WO2023151668A1 - 一种包含无菌pcl微球的美容产品 - Google Patents

一种包含无菌pcl微球的美容产品 Download PDF

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WO2023151668A1
WO2023151668A1 PCT/CN2023/075505 CN2023075505W WO2023151668A1 WO 2023151668 A1 WO2023151668 A1 WO 2023151668A1 CN 2023075505 W CN2023075505 W CN 2023075505W WO 2023151668 A1 WO2023151668 A1 WO 2023151668A1
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microspheres
pcl microspheres
sterile
pcl
irradiation
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English (en)
French (fr)
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张璇
王斌
张海茹
王卓
夏雯蓉
高健
董西健
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辛克莱制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the invention relates to the field of medical cosmetology or medical preparations, in particular to a cosmetic product containing sterile PCL microspheres and a preparation and sterilization method thereof.
  • Polycaprolactone (Polycaprolactone, PCL, CAS No.: 24980-41-4), also known as poly ⁇ -caprolactone, is formed by ring-opening polymerization of ⁇ -caprolactone monomer under the catalysis of metal anion complex catalyst
  • High molecular weight polymers can obtain different molecular weights by controlling the polymerization conditions. Its appearance is white solid powder or granules, non-toxic, insoluble in water, soluble in many polar organic solvents.
  • PCL has good biocompatibility, good organic polymer compatibility, and good biodegradability. It can be used as a cell growth support material. It is compatible with a variety of conventional plastics and can be completely degraded in the natural environment and in the body. .
  • PCL degradation The products of PCL degradation are CO 2 and H 2 O, which are non-toxic to human body.
  • PCL is used as a medical aesthetic filler for implanted materials in the body to stimulate collagen production.
  • PCL-based carboxymethylcellulose gel injection provides immediate and long-lasting plumping results.
  • PCL as a filler material is usually made into microspheres, and its size, shape, and smoothness have been proven to have a great influence on tissue response and determine the degree of response.
  • PCL microspheres are prepared into a regular spherical shape with a smooth surface, the occurrence of inflammation can be minimized; the smoother the formed spheres, the more conducive to subcutaneous injection, and the microspheres with high smoothness can stimulate fibroblasts under wrinkles While wrapping one by one, it can reduce the occurrence of foreign body reactions.
  • sterilization methods have been used in the field of medical device products and medical aesthetic injectable products, including steam treatment, heat/autoclave sterilization, ethylene oxide treatment, plasma sterilization, and electron beam irradiation ( beta irradiation), gamma irradiation or X-ray irradiation.
  • the sterilization method must be compatible with the material selection of the implant to avoid the ineffectiveness of the sterilization process and reduce its damage to the product.
  • PCL has a low melting temperature (about 60°C) and cannot withstand high temperatures. In the case of exceeding 50°C, PCL microspheres will melt, so PCL microspheres are not suitable for steam treatment and autoclaving techniques, which will damage the material or the structure of the implant.
  • Cassan et al. evaluated three low-temperature techniques for PCL sterilization, including beta-ray irradiation sterilization, gamma-ray irradiation sterilization, and X-ray irradiation sterilization, and found that irradiation led to a decrease in molecular weight and an increase in crystallinity.
  • the microspheres are also affected by irradiation, and the irradiation effect on the morphology is dose-dependent, with higher irradiation doses leading to stronger changes.
  • gamma irradiation seemed to be the least suitable method, while electron beam (beta irradiation) was less affected.
  • WO2011089173A1 mentions that the sterilization stage containing CMC microsphere suspension is usually processed by a back pressure autoclave.
  • Tichy et al. evaluated the effect of steam sterilization and the presence of electrolytes on cellulose derivatives Effects on the rheology and textile properties of carrageenan and xanthan-based hydrogels.
  • gases such as ethylene oxide cannot pass through the syringe, the assembled beauty injection products cannot be sterilized with ethylene oxide. Therefore, how to effectively sterilize the PCL microspheres while maintaining the spherical shape and surface smoothness of the microspheres, and preparing cosmetic products containing sterile PCL microspheres is an urgent problem in the field of medical cosmetology.
  • the present invention is a cosmetic product containing sterile PCL microspheres, which needs to be injected into the human body, so the product itself needs to be strictly sterilized, especially the PCL microspheres.
  • PCL microspheres By studying the effect of ⁇ -ray and ⁇ -ray irradiation sterilization on PCL microspheres, it was found that the sterilizing effect of ⁇ -ray is better than that of ⁇ -ray.
  • high doses of gamma rays and high doses of beta rays would have adverse effects on the PCL microspheres themselves, mainly because the surface smoothness of the PCL microspheres and the shape of the spheres changed. Therefore, when sterilizing PCL microspheres, the method of irradiation The formula and dose of radiation will be very important.
  • the present invention adopts Sysmex3000 particle shape and particle size analyzer, combined with flow particle image analysis technology (FPIA) to detect the roundness of PCL microspheres, and can characterize the spherical shape of microspheres by quantitative indicators , can take into account the smoothness of the surface, and can carry out quality control in actual production, effectively meeting the quality requirements for sterile PCL microspheres.
  • FPIA flow particle image analysis technology
  • the present invention relates to a cosmetic product containing sterile PCL microspheres, comprising sterile PCL microspheres, the sterile PCL microspheres are sterilized by irradiation, the surface of which is smooth and spherical, and the average particle size range is 26- 46 ⁇ m, preferably 28-42 ⁇ m, roundness ⁇ 0.96, preferably 0.97 or more, 0.98 or more, 0.99 or more.
  • the sterile PCL microspheres are sterilized by gamma or beta ray irradiation; According to the dose of 20kGy, 25kGy or 30kGy.
  • microspheres with a particle size of 25-50 ⁇ m account for ⁇ 65%, microspheres with a particle size of less than 25 ⁇ m account for ⁇ 20%, and microspheres with a particle size greater than 50 ⁇ m
  • the proportion of spheres is ⁇ 15%; preferably, the proportion of microspheres with a particle size of 25-50 ⁇ m is ⁇ 70%, the proportion of microspheres with a particle size of less than 20 ⁇ m is ⁇ 1%, and the proportion of microspheres with a particle size of 20-25 ⁇ m is ⁇ 15% , the proportion of microspheres with a particle size greater than 50 ⁇ m is ⁇ 14%.
  • the microspheres with the above particle size distribution can greatly reduce the incidence of foreign body granuloma and improve the biological safety of beauty products.
  • the weight average molecular weight of the sterile PCL microspheres is 8k-80,000 Da, such as 10,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 60,000 Da, 80,000 Da , preferably 1-40,000 Da.
  • the molecular weight distribution coefficient is 1.5-3.0, preferably 1.8-2.5.
  • the degradation time of the cosmetic products with the above-mentioned weight average molecular weight in the human body is longer than 12 months, 18 months, 24 months, and 48 months.
  • the change rate of the roundness of the PCL microspheres before and after irradiation is ⁇ 3.5%, preferably ⁇ 3%, 2.5% or 2%.
  • the cosmetic product as mentioned above also includes the CMC-Na gel obtained by moist heat sterilization.
  • Cosmetic products as described above also include sterile processed syringes.
  • a method for preparing the aseptic PCL microspheres in claim 1, the step of obtaining the aseptic PCL microspheres through irradiation sterilization is specifically as follows:
  • Step 1 Weigh an appropriate amount of PCL microspheres and place them in a centrifuge tube, and mark the irradiation dose and type of microspheres on the centrifuge tube;
  • Step 2 In order to ensure the sterility and not leak the sample during the test, seal the centrifuge tube with a parafilm;
  • Step 3 Put the centrifuge tube into the packing box, and affix a label on the outside of the box, marking the sterilization dose information;
  • Step 4 Receive gamma-ray or beta-ray irradiation.
  • the irradiation dose is 20kGy, 25kGy or 30kGy.
  • the ratio of the oil phase to the water phase is 1:5 to 1:50, preferably 1:8 to 1:30, more preferably 1:10 to 1:20;
  • the mass concentration of polycaprolactone in the oil phase is 10mg /ml ⁇ 50mg/ml, preferably 15mg/ml ⁇ 30mg/ml, more preferably 15mg/ml ⁇ 25mg/ml.
  • the speed of high-speed shearing is greater than 12000r/min, and the time is 1-5min.
  • the stirring speed is 800-1200r/min, and the time is 2-4h.
  • a method for preparing a beauty product containing aseptic PCL microspheres as described above, comprising the step of mixing the aseptic PCL microspheres with the CMC-Na gel obtained after being sterilized by a moist heat sterilization method.
  • the mixing step includes: mixing under vacuum conditions.
  • the mixing process adopts a planetary agitator or a vacuum stirring defoamer.
  • Step 1 After fabrication is complete, transfer the gel to a container and place it in an autoclave;
  • Step 2 High-pressure steam sterilization is adopted, wherein the steam pressure is 101-105 kPa, the temperature is 119-123° C., and the steam sterilization is carried out by maintaining for 15-20 minutes.
  • Step 1 Obtain aseptic PCL microspheres by irradiation sterilization; obtain CMC-Na gel by sterilizing by moist heat sterilization; sterilize the syringe with ethylene oxide;
  • Step 2 adding the sterilized PCL microspheres into the CMC-Na gel and mixing;
  • Step 3 encapsulate the mixed CMC-Na gel loaded with sterile PCL microspheres into the syringe.
  • the aseptic beauty product formed by mixing the aseptic PCL microspheres sterilized by irradiation with the CMC-Na gel obtained after damp heat sterilization and filling them into syringes sterilized by ethylene oxide can not only Meeting the needs of sterile injection can also better ensure product quality and reduce the occurrence of side effects such as inflammatory reactions.
  • a and b in Fig. 1 are scanning electron micrographs and micrographs of PCL microspheres in Example 1, respectively.
  • Figure 2 is a scanning electron micrograph of 10,000 Da weight average molecular weight PCL microspheres.
  • a to d are 10,000 Da weight-average molecular weight PCL, which are electron micrographs of microspheres sterilized by ⁇ -rays of 15, 20, 30 or 40 kGy.
  • Figure 4 is an electron micrograph of 40,000 Da weight-average molecular weight PCL microspheres sterilized by 30kGy gamma rays.
  • a to b are 10,000 Da weight-average molecular weight PCL, which is the electron micrograph of microspheres after being sterilized by ⁇ -rays of 30 or 40 kGy.
  • Embodiment 1 the preparation of PCL microsphere
  • Raw materials 500 mg of PCL materials with a weight average molecular weight of 10,000 Da and 40,000 Da, 2.5 mL of DCM, and 25 mL of 1% MC solution.
  • the prepared PCL microspheres are uniform in size and suitable in particle size.
  • Figure 1a and Figure 1b are scanning electron micrographs and micrographs (microscope BM200010x) of PCL microspheres, respectively.
  • Embodiment 2 the detection of PCL microsphere before sterilization
  • the PCL microspheres prepared by the method of Example 1 were first observed under a microscope, and it was found that the prepared PCL microspheres had a uniform and suitable particle size (see Fig. 1a, Fig. 2).
  • the particle size, molecular weight distribution coefficient and crystallization of 10,000 Da and 40,000 Da PCL microspheres before and after sterilization Degree, roundness and colony count were tested.
  • the particle size range of PCL microspheres was detected by morphology particle size analyzer (Topsizerplus); the molecular weight distribution coefficient of PCL microspheres was detected by gel permeation chromatography (GPC); the differential scanning calorimeter (DSC) was used to detect the Detect the crystallinity of PCL microspheres; use flow particle image analysis technology (FPIA) to detect the roundness and change rate of PCL microspheres; use the method of colony counting to detect the number of colonies of PCL microspheres before and after sterilization .
  • GPC gel permeation chromatography
  • DSC differential scanning calorimeter
  • Particle size detection use a particle size analyzer to detect the particle size range of PCL microspheres, the process is as follows:
  • the average particle size of PCL microspheres with a weight average molecular weight of 10,000 Da before sterilization was 35.40 ⁇ m
  • the PCL microspheres with a particle size of less than 20 ⁇ m accounted for 0.66%
  • the PCL microspheres with a particle size of 20-25 ⁇ m accounted for
  • the proportion of PCL microspheres with a particle size of 25-50 ⁇ m is 65.60%
  • the proportion of PCL microspheres with a particle size greater than 50 ⁇ m is 14.97%.
  • the average particle size of PCL microspheres with a weight-average molecular weight of 40,000 Da before sterilization is 37.12 ⁇ m, PCL microspheres with a particle size of less than 20 ⁇ m accounted for 0.62%, PCL microspheres with a particle size of 20-25 ⁇ m accounted for 14.73%, PCL microspheres with a particle size of 25-50 ⁇ m accounted for 70.67%, and PCL microspheres with a particle size greater than 50 ⁇ m Microspheres accounted for 13.98%.
  • the PCL microspheres with a weight average molecular weight of 10,000 Da before sterilization had a molecular weight distribution coefficient (PDI) of 2.1652; the PCL microspheres with a weight average molecular weight of 40,000 Da before sterilization had a molecular weight distribution coefficient ( PDI) is: 1.8001.
  • DSC differential scanning calorimeter
  • the test results are: the crystallinity of PCL microspheres with a weight average molecular weight of 10,000 Da is 70.2266%; the crystallinity of PCL microspheres with a weight average molecular weight of 40,000 Da is 64.9681%.
  • the PCL microspheres were dispersed in phosphate-buffered saline (PBS) to form a dilute dispersion suitable for FPIA analysis.
  • PBS phosphate-buffered saline
  • the flow particle image analysis software takes two-dimensional shots of these particles.
  • FPIA calculates the two-dimensional area of a particle and generates a perfect circle with the same two-dimensional area. It then calculates the circumference of this perfect circle and compares it to the actual total length of the grain's edges. Roundness is the ratio of the circumference of a perfect circle to the actual total length of the particle edge. If the sample particle is a perfect circle, the ratio is 1.
  • the sample average roundness calculated by this method is always between 0 and 1.
  • the software will automatically calculate the average roundness of 1000-2000 microspheres in a test, and calculate the average value. After testing, the roundness of the 10,000 Da and 40,000 Da PCL microspheres was higher than 0.99 (0.9951 and 0.9967, respectively).
  • the rate of change in roundness is the ratio of the change in roundness before and after sterilization to the roundness before sterilization: (roundness before sterilization - roundness after sterilization)/roundness before sterilization.
  • Step 1 Dilution of samples. Weigh a certain amount of PCL microsphere samples and place them in a sterile homogenizing cup filled with phosphate buffer saline or saline, and homogenize at 8000r/min to 10000r/min for 1min to 2min. Use a sterile pipette or a micropipette to draw a 1:10 sample homogeneous solution, slowly inject it into a sterile test tube containing the diluent along the tube wall (note that the tip of the straw or tip does not touch the surface of the diluent), shake the test tube or Use a sterile pipette to blow repeatedly to mix evenly to make a 1:100 homogenous sample solution.
  • a sterile pipette or tip For each incremental dilution, use a sterile pipette or tip. According to the estimation of sample contamination status, select 2 to 3 sample homogeneous solutions with appropriate dilution (liquid samples may include the original solution), and when performing 10-fold incremental dilution, pipette 1mL sample homogeneous solution into a sterile plate, each Make two plates for the dilution. at the same time, Pipette 1mL of the blank diluent and add it to two sterile plates as a blank control. Pour 15mL ⁇ 20mL plate count agar medium cooled to 46°C in time (it can be kept in a constant temperature water bath at 46°C ⁇ 1°C) and pour it into the plate, and rotate the plate to mix evenly.
  • Step 2 Cultivate. After the agar solidifies, turn the plate over and incubate at 36°C ⁇ 1 for 48h ⁇ 2h.
  • Step 3 Colony counting. Colony counts were expressed in colony-forming units (CFU). Select a plate with a colony number between 30CFU and 300CFU and no spreading colony growth to count the total number of colonies. Record the specific number of colonies on the plate below 30CFU, and record the number of colonies above 300CFU as too many. The number of colonies for each dilution should be the average of two plates. Colony counting: PCL microspheres with a molecular weight of 10,000 Da and 40,000 Da had colonies detected before sterilization, and the number of colonies was ⁇ 100 (grade).
  • Embodiment 3 utilize gamma ray to carry out sterilization to PCL microsphere
  • PCL microspheres The irradiation sterilization steps of PCL microspheres are as follows:
  • Step 1 Weigh an appropriate amount of microspheres and place them in a centrifuge tube, and mark the irradiation dose and type of microspheres on the centrifuge tube;
  • Step 2 In order to ensure the sterility and not leak the sample during the test, seal the centrifuge tube with a parafilm;
  • Step 3 Put the centrifuge tubes with the same irradiation dose that need to be sterilized into the packaging box, and put a label on the outside of the box, marking the sterilization dose information;
  • Step 4 The packaging groups of the encapsulated PCL microspheres are irradiated with different doses of gamma rays (Co60).
  • Figures 3a to 3d are 10,000 Da PCL microspheres irradiated by 15, 20, 30, 40kGy gamma rays Subsequent electron microscope photographs.
  • the roundness value of the microspheres irradiated by 15kGy gamma rays is 0.9892, and the roundness change rate is 0.59%. It can be seen from the electron microscope photo in Figure 3a that there are few The amount of cracks, but the overall change of the microspheres is not significant.
  • PCL microspheres with a weight average molecular weight of 40,000 Da are more susceptible to ⁇ -ray irradiation than PCL microspheres with a weight average molecular weight of 10,000 Da. This can be seen from the roundness value and roundness change rate in Table 1 and Table 2.
  • the PCL microspheres with a weight-average molecular weight of 10,000 Da are irradiated with ⁇ -rays of 30kGy.
  • the roundness is 0.9675, and the roundness change rate is 2.77%, while the roundness of 40,000 Da PCL microspheres is 0.9618, and the roundness change rate is 3.50% under 30kGy ⁇ -ray irradiation.
  • Figure 4 shows the electron microscope photos of 40,000 Da PCL microspheres irradiated by 30kGy gamma rays. It can be seen that there are more cracks and depressions on the surface of the microspheres than that of Wanda's PCL microspheres (Fig. 3c). These all confirm that high molecular weight PCL microspheres are more susceptible to irradiation.
  • Embodiment 4 Utilize beta ray sterilization to obtain sterile PCL microspheres
  • the method of irradiation sterilization of PCL microspheres is basically the same as the steps adopted in Example 3, the difference being that the radiation ray used for sterilization is beta ray (electron accelerator).
  • the impact on PCL microspheres is less than 30 kGy. repeat.
  • the sterilization effect of ⁇ -ray irradiation is not as good as that of ⁇ -ray.
  • 15-30kGy dose of ⁇ -rays is used for sterilization, the sterilization effect is not good, and there are still bacteria in the microspheres.
  • 40kGy high-dose ⁇ -rays are used to sterilize PCL microspheres, the roundness of the microspheres will be affected. The uniformity and surface smoothness have a great influence.
  • Embodiment 5 CMC-Na gel sterilization
  • Carrier gels based on carboxymethylcellulose sodium (CMC-Na) cannot withstand radiation sterilization because radiation sterilization has a severe effect on the viscosity of CMC-Na. Therefore, the CMC-Na gel is sterilized by hot and humid steam sterilization, that is, high-pressure steam sterilization, the steam pressure is about 101-105 kPa, the temperature is 119-123 ° C, and it is maintained for 15-20 minutes; the specific steps are as follows:
  • Step 1 After the CMC-Na gel is prepared, transfer it to a mixing container and place it in an autoclave;
  • Step 2 adopt high-pressure steam sterilization method, wherein the steam pressure is about 101-105 kPa, the temperature is 119-123° C., and the steam sterilization is carried out by maintaining for 15-20 minutes.
  • Embodiment 6 the CMC-Na gel that aseptic PCL microsphere and sterilizing process obtains is mixed to form aseptic cosmetic product
  • Step 1 Measure 10ml of gel, pour it into a centrifuge tube, use a scraper to carefully scrape off the gel adhered to the wall of the measuring cylinder, and transfer it to the centrifuge tube until the loss of the gel on the wall is minimal;
  • Step 2 Add 3g of weighed microspheres to the gel, and use a glass rod or a fine spatula for preliminary stirring.
  • the microsphere gel adhered to the stirring rod should be scraped back to the edge of the centrifuge tube to ensure the minimum loss;
  • Step 3 Prepare the preliminarily mixed sample, and use a vacuum stirring and degassing machine to perform stirring and degassing treatment.
  • Example 7 Inject cosmetic products into sterilized syringes to form sterile injectable cosmetic products
  • Syringes and needle caps were aseptically fed into the LAF Laminar Air Flow. pass
  • the priming method fills the syringe with cosmetic product to the correct volume and attaches the end cap to create a sterile barrier. All mixing and filling steps are performed aseptically in an ISO class 5 LAF unit, labeled resulting in sterile injectable cosmetic products and then stored.

Abstract

本发明涉及一种含有无菌PCL微球的美容产品，包含无菌PCL微球，所述无菌PCL微球采用辐照灭菌处理，其表面光滑，呈球形，平均粒径范围为26-46μm，圆整度≥0.96以上。所述无菌PCL微球采用γ或β射线辐照灭菌处理得到；所述CMC-Na凝胶通过湿热灭菌法灭菌处理得到。将辐照灭菌后的无菌PCL微球与湿热灭菌后得到的CMC-Na凝胶混匀后灌装到经环氧乙烷灭菌过的注射器后形成的无菌美容产品，不仅可以满足无菌注射的需要，也能更好地保证产品质量，减少炎症反应等副作用的产生。

Description

一种包含无菌PCL微球的美容产品 技术领域
本发明涉及医疗美容或医用制剂领域，具体涉及一种包含无菌PCL微球的美容产品及其制备、灭菌方法。
背景技术
聚己内酯(Polycaprolactone，PCL，CAS号：24980-41-4)又称聚ε-己内酯，是通过ε-己内酯单体在金属阴离子络合催化剂催化下开环聚合而成的高分子聚合物，通过控制聚合条件，可以获得不同的分子量。其外观为白色固体粉末或颗粒，无毒，不溶于水，易溶于多种极性有机溶剂。PCL具有良好的生物相容性、良好的有机高聚物相容性，以及良好的生物降解性，可用作细胞生长支持材料，可与多种常规塑料互相兼容，自然环境及体内可完全降解。PCL降解后的产物为CO2和H2O，对人体无毒。PCL用作体内植入材料的医美填充剂，可刺激胶原蛋白的生成。基于PCL的羧甲基纤维素凝胶注射后可提供即时和持续的丰盈效果。
作为填充剂原料的PCL通常被制成微球，其大小、形状、光滑度已被证明对组织反应有很大影响，并决定了反应的程度。当PCL微球被制备成规则的、具有光滑表面的球形，可以最大限度地减少炎症的发生；形成的球体越光滑，越有利于皮下注射，高光滑度的微球在刺激皱纹下成纤维细胞逐一包裹的同时，能够减少异物反应的发生。
由于注射美容产品的给药方式是直接注射入人体的皮下组织，如果产品表面或者内部携带病原体可导致严重的感染，引起患者严重的不良反应，因此用于人体植入的生物医学填充剂需要灭菌。灭菌不仅需要杀灭或除去微生物繁殖体和芽孢，最大限度提高注射剂的安全性和有效性，还需要保证注射美容产品的形态、稳定性以及临床疗效，因此选择适宜的灭菌方法对保证美容产品质量具有重要意义。过去几十年来，在医疗器械产品和医美注射产品领域已经采用了多种灭菌方法，包括蒸汽处理、加热/高压灭菌、环氧乙烷处理、等离子体灭菌以及电子束辐照(β辐照)、γ辐照或X射线辐照。灭菌方法须与植入物的材料选择相适应，以避免灭菌过程的无效性，降低其对产品的破坏性。
PCL具有较低的熔融温度(约为60℃)，不能耐受高温。在超过50℃的情况下，PCL微球会融化，因此PCL微球不适合蒸汽处理和高压灭菌技术，上述方式会损坏材料或者植入物的结构。Cassan等人评估了有关于PCL灭菌的三种低温技术，包括β射线辐照灭菌，γ射线辐照灭菌和X射线辐照灭菌，发现辐照会导致分子量降低，结晶度升高，同时微球也会受到辐照的影响，产生形态辐照效应是剂量依赖性的，较高的辐照剂量导致更强的变化。其中发现γ辐照似乎是最不合适的方法，而电子束(β辐照)的影响较小。
此外，维持注射微球的球体形状及表面光滑度十分重要，一方面可以提高凝胶的流动性，另一方面也可以防止不良异物反应，因为真皮填充物的总体生物相容性是由光滑和规则形状的微粒决定的，这些光滑微粒会刺激胶原蛋白生长，而粗糙、不规则形状的微粒被巨噬细胞和多核巨细胞包围，导致包膜纤维化，抑制胶原蛋白的产生，并且可能导致不良异物反应。
另外，含有PCL微球的注射美容产品的其他组成部分注射器部件和CMC-Na凝胶也需要灭菌。已知注射器材料如聚丙烯材料在接受辐照灭菌时会变色且变得发脆，因此不适合通过γ辐照进行灭菌，EP3685826B1提及对注射器和注射针以环氧乙烷、过氧化氢等进行灭菌。US10905786B2提及对注射器采用蒸发的过氧化氢方法灭菌。而关于CMC凝胶的灭菌方法，WO2011089173A1提及包含CMC微球悬浮液的灭菌阶段通常采用背压式高压灭菌器处理，Tichy等人评估了蒸汽灭菌和电解质的存在对纤维素衍生物、卡拉胶和黄原胶基水凝胶的流变学和纺织性能的影响。而注射器一旦装填了凝胶注射剂，由于环氧乙烷等气体无法穿过注射器，因此组装后的注射美容产品，无法再使用环氧乙烷灭菌。因此如何有效的对PCL微球进行灭菌，同时又能维持微球的球体形状和表面的光滑度，制备出包含无菌PCL微球的美容产品是医疗美容领域的一个亟需解决的问题。
发明内容
本发明是一种包含无菌PCL微球的美容产品，该产品需要注射到人体内，因此需要对产品本身进行严格的灭菌，特别是PCL微球。通过研究γ射线和β射线辐照灭菌对PCL微球的影响，发现γ射线的灭菌效果要好于β射线。此外还发现，高剂量的γ射线和高剂量的β射线都会对PCL微球本身产生不利的影响，主要是PCL微球的表面光滑度和球体的形状发生了变化。因此，在对PCL微球进行灭菌时，辐照的方 式和辐照的剂量将是十分重要的。
现有技术多是采用光学显微或电镜来观察微球的表面光滑度和球体的形状(例如观察微球表面光不光滑，球体圆不圆等)，主观性较强，没有一个量化的指标，实际生产过程中也难以做到实时监控和准确控制。本发明为了克服上述问题，采用了Sysmex3000颗粒形态和粒度分析仪，结合流动颗粒图像分析技术(FPIA)来检测PCL微球的圆整度，通过量化指标，既能对微球的球体形状进行表征，又能兼顾到表面光滑度，并且能够在实际生产中进行质量控制，有效的满足了对无菌PCL微球的质量要求。
本发明涉及一种含有无菌PCL微球的美容产品，包含无菌PCL微球，所述无菌PCL微球采用辐照灭菌处理，其表面光滑，呈球形，平均粒径范围为26-46μm，优选28-42μm，圆整度≥0.96，优选0.97以上、0.98以上、0.99以上。
如前所述的美容产品，所述无菌PCL微球采用γ或β射线辐照灭菌处理得到；优选采用γ射线辐照灭菌所述辐照的剂量为20kGy-30kGy，优选所述辐照的剂量为20kGy、25kGy或30kGy。
如前所述的美容产品，所述无菌PCL微球中粒径为25-50μm的微球占比≥65％，粒径小于25μm的微球占比≤20％，粒径大于50μm的微球占比≤15％；优选，粒径为25-50μm的微球占比≥70％，粒径小于20μm的微球占≤1％，粒径为20-25μm的微球占比≤15％，粒径大于50μm的微球占比≤14％。上述粒径分布的微球可以大幅减少异物肉芽肿的发生率，提高美容产品的生物安全性。
如前所述的美容产品，所述无菌PCL微球的重均分子量为8k-8万Da，例如1万Da、2万Da、3万Da、4万Da、6万Da、8万Da，优选1-4万Da。分子量分布系数为1.5-3.0，优选1.8-2.5。采用上述重均分子量的美容产品其在人体内的降解时间大于12个月、18个月、24个月、48个月。
如前所述的美容产品，辐照前后PCL微球的圆整度变化率≤3.5％，优选≤3％、2.5％或2％。
如前所述的美容产品，还包含湿热灭菌法灭菌处理得到的CMC-Na凝胶。
如前所述的美容产品，还包括无菌处理的注射器。
一种制备权利要求1中的无菌PCL微球的方法，经辐照灭菌处理得到无菌PCL微球的步骤具体如下：
步骤1：称取适量PCL微球放置于离心管中，并在离心管上做好辐照剂量与微球种类的标记；
步骤2：为保证无菌状态和检测中不泄露样品，使用封口膜对离心管进行密封；
步骤3：把离心管装入包装箱内，并在箱外贴上标签，标注灭菌剂量信息；
步骤4：接受γ射线或β射线辐照。
如前所述的方法，其中辐照剂量为20kGy、25kGy或30kGy。
一种制备如前所述的PCL微球的方法，具体步骤如下：
将PCL材料溶解于DCM中形成油相；将所述油相逐滴搅拌加入到MC溶液中，利用高速剪切搅拌机高速剪切，搅拌2-4h；水洗离心3次，筛分、干燥得到；其中，油相与水相比例为1∶5～1∶50，优选1∶8～1∶30，更优选1∶10～1∶20；所述油相中聚己内酯的质量浓度为10mg/ml～50mg/ml，优选15mg/ml～30mg/ml，更优选15mg/ml～25mg/ml。
如前所述的方法，针对4-8万重均分子量的PCL，在高速剪切的步骤之后还有超声的步骤；所述超声的时间为3-6分钟。
如前所述的方法，高速剪切的转速大于12000r/min，时间为1-5min。
如前所述的方法，搅拌时转速800-1200r/min，时间为2-4h。
一种制备如前所述的含有无菌PCL微球的美容产品的方法，包括将无菌PCL微球与通过湿热灭菌法灭菌后得到的CMC-Na凝胶混匀的步骤。
如前所述的制备方法，所述混匀步骤包括：在真空条件下进行混匀。
如前所述的制备方法，所述混匀过程采用行星搅拌仪或真空搅拌脱泡机。
一种制备如前所述的CMC-Na凝胶的方法，具体步骤如下：
步骤1：制造完成后，将凝胶转移到容器中，并将其放入高压灭菌器中；
步骤2：采用高压蒸汽灭菌法，其中蒸汽压为101-105千帕，温度为119-123℃，维持15-20分钟进行蒸汽灭菌。
一种制备如前所述的美容产品的方法，具体步骤如下：
步骤1：通过辐照灭菌得到无菌PCL微球；通过湿热灭菌法灭菌得到CMC-Na凝胶；对注射器采用环氧乙烷灭菌；
步骤2：将灭菌后的所述无菌PCL微球加入到CMC-Na凝胶中进行混匀；
步骤3：将混匀的负载有无菌PCL微球的CMC-Na凝胶封装于所述注射器中。
本发明有益的技术效果：
采用一定剂量的γ或β射线辐照得到的无菌PCL微球，菌落数检测值可达到“未检出”水平；尤其当采用20kGy-30kGy的γ或β射线对重均分子量1-4万道尔顿的 PCL微球进行辐照时，通过电镜照片可以看到其表面的凹陷和沟壑较少，对PCL微球的表面光滑度和圆整度的影响较小，不易引起皮肤肉芽肿和炎症反应。将辐照灭菌后的无菌PCL微球与湿热灭菌后得到的CMC-Na凝胶混匀后灌装到经环氧乙烷灭菌过的注射器后形成的无菌美容产品，不仅可以满足无菌注射的需要，也能更好地保证产品质量，减少炎症反应等副作用的产生。
附图说明
图1中a和b分别为实施例1中PCL微球的扫描电镜照片和显微镜照片。
图2为1万Da重均分子量PCL微球的扫描电镜照片。
图3中a至d为1万Da重均分子量PCL为微球经15、20、30或40kGy的γ射线灭菌后的电镜照片。
图4为4万Da重均分子量PCL微球经30kGy的γ射线灭菌后的电镜照片。
图5中a至b为1万Da重均分子量PCL为微球经30或40kGy的β射线灭菌后的电镜照片。
具体实施方式
以下通过实施例和对比例来进一步说明本发明，但并不限于此。
实施例1：PCL微球的制备
原料：重均分子量1万Da和4万Da的PCL材料各500mg，2.5mL DCM，25mL 1％MC溶液。
步骤：将500mg PCL材料涡旋溶解于2.5ml DCM中形成油相；将所述油相一边搅拌一边逐滴加入到25ml 1％MC溶液中，再搅拌30s，后利用高速剪切搅拌机(大龙兴创D-160)2档(14000r/min)剪切1min，超声5min，搅拌(1000r/min)3h；水洗离心(10000r/min)3次，每次15min，筛分，干燥24h。制得的PCL微球大小均匀、粒径合适。图1a和图1b分别为PCL微球的扫描电镜照片和显微镜照片(显微镜BM200010x)。
实施例2：灭菌前PCL微球的检测
利用实施例1的方法制备的PCL微球，首先在显微镜下观察，发现制备的PCL微球粒径大小均匀合适(参见图1a、图2)。
本发明对灭菌前后1万Da和4万Da的PCL微球的粒径、分子量分布系数、结晶 度、圆整度和菌落数进行了检测。分别利用形态粒度分析仪(Topsizerplus)对PCL微球的粒径范围进行检测；利用凝胶渗透色谱(GPC)对PCL微球的分子量分布系数进行检测；利用差示扫描量热仪(DSC)对PCL微球的结晶度进行检测；利用流动颗粒图像分析技术(FPIA)对PCL微球圆整度和变化率进行检测；利用菌落计数的方法对灭菌前、后PCL微球的菌落数进行检测。
(1)粒径检测：利用形态粒度分析仪对PCL微球的粒径范围进行检测，过程如下：
称取0.1g微球置于离心管中，加入10ml水，振荡、分散，超声处理30min，再分散到预装500mL去离子水的烧杯中，使用欧美克粒径仪测定粒径，测定三次，取平均值。
经检测分析发现，灭菌前重均分子量为1万Da的PCL微球的平均粒径为35.40μm，粒径小于20μm的PCL微球占比0.66％，粒径20-25μm的PCL微球占比18.77％，粒径25-50μm的PCL微球占比65.60％，粒径大于50μm的PCL微球占比14.97％；灭菌前重均分子量为4万Da的PCL微球的平均粒径为37.12μm，粒径小于20μm的PCL微球占比0.62％，粒径20-25μm的PCL微球占比14.73％，粒径25-50μm的PCL微球占比70.67％，粒径大于50μm的PCL微球占比13.98％。
(2)分子量分布系数检测：利用凝胶渗透色谱(GPC)对PCL微球的分子量分布系数进行检测，具体检测步骤如下：
仪器型号：Agilent 1260Infinity
检测器：Agilent 1260RID
色谱柱：Waters Styragel HR 5E(7.8*300mm，2000-400w)和Waters Styragel HR 2(7.8*300mm，500-2w)串联
柱温：35℃
软件：ChemStation for LC systems
标准品：PS(聚苯乙烯)。
称取一定量PCL微球用四氢呋喃溶剂溶解、过滤，待仪器稳定后用进样针筒注入进样口，待色谱柱淋洗后，得到完整的GPC曲线，根据测得的GPC曲线计算分子量及其分子量分布系数。
经检测分析发现，灭菌前重均分子量为1万Da的PCL微球，其分子量分布系数(PDI)为：2.1652；灭菌前重均分子量为4万Da的PCL微球，分子量分布系数(PDI)为：1.8001。
(3)结晶度检测：利用差示扫描量热仪(DSC)对PCL微球的结晶度进行检测，具体检测步骤如下：
使用差示扫描量热仪(CED-E-DSC-045DSC250)，先将PCL微球升温到80℃，恒温10min，然后以相同的速度冷却到-40℃，在氮气保护下，记录-40℃到80℃的升温扫描曲线，升温速度10℃/min，结晶度按比熔融热焓法计算：f＝△Hm/△Hm,c，其中△Hm为DSC分析曲线计算峰面积得到的熔融焓，△Hm,c为100％结晶PCL的熔融焓，依据文献报道的数值139.5J/g来计算。检测结果为：重均分子量1万Da的PCL微球，其结晶度为70.2266％；重均分子量4万Da的PCL微球，其结晶度为64.9681％。
(4)圆整度及其变化率的检测：利用Sysmex 3000颗粒形态和粒度分析仪(英国Malvern Instruments)，结合流动颗粒图像分析技术(FPIA)检测微球的圆整度。具体步骤为：
将PCL微球分散于磷酸盐缓冲盐水(PBS)中，形成稀释分散体，该分散体适用于FPIA分析。在近千个单颗粒通过Sysmex 3000颗粒形态和粒度分析仪流通池时，流动颗粒图像分析软件对这些颗粒进行二维拍摄。FPIA计算颗粒的二维面积，并生成一个具有相同二维面积的正圆。然后，它计算出该正圆的周长，将其与颗粒边缘的实际总长度进行对比。圆整度是正圆周长与颗粒边缘实际总长度的比值。如果样本颗粒是一个正圆，则该比值为1。颗粒形状越不规则，表面越不光滑，该比值就越来越小于1。该方法计算出的样本平均圆整度始终位于0和1之间。软件将自动计算出一次测试中1000-2000个微球的平均的圆整度，计算平均值。经检测，1万Da和4万Da的PCL微球的圆整度均高于0.99(分别达到了0.9951、0.9967)。
圆整度变化率为灭菌前后的圆整度变化量与灭菌前圆整度的比值:(灭菌前圆整度-灭菌后圆整度)/灭菌前圆整度。
(5)菌落数测定方法：
步骤1：样品的稀释。称取一定量的PCL微球样品置盛有磷酸盐缓冲液或生理盐水的无菌均质杯内，8000r/min～10000r/min均质1min～2min。用无菌吸管或微量移液器吸取1∶10样品匀液，沿管壁缓慢注于盛有稀释液的无菌试管中(注意吸管或吸头尖端不要触及稀释液面)，振摇试管或换用1支无菌吸管反复吹打使其混合均匀，制成1∶100的样品匀液。每递增稀释一次，换用1次无菌吸管或吸头。根据对样品污染状况的估计，选择2个～3个适宜稀释度的样品匀液(液体样品可包括原液)，在进行10倍递增稀释时，吸取1mL样品匀液于无菌平皿内，每个稀释度做两个平皿。同时， 分别吸取1mL空白稀释液加入两个无菌平皿内作空白对照。及时将15mL～20mL冷却至46℃的平板计数琼脂培养基(可放置46℃±1℃恒温水浴箱中保温)倾注平皿，并转动平皿使其混合均匀。
步骤2：培养。待琼脂凝固后，将平板翻转，36℃±1培养48h±2h。
步骤3：菌落计数。菌落计数以菌落形成单位(colony-formingunits，CFU)表示。选取菌落数在30CFU～300CFU之间、无蔓延菌落生长的平板计数菌落总数。低于30CFU的平板记录具体菌落数，大于300CFU的可记录为多不可计。每个稀释度的菌落数应采用两个平板的平均数。经菌落计数：灭菌前1万Da和4万Da分子量的PCL微球均有菌落检出，且菌落数＜100(等级)。
实施例3：利用γ射线对PCL微球进行灭菌
PCL微球的辐照灭菌步骤具体如下：
步骤1：称取适量微球放置于离心管中，并在离心管上做好辐照剂量与微球种类的标记；
步骤2：为保证无菌状态和检测途中不泄露样品，使用封口膜对离心管进行密封；
步骤3：把需要灭菌的相同辐照剂量的离心管装入包装箱内进行，并在箱外贴上标签，标注灭菌剂量信息；
步骤4：封装好的PCL微球的包装分组接受γ射线(Co60)不同剂量的辐照。
选用15、20、30、40kGy等4种剂量进行辐照，并对辐照后的PCL微球进行进一步地检测和分析，检测方法和步骤如实施例2中所示。
检测结果如下：
表1：1万Da重均分子量的无菌PCL微球

表2：4万Da重均分子量的无菌PCL微球

通过上述结果可以看出，经过γ射线辐照灭菌，PCL微球中的菌落数显著减少，这说明γ射线辐照可以有效地对PCL微球进行灭菌。
通过比较γ射线各个剂量的实验结果可以看出，当采用15kGy的γ射线对PCL微球进行辐照灭菌时，其菌落数(等级)降至＜5，虽然菌落数已经较低，但是对于一款注射入人体皮肤的美容产品而言，细菌污染将严重影响产品质量，未完全灭菌的产品也将会导致严重的炎症反应。因此，菌落数需控制在“未检出”的标准。通过表1-2可以看出，当γ射线的剂量达到20kGy以上时，菌落数检测即达到“未检出”水平，可以满足产品无菌的要求。
通过对PCL微球的分子量的分布系数、结晶度、圆整度、圆整度变化率和表面光滑度(显微镜及扫描电镜观察)进行分析后发现：γ射线灭菌后，PCL微球的分子量分布系数有所增大，结晶度也有所增大。特别是结晶度的变化，由于测量到的结晶度是一个平均值，是一个高低结晶度平均化的结果，猜测微球内部可能发生了不均匀的结晶度改变，而局部的结晶度升高，很可能会引起局部体积的塌陷，从而导致PCL微球的表面变得不光滑，形成一定裂痕或者小坑。这一点通过扫描电子显微镜也可以得到印证(如附图3a至3d及图4所示)，其中图3a至3d为1万Da的PCL微球经15、20、30、40kGy的γ射线辐照后的电镜照片。经15kGyγ射线辐照后的微球，圆整度值为0.9892，圆整度变化率为0.59％。通过电镜照片图3a可以看到，微球上出现少 量裂痕，但是微球整体变化不大。经过20kGy和30kGy的γ射线辐照后，微球的圆整度值分别降为0.9765和0.9675，圆整度变化率分别为1.87％和2.77％。如图3b和3c所示，PCL微球变化更加明显，微球表面出现了多条裂痕以及凹陷，但是通过电镜照片可以看到微球整体球形度变化不大，仍然维持了较好的球体形状。
当采用40kGy的γ射线辐照灭菌时，发现PCL微球的分子量分布系数、结晶度和圆整度值变化更大。圆整度值降为＜0.96(0.9547)，圆整度变化率达到4.06％。通过电镜照片图3d也可以看到，辐照后的PCL微球表面出现了大量的裂痕和凹陷，微球出现明显皱缩，很多皱缩已经导致球体形状发生改变，部分微球无法形成完整的球体。而本领域知晓，当微球的表面光滑度和球体形状较差时，粗糙和不规则形状的微球会被巨噬细胞和多核巨细胞包围，导致包膜纤维化，抑制胶原蛋白的产生，并且导致不良异物反应。
此外，重均分子量4万Da的PCL微球比1万Da的PCL微球，更易受到γ射线辐照的影响。这从表1和表2的圆整度值和圆整度变化率可以看出，对于不同分子量的PCL微球，重均分子量1万Da的PCL微球在30kGy的γ射线辐照下，圆整度为0.9675，圆整度变化率为2.77％，而4万Da的PCL微球在30kGy的γ射线辐照下，圆整度为0.9618，圆整度变化率为3.50％。通过电镜照片观察也证明了这一点，图4显示了4万Da的PCL微球在30kGy的γ射线辐照下的电镜照片，可以看到微球表面的裂痕和凹陷要多于同剂量下1万Da的PCL微球(图3c)。这些都印证了高分子量的PCL微球更易受到辐照的影响。
实施例4：利用β射线灭菌得到无菌PCL微球
PCL微球辐照灭菌的方法与实施例3所采用的步骤基本相同，不同点为灭菌所采用的辐照射线是β射线(电子加速器)。
选用15、20、30或40kGy四种剂量进行辐照，并对β射线辐照后的PCL微球进行分析和检测。其中，40kGy以上的剂量即无菌落检出。
表3：1万Da重均分子量的无菌PCL微球：


表4：4万Da重均分子量的无菌PCL微球：

通过上述结果可以看出，经过β射线辐照灭菌，PCL微球中的菌落数明显减少，随着剂量的增加，菌落数逐渐减少，其中1万Da或4万Da重均分子量的PCL微球均由灭菌前的＜100(等级)分别降低到＜10、＜10、＜5、未检出，这说明β射线辐照也可以对PCL微球进行灭菌，但是其灭菌效果并没有同剂量下的γ射线好。通过对PCL微球的分子量分布系数、结晶度、圆整度和圆整度变化率进行分析后发现：灭菌前后，PCL微球的分子量分布系数有所增大，结晶度也有所增大。通过表3-4可以看出，采用30kGy的β射线对1万Da分子量的PCL微球进行辐照灭菌时，PCL微球的分子量分布系数、结晶度和圆整度均受到了辐照影响，圆整度＞0.96(0.9691)，圆整度变化率为2.61％，而4万Da分子量的PCL微球，圆整度＞0.96(0.9662)，圆整度变化率为3.06％。通过附图5a可以看出，虽然1万Da分子量的PCL微球的表面也出现了凹陷和沟壑，但是球体形状维持较好，球体表面整体仍较为光滑。由于4万Da分子量的PCL微球，其辐照影响与1万Da分子量的PCL微球差异不大，只是破坏程度上有些许增加，简略起见不再附图展示。
当采用40kGy剂量的β射线对PCL微球进行辐照灭菌时，通过表3-4可以看出，不论是1万Da还是4万Da的PCL微球的表面光滑度和圆整度均受到了辐照较大的影响，圆整度均＜0.96(0.9568，0.9563)，圆整度变化率分别达到3.85％、4.05％。可 见，类似于γ辐照的情况，重均分子量4万Da的PCL微球比1万Da的PCL微球更易受到β射线辐照的影响。通过附图5b可以看出，对于1万Da的PCL微球，其表面已出现了明显的凹陷和沟壑，球体形状也发生了改变。这对保证PCL微球的表面光滑度和球体形状，减少异物不良反应是十分不利的。因此，尽管该剂量下可以实现达到“未检出”的灭菌要求，但本申请仍不建议采用40kGy剂量的β射线对PCL微球进行辐照灭菌。
此外，由于15和20kGy的β射线辐照无论是在分子量分布系数、结晶度、圆整度、圆整度变化率上对PCL微球的影响均小于30kGy，为避免重复描述，说明书中不再赘述。
综上，采用β射线辐照灭菌的效果不如γ射线。当采用15-30kGy剂量的β射线进行灭菌时，其灭菌效果欠佳，微球中仍有细菌残留，而采用40kGy高剂量的β射线对PCL微球灭菌时，对微球的圆整度和表面光滑度影响较大，综合来看，选择20-30kGy剂量的γ射线对重均分子量1-4万Da的PCL微球进行辐照灭菌是最有利的。
实施例5：CMC-Na凝胶灭菌
因为辐照灭菌对CMC-Na的粘度有严重影响，因而基于羧甲基纤维素钠(CMC-Na)的载体凝胶不能耐受射线辐照的灭菌。故所述CMC-Na凝胶灭菌选择湿热蒸汽灭菌，即高压蒸汽灭菌法，蒸汽压约为101-105千帕，温度为119-123℃，维持15-20分钟；具体步骤如下：
步骤1：CMC-Na凝胶制备完成后，将其转移到混合容器中，再将其放入高压灭菌器中；
步骤2：采用高压蒸汽灭菌法，其中蒸汽压约为101-105千帕，温度为119-123℃，维持15-20分钟进行蒸汽灭菌。
实施例6：将无菌PCL微球和灭菌处理得到的CMC-Na凝胶混合形成无菌美容产品
步骤1、量取10ml凝胶，倒入离心管中，使用刮刀仔细刮取量筒壁上粘附的凝胶，将其转移到离心管中，直至凝胶挂壁损失最小为止；
步骤2、向凝胶中加入称量好的3g的微球，并使用玻璃棒或细刮刀进行初步搅拌，粘附在搅拌棒上的微球凝胶要刮回离心管边缘以保证损失最小；
步骤3、准备好初步混合好的样品，使用真空搅拌脱泡机进行搅拌除气泡处理。
实施例7：将美容产品注入灭菌后的注射器中，形成无菌注射美容产品
注射器和针头帽以无菌方式进入LAF层流气流装置(Laminar Air Flow)。通过 灌注的方式将美容产品充入注射器，到达正确的体积，并连接端盖以形成无菌屏障。所有混合和填充步骤均在ISO 5级LAF装置中无菌进行，贴上标签，从而得到无菌注射美容产品，然后储存。

Claims (29)

  1. 一种含有无菌PCL微球的美容产品，其特征在于：包含无菌PCL微球，所述无菌PCL微球采用辐照灭菌处理，其表面光滑，平均粒径范围为26-46μm，圆整度≥0.96。
  2. 如权利要求1所述的一种含有无菌PCL微球的美容产品，其特征在于：所述的无菌PCL微球圆整度≥0.97。
  3. 如权利要求1所述的美容产品，其特征在于：所述无菌PCL微球采用γ或β射线辐照灭菌处理得到。
  4. 如权利要求3所述的美容产品，其特征在于：所述辐照灭菌采用γ射线，所述辐照的剂量为20kGy-30kGy。
  5. 如权利要求4所述的美容产品，其特征在于：所述辐照的剂量为20kGy、25kGy或30kGy。
  6. 如权利要求1所述的美容产品，其特征在于：所述无菌PCL微球中粒径为25-50μm的微球占比≥65％，粒径小于25μm的微球占比≤20％。
  7. 如权利要求6所述的美容产品，其特征在于：所述无菌PCL微球中粒径大于50μm的微球占比≤15％。
  8. 如权利要求1所述的美容产品，其特征在于：所述无菌PCL微球的重均分子量为8k-8万Da。
  9. 如权利要求8所述的美容产品，其特征在于：所述无菌PCL微球的重均分子量为1-4万Da。
  10. 如权利要求1所述的美容产品，其特征在于：所述无菌PCL微球的分子量分布系数PDI为1.5-3.0。
  11. 如权利要求10所述的美容产品，其特征在于：所述无菌PCL微球的分子量分布系数PDI为1.8-2.5。
  12. 如权利要求1所述的美容产品，其特征在于：所述无菌PCL微球的圆整度≥0.97。
  13. 如权利要求1所述的美容产品，其特征在于：辐照前后PCL微球的圆整度变化率≤3.5％。
  14. 如权利要求1所述的美容产品，其特征在于：辐照前后PCL微球的圆整度变化率≤2.5％。
  15. 如权利要求1所述的美容产品，其特征在于：还包含湿热灭菌法灭菌处理得 到的无菌CMC-Na凝胶。
  16. 如权利要求1所述的美容产品，其特征在于：还包括无菌处理的注射器。
  17. 一种制备如权利要求1所述的含有无菌PCL微球的美容产品的方法，其特征在于：包括将无菌PCL微球与通过湿热灭菌法灭菌后得到的无菌CMC-Na凝胶混匀的步骤。
  18. 一种制备如权利要求1中所述的无菌PCL微球的方法，其特征在于：经辐照灭菌处理得到无菌PCL微球的步骤具体如下：
    步骤1：称取适量PCL微球放置于离心管中，并在离心管上做好辐照剂量与微球种类的标记；
    步骤2：为保证无菌状态和检测中不泄露，使用封口膜对离心管进行密封；
    步骤3：把离心管装入包装箱内，并在箱外贴上标签，标注灭菌剂量信息；
    步骤4：接受γ射线或β射线辐照。
  19. 如权利要求17所述的方法，其中辐照剂量为20-30kGy。
  20. 如权利要求19所述的方法，其中辐照剂量为20kGy、25kGy或30kGy。
  21. 一种制备权利要求17所述的无菌CMC-Na凝胶的方法，其特征在于：具体步骤如下：
    步骤1：将凝胶转移到待灭菌容器中，并将其放入高压灭菌锅中；
    步骤2：采用高压蒸汽灭菌法，其中蒸汽压为101-105千帕，温度为119-123℃，维持15-20分钟进行蒸汽灭菌。
  22. 如权利要求21所述的制备方法，其特征在于：所述混匀步骤包括：在真空条件下进行混匀。
  23. 如权利要求21所述的制备方法，其特征在于：所述混匀步骤采用行星搅拌仪或真空搅拌脱泡机实施混匀。
  24. 一种制备权利要求1所述的美容产品的方法，其特征在于：具体步骤如下：
    步骤1：通过辐照灭菌得到无菌PCL微球；通过湿热灭菌法灭菌得到灭菌后的CMC-Na凝胶；对注射器采用环氧乙烷灭菌；
    步骤2：将灭菌后的所述无菌PCL微球加入到CMC-Na凝胶中进行混匀；
    步骤3：将混匀的负载有无菌PCL微球的CMC-Na凝胶封装于所述注射器中。
  25. 一种制备权利要求1中的PCL微球的方法，其特征在于：具体步骤如下：
    将PCL材料溶解于DCM中形成油相；将所述油相逐滴搅拌加入到MC溶液中， 利用高速剪切搅拌机高速剪切，搅拌2-4h；水洗离心3次，筛分、干燥得到PCL微球；其中，油相与水相比例为1:5～1:50；所述油相中聚己内酯的质量浓度为10mg/ml～50mg/ml。
  26. 如权利要求25所述的方法，其特征在于：在高速剪切的步骤之后还有超声的步骤。
  27. 如权利要求25所述的方法，其特征在于：高速剪切的转速大于12000r/min，时间为1-5min。
  28. 如权利要求25所述的方法，其特征在于：所述超声的时间为3-6分钟。
  29. 如权利要求25所述的方法，其特征在于：搅拌时转速800-1200r/min，时间为2-4h。
PCT/CN2023/075505 2022-02-10 2023-02-10 一种包含无菌pcl微球的美容产品 WO2023151668A1 (zh)

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