Preparation and application of microspheres fully loaded with palmitoyl blue copper peptide

Abstract

The invention discloses a preparation and application of microspheres fully loaded with palmitoyl blue copper peptide, belongs to the field of polypeptide preparations, and specifically relates to loaded palmitoyl blue copper peptide microspheres. The present invention adopts polyvinyl alcohol as a water-soluble polymer to make an aqueous phase solution, adopts a polycaprolactone-based polymer as an oil-soluble polymer to make an oil phase solution, and a sample solution is made of palmitoyl blue copper peptide and anhydrous ethanol, and then the loaded palmitoyl blue copper peptide microspheres are prepared by the aqueous phase solution, the oil phase solution and the sample solution; wherein the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, and the block polycaprolactone is obtained by reacting a polyhydroxy compound and anhydrous cyclohexanolactone, and the polyhydroxy compound is prepared by reacting 2,2-dihydroxypropionic acid and ethylene glycol bishydroxymethyl ether, and under the common use of a specific amount of polycaprolactone and block polycaprolactone, the loaded palmitoyl blue copper peptide microspheres prepared by the present invention have a better sustained release effect, and the effect of improving the content of melanin.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide preparations, and particularly relates to the preparation and application of microspheres fully loaded with palmitoyl blue copper peptide. Background Art

[0002] The amino acid sequence of the copper peptide is glycyl-L-histidyl-L-lysine, or GHK. This small molecule tripeptide can regulate the concentration of copper ions in cells. The tripeptide GHK in human plasma shows a strong affinity for Cu 2+ With strong binding force, they can form a complex copper peptide GHK-Cu. It has the effects of promoting wound healing, inhibiting inflammatory factors, promoting the growth of skin stratum corneum cells, promoting collagen synthesis, reducing light damage, and preventing hair loss. Copper peptide is a highly hydrophilic substance. According to the principle of similar dissolves in the skin, most hydrophilic substances are insoluble in phospholipids and cannot freely enter and exit the cell membrane. The skin's permeability and transport capacity for hydrophilic substances is limited, resulting in low bioavailability.

[0003] Studies have shown that the biological stability of GHK-Cu is very poor. When the ointment containing GHK-Cu is applied to wounds or damaged skin, only 0.05% to 0.15% is absorbed. Alternatively, when CHK-Cu is injected into the skin or to the edge of the wound, more than 95% will be quickly cleared within 1 minute. The half-life of GHK-Cu is about 20 minutes, and it will be rapidly degraded and then metabolized. Therefore, modifying the structure to improve the stability and permeability of the peptide has become the main direction of current research.

[0004] Efficacy of Palmitoyl Blue Copper Peptide: Zhejiang Paipeptide Biotech innovatively introduced the lipophilic group palmitoyl to structurally modify and transform the Lys side chain of GHK-Cu, and successfully synthesized a new copper peptide Pal-GHK-Cu with a lipophilic group.

[0005] Conventional blue copper peptides have the mechanism of regulating cell proliferation and migration, reducing inflammation, promoting angiogenesis, and promoting the synthesis and regulation of certain proteins. They are safe and have a low allergy rate, and have potential application prospects in the treatment of black hair. Blue copper peptides, as growth factors for various differentiated cells, have been shown to stimulate keratinocytes. Studies have shown that GHK-Cu can stimulate the elongation of hair follicles in vitro, increase the expression of Bcl-2 protein in hair papilla cells, reduce the expression of Bax, and promote cell proliferation. Researchers have confirmed that after human hair transplantation, the maintenance of GHK-Cu can accelerate hair growth. Hair papilla cells are specific fibroblasts and are essential for hair follicle growth. The access of palmitoyl groups protects short-chain amino acids and improves the lipophilicity and stability of GHK-Cu. Studies have verified that palmitoyl blue copper peptides have better bioavailability, stability and application safety than copper peptides. The dual characteristics of Pal-GHK indicate that it has broad application prospects in skin cell research and great potential in strengthening the extracellular matrix. In addition, palmitoyl-modified blue copper peptide not only has the ability to promote hair growth, but related studies have shown that it also has outstanding effects such as tanning and turning white hair black by activating the tyrosinase signaling pathway. It nourishes the scalp of middle-aged and elderly people and brings a compound effect of black hair growth. Currently, no ingredient has the above effects at the same time. However, the ideal active ingredient requires that it not only has specificity and pharmacological activity, but also can fully reach the target site to exert its effect. It is difficult for the single action of palmitoyl blue copper peptide to achieve the ideal effect.

[0006] Microspheres are skeleton-type tiny spherical entities formed by dissolving or dispersing active substances in polymer materials, and their particle size ranges from 1 to 250 μm. Using biodegradable polymers as delivery carriers for polypeptides and encapsulating polypeptides can increase the stability and targeting of drugs, reduce the toxic side effects of drugs on surrounding tissues and non-target tissue cells, etc. In addition, the targeting of microspheres can concentrate active ingredients in the target area, control the release rate, prolong the duration of action, and improve the efficacy. As a new type of carrier, microspheres have unique advantages and great development potential, and are one of the hot spots in the research of sustained-release preparations in recent years. The loading of palmitoyl blue copper peptide in microspheres can give medical devices and cosmetics more application scenarios.

[0007] Currently, many patents for the preparation of microspheres are mostly for loading hydrophilic active ingredients, such as water-soluble polypeptides, which require a W / O / W system, have a complicated preparation process, and have limited encapsulation efficiency. CN111836654A discloses a polycaprolactone microsphere filler containing vitamin C and a preparation method thereof, which specifically includes: (a) preparing a dispersed phase by dissolving polycaprolactone in a first solvent and vitamin C in a second solvent to prepare respective solutions, and then uniformly mixing the two solutions to prepare a single solution; (b) preparing an emulsion by mixing the dispersed phase with an aqueous solution (continuous phase) containing a surfactant; (c) forming microspheres by extracting an organic solvent from the dispersed phase in the emulsion prepared in step (b) into a continuous phase and evaporating the same; (d) recovering the microspheres from the continuous phase of step (c) to prepare polycaprolactone microspheres. This method is more complicated than the single emulsion method, especially during the production process, and the encapsulation rate is lower than that of the single emulsion method. In the late stage of the sustained release process, a burst release phenomenon will occur, which is not conducive to the function of the encapsulated components. CN118121719A discloses a drug sustained-release porous silica gel material and its preparation method and application. This method is the preparation of porous microspheres. First, microspheres are made with a placeholder, and then the placeholder is melted, and finally immersed in an aqueous solution to adsorb the target polypeptide. This method has a low loading rate and serious waste of precious drugs. In addition, due to the presence of porosity, the mutual adsorption is more of a van der Waals force, which is easy to release during the drug storage process, which poses a great challenge to storage. The porous loaded microspheres are likely to produce a burst release of drugs when they first enter the human body. Summary of the invention

[0008] The purpose of the present invention is to provide a preparation and application of microspheres fully loaded with palmitoyl blue copper peptide with good stability, good loading effect, good encapsulation effect and good sustained release effect.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0010] A method for preparing palmitoyl blue copper peptide-loaded microspheres, comprising:

[0011] The sample solution is mixed with the oil phase solution, and then the water phase solution is added to form an emulsion, which is vacuum sealed and finally separated to obtain the palmitoyl blue copper peptide loaded microspheres;

[0012] The sample solution contains palmitoyl blue copper peptide; the oil phase solution contains an oil-soluble polymer, the oil-soluble polymer includes at least one of a polycaprolactone-based polymer, a polylactic acid-based polymer, a polyhydroxyalkanoate-based polymer and a polyglycolide-lactide copolymer, and the polycaprolactone-based polymer includes polycaprolactone and / or block polycaprolactone;

[0013] The block polycaprolactone has a polyhydroxy structure formed by 2,2-dihydroxypropionic acid and ethylene glycol bishydroxymethyl ether, as well as a structure of polycaprolactone. The block polycaprolactone in the present invention has a branched structure, and the polycaprolactone is a linear structure. Within the optional range, the block polycaprolactone and the polycaprolactone are used as oil-soluble polymers, which can provide a coating site for palmitoyl blue copper peptide in the process of cross-entanglement with each other, and can better load the palmitoyl blue copper peptide, increase the stability of palmitoyl blue copper, and reduce the toxic and side effects of palmitoyl blue copper on surrounding tissues and non-target tissue cells. The targeting of the microspheres loaded with palmitoyl blue copper peptide can also concentrate the active ingredients in the target area, delay the release rate of palmitoyl blue copper peptide, prolong the action time, and improve the efficacy. Even if the release rate is slower, it is better concentrated in the target area under the coating of the block polycaprolactone and the polycaprolactone, and the effect of the loaded palmitoyl blue copper peptide microspheres is improved.

[0014] Preferably, the solvent of the sample solution is anhydrous ethanol; or, the solvent in the oil phase solution is DCM; or, the aqueous phase solution contains a water-soluble polymer and water.

[0015] More preferably, the content of palmitoyl blue copper peptide in the sample solution is 2-5000 ppm; or, the content of oil-soluble polymer in the oil phase solution is 1-25 wt %; or, the water-soluble polymer includes polyvinyl pyrrolidone or polyvinyl alcohol; or, the content of water-soluble polymer in the aqueous phase solution is 0.1-1.2 wt %.

[0016] Preferably, the volume ratio of the sample solution to the oil phase solution is 1:100-1:5; or, the volume ratio of the sample solution to the water phase solution is 1:1000-1:25.

[0017] Preferably, the oil-soluble polymer is a polycaprolactone-based polymer, and the polycaprolactone-based polymer includes polycaprolactone and block polycaprolactone.

[0018] More preferably, the usage ratio of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.5-3.

[0019] Preferably, in the process of mixing to form the emulsion, a homogenization process is used to form the emulsion; and, in the vacuum sealing process, a water pump is used to evacuate the mixture for 2-6 hours.

[0020] Preferably, in the separation process, the product of the vacuum sealing process is filtered, centrifuged, and freeze-dried.

[0021] Preferably, the preparation of microspheres loaded with palmitoyl blue copper peptide may also include the preparation of polyhydroxy compounds and the preparation of block polycaprolactone.

[0022] More preferably, in the preparation of the polyhydroxy compound, 2,2-dihydroxypropionic acid and ethylene glycol bis(hydroxymethyl) ether are mixed, a catalyst, a water-carrying agent and a polymerization inhibitor are added, and the mixture is reacted at a temperature of 100-130° C. for 6-24 hours. After the reaction is completed, the mixture is adjusted to neutral, washed with a saturated sodium chloride solution, and distilled under reduced pressure to obtain the polyhydroxy compound.

[0023] More preferably, in the preparation of the polyhydroxy compound, the amount of ethylene glycol bis(hydroxymethyl) ether used is 40-60 wt % of 2,2-dihydroxypropionic acid.

[0024] More preferably, in the preparation of the polyhydroxy compound, the catalyst is cerium sulfate, and the amount of the catalyst used is 0.5-3wt% of 2,2-dihydroxypropionic acid.

[0025] More preferably, in the preparation of the polyhydroxy compound, the water-carrying agent is cyclohexane, and the amount of the water-carrying agent used is 80-120 wt % of the 2,2-dihydroxypropionic acid.

[0026] More preferably, in the preparation of the polyhydroxy compound, the polymerization inhibitor is hydroquinone, and the amount of the polymerization inhibitor used is 1-3 wt % of 2,2-dihydroxypropionic acid.

[0027] More preferably, in the preparation of the blocked polycaprolactone, the polyhydroxy compound and the anhydrous cyclohexyl lactone are mixed, a catalyst is added, and the mixture is reacted at 100-130° C. for 10-48 hours under a nitrogen atmosphere. After the reaction is completed, the mixture is cooled, dichloromethane is added, and then glacial ether is added. After the precipitate is precipitated, it is filtered and dried to obtain the blocked polycaprolactone.

[0028] More preferably, in the preparation of the block polycaprolactone, the amount of the polyhydroxy compound used is 10-30 wt % of the anhydrous cyclohexanolactone.

[0029] More preferably, in the preparation of the block polycaprolactone, the catalyst is stannous octoate, and the amount of the catalyst used is 0.2-2 wt % of the anhydrous cyclohexanolactone.

[0030] More preferably, in the preparation of the block polycaprolactone, dichloromethane is used to dissolve the crude product, and glacial ether is used to precipitate. Dichloromethane and glacial ether can be used in appropriate amounts.

[0031] Preferably, in the preparation of the aqueous phase solution, the water-soluble polymer is added into water, dissolved and cooled at 80-90° C. to obtain the aqueous phase solution.

[0032] More preferably, in the preparation of the aqueous phase solution, the water-soluble polymer includes polyvinyl pyrrolidone or polyvinyl alcohol, and the content of the water-soluble polymer in the aqueous phase solution is 0.1-1.2 wt %.

[0033] Preferably, in the preparation of the oil phase solution, the oil-soluble polymer is added to the oil phase solvent and shaken to dissolve to obtain the oil phase solution.

[0034] More preferably, in the preparation of the oil phase solution, the oil-soluble polymer includes at least one of a polycaprolactone-based polymer, a polylactic acid-based polymer, a polyhydroxyalkanoate-based polymer and a polyglycolide-lactide copolymer.

[0035] More preferably, in the preparation of the oil phase solution, the oil phase solvent includes at least one of DCM, DMF and DMSO.

[0036] More preferably, in the preparation of the oil phase solution, the content of the oil-soluble polymer in the oil phase solution is 1-25 wt %.

[0037] More preferably, in the preparation of the oil phase solution, the polycaprolactone-based polymer includes polycaprolactone and / or block polycaprolactone.

[0038] More preferably, in the preparation of the oil phase solution, the usage ratio of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.5-3.

[0039] Preferably, in the preparation of the sample solution, the sample is added into a sample solvent and shaken to dissolve to obtain a sample solution.

[0040] More preferably, in the preparation of the sample solution, the sample includes palmitoyl blue copper peptide, the sample solvent includes anhydrous ethanol, and the content of the sample in the sample solution is 2-5000 ppm.

[0041] Preferably, in the preparation of palmitoyl blue copper peptide loaded microspheres, the sample solution is mixed with the oil phase solution, and then the aqueous phase solution is added, homogenized to form a blue copper peptide emulsion, which is sealed and vacuumed for 2-6 hours, filtered, washed, and freeze-dried to obtain palmitoyl blue copper peptide loaded microspheres.

[0042] More preferably, in the preparation of palmitoyl blue copper peptide loaded microspheres, the volume ratio of the sample solution to the oil phase solution is 1:100-1:5.

[0043] More preferably, in the preparation of palmitoyl blue copper peptide loaded microspheres, the volume ratio of the sample solution to the aqueous phase solution is 1:1000-1:25.

[0044] Preferably, in the present invention, sucrose polyoxypropylene ether can also be added in the preparation of the aqueous phase solution, and the amount of sucrose polyoxypropylene ether used is 0.5-4wt% of the water-soluble polymer. In the present invention, polyvinyl alcohol and sucrose polyoxypropylene ether can be used together in the aqueous phase solution, and polyvinyl alcohol and sucrose polyoxypropylene ether can make the palmitoyl blue copper peptide microspheres more reasonably coated in the microsphere sites formed by polycaprolactone and block polycaprolactone, thereby slowing down the release rate of palmitoyl blue copper peptide in the obtained palmitoyl blue copper peptide-loaded microspheres, but making the palmitoyl blue copper peptide better concentrated in the target area, thereby improving the effect of loading the palmitoyl blue copper peptide microspheres.

[0045] The invention discloses palmitoyl blue copper peptide loaded microspheres prepared by the preparation method.

[0046] The invention discloses use of the palmitoyl blue copper peptide loaded microspheres in preparing shampoo products and / or hair care products and / or scalp injection nourishing products and / or hair growth and anti-hair loss products and / or tanning products.

[0047] The present invention adopts polyvinyl alcohol as a water-soluble polymer to prepare an aqueous phase solution, adopts a polycaprolactone-based polymer as an oil-soluble polymer to prepare an oil phase solution, and prepares a sample solution from palmitoyl blue copper peptide and anhydrous ethanol, and then prepares the loaded palmitoyl blue copper peptide microspheres from the aqueous phase solution, the oil phase solution and the sample solution; wherein the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, the block polycaprolactone is obtained by reacting a polyhydroxy compound and anhydrous cyclohexanolactone, and the polyhydroxy compound is prepared by reacting 2,2-dihydroxypropionic acid and ethylene glycol bishydroxymethyl ether, and thus has the following beneficial effects: under the common use of a specific amount of polycaprolactone and block polycaprolactone, the loaded palmitoyl blue copper peptide microspheres prepared by the present invention have a better sustained release effect, and the effect of increasing the content of melanin. Therefore, the present invention is a preparation and application of microspheres fully loaded with palmitoyl blue copper peptide with good stability, good loading effect, good encapsulation effect, and good sustained release effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is an electron micrograph of microspheres loaded with palmitoyl blue copper peptide.

[0049] Figure 2 This is the relative gene expression diagram of dermal papilla cells.

[0050] Figure 3 The relative gene expression of A375 cells.

[0051] Figure 4 This is a graph of melanin content.

[0052] Figure 5 The release rate diagram. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0055] Example 1: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0056] Preparation of aqueous phase solution: adding a water-soluble polymer into water, dissolving and cooling at 85° C. to obtain an aqueous phase solution. The water-soluble polymer includes polyvinyl alcohol, and the content of the water-soluble polymer in the aqueous phase solution is 0.6 wt %.

[0057] Preparation of oil phase solution: adding an oil-soluble polymer to an oil phase solvent, shaking and dissolving, and obtaining an oil phase solution. The oil-soluble polymer is a polycaprolactone-based polymer, and the polycaprolactone-based polymer is polycaprolactone. The oil phase solvent is DCM. The content of the oil-soluble polymer in the oil phase solution is 15wt%.

[0058] Preparation of sample solution: Add the sample into the sample solvent, shake and dissolve to obtain the sample solution. The sample is palmitoyl blue copper peptide, the sample solvent is anhydrous ethanol, and the sample content in the sample solution is 1000ppm.

[0059] Preparation of palmitoyl blue copper peptide loaded microspheres: The sample solution was mixed with the oil phase solution, and then the aqueous phase solution was added, homogenized to form a blue copper peptide emulsion, sealed and vacuum treated for 4 hours, filtered, washed, and freeze-dried to obtain palmitoyl blue copper peptide loaded microspheres. The volume ratio of the sample solution to the oil phase solution was 1:10, and the volume ratio of the sample solution to the aqueous phase solution was 1:100.

[0060] Example 2: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0061] Compared with Example 1, this embodiment is different in the preparation of the oil phase solution. In the preparation of the oil phase solution, the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, and the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.8.

[0062] Preparation of polyhydroxy compounds: 2,2-dihydroxypropionic acid and ethylene glycol bis(hydroxymethyl)ether are mixed, a catalyst, a water-carrying agent and a polymerization inhibitor are added, and the mixture is reacted at a temperature of 120°C for 12 hours. After the reaction is completed, the mixture is adjusted to neutral, washed with a saturated sodium chloride solution, and distilled under reduced pressure to obtain a polyhydroxy compound. The amount of ethylene glycol bis(hydroxymethyl)ether used is 50wt% of 2,2-dihydroxypropionic acid, the catalyst is cerium sulfate, the amount of the catalyst used is 1.5wt% of 2,2-dihydroxypropionic acid, the water-carrying agent is cyclohexane, the amount of the water-carrying agent used is 100wt% of 2,2-dihydroxypropionic acid, and the polymerization inhibitor is hydroquinone, the amount of the polymerization inhibitor used is 2wt% of 2,2-dihydroxypropionic acid.

[0063] Preparation of block polycaprolactone: Mix polyhydroxy compounds and anhydrous cyclohexanolactone, add a catalyst, react at 120°C for 24 hours under a nitrogen atmosphere, cool after the reaction is completed, add dichloromethane, then add icy ether, filter after precipitation, and dry to obtain block polycaprolactone. The amount of polyhydroxy compound used is 20wt% of anhydrous cyclohexanolactone, the catalyst is stannous octoate, and the amount of catalyst used is 1.2wt% of anhydrous cyclohexanolactone. Dichloromethane is used to dissolve the crude product, and icy ether is used to precipitate. Dichloromethane and icy ether can be used in appropriate amounts.

[0064] Example 3: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0065] Compared with Example 2, this embodiment is different in the preparation of the oil phase solution. In the preparation of the oil phase solution, the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, and the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:2.4.

[0066] Example 4: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0067] The present embodiment is different from the embodiment 3 in that the aqueous phase solution is prepared, sucrose polyoxypropylene ether is further added to the aqueous phase solution, and the amount of sucrose polyoxypropylene ether used is 0.8 wt % of the water-soluble polymer.

[0068] Example 5: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0069] The present embodiment is different from the embodiment 4 in that the aqueous phase solution is prepared, sucrose polyoxypropylene ether is further added to the aqueous phase solution, and the amount of sucrose polyoxypropylene ether used is 3.2 wt % of the water-soluble polymer.

[0070] Comparative Example 1: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0071] The present embodiment is different from the embodiment 1 in the preparation of the oil phase solution. In the preparation of the oil phase solution, the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is a block polycaprolactone, and the synthesis of the block polycaprolactone is the same as that of the embodiment 2.

[0072] Comparative Example 2: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0073] Compared with Example 2, this embodiment is different in the preparation of the oil phase solution. In the preparation of the oil phase solution, the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, and the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.3.

[0074] A specific ratio has a good effect

[0075] Comparative Example 3: A method for preparing microspheres loaded with palmitoyl blue copper peptide

[0076] Compared with Example 2, this embodiment is different in the preparation of the oil phase solution. In the preparation of the oil phase solution, the oil-soluble polymer is a polycaprolactone-based polymer, the polycaprolactone-based polymer is polycaprolactone and block polycaprolactone, and the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:3.5.

[0077] Test example:

[0078] The present invention conducted electron microscopic characterization on the palmitoyl blue copper peptide loaded microspheres prepared in Example 1, and the results were as follows: Figure 1 As shown, the particle size of the palmitoyl blue copper peptide loaded microspheres prepared by the method of Example 1 of the present invention is between 10-50 μm.

[0079] 1. Regulation test of anti-hair loss and hair strengthening genes

[0080] The Luminex200 suspension chip multiplex detection system technology combined with the QGP method was used to analyze the effects of palmitoyl blue copper peptide microspheres on the expression of genes related to hair loss prevention and hair fixation in dermal papilla cells.

[0081] In this experiment, dermal papilla cells were used, and 6 × 10 4 The cells were plated in a 24-well plate at a density of 100 cells / well, with a volume of 0.5 mL per well. The cell well plate was cultured in an incubator for 24 h. The experiment was divided into a negative control group and a sample group, each group containing 3 replicate wells, and the sample group was the palmitoyl blue copper peptide loaded microspheres prepared in Example 1.

[0082] The present invention detected the effect of the palmitoyl blue copper peptide loaded microspheres prepared in Example 1 on the expression of 25 genes in hair papilla cells, and the two genes were FGF7, VEGFA, LPAR6, EDAR, IGF1, ITGA6, KRT75, FOXL2, VDR, SOX21, WNT4, BMP3, COL17A1, TGM3, TGFB1, DKK1, IL6, AR, TRPV3, SRD5A2, TNF, ESR1, ACE2, TBP, HPRT1, among which, it promoted the expression of genes such as FGF7, IGF1, KRT75, VDR, COL17A1, and could downregulate the relative expression of DKK1 and IL6 genes. The expression results of some genes are as follows: Figure 2 As shown, NC is the negative control group, and the sample is the palmitoyl blue copper peptide loaded microspheres prepared in Example 1. Compared with the negative control, the sample group can significantly upregulate the relative expression of FGF7, IGF1, FOXL2, WNT4, and BMP3 genes (p<0.01), and can significantly downregulate the relative expression of DKK1 and IL6 genes (p<0.01). The results show that the palmitoyl blue copper peptide loaded microspheres prepared by the present invention have a regulatory effect on some anti-hair loss and hair solidification genes.

[0083] According to current research, FGF7 is a member of the fibroblast growth factor (FGF) family. FGF family members are involved in a variety of biological processes such as embryonic development, cell growth, morphogenesis and tissue repair. FGF7 plays an important role in wound re-epithelialization, hair development and early lung organ formation. Dickkopf-related protein (DKKI) is an important antagonist of the Wnt signaling pathway, which plays an important regulatory role in the development and regeneration of hair follicles. Continuous overexpression of DKK1 during embryonic development will not generate hair follicles, and overexpression of DKK1 after birth will inhibit the growth of hair follicles. In addition, interleukins (ILs) include IL-1a, IL-1B, IL-6, etc., which affect the extracellular matrix and intracellular cAMP levels of dermal papilla cells to regulate hair follicle growth. ILs stimulation can cause malnutrition of hair follicles during the growth period, such as deformation and contraction of the hair papilla, abnormal vacuoles of hair matrix cells and abnormal keratinization of the hair bulb, inhibiting the growth of hair follicles and hair.

[0084] 2. Melanogenesis gene test

[0085] The present invention adopts the QGP method of the Luminex200 suspension chip multiplex detection system to analyze the effect of the palmitoyl blue copper peptide-loaded microspheres prepared in Example 1 on gene expression in melanoma cells A375.

[0086] The present invention uses A375 cells, with 6×10 4The cells were plated in a 24-well plate at a density of 100 cells / well, with a volume of 0.5 mL per well. The cell well plate was cultured in an incubator for 24 h. The experiment was divided into a negative control group and a sample group, each group containing 3 replicate wells, and the sample group was the palmitoyl blue copper peptide loaded microspheres prepared in Example 1.

[0087] This study detected the expression of 36 genes, including PTGS2, PTGER1, CREB1, ASIP, EDN3, COMMD3, MET, MCIR, HGF, DCT, F2RL1, OCA2, IL1RN, KIT, NQO1, HPRT1, TP53, CNR1, LEF1, MITF, RAB27A, PMEL, TNF, SLC45A2, IL1A, KLF6, TBP, CPX3, DSG1, LICAM, TYR, POMC, SOX10, TYRP1, TGFB1, and CDSN. The palmitoyl blue copper peptide microspheres prepared in Example 1 had a regulatory effect on TYP, KIT, POMC, PMEL, OCA2, PTGS2, IL1A and other related genes in melanoma cells A375. The expression results of some genes are shown in Figure 1. Figure 3 As shown, NC is a negative control group, and the sample is the palmitoyl blue copper peptide loaded microspheres prepared in Example 1. Compared with the negative control, the palmitoyl blue copper peptide loaded microspheres prepared in Example 1 can significantly increase the relative expression of DCT and POMC genes (P<0.01); the relative expression of EDN3 and TYR genes (P<0.05), and can significantly reduce the relative expression of IL1A gene (P<0.01). The results show that the palmitoyl blue copper peptide loaded microspheres prepared by the present invention can regulate the expression of different related genes in melanoma cells A375.

[0088] In summary, the palmitoyl blue copper peptide loaded microspheres prepared by the present invention can change the gene expression in melanoma cells A375, and have promoting and inhibiting effects on melanin synthesis related genes such as DCT, tyrosinase activity related genes such as TYP and other related genes.

[0089] 3. Melanin content test

[0090] The present invention explores the effect of palmitoyl blue copper peptide-loaded microspheres on the intracellular melanin content through human melanoma cells A375.

[0091] This experiment was divided into a negative control group, a positive control group and a sample group, wherein the negative control was a DMEM complete medium without any treatment, the positive control group was a DMEM complete medium containing 1 mg / mL kojic acid, and the sample group was a DMEM complete medium containing palmitoyl blue copper peptide microspheres loaded with the prepared in Example 1-5 or Comparative Example 1-3.

[0092] After the palmitoyl blue copper peptide-loaded microspheres are applied to human melanoma cells A375, the melanin content in the human melanoma cells A375 is as follows: Figure 4 As shown, NC is a negative control group, PC is a positive control group, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. Compared with the NC group, the human melanoma cells A375 treated with palmitoyl blue copper peptide microspheres loaded in Example 1 have a significantly increased melanin content in the human melanoma cells A375, indicating that the palmitoyl blue copper peptide microspheres loaded in Example 1 of the present invention have the ability to promote melanin production, while the melanin content in the human melanoma cells A375 in the PC group is greatly reduced; compared with Example 1, Examples 2-3 show that in When polycaprolactone and block polycaprolactone are used together as oil-soluble polymers, the prepared palmitoyl blue copper peptide loaded microspheres have a higher effect of increasing the melanin content, and in the use of polycaprolactone and block polycaprolactone, the increase in the proportion of block polycaprolactone can also increase the melanin content in human melanoma cells A375; compared with comparative examples 1-3, Example 1-3 shows that the joint use of polycaprolactone and block polycaprolactone is better than the single use of polycaprolactone or block polycaprolactone, and the usage of polycaprolactone and block polycaprolactone meets a specific proportional relationship, which has the effect of increasing the melanin content in human melanoma cells A375. Compared with Example 3, Example 4-5 shows that when preparing palmitoyl blue copper peptide loaded microspheres, when polyvinyl alcohol is present in the aqueous phase solution, sucrose polyoxypropylene ether can also be added. When polyvinyl alcohol and sucrose polyoxypropylene ether are used together, the palmitoyl blue copper peptide loaded microspheres are prepared together with the oil phase solution and the sample solution. The content of melanin in human melanoma cells A375 can be further increased, indicating that the joint use of polyvinyl alcohol and sucrose polyoxypropylene ether can also improve the performance of palmitoyl blue copper peptide microspheres.

[0093] 4. Release effect test of palmitoyl blue copper peptide

[0094] The test sample in this experiment is the palmitoyl blue copper peptide loaded microspheres prepared in Example 1-5 or Comparative Example 1-4. 0.2 g of the test sample is accurately weighed and placed in a 15 mL centrifuge tube containing 9.8 g of PSB solution. After sealing, it is placed in a shaking incubator at 37 ° C and constant temperature shaking at 150 r / min. 5 mL of the sample was centrifuged for 200 h, and the polypeptide content therein was detected by high performance liquid chromatography. The release rate of the palmitoyl blue copper peptide loaded microspheres was calculated.

[0095] The test results of the present invention on the release of palmitoyl blue copper peptide are as follows Figure 5As shown, wherein S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. Compared with Example 1, Examples 2-3 show that when polycaprolactone and block polycaprolactone are used together as oil-soluble polymers, the prepared palmitoyl blue copper peptide loaded microspheres have a higher sustained release effect, and in the use of polycaprolactone and block polycaprolactone, the increase in the proportion of block polycaprolactone can also improve the sustained release effect of the palmitoyl blue copper peptide loaded microspheres; Compared with Comparative Examples 1-3, Examples 1-3 show that the joint use of polycaprolactone and block polycaprolactone is better than the separate use of polycaprolactone or block polycaprolactone, and the amount of polycaprolactone and block polycaprolactone used satisfies a specific proportional relationship, which has the effect of improving the sustained release effect of the palmitoyl blue copper peptide loaded microspheres. Compared with Example 3, Example 4-5 shows that when preparing palmitoyl blue copper peptide-loaded microspheres, when polyvinyl alcohol is present in the aqueous phase solution, sucrose polyoxypropylene ether can also be added. When polyvinyl alcohol and sucrose polyoxypropylene ether are used together, the palmitoyl blue copper peptide-loaded microspheres are prepared together with the oil phase solution and the sample solution. The sustained release effect of the palmitoyl blue copper peptide-loaded microspheres can be further improved, indicating that the joint use of polyvinyl alcohol and sucrose polyoxypropylene ether can also improve the sustained release performance of palmitoyl blue copper peptide microspheres.

[0096] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0097] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing palmitoyl blue copper peptide loaded microspheres, comprising: The sample solution is mixed with the oil phase solution, and then the aqueous phase solution is added to form an emulsion, vacuum-sealed, and finally separated to obtain microspheres loaded with palmitoyl blue copper peptide; the content of palmitoyl blue copper peptide in the sample solution is 2-5000 ppm; The sample solution contains palmitoyl blue copper peptide; the oil phase solution contains an oil-soluble polymer, the oil-soluble polymer is a polycaprolactone-based polymer, and the polycaprolactone-based polymer includes polycaprolactone and block polycaprolactone; the usage amount of polycaprolactone and block polycaprolactone in the polycaprolactone-based polymer is 1:0.5-3; The block polycaprolactone has a polyhydroxy structure formed by 2,2-dihydroxypropionic acid and ethylene glycol bis(hydroxymethyl)ether, and a polycaprolactone structure; The aqueous phase solution contains a water-soluble polymer and water, and the water-soluble polymer includes polyvinyl pyrrolidone or polyvinyl alcohol.

2. The method for preparing palmitoyl blue copper peptide-loaded microspheres according to claim 1, characterized in that: The solvent of the sample solution is anhydrous ethanol; or, the solvent in the oil phase solution is DCM.

3. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 2, characterized in that: The content of the oil-soluble polymer in the oil phase solution is 1-25 wt %; or, the content of the water-soluble polymer in the water phase solution is 0.1-1.2 wt %.

4. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that: The volume ratio of the sample solution to the oil phase solution is 1:100-1:5; or, the volume ratio of the sample solution to the water phase solution is 1:1000-1:

25.

5. The method for preparing palmitoyl blue copper peptide loaded microspheres according to claim 1, characterized in that: In the mixing to form the emulsion, a homogenization treatment is used to form the emulsion; or, the vacuum sealing treatment is performed by vacuuming with a water pump; or, the vacuum sealing treatment is performed for 2-6 hours.

6. The method for preparing palmitoyl blue copper peptide-loaded microspheres according to claim 1, characterized in that: In the separation process, the vacuum-sealed product is filtered, centrifuged, and freeze-dried.

7. Palmitoyl blue copper peptide loaded microspheres prepared by any one of the preparation methods of claims 1-6.

8. Use of the palmitoyl blue copper peptide loaded microspheres according to claim 7 in the preparation of shampoo products and / or hair care products and / or scalp injection nourishing products and / or hair growth and anti-hair loss products and / or tanning products.

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