Polycaprolactone porous microspheres as well as preparation method and application thereof

Abstract

The invention relates to a polycaprolactone porous microsphere and a preparation method and application thereof, the particle size of the microsphere is 20-50 [mu] m, and the internal porous structure of the microsphere is formed by a hydrophobic pore-foaming agent through liquid-liquid phase separation; the preparation method comprises the following steps: dissolving polycaprolactone and a hydrophobic pore-forming agent in an organic solvent to obtain an oil phase, mixing and emulsifying the oil phase and a polyvinyl alcohol water phase solution to form an O / W type emulsion, stirring and heating to enable the organic solvent and the pore-forming agent to volatilize synchronously, separating the pore-forming agent, and forming pores in the polycaprolactone curing and balling process, so as to obtain the polycaprolactone / hydrophobic pore-forming polymer. And finally, washing, freeze-drying and screening to obtain a target product. The preparation method provided by the invention is simple in process, high in controllability and easy for large-scale production, and the obtained microspheres have high specific surface area, excellent dispersity and injectability and good biocompatibility, are particularly suitable for preparing injectable soft tissue filling agents and other medical instruments, have outstanding beneficial effects and remarkable progress, and have wide application prospects. The method has great popularization and application values.

Description

Technical Field

[0001] This invention relates to a polycaprolactone porous microsphere, its preparation method and application, belonging to the technical field of medical device preparation and application. Background Technology

[0002] Poly(ε-caprolactone); abbreviated as PCL, is a biodegradable polymer and a biomedical material approved by the U.S. Food and Drug Administration (FDA). Due to its excellent biocompatibility and controllable degradation properties, it has been widely used in the biomedical and medical device fields. Based on the properties of PCL, its microsphere products have been widely used in drug delivery systems, tissue repair scaffolds, and injectable fillers. Because PCL microspheres can stimulate collagen production through gradual degradation, thereby achieving skin filling and long-lasting repair, it shows great potential in tissue regeneration and cosmetic repair. Especially in the cosmetic field, injectable products with PCL microspheres as the core ingredient are widely used for facial contouring and soft tissue filling.

[0003] Because most of the polycaprolactone microspheres in existing cosmetic products are solid microspheres prepared by emulsification solvent evaporation, spray drying and other technologies, they have a high density. When preparing injectable fillers, they are prone to deposition and local aggregation, which leads to high resistance and local stress concentration during injection, easily triggering inflammatory reactions, uneven filling effect, and even causing additional pain to patients.

[0004] To address the deposition and aggregation issues of solid microspheres, studies have explored the fabrication of porous polycaprolactone microspheres. On one hand, the introduction of pores improves the dispersibility and injectability of the microspheres, avoiding the deposition problems inherent in traditional solid microspheres in solution. On the other hand, the porous structure increases the specific surface area, which helps increase filling volume and promote tissue repair. Therefore, porous polycaprolactone microspheres are considered an important development direction for next-generation absorbable and regenerative injectable fillers.

[0005] Currently, the main methods for preparing porous microspheres include double emulsion / solvent evaporation, phase separation, spray drying, and microfluidics. For example, Sheffield et al. (Thomas E. Paterson et al., J. Mater. Chem. B 2024, 12, 11746) prepared high-porosity PCL microspheres with interconnected pores using a double emulsion method (W / O / W) combined with a photocuring step. However, this process involves multiple complex steps, making it difficult to achieve large-scale continuous production. Seng Yeol Kim et al. (Journal of Colloid and Interface Science, 465(2016)18-25) used a phase separation-based "liquid template" method to prepare PCL porous microspheres with a particle size distribution of 200–600 μm, but this method requires a selective extraction step to remove ionic liquids, making the process sensitive and difficult to implement industrially. Zhongguo Chen et al. (Polymers, 14(2022) 2687) achieved precise control of microsphere size and pore size (1-30 μm) by combining isodensity emulsion (IDE) templates with microfluidic technology. However, this strategy relies on microfluidic devices to generate droplets and requires the use of specific mixed solvents and customized amphiphilic block copolymers (PEG-b-PCL) as stabilizers and pore-forming regulators. Therefore, it faces challenges in efficiency and cost control in large-scale continuous production.

[0006] Furthermore, similar challenges exist in publicly disclosed patent technologies. For example, Chinese invention patent application CN115536900 A, entitled "A Method for Preparing Bioactive Absorbable Open Porous Polyester Microspheres," provides a two-step method for preparing polyester microspheres with a hierarchical pore structure (macropores 10–60 μm, micropores <10 μm) through a combination of reemulsification-solvent evaporation and chemical surface hydrolysis post-treatment. Although this method effectively expands the pore size of the microspheres through subsequent chemical etching, it increases the complexity of the process steps, and its target product particle size is approximately 230 μm, primarily designed as a cell carrier rather than optimized for injectable soft tissue filling applications. For example, Chinese invention patent application CN111249524 A, entitled "High-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and its preparation method," describes the one-step preparation of PCL porous microspheres with a porosity as high as 90.73% and a pore size of 43–217 μm through precise control of the parameters of the double emulsion-solvent evaporation method. The large pore size distribution is beneficial for bone tissue ingrowth. However, this method requires extremely strict control of process parameters such as emulsification rate, temperature, and phase ratio, making batch stability control very difficult. Furthermore, the target microsphere particle size is 250–1000 μm, which also fails to meet the injectability requirements for fine filling of facial and soft tissue using fine needles.

[0007] In summary, the polycaprolactone porous microspheres obtained by existing technologies still have significant shortcomings: on the one hand, polycaprolactone porous microspheres prepared by multi-step processes such as re-emulsification, phase separation, or porogen leaching often suffer from complex processes, unstable porous structures, and large batch-to-batch variations, which are not conducive to large-scale production; on the other hand, the polycaprolactone porous microspheres reported for applications such as bone defect filling typically have particle sizes ranging from tens to hundreds of micrometers (typical values ​​of about 200 μm or larger), making them difficult to inject smoothly through fine needles and unsuitable for applications requiring precise filling of the face and soft tissues.

[0008] In addition, traditional facial fillers have the following problems: First, biodegradable hyaluronic acid fillers mainly rely on physical filling and are gradually degraded and absorbed in the body, resulting in a short duration of effect and requiring repeated injections, which increases the pain and inconvenience for patients. Second, synthetic non-degradable microspheres, such as polymethyl methacrylate (PMMA) microspheres, cannot be metabolized and absorbed by the body, and may form nodules or remain for a long time at the injection site, causing local inflammation and tissue damage. Third, using solid polycaprolactone microspheres as fillers can easily aggregate during injection and cause local inflammatory reactions. At the same time, due to their high density, the effective filling volume of a single injection is limited, and the collagen production process is slow, which negatively affects the sustainability of tissue repair.

[0009] In summary, existing processes for preparing porous polycaprolactone microspheres generally suffer from numerous steps, complex procedures, and difficulties in efficiently completing sphere formation and porous structure construction in a single step. Furthermore, the resulting microspheres often have a limited number of surface pores, uneven distribution, or unsuitable particle size for fine injection, making it difficult to simultaneously achieve good injectability, suspension stability, and ideal filling volume. Therefore, there is an urgent need for a simpler process that allows for efficient one-step emulsification to prepare porous polycaprolactone microspheres with abundant and uniformly distributed surface pores and a suitable particle size for injection, thereby improving the injectability and filling effect of the microspheres and enhancing their tissue repair performance. Summary of the Invention

[0010] The present invention aims to overcome the shortcomings of existing technologies, such as complex preparation process of polycaprolactone porous microspheres, poor batch stability, difficulty in large-scale production, and unsuitability of product particle size for fine injection, and provides a polycaprolactone porous microsphere with uniform particle size, excellent pore structure, and suitable for injection applications, as well as its efficient and stable preparation method.

[0011] The core of this invention lies in the direct one-step construction of porous structures inside microspheres within an O / W emulsion system by employing specific hydrophobic porogens and optimized emulsification solvent evaporation processes, utilizing a liquid-liquid phase separation mechanism. This fully demonstrates the advantages of simple process, mild conditions, and easy scale-up. Furthermore, the particle size of the final product can be precisely controlled within a narrow range of 20–50 μm through sieving, thereby significantly improving the dispersibility, injectability, and batch-to-batch consistency of the microspheres. This makes them suitable for medical applications such as soft tissue filling, especially injectable applications for tissue regeneration or repair.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] First, the present invention provides a porous polycaprolactone microsphere.

[0014] The microspheres have a particle size of 20–50 µm, and their internal porous structure is formed by a hydrophobic porogen through liquid-liquid phase separation.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned polycaprolactone porous microspheres.

[0016] The method includes the following steps:

[0017] Polycaprolactone (PCL) was dissolved in an organic solvent, a hydrophobic porogen was added and mixed thoroughly to obtain an oil phase solution.

[0018] Polyvinyl alcohol (PVA) is dissolved in water to obtain an aqueous solution;

[0019] The oil phase solution and the aqueous phase solution are mixed and emulsified to form an O / W type emulsion.

[0020] The O / W emulsion is stirred and heated to evaporate the organic solvent and the hydrophobic porogen, and the polycaprolactone is solidified into spheres to obtain a microsphere suspension.

[0021] The microsphere suspension was subjected to solid-liquid separation, washing and drying to obtain crude polycaprolactone porous microspheres, which were then added to an aqueous mannitol solution and mixed evenly to obtain a mixture.

[0022] The mixture is freeze-dried and then sieved to collect microspheres with a particle size of 20-50 µm, thus obtaining the polycaprolactone porous microspheres.

[0023] Preferably, in the above preparation method:

[0024] The polycaprolactone has a molecular weight of 50,000 to 150,000 Da, and the hydrophobic porogen is selected from at least one of petroleum ether, liquid paraffin, pinene, camphene, or juniperene.

[0025] The organic solvent is dichloromethane or trichloromethane, and the concentration of polycaprolactone in the organic solvent is 0.5-10 wt%, and the mass ratio of polycaprolactone to the hydrophobic porogen is 1:(0.2-1).

[0026] The concentration of the polyvinyl alcohol aqueous solution is 0.5–10 wt%;

[0027] The volume ratio of the oil phase solution to the aqueous phase solution is 1:(5-10), the emulsification method is mechanical stirring, the stirring speed is (500-1000) rpm, and the emulsification time is 1-10 hours;

[0028] The oil phase solution and the aqueous phase solution are mixed by slowly adding the oil phase solution dropwise into the aqueous phase solution with a peristaltic pump while simultaneously mechanically stirring, in order to achieve a more stable and controllable emulsification process.

[0029] The O / W type emulsion is continuously stirred at a temperature of 30-50 °C for 10-50 hours to complete the evaporation of solvent and hydrophobic porogen, and to solidify and shape the generated polycaprolactone porous microparticles.

[0030] The added mannitol aqueous solution has a concentration of 0.5-10 wt%, and its addition helps to protect the microsphere structure and prevent collapse and adhesion during the subsequent freeze-drying process.

[0031] In addition, the present invention provides an injectable soft tissue filler comprising polycaprolactone porous microspheres as described above, because the specific particle size and porous structure of such microspheres endow the filler with excellent injection extrusion properties and in vivo dispersibility.

[0032] Furthermore, the present invention also provides the application of the above-mentioned polycaprolactone porous microspheres in the preparation of medical devices for tissue regeneration or repair.

[0033] The microspheres can be used as active ingredients or carriers to prepare medical devices that promote the regeneration of soft tissue or bone tissue, such as injectable fillers and tissue engineering scaffolds. Compared with the prior art, the outstanding beneficial effects and significant progress of the present invention are as follows:

[0034] First, this invention provides a polycaprolactone porous microsphere with uniform particle size and excellent pore structure and its preparation method. The core of the invention lies in using a specific hydrophobic porogen to construct the porous structure inside the microsphere in one step through a liquid-liquid phase separation mechanism in an optimized O / W emulsification solvent evaporation process. The obtained microspheres can be precisely controlled within a narrow range of 20 to 50 μm by sieving. The process is simple, the conditions are mild, and it is easy to scale up.

[0035] Obviously, the preparation method provided by this invention, compared with the prior art, which usually involves complicated steps, sensitive conditions, poor batch stability, and is difficult to achieve large-scale continuous production, such as the double emulsion method, phase separation method, or etching treatment route after the combination of double emulsion and phase separation, not only greatly simplifies the process, improves production efficiency and process controllability, but also improves production efficiency and industrial application and reduces production costs because it can complete spheroidization and pore formation simultaneously through only one step of emulsification and solvent evaporation.

[0036] Secondly, the preparation method provided by this invention significantly improves the key performance of its products compared to existing technologies. Existing technologies typically produce porous microspheres with particle sizes exceeding 200 μm, failing to meet the injectability requirements for fine facial and soft tissue filling via fine needles. Furthermore, the uniformity and stability of the porous structure of the microspheres are often insufficient. However, this invention, through optimization of the type and ratio of pore-forming agents and process parameters, not only stably obtains porous microspheres with abundant and uniformly distributed surface pores, but also successfully controls the microsphere particle size precisely between 20 and 50 μm. This size is significantly smaller than the microspheres commonly used in bone filling and other applications in existing technologies, allowing them to pass smoothly through finer injection needles. This greatly improves the dispersibility, suspension stability, and injection pushing performance of the microspheres, effectively avoiding problems such as high injection resistance, stress concentration, inflammatory reactions, and uneven filling caused by the high density and easy deposition and local aggregation of traditional solid microspheres.

[0037] Furthermore, in terms of final application effects, compared with the shortcomings of hyaluronic acid fillers, such as short duration of action and the need for repeated injections, or the potential for long-term residue and hardening caused by non-degradable microspheres such as PMMA, the porous microspheres obtained in this invention have both biodegradability and excellent pore structure. Their moderately increased specific surface area not only provides a better filling volume, but also increases the filling volume and specific surface area at the same dosage, and provides effective mechanical support, enhances the tissue repair ability, and is more conducive to promoting the continuous generation of collagen and tissue ingrowth, thereby achieving a longer-lasting and natural tissue repair and regeneration effect.

[0038] Furthermore, the preparation method provided by this invention further ensures the integrity of the microsphere structure and effectively prevents adhesion by introducing mannitol-assisted treatment and sieving steps, thus ensuring the high uniformity of the particle size of the final product, thereby significantly improving the consistency between batches and the reliability and safety of clinical applications.

[0039] It can be seen that the polycaprolactone porous microspheres and their preparation method provided by this invention have made substantial progress in terms of process simplification, precise particle size control, improved injection performance, and enhanced tissue repair effect. They are particularly suitable as the core component of next-generation injectable soft tissue fillers and have outstanding application value in medical fields such as facial fine filling and soft tissue regeneration. Attached Figure Description

[0040] To more clearly illustrate the technical solution of the present invention and the technical effects of implementing the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0041] Obviously, the accompanying drawings described below are only some of the drawings in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0042] Figure 1 SEM image of polycaprolactone microspheres-a obtained in Example 1 of this invention;

[0043] Figure 2 SEM image of polycaprolactone porous microspheres-b obtained in Example 2 of this invention;

[0044] Figure 3 SEM image of polycaprolactone porous microspheres-c obtained in Example 3 of this invention;

[0045] Figure 4 SEM image of polycaprolactone porous microspheres-d obtained in Example 4 of this invention;

[0046] Figure 5SEM image of polycaprolactone porous microspheres-e obtained in Example 5 of this invention;

[0047] Figure 6 SEM image of the polycaprolactone porous microspheres-f obtained in Example 6 of this invention;

[0048] Figure 7 A collection of cell viability and mortality staining images after co-culturing SMs and PMs with L929 cells for 1, 3, and 5 days, as provided in Example 2 of the present invention;

[0049] Figure 8 The bar charts showing the changes in the activity of SMs and PMs co-cultured with L929 cells for 1, 3, and 5 days, as provided in Example 2 of the present invention, indicate the effects of the invention. In the charts, the horizontal axis represents the culture time (days), and the vertical axis represents the cell absorbance of 450nm light. "ns" indicates no statistically significant difference, and "***" indicates an extremely significant statistical difference. ;

[0050] Figure 9 The bar charts showing the cell viability of SMs and PMs co-cultured with L929 cells for 1, 3, and 5 days, as provided in Example 2 of the present invention, indicate the following: In the charts, the horizontal axis represents the culture time (days), and the vertical axis represents the cell viability (%). "ns" indicates no statistical difference, and "*" indicates a statistical difference. . Detailed Implementation

[0051] To make the technical solution, beneficial effects and significant progress of the present invention clearer and more comprehensive, the technical solution provided by the present invention will be clearly and completely described below through specific embodiments and their effects. Obviously, all embodiments and their effects described below are only some embodiments and effects of the present invention, and not all of them.

[0052] Based on the embodiments and effects provided by this invention, all other embodiments and effects obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0053] It should be noted that:

[0054] The terms "firstly," "secondly," etc., used in the claims, description, and examples and effects of the embodiments of this invention are only used to distinguish different objects and not to describe a specific order; furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion, for example, including not only a series of listed steps or units of a process, method, system, product, or device, but also optionally steps or units not listed, or optionally other operational steps or units inherent to these processes, methods, products, or devices.

[0055] What needs to be understood is:

[0056] In the description of the embodiments of the present invention, some basic operational terms commonly used in the art are used, such as "stirring" and "dissolving". These terms should be interpreted broadly, that is, they can refer to routine operations performed using various conventional equipment and instruments in the art, or operations performed using the latest equipment, such as program-controlled operations and unmanned automatic operations. Unless otherwise explicitly limited, those skilled in the art should understand the specific meaning of the above terms in the present invention according to the specific circumstances and adopt specific operating methods to achieve their operating objectives.

[0057] It should also be noted that:

[0058] The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some implementation cases and comparative examples;

[0059] In addition, all instruments, equipment, raw materials, reagents and standards involved in the following specific embodiments are commercially available unless otherwise specified.

[0060] The technical solution of the present invention will now be described in detail with reference to specific embodiments. Example 1

[0061] This embodiment provides a method for preparing polycaprolactone porous microspheres with a particle size of 20–50 µm.

[0062] The method for preparing polycaprolactone porous microspheres with a particle size of 20–50 µm provided in this embodiment includes the following steps:

[0063] Polycaprolactone was dissolved in an organic solvent, a hydrophobic porogen was added and mixed evenly to obtain an oil phase solution;

[0064] Polyvinyl alcohol is dissolved in water to obtain an aqueous solution;

[0065] The oil phase solution and the aqueous phase solution are mixed and emulsified to form an O / W type emulsion.

[0066] The O / W emulsion is stirred and heated to evaporate the organic solvent and the hydrophobic porogen, and the polycaprolactone is solidified into spheres to obtain a microsphere suspension.

[0067] The microsphere suspension was subjected to solid-liquid separation, washing and drying to obtain crude polycaprolactone porous microspheres, which were then added to an aqueous mannitol solution and mixed evenly to obtain a mixture.

[0068] The mixture is freeze-dried and then sieved to collect microspheres with a particle size of 20-50 µm, thus obtaining the polycaprolactone porous microspheres.

[0069] In the above preparation method:

[0070] The molecular weight of polycaprolactone is preferably 50,000 to 150,000 Da, and the hydrophobic porogen is selected from at least one of petroleum ether, liquid paraffin, pinene, camphene or juniperene.

[0071] The organic solvent is preferably dichloromethane or chloroform, and the concentration of polycaprolactone in the organic solvent is preferably 0.5 to 10 wt%, and the mass ratio of polycaprolactone to hydrophobic porogen is preferably 1:(0.2 to 1).

[0072] The concentration of the polyvinyl alcohol aqueous solution is preferably 0.5–10 wt%;

[0073] The preferred volume ratio of the oil phase solution to the aqueous phase solution is 1:(5-10). The emulsification method can be mechanical stirring, the preferred stirring speed is (500-1000) rpm, and the emulsification time is 1-10 hours.

[0074] Furthermore, the mixing of the oil phase solution and the aqueous phase solution can be achieved by slowly adding the oil phase solution dropwise into the aqueous phase solution using a peristaltic pump while simultaneously performing mechanical stirring.

[0075] The O / W type emulsion is continuously stirred at a temperature of 30-50 °C for 10-50 hours to complete the evaporation of solvent and hydrophobic porogen, and to solidify and shape the generated polycaprolactone porous microparticles.

[0076] The concentration of the added mannitol aqueous solution is preferably 0.5–10 wt%.

[0077] To further aid in understanding the technical solution provided in this embodiment, as well as the specific operation process and technical effects achievable, the preparation method provided in this embodiment will be further explained below through specific preparation examples.

[0078] Of course, those skilled in the art should understand that the preparation examples described below are illustrative rather than restrictive and should not be used to limit the scope of protection claimed by the present invention; and the preparation examples below only use some preferred proportioning parameters and operating parameters in this embodiment as examples. In fact, within the range of all optional materials, proportioning parameters and operating parameters provided in this embodiment, the same results can be obtained. This embodiment is only for the sake of simplicity and will not be described in detail. Preparation Example 1 (Preparation of a control sample without the addition of a hydrophobic porogen)

[0079] 3 g of polycaprolactone (PCL) with a molecular weight of 125000 Da was added to 100 ml of dichloromethane solution and magnetically stirred until completely dissolved to obtain the oil phase.

[0080] Prepare a 1 wt% polyvinyl alcohol (PVA) aqueous solution, and then slowly add the above oil phase dropwise to 600 ml of PVA solution using a peristaltic pump. During the dropwise addition, mechanical stirring is performed at a stirring rate of 600 rpm. After the dropwise addition is completed, continue stirring for 4 hours until complete emulsification.

[0081] The emulsion is heated to 40°C, and the outer cover of the reaction vessel is opened to allow dichloromethane to evaporate. During the evaporation of dichloromethane, the polycaprolactone microparticles in the emulsion begin to solidify into spheres.

[0082] After the dichloromethane has completely evaporated, remove the supernatant from the reaction vessel, retain the solidified microspheres at the bottom of the reaction vessel, wash them three times with deionized water, add a small amount of 3 wt% mannitol aqueous solution, mix well, and freeze-dry for 72 hours.

[0083] The PCL microspheres obtained after freeze-drying are sieved through a metal sieve with a pore size of 20–50 µm to obtain polycaprolactone microspheres-a with an average particle size of 39 ± 4 µm.

[0084] Electron microscopy scanning of this polycaprolactone microsphere-a yielded the following results: Figure 1 The image shown is a SEM image of the polycaprolactone microspheres-a obtained in Preparation Example 1 of the present invention.

[0085] Figure 1 The results show that polycaprolactone microspheres prepared by this method, i.e., without the addition of hydrophobic porogens during the preparation process, are solid microspheres without porous structures. Preparation Example 2 (using 1 wt% petroleum ether as a hydrophobic porogen)

[0086] Add 3g of polycaprolactone with a molecular weight of 50000 Da to 100ml of dichloromethane, and add petroleum ether, a hydrophobic porogen, at a concentration of 1wt% of the reaction system. Stir magnetically until completely dissolved to form the oil phase.

[0087] Prepare 600 ml of 1.5 wt% polyvinyl alcohol (PVA) aqueous solution in a round-bottom flask. Slowly add the oil phase dropwise to the PVA solution using a peristaltic pump. Mechanically stir at 800 rpm for 4 hours to complete emulsification and obtain an emulsion.

[0088] The emulsion was transferred to a 30°C electric heating mantle for curing, i.e., the reaction system was kept at 30°C under stirring to allow the solvent to evaporate; it can be seen that during the evaporation of dichloromethane and the curing of microspheres, the pore-forming agent petroleum ether aggregated on the surface and inside of the microspheres, and then evaporated together with the solvent, causing the polycaprolactone microspheres to form pores inside and on the surface during the curing process.

[0089] After the solvent has completely evaporated, the microspheres were washed and a 3 wt% mannitol aqueous solution was added, followed by freeze drying for 72 hours.

[0090] After freeze-drying, porous PCL microspheres are obtained. After sieving through a 20-50 μm metal sieve, polycaprolactone porous microspheres-b with an average particle size of 37±5 μm are obtained.

[0091] Electron microscopy was performed on these polycaprolactone porous microspheres-b to obtain the following results: Figure 2 SEM image of polycaprolactone porous microspheres-b obtained in Example 2 of this invention.

[0092] Figure 2 The results show that the polycaprolactone porous microspheres-b have pores on their surface, but the number of pores is small and the pores are unevenly distributed. Preparation Example 3 (using 3 wt% petroleum ether as a hydrophobic porogen)

[0093] The implementation steps of this preparation example are similar to those of preparation example 2, except that:

[0094] The added polycaprolactone has a molecular weight of 125,000 Da, the added petroleum ether has a concentration of 3 wt%, the PVA concentration is 3 wt%, the stirring speed is 600 rpm, the curing temperature is 40°C, and the resulting microspheres, namely polycaprolactone porous microspheres-c, have an average particle size of 37 ± 6 μm.

[0095] Electron microscopy scanning of this polycaprolactone porous microsphere-c yielded the following results: Figure 3 The image shown is a SEM image of the polycaprolactone porous microspheres-c obtained in Preparation Example 3 of the present invention.

[0096] Figure 3The results showed that polycaprolactone porous microspheres-c had more pores on the surface due to the increased concentration of pore-forming agent during the preparation process, but the distribution was still uneven. Preparation Example 4 (using 5 wt% petroleum ether as a hydrophobic porogen)

[0097] The implementation steps of this preparation example are similar to those of preparation example 2, except that:

[0098] The added polycaprolactone has a molecular weight of 150,000 Da, the concentration of petroleum ether is increased to 5 wt%, and the average particle size of the resulting polycaprolactone microspheres-d is 38 ± 6 μm.

[0099] Electron microscopy scanning of this polycaprolactone porous microsphere-d yields the following results: Figure 4 The image shown is a SEM image of the polycaprolactone porous microspheres-d obtained in Example 4 of the present invention.

[0100] Figure 4 The results show that polycaprolactone porous microspheres-d have increased surface pores due to the increased concentration of pore-forming agent during the preparation process, but the pores are unevenly distributed and have irregular shapes.

[0101] The three preparation examples 2, 3 and 4 above show that petroleum ether can be used as a porogen for polycaprolactone porous microspheres, but it is not the optimal porogen for forming a uniform porous structure. Example 5 (using 1 wt% camphene as a hydrophobic porogen)

[0102] 3g of polycaprolactone with a molecular weight of 50,000 Da was added to 100ml of dichloromethane, and camphene, a hydrophobic porogen, was added at a concentration of 1wt% of the reaction system. The mixture was magnetically stirred until completely dissolved to obtain the oil phase.

[0103] Prepare 600 ml of 3 wt% PVA aqueous solution. Slowly add the oil phase to the PVA aqueous solution using a peristaltic pump while stirring and emulsifying at 600 rpm for 4 hours to obtain an emulsion.

[0104] Turn on the electric heating mantle to heat the emulsion to 40 degrees Celsius. o Under C and stirring conditions, the reaction vessel opening is opened to volatilize dichloromethane. It can be seen that as dichloromethane volatilizes, the polycaprolactone microparticles in the emulsion begin to solidify into spheres. At the same time, the hydrophobic porogen camphene accumulates on the surface and inside of the microspheres that have not yet been completely solidified, and then volatilizes along with the solvent, causing the polycaprolactone microspheres in the solidification process to form pores inside and on their surface.

[0105] After the dichloromethane has completely evaporated, remove the supernatant from the reaction system, retain the microspheres that have been solidified at the bottom, wash them repeatedly with deionized water three times, add a small amount of 3 wt% mannitol aqueous solution, and freeze-dry for 72 hours.

[0106] After freeze-drying, porous PCL microspheres were obtained. After sieving through a 20-50 μm metal sieve, polycaprolactone porous microspheres-e with an average particle size of 37±6 μm were obtained.

[0107] Electron microscopy was performed on these polycaprolactone porous microspheres-e to obtain the following results: Figure 5 The image shown is a SEM image of the polycaprolactone porous microspheres-e obtained in Example 5 of the present invention.

[0108] Figure 5 The results show that polycaprolactone porous microspheres-e have a uniform distribution of pores on their surface and exhibit a dense, concave structure due to the use of 1 wt% of the hydrophobic porogen camphene in their preparation process. Preparation Example 6 (using 3 wt% camphene as a hydrophobic porogen)

[0109] The implementation steps of this preparation example are similar to those of preparation example 5, except that:

[0110] The added polycaprolactone has a molecular weight of 125,000 Da, and the added camphene has a concentration of 3 wt%. The resulting polycaprolactone porous microspheres-f have an average particle size of 37 ± 5 μm.

[0111] Electron microscopy was performed on these polycaprolactone porous microspheres-f to obtain the following results: Figure 6 The image shown is a SEM image of the polycaprolactone porous microspheres-f obtained in Example 6 of the present invention.

[0112] Figure 6 The results show that, compared with polycaprolactone porous microspheres-e, polycaprolactone porous microspheres-f have a further increase in surface pores, which are evenly distributed, with obvious concave structures and the best morphology.

[0113] It can be seen that camphene at a concentration of 3 wt% is a better hydrophobic porogen option for forming a uniform porous structure of polycaprolactone. Preparation Example 7 (using 5 wt% camphene as a hydrophobic porogen)

[0114] The implementation steps of this preparation example are similar to those of Preparation Example 4, except that the added polycaprolactone has a molecular weight of 150,000 Da and the added camphene has a concentration of 5 wt%.

[0115] Experiments revealed that during the preparation process, after the solvent dichloromethane had completely evaporated, the solidified microspheres and excess unevaporated camphene precipitated together, leading to difficulties in post-processing and making it impossible to obtain polycaprolactone porous microspheres with a uniform porous structure.

[0116] It needs to be stated again:

[0117] Although the above preparation examples are mainly prepared using the preferred technical solutions provided in this embodiment, it is fully understood by those skilled in the art that all technical solutions provided in this embodiment, including all optional materials and their ratios, and all operating parameter ranges, can yield the corresponding target product. However, due to different ratios and different operating control parameters, the product yield or product characteristics may fluctuate within a reasonable range. Therefore, this specification only provides a more detailed description of the preparation using the optimized method, and does not provide a detailed description of other possible preparation schemes.

[0118] To further help understand the technical effects that the technical solution provided in this embodiment can achieve, the following will use specific effect examples to conduct corresponding effect tests and comparisons on the polycaprolactone microspheres prepared in this embodiment, thereby further explaining the technical effects that can be obtained in this embodiment. Example 1 (characterization of porous structure)

[0119] The polycaprolactone microspheres prepared in Examples 1, 4 and 6 were subjected to mercury porosimetry to quantitatively analyze the porous structure of the obtained microparticles. The results are shown in Table 1. Table 1

[0120]

[0121] As can be seen from the data listed in Table 1:

[0122] The polycaprolactone microspheres prepared in Example 1 were solid microspheres with smooth surfaces and extremely low specific surface area (0.29 m² / g).

[0123] Polycaprolactone microspheres prepared using 5 wt% petroleum ether as a hydrophobic porogen are porous microspheres, and their specific surface area can be increased to 4.06 m² / g, but the increase in total pore volume and porosity is limited.

[0124] Polycaprolactone microspheres prepared using 3 wt% camphene as a hydrophobic porogen are uniformly porous microspheres, exhibiting the best porous structure. Their specific surface area (5.91 m² / g) is about 20 times that of solid microspheres, and their total pore volume and porosity (62.61%) are the highest among the three types of microspheres listed in the table. Example 2 (Biocompatibility Verification)

[0125] The polycaprolactone porous microspheres-f (PMs) obtained in Preparation Example 6 and the polycaprolactone solid microspheres-a (SMs) obtained in Preparation Example 1 were co-cultured with L929 cells for 1, 3, and 5 days, respectively. Then, cell staining was performed to detect cell viability (blue) and cell death (red). At the same time, cell toxicity (CCK-8) and proliferation performance tests were completed by detecting changes in cell activity and survival rate of co-cultured cells.

[0126] Through the above experiments, we can obtain the following results respectively: Figure 7 The image shown is a collection of cell staining images of SMs and PMs co-cultured with L929 cells for 1, 3, and 5 days, as provided in Example 2 of the present invention. Figure 8 The figures shown are bar charts illustrating the changes in the activity of SMs and PMs after co-culturing with L929 cells for 1, 3, and 5 days, as provided in Example 2 of the present invention. Figure 9 The bar chart shown is a graph of cell survival rates after 1, 3, and 5 days of co-culturing SMs and PMs with L929 cells provided in Example 2 of the present invention.

[0127] from Figure 7 As can be seen from the images, almost no dead cells (red) are visible in any of the images.

[0128] from Figure 8 and Figure 9 As can be seen, the OD value representing cell viability in the porous microsphere (PMs) group was not significantly different from that in the control group and the solid microsphere (SMs) group.

[0129] Example 2 demonstrates that the introduction of the hydrophobic porogen camphene and the resulting porous structure did not adversely affect the biocompatibility of the obtained polycaprolactone porous microspheres. Its toxicity is very low and can meet the biological safety requirements of injection filler materials.

[0130] It needs to be stated again:

[0131] Although the above comparative examples are limited experiments conducted on products prepared using the preferred technical solutions provided in this embodiment, it is fully understood by those skilled in the art that all products prepared using all technical solutions provided in this embodiment, including all optional materials and their proportions, and all operating parameter ranges, can achieve the same or similar technical effects, although they may fluctuate within a reasonable range. Therefore, this specification only provides a more detailed description of the effects of the products prepared using the optimized method, and does not provide a detailed description of the corresponding technical effects of products obtained by other possible preparation schemes.

[0132] In summary, the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

[0133] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features, and such modifications or substitutions do not cause the corresponding technical solutions to deviate from the spirit and scope of the technical solutions given in the embodiments of the present invention. Example 2

[0134] This embodiment provides an injectable soft tissue filler.

[0135] The soft tissue filler provided in this embodiment comprises polycaprolactone porous microspheres with a particle size of 20-50 µm prepared by the preparation method provided in Example 1 above, and whose internal porous structure is formed by hydrophobic porogen through liquid-liquid phase separation. Due to the specific particle size and porous structure of such microspheres, the filler has excellent injection extrusion performance and in vivo dispersibility, so it can be used as an injectable soft tissue filler. Example 3

[0136] This embodiment provides an application of the polycaprolactone porous microspheres provided in Example 1 above in the preparation of medical devices for tissue regeneration or repair.

[0137] Since the polycaprolactone porous microspheres provided in Example 1 have a particle size of 20-50 µm and their internal porous structure is formed by a hydrophobic porogen through liquid-liquid phase separation, as demonstrated in Example 2, they have very low toxicity and do not have an adverse effect on biocompatibility. They can meet the biological safety requirements of injectable filler materials. Therefore, they can be used as active ingredients or carriers to prepare medical devices that promote the regeneration and repair of soft tissue or bone tissue, such as injectable fillers, tissue engineering scaffolds, etc.

[0138] In conclusion, it can be seen that:

[0139] This invention provides a porous polycaprolactone microsphere suitable for injectable soft tissue filling and its one-step preparation process. The particle size of the porous microsphere is precisely controlled within the range of 20-50 μm. The porous structure inside is formed in situ during the curing process by co-dissolving a hydrophobic porogen (such as camphene, preferred) with polycaprolactone in the organic phase and utilizing a liquid-liquid phase separation mechanism. The preparation process adopts an optimized O / W single emulsion solvent evaporation technology. The key steps include oil phase emulsification, simultaneous evaporation of solvent and porogen, and final strict sieving. The entire process route is simple, the parameters are controllable, and it has good potential for large-scale production.

[0140] In addition, the present invention provides an injectable soft tissue filler comprising polycaprolactone porous microspheres as described above, because the specific particle size and porous structure of such microspheres endow the filler with excellent injection extrusion properties and in vivo dispersibility.

[0141] Furthermore, the present invention also provides the application of the above-mentioned polycaprolactone porous microspheres in the preparation of medical devices for tissue regeneration or repair. The microspheres can be used as active ingredients or carriers to prepare medical devices that promote the regeneration of soft tissue or bone tissue, such as injectable fillers, tissue engineering scaffolds, etc.

[0142] The main advantages of this invention are:

[0143] First, its one-step process avoids complex multi-step processing, significantly improving production efficiency and batch stability;

[0144] Secondly, the microspheres produced have both structural and functional properties. Their uniform porous structure allows the specific surface area to be about 20 times that of solid microspheres, and the porosity to be as high as 62.6%. The precise narrow particle size distribution ensures the product’s excellent dispersibility and injectability, and can effectively reduce injection resistance and the risk of local aggregation.

[0145] Furthermore, this material maintains good biocompatibility, successfully expanding the application of biodegradable polycaprolactone to the field of injectable soft tissue repair, demonstrating significant clinical translational value. Compared with existing technologies, it shows outstanding beneficial effects and significant progress, thus possessing great value for promotion and application.

[0146] In the description process of the above instruction manual:

[0147] The terms “this embodiment,” “this embodiment of the invention,” “this case,” “this comparative example,” “as shown,” “further,” etc., are used to indicate that the specific features, structures, materials, or characteristics described in the embodiment or case or comparative example are included in at least one embodiment or case or comparative example of the present invention.

[0148] In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiments, preparation examples, or effect examples. Moreover, the specific features, structures, materials, or characteristics described may be combined or combined in any suitable manner in one or more embodiments, preparation examples, or effect examples. Furthermore, without creating contradictions, those skilled in the art may combine or combine the different embodiments, preparation examples, or effect examples described in this specification, as well as the features in the different embodiments, preparation examples, or effect examples.

[0149] Finally, it should be noted that:

[0150] The above embodiments and comparative examples are only used to illustrate the technical solutions and technical effects of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, examples, and comparative examples, those skilled in the art should understand that modifications or supplements can still be made to the technical solutions or technical effects described in the foregoing embodiments, examples, and comparative examples, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the embodiments of the present invention. Non-essential improvements, adjustments, or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A porous polycaprolactone microsphere, characterized in that, The microspheres have a particle size of 20–50 µm, and their internal porous structure is formed by a hydrophobic porogen through liquid-liquid phase separation.

2. A method for preparing polycaprolactone porous microspheres as described in claim 1, characterized in that, Includes the following steps: Polycaprolactone was dissolved in an organic solvent, a hydrophobic porogen was added and mixed evenly to obtain an oil phase solution; Polyvinyl alcohol is dissolved in water to obtain an aqueous solution; The oil phase solution and the aqueous phase solution are mixed and emulsified to form an O / W type emulsion. The O / W type emulsion is stirred and heated to volatilize the organic solvent and the hydrophobic porogen, and to solidify and shape the generated polycaprolactone porous microspheres to obtain a microsphere suspension. The microsphere suspension was subjected to solid-liquid separation, washing and drying to obtain crude polycaprolactone porous microspheres, which were then added to an aqueous mannitol solution and mixed evenly to obtain a mixture. The mixture is freeze-dried and then sieved to collect microspheres with a particle size of 20-50 µm, thus obtaining the polycaprolactone porous microspheres.

3. The preparation method according to claim 2, characterized in that, The polycaprolactone has a molecular weight of 50,000 to 150,000 Da, and the hydrophobic porogen is selected from at least one of petroleum ether, liquid paraffin, pinene, camphene, or juniperene.

4. The preparation method according to claim 2 or 3, characterized in that, The organic solvent is dichloromethane or trichloromethane, and the concentration of polycaprolactone in the organic solvent is 0.5 to 10 wt%, and the mass ratio of polycaprolactone to the hydrophobic porogen is 1:(0.2 to 1).

5. The preparation method according to claim 2, characterized in that, The concentration of the polyvinyl alcohol aqueous solution is 0.5–10 wt%.

6. The preparation method according to claim 2, characterized in that, The volume ratio of the oil phase solution to the aqueous phase solution is 1:(5-10), the emulsification method is mechanical stirring, the stirring speed is 500-1000 rpm, and the emulsification time is 1-10 hours.

7. The preparation method according to claim 2, characterized in that, The oil phase solution and the aqueous phase solution are mixed by slowly adding the oil phase solution dropwise into the aqueous phase solution using a peristaltic pump, while simultaneously stirring using a mechanical device.

8. The preparation method according to claim 2, characterized in that: The O / W type emulsion is continuously stirred at a temperature of 30-50 °C for 10-50 hours to complete the evaporation of the solvent and the hydrophobic porogen, and to solidify and shape the generated polycaprolactone porous microparticles. The concentration of the added mannitol aqueous solution is 0.5–10 wt%.

9. An injectable soft tissue filler, characterized in that, It comprises the polycaprolactone porous microspheres as described in claim 1.

10. The use of the polycaprolactone porous microspheres as described in claim 1 in the preparation of medical devices for tissue regeneration or repair.

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