A highly stable, endotoxin-free PHA nanoemulsion and its preparation method

Abstract

This invention discloses a highly stable, endotoxin-free PHA nanoemulsion and its preparation method, belonging to the field of biodegradable polymer material compositions and their processing technology. This nanoemulsion has an endotoxin content of no more than 0.10 EU / g and a median particle size of d... 50 Characterized by a nanoparticle size of 100-200 nm and a PDI not exceeding 0.15, it exhibits long-term storage stability. Its preparation method is a continuous solvent-free process, including melt extrusion, high-pressure thermal emulsification, and deep endotoxin removal steps. This invention resolves the chemical incompatibility between efficient alkaline purification processes and the stable nanoemulsion system by employing specific pH-insensitive cationic polymer emulsifiers and crystallization inhibitors, and also inhibits physical aging, achieving unexpected technical effects. This invention also discloses an O / W type cream containing this nanoemulsion.

Description

Technical Field

[0001] This invention belongs to the field of biodegradable polymer material composition and processing technology, specifically relating to a highly stable, endotoxin-free PHA nanoemulsion and its preparation method. Background Technology

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

[0003] However, applying PHA to 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 have provided 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 drawback of this route is that it cannot fundamentally avoid the safety hazards caused by residual organic solvents, and the particles obtained are usually in the micron range, such as 0.2 to 500 microns disclosed in EP3560479A1, which cannot meet the requirements of high-end cosmetics for nanoscale skin feel and optical properties. In addition, although CN114409886A claims that its endotoxin can be lower than 0.005 EU / mg, or 5 EU / g, its process is complex and still depends on solvents, while this 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 enzymatic hydrolysis to break down cell walls and adding polyvinyl alcohol (PVA). The limitations of this method are: firstly, the introduction of additional, non-biologically derived polymeric stabilizer PVA affects the purity and natural properties of the final product; secondly, the product form is micron-sized particles of 0.5 to 1.8 micrometers, which is not the nanoemulsion sought in this invention; and thirdly, its endotoxin removal effect is limited, less than 0.5 EU / mL, far from achieving the ultra-low medical-grade level sought in this invention, i.e., no higher than 0.10 EU / g.

[0006] The third technical approach is upstream avoidance, which prevents endotoxin production at the source. For example, Chinese patent CN115976088A reduces endotoxin production by genetically engineering *Rhodotorula gravidarum*, while Chinese patent CN118255974A uses naturally non-endotoxin-producing Gram-positive bacteria for fermentation. A common problem with these methods is that they rely on specific, non-mainstream production strains, neglecting the application of PHA raw materials produced by conventional Gram-negative bacteria, such as *Rhodotorula gravidarum*, which have higher fermentation efficiency and wider applications in existing industries. This lack of applicability to existing industrial raw materials addresses this issue by providing a downstream processing technology capable of processing endotoxin-containing PHA raw materials from any source.

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

[0008] Therefore, a common technical dilemma and bias exists in this field: on the one hand, to obtain 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 usually used. However, the cationic properties of these conventional emulsifiers depend on an acidic environment, and they lose their charge or even precipitate under alkaline conditions, thus completely destroying the physical stability of the entire nanoemulsion system. This "chemical incompatibility" between the pursuit of ultra-low biological purity purification methods and the need to maintain the physical stability of nanoparticles constitutes a barrier that is difficult for those skilled in the art to overcome. Those skilled in the art have reason 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 stable PHA nanoemulsions under solvent-free conditions, enabling them to withstand subsequent rigorous purification processes, typically requiring alkaline conditions, while simultaneously ensuring the long-term physical stability of the product and preventing aging due to secondary crystallization. Combining these inherently challenging and even conflicting technical aspects yields unpredictable results for those skilled in the art. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a highly stable, endotoxin-free PHA nanoemulsion and its preparation method. By introducing a specific stabilization system and employing advanced preparation processes, this invention simultaneously resolves the inherent contradictions regarding particle size control, biopurity, long-term stability, and process chemical compatibility. Another objective of this invention is to provide a continuous, solvent-free preparation method for this nanoemulsion and its application in cosmetics.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[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 higher than 0.10 EU / g, based on PHA dry weight;

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

[0015] Multidispersion Index (PDI): not higher than 0.15;

[0016] Solid content: 20-35 wt%

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

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

[0019] The "pH-insensitive cationic polymer emulsifier" referred to in this invention is specifically and functionally defined as: a class of polymer emulsifiers capable of effectively maintaining the physical stability of the nanoemulsion through electrostatic or steric hindrance effects over a wide pH range of at least 6.0 to 8.5, without a significant decrease in the absolute value of its Zeta potential, and without any visible flocculation or precipitation of the emulsion. The statement in this invention that "the absolute value of the Zeta potential of the nanoemulsion is not less than 25 mV under pH 6.0 conditions" is one of the quantitative criteria for judging 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 molar fraction of the 3-hydroxyhexanoate 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 embodiment of the present invention, the median particle size d of the PHA nanoemulsion is... 50 The range 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 for the above-mentioned PHA nanoemulsion, comprising the following steps:

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

[0026] Step 2: The PHA molten stream is premixed with an aqueous phase containing a pH-insensitive cationic polymer emulsifier preheated to 70-85°C using an online mixing device, and then introduced into a high-pressure homogenizer consisting of at least two stages connected in series for high-pressure thermal emulsification to obtain a primary nanoemulsion.

[0027] The preferred online mixing device is 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 this invention.

[0028] Step 3: Perform vacuum flash evaporation and devolatilization treatment on the primary nanoemulsion.

[0029] Step 4: Deeply detoxify the emulsion treated in Step 3. The treatment includes ultrafiltration washing and weakly basic anion exchange resin adsorption treatment at pH 7.5-8.5.

[0030] Step 5: Aseptically filter the lotion and finally adjust the pH value to a range suitable for cosmetic application.

[0031] To further clarify the continuous operation feature of this invention, the process is achieved through direct physical coupling of 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 mixer outlet directly supplies the first-stage inlet of the high-pressure homogenizer, thereby realizing a continuous and uninterrupted material flow from raw material melting to the formation of the primary nanoemulsion. This integrated continuous design, compared with traditional batch operations, not only significantly improves production efficiency but also ensures high product consistency and quality stability by precisely controlling the residence time of materials at high temperatures.

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

[0033] As a preferred embodiment of the present invention, the pH-insensitive cationic polymer emulsifier is one or more polymers containing quaternary ammonium salt groups in their molecular structure, such as 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% based on the dry weight of PHA solids, 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%.

[0034] As a preferred technical solution of the present invention, the pH conditions for the weakly basic anion exchange resin adsorption treatment in step 4 are 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 invention provides an O / W type cream composition comprising 1-15 wt% of the PHA nanoemulsion according to the 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 a PHA nanoemulsion according to a first aspect of the present invention, wherein the method achieves at least one of the following effects by adding 1-15 wt% of the PHA nanoemulsion, based on the dry weight of PHA solids, to the composition:

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

[0038] Enhance skin barrier function;

[0039] Reduce transepidermal water loss.

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

[0041] Compared with the prior art, the following significant advantages can be obtained by using the present invention:

[0042] Overcoming technical biases and achieving process chemical compatibility: This invention resolves the inherent chemical contradiction between efficient alkaline purification processes and cationic nanoemulsion stabilization systems in existing technologies. By employing a specific class of pH-insensitive cationic polymer emulsifiers, the nanoemulsions can maintain physical stability without collapsing under subsequent efficient alkaline purification conditions. This design, which makes the originally mutually exclusive efficient purification and nanoemulsion stabilization compatible, is the key to realizing this invention and has achieved unexpected technical effects.

[0043] Achieving a balance between biocompatibility and long-term physical stability through synergistic effects: This invention uniquely combines a specific pH-insensitive emulsifier with a crystallization inhibitor, ensuring that the emulsion can withstand stringent purification processes while effectively inhibiting the long-term physical aging of PHA nanoparticles caused by secondary crystallization. This unique formulation synergistically achieves the seemingly independent and difficult-to-achieve goals of "ultra-high biocompatibility" and "excellent long-term physical stability," resulting in a synergistic effect greater than the sum of its parts.

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

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

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 illustrative of the invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention. Unless otherwise stated, the raw materials used in this embodiment are commercially available industrial products or can be prepared by conventional methods. Unless otherwise specified, performance testing methods are performed according to the standards described in the invention summary section.

[0047] Main reagents and materials:

[0048] Table 1. Main Reagent and Material 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 stator-rotor gap controlled at 0.2-1.0mm, which can efficiently pre-dispersettle the two phases online within a speed range of 10000-20000rpm.

[0054] Thin-film evaporator: UIC GmbH KD-6 type wiped-film molecular distillation unit.

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

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

[0057] Limulus amebocyte lysate (LAL) reagent dynamic turbidimeter: Charles River Endosafe nexgen-PTS.

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

[0059] SPF Analyzer: Labsphere UV-2000S Ultraviolet Transmittance Analyzer.

[0060] Viscometer: Brookfield DV3T rheometer.

[0061] Key performance testing standards:

[0062] Endotoxin content determination: according to the United States Pharmacopeia (USP) <85> The prescribed Limulus Amebocyte Lysate (LAL) reagent is used for dynamic turbidimetry or chromogenic matrix method.

[0063] Particle size and PDI determination: Dynamic light scattering (DLS) technology was used in accordance with ISO 22412:2020 standard.

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

[0065] SPF in vitro assay: 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 GreenPlanet) and 15 g of triethyl citrate were melt plasticized in a twin-screw extruder. The extruder temperatures were set as follows: feed zone 80℃, compression zone 130℃, melting zone 145℃, and die 140℃.

[0069] Step 2. High-Pressure Thermal Emulsification: The above-mentioned PHA molten stream was pumped out at a rate of 0.5 kg / h, while the aqueous phase, preheated to 80°C and pre-dissolved in polyquaternium-10 (7.5 g, equivalent to 0.75 wt% of the dry weight of PHA), was pumped out at a rate of 2.5 kg / h. After the two phases were premixed in an online high-shear mixing chamber maintained at 95°C, they were introduced into a two-stage series high-pressure homogenizer for emulsification. The first-stage homogenization pressure was set to 800 bar, and the second-stage pressure was set to 150 bar.

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

[0071] Step 4. Deep detoxification:

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

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

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

[0075] Product characterization: The measured solid content of the product was 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 being stored at 45°C for 6 months, the particle size changed by 4.2%.

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

[0077] Repeat the steps of Example 1, but in step 2, adjust the weight ratio of melt to water to 1:3.5, and in step 4, after ultrafiltration, further concentrate the emulsion to obtain a product with a final solid content of 35wt%.

[0078] Product characterization: The median particle size d of the product was measured. 50 The particle size was 155 nm; the PDI was 0.14; the endotoxin content was 0.08 EU / gPHA; and the zeta potential was +32 mV. After being stored at 45°C for 6 months, the particle size changed by 4.6%.

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

[0080] Repeat the steps of Example 1, but replace the PHA raw material used in step 1 with PHBHHx with a 3-hydroxyhexanoic acid molar fraction of 8 mol%.

[0081] Product characterization: The measured solid content of the product was 26.8 wt%; 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 being stored at 45°C for 6 months, the particle size changed by 3.9%.

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

[0083] Repeat the steps of Example 1, except that in step 2, the high-pressure thermal emulsification, the first-stage homogenization pressure is increased to 1000 bar, and the second-stage homogenization pressure is maintained at 200 bar.

[0084] Product characterization: The measured solid content of the product was 26.5 wt%; 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 being stored at 45°C for 6 months, the particle size changed by 4.8%.

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

[0086] Repeat the steps of Example 1, except that in step 2, the high-pressure thermal emulsification, the first-stage homogenization pressure is reduced to 600 bar and the second-stage homogenization pressure is reduced to 100 bar.

[0087] Product characterization: The measured solid content of the product was 27.5 wt%; 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 being stored at 45°C for 6 months, the particle size changed by 4.0%.

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

[0089] Repeat the steps of Example 1, except that in step 2, the weight ratio of the molten material to the aqueous phase is adjusted to 1:5.5.

[0090] Product characterization: The solid content of the product was measured to be 20.5 wt%; 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 being stored at 45°C for 6 months, the particle size changed by 4.1%.

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

[0092] Repeat the steps of Example 1, except that in step 2, the emulsifier polyquaternium salt-10 is replaced with an equal amount of polyquaternium salt-7.

[0093] Product characterization: The preparation process proceeded smoothly without any abnormalities. The measured solid content of the product was 27.0 wt%; 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 being stored at 45°C for 6 months, the particle size changed by 4.5%. This result indicates that other pH-insensitive cationic polymers are also suitable for this invention.

[0094] Comparative Example 1: PHA emulsion was prepared using the traditional solvent evaporation method.

[0095] Dissolve 10g PHBHHx in 100mL of dichloromethane, add it to an aqueous solution containing PVA, emulsify by high-speed shearing, and then remove the dichloromethane 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 being stored at 45°C for 6 months, the sample showed severe demulsification and precipitation, making accurate particle size measurement impossible, with an estimated particle size change of more than 30%.

[0097] Comparative Example 2: The present invention's nano-process was used, but all endotoxin removal steps were omitted.

[0098] Repeat steps 1-3 and 5 of Example 1, but completely omit the "deep endotoxin removal treatment" in step 4.

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

[0100] Comparative Example 3: The present invention employs nanotechnology, but only partially removes endotoxins.

[0101] Repeat the steps of Example 1, but perform only ultrafiltration washing in the deep endotoxin removal process of step 4, omitting the column chromatography adsorption step.

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

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

[0104] Repeat all the steps of Example 1, except that the PHA raw material in step 1 is replaced with 1.0 kg of pure P(3HB).

[0105] Process observation and product characterization: During the melt plasticizing process in step 1, a significant increase and fluctuation in extruder torque was observed, and the extrudate from the die head showed slight yellowing, indicating possible partial thermal degradation. The final product obtained had a median particle size d. 50 The particle size was 285 nm, the PDI was 0.31, and the endotoxin content was 0.09 EU / g PHA. After being stored at 45°C for 6 months, the sample showed partial precipitation, with a particle size change of more than 15%.

[0106] Comparative Example 5: A comparative experiment omitting the crystallization inhibitor.

[0107] Repeat all the steps of Example 1, except that triethyl citrate is 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 was measured. 50 The median particle size was 148 nm, and the PDI was 0.14. However, after being stored at 45°C for 6 months, the median particle size d... 50 The particle size increased significantly to 175 nm, with a change of up to 18.2%, far exceeding the 5% stability threshold, and a small amount of white precipitate was observed at the bottom of the sample vial.

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

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

[0111] Process observation and product characterization: Steps 1 to 3 of the preparation process, as well as the ultrafiltration washing in step 4, proceeded normally, and the emulsion obtained after ultrafiltration had a good appearance. However, when preparing for the column chromatography adsorption process in step 4, during the adjustment of the pH of the emulsion to 8.0 with triethanolamine, the emulsion immediately underwent irreversible flocculation and rapidly formed a large amount of precipitate, causing the system to completely collapse. Therefore, it was impossible to pump the sample into the chromatography column for subsequent purification operations.

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

[0113]

[0114] As can be seen from the comparative data in Table 2, the PHA nanoemulsion prepared in the embodiments of the present invention achieved all the expected targets in terms of key performance parameters, including the ideal particle size range, low PDI, ultra-low endotoxin content, and excellent long-term stability. In stark contrast, the products in the comparative examples showed significant defects in at least one key performance aspect, fully demonstrating the innovation and effectiveness of the technical solution of the present invention.

[0115] Application example:

[0116] The basic formulations for application examples are shown in Table 3 below:

[0117]

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

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

[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 emulsion.

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

[0123] Product review: The product has a light, thin lotion texture and feels refreshing on the skin.

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

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

[0126] Product evaluation: The product has a rich, creamy texture and provides good sealing.

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

[0128] The 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 made up by deionized water in phase C.

[0129] Product review: The cream has a slightly thin texture and spreads only moderately.

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

[0131] Table 4 shows a comparison of the effectiveness of application examples:

[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 cosmetic compositions incorporating the PHA nanoemulsion of this invention on improving skin barrier function and enhancing the water resistance of sunscreen products. Compared to placebo formulations without PHA nanoemulsion, the addition of the nanoemulsion of this invention significantly improved the TEWL reduction rate and SPF maintenance rate, with statistically significant differences, 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 regarding the technical solution of the present invention:

[0136] Superiority of the Process and Uniqueness of the Product: The core process of this invention, namely the continuous solvent-free melt emulsification technology, has been proven to stably prepare PHA nanoemulsions with a unique combination of properties. These properties include a controllable particle size of 100-200 nm, excellent uniformity (PDI) not exceeding 0.15, and ultra-high biopurity not exceeding 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, including particle size control, distribution uniformity, biosafety, and chemical purity. This indicates that the proposed solvent-free melt emulsification pathway is an effective technical solution for achieving this specific high-performance nanoemulsion, and possesses significant innovation.

[0137] Synergistic Effects and Necessity of Deep Endotoxin Removal Strategy: Experimental data clearly reveal the necessity of deep endotoxin removal treatment. Completely omitting this step results in products carrying up to 185 EU / g of endotoxin, triggering a strong in vitro inflammatory response. Comparative Example 3 further demonstrates that even with conventional ultrafiltration washing, the endotoxin content only decreases to 35 EU / g, and while the induced inflammation level decreases, it remains high. Only by combining ultrafiltration washing with column chromatography adsorption can the endotoxin content be synergistically controlled to an ultra-low level below 0.10 EU / g, and the inflammatory response reduced to baseline. This comparison powerfully demonstrates that this unique two-step deep purification strategy is a key technical feature for achieving ultra-high product biosafety, with unexpected synergistic effects. It should be noted that IL-8 release, as a direct indicator of endotoxin-induced inflammatory response, is mainly used to assess the effectiveness of endotoxin removal treatment; therefore, this data is only provided in examples and comparative examples related to endotoxin content.

[0138] Necessity and Advantages of Each Component in the Formulation System: The formulation system adopted in this technical solution is key to achieving the entire process flow. Comparative Example 6 decisively demonstrates the necessity of using a pH-insensitive cationic polymer emulsifier; when pH-sensitive chitosan was used instead, the system immediately collapsed during the alkaline purification step, revealing the chemical contradictions faced by conventional emulsifiers in this special process. Example 7 further demonstrates that the technical solution of this invention is not limited to the specific polyquaternium-10; other cationic polymers with similar pH-insensitive properties, such as polyquaternium-7, can also successfully implement this invention, thus supporting the functionally defined scope. In addition, Comparative Example 5 confirms the necessity of a crystallization inhibitor from the opposite perspective: the lack of triethyl citrate leads to severe physical instability of the product after accelerated aging, with particle size drift as high as 18.2%. Meanwhile, Comparative Example 4 also highlights the superiority of choosing PHBHHx copolymer raw materials; using highly crystalline pure PHB raw materials leads to a significant deterioration in product particle size and uniformity.

[0139] Application Value and Wide Applicability of the Product: Application examples demonstrate the significant application value of the PHA nanoemulsion prepared by this technical solution. Within a wide concentration range of 1.5wt% to 12.0wt%, this nanoemulsion can be successfully applied to cosmetics of different textures, exhibiting a positive correlation between concentration and efficacy. Compared to a placebo formulation without this nanoemulsion, the formulation with this emulsion showed undeniable superiority in reducing skin moisture loss (e.g., a TEWL reduction rate of 25.4%±3.1% compared to the placebo group of 4.1%±1.5%) and improving the water resistance of sunscreen products (e.g., an SPF retention rate of 88.2%±4.5%, significantly higher than the placebo group of 65.1%±5.2%). These data indicate that the efficacy truly originates from the PHA nanoemulsion itself, rather than from the matrix, and the effect is statistically significant.

[0140] To ensure the robustness and controllability of this technical solution, we conducted extensive experimental studies on the effects of key components and process parameters at different levels. The research results systematically revealed the patterns of their influence on the final product performance, and the following trends were drawn:

[0141] 3-Hydroxyhexanoic acid monomer content: Experimental results show that the molar fraction of 3-hydroxyhexanoic acid monomer in PHBHHx copolymers is a key factor affecting the final emulsion properties. Systematic adjustment of this monomer's molar fraction revealed that moderately increasing its content effectively reduces the polymer's crystallinity and melting point. This characteristic makes the polymer easier to shear and disperse during melt emulsification, thus exhibiting a clear trend towards obtaining smaller particle sizes, or achieving the same particle size with lower homogenization energy consumption.

[0142] Crystallization inhibitor: Gradient experiments on the addition of crystallization inhibitor, triethyl citrate, revealed a direct correlation between its dosage and the long-term physical stability of the product. Studies confirmed that within the publicly available formulation system, increasing the dosage of crystallization inhibitor more effectively inhibits secondary crystallization and Austmann ripening effects of PHA nanoparticles during long-term storage, thereby significantly enhancing the physical stability of the product, manifested as an effective reduction in long-term particle size drift.

[0143] Emulsifier dosage: Systematic studies on the concentration of pH-insensitive cationic polymer emulsifiers have confirmed that their dosage is one of the core variables determining the initial particle size and stability of nanoemulsions. Experimental data show that increasing the emulsifier concentration within the effective concentration range allows for faster and more complete coating of the newly formed nanoparticle surface. This directly leads to smaller initial particle size and higher Zeta potential, thereby enhancing the immediate stability of the emulsion during preparation.

[0144] Solid content: Studies on the solid content of emulsions covered the entire range of 20-35 wt%, and the results showed that solid content has a significant impact on the processing. Rheological and processing data analysis revealed that when the solid content was adjusted towards the higher values ​​within this range, the overall viscosity of the system increased accordingly, which reduced the energy transfer efficiency during high-pressure homogenization. Therefore, experimental conclusions indicate that higher solid content requires higher homogenization pressure to achieve the same target particle size, or that under the same homogenization pressure, the final particle size tends to increase.

[0145] In summary, this technical solution successfully addresses the core challenges in existing PHA aqueous dispersion preparation technologies, such as residual organic solvents, poor particle size control, poor long-term physical stability, and endotoxin contamination, through an innovative, continuous, solvent-free preparation method. The defined PHA nanoemulsion, with its unique formulation system and parameter combination, exhibits extremely high biocompatibility, uniform nanoscale particle size, and excellent physicochemical stability, demonstrating clear practical application value.

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

Claims

1. A polyhydroxyalkanoate nanoemulsion, characterized in that, The nanoemulsion simultaneously satisfies all of the following limiting parameters: Endotoxin content: not higher than 0.10 EU / g, based on the dry weight of polyhydroxyalkanoates; Median particle size d 50 100-200nm; Multidispersion Index (PDI): not higher than 0.15; Solid content: 20-35 wt% The nanoemulsion is stabilized by a pH-insensitive cationic polymer emulsifier, and the absolute value of the zeta potential of the nanoemulsion is not less than 25 mV at pH 6.

0. After being stored at 45°C for 6 months under accelerated aging conditions, the median particle size d of the nanoemulsion was... 50 The drift is no higher than 5%; The polyhydroxy fatty acid ester is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), wherein the molar fraction of 3-hydroxyhexanoate monomer is 8-12 mol%. Furthermore, the nanoemulsion is prepared by a continuous solvent-free preparation method, the method comprising the following steps: Step 1: Polyhydroxyalkanoate granules and a crystallization inhibitor are melt-plasticized in a twin-screw extruder at 110-150℃ to form a polyhydroxyalkanoate melt stream; Step 2: The polyhydroxy fatty acid ester melt stream is premixed with an aqueous phase containing a pH-insensitive cationic polymer emulsifier preheated to 70-85°C using an online mixing device, and then introduced into a high-pressure homogenizer with at least two stages connected in series for high-pressure thermal emulsification to obtain a primary nanoemulsion. Step 3: Perform vacuum flash evaporation and devolatilization on the primary nanoemulsion; Step 4: The emulsion treated in Step 3 is subjected to deep endotoxin removal treatment, which includes ultrafiltration washing and weakly basic anion exchange resin adsorption treatment at pH 7.5-8.

5. Step 5: Aseptically filter the emulsion and adjust the pH value; The pH-insensitive cationic polymer emulsifier is one or more polymers containing quaternary ammonium salt groups in their molecular structure, and its addition amount is 0.5-5.0 wt% based on the dry weight of polyhydroxy fatty acid ester solids.

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

3. The polyhydroxyalkanoate nanoemulsion according to claim 1, characterized in that, The polymer of the quaternary ammonium salt group is polyquaternary ammonium salt-10 or polyquaternary ammonium salt-7.

4. The polyhydroxyalkanoate nanoemulsion according to claim 1, characterized in that, The weakly basic anion exchange resin adsorption treatment in step 4 is a column chromatography purification step.

5. An O / W type cream composition, characterized in that, Contains 1-15 wt% of the polyhydroxyalkanoate nanoemulsion according to claim 1 by solid dry weight.

6. A method for improving the properties of a cosmetic composition using the polyhydroxyalkanoate nanoemulsion according to claim 1, characterized in that, Adding 1-15 wt% of the polyhydroxyalkanoate nanoemulsion, based on the dry weight of polyhydroxyalkanoate solids, to the composition achieves at least one of the following effects: Improve the water resistance of O / W type sunscreen compositions; Enhance skin barrier function; Reduce transepidermal water loss.

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