Amphiphilic block copolymer nano-micelle loaded with PDRN as well as preparation method and application of amphiphilic block copolymer nano-micelle

Abstract

The invention provides an amphiphilic block copolymer nano-micelle loaded with PDRN as well as a preparation method and application of the amphiphilic block copolymer nano-micelle, and belongs to the technical field of medical cosmetology. The preparation method of the amphiphilic block copolymer nano-micelle loaded with PDRN comprises the following steps: S1, providing a buffer solution containing the amphiphilic block copolymer nano-micelle and PDRN at the same time; s2, the buffer solution is subjected to shearing treatment at the temperature of 0-25 DEG C at the speed of 8000-15000 rpm and then mixed treatment at the speed of 100-500 rpm, the problem that the binding rate of the amphiphilic block copolymer and PDRN is low can be effectively solved through the preparation method, meanwhile, the amphiphilic block copolymer nano-micelle loaded with PDRN prepared through the method further has the advantages of being high in drug slow release function and membrane permeation capacity, and the amphiphilic block copolymer nano-micelle loaded with PDRN can be used for preparing the amphiphilic block copolymer nano-micelle loaded with PDRN and the preparation method of the amphiphilic block copolymer nano-micelle loaded with PDRN and the preparation method of the amphiphilic block copolymer nano-micelle loaded with PDRN. Therefore, the PDRN can continuously and efficiently play a role.

Description

Technical Field

[0001] This application relates to the field of medical aesthetics technology, and more specifically, to an amphiphilic block copolymer nanomicelle loaded with PDRN, its preparation method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are bioactive substances extracted from salmon sperm DNA. Their biological functions include, but are not limited to, stimulating fibroblast proliferation, promoting collagen and hyaluronic acid synthesis, anti-inflammatory effects, and angiogenesis, leading to their widespread application in the medical and cosmetic fields. However, PDRNs, primarily composed of polydeoxyribonucleotides, are easily and rapidly degraded by nucleases in vivo, resulting in a short duration of action. Furthermore, their large molecular weight leads to poor membrane absorption, further hindering their effectiveness.

[0003] Meanwhile, the construction of drug delivery systems based on amphiphilic block copolymer nanomicelles is currently a research hotspot. The basic function of this technology is to effectively prolong the degradation cycle of drugs and enhance their transmembrane absorption capacity. However, in the process of constructing amphiphilic block copolymer nanomicelle delivery systems loaded with PDRN, researchers have discovered a low binding rate between the amphiphilic block copolymer nanomicelles and PDRN, which limits the promotion and application of PDRN in the field of medical aesthetics. Summary of the Invention

[0004] The purpose of this application is to provide an amphiphilic block copolymer nanomicelle loaded with PDRN, its preparation method and application. The preparation method can effectively improve the problem of low binding rate of amphiphilic block copolymer nanomicelles with PDRN. At the same time, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by this method also have the advantages of drug sustained release function and strong membrane permeability, so that PDRN can play a continuous and efficient role.

[0005] The embodiments of this application are implemented as follows: In a first aspect, embodiments of this application provide a method for preparing 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, and the molecular weight of PDRN is not greater than 1000 KDa; and the mass ratio of the amphiphilic block copolymer nanomicelles to PDRN is 100:(0.1~2); S2 shearing the buffer solution at 8000~15000 rpm at 0~25℃ and then mixing it at 100~500 rpm to obtain amphiphilic block copolymer nanomicelles loaded with PDRN.

[0006] In the above technical solution, on the one hand, under the aforementioned temperature conditions, a high-speed shearing process followed by a low-speed mixing process is performed. The temperature conditions effectively maintain the structural stability and physiological activity of PDRN. The high-speed shearing process first disrupts at least some of the structure of the nanomicelles, facilitating the entry of PDRN into the nanomicelles and their stable and reliable bonding through chain entanglement and van der Waals forces. The low-speed mixing process then reassembles the structurally damaged nanomicelles into complete nanomicelles. Simultaneously, PDRN is loaded onto the surface of the nanomicelles, ensuring bonding both inside and outside the nanomicelles. The presence of PDRN enhances the binding rate between the nanomicelles and PDRN. Furthermore, limiting the molecular weights of the hydrophilic and hydrophobic polymers and the molecular weight of PDRN in the amphiphilic block copolymer nanomicelles to the aforementioned ranges, as well as limiting the mass ratio of the amphiphilic block copolymer nanomicelles to PDRN to the aforementioned ranges, can also effectively improve the binding rate between the amphiphilic block copolymer nanomicelles and PDRN. Secondly, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by this method also have the advantages of drug sustained-release function and strong membrane permeability, enabling PDRN to be effectively enriched in the action area and exert its effect continuously and efficiently.

[0007] In some optional embodiments, step S2 includes: S21 adding ice to the buffer solution to obtain an ice-water mixture; S22 first shearing the ice-water mixture at 8000~15000 rpm for 5~20 min; S23 then stirring the ice-water mixture at 100~500 rpm for 2~3 h.

[0008] In the above technical solution, adding ice to the buffer solution can conveniently and stably control the temperature of the buffer solution within the range of 0~25℃. In addition, limiting the duration of shearing and mixing treatments within the above ranges ensures that more PDRN is bound inside and outside the nanomicelles, thereby helping to further improve the binding rate of nanomicelles and PDRN.

[0009] In some alternative embodiments, 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.

[0010] In the above technical solution, limiting the molecular weight of the hydrophilic polymer, the molecular weight of the hydrophobic polymer, and the molecular weight of PDRN to the above ranges helps to further improve the binding rate of amphiphilic block copolymer nanomicelles and PDRN.

[0011] In some alternative embodiments, the hydrophilic polymer has a molecular weight of 3000 Da, and the hydrophobic polymer has a molecular weight of 10000~15000 Da.

[0012] In the above technical solution, limiting the molecular weights of the hydrophilic polymer and the hydrophobic polymer to the above range can more effectively improve the binding rate of the amphiphilic block copolymer nanomicelles and PDRN.

[0013] In some alternative embodiments, the mass-to-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 phosphate buffer with a pH of 6.5~7.

[0014] In the above technical solution, limiting the mass-to-volume ratio of the amphiphilic block copolymer nanomicelles and the buffer solution to the above range enables both the amphiphilic block copolymer nanomicelles and PDRN to dissolve rapidly and disperse uniformly in the buffer solution. At the same time, it also ensures that the buffer solution contains a suitable concentration of nanomicelles and PDRN, which helps to improve the binding rate of the two. In addition, using a phosphate buffer solution with a pH of 6.5-7 can effectively dissolve and disperse the nanomicelles and PDRN while maintaining the structural stability and activity of PDRN.

[0015] In some alternative embodiments, the amphiphilic block copolymer nanomicelles are diblock amphiphilic block copolymer nanomicelles, wherein the hydrophilic polymer is selected from at least one of methoxy polyethylene glycol, monoalkoxy polyethylene glycol and monoacyl polyethylene glycol, and the hydrophobic polymer is selected from polycaprolactone.

[0016] In the above technical solutions, there are many ways to combine hydrophilic polymers and hydrophobic polymers, which can provide a variety of implementation methods, thereby facilitating the promotion and application of the technical solutions provided in the embodiments of this application. At the same time, the above materials also have the advantage of good biocompatibility.

[0017] In some alternative embodiments, the hydrophilic polymer is selected from methoxy polyethylene glycol, and the hydrophobic polymer is selected from polycaprolactone.

[0018] In the above technical solution, the amphiphilic block copolymer nanomicelles formed by combining the two polymer materials are used as PDRN carriers. Since this carrier also has the effect of promoting collagen regeneration, it can work together with PDRN to achieve better therapeutic effects.

[0019] In some alternative implementations, step S1 includes: S11 The amphiphilic block copolymer is dissolved in the first buffer solution and sheared to allow the amphiphilic block copolymer to self-assemble into nanomicelles, resulting in a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles; S12 PDRN is dissolved in the second buffer solution to obtain a PDRN solution; S13 The polymer nanomicelle solution and the PDRN solution are mixed.

[0020] In the above technical solution, a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles and a PDRN solution are first prepared separately, and then the two are mixed. That is, the mixing process is carried out after both are effectively dissolved and uniformly dispersed, which helps to improve the binding rate of the two. At the same time, in order to enable the amphiphilic block copolymer to quickly form nanomicelles through self-assembly, the self-assembly process usually needs to be carried out under heating conditions. The stepwise dissolution and mixing method can effectively protect the structural integrity and activity of PDRN.

[0021] In some alternative embodiments, step S11 includes: adding the amphiphilic block copolymer to a first buffer solution at 65-80°C for shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly, thereby obtaining a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles.

[0022] In the above technical solution, shearing at a suitable temperature can provide a suitable driving force for self-assembly, thereby facilitating the efficient and thorough formation of nanomicelles by amphiphilic block copolymers.

[0023] In some alternative embodiments, the mass-to-volume ratio of the amphiphilic block copolymer to the first buffer is 1 g: (3.5~4.5) mL.

[0024] In the above technical solution, limiting the mass-volume ratio of the amphiphilic block copolymer and the first buffer solution to the above range helps the amphiphilic block copolymer to dissolve quickly and disperse evenly, and also helps it to form nanomicelles through self-assembly.

[0025] In some alternative embodiments, the amphiphilic block copolymer is added to a first buffer solution at 65-80°C for shear treatment, with a stirring speed of 8000-15000 rpm and a stirring time of 1-3 h.

[0026] In the above technical solution, shearing under high-speed conditions enables the amphiphilic block copolymer to form a large number of small and relatively uniform nanomicelles. On the one hand, the small nanomicelles have a large specific surface area, which helps to improve the binding rate of nanomicelles with PDRN. On the other hand, the relatively uniform particle size of the nanomicelles helps to improve the stability of the nanomicelles.

[0027] Secondly, embodiments of this application provide an amphiphilic block copolymer nanomicelle loaded with PDRN, which is prepared using the preparation method provided in the first aspect embodiment.

[0028] In the above technical solution, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by the preparation method provided in the first aspect embodiment have the advantages of drug sustained release and strong gas / membrane permeability, so that PDRN can exert a continuous and efficient therapeutic effect.

[0029] Thirdly, embodiments of this application provide the application of PDRN-loaded amphiphilic block copolymer nanomicelles as provided in the second aspect embodiments in the preparation of drugs that promote tissue repair, drugs that promote tissue regeneration, or anti-inflammatory drugs. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a process flow diagram of a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN, provided in an embodiment of this application. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] It should be noted that the terms "and / or" in this application, such as "feature 1 and / or feature 2", all refer to the three cases of "feature 1" alone, "feature 2" alone, and "feature 1" plus "feature 2".

[0034] In addition, in the description of this application, unless otherwise stated, "one or more" means two or more; the range of "numerical value a to numerical value b" includes the two endpoints "a" and "b"; and "unit of measurement" in "numerical value a to numerical value b + unit of measurement" represents the "unit of measurement" of both "numerical value a" and "numerical value b".

[0035] Currently, nucleic acid delivery systems based on amphiphilic block copolymers are typically designed for nucleic acid substances such as mRNA, siRNA, miRNA, or tRNA. However, there is relatively little research on delivery systems based on PDRN (a type of high molecular weight nucleic acid). Furthermore, researchers have found that amphiphilic block copolymers have a low binding rate with PDRN when attempting to construct PDRN-based amphiphilic block copolymer delivery systems.

[0036] Based on this, the inventors discovered that by using amphiphilic block copolymer nanomicelles as a delivery carrier for PDRN, the problem of low binding rate between nanocarriers and PDRN can be effectively improved.

[0037] The following is a detailed description of an amphiphilic block copolymer nanomicelle loaded with PDRN, its preparation method, and its application, based on embodiments of this application.

[0038] In a first aspect, embodiments of this application provide a method for preparing 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, in the amphiphilic block copolymer nanomicelles, the molecular weight of the hydrophilic polymer is 2000~5000 Da (e.g., but not limited to any one of 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da and 5000 Da or any range between the two), the molecular weight of the hydrophobic polymer is 5000~20000 Da (e.g., but not limited to any one of 5000 Da, 7500 Da, 10000 Da, 12500 Da, 15000 Da, 17500 Da and 20000 Da or any range between the two), and the molecular weight of PDRN is not greater than 1000 kDa (e.g., but not limited to a molecular weight of 100 kDa). The values ​​are: kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, and 1000 kDa, or any value between any two; the mass ratio of amphiphilic block copolymer nanomicelles to PDRN is 100:(0.1~2), for example, but not limited to, any value between any two of the mass ratios of 100:0.1, 100:0.5, 100:1, 100:1.5, and 100:2; S2 The buffer solution is heated at 0~25°C (for example, but not limited to, any value between any two of the temperatures of 0°C, 2°C, 5°C, 8°C, 10°C, 12°C, 15°C, 17°C, 20°C, and 25°C, or any value between any two) at 8000~15000 rpm (for example, but not limited to, a rotation speed of 8000 rpm, 9000 rpm, etc.). After shearing at any one of 10000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm and 15000 rpm or any range between two rpm, the mixture is then subjected to mixing at 100~500 rpm (e.g., but not limited to any one of 100 rpm, 200 rpm, 300 rpm, 400 rpm and 500 rpm or any range between two rpm) to obtain amphiphilic block copolymer nanomicelles loaded with PDRN.

[0039] It should be noted that if the rotational speed of the shearing process is too low, it will be difficult to effectively destroy the nanomicelle structure, which will result in the inability to effectively utilize the internal space of the nanomicelles to load PDRN.

[0040] It should be emphasized that the technical solution of using amphiphilic block copolymer nanomicelles as a carrier for assisted delivery of PDRN is rarely reported in this application.

[0041] In this application, on the one hand, under the aforementioned temperature conditions, a high-speed shearing process is first performed followed by a low-speed mixing process. The aforementioned temperature conditions effectively maintain the structural stability and physiological activity of PDRN. The high-speed shearing process first disrupts at least a portion of the structure of the nanomicelles, thereby facilitating the entry of PDRN into the nanomicelles and their stable and reliable bonding through chain entanglement and van der Waals forces. The low-speed mixing process then reassembles the structurally damaged nanomicelles into complete nanomicelles. Simultaneously, PDRN is loaded onto the surface of the nanomicelles, ensuring that PDRN is bound both inside and outside the nanomicelles. This method improves the binding rate of PDRN to the amphiphilic block copolymer nanomicelles. Furthermore, limiting the molecular weights of the hydrophilic and hydrophobic polymers and the molecular weight of PDRN within the aforementioned ranges, as well as the mass ratio of the amphiphilic block copolymer nanomicelles to PDRN within the aforementioned ranges, also effectively improves the binding rate of PDRN to the amphiphilic block copolymer nanomicelles. Secondly, the PDRN-loaded amphiphilic block copolymer nanomicelles prepared by this method also possess advantages such as drug sustained-release function and strong membrane permeability, enabling PDRN to be effectively enriched in the target area and exert its effects continuously and efficiently.

[0042] As an example, step S2 includes: S21 adding ice to the buffer solution to obtain an ice-water mixture; S22 first shearing the ice-water mixture at 8000~15000 rpm, wherein the shearing time is 5~20 min (e.g., but not limited to any one of 5 min, 10 min, 15 min and 20 min or any range between the two); S23 then stirring the ice-water mixture at 100~500 rpm, wherein the stirring time is 2~3 h (e.g., 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 any range between the two).

[0043] In this embodiment, adding ice to the buffer solution allows for convenient and stable temperature control within the range of 0~25°C. Furthermore, limiting the duration of shearing and mixing treatments to the above ranges ensures that a large number of PDRN molecules are bound both inside and outside the nanomicelles, thereby helping to further improve the binding rate of nanomicelles and PDRN.

[0044] As an example, the ratio of the volume of ice added to the buffer to the volume of the buffer is 1:(8~10), for example, but not limited to any one of the ratios of 1:8, 1:8.5, 1:9, 1:9.5 and 1:10 or any range between the two.

[0045] In this embodiment, by limiting the amount of ice added to the above range, the concentration of nanomicelles and PDRN in the solution can be maintained within a suitable range while controlling the temperature, so that the two can combine efficiently. As an example, the molecular weight of the hydrophilic polymer is 3000 to 4000 Da (e.g., but not limited to any one of 3000 Da, 3200 Da, 3400 Da, 3600 Da, 3800 Da, and 4000 Da, or any range between two), the molecular weight of the hydrophobic polymer is 10000 to 15000 Da (e.g., but not limited to any one of 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, and 15000 Da, or any range between two), and the molecular weight of the PDRN is 200 to 500 KDa (e.g., but not limited to any one of 200 KDa, 250 KDa, 300 KDa, 350 KDa, 400 KDa, 450 KDa, and 500 KDa, or any range between two).

[0046] In this embodiment, limiting the molecular weight of the hydrophilic polymer, the molecular weight of the hydrophobic polymer, and the molecular weight of PDRN to the above-mentioned ranges helps to further improve the binding rate of the amphiphilic block copolymer nanomicelles and PDRN.

[0047] As an example, the hydrophilic polymer has a molecular weight of 3000 Da, and the hydrophobic polymer has a molecular weight of 10000 to 15000 Da (e.g., but not limited to any one of 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da and 15000 Da or any range between two).

[0048] In this embodiment, limiting the molecular weights of the hydrophilic polymer and the hydrophobic polymer to the above-mentioned ranges can more effectively improve the binding rate of the amphiphilic block copolymer nanomicelles and PDRN.

[0049] It should be noted that the mass ratio of amphiphilic block copolymer nanomicelles to PDRN is not limited and can be adjusted according to actual needs.

[0050] As an example, in the buffer solution, the mass-to-volume ratio of the amphiphilic block copolymer nanomicelles to the buffer solution is 1 g: (3~6) mL, for example, but not limited to any one of the mass-to-volume ratios 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 any range between the two.

[0051] In this embodiment, limiting the mass-to-volume ratio of the amphiphilic block copolymer nanomicelles to the buffer solution within the aforementioned range enables both the amphiphilic block copolymer nanomicelles and PDRN to dissolve rapidly and disperse uniformly in the buffer solution. Simultaneously, it also ensures that the buffer solution contains a suitable concentration of nanomicelles and PDRN, which helps to improve their binding rate.

[0052] It should be noted that the type of buffer solution is not limited and can be selected and set in accordance with the conventional methods used in this field.

[0053] As an example, the buffer solution is selected from phosphate buffer solutions with a pH of 6.5 to 7 (e.g., but not limited to any one of pH values ​​of 6.5, 6.6, 6.7, 6.8, 6.9 and 7, or any range between two of them).

[0054] In this embodiment, a phosphate buffer solution with a pH of 6.5-7 can effectively dissolve and disperse the nanomicelles and PDRN while maintaining the structural stability and activity of PDRN.

[0055] It should be noted that there are no restrictions on the preparation method of phosphate buffer. It can be prepared in accordance with the conventional methods in the field. For example, it can be prepared from disodium hydrogen phosphate and potassium dihydrogen phosphate, or it can be prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0056] As an example, the amphiphilic block copolymer nanomicelles are biblock amphiphilic block copolymer nanomicelles, wherein the hydrophilic polymer is selected from at least one of methoxy polyethylene glycol, monoalkoxy polyethylene glycol and monoacyl polyethylene glycol, and the hydrophobic polymer is selected from polycaprolactone.

[0057] In this embodiment, there are many ways to combine hydrophilic polymers and hydrophobic polymers, which can provide a variety of implementation methods, thereby facilitating the promotion and application of the technical solutions provided in this application. At the same time, the above-mentioned materials also have the advantage of good biocompatibility.

[0058] As an example, the hydrophilic polymer is made of methoxy polyethylene glycol, and the hydrophobic polymer is made of polycaprolactone.

[0059] In this embodiment, the amphiphilic block copolymer nanomicelles formed by combining the two polymer materials are used as PDRN carriers. Since the carriers also have the effect of promoting collagen regeneration, they can work together with PDRN to achieve better therapeutic effects.

[0060] As an example, step S1 includes: S11 The amphiphilic block copolymer is dissolved in the first buffer solution and sheared to allow the amphiphilic block copolymer to self-assemble into nanomicelles, resulting in a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles; S12 PDRN is dissolved in the second buffer solution to obtain a PDRN solution; S13 The polymer nanomicelle solution and the PDRN solution are mixed.

[0061] In this embodiment, a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles and a PDRN solution are first prepared separately, and then the two are mixed. That is, the mixing process is carried out after both are effectively dissolved and uniformly dispersed, which helps to improve the binding rate of the two. At the same time, in order to enable the amphiphilic block copolymer to quickly form nanomicelles through self-assembly, the self-assembly process usually needs to be carried out under heating conditions. The stepwise dissolution and mixing method can effectively protect the structural integrity and activity of PDRN.

[0062] As an example, step S11 includes: adding the amphiphilic block copolymer to a first portion of buffer solution at 65-80°C (e.g., but not limited to any one of 65°C, 70°C, 75°C and 80°C or any range between two) for shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly, thereby obtaining a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles.

[0063] In this embodiment, shearing at a suitable temperature provides a suitable driving force for self-assembly, thereby facilitating the efficient and thorough formation of nanomicelles from the amphiphilic block copolymer.

[0064] As an example, the mass-to-volume ratio of the amphiphilic block copolymer to the first buffer is 1 g: (3.5~4.5) mL, for example, but not limited to any one of the mass-to-volume ratios 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, or any range between the two.

[0065] In this embodiment, limiting the mass-to-volume ratio of the amphiphilic block copolymer and the first buffer solution to the above-mentioned range helps the amphiphilic block copolymer to dissolve rapidly and disperse uniformly, and also helps it to form nanomicelles through self-assembly.

[0066] As an example, in the step of adding the amphiphilic block copolymer to a first buffer solution at 65-80°C for shear treatment, the stirring speed is 8000-15000 rpm (e.g., 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 any range between two), and the stirring time is 1-3 h (e.g., but not limited to any one of 1 h, 1.5 h, 2 h, 2.5 h and 3 h or any range between two).

[0067] In this embodiment, the shearing process under high-speed conditions enables the amphiphilic block copolymer to form a large number of small and relatively uniform nanomicelles. On the one hand, the small nanomicelles have a large specific surface area, which helps to improve the binding rate of nanomicelles with PDRN. On the other hand, the relatively uniform particle size of the nanomicelles helps to improve the stability of the nanomicelles.

[0068] It should be noted that, unless otherwise specified or limited, the processes or steps in the preparation of PDRN-loaded amphiphilic block polymer nanomicelles can be set according to conventional methods in the art.

[0069] It should be noted that the nanomicelles in the embodiments of this application are spherical in shape and have an average particle size of 100~300nm.

[0070] As an example, a process flow diagram for the preparation of PDRN-loaded amphiphilic block polymer nanomicelles is exemplarily provided. Figure 1 .

[0071] Secondly, embodiments of this application provide an amphiphilic block copolymer nanomicelle loaded with PDRN, which is prepared using the preparation method provided in the first aspect embodiment.

[0072] In this application, the amphiphilic block copolymer nanomicelles loaded with PDRN prepared by the preparation method provided in the first aspect of the embodiment have the advantages of drug sustained release and strong gas / membrane permeability, so that PDRN can exert a continuous and efficient therapeutic effect.

[0073] As an example, the PDRN-loaded amphiphilic block copolymer nanomicelles have an average particle size of 80–300 nm (e.g., but not limited to point values ​​or ranges between any two of the average particle sizes of 80 nm, 100 nm, 150 nm, 200 nm, 250 nm, and 300 nm), and / or the PDI of the PDRN-loaded amphiphilic block copolymer nanomicelles is less than 0.3.

[0074] As an example, in amphiphilic block copolymer nanomicelles loaded with PDRN, the PDRN loading is 0.1 to 1 wt%, for example, but not limited to any one of 0.1 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8 wt%, and 1.0 wt%, or any range between two of them.

[0075] Thirdly, embodiments of this application provide the application of PDRN-loaded amphiphilic block copolymer nanomicelles as provided in the second aspect embodiments in the preparation of drugs that promote tissue repair, drugs that promote tissue regeneration, or anti-inflammatory drugs.

[0076] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0077] Example 1 This application provides a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN, comprising the following steps: S1 provides a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN: S11 200 g of amphiphilic block copolymer MPEG3000-PCL10000 was added to 800 mL of phosphate buffer (pH 6.8) at 80℃ and mixed evenly. The mixture was then subjected to high-speed shearing at 10000 rpm for 2 h to allow the amphiphilic block copolymer to self-assemble into nanomicelles, resulting in a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles. The solution was then cooled to room temperature for later use.

[0078] S12 Add 2 g of PDRN with a molecular weight of 235 kDa to 100 mL of phosphate buffer (pH 6.8) and stir at 150 rpm until the PDRN is completely dissolved to obtain a PDRN solution.

[0079] S13. PDRN solution is slowly added to polymer nanomicelle solution to obtain buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN.

[0080] S2 prepared PDRN-loaded amphiphilic block copolymer nanomicelles using a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN: S21 Add ice to the above buffer solution, wherein the ratio of the volume of ice added to the buffer solution to the volume of the buffer solution is 1:10, to obtain an ice-water mixed solution.

[0081] S22 Then, the ice-water mixture was sheared at a high speed of 10,000 rpm for 10 min.

[0082] S23 Then, the ice-water mixture was stirred and mixed at a low speed of 150 rpm for 1 h to obtain amphiphilic block copolymer nanomicelles loaded with PDRN.

[0083] (1) In order to better illustrate the importance of simultaneously limiting the hydrophilic polymer and hydrophobic polymer and PDRN in the amphiphilic block copolymer nanomicelles to their respective suitable ranges, corresponding examples and comparative examples were designed below (the process steps are the same as in Example 1), and the binding rate of nanomicelles and PDRN in each example and comparative example was statistically analyzed. The formula for calculating the binding rate is: binding rate = [(PDRN addition amount - PRDN unloaded amount) / PDRN addition amount] × 100%. For details, please refer to Table 1.

[0084] Table 1

[0085] Referring to Table 1, the test results of Examples 1-18 and Comparative Examples 1-4 show that when the molecular weights of the hydrophilic polymer, the hydrophobic polymer, and PDRN in the nanomicelles are all limited to the suitable range of the embodiments of this application, the nanomicelles have a high binding rate with PDRN.

[0086] The test results from Examples 1-8 and Examples 9-18 show that when the hydrophilic polymer and hydrophobic polymer in the nanomicelles are limited to 3000-4000 Da and 10000-15000 Da, respectively, the nanomicelles corresponding to the former have a higher binding rate with PDRN compared to those not within the limited range. In particular, when the hydrophilic polymer and hydrophobic polymer in the nanomicelles are limited to 3000 Da and 10000-15000 Da, respectively, the binding rate of the corresponding nanomicelles with PDRN is as high as 9%, which is significantly higher than other molecular weight combinations.

[0087] (2) In order to better illustrate the importance of limiting the mass ratio of nanomicelles and PDRN within a specific range, the following corresponding examples were designed (except for the mass ratio of nanomicelles and PDRN, the preparation process is the same as in Example 1), and the binding rate of nanomicelles and PDRN in each example was statistically analyzed. For details, please refer to Table 2.

[0088] Table 2

[0089] Referring to Table 2, the test results of Examples 19-21 show that when the mass ratio of nanomicelles to PDRN is limited to the suitable range provided in the embodiments of this application, both nanomicelles and PDRN have high binding rates. In particular, when the mass ratio of nanomicelles to PDRN is 100:1, the corresponding binding rate of nanomicelles to PDRN is as high as 9.5%, which is significantly higher than other mass ratios.

[0090] (3) In order to better illustrate the rationality of the process steps in the preparation of drug-loaded nanomicelles, corresponding examples and comparative examples are designed below, and the binding rate of nanomicelles and PDRN in each example and comparative example is statistically analyzed. For details, please refer to Examples 22-23 and Comparative Examples 5-6.

[0091] Example 22 This application provides a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN. The only difference between this method and Example 1 is that in step S22, the rotation speed of the shearing process is 8000 rpm, and the corresponding binding rate of the nanomicelles and PDRN is 5.3%.

[0092] The test results of Examples 1 and 22 show that limiting the rotation speed of the shearing process to a suitable range in the step of shearing the buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN helps to improve the binding rate of nanomicelles and PDRN.

[0093] Example 23 This application provides a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN, comprising the following steps: S1 200 g of amphiphilic block copolymer MPEG3000-PCL10000 was added to 900 mL of phosphate buffer (pH 6.8) at 80℃ and mixed evenly. The mixture was then subjected to high-speed shearing at 10000 rpm for 2 h to allow the amphiphilic block copolymer to form nanomicelles through self-assembly, resulting in a polymer nanomicelle solution containing amphiphilic block copolymer nanomicelles. The solution was then cooled to room temperature for later use.

[0094] S2 2 g of PDRN with a molecular weight of 235 kDa was slowly added to a 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 volume ratio of ice added to the buffer solution was 1:10, to obtain an ice-water mixture. The ice-water mixture was then sheared at a high speed of 10,000 rpm for 10 min. The mixture was then stirred at a low speed of 150 rpm for 1 h to obtain amphiphilic block copolymer nanomicelles loaded with PDRN, wherein the binding rate of nanomicelles and PDRN was 4.2%.

[0095] As can be seen from the test results of Examples 1 and 23, first preparing polymer nanomicelle solutions containing amphiphilic block copolymer nanomicelles and PDRN solutions respectively, and then mixing the two, that is, mixing them after both are effectively dissolved and uniformly dispersed, helps to improve the binding rate of the two.

[0096] Comparative Example 5 This application provides a comparative example of a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN. The only difference between this method and Example 1 is that in step S22, the ice-water mixture was not sheared at a high speed of 10,000 rpm, and the corresponding binding rate of nanomicelles and PDRN was 1.5%.

[0097] As can be seen from the test results of Example 1 and Comparative Example 5, performing shearing treatment at high speed followed by mixing treatment at low speed, so that PDRN is loaded both inside and outside the nanomicelles, helps to improve the binding rate of nanomicelles and PDRN.

[0098] Comparative Example 6 This application provides a comparative example of a method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN. The only difference between this method and Example 1 is that in step S22, the ice-water mixture is sheared at a speed of 6000 rpm, and the corresponding binding rate of nanomicelles and PDRN is 3.6%.

[0099] As can be seen from the test results of Examples 1, 22 and Comparative Example 6, limiting the shearing speed within the appropriate range of the embodiments of this application in the step of shearing the buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN can effectively improve the binding rate of nanomicelles and PDRN.

[0100] Test case Cell proliferation and sustained-release function test Test steps: (1) Cell culture: Fibroblasts were seeded in 96-well plates at a density of 5000 cells per well. DMEM medium containing 10% fetal bovine serum was used and the cells were cultured at 37°C and 5% carbon dioxide for 24 h to allow the cells to adhere fully.

[0101] (2) Grouping and administration: Each cell type was divided into four groups: Blank control group: containing only cell culture solvent (PBS buffer); Carrier group: Add the MPEG-PCL carrier solution prepared in Example 1 (concentration of 10 μg / mL); Free PDRN group: Add free PDRN solution (concentration of 10 μg / mL); Sample group: Add the MPEG-PCL-PDRN composite solution (concentration of 10 μg / mL) prepared in Example 1.

[0102] Each group has 5 duplicate wells, and the tests are performed at 24 h, 48 h and 72 h.

[0103] (3) Cell viability detection: At each time point, CCK-8 reagent (10 μL / well) was added to each well and cultured for another 2 h. The absorbance (OD value) at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the relative proliferation rate of each group of cells relative to the blank control group (set as 100%) was calculated.

[0104] (4) Data statistics: SPSS 22.0 software was used for one-way ANOVA. Tukey test was used for comparison between groups. p<0.05 was considered to be statistically significant.

[0105] For statistical analysis of the experimental data, please refer to Table 3. Table 3

[0106] It should be noted that in Table 3, each result is obtained by averaging the corresponding 5 data points in the table; "*" indicates that compared with the blank control group at the same time point, p<0.05, indicating a significant difference; "#" indicates that compared with the free PDRN group at the same time point, p<0.05, indicating a significant difference.

[0107] 2.3 Experimental Results (1) At 72 h, the cell viability values ​​of the free PDRN group (130.2%) and the sample group (150.5%) were significantly higher than those of the control group (100.0%), indicating that PDRN has a significant effect on promoting cell proliferation.

[0108] (2) At 48 h, the cell viability of the sample group (132.6%) was significantly higher than that of the free PDRN group (125.7%). At the same time, at 72 h, the cell viability of the sample group (150.5%) was also significantly higher than that of the free PDRN group (130.2%). This indicates that the amphiphilic block copolymer nanomicelles provided in the embodiments of this application can enable PDRN to exert its effect better.

[0109] (3) The cell activity results of the blank group and the vector group were basically the same throughout the process, indicating that the amphiphilic block copolymer nanomicelles provided in the embodiments of this application have good biocompatibility.

[0110] (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%) surpassed that of the free PDRN group (125.7%). Furthermore, 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%). This indicates that the amphiphilic block copolymer nanomicelles loaded with PDRN provided in the embodiments of this application have the effect of sustained-release of PDRN, which can prolong the degradation cycle of PDRN, so that PDRN can play a long-term and efficient role.

[0111] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for preparing amphiphilic block copolymer nanomicelles loaded with PDRN, characterized in that, Includes the following steps: S1 provides a buffer solution containing both amphiphilic block copolymer nanomicelles and PDRN, wherein the amphiphilic block copolymer nanomicelles have a molecular weight of 2000-5000 Da for the hydrophilic polymer and 5000-20000 Da for the hydrophobic polymer, and 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 The buffer solution is sheared at 8000-15000 rpm at 0-25°C and then mixed at 100-500 rpm to obtain amphiphilic block copolymer nanomicelles loaded with PDRN.

2. The preparation method according to claim 1, characterized in that, Step S2 includes: S21 Add ice to the buffer solution to obtain an ice-water mixture; S22 First, the ice-water mixture is sheared at 8000~15000 rpm for 5~20 min. S23 Then the ice-water mixture is stirred at 100~500 rpm for 2~3 hours.

3. The preparation method according to claim 1 or 2, characterized in that, 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 preparation method according to claim 3, characterized in that, The hydrophilic polymer has a molecular weight of 3000 Da, and the hydrophobic polymer has a molecular weight of 10000~15000 Da.

5. The preparation method according to claim 1 or 2, characterized in that, In the buffer solution, the mass-to-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 phosphate buffer solutions with a pH of 6.5 to 7; Or / and, the amphiphilic block copolymer nanomicelles are biblock amphiphilic block copolymer nanomicelles, wherein the hydrophilic polymer is selected from at least one of methoxy polyethylene glycol, monoalkoxy polyethylene glycol and monoacyl polyethylene glycol, and the hydrophobic polymer is selected from polycaprolactone.

6. The preparation method according to claim 1 or 2, characterized in that, Step S1 includes: S11 The amphiphilic block copolymer is dissolved in the first buffer solution and sheared to allow the amphiphilic block copolymer to self-assemble into nanomicelles, thereby obtaining a polymer nanomicelle solution containing the amphiphilic block copolymer nanomicelles. S12 Dissolves PDRN in the second buffer solution to obtain a PDRN solution; S13 The polymer nanomicelle solution and the PDRN solution are mixed.

7. The preparation method according to claim 6, characterized in that, Step S11 includes: An amphiphilic block copolymer is added to a first buffer solution at 65-80°C for shearing treatment, so that the amphiphilic block copolymer forms nanomicelles through self-assembly, resulting in a polymer nanomicelle solution containing the amphiphilic block copolymer nanomicelles.

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

9. A type of amphiphilic block copolymer nanomicelles loaded with PDRN, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 8.

10. The use of the PDRN-loaded amphiphilic block copolymer nanomicelles as described in claim 9 in the preparation of drugs that promote tissue repair, drugs that promote tissue regeneration, or anti-inflammatory drugs.

Images