The arginine of the present invention is added to competitively inhibit the physicochemical interaction between the peptide and the polyester polymer; if the amount of arginine included in the dispersion phase of the present invention is less than 2 times the molar ratio of the peptide, it is difficult to effectively inhibit the physicochemical interaction between the peptide and the polyester polymer, and if it exceeds 25 times, it forms a large number of pores on the surface, making it difficult to control initial release.

[0051] In addition, hydroxypropyl-β-cyclodextrin (HP-β-CD) additionally included in the dispersion phase refers to a modified cyclic sugar molecule derived from β-cyclodextrin, and may refer to a cyclic oligosaccharide used to improve the solubility and stability of drugs and other substances, i.e., a molecule acting as a pharmaceutical excipient. The hydroxypropyl-β-cyclodextrin of the present invention may be added to effectively inhibit non-specific interactions between the peptide and the polyester polymer by influencing the structural environment of the peptide. Furthermore, the dispersion phase can be maintained stably by improving interfacial stability through the interaction of hydroxypropyl-β-cyclodextrin with the hydrophobic sites of the peptide.

[0052] For dissolving the peptide in the dispersion phase, the solvent used in the primary water phase may be selected from, for example, water for injection, purified water, water-soluble buffer solution, acidic solution such as acetic acid, hydrochloric acid, or a combination thereof, and for the secondary oil phase to use the polymer, a highly volatile organic solvent with low miscibility with water may be used, such as, for example, a solvent selected from dichloromethane, chloroform, ethyl acetate, or a combination thereof, but is not limited thereto.

[0053] In the present invention, the peptide may be samaglutide or liraglutide, but is not limited thereto.

[0054] The present invention has technical significance in that the composition of the dispersed phase for manufacturing peptide-containing microspheres inhibits physicochemical interactions between the peptide and the polyester polymer during the manufacturing process, thereby ensuring that the dispersed phase remains stable and is suitable for manufacturing uniform microspheres, as well as facilitating the drug release of the final microsphere formulation.

[0055]

[0056] According to another aspect of the present invention,

[0057] The present invention provides a method for manufacturing peptide-containing microspheres using a dispersed phase composition for manufacturing the peptide-containing microspheres.

[0058] The method for manufacturing the peptide-containing microspheres of the present invention is an invention of an article and a method comprising common components, and the content regarding the common components between each invention can be applied in the same way.

[0059] Hereinafter, a method for manufacturing peptide-containing microspheres using a dispersed phase composition for manufacturing peptide-containing microspheres according to the present invention will be described in detail.

[0060] The method for manufacturing peptide-containing microspheres of the present invention may include the following steps.

[0061] i) a step of preparing a water phase by dissolving the peptide and arginine in a water phase solvent;

[0062] ii) A step of preparing a secondary oil phase by dissolving a polyester-based polymer in an organic solvent which is a secondary oil phase solvent;

[0063] iii) a step of homogeneously mixing the primary aqueous phase and the secondary oil phase to prepare a dispersed phase that is an emulsion;

[0064] iv) a step of reacting the dispersed phase in a static manner; and

[0065] v) a step of injecting the dispersed phase into one microfluidic channel (MFC) and injecting a continuous phase containing a surfactant into a separate microfluidic channel different from the dispersed phase to generate microsphere droplets from the interface of the two microfluidic channels.

[0066] In the present invention, the step of preparing the primary aqueous phase may be to dissolve a peptide, arginine, and hydroxypropyl-beta-cyclodextrin in a primary aqueous phase solvent.

[0067] In the manufacturing method of the present invention, steps i) and ii) may each be performed independently. Additionally, steps i) and ii) may be performed simultaneously or sequentially, or in reverse order.

[0068] The definitions and amounts of "peptide," "arginine," "hydroxypropyl-beta-cyclodextrin," "polyester-based polymer," "primary water phase solvent," and "secondary oil phase solvent" in steps i) and ii) above are as described above in the composition of the dispersion phase for manufacturing the peptide-containing microspheres.

[0069] In step iii) above, methods for homogeneously mixing the primary water phase of step i) and the secondary oil phase of step ii) to form an emulsion may include homogenization using a high-speed stirrer (Homogenizer), ultrasonic fragmentation using sonication, or vibration stirring using a vortex, but are not particularly limited as long as they do not impair the structural stability of the drug.

[0070] Step iv) above is a step necessary for lowering the viscosity of the dispersed phase prepared in step iii), and while the time and temperature conditions required for the standing reaction are highly related to the amount and concentration of the polyester polymer and peptide, it must be set at least for 2 hours within the range of at least 2 to 35°C.

[0071] Specifically, the above static reaction may be performed for 2 to 30 hours, 2 to 25 hours, 2 to 20 hours, 2 to 15 hours, 2 to 10 hours, 4 to 30 hours, 4 to 25 hours, 4 to 20 hours, 4 to 15 hours, 4 to 10 hours, 6 to 30 hours, 6 to 25 hours, 6 to 20 hours, 6 to 15 hours, 6 to 10 hours, 8 to 30 hours, 8 to 25 hours, 8 to 20 hours, or 8 to 15 hours, for example, for 8 to 10 hours, and more specifically, for 10 hours.

[0072] In addition, the above static reaction may be performed under temperature conditions of 2 to 35°C, 2 to 32°C, 2 to 29°C, 2 to 26°C, 2 to 23°C, 2 to 20°C, 6 to 35°C, 6 to 32°C, 6 to 29°C, 6 to 26°C, 6 to 23°C, 6 to 20°C, 10 to 35°C, 10 to 32°C, 10 to 29°C, 10 to 26°C, 10 to 23°C, 10 to 20°C, 15 to 35°C, 15 to 32°C, 15 to 29°C, 15 to 26°C, or 15 to 23°C, depending on the amount and concentration of the polyester-based polymer and peptide; for example, it may be performed under temperature conditions of 15 to 20°C, and more specifically, It may be performed under temperature conditions of 20℃.

[0073] It was confirmed that by including arginine and / or hydroxypropyl-beta-cyclodextrin in the above dispersion phase, arginine alleviates electrostatic interactions with polyester polymers and / or hydroxypropyl-beta-cyclodextrin interacts with some hydrophobic sites of peptides to improve interfacial stability, and accordingly, the kinetic stability of the dispersion phase is improved, thereby having the effect of suppressing phase separation and aggregation.

[0074] In addition, if the dispersed phase is prepared and subjected to a set time reaction, the viscosity of the dispersed phase can be lowered to facilitate the production of microspheres using the microfluidic method. This is technically significant in that it facilitates stable injection into microfluidic channels for a long period of time when producing microspheres using the composition of the dispersed phase for producing peptide-containing microspheres according to the present invention.

[0075] In the step of generating microsphere droplets from the contact of the two microfluidic channels by injecting the dispersed phase of step iv) into one microfluidic channel and injecting a continuous phase containing a surfactant into a separate microfluidic channel different from the dispersed phase in the microfluidic device in step v) above, the "continuous phase" refers to an aqueous solution in which a surfactant is dissolved, which is a solution used to produce microspheres by the microfluidic method and is injected into a microfluidic channel different from the dispersed phase. The surfactant used in the continuous phase may be, for example, a surfactant selected from polyvinyl alcohol, sodium lauryl sulfate, Tween 80, Tween 20, ethylenediaminetetraacetic acid, or a combination thereof, but is not limited thereto.

[0076] The term "microfluidic method" in this invention refers to a technique capable of generating uniform spherical particles by injecting a dispersed phase and a continuous phase through respective microfluidic channels and forming the two channels to intersect or meet.

[0077] The term "microfluidic channel (MFC)" in the present invention may refer to a microfluidic channel within a microfluidic device capable of producing particles having a particle size of 1 mm or less. By injecting the dispersed phase and the continuous phase at a constant speed through separate microfluidic channels, a microparticle droplet of uniform shape and / or size can be formed at the contact point between the microfluidic channel for injecting the dispersed phase and the microfluidic channel for injecting the continuous phase. To this end, the microfluidic device is provided with three or more separately configured microfluidic channels sharing a single contact point, so that the dispersed phase and the continuous phase are injected into two channels respectively, and the microparticle droplet formed by the contact between the dispersed phase and the continuous phase at the contact point can be transferred to a second aqueous phase through another channel, wherein the channels for injecting the dispersed phase and / or the continuous phase may each be two or more as needed. The manufacturing method of the present invention may use a multi-channel microfluidic device comprising a plurality of structures for continuous phase injection, dispersed phase injection, contacts thereof, and droplet transport, in order to increase production volume, but is not limited thereto.

[0078] The term "droplet" in the present invention refers to a dispersed phase droplet dispersed in an aqueous phase in a colloidal form. The terms "droplet" and "microsphere droplet" in the present invention may be used interchangeably as having the same meaning. The microsphere droplet of the present invention is an intermediate material obtained in the manufacturing step of peptide-containing microspheres.

[0079] In preparing peptide-containing microspheres using the microfluidic method in step v) above, the present invention has technical significance in that it provides a technical advantage of being able to stably produce droplets formed by the microfluidic method, thereby minimizing drug loss.

[0080] In the present invention, vi) the step of dispersing the generated microsphere droplets in a secondary water phase containing a surfactant while stirring may be further included.

[0081] The term "secondary water phase" in the present invention may refer to a fluid in which microparticle droplets prepared at the interface of a microfluidic channel are dispersed.

[0082] In step vi) above, the surfactant may be used, for example, a surfactant selected from polyvinyl alcohol, sodium lauryl sulfate, Tween 80, Tween 20, ethylenediaminetetraacetic acid, or a combination thereof, but is not limited thereto.

[0083] In the present invention, after step vi), the method may further include one or more processes selected from the group consisting of (a) removing an organic solvent from the dispersed microsphere droplets to cure them, (b) washing the cured microspheres, and (c) obtaining and drying the microspheres.

[0084] In the above step (a), the method for removing the organic solvent may include volatilization of the organic solvent by heating, removal of the organic solvent by reduced pressure, and removal of the organic solvent by using a co-solvent capable of precipitating the particles, but is not particularly limited as long as it is a method capable of removing the particles while maintaining a spherical shape.

[0085] As a solvent for washing the microspheres cured in step (b) above, a solvent selected from water for injection, purified water, a buffer solution, an aqueous sodium chloride solution, or a combination thereof may be used, but is not particularly limited as long as it is a method capable of removing particles while maintaining a spherical shape.

[0086] The method for obtaining microspheres in step (c) above may include obtaining them using a membrane filter or filtration using a sieve, but is not limited thereto.

[0087] In step (c) above, drying methods such as vacuum drying, natural drying, freeze-drying, heat drying, or air drying may be used, and specifically, freeze-drying may be used. As a cryopreservative for freeze-drying, salts selected from, for example, mannitol, sucrose, cellulose, glucose, sodium chloride, potassium chloride, or combinations thereof may be additionally included, but are not limited thereto.

[0088]

[0089] According to another aspect of the present invention,

[0090] The present invention provides peptide-containing microspheres comprising peptides, arginine, and polyester-based polymers.

[0091] In the present invention, the peptide-containing microspheres may further comprise hydroxypropyl-beta-cyclodextrin.

[0092] The definitions of the above "peptide," "arginine," "hydroxypropyl-beta-cyclodextrin," and "polyester-based polymer" are as described above in the composition of the dispersion phase for manufacturing the above peptide-containing microspheres.

[0093] The term "microsphere" in the present invention refers to a capsule composed of an outer inactive shell and an inner active core, which can be classified into spherical, crystalline, emulsions, etc., depending on its shape, and may have a particle size in the range of several μm to several mm, and generally, the particle size of the microsphere may be 1 to 1,000 μm. In addition, the microspheres may be classified according to their structure into core / shell capsules and mononuclear encapsulates, such as a mononuclear type (core / shell type) in which a single shell surrounds a core, a polynuclear type in which multiple cores are surrounded by a shell, a matrix type in which a core is uniformly dispersed within a matrix inside a shell and is known to be most commonly used in the pharmaceutical and food industries, a multi-wall type in which a structure consisting of two or more coating layers, or a coated matrix type in which a matrix type and a mononuclear type are combined, but are not limited thereto (see Agnieszka Klosowska et al., "Microencapsulation as a Route for Obtaining Encapsulated Flavors and Fragrances", Cosmetics 2023, 10, 26).

[0094] According to one embodiment of the present invention, the peptide-containing microspheres of the present invention may have a peptide and arginine dispersed within a polyester-based polymer matrix, or a peptide, arginine, and hydroxypropyl-beta-cyclodextrin dispersed within a polyester-based polymer matrix.

[0095] In the present invention, the particle size of the peptide-containing microspheres may be 5 to 100 μm, 5 to 90 μm, 5 to 80 μm, 5 to 70 μm, 5 to 60 μm, 5 to 50 μm, 10 to 100 μm, 10 to 90 μm, 10 to 80 μm, 10 to 70 μm, 10 to 60 μm, 10 to 50 μm, 15 to 100 μm, 15 to 90 μm, 15 to 80 μm, 15 to 70 μm, 15 to 60 μm, 15 to 50 μm, 20 to 100 μm, 20 to 90 μm, 20 to 80 μm, or 20 to 70 μm, and specifically 20 to The particle size may be 60 μm, more specifically 20 to 50 μm, but is not limited thereto. When peptide-containing microspheres are manufactured with the above particle size, they are most suitable for use in injection into the human body, and drug release from the particles can be facilitated. In particular, the technical feature of the present invention is that the peptide-containing microspheres of the present invention provide microspheres having a uniform size with a coefficient of variation (CV) of within 10%.

[0096] The peptide-containing microspheres of the present invention contain a drug content of 10% to 40% within the microspheres. Since the peptide-containing microspheres of the present invention have a content of 10% or more, the dosage is lower compared to conventional technology, making it easy to administer to patients and highly suitable for industrial use.

[0097] In the present invention, the peptide-containing microspheres may be used for the treatment of obesity, diabetes, or non-alcoholic fatty liver disease, but are not limited thereto.

[0098]

[0099] According to another aspect of the present invention,

[0100] i) a primary water phase containing peptides and arginine; and

[0101] ii) Provides a use for preparing peptide-containing microspheres of a dispersed phase composition comprising a polyester-based polymer and a secondary oil phase comprising an organic solvent.

[0102] In the present invention, the primary aqueous phase may additionally comprise hydroxypropyl-beta-cyclodextrin (HP-β-CD).

[0103] The definitions of the above "peptide," "arginine," "hydroxypropyl-beta-cyclodextrin," "polyester-based polymer," and "microsphere" are as described above regarding the peptide-containing microspheres.

[0104]

[0105] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0106]

[0107] Example 1: Preparation of Peptide-Containing Dispersed Phase - 1

[0108] 25 mg of semaglutide (Manufacturer: Bachem) and 15 mg of arginine (Manufacturer: Sigma Aldrich) were dissolved in 300 µl of purified water and stirred to completely dissolve them. Separately, 100 mg of PLGA (RG503H, Manufacturer: Evonik) was completely dissolved in 1.7 ml of dichloromethane. The purified water containing the dissolved semaglutide and arginine and the dichloromethane containing the dissolved PLGA were mixed, stirred using a vortex for 5 minutes, and sonicated to prepare a semaglutide-containing dispersed phase.

[0109]

[0110] Example 2: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 1

[0111] The semaglutide-containing dispersed phase prepared in Example 1 was left standing at 20°C for 18 hours and then injected into a microfluidic device. A 0.5% aqueous polyvinyl alcohol solution was injected into the microfluidic device as a continuous phase through a separate channel, and droplets were formed at the contact points within the microfluidic device. Subsequently, the formed droplets were completely dispersed in an aqueous solution containing 0.5% polyvinyl alcohol, and the organic solvent was evaporated by raising the temperature of the polyvinyl alcohol solution containing the dispersed droplets to 35°C or higher. The cured microspheres remaining after the organic solvent evaporated were obtained using a 0.45 μm PVDF membrane filter (Manufacturer: Hyundai Micro), washed with purified water, and freeze-dried.

[0112]

[0113] Example 3: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 2

[0114] Peptide-containing microspheres were prepared in the same manner as in Example 2 above, except that the polyester-based polymer was changed to PLA (R2O3H, manufacturer: Evonik).

[0115]

[0116] Example 4: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 3

[0117] 25 mg of semaglutide (Manufacturer: PolyPeptide Lab) and 15 mg of arginine (Manufacturer: Sigma Aldrich) were dissolved in 300 µl of purified water and stirred to completely dissolve them. Separately, 100 mg of PLGA (RG653H, Manufacturer: Evonik) was completely dissolved in 1.7 ml of dichloromethane. The purified water containing the dissolved semaglutide and arginine and the dichloromethane containing the dissolved PLGA were mixed, stirred using a vortex for 5 minutes, and sonicated to prepare a semaglutide-containing dispersed phase. Subsequently, peptide-containing microspheres were prepared using the dispersed phase in the same manner as in Example 2.

[0118]

[0119] Example 5: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 4

[0120] 50 mg of semaglutide (Manufacturer: Bachem), 14 mg of arginine (Manufacturer: Sigma aldrich), and 25 mg of hydroxypropyl-beta-cyclodextrin (Manufacturer: Sigma aldrich) were dissolved in 500 µl of purified water and stirred to completely dissolve them. Separately, 200 mg of PLGA RG502H (Manufacturer: Evonik) was completely dissolved in 1.5 ml of dichloromethane. The purified water containing the dissolved semaglutide, arginine, and hydroxypropyl-beta-cyclodextrin was mixed with the dichloromethane containing the dissolved PLGA RG502H, stirred using a vortex for 5 minutes, and sonicated to prepare a semaglutide-containing dispersed phase. Subsequently, peptide-containing microspheres were prepared using the dispersed phase in the same manner as in Example 2.

[0121]

[0122] Example 6: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 5

[0123] Peptide-containing microspheres were prepared in the same manner as in Example 5 above, except that the hydroxypropyl-beta-cyclodextrin was changed to 37.5 mg.

[0124]

[0125] Example 7: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 6

[0126] Peptide-containing microspheres were prepared in the same manner as in Example 5 above, except that the polyester-based polymer was changed to PLGA RG752H.

[0127]

[0128] Example 8: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 7

[0129] Peptide-containing microspheres were prepared in the same manner as in Example 5 above, except that the polyester-based polymer was changed to PLGA RG752H and the hydroxypropyl-beta-cyclodextrin was changed to 37.5 mg.

[0130]

[0131] Comparative Example 1: Preparation of Peptide-Containing Dispersed Phase - 2

[0132] A peptide-containing dispersed phase was prepared in the same manner as in Example 1 above, except that arginine was not added to the dispersed phase.

[0133]

[0134] Comparative Example 2: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 8

[0135] Peptide-containing microspheres were prepared in the same manner as in Example 2 above, except that there was no settling time (0 hours) after the preparation of the dispersed phase.

[0136]

[0137] Comparative Example 3: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 9

[0138] Peptide-containing microspheres were prepared in the same manner as in Example 2, except that the dispersed phase of Comparative Example 1, which did not contain arginine, was used instead of the dispersed phase of Example 1, and there was no settling time (0 hours) after the preparation of the dispersed phase.

[0139]

[0140] Comparative Example 4: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 10

[0141] Peptide-containing microspheres were prepared in the same manner as in Example 2, except that the dispersed phase of Comparative Example 1, which did not contain arginine, was used instead of the dispersed phase of Example 1.

[0142]

[0143] Comparative Example 5: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 11

[0144] Peptide-containing microspheres were prepared in the same manner as in Example 5 above, except that hydroxypropyl-beta-cyclodextrin was not added to the dispersed phase.

[0145]

[0146] Comparative Example 6: Preparation of peptide-containing microspheres using a peptide-containing dispersed phase - 12

[0147] Peptide-containing microspheres were prepared in the same manner as in Example 5 above, except that the polyester-based polymer in the dispersed phase was changed to PLGA RG752H and hydroxypropyl-beta-cyclodextrin was not added to the dispersed phase.

[0148]

[0149] Experimental Example 1: Test for Confirmation of Dispersed Phase Colloid Stability with Addition of Arginine

[0150] The dispersion phase of Example 1 and the dispersion phase of Comparative Example 1 were each left to stand at 20°C for 18 hours, and the stability of the dispersed phase colloid was confirmed, as shown in Figure 1.

[0151] As a result, as can be seen in Figure 1, in the case of Example 1, no phase separation was observed even after 18 hours, making it easy to manufacture microspheres by the microfluidic method, whereas in the case of Comparative Example 1, phase separation was observed, confirming that it is not easy to manufacture microspheres by the microfluidic method. This means that by adding arginine to the dispersed phase, microspheres can be manufactured by the microfluidic method over a long period of time without affecting the shape of the microspheres or the drug encapsulation rate.

[0152]

[0153] Experimental Example 2: Test of viscosity reduction according to the standing reaction time of the dispersed phase with added arginine

[0154] The shear stress of the dispersed phase over time was measured using a viscometer (Brookfield, Model: DV-II, Spindle No: 21) under a fixed rotational condition of 50 rpm while the dispersed phase of Example 1 underwent a static reaction at a temperature of 20°C, and this is shown in Figure 2.

[0155] As a result, as can be seen in Figure 2, it was confirmed that the shear stress is significantly reduced depending on the settling reaction time after the preparation of the dispersed phase. This means that the manufacturing method of the present invention, that is, the method of preparing the dispersed phase and then undergoing a settling reaction for an appropriate time, lowers the viscosity of the dispersed phase, thereby enabling the dispersed phase to be effectively injected into microfluidic channels for a long period of time when manufacturing microspheres using microfluidic technology.

[0156]

[0157] Experimental Example 3: Test to Confirm Differences in Microsphere Characteristics According to Settling Time After Dispersed Phase Preparation

[0158] The difference in the characteristics of the microspheres of Example 2 and Comparative Example 2 was confirmed using a scanning electron microscope (manufacturer: SEC, model name: SNE-3000MS), respectively, and this is shown in Fig. 3.

[0159] As a result, as can be seen in Figure 3, in the case of Example 2, which underwent a settling reaction after the preparation of the dispersed phase, spherical microspheres of uniform size were obtained, whereas in the case of Comparative Example 2, which did not undergo a settling reaction after the preparation of the dispersed phase, the size of the microspheres was not uniform and some non-spherical microspheres were obtained, indicating that the characteristics of the microspheres were deformed. This means that the manufacturing method of the present invention is effective in forming spherical and uniform microspheres when manufacturing microspheres by the microfluidic method by lowering the viscosity of the dispersed phase.

[0160]

[0161] Experimental Example 4: Test to Confirm Differences in Microsphere Characteristics Due to Addition of Arginine to the Dispersed Phase

[0162] The microspheres of Example 2, Comparative Example 3, and Comparative Example 4 were each examined using a scanning electron microscope (Manufacturer: SEC, Model: SNE-3000MS), and the characteristics of the microspheres were confirmed, as shown in Fig. 4.

[0163] As a result, as can be seen in FIG. 4, it was confirmed that in the case of microspheres prepared using a dispersed phase containing arginine and subjected to a static reaction for 18 hours according to the present invention (Example 2), the stability of the dispersed phase colloid was high and uniform spherical microspheres were formed. In contrast, when microspheres were prepared using a dispersed phase that did not contain arginine and did not undergo a static reaction (Comparative Example 3), and when microspheres were prepared using a dispersed phase that did not contain arginine and underwent a static reaction (Comparative Example 4), some particles did not form a spherical shape or exhibited non-uniform particle sizes. From this, it was specifically confirmed that the feature of the present invention, which includes a dispersed phase containing arginine and a step of subjecting to a static reaction after preparing the dispersed phase, helps in obtaining uniform spherical microspheres.

[0164]

[0165] Experimental Example 5: Comparative test of initial elution according to the addition of arginine to the dispersed phase

[0166] 20 mg each of the microspheres of Example 2 and Comparative Example 4 were taken and placed into a Lobind tube (Manufacturer: Eppendorf), then 20 mL of eluent (0.01 M Phosphate buffered saline, pH 7.4) was added, and a dissolution test was conducted using a shaking water bath (Manufacturer: Daehan Science) at 37°C and 50 rpm. After 24 hours, samples were collected, and the amount of eluted drug was quantitatively analyzed using HPLC, and the results are shown in Table 1 below.

[0167] As a result, as can be seen in Table 1 below, it was specifically confirmed that while no drug was released in Comparative Example 4, which did not contain arginine, in Example 2, which contained arginine, the interaction between the polymer and semaglutide was blocked, allowing semaglutide to be smoothly released from the microspheres.

[0168] 24-hour dissolution rate (%) Example 221.8 Comparative Example 40.0

[0169]

[0170] Experimental Example 6: Comparative test of particle shape according to polymer type

[0171] The characteristics of the microspheres in Example 2, Example 3, and Example 4 were confirmed using a scanning electron microscope (manufacturer: SEC, model name: SNE-3000MS), respectively, and are shown in Fig. 5.

[0172] As a result, as can be seen in Fig. 5, when comparing microspheres prepared using a dispersed phase containing a PLGA polymer (Examples 2 and 4) with microspheres prepared using a dispersed phase containing a PLA polymer (Example 3), there was no significant difference in the shape of the microspheres. Therefore, it was specifically confirmed that regardless of the type of polymer used, when microspheres are prepared using the method for preparing peptide-containing microspheres according to the present invention, uniform spherical microspheres can be obtained.

[0173]

[0174] Experimental Example 7: Test to Confirm Differences in Microsphere Characteristics Due to Addition of Hydroxypropyl-Beta-Cyclodextrin to the Dispersed Phase

[0175] The microspheres of Examples 5, 6, 7, and 8, and the microspheres of Comparative Examples 5 and 6 were each examined using a scanning electron microscope (manufacturer: SEC, model name: SNE-3000MS), and the characteristics of the microspheres were shown in Figures 6 and 7.

[0176] As a result, as can be seen in FIGS. 6 and 7, it was confirmed that microspheres prepared using a dispersion phase containing hydroxypropyl-beta-cyclodextrin (Examples 5, 6, 7, and 8) formed porous, uniform spherical microspheres with some pores on the surface, regardless of the type of polymer used; however, in contrast, when microspheres were prepared using a dispersion phase not containing hydroxypropyl-beta-cyclodextrin (Comparative Examples 5 and 6), non-porous microspheres were formed. From this, it was specifically confirmed that the feature of the present invention using a dispersion phase containing hydroxypropyl-beta-cyclodextrin helps in obtaining porous, uniform spherical microspheres.

[0177]

[0178] Experimental Example 8: Comparative test of initial elution according to the addition of hydroxypropyl-beta-cyclodextrin to the dispersed phase

[0179] 20 mg each of the microspheres from Examples 5, 6, 7, and 8, and the microspheres from Comparative Examples 5 and 6 were placed in a Lobind tube (Manufacturer: Eppendorf), 20 mL of eluent (0.01 M Phosphate buffered saline containing 0.2% Sodium Dodecyl Sulfate, pH 7.4) was added, and a dissolution test was conducted using a Shaking Water Bath (Manufacturer: Daehan Science) at 37°C and 50 rpm. After each designated time elapsed, samples were collected and the amount of eluted drug was quantitatively analyzed using HPLC. The time according to the drug dissolution rate is shown in Table 2 below.

[0180] As a result, as can be seen in Table 2 below, in the microspheres of Comparative Example 5 prepared with a dispersed phase not containing hydroxypropyl-beta-cyclodextrin, it took 14 days to reach an initial drug release rate of 10% and 70 days to reach a drug release rate of 85%. On the other hand, in the microspheres of Examples 5 and 6 prepared with a dispersed phase containing hydroxypropyl-beta-cyclodextrin, it took 1 to 3 days (10%) and 49 to 56 days (85%) to reach the same drug release rates, showing a significantly faster initial release pattern compared to the microspheres of Comparative Example 5. These results indicate that the hydrophobic interaction between the polymer and semaglutide is blocked as the hydrophobic portion of semaglutide interacts with the hydrophobic structure of the cyclodextrin (becomes encapsulated within the hydrophobic structure) upon the addition of hydroxypropyl-beta-cyclodextrin, thereby allowing semaglutide to be released more easily from the microspheres.

[0181] In addition, it was confirmed that the initial drug release rate increased in the microspheres of Examples 7 and 8, which were prepared by changing the polyester-based polymer to PLGA RG752H with higher hydrophobicity and using a dispersed phase containing hydroxypropyl-beta-cyclodextrin, compared to the microspheres of Comparative Example 6. This specifically confirmed that the above effect can be consistently exhibited regardless of the type of polymer.

[0182] Time according to drug dissolution rate 0~10% 15~30% 40~55% 70~85% Example 5 3 days 14 days 28 days 56 days Example 6 1 day 7 days 21 days 49 days Comparative Example 5 14 days 28 days 35 days 70 days Example 71 days 28 days 56 days 77 days Example 8 3 days 7 days 49 days 70 days Comparative Example 6 14 days 35 days 63 days 91 days

[0183]

[0184] From the foregoing description, those skilled in the art to which this application pertains will understand that this application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of this application should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. i) a primary phase comprising a peptide, arginine, and a primary phase solvent; and ii) A composition of a dispersed phase for preparing peptide-containing microspheres comprising a polyester-based polymer and a secondary oil phase comprising an organic solvent which is a secondary oil phase solvent.

2. A composition of a dispersed phase for manufacturing peptide-containing microspheres, wherein the primary aqueous phase further comprises hydroxypropyl-β-cyclodextrin, in accordance with claim 1.

3. A composition of a dispersed phase for manufacturing peptide-containing microspheres, wherein, in claim 1, the peptide has glycine and arginine at the C-terminus.

4. A composition of a dispersed phase for manufacturing peptide-containing microspheres according to claim 1, wherein the peptide comprises 10 to 40% (w / w%) of the total weight of the peptide, the polyester polymer, and the arginine.

5. A composition of a dispersed phase for manufacturing peptide-containing microspheres according to claim 2, wherein the peptide comprises 10 to 40% (w / w%) of the total weight of the peptide, polyester polymer, arginine, and hydroxypropyl-beta-cyclodextrin.

6. A composition of a dispersed phase for manufacturing peptide-containing microspheres according to claim 1, wherein the polyester-based polymer is one or more selected from the group consisting of polylactic-co-glycolide copolymer, polylactic-co-glycolic acid, polylactide, polylactic acid, and polycaprolactone.

7. A composition of a dispersed phase for manufacturing peptide-containing microspheres, wherein, in claim 1, the polyester-based polymer comprises 5 to 25% (w / v%) of the total volume of the organic solvent.

8. A composition of a dispersed phase for producing peptide-containing microspheres according to claim 1, wherein the arginine is one or more selected from the group consisting of arginine, pharmaceutically acceptable salts thereof, arginine derivatives, and polyarginine.

9. A dispersed phase composition for preparing peptide-containing microspheres according to claim 1, wherein the arginine is contained in a molar ratio of 2 to 25 times the molar number of the peptide based on a single molecule thereof.

10. A composition of a dispersed phase for preparing peptide-containing microspheres, wherein the organic solvent according to claim 1 is one or more selected from the group consisting of dichloromethane, chloroform, and ethyl acetate.

11. A composition of a dispersed phase for manufacturing peptide-containing microspheres, wherein, in claim 1, the peptide is semaglutide or liraglutide. 12.i) A step of preparing a water phase by dissolving the peptide and arginine in a water phase solvent; ii) A step of preparing a secondary oil phase by dissolving a polyester-based polymer in an organic solvent which is a secondary oil phase solvent; iii) a step of homogeneously mixing the primary aqueous phase and the secondary oil phase to prepare a dispersed phase that is an emulsion; iv) a step of reacting the dispersed phase in a static manner; and v) A method for producing peptide-containing microspheres comprising the step of injecting the dispersed phase into one microfluidic channel (MFC) and injecting a continuous phase containing a surfactant into a separate microfluidic channel different from the dispersed phase to generate microsphere droplets from the interface of the two microfluidic channels.

13. A method for preparing peptide-containing microspheres according to claim 12, wherein the step of preparing the primary aqueous phase is to dissolve a peptide, arginine, and hydroxypropyl-beta-cyclodextrin in a primary aqueous phase solvent.

14. A method for preparing peptide-containing microspheres according to claim 12, wherein the static reaction is performed on the dispersed phase at a temperature of 2 to 35°C for 2 to 20 hours.

15. A method for producing peptide-containing microspheres according to claim 12, wherein vi) the step of dispersing the generated microsphere droplets in a secondary water phase containing a surfactant while stirring.

16. A method for producing peptide-containing microspheres according to claim 15, wherein, after step vi), one or more processes selected from the group consisting of (a) removing an organic solvent from a dispersed microsphere droplet to cure it, (b) washing the cured microspheres, and (c) obtaining and drying the microspheres.

17. Peptide-containing microspheres comprising peptide, arginine, and polyester-based polymer.

18. The peptide-containing microspheres of claim 17, wherein the peptide-containing microspheres further comprise hydroxypropyl-beta-cyclodextrin.

19. In claim 17, the peptide-containing microspheres having glycine and arginine at the C-terminus.

20. Peptide-containing microspheres according to claim 17, wherein the peptide is semaglutide or liraglutide.

21. Peptide-containing microspheres according to claim 17, wherein the particle size of the microspheres is 5 to 100 μm.

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