Facial skin filler and preparation method thereof

Abstract

The invention relates to a facial skin filler and a preparation method thereof. The method comprises the following steps: S1-a: dissolving collagen in water, then adding hydroxyapatite microspheres, and mixing to obtain a first mixture; s1-b: adding the first mixture and an emulsifier into the oil phase, keeping the temperature constant, then stirring to obtain a second mixture, and then carrying out solid-liquid separation to obtain intermediate microspheres; s1-c: adding the intermediate microspheres into a cross-linking agent solution for cross-linking to obtain a third mixture, and then performing solid-liquid centrifugal separation to obtain composite microspheres; s2, dissolving first sodium hyaluronate with the molecular weight of 120-150 million Daltons and second sodium hyaluronate with the molecular weight of 20-40 million Daltons in water to obtain a sodium hyaluronate aqueous solution; s3, mixing the composite microspheres with a sodium hyaluronate aqueous solution to obtain the facial skin filling agent. The facial skin filler has improved biocompatibility, prolonged retention time, and improved injection performance.

Description

Technical Field

[0001] The present invention belongs to the field of medical cosmetic surgery technology, and specifically relates to a facial skin filler and a preparation method thereof. The facial skin filler comprises composite microspheres (having a core-shell structure formed by collagen and hydroxyapatite microspheres) and a sodium hyaluronate aqueous solution. Background Art

[0002] Facial skin aging is a multifactorial biological process, primarily characterized by degradation of the dermal extracellular matrix (ECM), atrophy and displacement of the subcutaneous fat pad, and weakening of the supporting structure due to bone resorption. These changes collectively lead to signs of aging, such as sagging skin, deepening wrinkles, and a loss of facial contours. To address these issues, the use of hydroxyapatite, sodium hyaluronate, poly-L-lactic acid, polycaprolactone, collagen, and chitosan as facial fillers has been studied to improve skin condition by providing volume, mechanical support, and collagen stimulation.

[0003] Hydroxyapatite microspheres have a porous spherical structure that provides mechanical support and are primarily used to improve nasolabial folds, temple hollows, and contour shaping. However, hydroxyapatite microspheres have drawbacks such as easy aggregation (resulting in poor injectability), a hard feel, and uncontrollable degradation (resulting in a short maintenance period). Furthermore, their biocompatibility needs further improvement.

[0004] Sodium hyaluronate is a linear polysaccharide composed of D-glucuronic acid and N-acetylglucosamine disaccharide units, which is highly hydrophilic and viscoelastic. Low molecular weight sodium hyaluronate (molecular weight of 200,000 to 400,000 Daltons) is commonly used as a filler. As a biodegradable filler, it is mainly used to improve facial wrinkles, lip volume and contour shaping. Sodium hyaluronate can immediately fill tissue volume while binding water to increase skin elasticity. However, the above-mentioned low molecular weight sodium hyaluronate has problems such as rapid enzymatic hydrolysis, resulting in a short maintenance time.

[0005] Hydroxyapatite microspheres are often combined with carriers such as sodium hyaluronate to improve injectability. However, the resulting composite fillers still have the above-mentioned drawbacks.

[0006] Collagen, typically derived from animal sources (such as bovine collagen) or recombinant human collagen, possesses a natural triple-helix structure. As an absorbable filler, collagen is primarily used to improve facial fine lines, sculpt lips, and repair skin texture. Collagen is typically used alone. However, its support is weak and it is easily degraded (with a short shelf life), making it unsuitable for deep fillers.

[0007] Therefore, there is a need to develop a facial dermal filler that can simultaneously enhance biocompatibility, prolong duration, and improve injectability. Summary of the Invention

[0008] Technical issues

[0009] The first object of the present invention is to provide a method for preparing facial dermal fillers, which is simple to operate, has mild conditions, good reproducibility, and is convenient for large-scale production.

[0010] A second object of the present invention is to provide a facial dermal filler comprising: composite microspheres (comprising collagen and hydroxyapatite microspheres); and an aqueous sodium hyaluronate solution. The facial dermal filler has enhanced biocompatibility, prolonged duration, and improved injectability.

[0011] Technical Solution

[0012] According to a first aspect of the present invention, there is provided a method for preparing a facial dermal filler, the method comprising the following steps:

[0013] S1-a: dissolving collagen in water, then adding hydroxyapatite microspheres and mixing to obtain a first mixture;

[0014] S1-b: adding the first mixture and the emulsifier to the oil phase and maintaining a constant temperature, followed by stirring to obtain a second mixture, and then performing solid-liquid separation to obtain intermediate microspheres;

[0015] S1-c: adding the intermediate microspheres to a crosslinking agent solution for crosslinking to obtain a third mixture, and then performing solid-liquid centrifugation to obtain composite microspheres;

[0016] S2: dissolving a first sodium hyaluronate having a molecular weight of 1.2 to 1.5 million daltons and a second sodium hyaluronate having a molecular weight of 0.2 to 0.4 million daltons in water to obtain a sodium hyaluronate aqueous solution;

[0017] S3: mixing the composite microspheres and the sodium hyaluronate aqueous solution to obtain the facial skin filler.

[0018] In one embodiment, in step S1-a,

[0019] The water is at least one of deionized water, ion exchange water, distilled water and pure water,

[0020] The average particle size of the hydroxyapatite microspheres is 5 to 50 μm.

[0021] The mass ratio of the collagen to the water is (1-5):100,

[0022] The mass ratio of the collagen to the hydroxyapatite microspheres is (1-5):1.

[0023] In one embodiment, in step S1-b,

[0024] The mass ratio of the first mixture: the emulsifier: the oil phase is (3-10): (1-10): 100, and the emulsifier is one or more of sorbitan trioleate, sorbitan monooleate and sorbitan monostearate.

[0025] The oil phase is one or more of olive oil, peanut oil and soybean oil,

[0026] The constant temperature is 15-60°C,

[0027] The stirring rate is 500-1000 rpm, and the stirring time is 1-8 hours.

[0028] In one embodiment, in step S1-c,

[0029] The cross-linking agent is one or more of pentanediol, a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and 1,4-butanediol diglycidyl ether.

[0030] The solvent of the cross-linking agent solution is water, and the water is at least one of deionized water, ion exchange water, distilled water and pure water.

[0031] The volume ratio of the cross-linking agent to the water is (1-5):100,

[0032] The mass ratio of the intermediate microspheres to the crosslinking agent solution is (0.5-1.5):100.

[0033] The cross-linking is carried out at 20-50°C for 1-12 hours.

[0034] The centrifugal speed of the solid-liquid centrifugal separation is 1000-5000 rpm, and the centrifugal time is 1-10 minutes.

[0035] In one embodiment, in step S2,

[0036] The mass ratio of the first sodium hyaluronate to the second sodium hyaluronate is 1:(1-10),

[0037] The mass ratio of the combination of the first sodium hyaluronate and the second sodium hyaluronate to water is (0.5-3):100,

[0038] The water is at least one of deionized water, ion exchanged water, distilled water and pure water.

[0039] In one embodiment, in step S3, the mass ratio of the composite microspheres to the sodium hyaluronate aqueous solution is (3-40):100.

[0040] According to a second aspect of the present invention, there is provided a facial dermal filler prepared by the above method, the facial dermal filler comprising:

[0041] Composite microspheres comprising collagen and hydroxyapatite microspheres, and

[0042] Sodium hyaluronate aqueous solution;

[0043] The composite microspheres have a core-shell structure, wherein collagen as a shell covers hydroxyapatite microspheres as a core.

[0044] In one embodiment, the average particle size of the composite microspheres is 20 to 70 μm.

[0045] In one embodiment, the content of the collagen is 5 to 60 wt % relative to the total weight of the composite microspheres.

[0046] Beneficial effects

[0047] The preparation method of the present invention has the significant advantages of being simple to operate, mild in conditions, and having good reproducibility. Furthermore, the preparation method of the present invention has a short reaction time and does not require complicated post-processing.

[0048] Furthermore, compared to conventional facial dermal fillers prepared solely with hydroxyapatite microspheres and sodium hyaluronate, the facial dermal filler of the present invention, comprising three specific components, exhibits excellent synergistic effects. Specifically, by forming a "soft-in-hard" core-shell structure with collagen and hydroxyapatite (with a crosslinker used to secure their mutual bonding), and by combining the synergistic effects of two sodium hyaluronates of varying molecular weights, the facial dermal filler of the present invention addresses the problems of existing single fillers, such as the low biocompatibility, poor injectability, and hard feel of hydroxyapatite, and the short maintenance of collagen and low-molecular-weight sodium hyaluronate. Furthermore, the facial dermal filler exhibits enhanced biocompatibility, prolonged in vivo maintenance, and improved injectability, thereby combining the three advantages of rapid onset of effect, gradual improvement, and long-term maintenance. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a photo of the composite microspheres prepared in Example 1 under an optical microscope.

[0050] Figure 2 This is a scanning electron microscope (SEM) photograph of the composite microspheres prepared in Example 1. DETAILED DESCRIPTION

[0051] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

[0052] Unless otherwise defined, all terms used herein, including technical or scientific terms, should be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Therefore, unless explicitly defined, terms such as those defined in commonly used dictionaries should not be interpreted in an ideal or overly formal sense.

[0053] When the conditions and methods for measuring a property or parameter described in this specification are not specifically described, the property or parameter can be measured using measurement conditions and methods generally used by those skilled in the art.

[0054] As used herein, the average particle size (D 50 ) refers to the point at which 50% of the volume cumulative distribution of particle size is obtained (D 50 The average particle size (D 50 ).

[0055] Unless otherwise specified, "%" used herein refers to weight %.

[0056] Methods of preparing facial dermal fillers

[0057] According to a first aspect of the present invention, there is provided a method for preparing a facial dermal filler, the method comprising the following steps:

[0058] S1-a: dissolving collagen in water, then adding hydroxyapatite microspheres and mixing to obtain a first mixture;

[0059] S1-b: adding the first mixture and the emulsifier to the oil phase and maintaining a constant temperature, followed by stirring to obtain a second mixture, and then performing solid-liquid separation to obtain intermediate microspheres;

[0060] S1-c: adding the intermediate microspheres to a crosslinking agent solution for crosslinking to obtain a third mixture, and then performing solid-liquid centrifugation to obtain composite microspheres;

[0061] S2: dissolving a first sodium hyaluronate having a molecular weight of 1.2 to 1.5 million daltons and a second sodium hyaluronate having a molecular weight of 0.2 to 0.4 million daltons in water to obtain a sodium hyaluronate aqueous solution;

[0062] S3: mixing the composite microspheres and the sodium hyaluronate aqueous solution to obtain the facial skin filler.

[0063] Hereinafter, the method of the present invention will be described in detail with respect to each step.

[0064] Among them, steps S1-a, S1-b and S1-c can be collectively referred to as step S1, which is used to prepare composite microspheres.

[0065] Furthermore, steps S1 and S2 can be performed independently, and there is no particular restriction on the order in which they are performed. That is, one step can be performed first and then the other; or, steps S1 and S2 can be performed simultaneously.

[0066] Step S1-a: preparing a first mixture

[0067] Collagen is dissolved in water, and then hydroxyapatite microspheres are added and mixed to obtain a first mixture.

[0068] Step S1-a can be performed by conventional processes.

[0069] Collagen is a commonly used material for facial dermal fillers. To facilitate its dissolution, it is typically dissolved in water before adding hydroxyapatite microspheres to produce a collagen aqueous solution. Collagen is then used to coat the hydroxyapatite microspheres in the subsequent process, effectively shielding their surface charge, improving biocompatibility, and preventing aggregation.

[0070] There is no particular limitation on the type of water as long as it is water that can be used for skin injection. For example, the water may be at least one of deionized water, ion-exchanged water, distilled water, and pure water.

[0071] Hydroxyapatite microspheres are round particles used for mechanical support. The average particle size of hydroxyapatite microspheres (D 50 ) can be 5 to 50 μm, specifically 15 to 40 μm. If the average particle size is too small, the composite microspheres obtained subsequently are easily and rapidly cleared by macrophages. If the average particle size is too large, injection of the composite microspheres becomes difficult. Furthermore, hydroxyapatite microspheres are commercially available, and particles of uniform size are selected to avoid the above-mentioned problems.

[0072] The mass ratio of collagen to water may be (1-5):100, preferably (2-4):100, and more preferably (2.5-3.5):100.

[0073] The mass ratio of collagen to hydroxyapatite microspheres may be (1-5):1, preferably (2-4):1, and more preferably (2.5-3.5):1.

[0074] If the amount of water used is too large, the subsequent composite microspheres will have insufficient support and a short duration. If the amount of water used is too small, the local collagen concentration will be too high, easily agglomerating into clumps, which will lead to difficulty in injection.

[0075] If the amount of collagen used is too high, the process cost will be too high, and the subsequent composite microspheres will be too large, making injection difficult. If the amount is too low, the subsequent collagen coating of the hydroxyapatite microspheres will not be complete, and the collagen function will not be fully exerted.

[0076] If the amount of hydroxyapatite microspheres used is too large, the collagen will not completely coat the hydroxyapatite microspheres in the subsequent step S1-b, and the resulting composite microspheres will be hard, making injection difficult. If the amount is too small, the resulting composite microspheres will have insufficient support and will be easily degraded and absorbed quickly, resulting in a shortened maintenance period and poor filling effect.

[0077] Hydroxyapatite microspheres are added to the collagen aqueous solution obtained above and mixed. There is no particular limitation on the mixing method, as long as the components can be mixed evenly. For example, a stirrer can be used for mechanical stirring and mixing. In addition, mixing can be carried out at room temperature (e.g., 20-25°C), normal pressure (e.g., one atmosphere), and air atmosphere. The mixing time can generally be 10 to 60 minutes, preferably 10 to 50 minutes, and more preferably 20 to 40 minutes.

[0078] The first mixture can be obtained by the above mixing, which is a solid-liquid mixture in which the hydroxyapatite microspheres as solids are uniformly dispersed.

[0079] Step S1-b: Preparation of intermediate microspheres

[0080] The first mixture and the emulsifier are added to the oil phase and kept at a constant temperature, and then stirred to obtain a second mixture, which is then subjected to solid-liquid separation to obtain intermediate microspheres.

[0081] There is no particular restriction on the order in which the first mixture and the emulsifier are added to the oil phase. For example, the first mixture may be added first, followed by the emulsifier.

[0082] Under the action of an emulsifier, stirring is performed to form a water-in-oil (W / O) emulsion system with the first mixture and the oil phase. The oil phase is the continuous phase, and the first mixture (i.e., the aqueous phase) is the dispersed phase, forming uniformly dispersed droplets within the oil phase. Furthermore, the use of an emulsifier and stirring prevent premature aggregation of the droplets and allow a collagen layer of sufficient thickness to grow on the surface of the hydroxyapatite microspheres within the droplets, forming intermediate microspheres with a core-shell structure.

[0083] The oil phase can be one or more of olive oil, peanut oil and soybean oil.

[0084] The emulsifier may be one or more of sorbitan trioleate, sorbitan monooleate, and sorbitan monostearate.

[0085] The first mixture can be used in an amount of 3 to 10 parts by mass, for example, 5 to 8 parts by mass, relative to 100 parts by mass of the oil phase. If the amount is too large, the viscosity of the oil phase decreases, the first mixture droplets tend to aggregate, and it becomes difficult to form a stable water-in-oil (W / O) system. If the amount is too small, the viscosity of the oil phase becomes too high, making dispersion difficult and reducing the yield of the intermediate microspheres.

[0086] The amount of emulsifier used can be 1 to 10 parts by mass, for example, 2 to 8 parts by mass, or 5 to 7 parts by mass, relative to 100 parts by mass of the oil phase. If the amount is too large, the surface activity of the intermediate microspheres will be reduced, affecting their biocompatibility, and residual emulsifier will affect the purity of the intermediate microspheres. If the amount is too small, the oil-water interfacial tension will not be effectively reduced, and the intermediate microsphere formation rate will be low.

[0087] During the stirring process, the stability of the system can be ensured by maintaining a constant temperature. The constant temperature can be 15-60°C, preferably 20-50°C. If the temperature is too high, side reactions may increase and the stability of the system may decrease. If the temperature is too low, the reaction may be incomplete and the first mixture may be unevenly dispersed in the oil phase.

[0088] The stirring may be mechanical stirring by using a stirrer.

[0089] The stirring time can be 1 to 8 hours, preferably 2 to 6 hours. If the stirring time is too long, the surface of the intermediate microspheres will be worn, increasing time and manufacturing costs. If the stirring time is too short, the first mixture will be unevenly dispersed in the oil phase.

[0090] The stirring rate directly affects the degree of droplet breakup, thereby regulating the particle size distribution of the intermediate microspheres. The stirring rate can be 500-1000 rpm, preferably 600-800 rpm. If the stirring rate is too fast, the hydroxyapatite microspheres will fall off from the collagen coating, destroying the core-shell structure. If the stirring rate is too slow, it will lead to incomplete dispersion, unable to form a stable oil-in-water (W / O) emulsion system, and the particle size of the intermediate microspheres will be too large.

[0091] During the continuous stirring process, a collagen shell of the desired thickness gradually forms on the surface of the hydroxyapatite microspheres serving as cores in each dispersed aqueous phase (droplet). Specifically, a portion of the collagen particles dissolved in water continuously precipitate on the surface of the hydroxyapatite microspheres to form the shell. The shell has pores, i.e., a porous structure. Thus, a second mixture containing the intermediate microspheres is formed. The second mixture is a uniformly mixed solid-liquid mixture.

[0092] By adjusting the type of oil phase, emulsifier and stirring parameters, the particle size of the intermediate microspheres can be precisely controlled and their surface properties can be optimized.

[0093] Then, the second mixture can be subjected to solid-liquid separation. There is no particular limitation on the method of solid-liquid separation, as long as the intermediate microspheres can be separated from the liquid phase (including the oil phase and the aqueous phase). For example, a standing method can be used to separate the intermediate microspheres from the liquid in the second mixture by natural sedimentation. Standing can be carried out at room temperature (e.g., 20-25°C), normal pressure (e.g., one atmosphere), and air atmosphere. The standing time can generally be 1 to 6 hours, preferably 2 to 5 hours, and more preferably 3 to 4 hours.

[0094] Individual intermediate microspheres can be obtained by solid-liquid separation, in which the collagen shell has a weak binding force with the hydroxyapatite microspheres.

[0095] Step S1-c: Preparation of composite microspheres

[0096] The intermediate microspheres are added to a crosslinking agent solution for crosslinking to obtain a third mixture, which is then subjected to solid-liquid centrifugal separation to obtain composite microspheres.

[0097] The crosslinking agent solution refers to a solution formed by dissolving a crosslinking agent in a solvent.

[0098] The cross-linking agent may be one or more of pentanediol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and 1,4-butanediol diglycidyl ether.

[0099] The solvent may be an organic solvent such as ethanol, acetone, dimethyl sulfoxide, or water. Preferably, the solvent is water. The water may be at least one of deionized water, ion-exchanged water, distilled water, and pure water.

[0100] The amount of crosslinker used can be 1 to 5 parts by volume, preferably 2 to 4 parts by volume, and more preferably 2.5 to 3.5 parts by volume, relative to 100 parts by volume of water. If the amount is too large, excessive crosslinking will form a dense network structure, resulting in reduced material elasticity. Furthermore, excessive amounts of unreacted crosslinker residue will affect the biocompatibility of the composite microspheres. If the amount is too small, the degree of crosslinking will be insufficient, resulting in poor structural stability of the composite microspheres.

[0101] The cross-linking is chemical cross-linking. As known to those skilled in the art, chemical cross-linking refers to a method of connecting the molecules in a polymer chain, microspheres or gel by covalent bonds or strong interactions (such as ionic bonds) to form a three-dimensional network structure. In the present invention, the cross-linking agent interacts with the collagen shell and the hydroxyapatite microsphere core respectively, thereby firmly connecting the two. Specifically, the cross-linking agent solution can be infiltrated into the collagen shell and play the above-mentioned connection role, thereby obtaining a composite microsphere in which the core-shell is firmly combined.

[0102] The amount of the intermediate microspheres used can be 0.5 to 1.5 parts by mass, preferably 0.8 to 1.2 parts by mass, and more preferably 1 to 1.2 parts by mass relative to 100 parts by mass of the crosslinker solution. If the amount is too large, the intermediate microspheres are prone to stacking, resulting in local shielding and uneven crosslinking. If the amount is too small, multiple solid-liquid centrifugation operations are required to obtain separate intermediate microspheres, resulting in increased costs.

[0103] The crosslinking reaction time can be 1 to 12 hours, preferably 2 to 8 hours. If the crosslinking reaction time is too long, excessive crosslinking will form a dense network structure, resulting in reduced material elasticity and increased crosslinking side reactions. If the crosslinking reaction time is too short, incomplete crosslinking will occur, resulting in an uneven product structure.

[0104] The crosslinking reaction temperature can be 20-50°C, preferably 25-40°C. If the crosslinking temperature is too high, the reaction will run away, and crosslinking side reactions will increase, resulting in an increase in by-products. If the crosslinking temperature is too low, the reaction rate will slow down and crosslinking will be insufficient.

[0105] The cross-linking reaction can produce a third mixture, which is a solid-liquid mixture. The third mixture can be subjected to solid-liquid centrifugal separation, wherein the centrifugal force generated by high-speed rotation of the centrifuge causes the solid particles (i.e., composite microspheres) in the solid-liquid mixture to settle toward the outside of the centrifugal field under the action of the centrifugal force, thereby achieving separation of the solid particles from the liquid.

[0106] The centrifugal speed can be 1000-5000 rpm, preferably 2000-4000 rpm. If the centrifugal speed is too fast, the structure of the composite microspheres may be destroyed, and the separated composite microspheres may be adsorbed on the wall of the centrifuge tube, resulting in loss. If the centrifugal speed is too slow, the separation efficiency will be low, the microspheres will not be completely separated, and the time cost will be increased.

[0107] The centrifugation time can be 1 to 10 minutes, preferably 2 to 8 minutes. If the centrifugation time is too long, the structure of the composite microspheres will be destroyed and the equipment will be worn out. If the centrifugation time is too short, the separation will be incomplete and the separation effect will be poor.

[0108] Composite microspheres can be obtained by solid-liquid centrifugal separation, and their average particle size (D 50 ) can be in the range of 20-70 μm, specifically 30-50 μm. By controlling the above average particle size, the composite microspheres can be prevented from being phagocytosed too quickly by macrophages, the effect can be prolonged, and the injection smoothness and good biocompatibility can be ensured.

[0109] In addition, the particle size of the composite microspheres is kept uniform, which helps to avoid local accumulation, prolong the effect duration and improve safety.

[0110] In the composite microspheres, the thickness of the collagen shell may generally be 2.5-20 μm, specifically 5-15 μm, or 8-12 μm.

[0111] The present invention wraps hydroxyapatite microspheres with collagen and uses a cross-linking agent to firmly combine them with each other, thereby obtaining composite microspheres having a "soft-encapsulated hard" core-shell structure.

[0112] Compared with hydroxyapatite microspheres alone, the composite microspheres of the present invention have enhanced mechanical strength.

[0113] In addition, collagen encapsulation can effectively shield the surface charge of hydroxyapatite, improving biocompatibility. Moreover, the core-shell structure can prevent hydroxyapatite from agglomerating through steric hindrance and chemical bond locking.

[0114] Furthermore, the hydroxyapatite microspheres in the composite microspheres can provide long-lasting support and continuously release calcium ions during the degradation process of the composite microspheres to stimulate collagen regeneration, effectively enhancing the duration of the composite microspheres' effects in the body.

[0115] The above combination can not only avoid the defect of short maintenance time of simple collagen, but also make up for the deficiency of hydroxyapatite microspheres in the initial hard touch, and at the same time reduce the risk of inflammation after hydroxyapatite microsphere injection through the buffering effect of collagen.

[0116] Step S2: Preparation of sodium hyaluronate aqueous solution

[0117] A first sodium hyaluronate having a molecular weight of 1.2 to 1.5 million daltons and a second sodium hyaluronate having a molecular weight of 200,000 to 400,000 daltons are dissolved in water to obtain a sodium hyaluronate aqueous solution.

[0118] Step S2 can be performed by conventional processes.

[0119] Sodium hyaluronate is usually white or off-white in powder or granular form and is easily soluble in water. Upon dissolution in water, it forms a transparent, viscoelastic, homogeneous solution.

[0120] In conventional facial dermal fillers, only the aforementioned low-molecular-weight second sodium hyaluronate is generally used, which has the problem of a short maintenance time.

[0121] To this end, the present invention uses a combination of two sodium hyaluronates with different molecular weights, wherein the high molecular weight sodium hyaluronate (the first sodium hyaluronate) can increase the viscosity of the resulting sodium hyaluronate aqueous solution, enhance the supporting effect of the composite microspheres, prolong the maintenance time, and improve biocompatibility, thereby achieving the dual effects of immediate filling and long-term shaping; while the low molecular weight sodium hyaluronate (the second sodium hyaluronate) can improve lubricity and reduce resistance during injection.

[0122] The mass ratio of the first sodium hyaluronate to the second sodium hyaluronate may be 1:(1-10), preferably 1:(2-8).

[0123] If the amount of the first sodium hyaluronate used is too large, the viscosity of the solution increases, making injection difficult.

[0124] If the amount of the first sodium hyaluronate used is too small, the mechanical support will be insufficient, the structural stability will be poor, and it will not be able to provide effective immediate filling and long-term shaping effects.

[0125] There is no particular limitation on the type of water. For example, it can be at least one of deionized water, ion-exchanged water, distilled water, and pure water, preferably deionized water.

[0126] The total amount of the first and second sodium hyaluronates can be 0.5 to 3 parts by mass, preferably 1 to 2 parts by mass, relative to 100 parts by mass of water. If the total amount is too large, it will lead to increased injection resistance, reduced tissue compatibility, and a stiff feel. If the total amount is too small, it will lead to insufficient support force, too rapid diffusion, and too short a maintenance time.

[0127] There are no particular restrictions on the method of dissolving in water, as long as the components are evenly distributed. For example, a stirrer can be used for mechanical stirring to quickly dissolve the first sodium hyaluronate and the second sodium hyaluronate in water. This can be carried out at room temperature (e.g., 20-25°C), normal pressure (e.g., 1 atmosphere), and air. The stirring time can generally be 1 to 6 hours, preferably 2 to 5 hours, and more preferably 3 to 4 hours.

[0128] The sodium hyaluronate aqueous solution obtained by dissolution is a homogeneous aqueous solution.

[0129] Step S3: Preparation of facial dermal fillers

[0130] The composite microspheres and the sodium hyaluronate aqueous solution are mixed to obtain a facial dermal filler.

[0131] Step S3 can be performed by conventional processes.

[0132] Combining the rheological properties of sodium hyaluronate with different molecular weights (high molecular weight sodium hyaluronate thickens and prevents sedimentation, low molecular weight sodium hyaluronate lubricates the needle), sodium hyaluronate aqueous solution can inhibit the sedimentation of composite microspheres through viscoelastic differences, ensuring the uniformity and stability of the filler, and significantly reducing injection resistance and avoiding needle clogging.

[0133] The amount of composite microspheres used can be 3 to 40 parts by mass per 100 parts by mass of the sodium hyaluronate aqueous solution. If the amount is too large, the composite microspheres will not be evenly dispersed in the sodium hyaluronate aqueous solution, resulting in difficulty in injection. If the amount is too small, the supporting force will be insufficient, the metabolism rate will be rapid, and the filling effect will be insignificant.

[0134] There are no particular limitations on the mixing method, as long as the components are uniformly mixed. For example, a stirrer can be used for mechanical stirring and mixing. The stirring and mixing can be carried out at room temperature (e.g., 20-25° C.), normal pressure (e.g., 1 atmosphere), and air atmosphere. The mixing time can generally be 1 to 15 hours, preferably 2 to 10 hours.

[0135] In the obtained facial skin filler, the composite microspheres are uniformly dispersed in the sodium hyaluronate aqueous solution in the form of a solid-liquid mixture.

[0136] facial dermal fillers

[0137] According to a second aspect of the present invention, a facial dermal filler is provided, which can be produced by the above method. The facial dermal filler comprises composite microspheres (comprising collagen and hydroxyapatite microspheres) and an aqueous sodium hyaluronate solution. The composite microspheres have a core-shell structure, wherein the collagen as the shell encapsulates the hydroxyapatite microspheres as the core.

[0138] The description of each component can be found in the previous text and will not be repeated here.

[0139] The average particle size of the composite microspheres may be 20 to 70 μm, preferably 30 to 60 μm.

[0140] In addition, relative to the total weight of the composite microspheres, the content of the collagen is 5 to 60 wt%, preferably 10 to 40 wt%. The content of the collagen can be measured by conventional thermogravimetric analysis (TGA).

[0141] The facial dermal filler of the present invention utilizes collagen and hydroxyapatite microspheres to form intermediate microspheres with a "soft-encapsulated hard" core-shell structure. A crosslinker is then used to securely bond these microspheres together, creating composite microspheres. This, combined with the synergistic effect of two different molecular weight sodium hyaluronates, addresses existing single fillers, including the low biocompatibility, poor injectability, and hard feel of hydroxyapatite microspheres, and the short maintenance of collagen and low-molecular-weight sodium hyaluronate. Furthermore, the facial dermal filler exhibits enhanced biocompatibility, prolonged in vivo maintenance, and improved injectability.

[0142] Example

[0143] Hereinafter, the present invention will be described in detail with reference to Examples to specifically describe the present invention. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention should not be interpreted as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

[0144] Unless otherwise specified, the experimental methods in the following examples are generally based on conventional conditions in the art or conditions recommended by the manufacturer; the raw materials and equipment used are all commercially available from conventional markets, unless otherwise specified.

[0145] Unless otherwise specified, all steps in the Examples and Comparative Examples were carried out at room temperature (25° C.), atmospheric pressure, and air atmosphere.

[0146] Example 1

[0147] Facial dermal fillers were prepared by the following steps.

[0148] Step S1-a

[0149] 3 g of collagen was completely dissolved in 100 g (ie, 100 ml) of deionized water, and then 1 g of hydroxyapatite microspheres with an average particle size of 17 μm was added, followed by stirring for 30 minutes to uniformly mix the components to obtain a first mixture.

[0150] Step S1-b

[0151] 5 g of the first mixture (as the aqueous phase) and 2 g of sorbitan trioleate (as an emulsifier) ​​were added to 100 g of olive oil (as the oil phase),

[0152] Then, the mixture was stirred at a constant temperature of 37° C. for 3 hours using a stirrer at 800 rpm (OES-60 electric stirrer, Hangzhou Qinlai Experimental Instrument Co., Ltd.) to obtain a second mixture.

[0153] Next, the mixture was allowed to stand at room temperature of 25° C. for 3 hours to perform solid-liquid separation, thereby obtaining intermediate microspheres.

[0154] Step S1-c

[0155] 2.5 ml of pentanediol (as a cross-linking agent) was added to 100 ml of deionized water to prepare a pentanediol aqueous solution (as a cross-linking agent solution).

[0156] Then, 1 g of the intermediate microspheres was added to 100 g of pentanediol aqueous solution and cross-linked at a constant temperature of 37° C. for 3 hours to obtain a third mixture.

[0157] Next, the mixture was centrifuged at 2000 rpm for 5 minutes (SN-TGL-21 desktop high-speed centrifuge, Shanghai Shangpu Instrument Equipment Co., Ltd.) to perform solid-liquid centrifugal separation to obtain composite microspheres.

[0158] The collagen content in the composite microspheres was measured by thermogravimetric analysis (TGA) to be 27.9 wt %.

[0159] Step S2

[0160] 1 g of a first sodium hyaluronate having a molecular weight of 1.3 million daltons and 4 g of a second sodium hyaluronate having a molecular weight of 250,000 daltons were added to 500 g (i.e., 500 ml) of deionized water, and then stirred for 3 hours to fully dissolve them, thereby obtaining a sodium hyaluronate aqueous solution.

[0161] Step S3

[0162] 10 g of the composite microspheres were added to 100 g of the sodium hyaluronate aqueous solution, and then stirred for 4 hours to uniformly disperse the composite microspheres in the sodium hyaluronate aqueous solution in the form of a solid-liquid mixture, thereby obtaining a facial skin filler.

[0163] Example 2 (reducing the amount of collagen)

[0164] A facial dermal filler was prepared in the same manner as in Example 1, except that the amount of collagen used in step S1-a was changed to 1 g.

[0165] Example 3 (Increasing the amount of collagen)

[0166] A facial dermal filler was prepared in the same manner as in Example 1, except that the amount of collagen used in step S1-a was changed to 5 g.

[0167] Example 4 (Changing the type of emulsifier)

[0168] A facial dermal filler was prepared in the same manner as in Example 1, except that sorbitan monooleate was used as an emulsifier in step S1-b.

[0169] Comparative Example 1 (using hydroxyapatite microspheres instead of composite microspheres, omitting collagen and cross-linking agent)

[0170] A facial dermal filler was prepared in the same manner as in Example 1, except that steps S1-a to S1-c were omitted, and the hydroxyapatite microspheres in step S1-a were used in step S3 instead of the composite microspheres.

[0171] Comparative Example 2 (crosslinking agent omitted)

[0172] A facial dermal filler was prepared in the same manner as in Example 1, except that step S1-c was omitted.

[0173] Comparative Example 3 (omitting the first sodium hyaluronate)

[0174] A facial dermal filler was prepared in the same manner as in Example 1, except that the first sodium hyaluronate (high molecular weight sodium hyaluronate) was omitted in step S2.

[0175] Experimental example

[0176] Experimental Example 1 Morphology Observation

[0177] The composite microspheres obtained in step S1-c of Example 1 were photographed using an optical microscope (MSD1125 optical microscope, Maishidi Technology Co., Ltd.). Figure 1 In. Figure 1 In the upper right corner of the image, you can see a white ring surrounding a black sphere, corresponding to the hydroxyapatite microspheres being encapsulated by collagen. This is because hydroxyapatite particles are opaque to light and appear as black spheres, while collagen is transparent to light and appears as a white ring. This confirms that the outer surface of the hydroxyapatite microspheres is completely covered by collagen.

[0178] In addition, the composite microspheres of Example 1 were photographed by scanning electron microscopy (SU8020 cold field emission scanning electron microscope, HITACHI), and the obtained photographs are shown in FIG. Figure 2 In. From Figure 2 It can be seen that the surface of the composite microspheres is relatively rough, which means that it can be confirmed that the hydroxyapatite microspheres are completely covered by collagen.

[0179] Experimental Example 2 Degradation Test (Maintenance Time Test)

[0180] In accordance with the "Biological Evaluation of Medical Devices" (GB / T 16886), various facial dermal fillers from the Examples and Comparative Examples were placed in dialysis bags and immersed in 250 mL of PBS solution (pH 7.4) for degradation testing. The degradation rates (%) were obtained. The results are shown in Table 1.

[0181] Table 1 Degradation rate of facial dermal fillers at different times (%)

[0182] Testing time 14 days 28 days 42 days Example 1 2.9 8.9 15.6 Example 2 3.2 9.7 16.3 Example 3 2.1 7.4 13.8 Example 4 3.4 9.8 14.3 Comparative Example 1 8.7 14.4 21.7 Comparative Example 2 4.1 11.3 17.9 Comparative Example 3 7.9 13.2 20.4

[0183] As can be seen from Table 1, the degradation rate of the facial dermal filler in each example is significantly lower than that in each comparative example, that is, the maintenance time is prolonged.

[0184] Specifically, the degradation rate of the facial dermal filler of Comparative Example 1 (where hydroxyapatite microspheres were used instead of composite microspheres) was significantly increased compared to Example 1. This confirms that the collagen in Example 1 is beneficial for prolonging the in vivo maintenance time.

[0185] The degradation rate of the facial dermal filler of Comparative Example 2 (in which the cross-linking agent was omitted) increased compared to Example 1. This confirms that the cross-linking agent in Example 1 is beneficial for prolonging the in vivo maintenance time.

[0186] The degradation rate of the facial dermal filler in Comparative Example 3 (in which the first sodium hyaluronate was omitted) increased compared to Example 1. This confirms that the first sodium hyaluronate in Example 1 is beneficial for prolonging the in vivo maintenance time.

[0187] Experimental Example 3 Biocompatibility Test

[0188] In accordance with the "Biological Evaluation of Medical Devices" (GB / T 16886), biocompatibility tests were conducted using the CCK-8 assay for the control group (pure culture medium, without dermal filler), the various facial dermal fillers in the examples, and the comparative examples. Cell viability (%) was determined. The results are shown in Table 2.

[0189] Table 2

[0190] Cell survival rate (%) control group 100 Example 1 98.9 Example 2 98.6 Example 3 99.0 Example 4 98.4 Comparative Example 1 89.6 Comparative Example 2 98.8 Comparative Example 3 96.8

[0191] As can be seen from Table 2, the cell survival rate of the facial dermal fillers of each Example is significantly higher than that of each Comparative Example.

[0192] Specifically, the cell viability (%) of the facial dermal filler of Comparative Example 1 (where hydroxyapatite microspheres were used instead of composite microspheres) was significantly lower than that of Example 1. This confirmed that the collagen in Example 1 was beneficial in improving biocompatibility.

[0193] Furthermore, the cell viability (%) of the facial dermal filler of Comparative Example 3 (in which the first sodium hyaluronate was omitted) was lower than that of Example 1. This confirmed that the first sodium hyaluronate in Example 1 is beneficial for improving biocompatibility.

[0194] Experimental Example 4 Injection Performance Test

[0195] 0.6 ml of each facial dermal filler from the examples and comparative examples was drawn into a 1 mL syringe, which was then fitted with a 27G needle. The syringe was pushed at a constant rate of 1 mL / min to observe for clogging.

[0196] In addition, the microspheres were collected after injection and examined for changes (such as fragmentation or aggregation) using a scanning electron microscope (SU8020 cold-field emission scanning electron microscope, HITACHI) to determine whether the facial dermal filler had good injectability.

[0197] The results are shown in Table 3.

[0198] Table 3

[0199] Is it blocked? Microsphere changes Example 1 Not blocked No change Example 2 Not blocked No change Example 3 Not blocked No change Example 4 Not blocked No change Comparative Example 1 Partial blockage No change Comparative Example 2 Not blocked Some microspheres were broken Comparative Example 3 Not blocked No change

[0200] As can be seen from Table 3, the injection performance of the facial dermal fillers of each example is better than that of Comparative Examples 1 and 2.

[0201] Specifically, the facial dermal filler of Comparative Example 1 (in which hydroxyapatite microspheres were used instead of composite microspheres) was clogged during injection compared to Example 1. This confirms that the collagen in Example 1 is beneficial in preventing clogging.

[0202] Furthermore, the facial dermal filler of Comparative Example 2 (in which the crosslinking agent was omitted) exhibited microsphere breakage after injection compared to Example 1. This confirms that the crosslinking agent in Example 1 contributes to improving the supportability of the composite microspheres.

[0203] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

1. A method for preparing a facial dermal filler, the method comprising the following steps: S1-a: dissolving collagen in water, then adding hydroxyapatite microspheres and mixing to obtain a first mixture; S1-b: adding the first mixture and the emulsifier to the oil phase and maintaining a constant temperature, followed by stirring to obtain a second mixture, and then performing solid-liquid separation to obtain intermediate microspheres; S1-c: adding the intermediate microspheres to a crosslinking agent solution for crosslinking to obtain a third mixture, and then performing solid-liquid centrifugation to obtain composite microspheres; S2: dissolving a first sodium hyaluronate having a molecular weight of 1.2 to 1.5 million daltons and a second sodium hyaluronate having a molecular weight of 0.2 to 0.4 million daltons in water to obtain a sodium hyaluronate aqueous solution; S3: mixing the composite microspheres and the sodium hyaluronate aqueous solution to obtain the facial skin filler.

2. The method according to claim 1, wherein In step S1-a, The water is at least one of deionized water, ion exchange water, distilled water and pure water, The average particle size of the hydroxyapatite microspheres is 5 to 50 μm. The mass ratio of the collagen to the water is (1-5):100, The mass ratio of the collagen to the hydroxyapatite microspheres is (1-5):

1.

3. The method according to claim 1, wherein In step S1-b, The mass ratio of the first mixture: the emulsifier: the oil phase is (3-10): (1-10): 100, The emulsifier is one or more of sorbitan trioleate, sorbitan monooleate and sorbitan monostearate. The oil phase is one or more of olive oil, peanut oil and soybean oil, The constant temperature is 15-60°C, The stirring rate is 500-1000 rpm, and the stirring time is 1-8 hours.

4. The method according to claim 1, wherein In step S1-c, The cross-linking agent is one or more of pentanediol, a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and 1,4-butanediol diglycidyl ether. The solvent of the cross-linking agent solution is water, and the water is at least one of deionized water, ion exchange water, distilled water and pure water. The volume ratio of the cross-linking agent to the water is (1-5):100, The mass ratio of the intermediate microspheres to the crosslinking agent solution is (0.5-1.5):

100. The cross-linking is carried out at 20-50°C for 1-12 hours. The centrifugal speed of the solid-liquid centrifugal separation is 1000-5000 rpm, and the centrifugal time is 1-10 minutes.

5. The method according to claim 1, wherein In step S2, The mass ratio of the first sodium hyaluronate to the second sodium hyaluronate is 1:(1-10), The mass ratio of the combination of the first sodium hyaluronate and the second sodium hyaluronate to water is (0.5-3):100, The water is at least one of deionized water, ion exchanged water, distilled water and pure water.

6. The method according to claim 1, wherein In step S3, the mass ratio of the composite microspheres to the sodium hyaluronate aqueous solution is (3-40):

100.

7. A facial dermal filler prepared by the method of claim 1, comprising: Composite microspheres comprising collagen and hydroxyapatite microspheres, and Sodium hyaluronate aqueous solution; in, The composite microspheres have a core-shell structure, wherein collagen as a shell covers hydroxyapatite microspheres as a core.

8. The facial dermal filler according to claim 7, wherein The average particle size of the composite microspheres is 20 to 70 μm.

9. The facial dermal filler according to claim 7, wherein The content of the collagen is 5 to 60 wt % relative to the total weight of the composite microspheres.

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