An amphiphilic block copolymer nanomicelle loaded with PDRN, and a preparation method and application thereof

Abstract

The application provides a kind of amphiphilic block copolymer nanomicelles loaded with PDRN and its preparation method and application, belong to the medical cosmetic technical field.The preparation method of the amphiphilic block copolymer nanomicelles loaded with PDRN includes the following steps: S1 provides the buffer solution containing amphiphilic block copolymer nanomicelles and PDRN simultaneously;S2 the buffer solution is sheared at 0~25 ℃ with 8000~15000 rpm, then mixed with 100~500 rpm, the preparation method can effectively improve the problem that the combination rate of amphiphilic block copolymer and PDRN is low, at the same time, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by the method also has the advantages of drug sustained release function and strong membrane permeability, so that PDRN can continuously and efficiently play a role.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical cosmetology, in particular to an amphiphilic block copolymer nanomicelle loaded with PDRN, a preparation method and application thereof. BACKGROUND

[0002] Polydeoxyribonucleotide (PDRN for short) is a bioactive substance extracted from salmon sperm DNA, and its biological functions include but are not limited to stimulating fibroblast proliferation, promoting collagen and hyaluronic acid synthesis, anti-inflammatory and pro-angiogenic effects, etc. Many functions make it widely used in the field of medical cosmetology. However, the main component of PDRN is polydeoxyribonucleotide, which is easily degraded by nucleases in vivo, resulting in a short action time. Moreover, due to its large molecular weight, it has poor transmembrane absorption, which further leads to its difficulty in effectively playing a role.

[0003] At the same time, the construction of a drug delivery system based on amphiphilic block copolymer nanomicelles is one of the current research hotspots. The basic function of this technical means is to effectively prolong the degradation period of the drug and enhance its transmembrane absorption capacity. However, in the process of constructing an amphiphilic block copolymer nanomicelle delivery system loaded with PDRN, the technical personnel found that there was a problem of low combination rate of the amphiphilic block copolymer nanomicelle and PDRN, which further led to the limitation of the promotion and application of PDRN in the field of medical cosmetology. SUMMARY

[0004] The purpose of the present application is to provide an amphiphilic block copolymer nanomicelle loaded with PDRN, a preparation method and application thereof. The preparation method can effectively improve the problem of low combination rate of the amphiphilic block copolymer nanomicelle and PDRN, and the amphiphilic block copolymer nanomicelle loaded with PDRN prepared by the method also has the advantages of drug sustained release function and strong transmembrane capacity, so that PDRN can continuously and efficiently play a role.

[0005] The embodiments of the present application are implemented as follows:

[0006] In a first aspect, the embodiments of the present application provide a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, comprising the following steps: S1, providing a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN, wherein the molecular weight of the hydrophilic polymer in the amphiphilic block copolymer nanomicelles is 2000-5000 Da, the molecular weight of the hydrophobic polymer is 5000-20000 Da, the molecular weight of the PDRN is not more than 1000 KDa, and the mass ratio of the amphiphilic block copolymer nanomicelles to the PDRN is 100:(0.1-2); S2, shearing the buffer solution at 8000-15000 rpm at 0-25°C and then mixing it at 100-500 rpm to obtain the amphiphilic block copolymer nanomicelles loaded with PDRN.

[0007] In the above technical solution, on the one hand, the shearing treatment at a high speed and then the mixing treatment at a low speed under the above temperature condition can effectively maintain the structural stability and physiological activity of the PDRN. The shearing treatment at a high speed can cause the partial structure of at least part of the nanomicelles to be damaged, thereby helping the PDRN to enter the inside of the nanomicelles and to be stably and firmly combined with the nanomicelles through the mutual entanglement of chains and van der Waals force. Then, the mixing treatment at a low speed can cause the nanomicelles with damaged structures to be reassembled into nanomicelles with complete structures. At the same time, the surface of the nanomicelles can also be loaded with PDRN, so that PDRN is combined inside and outside the nanomicelles, thereby improving the combination rate of the nanomicelles and the PDRN. In addition, the molecular weight of the hydrophilic polymer and the hydrophobic polymer in the amphiphilic block copolymer nanomicelles and the molecular weight of the PDRN are respectively limited in the above ranges, and the mass ratio of the amphiphilic block copolymer nanomicelles to the PDRN is limited in the above range, which can also effectively improve the combination rate of the amphiphilic block copolymer nanomicelles and the PDRN. In a second aspect, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by the method also have the advantages of drug release function and strong membrane permeability, so that the PDRN can be effectively enriched in the action area and continuously and efficiently play a role.

[0008] In some optional embodiments, step S2 comprises: S21, adding ice to the buffer solution to obtain an ice-water mixed solution; S22, shearing the ice-water mixed solution at 8000-15000 rpm, wherein the shearing time is 5-20 min; and S23, then stirring the ice-water mixed solution at 100-500 rpm, wherein the stirring time is 2-3 h.

[0009] In the technical solution, the temperature of the buffer solution can be conveniently and stably controlled in the range of 0-25 DEG C by adding ice to the buffer solution; in addition, the shearing treatment and mixing treatment are limited in the above range, so that more PDRN is combined in and outside the nanomicelles, thereby further improving the combination rate of the nanomicelles and the PDRN.

[0010] In some optional embodiments, the molecular weight of the hydrophilic polymer is 3000-4000 Da, the molecular weight of the hydrophobic polymer is 10000-15000 Da, and the molecular weight of the PDRN is 200-500 KDa.

[0011] In the technical solution, the molecular weight of the hydrophilic polymer, the molecular weight of the hydrophobic polymer, and the molecular weight of the PDRN are limited in the above range, which further improves the combination rate of the amphiphilic block copolymer nanomicelles and the PDRN.

[0012] In some optional embodiments, the molecular weight of the hydrophilic polymer is 3000 Da, and the molecular weight of the hydrophobic polymer is 10000-15000 Da.

[0013] In the technical solution, the molecular weight of the hydrophilic polymer and the molecular weight of the hydrophobic polymer are limited in the above range, which more effectively improves the combination rate of the amphiphilic block copolymer nanomicelles and the PDRN.

[0014] In some optional embodiments, in the buffer solution, the mass-volume ratio of the amphiphilic block copolymer nanomicelles to the buffer solution is 1 g:(3-6) mL; or / and, the buffer solution is selected from a phosphate buffer solution with a pH of 6.5-7.

[0015] In the technical solution, the mass-volume ratio of the amphiphilic block copolymer nanomicelles to the buffer solution is limited in the above range, which enables the amphiphilic block copolymer nanomicelles and the PDRN to be quickly dissolved and uniformly dispersed in the buffer solution, and also enables the buffer solution to have a relatively appropriate concentration of the nanomicelles and the PDRN, thereby further improving the combination rate of the two; in addition, the use of the phosphate buffer solution with a pH of 6.5-7 can effectively dissolve and disperse the nanomicelles and the PDRN while maintaining the structural stability and activity of the PDRN.

[0016] In some optional embodiments, the amphiphilic block copolymer nanomicelles are two-block amphiphilic block copolymer nanomicelles, wherein the material of the hydrophilic polymer is selected from at least one of methoxypolyethylene glycol, monoalkyloxypolyethylene glycol, and monoacylpolyethylene glycol, and the material of the hydrophobic polymer is selected from polycaprolactone.

[0017] In the technical solution, the hydrophilic polymer and the hydrophobic polymer can be combined in many ways, which can provide more implementation manners, thereby facilitating the popularization and application of the technical solution provided by the embodiments of the application, and the material has the advantage of good biocompatibility.

[0018] In some optional embodiments, the material of the hydrophilic polymer is selected from methoxypolyethylene glycol, and the material of the hydrophobic polymer is selected from polycaprolactone.

[0019] In the technical solution, the amphiphilic block copolymer nanomicelles formed by combining the two polymer materials are used as the PDRN carrier, and the carrier also has the effect of promoting collagen regeneration, which can act together with the PDRN, thereby achieving better curative effect.

[0020] In some optional embodiments, the step S1 comprises:

[0021] S11, the amphiphilic block copolymer is dissolved in the first part of the buffer solution and subjected to shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly to obtain a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles; S12, the PDRN is dissolved in the second part of the buffer solution to obtain a PDRN solution; and S13, the polymer nanomicelle solution and the PDRN solution are mixed.

[0022] In the technical solution, the polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles and the PDRN solution are prepared separately, and then mixed, that is, the mixing is performed after the two are effectively dissolved and uniformly dispersed, which helps to improve the combination rate of the two; at the same time, in order to make the amphiphilic block copolymer quickly form nanomicelles through self-assembly, the self-assembly process is usually performed under heating conditions, and the step-by-step dissolution and mixing method can effectively protect the structural integrity and activity of the PDRN.

[0023] In some optional embodiments, the step S11 comprises: the amphiphilic block copolymer is added to the first part of the buffer solution at 65-80°C and subjected to shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly to obtain a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles.

[0024] In the technical solution, the shearing treatment is performed at a suitable temperature, which can provide a suitable driving force for self-assembly, thereby facilitating the amphiphilic block copolymer to form nanomicelles more efficiently and completely.

[0025] In some optional embodiments, the mass-to-volume ratio of the amphiphilic block copolymer to the first part of the buffer solution is 1 g: (3.5-4.5) mL.

[0026] In the technical solution, the mass / volume ratio of the amphiphilic block copolymer and the first part of the buffer is limited in the range, which helps the amphiphilic block copolymer to be quickly dissolved and uniformly dispersed, and also helps the amphiphilic block copolymer to form nanomicelles through self-assembly.

[0027] In some optional embodiments, the amphiphilic block copolymer is added to the first part of the buffer at 65-80°C for shearing treatment, the stirring speed is 8000-15000 rpm, and the stirring time is 1-3 h.

[0028] In the technical solution, the shearing treatment is carried out under high-speed conditions, which can make the amphiphilic block copolymer form a large number of nanomicelles with small particle size and uniform particle size. On the one hand, the nanomicelles with small particle size have a large specific surface area, which helps to improve the combination rate of the nanomicelles and the PDRN. On the other hand, the particle size of the nanomicelles is uniform, which helps to improve the stability of the nanomicelles.

[0029] In a second aspect, the embodiments of the present application provide an amphiphilic block copolymer nanomicelle loaded with PDRN, which is prepared by the preparation method provided in the first aspect.

[0030] In the technical solution, the amphiphilic block copolymer nanomicelle loaded with PDRN prepared by the preparation method provided in the first aspect has the advantages of drug release and strong air permeability / membrane permeability, so that the PDRN can continuously and efficiently play a therapeutic role.

[0031] In a third aspect, the embodiments of the present application provide a use of the amphiphilic block copolymer nanomicelle loaded with PDRN provided in the second aspect in the preparation of a drug for promoting tissue repair, a drug for promoting tissue regeneration, or an anti-inflammatory drug. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0033] Figure 1 A process flow chart of the preparation method of the amphiphilic block copolymer nanomicelle loaded with PDRN provided in the embodiments of the present application. DETAILED DESCRIPTION

[0034] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased in the market are adopted.

[0035] It should be noted that, in the present application, "and / or", such as "feature 1 and / or feature 2", means "feature 1" alone, "feature 2" alone, or "feature 1" plus "feature 2".

[0036] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" is two or more; the range of "value a~value b" includes both end values "a" and "b", and the "unit of measurement" in "value a~value b+unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0037] At present, the nucleic acid delivery system based on amphiphilic block copolymer is usually for mRNA, siRNA, miRNA or tRNA and other nucleic acid substances, and there are few related researches on the construction of delivery system for PDRN (which belongs to a macromolecular nucleic acid substance); and when the skilled person tries to construct the amphiphilic block copolymer delivery system based on PDRN, it is also found that there is a problem of low combination rate of amphiphilic block copolymer and PDRN.

[0038] Based on this, the inventors found that by using amphiphilic block copolymer nanomicelles as the delivery carrier of PDRN, the problem of low combination rate of nanocarrier and PDRN can be effectively improved.

[0039] The following will specifically describe one kind of amphiphilic block copolymer nanomicelles loaded with PDRN, its preparation method and application.

[0040] In a first aspect, the embodiments of the present application provide a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, comprising the following steps: S1, providing a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN, wherein the molecular weight of the hydrophilic polymer in the amphiphilic block copolymer nanomicelles is 2000-5000 Da (for example, but not limited to, any one of 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da and 5000 Da, or a range value between any two of them), the molecular weight of the hydrophobic polymer is 5000-20000 Da (for example, but not limited to, any one of 5000 Da, 7500 Da, 10000 Da, 12500 Da, 15000 Da, 17500 Da and 20000 Da, or a range value between any two of them), and the molecular weight of the PDRN is not more than 1000 KDa (for example, but not limited to, any one of 100 KDa, 200 KDa, 300 KDa, 400 KDa, 500 KDa, 600 KDa, 700 KDa, 800 KDa, 900 KDa and 1000 KDa, or a range value between any two of them); the mass ratio of the amphiphilic block copolymer nanomicelles to the PDRN is 100:(0.1-2), for example, but not limited to, any one of 100:0.1, 100:0.5, 100:1, 100:1.5 and 100:2, or a range value between any two of them; S2, shearing the buffer solution at 0-25℃ (for example, but not limited to, any one of 0℃, 2℃, 5℃, 8℃, 10℃, 12℃, 15℃, 17℃, 20℃ and 25℃, or a range value between any two of them) at a speed of 8000-15000 rpm (for example, but not limited to, any one of 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm and 15000 rpm, or a range value between any two of them), and then mixing at a speed of 100-500 rpm (for example, but not limited to, any one of 100 rpm, 200 rpm, 300 rpm, 400 rpm and 500 rpm, or a range value between any two of them), to obtain the amphiphilic block copolymer nanomicelles loaded with PDRN.

[0041] It should be noted that if the shearing speed is too low, it is difficult to effectively destroy the nanomicelle structure, thereby failing to effectively utilize the internal space of the nanomicelles to load PDRN.

[0042] It needs to be emphasized that the technical solution of using amphiphilic block copolymer nanomicelles as carriers to assist the delivery of PDRN is rarely reported in the prior art.

[0043] In the present application, on the one hand, the shearing treatment at high speed and the mixing treatment at low speed under the above temperature condition can effectively maintain the structural stability and physiological activity of PDRN; the shearing treatment at high speed can cause the partial structure of at least part of the nanomicelles to be damaged, thereby helping PDRN to enter the nanomicelles and stably and firmly combine with them through the mutual entanglement of chains and van der Waals force, and the mixing treatment at low speed can cause the nanomicelles with damaged structures to reassemble into nanomicelles with complete structures, while the surface of the nanomicelles also loads PDRN, so that PDRN is combined inside and outside the nanomicelles, thereby improving the combination rate of the nanomicelles and PDRN; in addition, limiting the molecular weight of the hydrophilic polymer and the hydrophobic polymer in the amphiphilic block copolymer nanomicelles and the molecular weight of PDRN within the above ranges, and limiting the mass ratio of the amphiphilic block copolymer nanomicelles and PDRN within the above range, can also effectively improve the combination rate of the amphiphilic block copolymer nanomicelles and PDRN; on the other hand, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by the method also have the advantages of drug sustained-release function and strong membrane permeability, so that PDRN can be effectively enriched in the action area and continuously and efficiently exert its function.

[0044] As an example, step S2 includes: S21 adding ice to the buffer solution to obtain an ice-water mixed solution; S22 shearing the ice-water mixed solution at 8000-15000 rpm, wherein the shearing time is 5-20 min (for example, but not limited to, any one of 5 min, 10 min, 15 min and 20 min or a range value between any two of them); S23 then stirring the ice-water mixed solution at 100-500 rpm, wherein the stirring time is 2-3 h (for example, but not limited to, any one of 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h and 3 h or a range value between any two of them).

[0045] In this embodiment, the temperature of the buffer solution can be conveniently and stably controlled in the range of 0-25℃ by adding ice to the buffer solution; in addition, the shearing time and the mixing time are limited within the above ranges, so that more PDRN is combined inside and outside the nanomicelles, thereby further improving the combination rate of the nanomicelles and PDRN.

[0046] As an example, the ratio of the volume of ice added to the buffer solution to the volume of the buffer solution is 1: (8~10), such as but not limited to any one of 1:8, 1:8.5, 1:9, 1:9.5, and 1:10, or a range value between any two of them.

[0047] In this embodiment, the amount of ice added is limited within the above range, which can maintain the concentration of nanomicelles and the concentration of PDRN in the solution within a suitable range while controlling the temperature, so that the two can be efficiently combined. As an example, the molecular weight of the hydrophilic polymer is 3000~4000 Da (such as but not limited to any one of 3000 Da, 3200 Da, 3400 Da, 3600 Da, 3800 Da, and 4000 Da, or a range value between any two of them), the molecular weight of the hydrophobic polymer is 10000~15000 Da (such as but not limited to any one of 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, and 15000 Da, or a range value between any two of them), and the molecular weight of the PDRN is 200~500 KDa (such as but not limited to any one of 200 KDa, 250 KDa, 300 KDa, 350 KDa, 400 KDa, 450 KDa, and 500 KDa, or a range value between any two of them).

[0048] In this embodiment, the molecular weight of the hydrophilic polymer, the molecular weight of the hydrophobic polymer, and the molecular weight of the PDRN are respectively limited within the above ranges, which helps to further improve the combination rate of the amphiphilic block copolymer nanomicelles and the PDRN.

[0049] As an example, the molecular weight of the hydrophilic polymer is 3000 Da, and the molecular weight of the hydrophobic polymer is 10000~15000 Da (such as but not limited to any one of 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, and 15000 Da, or a range value between any two of them).

[0050] In this embodiment, the molecular weight of the hydrophilic polymer and the molecular weight of the hydrophobic polymer are respectively limited within the above ranges, which can more effectively improve the combination rate of the amphiphilic block copolymer nanomicelles and the PDRN.

[0051] It should be noted that the mass ratio of the amphiphilic block copolymer nanomicelles and the PDRN is not limited, and can be adaptively adjusted according to actual needs.

[0052] As an example, the mass-volume ratio of the amphiphilic block copolymer nanomicelles and the buffer solution is 1 g: (3-6) mL, for example but not limited to any one of 1 g:3 mL, 1 g:3.5 mL, 1 g:4 mL, 1 g:4.5 mL, 1 g:5 mL, 1 g:5.5 mL and 1 g:6 mL or a range value between any two of them.

[0053] In this embodiment, the mass-volume ratio of the amphiphilic block copolymer nanomicelles and the buffer solution is limited within the above range, which can make the amphiphilic block copolymer nanomicelles and the PDRN both quickly dissolve and uniformly disperse in the buffer solution, and at the same time, the buffer solution has a relatively appropriate concentration of the nanomicelles and the PDRN, which also helps to improve the combination rate of the two.

[0054] It should be noted that the type of the buffer solution is not limited and can be set according to the conventional selection in the art.

[0055] As an example, the buffer solution is selected from a phosphate buffer solution with a pH of 6.5-7 (for example but not limited to any one of 6.5, 6.6, 6.7, 6.8, 6.9 and 7 or a range value between any two of them).

[0056] In this embodiment, the phosphate buffer solution with a pH of 6.5-7 can effectively dissolve and disperse the nanomicelles and the PDRN while maintaining the structural stability and activity of the PDRN.

[0057] It should be noted that the configuration mode of the phosphate buffer solution is not limited and can be configured according to the conventional selection in the art, for example, it can be configured by disodium hydrogen phosphate and potassium dihydrogen phosphate, or it can be configured by disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0058] As an example, the amphiphilic block copolymer nanomicelles are two-block amphiphilic block copolymer nanomicelles, wherein the material of the hydrophilic polymer is selected from at least one of methoxypolyethylene glycol, monoalkoxy polyethylene glycol and monoacyl polyethylene glycol, and the material of the hydrophobic polymer is selected from polycaprolactone.

[0059] In this embodiment, the combination of the hydrophilic polymer and the hydrophobic polymer is various, which can provide more implementable embodiments, thereby facilitating the popularization and application of the technical solutions provided by the embodiments of the present application, and at the same time, the above-mentioned materials also have the advantage of good biocompatibility.

[0060] As an example, the material of the hydrophilic polymer is selected from methoxypolyethylene glycol, and the material of the hydrophobic polymer is selected from polycaprolactone.

[0061] In this embodiment, the amphiphilic block copolymer nanomicelles formed by the combination of the two polymer materials described above are used as the PDRN carrier. Since the carrier also has the effect of promoting collagen regeneration, it can act together with the PDRN, thereby achieving better curative effect.

[0062] As an example, step S1 comprises:

[0063] S11 dissolving the amphiphilic block copolymer in the first portion of buffer solution and performing shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly, thereby obtaining a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles; S12 dissolving PDRN in the second portion of buffer solution, thereby obtaining a PDRN solution; and S13 mixing the polymer nanomicelle solution and the PDRN solution.

[0064] In this embodiment, the polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles and the PDRN solution are prepared separately, and then mixed, i.e., mixed after both are effectively dissolved and uniformly dispersed, which helps to improve the combination rate of the two; at the same time, in order to make the amphiphilic block copolymer quickly form nanomicelles through self-assembly, the self-assembly process usually needs to be carried out under heating conditions, and the step-by-step dissolution and mixing method can effectively protect the structural integrity and activity of PDRN.

[0065] As an example, step S11 comprises: adding the amphiphilic block copolymer to the first portion of buffer solution at 65-80°C (for example, but not limited to, any one point value or a range value between any two of 65°C, 70°C, 75°C and 80°C) and performing shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly, thereby obtaining a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles.

[0066] In this embodiment, shearing treatment at an appropriate temperature can provide an appropriate driving force for self-assembly, thereby facilitating the amphiphilic block copolymer to form nanomicelles more efficiently and completely.

[0067] As an example, the mass-to-volume ratio of the amphiphilic block copolymer and the first portion of buffer solution is 1 g: (3.5-4.5) mL, for example, but not limited to, any one point value or a range value between any two of 1 g: 3.5 mL, 1 g: 3.75 mL, 1 g: 4 mL, 1 g: 4.25 mL and 1 g: 4.5 mL.

[0068] In the embodiment, the mass / volume ratio of the amphiphilic block copolymer and the first portion of the buffer is limited in the above range, which helps the amphiphilic block copolymer to be quickly dissolved and uniformly dispersed, and also helps the amphiphilic block copolymer to form nanomicelles through self-assembly.

[0069] As an example, the amphiphilic block copolymer is added to the first portion of the buffer at 65-80°C for shearing treatment, the stirring speed is 8000-15000 rpm (for example, but not limited to, any one point value or a range value between any two of 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm and 15000 rpm), and the stirring time is 1-3 h (for example, but not limited to, any one point value or a range value between any two of 1 h, 1.5 h, 2 h, 2.5 h and 3 h).

[0070] In the embodiment, the shearing treatment is performed under high-speed conditions, which can make the amphiphilic block copolymer form a large number of nanomicelles with small particle size and uniform particle size. On the one hand, the nanomicelles with small particle size have a large specific surface area, which helps to improve the combination rate of the nanomicelles and the PDRN. On the other hand, the particle size of the nanomicelles is uniform, which helps to improve the stability of the nanomicelles.

[0071] It should be noted that the processes or steps not specially mentioned or limited in the preparation process of the amphiphilic block copolymer nanomicelles loaded with PDRN can be set according to the conventional selection in the art.

[0072] It should be noted that the shape of the nanomicelles in the embodiments of the present application is a sphere, and the average particle size is 100-300 nm.

[0073] As an example, the process flow chart of the preparation method of the amphiphilic block copolymer nanomicelles loaded with PDRN is exemplarily shown in Figure 1 .

[0074] In a second aspect, the embodiments of the present application provide an amphiphilic block copolymer nanomicelle loaded with PDRN, which is prepared by the preparation method provided in the first aspect.

[0075] In the present application, the amphiphilic block copolymer nanomicelle loaded with PDRN prepared by the preparation method provided in the first aspect has the advantages of drug release and strong air permeability / membrane permeability, so that the PDRN can continuously and efficiently play a therapeutic role.

[0076] As an example, the average particle size of the amphiphilic block copolymer nanomicelle loaded with PDRN is 80-300 nm (for example, but not limited to, any one of the average particle sizes of 80 nm, 100 nm, 150 nm, 200 nm, 250 nm and 300 nm or a range value between any two of them), or / and the PDI of the amphiphilic block copolymer nanomicelle loaded with PDRN is less than 0.3.

[0077] As an example, the drug loading amount of PDRN in the amphiphilic block copolymer nanomicelle loaded with PDRN is 0.1-1 wt%, for example, but not limited to, any one of the drug loading amounts of 0.1 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8 wt% and 1.0 wt% or a range value between any two of them.

[0078] In a third aspect, the embodiments of the present application provide a use of the amphiphilic block copolymer nanomicelle loaded with PDRN as provided in the embodiments of the second aspect in the preparation of a drug for promoting tissue repair, a drug for promoting tissue regeneration or an anti-inflammatory drug.

[0079] The features and performances of the present application are further described in detail below in combination with embodiments.

[0080] Embodiment 1

[0081] The embodiments of the present application provide a preparation method of the amphiphilic block copolymer nanomicelle loaded with PDRN, which comprises the following steps:

[0082] S1 provides a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN:

[0083] S11 adds 200 g of amphiphilic block copolymer MPEG3000-PCL10000 into 800 mL of phosphate buffer solution (pH 6.8) at 80°C, mixes uniformly, and then performs high-speed shearing treatment at 10000 rpm for 2 h to make the amphiphilic block copolymer form nanomicelles through self-assembly, to obtain a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles, and then cools to room temperature for standby.

[0084] S12 adds 2 g of PDRN with a molecular weight of 235 KDa into 100 mL of phosphate buffer solution (pH 6.8) and stirs at 150 rpm until the PDRN is completely dissolved, to obtain a PDRN solution.

[0085] S13 slowly adds the PDRN solution into the polymer nanomicelle solution, to obtain a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN.

[0086] S2 preparing amphiphilic block copolymer nanomicelles loaded with PDRN in the buffer containing both amphiphilic block copolymer nanomicelles and PDRN:

[0087] S21 adding ice to the above buffer, wherein the ratio of the volume of ice added to the volume of the buffer is 1:10, to obtain an ice-water mixed solution.

[0088] S22 then shearing the ice-water mixed solution at a high rotation speed of 10000 rpm for 10 min.

[0089] S23 then stirring and mixing the ice-water mixed solution at a low rotation speed of 150 rpm for 1 h, to obtain amphiphilic block copolymer nanomicelles loaded with PDRN.

[0090] (1) In order to better illustrate the importance of simultaneously limiting the molecular weights of the hydrophilic polymer and the hydrophobic polymer in the amphiphilic block copolymer nanomicelles and PDRN within appropriate ranges, the following corresponding examples and comparative examples (the process steps are the same as those of Example 1) are designed, and the combination rates of nanomicelles and PDRN in each example and comparative example are counted, wherein the calculation formula of the combination rate is: combination rate = [(PDRN added amount - PDRN unloaded amount) / PDRN added amount] x 100%, and the specific content can be seen in Table 1.

[0091] Table 1

[0092]

[0093] Referring to Table 1, it can be seen from the test results of Examples 1-18 and Comparative Examples 1-4 that when the molecular weights of the hydrophilic polymer and the hydrophobic polymer in the nanomicelles and PDRN are simultaneously limited within the appropriate ranges of the present application, the nanomicelles have a higher combination rate with PDRN.

[0094] It can be seen from the test results of Examples 1-8 and Examples 9-18 that when the hydrophilic polymer and the hydrophobic polymer in the nanomicelles are limited to 3000-4000 Da and 10000-15000 Da respectively, the corresponding nanomicelles have a higher combination rate with PDRN than when they are not limited within the ranges; wherein when the hydrophilic polymer and the hydrophobic polymer in the nanomicelles are limited to 3000 Da and 10000-15000 Da respectively, the corresponding nanomicelles have a combination rate with PDRN as high as 9%, which is significantly higher than that of other combinations of molecular weights.

[0095] (2) In order to better illustrate the importance of limiting the mass ratio of nanomicelles and PDRN to a specific range, the following corresponding examples are designed (except for the mass ratio of nanomicelles and PDRN, the preparation process is the same as that of Example 1), and the combination rate of nanomicelles and PDRN in each example is counted. For details, see Table 2.

[0096] Table 2

[0097]

[0098] Referring to Table 2, according to the test results of Examples 19-21, when the mass ratio of nanomicelles and PDRN is limited to the appropriate range provided in the embodiments of the present application, the nanomicelles and PDRN both have a high combination rate; in particular, when the mass ratio of nanomicelles and PDRN is 100:1, the corresponding combination rate of nanomicelles and PDRN is as high as 9.5%, which is significantly higher than other mass ratios.

[0099] (3) In order to better illustrate the rationality of the process step design of the drug-loaded nanomicelles in the preparation process, the following corresponding examples and comparative examples are designed, and the combination rate of nanomicelles and PDRN in each example and comparative example is counted. For details, see Examples 22-23 and Comparative Examples 5-6.

[0100] Example 22

[0101] The embodiments of the present application provide a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, which is only different from Example 1 in that in step S22, the rotation speed of shearing treatment is 8000 rpm, and the corresponding combination rate of nanomicelles and PDRN is 5.3%.

[0102] According to the test results of Example 1 and Example 22, in the step of shearing treatment on the buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN, limiting the rotation speed of shearing treatment to an appropriate range helps to improve the combination rate of nanomicelles and PDRN.

[0103] Example 23

[0104] The embodiments of the present application provide a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, which includes the following steps:

[0105] S1 200 g of amphiphilic block copolymer MPEG3000-PCL10000 was added into 900 mL of phosphate buffer (pH 6.8) at 80 ℃, mixed uniformly, and then treated by high-speed shearing at 10,000 rpm for 2 h to make the amphiphilic block copolymer form nanomicelles by self-assembly, to obtain a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles, and then cooled to room temperature for standby.

[0106] S2 2 g of PDRN with a molecular weight of 235 KDa was slowly added into the polymer nanomicelle solution to obtain a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN; ice was added to the buffer solution, wherein the ratio of the volume of ice added to the buffer solution to the volume of the buffer solution was 1:10, to obtain an ice-water mixed solution; then the ice-water mixed solution was first treated by shearing at a high speed of 10,000 rpm for 10 min; and then the ice-water mixed solution was stirred and mixed at a low speed of 150 rpm for 1 h, to obtain amphiphilic block copolymer nanomicelles loaded with PDRN, wherein the combination rate of the nanomicelles and PDRN was 4.2%.

[0107] It can be known from the test results of Example 1 and Example 23 that, the polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles and the PDRN solution are prepared respectively, and then mixed, that is, mixed after both are effectively dissolved and uniformly dispersed, which helps to improve the combination rate of the two.

[0108] Comparative Example 5

[0109] The comparative example of the present application provides a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, which is only different from Example 1 in that, in step S22, the ice-water mixed solution is not treated by shearing at a high speed of 10,000 rpm, and the corresponding combination rate of the nanomicelles and PDRN is 1.5%.

[0110] It can be known from the test results of Example 1 and Comparative Example 5 that, the shearing treatment at a high speed and the mixing treatment at a low speed are performed in sequence, so that the inside and outside of the nanomicelles are both loaded with PDRN, which helps to improve the combination rate of the nanomicelles and PDRN.

[0111] Comparative Example 6

[0112] The comparative example of the present application provides a preparation method of amphiphilic block copolymer nanomicelles loaded with PDRN, which is only different from Example 1 in that, in step S22, the ice-water mixed solution is treated by shearing at a speed of 6,000 rpm, and the corresponding combination rate of the nanomicelles and PDRN is 3.6%.

[0113] From the test results of Example 1, Example 22 and Comparative Example 6, it can be seen that, in the step of shearing the buffer solution containing both the amphiphilic block copolymer nanomicelles and the PDRN, limiting the shearing rotation speed within the appropriate range of the present application can effectively improve the combination rate of the nanomicelles and the PDRN.

[0114] Test Example

[0115] Cell proliferation and sustained-release function test

[0116] Test procedure:

[0117] (1) Cell culture: fibroblasts were inoculated in a 96-well plate at a density of 5000 cells per well, and cultured in a DMEM medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide for 24 h to allow the cells to adhere fully.

[0118] (2) Grouping and dosing: each type of cell was divided into four groups:

[0119] Blank control group: containing only the cell culture solvent (PBS buffer solution);

[0120] Carrier group: adding the MPEG-PCL carrier solution prepared in Example 1 (concentration of 10 μg / mL);

[0121] Free PDRN group: adding a free PDRN solution (concentration of 10 μg / mL);

[0122] Sample group: adding the MPEG-PCL-PDRN complex solution prepared in Example 1 (concentration of 10 μg / mL).

[0123] Five replicates were set up for each group, and detection was performed at 24 h, 48 h and 72 h.

[0124] (3) Cell activity detection: at each time point, CCK-8 reagent (10 μL / well) was added to each well, and after 2 h of further incubation, the absorbance (OD value) at 450 nm was measured using a microplate reader, and the relative proliferation rate of each group of cells relative to the blank control group (set as 100%) was calculated.

[0125] (4) Data statistics: single factor ANOVA (ANOVA) was performed using SPSS 22.0 software, and Tukey's test was used for comparison between groups, with p<0.05 considered to be statistically significant.

[0126] Statistical analysis of experimental data, see Table 3

[0127] Table 3

[0128]

[0129] It should be noted that in Table 3, each result is obtained by averaging the corresponding 5 data in the table; "*" indicates that p < 0.05, has significant difference compared with the blank control group at the same time point; "#" indicates that p < 0.05, has significant difference compared with the free PDRN group at the same time point.

[0130] 2.3 Experimental results

[0131] (1) At 72 h, the cell activity values of the free PDRN group (130.2%) and the sample group (150.5%) were significantly higher than that of the control group (100.0%), indicating that PDRN had obvious effect of promoting cell proliferation.

[0132] (2) At 48 h, the cell activity of the sample group (132.6%) was significantly higher than that of the free PDRN group (125.7%), and at the same time, at 72 h, the cell activity of the sample group (150.5%) was also significantly higher than that of the free PDRN group (130.2%), indicating that the PDRN delivered by the amphiphilic block copolymer nanomicelle provided by the application could better exert the effect of PDRN.

[0133] (3) The cell activity results of the blank group and the carrier group were basically the same throughout the process, indicating that the amphiphilic block copolymer nanomicelle provided by the application had good biocompatibility.

[0134] (4) At 24 h, the cell activity of the sample group (110.2%) was lower than that of the free PDRN group (115.4%), however, at 48 h, the cell activity of the sample group (132.6%) was higher than that of the free PDRN group (125.7%), and at 72 h, the cell activity of the sample group (150.5%) was also significantly higher than that of the free PDRN group (130.2%), indicating that the amphiphilic block copolymer nanomicelle loaded with PDRN provided by the application had the function of releasing PDRN, and could prolong the degradation period of PDRN, so that PDRN could play a long-term and efficient role.

[0135] The above-described embodiments are part of the embodiments of the application, rather than all the embodiments. The detailed description of the embodiments of the application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the application.

Claims

1. A method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN, characterized in that, The method comprises the following steps: S1, providing a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN, wherein the amphiphilic block copolymer nanomicelles have a hydrophilic polymer with a molecular weight of 2000-5000 Da and a hydrophobic polymer with a molecular weight of 5000-20000 Da, the PDRN has a molecular weight of no more than 1000 KDa, and the mass ratio of the amphiphilic block copolymer nanomicelles to the PDRN is 100: (0.1-2); S2, shearing the buffer solution at 8000-15000 rpm and then mixing at 100-500 rpm at 0-25℃ to obtain amphiphilic block copolymer nanomicelles loaded with PDRN; The amphiphilic block copolymer nanomicelles are two-block amphiphilic block copolymer nanomicelles, wherein the hydrophilic polymer is made of methoxy polyethylene glycol, and the hydrophobic polymer is made of polycaprolactone.

2. The production method according to claim 1, characterized by, Step S2 comprises: S21, adding ice to the buffer solution to obtain an ice-water mixed solution; S22, shearing the ice-water mixed solution at 8000-15000 rpm, wherein the shearing time is 5-20 min; S23, then stirring the ice-water mixed solution at 100-500 rpm, wherein the stirring time is 2-3 h.

3. The production method according to claim 1 or 2, characterized by, The hydrophilic polymer has a molecular weight of 3000-4000 Da, the hydrophobic polymer has a molecular weight of 10000-15000 Da, and the PDRN has a molecular weight of 200-500 KDa.

4. The production method according to claim 3, characterized by, The hydrophilic polymer has a molecular weight of 3000 Da, and the hydrophobic polymer has a molecular weight of 10000-15000 Da.

5. The production method according to claim 1 or 2, characterized by, In the buffer solution, the mass-volume ratio of the amphiphilic block copolymer nanomicelles to the buffer solution is 1 g: (3-6) mL. Or / and, the buffer solution is selected from a phosphate buffer solution with a pH of 6.5-7.

6. The production method according to claim 1 or 2, characterized by, Step S1 comprises: S11, dissolving amphiphilic block copolymers in a first portion of buffer solution and shearing to form nanomicelles through self-assembly, to obtain a polymer nanomicelle solution containing the amphiphilic block copolymer nanomicelles; S12, dissolving PDRN in a second portion of buffer solution to obtain a PDRN solution; S13, mixing the polymer nanomicelle solution and the PDRN solution.

7. The preparation method according to claim 6, characterized in that, The step S11 comprises: adding amphiphilic block copolymers to a first portion of buffer solution at 65-80℃ for shearing to form nanomicelles through self-assembly, to obtain a polymer nanomicelle solution containing the amphiphilic block copolymer nanomicelles.

8. The production method according to claim 7, characterized by, The mass-volume ratio of the amphiphilic block copolymer and the first part of the buffer is 1 g: (3.5~4.5) mL; or / and, in the step of adding the amphiphilic block copolymer to the first part of the buffer at 65~80℃ for shearing treatment, the stirring speed is 8000~15000 rpm, and the stirring time is 1~3 h.

9. An amphiphilic block copolymer nanomicelle loaded with PDRN, characterized in that, The preparation method is prepared by using any one of claims 1~8.

10. The use of the PDRN-loaded amphiphilic block copolymer nanomicelles as claimed in claim 9 in the preparation of a drug for promoting tissue repair, a drug for promoting tissue regeneration, or an anti-inflammatory drug.

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