High-stability endotoxin-free PHA nano-emulsion and preparation method thereof

Abstract

The invention discloses a high-stability endotoxin-free PHA nano-emulsion and a preparation method thereof, and belongs to the technical field of biodegradable high polymer material compositions and processing thereof. The nano-emulsion is characterized in that the endotoxin content is not higher than 0.10 EU / g, the median particle size d50 is 100-200 nm, and the PDI is not higher than 0.15, and the nano-emulsion is stable in long-term storage. The preparation method is a continuous solvent-free process and comprises the steps of melt extrusion, high-pressure thermal emulsification and deep endotoxin removal. By adopting the specific pH-insensitive cationic polymer emulsifier and the crystallization inhibitor, the chemical contradiction that an efficient alkaline purification process is incompatible with a nano-emulsion stable system is solved, physical aging is inhibited, and an unexpected technical effect is obtained. The invention also discloses an O / W type cream containing the nano emulsion.

Description

Technical Field

[0001] The present invention belongs to the technical field of biodegradable polymer material compositions and processing thereof, and particularly relates to a high-stability endotoxin-free PHA nanoemulsion and a preparation method thereof. Background Art

[0002] Globally, tightening regulations prohibiting non-biodegradable microplastics in personal care products are prompting the industry to actively seek environmentally friendly and safe alternatives to functional polymers. Polyhydroxyalkanoates (PHAs), a class of aliphatic copolyesters synthesized by microbial fermentation, are recognized as one of the most promising alternative materials due to their completely biobased origin and ability to fully degrade in a variety of environments.

[0003] However, the application of PHA in high-end cosmetics, especially sensitive skin care products, faces multiple and mutually restrictive technical challenges. Existing technologies have explored several different technical routes, but none of them provides an ideal comprehensive solution.

[0004] The first technical route is the solvent method. For example, European Patent EP3560479A1 and Chinese Patent CN114409886A both disclose the use of organic solvents such as chloroform and dichloromethane to dissolve and purify PHA. The fundamental flaw of this route is that it cannot fundamentally avoid the safety hazards caused by organic solvent residues, and the particles usually obtained are micron-sized, such as 0.2 to 500 microns disclosed in EP3560479A1, which cannot meet the requirements of high-end cosmetics for nano-scale skin feel and optical properties. In addition, although CN114409886A claims that its endotoxin can be lower than 0.005EU / mg, that is, 5EU / g, its process is complicated and still relies on solvents. The present invention aims to achieve better biological purity through a completely different solvent-free route.

[0005] The second technical route is aqueous enzymatic hydrolysis. For example, Chinese patent CN117384362A discloses a method for stabilizing PHA particles by enzymatically breaking the wall and adding polyvinyl alcohol (PVA). The limitations of this method are: first, the introduction of an additional, non-biological polymer stabilizer, PVA, affects the purity and natural properties of the final product; second, the product form is micron-sized particles ranging from 0.5 to 1.8 microns, which is not the nanoemulsion sought by the present invention; and third, its endotoxin removal effect is limited, less than 0.5 EU / mL, far from the medical-grade ultra-low level sought by the present invention, i.e., no more than 0.10 EU / g.

[0006] The third technical route is the upstream avoidance method, which is to avoid the production of endotoxins from the source. For example, Chinese patent CN115976088A reduces endotoxin production by genetically engineering E. rhodesi, while Chinese patent CN118255974A uses Gram-positive bacteria that do not naturally produce endotoxins for fermentation. The common problem of these methods is that they rely on specific, non-mainstream production strains, and abandon the use of PHA raw materials produced by conventional Gram-negative bacteria in the existing industry, such as E. rhodesi, which have higher fermentation efficiency and wider application. They lack universal applicability to existing industrial raw materials. The present invention provides a downstream processing technology that can process PHA raw materials containing endotoxins from any source.

[0007] The fourth technical approach is simple physical dispersion. For example, Chinese patent CN114555046A, and its equivalent patent WO2021067158A1, disclose the direct use of micron-sized PHA powder in cosmetic formulations. This approach yields only a simple physical mixture, not the stable nanoemulsion system defined in this invention, which possesses excellent film-forming properties, skin feel, and enhanced efficacy.

[0008] Therefore, a common technical dilemma and bias exists in this field: On the one hand, to achieve medical-grade ultra-low endotoxin levels, one of the most efficient purification methods is to use negatively charged adsorption media, such as anion exchange resins, which specifically adsorb negatively charged endotoxins under alkaline pH conditions (e.g., pH 7.5-8.5). On the other hand, to prepare stable cationic nanoemulsions, positively charged cationic emulsifiers, such as chitosan, are often used. However, the cationic properties of these conventional emulsifiers rely on an acidic environment and they lose their charge or even precipitate under alkaline conditions, completely destroying the physical stability of the entire nanoemulsion system. This "chemical incompatibility" between the pursuit of ultra-low biological purity and the need to maintain the physical stability of nanoparticles presents an insurmountable obstacle for those skilled in the art. It is reasonable to expect that attempts to combine efficient alkaline purification processes with conventional cationic nanoemulsion systems will fail.

[0009] The challenge of this invention lies in how to prepare a stable PHA nanoemulsion under solvent-free conditions and enable it to withstand the rigorous subsequent purification treatment, which usually requires alkaline conditions, while also ensuring the long-term physical stability of the product and avoiding degradation caused by secondary crystallization. Combining these inherently challenging and even conflicting technical points can lead to unexpected results for those skilled in the art. Summary of the Invention

[0010] The present invention aims to overcome the shortcomings of the prior art by providing a highly stable, endotoxin-free PHA nanoemulsion and its preparation method. By introducing a specific stabilization system and employing advanced preparation techniques, the present invention simultaneously addresses the inherent contradictions between particle size control, biological purity, long-term stability, and process chemical compatibility. Another object of the present invention is to provide a continuous, solvent-free preparation method for this nanoemulsion and its use in cosmetics.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] A first aspect of the present invention provides a PHA nanoemulsion defined by its unique combination of physicochemical parameters. Specifically, the PHA nanoemulsion simultaneously satisfies all of the following defining parameters:

[0013] Endotoxin content: not more than 0.10EU / g, based on PHA dry weight;

[0014] Median particle size d 50 :100-200nm;

[0015] Polydispersity index PDI: not higher than 0.15;

[0016] Solid content: 20-35wt%;

[0017] The nanoemulsion is stabilized by a pH-insensitive cationic polymer emulsifier, and at pH 6.0, the absolute value of the zeta potential of the nanoemulsion is not less than 25 mV;

[0018] After being stored at 45°C for 6 months under accelerated aging conditions, the median particle size d 50 The drift is no more than 5%.

[0019] The "pH-insensitive cationic polymer emulsifier" referred to in this invention is specifically and functionally defined as a polymer emulsifier that effectively maintains the physical stability of the nanoemulsion through electrostatic or steric hindrance over a wide pH range from at least 6.0 to 8.5, without a significant decrease in the absolute value of its zeta potential and without visible flocculation or precipitation. The phrase "at pH 6.0, the absolute value of the zeta potential of the nanoemulsion is not less than 25 mV" used in this invention serves as one of the quantitative criteria for determining the stabilizing effect of this type of emulsifier.

[0020] In some embodiments of the present invention, the PHA is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), wherein the mole fraction of 3-hydroxyhexanoic acid monomer is 8-12 mol %.

[0021] The PHA is PHBHHx, wherein the molar fraction of the 3-hydroxyhexanoic acid monomer is 8-12 mol%, such as 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol% or 12 mol%.

[0022] As a preferred technical solution of the present invention, the median particle size d of the PHA nanoemulsion is 50 It is 100-200nm, such as 100nm, 110nm, 120nm, 130nm, 135nm, 145nm, 150nm, 155nm, 160nm, 170nm, 180nm, 190nm or 200nm, etc.

[0023] As a preferred technical solution of the present invention, the solid content of the PHA nanoemulsion is 20-35wt%, such as 20wt%, 20.5wt%, 22wt%, 24wt%, 25wt%, 26.5wt%, 27.2wt%, 28wt%, 30wt%, 32wt%, 34wt%, 35wt%, etc.

[0024] A second aspect of the present invention provides a continuous solvent-free preparation method of the above-mentioned PHA nanoemulsion, comprising the following steps:

[0025] Step 1: PHA pellets and a crystallization inhibitor are melted and plasticized at 110-150°C in a twin-screw extruder to form a PHA melt flow.

[0026] Step 2: The PHA melt stream is pre-mixed with an aqueous phase preheated to 70-85° C. and containing a pH-insensitive cationic polymer emulsifier through an in-line mixing device, and then introduced into at least two high-pressure homogenizers connected in series for high-pressure hot emulsification to obtain a primary nanoemulsion.

[0027] The online mixing device is preferably an online temperature-controlled high-shear mixing chamber. Other online mixing devices that can effectively pre-disperse the molten phase and the aqueous phase before entering the high-pressure homogenizer, such as static mixers, can also be applied to the present invention.

[0028] Step 3: subjecting the primary nanoemulsion to a vacuum flash devolatilization treatment.

[0029] Step 4: The emulsion treated in step 3 is subjected to a deep endotoxin removal treatment, which includes an ultrafiltration washing treatment and a weakly basic anion exchange resin adsorption treatment under a pH condition of 7.5-8.5.

[0030] Step 5: The emulsion is sterile filtered and the pH value is finally adjusted to a range suitable for cosmetic applications.

[0031] To further clarify the continuous characteristics of the present invention, the process is achieved by physically directly coupling the various unit operations. Specifically, the die outlet of the twin-screw extruder is directly connected to the inlet of the online high-shear mixing chamber, and the outlet of the mixer is directly supplied to the first-stage inlet of the high-pressure homogenizer, thereby achieving a continuous and uninterrupted material flow from raw material melting to the formation of primary nanoemulsion. Compared with traditional batch operations, this integrated continuous design not only significantly improves production efficiency, but also ensures high product consistency and quality stability by precisely controlling the residence time of the material at high temperature.

[0032] As a preferred technical solution of the present invention, the crystallization inhibitor is triethyl citrate, and its addition amount is 0.5-5wt% based on the dry weight of PHA solids, preferably 1-3wt%, such as 0.5wt%, 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, 3.0wt%, 4.0wt% or 5.0wt%.

[0033] As a preferred technical solution of the present invention, the pH-insensitive cationic polymer emulsifier is one or more polymers containing quaternary ammonium groups in their molecular structure, and can be polyquaternium-10 or polyquaternium-7. In some preferred embodiments, the amount of the pH-insensitive cationic polymer emulsifier added is 0.5-5.0 wt%, preferably 1.0-2.5 wt%, such as 0.5 wt%, 0.9 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 4.0 wt% or 5.0 wt%, based on the dry weight of the PHA solids.

[0034] As a preferred technical solution of the present invention, the pH condition of the weakly basic anion exchange resin adsorption treatment in step 4 is 7.5-8.5, such as 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.

[0035] A third aspect of the present invention provides an O / W cream composition comprising 1-15 wt % of the PHA nanoemulsion according to the present invention, based on the dry weight of PHA solids.

[0036] A fourth aspect of the present invention provides a method for improving the performance of a cosmetic composition using the PHA nanoemulsion according to the first aspect of the present invention, wherein the method comprises adding 1-15 wt % of the PHA nanoemulsion to the composition, based on the dry weight of PHA solids, to achieve at least one of the following effects:

[0037] Improve the water resistance of O / W sunscreen compositions;

[0038] Enhance skin barrier function;

[0039] Reduces transepidermal water loss.

[0040] As a preferred technical solution of the present invention, the O / W cream composition comprises 1-15 wt% of the PHA nanoemulsion according to the present invention, based on the dry weight of PHA solids, such as 1 wt%, 1.5 wt%, 3 wt%, 5 wt%, 8 wt%, 10 wt%, 12 wt% or 15 wt%.

[0041] Compared with the prior art, the present invention can achieve the following significant beneficial effects:

[0042] Overcoming technical biases and achieving process chemical compatibility: This invention resolves the inherent chemical conflict between the existing high-efficiency alkaline purification process and the cationic nanoemulsion stabilization system. By employing a specific class of pH-insensitive cationic polymer emulsifiers, the nanoemulsion remains physically stable and prevents collapse under subsequent high-efficiency alkaline purification conditions. This design, which enables the previously mutually exclusive high-efficiency purification and nanoemulsion stabilization, is the key to the present invention and achieves unexpected technical benefits.

[0043] Synergistic Effects Achieve Unified Biosafety and Long-Term Physical Stability: This invention uniquely combines a specific pH-insensitive emulsifier with a crystallization inhibitor, ensuring the emulsion's ability to withstand rigorous purification processes while effectively inhibiting the long-term physical degradation of PHA nanoparticles caused by secondary crystallization. This unique combination synergistically achieves the seemingly independent and challenging goals of "ultra-high biological purity" and "excellent long-term physical stability," creating a synergistic effect that is greater than 2.

[0044] Solvent-free process and product purity: The continuous, completely solvent-free melt emulsification process employed in this invention completely eliminates the risk of organic solvent residues at the source, ensuring the ultimate purity of the product. Compared to existing technologies that rely on organic solvents, this process significantly reduces production energy consumption, improving economic efficiency and environmental sustainability.

[0045] Excellent and uniform physical properties: The nanoemulsion prepared by the present invention has a median particle size d of 100-200 nm. 50 and a PDI of no more than 0.15, giving the final cosmetics an excellent translucent appearance or uniform milky white color and a delicate and smooth skin feel. DETAILED DESCRIPTION

[0046] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the present embodiment are all commercially available industrial products or can be prepared by conventional methods. Performance test methods, unless otherwise specified, are carried out in accordance with the standards described in the summary of the invention.

[0047] Main reagents and materials:

[0048] Table 1 Main reagents and materials categories, names, product models / names and representative manufacturers:

[0049]

[0050] Main analytical and testing instruments:

[0051] Twin-screw extruder: Thermo Scientific Process 11 parallel co-rotating twin-screw extruder.

[0052] High-pressure homogenizer: ATS Engineering Inc. AH-100D two-stage ceramic valve high-pressure homogenizer.

[0053] Online temperature-controlled high-shear mixing chamber: It adopts a customized stator-rotor structure with the gap between the stator and the rotor controlled at 0.2-1.0mm, which can perform efficient online pre-dispersion of the two phases within the speed range of 10,000-20,000rpm.

[0054] Thin film evaporator: UIC GmbH KD-6 wiped film molecular distiller.

[0055] Dynamic light scattering (DLS) and zeta potential analyzer: Malvern Zetasizer Ultra.

[0056] Gas chromatography-flame ionization detector (GC-FID): Agilent 8890 GC system.

[0057] Limulus amebocyte lysate kinetic turbidimeter: Charles River Endosafe nexgen-PTS.

[0058] Skin function tester: Corneometer CM825, Tewameter TM300 (Courage+Khazaka).

[0059] SPF analyzer: Labsphere UV-2000S UV transmittance analyzer.

[0060] Viscometer: Brookfield DV3T Rheometer.

[0061] Main performance test standards:

[0062] Endotoxin content determination: according to the United States Pharmacopoeia USP <85> Specified Limulus Amebocyte Lysate (LAL) kinetic turbidimetric method or chromogenic matrix method.

[0063] Particle size and PDI determination: According to ISO 22412:2020 standard, dynamic light scattering (DLS) technology is used.

[0064] Determination of residual organic solvents: refer to the gas chromatography method in the "Technical Specifications for Safety of Cosmetics" (2015 edition).

[0065] SPF in vitro determination: according to ISO 24443:2021 standard.

[0066] Water resistance test: According to ISO 18861:2020 standard.

[0067] Example 1: Preparation of PHBHHx (3-hydroxyhexanoic acid = 12 mol%) nanoemulsion.

[0068] Step 1. Melt Plasticization: 1.0 kg of pre-dried PHBHHx (Biodegradable Polymer Green Planet) and 15 g of triethyl citrate were melt plasticized in a twin-screw extruder. The extruder zone temperatures were set at 80°C in the feed zone, 130°C in the compression zone, 145°C in the melt zone, and 140°C in the die.

[0069] Step 2. High-Pressure Hot Emulsification: The PHA melt was pumped out at a rate of 0.5 kg / h. Simultaneously, an aqueous phase preheated to 80°C and containing 7.5 g of polyquaternium-10 (equivalent to 0.75 wt% of the dry weight of the PHA) was pumped out at a rate of 2.5 kg / h. The two phases were premixed in an inline high-shear mixing chamber maintained at 95°C and then emulsified in a two-stage series high-pressure homogenizer. The first stage homogenization pressure was set at 800 bar, and the second stage at 150 bar.

[0070] Step 3. Vacuum devolatilization: The primary nanoemulsion was continuously pumped into the thin film evaporator, the jacket temperature was set to 85° C., and the operating absolute pressure was not higher than 10 kPa.

[0071] Step 4. Deep endotoxin removal:

[0072] Ultrafiltration washing: The devolatilized emulsion was cooled to 40°C and washed four times with injection-grade pyrogen-free water using a 30 kDa MWCO ultrafiltration membrane, replacing 2 times the emulsion volume each time.

[0073] Column Chromatography Adsorption: The emulsion after ultrafiltration and washing was adjusted to pH 8.0 with pyrogen-free 20 mM Tris-HCl buffer. A chromatography column was prepared, packed with a weakly basic anion exchange resin that had been previously treated with 0.5 M NaOH and equilibrated with the aforementioned pyrogen-free buffer. The pH-adjusted emulsion was pumped through the column at a controlled flow rate to ensure a residence time of 90 minutes. The column temperature was maintained at 30°C. The purified emulsion was collected.

[0074] Step 5. Post-treatment: The purified emulsion was sterile filtered through sterilizing-grade filters with pore sizes of 0.45 µm and 0.22 µm, respectively. The pH of the emulsion was adjusted to 6.0 using citric acid.

[0075] Product characterization: The solid content of the product was measured to be 27.2 wt%; the median particle size d 50 The particle size was 145 nm, the PDI was 0.13, the endotoxin content was 0.07 EU / g PHA, and the zeta potential was +35 mV. After storage at 45°C for 6 months, the particle size change was 4.2%.

[0076] Example 2: Preparation of high solid content PHBHHx nanoemulsion.

[0077] The steps of Example 1 were repeated, except that the weight ratio of the melt to water was adjusted to 1:3.5 in step 2, and the emulsion was further concentrated after ultrafiltration in step 4 to obtain a product with a final solid content of 35 wt %.

[0078] Product characterization: Measure the product median particle size d 50 The particle size was 155 nm, the PDI was 0.14, the endotoxin content was 0.08 EU / g PHA, and the zeta potential was +32 mV. After storage at 45°C for 6 months, the particle size change was 4.6%.

[0079] Example 3: Preparation of PHBHHx nanoemulsion with low 3-hydroxyhexanoic acid content.

[0080] The steps of Example 1 were repeated, except that the PHA raw material used in Step 1 was replaced with PHBHHx having a molar fraction of 8 mol % of 3-hydroxyhexanoic acid.

[0081] Product characterization: The solid content of the product was measured to be 26.8wt%; the median particle size d 50The particle size was 130 nm, the PDI was 0.11, the endotoxin content was 0.06 EU / g PHA, and the zeta potential was +38 mV. After storage at 45°C for 6 months, the particle size change was 3.9%.

[0082] Example 4: Preparation of small-particle PHBHHx nanoemulsion.

[0083] The steps of Example 1 were repeated, except that in step 2, high-pressure hot emulsification, the first-stage homogenization pressure was increased to 1000 bar and the second-stage homogenization pressure was maintained at 200 bar.

[0084] Product characterization: The solid content of the product was measured to be 26.5wt%; the median particle size d 50 The particle size was 100 nm, the PDI was 0.14, the endotoxin content was 0.08 EU / g PHA, and the zeta potential was +29 mV. After storage at 45°C for 6 months, the particle size change was 4.8%.

[0085] Example 5: Preparation of large-particle PHBHHx nanoemulsion.

[0086] The steps of Example 1 were repeated, except that in step 2, high-pressure hot emulsification, the first-stage homogenization pressure was reduced to 600 bar and the second-stage homogenization pressure was reduced to 100 bar.

[0087] Product characterization: The solid content of the product was measured to be 27.5wt%; the median particle size d 50 The particle size was 200 nm, the PDI was 0.13, the endotoxin content was 0.07 EU / g PHA, and the zeta potential was +36 mV. After storage at 45°C for 6 months, the particle size change was 4.0%.

[0088] Example 6: Preparation of low solid content PHBHHx nanoemulsion.

[0089] The steps of Example 1 were repeated, except that in step 2, the weight ratio of the molten stream to the aqueous phase was adjusted to 1:5.5.

[0090] Product characterization: The solid content of the product was measured to be 20.5wt%; the median particle size d 50 The particle size was 135 nm, the PDI was 0.12, the endotoxin content was 0.07 EU / g PHA, and the zeta potential was +33 mV. After storage at 45°C for 6 months, the particle size change was 4.1%.

[0091] Example 7: Preparation of PHBHHx nanoemulsion using polyquaternium-7.

[0092] The steps of Example 1 were repeated, except that in step 2 the emulsifier Polyquaternium-10 was replaced with an equal amount of Polyquaternium-7.

[0093] Product Characterization: The preparation process went smoothly and no abnormalities were observed. The solid content of the product was measured to be 27.0wt%; the median particle size d 50 The particle size was 152 nm; the PDI was 0.14; the endotoxin content was 0.08 EU / g PHA; and the zeta potential was +31 mV. After 6 months of storage at 45°C, the particle size change was 4.5%. These results indicate that other pH-insensitive cationic polymers are also suitable for use in the present invention.

[0094] Comparative Example 1: PHA emulsion was prepared by conventional solvent evaporation method.

[0095] 10 g of PHBHHx was dissolved in 100 mL of dichloromethane and added to the aqueous solution containing PVA. After high-speed shear emulsification, the dichloromethane was removed by rotary evaporation.

[0096] Product Characterization: Average Particle Size d 50 The particle size was 450 nm, the PDI was 0.38, the endotoxin content was >50 EU / g PHA, and the residual dichloromethane was 150 ppm. After 6 months of storage at 45°C, the sample showed severe demulsification and precipitation, making it impossible to accurately measure the particle size. The estimated particle size change was greater than 30%.

[0097] Comparative Example 2: The nano-sizing process of the present invention was adopted but all endotoxin removal steps were omitted.

[0098] Steps 1-3 and 5 of Example 1 were repeated, but the "deep endotoxin removal treatment" of step 4 was completely omitted.

[0099] Product Characterization: Endotoxin content reached 185 ± 25 EU / g PHA. In vitro cell experiments showed that it induced IL-8 release of 250 ± 28 pg / mL. After 6 months of storage at 45°C, the particle size change was 4.3%.

[0100] Comparative Example 3: The nano-process of the present invention was used but only endotoxin was partially removed.

[0101] The steps of Example 1 were repeated, except that in step 4, only ultrafiltration washing was performed for the deep endotoxin removal treatment, and the column chromatography adsorption step was omitted.

[0102] Product Characterization: Endotoxin content is 35 ± 10 EU / g PHA. In vitro cell experiments showed that it induced IL-8 release of 85 ± 15 pg / mL. After 6 months of storage at 45°C, the particle size change was 4.4%.

[0103] Comparative Example 4: A comparative experiment using pure PHB as raw material.

[0104] All the steps of Example 1 were repeated, except that the PHA raw material in step 1 was replaced by 1.0 kg of pure P(3HB).

[0105] Process observation and product characterization: During the melt plasticization process in step 1, the extruder torque was observed to increase significantly and fluctuate, and the die extrudate showed slight yellowing, indicating that some thermal degradation may have occurred. The final product had a median particle size of d 50 The particle size was 285 nm, the PDI was 0.31, and the endotoxin content was 0.09 EU / g PHA. After storage at 45°C for 6 months, the sample showed some precipitation, and the particle size changed by more than 15%.

[0106] Comparative Example 5: A comparative experiment in which the crystallization inhibitor was omitted.

[0107] All the steps of Example 1 were repeated except that triethyl citrate was not added in step 1.

[0108] Product characterization: The initial product characterization was similar to that in Example 1, and the median particle size d 50 The median particle size d is 148 nm and the PDI is 0.14. However, after storage at 45 °C for 6 months, the median particle size d 50 The particle size increased significantly to 175 nm, with a particle size change of up to 18.2%, far exceeding the stable threshold of 5%, and a small amount of white precipitate was observed at the bottom of the sample bottle.

[0109] Comparative Example 6: A comparative experiment using a pH-sensitive cationic polymer emulsifier.

[0110] The steps of Example 1 were repeated, except that the emulsifier polyquaternium-10 was replaced with an equal amount of chitosan (Chitosan 95 / 500 S) in step 2. Chitosan was dissolved in 0.5% acetic acid aqueous solution before use.

[0111] Process Observations and Product Characterization: The preparations from steps 1 to 3, as well as the ultrafiltration wash in step 4, proceeded normally, and the emulsion obtained after ultrafiltration had a good appearance. However, during preparation for column chromatography adsorption in step 4, during the adjustment of the emulsion's pH to 8.0 with triethanolamine, the emulsion immediately underwent irreversible flocculation and rapidly formed a large amount of precipitate, resulting in complete system collapse and the inability to pump the sample into the chromatography column for subsequent purification.

[0112] Table 2 compares the key performance parameters of the embodiment and the comparative example as follows:

[0113]

[0114] The comparative data in Table 2 demonstrate that the PHA nanoemulsions prepared in the Examples of the present invention meet all key performance targets, including an ideal particle size range, low PDI, ultra-low endotoxin content, and excellent long-term stability. In stark contrast, the products in the Comparative Examples exhibit significant deficiencies in at least one key performance factor, fully demonstrating the innovativeness and effectiveness of the present invention's technical solutions.

[0115] Application examples:

[0116] The basic formula of the application example in Table 3 is as follows:

[0117]

[0118] Application Example 1: Preparation and evaluation of standard O / W sunscreen cream.

[0119] A cream was prepared according to the basic formula shown in Table 3, wherein the amount of the PHA nanoemulsion prepared in Example 1 in phase B was 18.45 wt %, corresponding to a dry weight of PHA of 5.0 wt %.

[0120] Product review: The cream has a rich texture and excellent spreadability.

[0121] Application Example 2: Preparation and evaluation of low-concentration PHA moisturizing essence lotion.

[0122] An emulsion was prepared according to the basic formula shown in Table 3, wherein the addition amount of the PHA nanoemulsion prepared in Example 1 in phase B was adjusted to 5.52 wt % corresponding to a PHA dry weight of 1.5 wt %, and the amount of xanthan gum in phase C was increased to 0.5%.

[0123] Product evaluation: The product is in the form of a light lotion and feels refreshing on the skin.

[0124] Application Example 3: Preparation and evaluation of high-concentration PHA repair barrier cream.

[0125] A cream was prepared according to the basic formula shown in Table 3, wherein the amount of the PHA nanoemulsion prepared in Example 1 in phase B was adjusted to 44.28 wt %, corresponding to a PHA dry weight of 12.0 wt %.

[0126] Product review: The product is in the form of a rich paste and has a good sealing feel.

[0127] Comparative Application Example 1: Placebo sunscreen cream without PHA nanoemulsion.

[0128] A cream was prepared according to the basic formula shown in Table 3, but without adding the PHA nanoemulsion prepared in Example 1 in Phase B, and its weight was supplemented by deionized water in Phase C.

[0129] Product evaluation: The texture of the cream is slightly thin and the spreadability is average.

[0130] To evaluate the effectiveness of the nanoemulsion in cosmetics, we recruited 20 healthy female subjects aged 25-45 and conducted a 28-day half-face comparison test. All data are presented as mean ± standard deviation (mean ± SD) and analyzed using the Student's t-test, with p < 0.05 considered statistically significant.

[0131] Table 4 Application Examples Efficacy Comparison is as follows:

[0132]

[0133] Note: * indicates that the difference is statistically significant compared with comparative application example 1 (p<0.05).

[0134] Table 4 clearly demonstrates the significant effects of the cosmetic composition containing the PHA nanoemulsion of the present invention in improving skin barrier function and enhancing the water resistance of sunscreen products. Compared with a placebo formulation without PHA nanoemulsion, the addition of the nanoemulsion of the present invention significantly increased the TEWL reduction rate and SPF maintenance rate, and the differences were statistically significant, strongly demonstrating its practical efficacy in cosmetic applications.

[0135] Through a systematic analysis of the above embodiments, comparative examples and application examples, the following conclusions can be drawn about the technical solution of the present invention:

[0136] Process Superiority and Product Uniqueness: The core process flow of this invention, namely the continuous solvent-free melt emulsification technology, has been demonstrated to be able to stably prepare PHA nanoemulsions with a unique combination of properties. These properties include a controllable particle size of 100-200 nm, an excellent uniformity index (PDI) of no more than 0.15, and an ultra-high biopurity of no more than 0.10 EU / g. Compared with the traditional solvent method used in Comparative Example 1, this technical solution demonstrates fundamental advantages in all key dimensions of the product, including particle size control, distribution uniformity, biosafety, and chemical purity. This demonstrates that the proposed solvent-free melt emulsification route is an effective technical solution for achieving this specific high-performance nanoemulsion and is significantly innovative.

[0137] The synergistic effect and necessity of the deep endotoxin removal strategy: The experimental data clearly reveal the necessity of deep endotoxin removal treatment. If this treatment step is completely omitted, the product will carry up to 185EU / g of endotoxins and induce a strong in vitro inflammatory response. Comparative Example 3 further proves that even with conventional ultrafiltration washing, the endotoxin content can only be reduced to 35EU / g, and the induced inflammation level, although it has decreased, is still high. Only by combining the two steps of "ultrafiltration washing" and "column chromatography adsorption" can the endotoxin content be effectively controlled to an ultra-low level below 0.10EU / g and the inflammatory response be reduced to the baseline. This set of comparisons strongly proves that the original "two-step" deep purification strategy is a key technical feature for achieving ultra-high biosafety of the product, and its effect has an unexpected synergistic effect. It should be pointed out that the IL-8 release amount, as a direct indicator of endotoxin-induced inflammatory response, is mainly used to evaluate the effectiveness of endotoxin removal treatment. Therefore, this data is only provided in the examples and comparative examples related to endotoxin content.

[0138] Necessity and advancement of each component of the formulation system: The formulation system adopted in this technical solution is the key to realizing the entire process flow. Comparative Example 6 decisively proves the necessity of using a pH-insensitive cationic polymer emulsifier; when pH-sensitive chitosan is used instead, the system immediately collapses in the alkaline purification step, revealing the chemical contradictions faced by conventional emulsifiers in this special process. Example 7 further proves that the technical solution of the present invention is not limited to a specific polyquaternium-10. Other cationic polymers with similar pH-insensitive properties, such as polyquaternium-7, can also successfully implement the present invention, thereby supporting the scope of this functional limitation. In addition, Comparative Example 5 confirms the necessity of a crystallization inhibitor from the opposite side: the lack of triethyl citrate will cause the product to have severe physical instability after accelerated aging, with a particle size drift of up to 18.2%. At the same time, Comparative Example 4 also highlights the superiority of selecting PHBHHx copolymer raw materials. The use of pure PHB raw materials with high crystallinity will lead to significant deterioration of product particle size and uniformity.

[0139] Application value and wide range applicability of the product: The application examples confirm that the PHA nanoemulsion prepared by this technical solution has significant application value. Within a wide concentration range of 1.5wt% to 12.0wt%, the nanoemulsion can be successfully applied to cosmetics of different textures and shows efficacy that is positively correlated with concentration. Compared with the placebo formula without this nanoemulsion, the formula with the addition of this emulsion showed irrefutable superiority in reducing skin water loss, such as the TEWL reduction rate of 25.4%±3.1% compared to 4.1%±1.5% in the placebo group, and improving the water resistance of sunscreen products, such as the SPF maintenance rate of 88.2%±4.5%, which was significantly higher than 65.1%±5.2% in the placebo group. These data show that the efficacy is indeed derived from the PHA nanoemulsion itself, rather than the contribution of the matrix, and the effect is statistically significant.

[0140] To ensure the robustness and controllability of this technical solution, we conducted extensive experimental research on the effects of various key components and process parameters at different levels. The results systematically revealed their influence on the performance of the final product, and the trend conclusions drawn are as follows:

[0141] 3-Hydroxyhexanoic Acid Monomer Content: Experimental results indicate that the mole fraction of 3-Hydroxyhexanoic Acid in PHBHHx copolymers is a key factor influencing the final emulsion properties. Systematically adjusting the mole fraction of this monomer revealed that moderately increasing its content effectively reduces the polymer's crystallinity and melting point. This property makes the polymer more easily sheared and dispersed during the melt emulsification process, resulting in a clear trend toward smaller particle sizes or achieving the same particle size with lower homogenization energy consumption.

[0142] Crystallization inhibitor: Gradient experiments with the addition of triethyl citrate, a crystallization inhibitor, revealed a direct correlation between its dosage and the long-term physical stability of the product. The study confirmed that increasing the amount of crystallization inhibitor within a publicly available formulation more effectively inhibits secondary crystallization and Ostwald ripening of PHA nanoparticles during long-term storage, significantly enhancing the product's physical stability, as evidenced by a significant reduction in long-term particle size drift.

[0143] Emulsifier Dosage: A systematic study of pH-insensitive cationic polymer emulsifier concentrations confirmed that dosage is a key variable in determining the initial particle size and stability of nanoemulsions. Experimental data demonstrate that increasing the emulsifier concentration within the effective concentration range allows for faster and more complete surface coating of newly formed nanoparticles. This directly leads to smaller initial particle sizes and higher zeta potentials, thereby enhancing the immediate stability of the emulsion during preparation.

[0144] Solids Content: Studies of emulsion solids content over the entire range of 20-35wt% have shown a significant impact on processing. Analysis of rheological and processing data indicates that as the solids content approaches the higher end of this range, the overall viscosity of the system increases, reducing the efficiency of energy transfer during high-pressure homogenization. Consequently, the experimental conclusions suggest that higher solids content requires higher homogenization pressures to achieve the same target particle size, or that at the same homogenization pressure, the final particle size tends to increase.

[0145] In summary, this technical solution successfully addresses the core pain points of existing PHA aqueous dispersion preparation techniques, including residual organic solvents, poor particle size control, poor long-term physical stability, and endotoxin contamination, through an innovative, continuous, solvent-free preparation method. The resulting PHA nanoemulsion, with its unique formulation and parameter combination, exhibits ultra-high biosafety, uniform nanoparticle size, and excellent physicochemical stability, demonstrating clear practical application value.

[0146] Those skilled in the art will appreciate that the above embodiments are merely exemplary and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, or improvements to the technical solutions of the present invention that fall within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A polyhydroxyalkanoate nanoemulsion, characterized in that: The nanoemulsion satisfies all of the following limiting parameters simultaneously: Endotoxin content: not more than 0.10EU / g, based on the dry weight of polyhydroxyalkanoate; Median particle size d 50 :100-200nm; Polydispersity index PDI: not higher than 0.15; Solid content: 20-35wt%; The nanoemulsion is stabilized by a pH-insensitive cationic polymer emulsifier, and at pH 6.0, the absolute value of the zeta potential of the nanoemulsion is not less than 25 mV; After being stored at 45°C for 6 months under accelerated aging conditions, the median particle size d 50 The drift is no more than 5%.

2. The polyhydroxyalkanoate nanoemulsion according to claim 1, wherein The polyhydroxyalkanoate is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), wherein the molar fraction of the 3-hydroxyhexanoic acid monomer is 8-12 mol %.

3. A continuous solvent-free preparation method of polyhydroxyalkanoate nanoemulsion according to claim 1 or 2, characterized in that: The following steps are involved: Step 1: Melting and plasticizing polyhydroxyalkanoate pellets and a crystallization inhibitor in a twin-screw extruder at 110-150° C. to form a polyhydroxyalkanoate melt stream; Step 2: pre-mixing the polyhydroxyalkanoate melt with an aqueous phase preheated to 70-85° C. and containing a pH-insensitive cationic polymer emulsifier through an in-line mixing device, and then introducing the mixture into at least two high-pressure homogenizers connected in series for high-pressure heat emulsification to obtain a primary nanoemulsion; Step 3: subjecting the primary nanoemulsion to a vacuum flash devolatilization treatment; Step 4: performing a deep endotoxin removal treatment on the emulsion treated in step 3, the treatment comprising ultrafiltration washing treatment and weakly basic anion exchange resin adsorption treatment at pH 7.5-8.5; Step 5: The emulsion is sterile filtered and the pH value is adjusted.

4. The preparation method according to claim 3, characterized in that The crystallization inhibitor in step 1 is triethyl citrate, and its addition amount is 0.5-5 wt % based on the solid dry weight of polyhydroxyalkanoate.

5. The preparation method according to claim 3, characterized in that The pH-insensitive cationic polymer emulsifier in step 2 is one or more polymers containing quaternary ammonium salt groups in their molecular structure, and the added amount thereof is 0.5-5.0 wt % based on the dry weight of the polyhydroxyalkanoate solid.

6. The preparation method according to claim 5, characterized in that The polymer of the quaternary ammonium salt group is polyquaternium-10 or polyquaternium-7.

7. The preparation method according to claim 3, characterized in that The weakly basic anion exchange resin adsorption treatment in step 4 is a column chromatography purification step.

8. An O / W cream composition, characterized in that: The method comprises the polyhydroxyalkanoate nanoemulsion according to claim 1 or 2 in an amount of 1-15 wt % by weight of the polyhydroxyalkanoate solid dry weight.

9. A method for improving the performance of a cosmetic composition using the polyhydroxyalkanoate nanoemulsion according to claim 1 or 2, characterized in that: The polyhydroxyalkanoate nanoemulsion is added to the composition in an amount of 1-15 wt % based on the dry weight of polyhydroxyalkanoate solids to achieve at least one of the following effects: Improve the water resistance of O / W sunscreen compositions; Enhance skin barrier function; Reduces transepidermal water loss.

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