High polymer material hollow microsphere as well as preparation method and application thereof

Abstract

Compared with a porous microsphere in the prior art, the polymer material microsphere with the closed hollow structure is provided with the closed hollow structure or a gas chamber, the surface of the microsphere is smooth on the whole, and acute inflammatory reaction can be avoided. By adopting the microspheres prepared by the invention, the total material implantation amount can be reduced when the same regenerative stimulation point is implanted; and the preparation process is simple, and large-scale production is facilitated.

Description

Technical Field

[0001] The present application relates to the field of medical cosmetology, and in particular, to a hollow microsphere made of a polymer material, and a preparation method and application thereof. Background Art

[0002] In recent years, medical cosmetology has evolved from simple filling to regeneration, that is, by injecting microspheres subcutaneously to stimulate the recipient to produce collagen, so as to achieve the purpose of long-term wrinkle removal. Biodegradable polycaprolactone has been developed into an injection. After subcutaneous injection, each microsphere becomes a collagen regeneration stimulation point, and new collagen is produced around the microsphere, achieving a gradual and natural anti-aging effect. Studies have shown that the rough surface of the microsphere will increase the degree of inflammatory response. Therefore, the two PCL microsphere products currently approved in China are both smooth in surface.

[0003] The collagen regeneration capacity is positively correlated with the number of implanted microspheres, but a high implantation amount of microspheres means the implantation of more exogenous substances, which may lead to increased inflammatory response during the degradation process. Porous microspheres can reduce the amount of polymer implanted while ensuring the number of implanted microspheres, but the surface of porous microspheres is not smooth enough and is prone to acute inflammation after injection.

[0004] Polymer microspheres are also often used in arterial or venous embolism to treat diseases such as aneurysms by blocking or reducing blood flow.

[0005] Porous microspheres are often prepared by the double emulsion method: the inner aqueous phase is dispersed in the polymer solution (oil phase) in the form of droplets to prepare a W / O emulsion, which is then added to the outer aqueous phase containing an emulsifier to obtain a W / O / W emulsion after emulsification. The organic solvent in the oil phase is removed to obtain porous microspheres. The stability of the inner aqueous phase droplets is the key to the preparation of porous microspheres. Adding an emulsifier is an effective method to stabilize the inner aqueous phase droplets, but the emulsifier will be encapsulated inside the microspheres and is difficult to remove, increasing the toxicity risk of the microspheres. Osmotic pressure active substances are also often added to the inner aqueous phase to generate an osmotic pressure difference between the inner aqueous phase and the outer aqueous phase, driving the penetration of the outer aqueous phase into the inner aqueous phase, and preparing open-pored microspheres with internal and external through-channel structures. The volatilization rate of the oil phase solvent directly affects the size of the micropores. If the volatilization rate is too slow, the oil phase will be in a low viscosity state for a long time, and the inner aqueous phase droplets may merge. In addition, the penetration of the external water phase into the internal water phase driven by the osmotic pressure difference will also increase the pore size of the porous microspheres.

[0006] Patent 202010061829.8 reports a high-porosity polycaprolactone porous microsphere scaffold for bone tissue regeneration and its preparation method. The characteristics of the polycaprolactone porous microspheres are spherical particles with interconnected pores on the surface and inside, large pore size (43-217μm), and high porosity (75.28-90.73%). Patent 202210476085.5 discloses a method for preparing an emulsion template and a polymer porous microsphere derived therefrom, including polycaprolactone porous microspheres. The method is characterized by using composite dissolution to prepare an oil phase with the same density as the water phase, improving the suspension stability of the dispersed phase in the continuous phase, and reducing the merging of emulsion droplets. It has been reported in the literature that camphene or kerosene is used as a porogen, and the emulsification-solvent evaporation method is used to prepare polycaprolactone open-cell microspheres with interconnected pores. Summary of the invention

[0007] In order to solve the above problems, the present application proposes a polymer material microsphere, wherein the polymer material is one or more polymers, the melting temperature of the polymer is less than 100° C., and the microsphere has one or more closed hollow structures inside.

[0008] Preferably, the polymer is a homopolymer of caprolactone or a copolymer with caprolactone as the main monomer; "main monomer" means that the weight content of the caprolactone monomer in the copolymer is the highest.

[0009] Preferably, the tap density of the polymer material microspheres is less than 0.70 g / ml, preferably less than 0.65 g / ml, more preferably less than 0.50 g / ml, and most preferably less than 0.45 g / ml.

[0010] Preferably, the internal porosity of the polymer material microspheres is greater than 6%, preferably greater than 10%, more preferably greater than 24%, more preferably greater than 36%, and most preferably greater than 40%.

[0011] Preferably, the particle density of the polymer material microspheres is less than 0.95 g / ml, more preferably less than 0.90 g / ml, more preferably less than 0.78 g / ml, more preferably less than 0.65 g / ml, and most preferably less than 0.60 g / ml.

[0012] Preferably, the D50 of the microspheres is 10-800 μm.

[0013] Preferably, the closed hollow structure has a diameter of 0.1-10 µm.

[0014] The present application also provides an injection, characterized in that the injection comprises the polymer material microspheres, and the injection is used for tissue filling, venous embolism or arterial embolism.

[0015] The present application also provides the use of the polymer material microspheres, which is the preparation of medical cosmetic injections, venous embolic agents or arterial embolic agents.

[0016] The present application further provides a method for preparing the polymer material microspheres, comprising the following steps: Step (1), preparing a polymer containing a first aqueous phase, the specific method is: Adding the first aqueous phase into the polymer, emulsifying the polymer at a temperature above the melting temperature of the polymer, and obtaining a polymer containing the first aqueous phase; Step (2), mixing the polymer containing the first aqueous phase prepared in step (1) with the second aqueous phase containing an emulsifier, emulsifying at a temperature above the melting temperature of the polymer, and cooling to obtain hollow microspheres.

[0017] Preferably, the first aqueous phase contains a porogen, and the porogen is preferably one or more of sodium chloride, mannitol, and polyethylene glycol.

[0018] Preferably, the emulsifier is selected from one or more of polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, gum arabic, and Tween; the concentration is 0.05%-5%, more preferably 0.1%-3%.

[0019] The closed mentioned in the present application means that no holes penetrating to the surface of the microspheres can be seen in the hollow structure under SEM, and the hollow mentioned in the present application means that there is gas therein.

[0020] Compared with the porous microspheres in the prior art, the microspheres obtained in the present application have a closed hollow structure, or air chamber, and the surface of the microspheres is smooth as a whole with a small number of smaller pores. This specific structure cannot be obtained by using the process of the prior art. The process of the present application must be used, that is, first preparing a melt containing the first aqueous phase, and then performing a second stage of emulsification in a molten state of the polymer, and obtaining it by cooling and solidification. The process preparation time of the present application is short, and emulsification and solidification are completed within 20 minutes, which avoids the second aqueous phase from penetrating into the first aqueous phase to form mutually penetrating pores due to the long solidification time and the osmotic pressure difference between the first and second aqueous phases in the prior art. Moreover, the polymer melted on the surface of the microspheres can block the open pores on the surface of the microspheres, thereby forming a closed structure.

[0021] The microspheres obtained in the examples of the present application have similar particle sizes, which is due to the use of a membrane with a specific pore size of 40 μm during emulsification in the examples. It is well known in the art that microspheres with different particle sizes can be obtained by emulsifying using membranes with different pore sizes, such as microspheres of 10 μm, 50 μm, 100 μm, 300 μm, 500 μm, and 800 μm.

[0022] When injecting cosmetic medicine, microspheres of 20-100 μm are generally used, and when arterial or venous embolism is performed, microspheres of 60 μm or more are generally used. Those skilled in the art can select a membrane with a suitable pore size to prepare microspheres of the desired particle size.

[0023] The true density of polycaprolactone is 1.0199 g / cm³. After being prepared into microspheres, it has particle density and tap density.

[0024] In the present application, polycaprolactone is taken as an example, and the determination method of each parameter is as follows, which can be referred to when other polymers are used.

[0025] (1) Microsphere particle size and particle size distribution 0.5 g of hollow microspheres were accurately weighed and dispersed in 10 mL of an aqueous solution containing 1.0 wt % polyvinyl alcohol. The particle size and particle size distribution of the microspheres were tested using a particle size analyzer under ultrasound.

[0026] (2) Surface and cross-sectional morphology of hollow microspheres After the hollow microspheres were sprayed with gold, the surface morphology of the microspheres was observed by scanning electron microscopy; cross-sectional morphology observation: the microspheres were dispersed in epoxy resin, and after curing for 48 hours, they were frozen in liquid nitrogen and brittle fractured. The brittle fracture morphology of the microspheres was observed by scanning electron microscopy, and the diameter of the air chamber was measured and calculated according to the scale. When the shape of the air chamber is irregular, its maximum diameter is taken.

[0027] (3) Tap density Weigh an appropriate amount of microspheres and add them to a 10 mL measuring cylinder. Tap until the volume does not change. Record the volume of the microspheres and calculate the tap density ρ. v .

[0028] (4) True density of polycaprolactone Weigh 20g of polycaprolactone and add it to a 25mL volumetric flask. Melt the polycaprolactone in a 105°C oven until it forms a transparent liquid. Slowly cool to room temperature. Add purified water to the volumetric flask to make it 25mL. Calculate the volume of polycaprolactone after removing the volume of the added water. Calculate the true density of polycaprolactone based on the mass and volume ratio of the added polycaprolactone.

[0029] After measurement and calculation, the true density of polycaprolactone used in the examples of the present application is 1.0199 g / ml.

[0030] (5) Calculation method of hollow microsphere particle density and internal porosity ①Porosity in microspheres: φ 内 =1-ρ v / ρ sv ② Microsphere particle density: ρ p =(1-φ 内 )×ρ t Among them, φ 内 is the pore volume inside the microsphere / microsphere volume; ρ v is the tap density of hollow microspheres; ρ sv is the tap density of solid microspheres; ρ t is the true density of polycaprolactone.

[0031] ρ sv Measurement: Comparative Example 1 Determination of ρ sv is 0.75.

[0032] Note: Solid and hollow microspheres with similar particle sizes and particle size distributions have similar volumes and external porosities. When precise measurements are not possible, this application performs approximate calculations.

[0033] The advantages of the technical solution of this application are: (1) Compared with porous microspheres, the microspheres of the present application have a smoother surface and reduce inflammatory response; (2) Compared with solid microspheres, the microspheres of the present application contain closed chambers, which can reduce the amount of polymer matrix implanted while ensuring the number of implanted microspheres, thereby improving biosafety; (3) The polymer melt microsphere preparation technology is simple, efficient, green and environmentally friendly, and more suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 Example 8 Preparation of the particle size and distribution curve of hollow microspheres; Figure 2 SEM image of hollow microspheres in Example 8; Figure 3 Enlarged SEM image of hollow microspheres in Example 8; Figure 4 Example 8: SEM image of the brittle fracture surface of hollow microspheres; Figure 5 The hollow microsphere composite gel implants prepared in Examples 12-14 (Examples 12-14 are No. 1-3, respectively); Figure 6 SEM image of porous microspheres prepared in Comparative Example 2; Figure 7 SEM magnified image of porous microspheres prepared in Comparative Example 2; Figure 8 Pathological section of the implantation site of the gel injection of Example 12. DETAILED DESCRIPTION

[0035] In order to better understand the present application, the technical solution of the present application will be described in more detail with reference to the accompanying drawings. It should be understood that these detailed descriptions are only descriptions of exemplary embodiments of the present application and are not intended to limit the scope of the present application in any way. Throughout the specification, the same reference numerals refer to the same elements. The expression "and / or" includes any combination or all combinations of one or more items in the associated listed items.

[0036] It should be noted that in this specification, expressions such as "first", "second", "third", etc. are only used to distinguish one feature from another feature, and do not represent any limitation on the features. Therefore, without departing from the teaching of this application, the first XXX discussed below may also be referred to as the second XXX. And vice versa.

[0037] In the accompanying drawings, the size, proportions and shapes of the figures have been slightly adjusted for ease of illustration. The drawings are for illustration only and are not drawn strictly to scale. As used herein, the terms "substantially," "approximately," and similar terms are used as terms of approximation, not degree, and are intended to account for the inherent deviations in measurements or calculations that would be recognized by one of ordinary skill in the art.

[0038] It should also be understood that expressions such as "including", "comprising", "having", "containing" and / or "comprising" are open rather than closed expressions in this specification, which indicate the presence of the stated features, elements and / or components, but do not exclude the presence of one or more other features, elements, components and / or combinations thereof. In addition, when expressions such as "at least one of..." appear after a list of listed features, they modify the entire list of features rather than just a single feature in the list. In addition, when describing embodiments of the present application, "may" is used to mean "one or more embodiments of the present application". And, the wording "exemplary" is intended to refer to an example or illustration.

[0039] Unless otherwise defined, all words (including engineering terms and scientific and technological terms) used in this article have the same meaning as those commonly understood by ordinary technicians in the field to which this application belongs. It should also be understood that unless there is a clear explanation in this application, words defined in commonly used dictionaries should be interpreted as having the same meaning as their meaning in the context of the relevant technology, and should not be interpreted in an idealized or overly formal sense.

[0040] It should be noted that, in the absence of conflict, the features in the embodiments and examples of the present application can be combined with each other. In addition, unless explicitly limited or contradictory to the context, the specific steps included in the method described in the present application are not necessarily limited to the order described, but can be performed in any order or in parallel. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0041] Example 1 The first aqueous phase (2.5 mL of pure water) was weighed separately and added to 30 g of preheated molten (100 ° C) polycaprolactone melt, and then emulsified at 24000 rpm for 30 s, and then poured into 500 mL of water containing 1.0 wt% polyvinyl alcohol preheated to 95 ° C. Under a pressure difference of 0.07 MPa, the emulsified solution was passed through a 40 µm SPG membrane for 5 times. Finally, the emulsion was cooled and solidified through a 2 ° C condenser and collected in a beaker. After centrifugal solid-liquid separation and water washing 5 times, the microspheres were immersed in 100 mL of anhydrous ethanol overnight, the supernatant was removed, and the microspheres were dried at 35 ° C for 12 h to obtain dry hollow microspheres.

[0042] Example 2 The steps of Example 1 were repeated, but the first aqueous phase was changed to 5 mL.

[0043] Example 3 The procedure of Example 1 was repeated, but the first aqueous phase was changed to 7.5 mL.

[0044] Example 4 The steps of Example 1 were repeated, but the first aqueous phase was changed to 10 mL.

[0045] Example 5 The preparation method of the polycaprolactone melt containing the first aqueous phase is as in Example 4, and then it is placed in a refrigerator to be cooled to below the melting temperature, crushed into coarse powder and added to water containing 1.0 wt% polyvinyl alcohol at 95°C, and kept warm for 5 min until the polycaprolactone becomes molten, and emulsified 5 times by the method of Example 4, and dried after solid-liquid separation, water washing, alcohol washing, and drying to obtain dry hollow microspheres.

[0046] Example 6 Example 1 was repeated, but the first aqueous phase was changed to 10 mL of 1% sodium chloride aqueous solution.

[0047] Example 7 Example 1 was repeated, but the first aqueous phase was changed to 10 mL of 2% sodium chloride aqueous solution.

[0048] Example 8 Example 1 was repeated, but the first aqueous phase was changed to 10 mL of 3% sodium chloride aqueous solution.

[0049] Example 9 Example 1 was repeated, but the first aqueous phase was changed to 2.5 mL of 3% sodium chloride aqueous solution.

[0050] Example 10 Example 1 was repeated, except that the first aqueous phase was changed to 5 mL of 3% sodium chloride aqueous solution.

[0051] Embodiment 11 Example 1 was repeated, but the first aqueous phase was changed to 7.5 mL of 3% sodium chloride aqueous solution.

[0052] Example 12 The hollow microspheres prepared in Example 11 were dispersed in 2% sodium carboxymethyl cellulose under mechanical stirring and filled into a prefilled syringe to prepare a hollow microsphere composite gel implant.

[0053] Example 13 Refer to Example 12, but use 2% sodium hyaluronate gel instead.

[0054] Embodiment 14 Refer to Example 12, but use 0.1% agarose gel instead.

[0055] Comparative Example 1 Solid polycaprolactone microspheres (polycaprolactone melt microsphere preparation method): Weigh 30 g of polycaprolactone raw material, disperse it in 500 mL of an aqueous solution containing 1% polyvinyl alcohol, keep the temperature at 95°C and maintain for 10 min, then pass through a microporous membrane for emulsification 5 times (membrane pressure 0.07 MPa), and cool it at 2°C to obtain a dispersion of solid polycaprolactone microspheres. After the microsphere dispersion is settled, the supernatant is removed, washed with water 5 times, and freeze-dried after solid-liquid separation to obtain solid polycaprolactone microspheres.

[0056] Comparative Example 2 Polycaprolactone porous microspheres: Weigh 10 g of polycaprolactone and dissolve it in 50 mL of dichloromethane to prepare an oil phase solution; weigh 3 g of sodium chloride and dissolve it in 100 mL of water for injection to prepare a first aqueous phase solution with a sodium chloride concentration of 3%; weigh 10 g of polyvinyl alcohol (emulsifier) ​​and dissolve it in 1 L of water for injection to prepare a second aqueous phase solution with an emulsifier concentration of 1%. Add 5 ml of the first aqueous phase to the oil phase and homogenize at 24,000 rpm for 30 s to obtain a W / O emulsion; add the W / O emulsion to 1 L of the second aqueous phase and mechanically stir at 1500 rpm for 15 min to obtain a W / O / W type emulsion. Reduce the stirring speed to 150 rpm and stir the emulsion for 15 min. Then it was added to 3L of the second aqueous phase solution, stirred at 150 rpm for 30 min, and then 5L of injection water was added. After mechanical stirring for 30 min, it was allowed to stand for 1 h. After removing the supernatant, 1L of water was added to wash 5 times. After centrifugation to remove the supernatant, the porous microspheres were dried.

[0057] Results and discussion: The microspheres obtained in the above examples and comparative examples were tested, and the data are shown in Table 1.

[0058] Table 1 Parameters of the products of the embodiments and comparative examples

[0059] Typical particle size distribution of hollow microspheres (Example 8) is as follows Figure 1 As shown. It can be seen that the particle size of the microspheres conforms to the normal distribution, D50 = 32.01 µm, and the span is 0.851. There are a few open pores on the surface of the microspheres ( Figure 2 , 3 ), there are non-open pores inside. Figure 4 The brittle cross-section morphology shows that there are a large number of closed spherical chambers inside the microspheres, with pore sizes less than 10 µm. Figure 6 , 7 ) There are more open pores on the surface, and no closed pores are observed. Figure 6 It can be seen from the broken microspheres that the pores in the microspheres are interconnected, and the inside of the microspheres is looser. We also observed that the microspheres are more easily broken during the stirring process of the gel composite process. The hollow microspheres show a lower tap density (as shown in Table 1). When the first aqueous phase is pure water, the tap density decreases slightly with the addition of the first aqueous phase, but the addition of NaCl to the first aqueous phase significantly reduces the tap density of the microspheres, and increasing the concentration of NaCl in the first aqueous phase helps to prepare hollow microspheres with lower tap density. For example, the tap density of Example 8 is reduced to 0.44 g / mL (the true density of polycaprolactone is 1.0199 g / mL, and the density of solid microspheres is 0.75 g / mL). The microspheres prepared by polycaprolactone melt membrane emulsification have good batch-to-batch repeatability. As shown in Table 1, D50 is controlled at 31.28-33.85, and the span is 0.851-0.866. Therefore, when calculating the porosity between microsphere particles (i.e., external porosity), the porosity between solid microsphere particles is used instead of the porosity between hollow microsphere particles. The calculated internal porosity and particle density of the hollow microspheres are shown in Table 1. The maximum internal porosity (Example 8) is 41.33%. Hollow microsphere composite gel implant ( Figure 5 )The gel was smooth and delicate with better whiteness. No stratification was observed after centrifugation at 4000 rpm for 3 min, indicating good stability. The 27G thin-walled needle did not have needle blockage and had good needle permeability.

[0060] Animal implantation experiment: Rabbits with healthy skin (about 2.5 kg) were depilated on the back. 0.2 mL of the gel of Example 12 was injected subcutaneously using a 27 G thin-walled needle. Each rabbit was then raised individually without dietary restriction. Six months after the injection, the rabbits were killed by air embolism, and the skin tissues at the injection sites were taken, fixed with 4% paraformaldehyde, and trimmed, dehydrated, embedded, sliced, stained, and sealed according to the pathological laboratory detection SOP procedure for pathological section examination.

[0061] Rabbit subcutaneous implantation 6 months later Figure 8 ) It can be seen from the observation that there is a small amount of connective tissue hyperplasia wrapped around the filler in the subcutaneous tissue, and there is a large amount of connective tissue hyperplasia and segmentation inside the filler. A large number of macrophages, multinucleated giant cells and scattered lymphocytes can be seen in the filler. It proves that the microspheres of this application can be used for injection filling and stimulating collagen production in medical cosmetology.

[0062] The above description is only an implementation method of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of protection involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the technical concept. For example, the above features are replaced with the technical features with similar functions disclosed in this application (but not limited to) by each other.

Claims

1. A polymer material microsphere, characterized in that: The polymer material is one or more polymers, the melting temperature of the polymer is less than 100° C., and the microsphere has one or more closed hollow structures inside.

2. The polymer microsphere according to claim 1, characterized in that: At least one of the polymers is a homopolymer of caprolactone or a copolymer with caprolactone as a main monomer.

3. The polymer microsphere according to claim 1, characterized in that: The tap density of the polymer material microspheres is less than 0.70 g / ml.

4. The polymer microsphere according to claim 1, characterized in that: The internal porosity of the polymer material microspheres is greater than 6%.

5. The polymer microsphere according to claim 1, characterized in that: The D50 of the polymer material microspheres is 10-800 μm.

6. The polymer microsphere according to claim 1, characterized in that: The closed hollow structure has a diameter of 0.1-10 µm.

7. An injection, characterized in that: The injection comprises the polymer material microspheres according to any one of claims 1 to 6, and the injection is used for tissue filling, venous embolism or arterial embolism.

8. Use of the polymer material microspheres according to any one of claims 1 to 6, wherein the use is for preparing medical cosmetic injections, venous embolic agents or arterial embolic agents.

9. A method for preparing the polymer microspheres according to any one of claims 1 to 6, characterized in that The steps include: Step (1), preparing a polymer containing a first aqueous phase, the specific method is: Adding the first aqueous phase into the polymer, emulsifying the polymer at a temperature above the melting temperature of the polymer, and obtaining a polymer containing the first aqueous phase; Step (2), mixing the polymer containing the first aqueous phase prepared in step (1) with the second aqueous phase containing an emulsifier, emulsifying at a temperature above the melting temperature of the polymer, and cooling to obtain hollow microspheres.

10. The method according to claim 9, characterized in that The first aqueous phase contains a porogen, and the porogen is preferably one or more of sodium chloride, mannitol, and polyethylene glycol.

11. The method according to claim 9, characterized in that The emulsifier is selected from one or more of polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, gum arabic, and Tween; the concentration is 0.05%-5%, more preferably 0.1%-3%.

Images