Bee venom-containing skin essence with small irritation and preparation method of bee venom-containing skin essence
Patent Information
- Application Number
- CN202510627579.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-29
Smart Images
Figure SMS_1 
Figure SMS_2
Abstract
Description
Technical Field
[0001] The invention relates to the field of skin essence preparation, in particular to a skin essence containing bee venom with low irritation and a preparation method thereof. Background Art
[0002] Skin aging is caused by both endogenous and exogenous factors. Endogenous factors, including excessive free radical production and mitochondrial DNA damage, are unavoidable processes. Exogenous factors, such as UV exposure and air pollution, can induce the appearance of coarse wrinkles, irregular pigmentation, and age spots. Both endogenous and exogenous factors lead to cumulative changes in skin structure, function, and appearance. Continuous exploration in the field of skincare has led to the discovery of the potential applications of melittin and snake venom-like peptides.
[0003] Melittin and venom-like peptides are two peptides with significant biological activity that have been widely used in anti-aging and skincare applications in recent years. Melittin can reduce wrinkles by promoting collagen production and inhibiting muscle contraction, but its strong irritation limits its direct application. Venom-like peptides, on the other hand, effectively soothe dynamic wrinkles by mimicking the neuromuscular blocking effects of snake venom proteins.
[0004] However, both peptides suffer from low transdermal absorption, easy degradation, and irritation, necessitating the development of novel delivery systems that achieve efficient and safe skin penetration. The development of nanocarrier technology offers a solution to this problem.
[0005] While traditional liposomes and polymer nanoparticles (such as PLGA) can improve peptide stability, single carriers have limitations: low liposome encapsulation efficiency and a significant burst release effect, while PLGA degradation can trigger a local acidic microenvironment and exacerbate skin irritation. In recent years, hybrid nanosystems (such as PLGA-liposome complexes) have emerged as an effective strategy to address these issues by combining the biocompatibility of liposomes with the sustained-release properties of polymers. Polyethylene glycol (PEG) modification can enhance the skin permeability of nanoparticles and reduce immunogenicity, but optimizing the carrier assembly process to achieve coordinated peptide delivery remains to be explored.
[0006] At the same time, poloxamer 407 is a thermosensitive gel matrix that is liquid at room temperature for easy application. After contacting the skin (≥25°C), it forms a gel to achieve in situ sustained release. However, its high concentration (>18%) may cause dry skin, and physical encapsulation of the polypeptide makes it difficult to avoid rapid release. In the existing technology, the "double sustained-release" system of embedding nanoparticles into poloxamer gel can prolong the drug's duration of action, but the compatibility, stability and irritation control of nanoparticles and gels remain technical difficulties. Summary of the Invention
[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to provide a skin essence containing bee venom with low irritation and a preparation method thereof.
[0008] In order to achieve the above object, the present invention adopts the following technical solutions:
[0009] The present invention first provides a low-irritation bee venom-containing skin essence, comprising the following raw materials in parts by weight:
[0010] 3 g of PLGA-PEG / liposome nanoparticles;
[0011] 19-23g of thermosensitive gel matrix, the thermosensitive gel matrix is poloxamer 407;
[0012] 5-8g of protective agent, the protective agent is mannitol;
[0013] 0.1-0.5g of antioxidant, the antioxidant is vitamin E;
[0014] 20-25 ml of stabilizer, which is a phosphate buffered saline solution with a pH of 7.4 and a molar concentration of 0.01 mol / L, i.e., PBS solution;
[0015] 80ml of ultrapure water;
[0016] The preparation process of the PLGA-PEG / liposome nanoparticles comprises the following steps:
[0017] S1. Preparation of hydrophobized melittin:
[0018] Melittin lyophilized powder was dissolved in borate buffer at pH 8.5, and N-hydroxysuccinimide palmitic acid ester (NHS-palmitate, manufactured by MERCK, purity 98%+) of the same mass as the melittin lyophilized powder was added. The mixture was reacted at room temperature for 4 hours to completely dissolve the melittin lyophilized powder and NHS-palmitate. Free palmitic acid was removed by dialysis at a MWCO of 1 kDa, and the hydrophobized melittin was obtained by lyophilization.
[0019] Melittin itself is hydrophilic. The amino group (-NH2, usually from the N-terminus or lysine side chain) of melittin reacts with the active ester group of NHS-palmitate to form an amide bond (-CO-NH-), thereby introducing a hydrophobic long chain of palmitic acid. This enhances compatibility with the hydrophobic core - PLGA-PEG, increases drug loading, reduces direct contact between free melittin and the human body, and reduces irritation.
[0020] Hydrophobicized melittin reaction formula:
[0021] Melittin-NH2+NHS-palmitate→Melittin-NH-CO-(CH2)14 -CH3+NHS
[0022] S2, PLGA-PEG loaded with melittin:
[0023] Use mPEG-PLGA (methyl polyethylene glycol-poly(lactic-co-glycolic acid), produced by Ruixi Biotechnology, purity 95%+) as the backbone material of the nanoparticles. Dissolve 0.79-1 g of mPEG-PLGA in 25 ml of chloroform and vortex thoroughly to dissolve.
[0024] Then, 0.12-1.3 g of hydrophobized melittin was dissolved in 25 ml of a chloroform-methanol mixture, the mPEG-PLGA solution and the hydrophobized melittin solution were mixed, and 250 ml of a 5% by mass PVA solution was added to stabilize the emulsion.
[0025] Ultrasonic emulsification was performed at 100W for 2 minutes to form a water-in-oil (W / O) colostrum. The colostrum was then poured into 400ml of PVA solution and mechanically stirred overnight (approximately 12 hours) to evaporate the organic solvents (chloroform and methanol) and form solid nanoparticles.
[0026] During this process, PVA wraps the surface of the nanoparticles to prevent aggregation, forming a stable water-in-oil-in-water (W / O / W) emulsion.
[0027] The double emulsion was centrifuged at 12,000 rpm to remove unencapsulated hydrophobized melittin and free PVA, thereby obtaining a double emulsion centrifuge and a precipitate. The precipitate was washed with distilled water, and the washing solution was mixed with the double emulsion centrifuge, and then centrifuged at 3,000 rpm to remove large impurities, retaining the supernatant containing the nanoparticles.
[0028] The supernatant was mixed with an equal volume of a 2% PEG aqueous solution, lyophilized, and resuspended in 20 ml of PBS solution to obtain a PLGA-PEG nanoparticle solution;
[0029] S3. Liposome-loaded snake venom peptides:
[0030] Dissolve 0.79-1g of liposomes in 25ml of chloroform and dissolve in a 40°C water bath until clear. Rotate at 80-100rpm at 40°C and a vacuum of -0.09MPa to form a film. Place the film in a desiccator and evacuate at -0.1MPa for 2h to completely remove any chloroform residues, thereby obtaining a lipid film.
[0031] Use 50 ml of 300 mmol / L ammonium sulfate solution to combine the lipid film with water molecules to form hydrates, and vortex for 30 min until the film is completely detached to obtain a crude liposome suspension;
[0032] The liposome crude suspension was extruded through a polycarbonate membrane with a pore size of 100 nm using a microfluidic membrane extrusion process to obtain a blank liposome mixture, which was then dialyzed against PBS solution.
[0033] After dialysis, the outer aqueous phase (NH4)2SO4 is removed, and the inner aqueous phase (NH4)2SO4 is retained, forming an ammonium sulfate gradient with high inside and low outside, which decomposes into NH3 (escaped) and H in the liposome. + , causing the pH of the inner cavity to drop to 4.0, while the pH of the outer aqueous phase is 7.4, forming a transmembrane proton gradient, adding 10 ml of PBS solution containing 0.12-1.3 g of snake venom peptide, incubating at 50-60 ° C with intermittent vortex mixing for 30 minutes, and cooling in an ice bath to obtain 20 ml of drug-loaded liposome solution;
[0034] After dialysis with ammonium sulfate (inner aqueous phase), a pH gradient is formed. The snake venom-like peptide (weak base) passes through the membrane as a neutral molecule at 55°C and is captured in the liposome after being protonated. The snake venom-like peptide passes through the lipid membrane in a non-protonated form (hydrophobic) in a neutral PBS solution. After entering the liposome, due to the low pH of the inner aqueous phase, the peptide is protonated (positively charged) and is captured in the inner aqueous phase.
[0035] The basic principle of preparing nanoparticles by microfluidic membrane extrusion:
[0036] Microfluidic membrane extrusion uses microfluidic chips to manipulate fluids in micron-scale channels. After rapidly mixing the organic phase containing drugs and lipids with the aqueous phase in the chip, the mixture is pushed through a polycarbonate membrane with a pore size of 100nm. Large particles or multi-compartment liposomes are sheared and ruptured by the membrane pores and then reaggregated into smaller nanoparticles, achieving particle size reduction and uniform distribution.
[0037] S4. PLGA-PEG / liposome hybrid assembly:
[0038] The PLGA-PEG nanoparticle solution and the drug-loaded liposome solution were mixed at a volume ratio of 1:1, and the mixture of the PLGA-PEG nanoparticle solution and the drug-loaded liposome solution was extruded through a polycarbonate membrane with a pore size of 100 nm using a microfluidic membrane extrusion process to form a hybrid nanoparticle mixture;
[0039] The hybrid nanoparticle mixture was ultrafiltrated and centrifuged at a MWCO of 10 kDa to remove free peptides, and the hybrid particles were collected and freeze-dried, namely PLGA-PEG / liposome nanoparticles;
[0040] PLGA-PEG / liposomes encapsulate melittin, reducing direct contact with the skin and delaying the duration of action of melittin, allowing its medicinal effects to be better exerted.
[0041] Preferably, the liposome is prepared from soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol with an average molecular weight of 2000, and cholesterol in a mass ratio of 70:25:5.
[0042] Preferably, the microfluidic membrane extrusion process of S3 comprises the following specific steps:
[0043] The liposome crude suspension preheated to 60°C is injected into the automatic extruder LIPEX TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min;
[0044] Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
[0045] Preferably, the dialysis in S3 comprises the following steps:
[0046] Use a cellulose dialysis bag with a MWCO of 10 kDa and place the blank liposome mixture into the bag, leaving about 20% of the volume free to prevent expansion and rupture. Seal both ends with dialysis clips to ensure no leakage.
[0047] The dialysis bag was completely immersed in 4000 ml of PBS solution and stirred magnetically at 100 rpm. The whole process was carried out in an ice bath at 4°C to prevent liposome aggregation or drug leakage.
[0048] After 1 hour, the PBS solution was replaced for the first time, and then the PBS solution was replaced every 4–6 hours, for a total of 3–4 times, for a total of 24 hours of dialysis;
[0049] After dialysis is stopped, the dialysate is taken and the SO4 is detected with BaCl2 solution. 2- If there is no white precipitate, it indicates that the dialysis is complete;
[0050] Preferably, the microfluidic membrane extrusion process of S4 comprises the following specific steps:
[0051] The mixture of PLGA-PEG nanoparticle solution and drug-loaded liposome solution preheated to 60℃ was injected into the automatic extruder LIPEX TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min;
[0052] Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
[0053] Preferably, the melittin freeze-dried powder is prepared by citing the preparation method of the document "Isolation and Purification of Melittin from Bee Venom" using self-produced crude bee venom as raw material, and the specific steps are:
[0054] Crude bee venom (homemade) was dissolved in purified water and centrifuged to obtain a centrifuge; the centrifuge was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain a bee venom ultrafiltrate, which was then subjected to preparative chromatography;
[0055] The bee venom ultrafiltrate was passed through a preparative chromatography column equipped with polystyrene microspheres Uni PS20-300. The mobile phase A was a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The mobile phase B was gradient eluted with a concentration of phase B of 35%-55%. At 0.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 15.00 min, the mobile phase A was 50% and the mobile phase B was 50%; at 15.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 20.00 min, the mobile phase A was 65% and the mobile phase B was 35%; by monitoring the chromatogram, the chromatographic peak eluate of melittin was identified and collected. Ensure that the collected eluate corresponds to the peak of melittin and has a purity of more than 99% to obtain the melittin eluate;
[0056] The melittin eluate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. After continuous constant-solubility ultrafiltration (ensuring continuous replenishment of the solvent, i.e., water, to maintain a substantially constant solute concentration on the membrane surface), the melittin eluate was concentrated to approximately 150 mg / mL. The ultrafiltration was stopped, and the concentrated melittin eluate was collected and lyophilized to obtain a melittin lyophilized powder.
[0057] The present invention also provides a method for preparing the low-irritation bee venom-containing skin essence, comprising the following steps:
[0058] 1) Prepare Poloxamer 407 solution:
[0059] Disperse 19-23 g of poloxamer 407 in 80 ml of 4°C ultrapure water, stir magnetically at 300 rpm until completely transparent, and keep in an ice bath throughout the process to obtain a poloxamer 407 solution;
[0060] 2) Nanoparticle resuspension:
[0061] The PLGA-PEG / liposome nanoparticles were resuspended in 20-25 ml of a 4° C. PBS solution containing 5-8 g of mannitol, and poloxamer 407 solution was slowly added. 0.1-0.5 g of vitamin E was then added. The mixture was magnetically stirred at 4° C. and 300 rpm for 10 min. The nanoparticles were embedded in the poloxamer. After the poloxamer gelled, the gel network physically blocked the diffusion of the nanoparticles. At body temperature (about 32° C.), the gel gradually dissolved, releasing the nanoparticles and free drugs simultaneously, thereby achieving dual sustained-release, thereby obtaining a thermosensitive skin essence stock solution with dual sustained-release capabilities.
[0062] 3) Filling and storage:
[0063] The original skin essence liquid is placed in a light-proof sterile bottle, filled with nitrogen for protection, stored in liquid form at 4°C, and restored to room temperature (25°C for best fluidity) before use to obtain the product - a skin essence containing bee venom with low irritation.
[0064] Preferably, in the bee venom-containing skin essence with low irritation, the PLGA-PEG / liposome nanoparticles are stably dispersed in water in the form of colloid, forming a uniform opalescent suspension and not separating into layers upon standing.
[0065] Both bee venom peptides and snake venom peptides can be used in skin care products. The principle is:
[0066] The target of snake venom-like peptides is the acetylcholine receptor, which it inhibits and simulates the neuromuscular blocking function of snake venom proteins, thereby reducing muscle contraction signal transmission and relieving dynamic wrinkles such as expression lines. Its effect is similar to botulinum toxin, but it is reversible and non-toxic.
[0067] The target of bee venom peptide is the TRPV1 channel and MAPK / ERK pathway of fibroblasts. By activating the TRPV1 channel and MAPK / ERK pathway of skin fibroblasts, it promotes the synthesis of collagen (type I / III) and elastic fibers, repairs static wrinkles; inhibits matrix metalloproteinases, and reduces collagen degradation.
[0068] Muscle relaxation and collagen remodeling work together to perform dual intervention on dynamic and static wrinkles. That is, snake venom peptides instantly inhibit muscle contraction, and bee venom peptides promote dermal reconstruction in the long term, forming a three-dimensional anti-wrinkle network of "short-term relief + long-term repair".
[0069] At the same time, the ERK pathway activated by bee venom peptide can enhance the sensitivity of fibroblasts to snake venom peptides and improve anti-wrinkle efficiency.
[0070] Melittin has a strong irritating effect on the human body. Although the PEG chains on the surface of PLGA-PEG reduce the electrostatic repulsion between nanoparticles and the skin's stratum corneum and promote penetration through hair follicles or intercellular spaces, bee venom peptide must wait for PLGA to degrade before it can be released. The sustained-release system thus constructed avoids erythema or burning pain caused by sudden release.
[0071] The phospholipid bilayer structure of liposomes is similar to the stratum corneum of the skin, which enhances the transdermal efficiency of snake venom-like peptides through fusion. At the same time, after being encapsulated by liposomes, the snake venom-like peptides block their nonspecific binding to nerve receptors, reducing facial stiffness.
[0072] The PLGA-PEG / liposome nanoparticles implemented in the present invention are an efficient and safe way to utilize two peptides.
[0073] Compared with the prior art, the present invention has the following beneficial effects:
[0074] 1. The present invention simultaneously utilizes the anti-wrinkle effects of melittin and snake venom-like peptides on the skin, and can perform dual intervention on dynamic and static wrinkles on the skin. That is, snake venom-like peptides immediately inhibit muscle contraction, and melittin promotes dermal reconstruction in the long term, forming a three-dimensional anti-wrinkle network of "short-term relief + long-term repair", achieving a synergistic effect of the two anti-wrinkle components and improving skin care effects.
[0075] 2. Melittin and snake venom-like peptides both have problems such as low transdermal absorption rate, easy degradation and irritation. Although traditional single carriers (such as liposomes or PLGA) can partially improve these problems, they still have defects such as low encapsulation efficiency and local irritation. The present invention designs a PLGA-PEG / liposome nanoparticle: first, melittin is hydrophobized to enhance the compatibility of melittin and the hydrophobic core - PLGA-PEG; secondly, the liposome is used to carry the snake venom-like peptide alone, avoiding the competitive loading of the two peptides by a single carrier, achieving high drug loading and coordinated delivery; through encapsulation and sustained release, irritation is reduced and the long-term efficacy of the drug is improved;
[0076] 3. The present invention uses PLGA-PEG / liposome nanoparticles in combination with poloxamer solution to construct a skin product with a dual sustained-release drug system: at 32°C (close to skin temperature), the poloxamer solution (15%-17%) will quickly transform into a gel. When applied to the human face, it usually completes the phase transition from liquid to semi-solid within 30 seconds to 1 minute.
[0077] Embedding PLGA-PEG / liposome nanoparticles into poloxamer 407 thermosensitive gel achieves a stepped release through the synergistic effect of "nanocarrier-gel matrix", further prolonging the duration of action;
[0078] On the one hand, the formed gel film itself has water-locking properties. On the other hand, even if the PLGA-PEG / liposome nanoparticles accidentally rupture, allowing bee venom peptide and snake venom-like peptide to leak out, the second sustained-release system formed by poloxamer can better protect the user, greatly improving the safety of the product.
[0079] In summary, the present invention provides an efficient and safe solution for the skin delivery of polypeptide active ingredients by integrating nanohybrid technology with the properties of thermosensitive gel, significantly improving the skin care function of the product. DETAILED DESCRIPTION
[0080] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0081] Example 1:
[0082] A low-irritation bee venom-containing skin essence and a preparation method thereof, comprising the following steps:
[0083] 1) Preparation of PLGA-PEG / liposome nanoparticles:
[0084] S1. Preparation of hydrophobized melittin:
[0085] Crude bee venom (homemade) was dissolved in purified water and centrifuged to obtain a centrifuge; the centrifuge was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain a bee venom ultrafiltrate, which was then subjected to preparative chromatography;
[0086] The bee venom ultrafiltrate was passed through a preparative chromatography column equipped with polystyrene microspheres Uni PS20-300. The mobile phase A was a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. The mobile phase B concentration was 35% to 55% for gradient elution. At 0.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 15.00 min, the mobile phase A was 50% and the mobile phase B was 50%; at 15.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 20.00 min, the mobile phase A was 65% and the mobile phase B was 35%; by monitoring the chromatogram, the chromatographic peak eluate of melittin was identified and collected. Ensure that the collected eluate corresponds to the peak of melittin and has a purity of more than 99% to obtain the melittin eluate;
[0087] The melittin eluate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. After continuous constant-solubility ultrafiltration (ensuring continuous replenishment of the solvent, i.e., water, to maintain a substantially constant solute concentration on the membrane surface), the melittin eluate was concentrated to approximately 150 mg / mL. The ultrafiltration was stopped, and the concentrated melittin eluate was collected and lyophilized to obtain a melittin lyophilized powder.
[0088] Melittin lyophilized powder (homemade) was dissolved in borate buffer at pH 8.5, and N-hydroxysuccinimide palmitate (NHS-palmitate, manufactured by MERCK, purity 98%+) of the same mass as the melittin lyophilized powder was added. The mixture was reacted at room temperature for 4 h to completely dissolve the melittin lyophilized powder and NHS-palmitate. Free palmitic acid was removed by dialysis at a MWCO of 1 kDa, and the hydrophobized melittin was obtained by lyophilization.
[0089] Melittin itself is hydrophilic. The amino group (-NH2, usually from the N-terminus or lysine side chain) of melittin reacts with the active ester group of NHS-palmitate to form an amide bond (-CO-NH-), thereby introducing a hydrophobic long chain of palmitic acid. This enhances compatibility with the hydrophobic core - PLGA-PEG, increases drug loading, reduces direct contact between free melittin and the human body, and reduces irritation.
[0090] Hydrophobicized melittin reaction formula:
[0091] Melittin-NH2+NHS-palmitate→Melittin-NH-CO-(CH2) 14 -CH3+NHS
[0092] S2, PLGA-PEG loaded with melittin:
[0093] mPEG-PLGA (methyl polyethylene glycol-poly(lactic-co-glycolic acid), produced by Ruixi Biotechnology, purity 95%+) was used as the framework material of the nanoparticles. 1 g of mPEG-PLGA was dissolved in 25 ml of chloroform and vortexed thoroughly to dissolve.
[0094] Subsequently, 0.5 g of hydrophobized melittin was dissolved in 25 ml of a mixed solution of chloroform and methanol, the mPEG-PLGA solution and the hydrophobized melittin solution were mixed, and 250 ml of a 5% by mass PVA solution was added to stabilize the emulsion;
[0095] Ultrasonic emulsification was performed at 100W for 2 minutes to form a water-in-oil (W / O) colostrum. The colostrum was then poured into 400ml of PVA solution and mechanically stirred overnight (approximately 12 hours) to evaporate the organic solvents (chloroform and methanol) and form solid nanoparticles.
[0096] During this process, PVA wraps the surface of the nanoparticles to prevent aggregation, forming a stable water-in-oil-in-water (W / O / W) emulsion.
[0097] The double emulsion was centrifuged at 12,000 rpm to remove unencapsulated hydrophobized melittin and free PVA, thereby obtaining a double emulsion centrifuge and a precipitate. The precipitate was washed with distilled water, and the washing solution was mixed with the double emulsion centrifuge, and then centrifuged at 3,000 rpm to remove large impurities, retaining the supernatant containing the nanoparticles.
[0098] The supernatant was mixed with an equal volume of a 2% PEG aqueous solution, lyophilized, and resuspended in 20 ml of PBS solution to obtain a PLGA-PEG nanoparticle solution. This step was repeated to obtain a PLGA-PEG nanoparticle solution for subsequent testing.
[0099] S3. Liposome-loaded snake venom peptides:
[0100] Dissolve 1 g of liposomes in 25 ml of chloroform and dissolve in a 40°C water bath until clear. Rotate the liposomes at 80–100 rpm at 40°C and a vacuum of -0.09 MPa to form a film. Place the film in a desiccator and evacuate at -0.1 MPa for 2 h to completely remove any chloroform residues, thereby obtaining a lipid film.
[0101] Use 50 ml of 300 mmol / L ammonium sulfate solution to combine the lipid film with water molecules to form hydrates, and vortex for 30 min until the film is completely detached to obtain a crude liposome suspension;
[0102] Using microfluidic membrane extrusion technology, the liposome crude suspension preheated to 60°C is injected into the automatic extruder LIPEX TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min;
[0103] Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
[0104] The crude liposome suspension was extruded through a polycarbonate membrane with a pore size of 100 nm to obtain a blank liposome mixture, which was dialyzed against PBS solution;
[0105] Use a cellulose dialysis bag with a MWCO of 10 kDa and place the blank liposome mixture into the bag, leaving about 20% of the volume free to prevent expansion and rupture. Seal both ends with dialysis clips to ensure no leakage.
[0106] The dialysis bag was completely immersed in 4000 ml of PBS solution and stirred magnetically at 100 rpm. The whole process was carried out in an ice bath at 4°C to prevent liposome aggregation or drug leakage.
[0107] After 1 hour, the PBS solution was replaced with fresh one, and then every 4–6 hours, for a total of 3–4 times (a total of 24 hours of dialysis).
[0108] After dialysis is stopped, the dialysate is taken and the SO4 is detected with BaCl2 solution. 2- If there is no white precipitate, it indicates that the dialysis is complete;
[0109] After dialysis, the outer aqueous phase (NH4)2SO4 is removed, and the inner aqueous phase (NH4)2SO4 is retained, forming an ammonium sulfate gradient with high inside and low outside, which decomposes into NH3 (escaped) and H in the liposome. +, causing the pH of the inner cavity to drop to 4.0, while the pH of the outer aqueous phase was 7.4, forming a transmembrane proton gradient, adding 10 ml of PBS solution containing 0.5 g of snake venom peptide, incubating at a temperature of 50-60 ° C with intermittent vortex mixing, incubating for 30 minutes, and cooling in an ice bath to obtain 20 ml of drug-loaded liposome solution;
[0110] After dialysis against ammonium sulfate (inner aqueous phase), a pH gradient is formed. The venom-like peptide (weak base) passes through the membrane as a neutral molecule at 55°C and is protonated and captured within the liposomes. The venom-like peptide (weak base, pKa ~8.0) passes through the lipid membrane in a non-protonated form (hydrophobic) in a neutral PBS solution. After entering the liposomes, the peptide is protonated (positively charged) and captured in the inner aqueous phase due to the low pH of the inner aqueous phase. This step is repeated to prepare a drug-loaded liposome solution for subsequent detection.
[0111] S4. PLGA-PEG / liposome hybrid assembly:
[0112] The PLGA-PEG nanoparticle solution and the drug-loaded liposome solution were mixed at a volume ratio of 1:1. The mixture of the PLGA-PEG nanoparticle solution and the drug-loaded liposome solution preheated to 60°C was injected into the automatic extruder LIPEX using a microfluidic membrane extrusion process. TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min;
[0113] Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
[0114] The mixture of PLGA-PEG nanoparticle solution and drug-loaded liposome solution was extruded through a polycarbonate membrane with a pore size of 100 nm to form a hybrid nanoparticle mixture;
[0115] The hybrid nanoparticle mixture was ultrafiltrated and centrifuged at a MWCO of 10 kDa to remove free peptides, and the hybrid particles were collected and freeze-dried, namely PLGA-PEG / liposome nanoparticles;
[0116] PLGA-PEG / liposomes encapsulate melittin, reducing direct contact with the skin and delaying the duration of action of melittin, allowing it to exert its desired effect better.
[0117] 2) Prepare Poloxamer 407 solution:
[0118] Disperse 21 g of poloxamer 407 in 80 ml of ultrapure water at 4°C, and stir magnetically at 300 rpm until completely transparent, with an ice bath throughout the process, to obtain a poloxamer 407 solution;
[0119] Poloxamer 407 is a thermosensitive nonionic triblock copolymer composed of 70% polyoxyethylene (PEO) and 30% polyoxypropylene (PPO).
[0120] Poloxamer 407 is liquid at low temperatures and can quickly form a gel when the temperature rises to body temperature or above, and the gelation temperature decreases with increasing its concentration.
[0121] 3) Nanoparticle resuspension:
[0122] The PLGA-PEG / liposome nanoparticles were resuspended in 22 ml of a 4° C. PBS solution containing 6 g of mannitol, and poloxamer 407 solution was slowly added. 0.3 g of vitamin E was then added. The mixture was magnetically stirred at 4° C. and 300 rpm for 10 min. The nanoparticles were embedded in the poloxamer. After the poloxamer gelled, the gel network physically blocked the diffusion of the nanoparticles. At body temperature (32° C.), the gel gradually dissolved, releasing the nanoparticles and free drugs synchronously, thereby achieving dual sustained-release, thereby obtaining a thermosensitive skin essence stock solution with dual sustained-release capabilities.
[0123] 4) Filling and storage:
[0124] The skin essence stock solution was placed in a sterile, light-proof bottle, protected by nitrogen, and stored at 4°C in liquid form. It was then allowed to return to room temperature (25°C offers optimal fluidity) before use. This resulted in a mildly irritating skin essence containing bee venom. In the skin essence, the PLGA-PEG / liposome nanoparticles were stably dispersed in water as a colloid, forming a homogeneous, opalescent suspension that did not separate upon standing.
[0125] Different from Example 1, according to the different formulations and experimental conditions, Examples 2 and 3, and Comparative Examples 1-15 were designed and completed:
[0126] Table 1. Skin essence formula and its heat preservation conditions
[0127]
[0128] Based on the formula and experimental conditions given in the chart, complete the experiment and test the product's skin irritation, fluidity, appearance, stability, moisturizing properties, antioxidant / whitening efficacy, anti-wrinkle / firming effect, residual solvents, and preservative content, and summarize the data into a table:
[0129] Table 2. Comparison of skin essence test results
[0130]
[0131] 1. Skin irritation, detected by human patch test:
[0132] Thirty healthy volunteers were recruited and a closed patch test (48 hours) was performed. The erythema, edema and other reactions were observed under full-spectrum lighting (scored according to ISO 10993-10 standard).
[0133] 2. Fluidity, test viscosity and rheological properties:
[0134] Use a rheometer to measure the viscosity changes at 4°C (refrigerated), 25°C (room temperature), and 32°C (skin surface temperature) after stabilization for 1 minute to verify temperature sensitivity (the gel-sol transition temperature of Poloxamer 407 should be 25-32°C).
[0135] 3. Appearance, visual inspection
[0136] Color (should be uniform milky white), transparency (opalescent suspension), and presence of stratification or precipitation.
[0137] 4. Stability (Physical)
[0138] Static stability: Place at 4°C for 30 days to observe whether there is stratification or particle aggregation.
[0139] 5. Residual solvent detection
[0140] Gas chromatography (GC) was used to detect residual chloroform and methanol (which must comply with ICH Q3C standards: chloroform ≤ 0.006% and methanol ≤ 0.3%).
[0141] 6. Moisturizing properties
[0142] In vitro test: Corneometer was used to measure the changes in the moisture content of isolated pig skin (8 hours after application).
[0143] 7. Antioxidant / whitening effects
[0144] Antioxidant (in vitro): DPPH / ABTS free radical scavenging rate (compared with VC, IC50 value evaluation).
[0145] Whitening (clinical): Mexameter was used to measure the changes in the melanin index (MI) on the face of volunteers (decreased by ≥10% after 28 days).
[0146] 8. Anti-wrinkle / firming effect:
[0147] VISIA image analysis of eye corner / forehead wrinkle depth (improved ≥5% after 28 days).
[0148] 9. Preservative content detection
[0149] In theory, high-performance liquid chromatography (HPLC) should be used to quantitatively test preservatives (such as phenoxyethanol and parabens) in the formula, and the results must meet the limits set in the "Technical Specifications for Safety of Cosmetics" (e.g., phenoxyethanol ≤ 1%). It is worth noting that this product should be stored at low temperatures due to the presence of melittin, parabens, and poloxamers, and no additional preservatives are added during production, so this test can be omitted.
[0150] In combination with Example 1, Comparative Examples 9 and 10, when the amount of PLGA-PEG / liposome nanoparticle carrier remains unchanged, simply increasing the amount of peptide has limited improvement in product performance. This is because during the production process, unloaded melittin and snake venom-like peptides will be discarded during the separation and purification of the nanoparticles, resulting in serious drug waste. The lack of melittin and snake venom-like peptides leads to a gap in the product's efficacy.
[0151] Changing the carrier dosage, as shown in Example 1, Comparative Example 1, and Comparative Example 2, can lead to nanoparticle aggregation and localized concentration unevenness, resulting in a powdery or grainy feel that affects skin feel. Excessive nanoparticles can also lead to an imbalanced release, forming an overly dense carrier network, slowing the release rate of melittin and venom-like peptides, and even preventing the active ingredients from being released within the effective timeframe. However, too low a carrier concentration reduces the amount of drug that can be loaded, resulting in a less effective serum.
[0152] 1. Skin irritation
[0153] Melittin stimulates by disrupting cell membranes, while nanoparticle encapsulation (PLGA-PEG / liposomes) can delay release and reduce direct contact. In all examples and comparative examples, due to the drug-loaded PLGA-PEG / liposome nanoparticles, no erythema or edema similar to that caused by direct human contact with melittin occurred, unless improper storage (Comparative Example 8) caused nanoparticle aggregation and drug leakage, weakening the protective effect. Snake venom-like peptides work by blocking nerve signal transmission and do not irritate the skin.
[0154] Best Performance:
[0155] Example 3 (melittin 0.10%, anhingasin 0.97%) was the least irritating, with no erythema or edema appearing in the patch test. The anhingasin in the product inhibited the release of acetylcholine rather than directly destroyed the cell membrane, resulting in lower irritation.
[0156] 2. Liquidity
[0157] The thermosensitivity of poloxamer 407 depends on its critical micelle temperature (LCST). When its concentration is 15%-17% and the temperature is below 25°C, it will always be in a highly fluid solution state. During use, due to the human facial temperature of approximately 32°C and the volatilization of the solvent, poloxamer will rapidly gel within 30 seconds to one minute, forming a uniform film. If the concentration is too high (greater than 18%), it will gel prematurely, while if the concentration is too low (less than 15%), the gelation time will be too long, making it difficult to apply well on the face.
[0158] During the test, except for Comparative Example 3 (20.10%), Comparative Example 5 (13.97%), and Comparative Example 6 (9.73%), the proportion of Poloxamer 407 in the remaining groups was moderate (15%-17%), and an ideal sol-gel transition was formed at 32°C.
[0159] Comparative Example 3: Poloxamer was excessive, and the viscosity was too high at room temperature, making it difficult to flow.
[0160] Comparative Example 5: The amount of PBS solution added was small, resulting in slightly less poloxamer, which could not gel quickly and affected the user experience.
[0161] Comparative Example 6: There is too little poloxamer. This concentration of poloxamer is difficult to gel even at 32°C and cannot be used as a product formulation.
[0162] In addition, in Comparative Example 8, the poloxamer was not stored at low temperatures and solidified as a thermosensitive gel, making it difficult to flow.
[0163] 3. Appearance
[0164] The proper concentration of PLGA-PEG / liposome nanoparticles is key to this skin serum's ability to maintain a uniform, opalescent sheen and resist stratification over extended periods. Mannitol, added during production, hydrogen bonds with water molecules, maintaining the colloidal stability of the nanoparticles; its absence can easily lead to Ostwald ripening.
[0165] Examples 1-3, Comparative Example 7, Comparative Example 9, Comparative Example 13: The nanoparticle ratio is balanced, the mannitol content meets the standard, and the dispersion is stable.
[0166] Mannitol and vitamin E exist in the product as a protective agent and antioxidant respectively, but excessive addition will not improve product performance.
[0167] Comparative Example 14: without mannitol, the nanoparticles aggregated and separated into layers due to lack of steric hindrance.
[0168] Comparative Example 8: Without cryopreservation, the PLGA-PEG / liposome nanoparticles in the essence were aggregated and precipitated.
[0169] 4. Stability (4°C, 30 days)
[0170] Low temperature inhibits the Brownian motion of nanoparticles, and mannitol reduces particle collisions; PLGA-PEG may hydrolyze when not exposed to low temperature.
[0171] 5. Moisturizing properties
[0172] Mannitol is a small molecule moisturizer, and the gel network formed by poloxamer also delays water evaporation.
[0173] Good moisturizing effect:
[0174] Example 1-3: Mannitol (5-8 g) absorbs water and moisturizes, while poloxamer forms a film to lock in moisture.
[0175] Poor moisturizing effect:
[0176] Comparative Example 3 (poloxamer concentration of 20.10%): The product itself provides insufficient moisture to the poloxamer film, and even causes the poloxamer to absorb moisture from the skin.
[0177] Comparative Example 4 (without mannitol): lack of protective agent;
[0178] Comparative Example 5 (PBS excess): active ingredient dilution.
[0179] 6. Antioxidant effect
[0180] The sulfhydryl group (-SH) of bee venom peptide and the phenolic hydroxyl group of vitamin E are both antioxidant active groups.
[0181] Best Antioxidant:
[0182] Example 2: Melittin (0.97%) directly scavenges free radicals (such as ·OH).
[0183] Worst antioxidant:
[0184] Comparative Example 8: Without low temperature storage, vitamin E was oxidized and lost its effectiveness, and the PLGA-PEG / liposome nanoparticles in the essence were aggregated and precipitated, causing drug leakage. The beneficial ingredient melittin also deteriorated and lost its antioxidant effect.
[0185] Comparative Example 10: There is no melittin. Although vitamin E is present, the content is too low, and other components such as snake venom peptides have no antioxidant effect. The antioxidant effect of the prepared product is extremely weak.
[0186] 7. Whitening effect
[0187] Snake venom-like peptides mimic snake venom tripeptides (such as acetyl hexapeptide-8), blocking nerve signal transduction and indirectly inhibiting melanocyte activation.
[0188] Best Whitening:
[0189] Example 2: High snake venom peptide (0.97%) inhibits tyrosinase activity and reduces melanin synthesis.
[0190] Worst whitening:
[0191] Comparative Example 9: There is no snake venom peptide, and other components such as melittin have no whitening effect, so the prepared product has no whitening effect.
[0192] 8. Anti-wrinkle / firming effect
[0193] Snake venom peptides are neurotransmitter antagonists, while bee venom peptides promote collagen synthesis.
[0194] The synergistic effect of the two can make the product have the characteristics of timely response and long-lasting effect. By comparing Example 1 and Example 3, it can be seen that the amount of snake venom peptide in Example 1 is less than half of that in Example 3, but the anti-wrinkle effect is not much worse.
[0195] In summary, the essence prepared in Example 1 exhibits balanced irritation, stability, and efficacy. Alternatively, a formula with a high proportion of snake venom peptides, such as in Example 3, can be used to create a whitening essence that focuses more on anti-aging and whitening effects; or a formula with a high proportion of melittin, such as in Example 2, can be used to create an antioxidant essence that focuses more on antioxidant and repair capabilities.
[0196] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A low-irritation bee venom-containing skin essence, characterized in that: The composition comprises the following raw materials in parts by weight: 3 g of PLGA-PEG / liposome nanoparticles; 19-23g of thermosensitive gel matrix, the thermosensitive gel matrix is poloxamer 407; 5-8g of protective agent, the protective agent is mannitol; 0.1-0.5g of antioxidant, the antioxidant is vitamin E; 20-25 ml of stabilizer, which is a phosphate buffered saline solution with a pH of 7.4 and a molar concentration of 0.01 mol / L, i.e., PBS solution; 80ml of ultrapure water; The preparation process of the PLGA-PEG / liposome nanoparticles comprises the following steps: S1. Preparation of hydrophobized melittin: Melittin lyophilized powder was dissolved in borate buffer at pH 8.5, and the same mass of palmitic acid N-hydroxysuccinimide ester as that of melittin lyophilized powder was added. The mixture was reacted at room temperature for 4 hours to completely dissolve the melittin lyophilized powder and palmitic acid N-hydroxysuccinimide ester. Free palmitic acid was removed by dialysis under MWCO of 1 kDa, and the hydrophobized melittin was obtained by lyophilization. S2, PLGA-PEG loaded with melittin: Dissolve 0.79-1 g of mPEG-PLGA in 25 ml of chloroform and vortex thoroughly to dissolve; Subsequently, 0.12-1.3 g of hydrophobized melittin was dissolved in 25 ml of a chloroform-methanol mixture with a volume ratio of 1:
1. The mPEG-PLGA solution and the hydrophobized melittin solution were mixed, and 250 ml of a 5% PVA solution was added. Ultrasonic emulsification was performed at a power of 100 W for 2 minutes to form a water-in-oil colostrum. Pour the colostrum into 400 ml of PVA solution and mechanically stir for about 12 hours to allow the organic solvent to fully evaporate and form solid nanoparticles; During this process, PVA wraps the surface of the nanoparticles to form a stable water-in-oil-in-water emulsion; The emulsion was centrifuged at 12,000 rpm to remove unencapsulated hydrophobized melittin and free PVA to obtain a precipitate; the precipitate was washed with distilled water, and the resulting washing solution was centrifuged at 3,000 rpm to remove large particle impurities, retaining the supernatant containing the suspended nanoparticles; The supernatant was mixed with an equal volume of a 2% PEG aqueous solution, lyophilized, and resuspended in 20 ml of PBS solution to obtain a PLGA-PEG nanoparticle solution; S3. Liposome-loaded snake venom peptides: Dissolve 0.79-1g of liposomes in 25ml of chloroform and dissolve in a 40°C water bath until clear. Rotate at 80-100rpm at 40°C and a vacuum of -0.09MPa to form a film. Place the film in a desiccator and evacuate at -0.1MPa for 2h to completely remove any chloroform residues, thereby obtaining a lipid film. Use 50 ml of 300 mmol / L ammonium sulfate solution to combine the lipid film with water molecules to form hydrates, and vortex for 30 min until the film is completely detached to obtain a crude liposome suspension; The liposome crude suspension was extruded through a polycarbonate membrane with a pore size of 100 nm using a microfluidic membrane extrusion process to obtain a blank liposome mixture, which was then dialyzed against PBS solution. After dialysis, the outer aqueous phase (NH4)2SO4 is removed, and the inner aqueous phase (NH4)2SO4 is retained, forming an ammonium sulfate gradient with high inside and low outside, which decomposes into escaped NH3 and H in the liposome. + , causing the pH of the inner cavity to drop to 4.0, while the pH of the outer aqueous phase is 7.4, forming a transmembrane proton gradient, adding 10 ml of PBS solution containing 0.12-1.3 g of snake venom peptide, at a temperature of 50-60 ° C, intermittently vortexing and incubating for 30 minutes, and cooling in an ice bath to obtain 20 ml of drug-loaded liposome solution; S4. PLGA-PEG / liposome hybrid assembly: The PLGA-PEG nanoparticle solution and the drug-loaded liposome solution were mixed at a volume ratio of 1:1, and the mixture of the PLGA-PEG nanoparticle solution and the drug-loaded liposome solution was extruded through a polycarbonate membrane with a pore size of 100 nm using a microfluidic membrane extrusion process to form a hybrid nanoparticle mixture; The hybrid nanoparticle mixture was ultrafiltrated and centrifuged under the condition of MWCO 10 kDa to remove free peptides, and the hybrid particles were collected and freeze-dried, namely PLGA-PEG / liposome nanoparticles.
2. The low-irritation bee venom-containing skin essence according to claim 1, characterized in that: The liposome is prepared from soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol with an average molecular weight of 2000, and cholesterol in a mass ratio of 70:25:
5.
3. The low-irritation bee venom-containing skin essence according to claim 1, characterized in that: The specific steps of the microfluidic membrane extrusion process of S3 are: The liposome crude suspension preheated to 60°C is injected into the automatic extruder LIPEX TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min; Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
4. The low-irritation bee venom-containing skin essence according to claim 1, characterized in that: The dialysis of S3 comprises the following specific steps: Use a cellulose dialysis bag with a MWCO of 10 kDa and place the blank liposome mixture into the bag, leaving about 20% of the volume free to prevent expansion and rupture. Seal both ends with dialysis clips to ensure no leakage. The dialysis bag was completely immersed in 4000 ml of PBS solution and stirred magnetically at 100 rpm. The whole process was carried out in an ice bath at 4°C to prevent liposome aggregation or drug leakage. After 1 hour, the PBS solution was replaced for the first time, and then the PBS solution was replaced every 4–6 hours, for a total of 3–4 times, for a total of 24 hours of dialysis; After dialysis is stopped, the dialysate is taken and the SO4 is detected with BaCl2 solution. 2- If there is no white precipitate, it indicates that the dialysis is complete.
5. The low-irritation bee venom-containing skin essence according to claim 1, characterized in that: The specific steps of the microfluidic membrane extrusion process of S4 are: The mixture of PLGA-PEG nanoparticle solution and drug-loaded liposome solution preheated to 60℃ was injected into the automatic extruder LIPEX TM Start the circulation pump of the extruder feed tank at a flow rate of 15 mL / min; Extrusion parameters were set as follows: extrusion pressure was 50–100 bar, monitored by online DLS until the particle size reached 1000 nm, and the outlet temperature was controlled at 60 °C.
6. The low-irritation bee venom-containing skin essence according to claim 1, characterized in that: The preparation process of the melittin freeze-dried powder comprises the following steps: The bee venom ultrafiltrate was passed through a preparative chromatographic column equipped with polystyrene microspheres Uni PS20-300, with mobile phase A being a 0.1% trifluoroacetic acid aqueous solution and mobile phase B being an acetonitrile solution containing 0.1% trifluoroacetic acid, and gradient elution was performed with a B phase concentration of 35% to 55%. At 0.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 15.00 min, the mobile phase A was 50% and the mobile phase B was 50%; at 15.01 min, the mobile phase A was 65% and the mobile phase B was 35%; at 20.00 min, the mobile phase A was 65% and the mobile phase B was 35%; by monitoring the chromatogram, the chromatographic peak eluate of melittin was identified and collected; ensuring that the collected eluate corresponded to the peak of melittin and had a purity of more than 99%, thereby obtaining a melittin eluate; The melittin eluate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and continuous constant-solubility ultrafiltration was performed to ensure continuous replenishment of the solvent, i.e., water, to maintain a substantially constant solute concentration on the membrane surface. When the melittin eluate was concentrated to approximately 150 mg / mL, the ultrafiltration was stopped, and the concentrated melittin eluate was collected and freeze-dried to obtain a melittin freeze-dried powder.
7. A method for preparing the low-irritation bee venom-containing skin essence according to any one of claims 1 to 6, comprising the following steps: 1) Prepare Poloxamer 407 solution: Disperse 19-23 g of poloxamer 407 in 80 ml of ultrapure water at 4°C and stir magnetically at 300 rpm until completely transparent, with an ice bath throughout the process, to obtain a poloxamer 407 solution. 2) Nanoparticle resuspension: The PLGA-PEG / liposome nanoparticles according to claim 1 are resuspended in 20-25 ml of a 4°C PBS solution containing 5-8 g of mannitol, and a poloxamer 407 solution is slowly added, followed by 0.1-0.5 g of vitamin E. The mixture is magnetically stirred at 4°C and 300 rpm for 10 minutes to allow the nanoparticles to be embedded in the poloxamer, thereby obtaining a thermosensitive skin essence stock solution with dual sustained-release capabilities. 3) Filling and storage: The skin essence stock solution was placed in a light-proof sterile bottle, filled with nitrogen for protection, stored in liquid form at 4°C, and returned to room temperature before use to obtain the skin essence product.
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CN121243027A