PDRN porous microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a PDRN porous microsphere as well as a preparation method and application thereof. The PDRN porous microspheres are prepared from the following raw materials: PDRN, an oil phase matrix, a curing agent and metal salt, the curing agent is at least one of polyethylene glycol acrylate and polylysine; the metal salt is at least one of calcium chloride, zinc chloride, magnesium chloride and ferric chloride. The PDRN porous microsphere has the advantages of being controllable in size, excellent in disintegration resistance and excellent in swelling resistance, is high in moist heat sterilization tolerance and strength, can be used as a filling material for medical beauty or an ideal candidate material of a tissue engineering scaffold, and has potential application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to a PDRN porous microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a bioactive polymer composed of deoxyribonucleotides, and has characteristics such as promoting tissue regeneration, reducing inflammatory responses, enhancing angiogenesis, moisturizing, and antioxidation, and has received extensive attention in the fields of tissue engineering, medical aesthetics, etc.

[0003] Due to its porous structure, the porous microsphere has a larger specific surface area and space capacity. The increase in the specific surface area will attach more tissue cells, facilitating the growth and migration of tissue cells; at the same time, the porous structure is also conducive to the adsorption of proteins, the exclusion of metabolic wastes, the transport of nutrients, and the delivery of biological factors, and thus has received much attention in the fields of medicine, materials science, etc. For example, by compounding different suspension stabilizers, the porous microsphere can be made into products such as subcutaneous filling and wrinkle removal, or medical products for tissue defect repair can be obtained through 3D printing technology, and has important application value in the fields of tissue engineering and medical aesthetics.

[0004] Although the microsphere preparation technology has become mature, there is no relevant report on the preparation of PDRN porous microspheres. In addition, traditional microsphere preparation technologies mostly rely on traditional cross-linking agents such as glutaraldehyde or 1,4-butanediol diglycidyl ether (BDDE), and the residues of these traditional cross-linking agents may cause cytotoxicity problems, which do not meet the safety requirements of medical aesthetic products.

[0005] Therefore, it is an urgent problem to be solved to explore the preparation of PDRN porous microspheres without using traditional cross-linking agents such as glutaraldehyde and 1,4-butanediol diglycidyl ether. Summary of the Invention

[0006] The present invention provides a PDRN porous microsphere, which does not need to rely on traditional cross-linking agents such as glutaraldehyde and 1,4-butanediol diglycidyl ether during the preparation process, and has the advantages of controllable size, excellent anti-disintegration performance, and excellent anti-swelling property; and it has strong tolerance to moist heat sterilization and has the advantage of high strength, and can be an ideal candidate material for medical aesthetic filling materials or tissue engineering scaffolds, and has potential application value.

[0007] The present invention also provides a method for preparing PDRN porous microspheres. This method can prepare the above-mentioned PDRN porous microspheres with controllable size, excellent anti-disintegration performance and excellent anti-swelling property without relying on traditional cross-linking agents such as glutaraldehyde or 1,4-butanediol diglycidyl ether. Moreover, the PDRN porous microspheres have strong tolerance to moist heat sterilization and good thermal stability. The preparation method is convenient to operate and can be widely promoted and applied.

[0008] The present invention also provides an application of the PDRN porous microspheres in the preparation of aesthetic medical filling materials or tissue engineering scaffolds. Research by the inventors of the present invention shows that the PDRN porous microspheres have the advantages of controllable size, excellent anti-disintegration performance, excellent anti-swelling property and strong tolerance to moist heat sterilization. Therefore, they can be used as ideal candidate materials for aesthetic medical filling materials or tissue engineering scaffolds.

[0009] In the first aspect of the present invention, a PDRN porous microsphere is provided. The raw materials for preparing the PDRN porous microsphere include: PDRN, oil phase matrix, curing agent, metal salt;

[0010] The curing agent is at least one of polyethylene glycol acrylate and polylysine;

[0011] The metal salt is at least one of calcium chloride, zinc chloride, magnesium chloride, and iron chloride.

[0012] For the PDRN porous microsphere as described above, the particle size of the PDRN porous microsphere is 20 - 60 μm.

[0013] For the PDRN porous microsphere as described above, the volume-mass ratio of the PDRN, the oil phase matrix, the curing agent, and the metal salt is (0.1 - 4 g) : (100 - 400 mL) : (0.5 - 5 g) : (0.05 - 2 g).

[0014] For the PDRN porous microsphere as described above, the oil phase matrix is any one of liquid paraffin, coconut oil, silicone oil, oleic acid, and rapeseed oil.

[0015] For the PDRN porous microsphere as described above, the PDRN porous microsphere is obtained by a preparation method including the following process:

[0016] Dissolve PDRN in water to obtain an aqueous phase, and mix the oil phase matrix with a surfactant to obtain an oil phase; add the aqueous phase to the oil phase for emulsification treatment, then cool the emulsified product and add a curing agent for cross-linking and curing, then add a metal salt for metal ion cross-linking, and then perform precipitation treatment on the product after metal ion cross-linking to obtain the precipitate, which is the PDRN porous microsphere;

[0017] The surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

[0018] The second aspect of the present invention provides a method for preparing the PDRN porous microspheres as described above, comprising the following steps:

[0019] Dissolve PDRN in water and adjust the pH to 8 - 9 to obtain an aqueous phase;

[0020] Mix the oil phase matrix at the first temperature with the surfactant and then perform stirring treatment to obtain an oil phase;

[0021] Add the aqueous phase to the oil phase for emulsification treatment, and cool to the second temperature to obtain an emulsion;

[0022] Add a curing agent to the emulsion for crosslinking and curing to obtain a preliminarily crosslinked emulsion;

[0023] Add a metal salt solution to the preliminarily crosslinked emulsion, perform metal ion crosslinking under stirring, and obtain PDRN porous microspheres after precipitation treatment.

[0024] In the method for preparing PDRN porous microspheres as described above, the concentration of PDRN in the aqueous phase is 10 - 50 mg / mL;

[0025] The metal salt solution is prepared by mixing a metal salt with water, and the concentration of the metal salt in the metal salt solution is 5 - 15 mg / mL.

[0026] In the method for preparing PDRN porous microspheres as described above, the first temperature is 40 - 50 °C;

[0027] And / or, in the stirring treatment, the rotation speed is 300 - 600 r / min, and the time is 10 - 30 min;

[0028] And / or, the time for the emulsification treatment is 30 - 60 min;

[0029] And / or, the second temperature is a temperature less than or equal to 4 °C;

[0030] And / or, the time for the crosslinking and curing is 6 - 12 h;

[0031] And / or, the time for the metal ion crosslinking is 12 - 24 h.

[0032] In the method for preparing PDRN porous microspheres as described above, the volume - mass ratio of the oil phase matrix to the surfactant is (100 - 400 mL) : (0.5 - 5 g);

[0033] The surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

[0034] The third aspect of the present invention provides an application of the described PDRN porous microspheres or PDRN porous microspheres prepared by the preparation method of the described PDRN porous microspheres in the preparation of aesthetic medical filling materials or tissue engineering scaffolds.

[0035] The solution of the present invention has at least the following effects:

[0036] The PDRN porous microspheres provided by the present invention, the raw materials for their preparation include: PDRN, an oil phase matrix, a curing agent, and a metal salt. The curing agent is at least one of polyethylene glycol acrylate and polylysine, and the metal salt is at least one of calcium chloride, zinc chloride, magnesium chloride, and iron chloride; the present invention uses polyethylene glycol acrylate and / or polylysine as a curing agent to combine with PDRN to enhance the stability (anti-disintegration performance and anti-swelling property) of the porous microspheres, and then conducts metal ion cross-linking through the metal salt to further enhance the strength of the porous microspheres, so that the finally prepared PDRN porous microspheres have the advantages of controllable size, excellent anti-disintegration performance, and excellent anti-swelling property; and the PDRN porous microspheres have strong tolerance to moist heat sterilization and have the advantage of high strength; in addition, the PDRN porous microspheres provided by the present invention do not need to rely on traditional cross-linking agents such as glutaraldehyde and 1,4-butanediol diglycidyl ether, and overcome the adverse reaction problems such as inflammatory reactions and tissue damage caused by the residues of traditional cross-linking agents. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 SEM image of the PDRN porous microspheres in Example 1 of the present invention;

[0039] Figure 2 SEM image of the PDRN porous microspheres in Example 2 of the present invention;

[0040] Figure 3 SEM image of the PDRN porous microspheres in Example 3 of the present invention;

[0041] Figure 4 SEM image of the PDRN porous microspheres in Example 4 of the present invention;

[0042] Figure 5 SEM image of the PDRN porous microspheres in Example 5 of the present invention;

[0043] Figure 6 SEM image of the PDRN porous microspheres in Comparative Example 1 of the present invention;

[0044] Figure 7 Strength test of the PDRN porous microspheres in Example 1 of the present invention, where Figure 7 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 1, Figure 7 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Example 1;

[0045] Figure 8 Strength test of the PDRN porous microspheres in Example 2 of the present invention, where Figure 8 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 2, Figure 8 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Example 2;

[0046] Figure 9 Strength test of the PDRN porous microspheres in Example 3 of the present invention, where Figure 9 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 3, Figure 9 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Example 3;

[0047] Figure 10 Strength test of the PDRN porous microspheres in Example 4 of the present invention, where Figure 10 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 4, Figure 10 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Example 4;

[0048] Figure 11 Strength test of the PDRN porous microspheres in Example 5 of the present invention, where Figure 11 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 5, Figure 11 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Example 5;

[0049] Figure 12 Strength test of the PDRN porous microspheres in Comparative Example 1 of the present invention, where Figure 12 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Comparative Example 1, Figure 12 b is the optical microscope image of the PDRN porous microsphere solution after sterilization in Comparative Example 1. Detailed implementation method

[0050] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0051] For those technologies or conditions not specified in the embodiments of the present invention, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0052] It should be noted that the descriptions involving "first", "second", etc. in the present invention are used to distinguish similar objects, rather than to describe a specific order or sequence, and therefore should not be construed as a limitation to the present invention.

[0053] The first aspect of the present invention provides a PDRN porous microsphere. The raw materials for preparing the PDRN porous microsphere include: PDRN, an oil-phase matrix, a curing agent, and a metal salt; the curing agent is at least one of polyethylene glycol acrylate and polylysine; the metal salt is at least one of calcium chloride, zinc chloride, magnesium chloride, and iron chloride.

[0054] The present invention does not make special limitations on the sources of the above raw materials, which can be obtained through commercial channels or prepared by methods known in the art.

[0055] In the present invention, a curing agent is used to combine with PDRN to enhance the stability (anti-degradation performance and anti-swelling property) of the porous microsphere, and a metal salt is used for metal ion cross-linking to enhance the strength of the porous microsphere. Finally, the prepared PDRN porous microsphere has the advantages of controllable size, excellent anti-disintegration performance, and excellent anti-swelling property; and the PDRN porous microsphere has strong tolerance to moist heat sterilization and has the advantage of high strength.

[0056] In a specific embodiment, the particle size of the above PDRN porous microsphere is 20 - 60 μm, and further preferably 40 - 50 μm.

[0057] When the particle size of the PDRN porous microsphere is within the above range, a pore structure can be seen on the surface of the PDRN porous microsphere, the sphericity is consistent, the monodispersity is good, and the size distribution is narrow.

[0058] In a specific embodiment, the volume mass ratio of the above-mentioned PDRN, the above-mentioned oil phase matrix, the above-mentioned curing agent and the above-mentioned metal salt is (0.1 - 4 g):(100 - 400 mL):(0.5 - 5 g):(0.05 - 2 g), and it can be preferably 0.5 g:200 mL:(0.5 - 2 g):(0.05 - 1 g).

[0059] When the parameters of the mass ratio of PDRN, the oil phase matrix, the curing agent and the metal salt are within the above ranges, PDRN porous microspheres with controllable size, excellent anti-disintegration performance, excellent anti-swelling property and strong tolerance to moist heat sterilization can be prepared.

[0060] In a specific embodiment, the above-mentioned oil phase matrix is any one of liquid paraffin, coconut oil, silicone oil, oleic acid, rapeseed oil, and can be preferably liquid paraffin.

[0061] When the above substances are selected as the oil phase matrix, the oil phase matrix can be mixed with a surfactant to prepare an oil phase, and this oil phase can smoothly form a water-in-oil system with the water phase (PDRN solution).

[0062] In a specific embodiment, the above-mentioned PDRN porous microspheres are obtained by a preparation method including the following process:

[0063] Dissolve PDRN in water to obtain a water phase, and mix the oil phase matrix with a surfactant to obtain an oil phase; add the water phase to the oil phase for emulsification treatment, then cool the emulsified product and add a curing agent for crosslinking and curing, then add a metal salt for metal ion crosslinking, and then perform precipitation treatment on the product after metal ion crosslinking to obtain the precipitate, which is the PDRN porous microspheres;

[0064] The surfactant is any one of Span - 60, Span - 80, Tween - 60, Tween - 80.

[0065] The second aspect of the present invention provides a preparation method of the above-mentioned PDRN porous microspheres, including the following steps:

[0066] Dissolve PDRN in water and adjust the pH to 8 - 9 to obtain a water phase;

[0067] Mix the oil phase matrix at a first temperature with a surfactant and then perform stirring treatment to obtain an oil phase;

[0068] Add the water phase to the oil phase for emulsification treatment, and cool to a second temperature to obtain an emulsion;

[0069] Add a curing agent to the emulsion for crosslinking and curing to obtain a preliminarily crosslinked emulsion;

[0070] A metal salt solution is added to the initially crosslinked emulsion, and metal ion crosslinking is carried out under stirring. After precipitation treatment, PDRN porous microspheres are obtained.

[0071] The present invention does not particularly limit the specific manner of the above-mentioned stirring treatment. In some embodiments, an oil phase matrix and a surfactant at a first temperature can be mixed and then placed in a blender for mechanical stirring.

[0072] The object of preparation in the present invention is PDRN porous microspheres. Specifically, first, PDRN is dissolved in water and the pH is adjusted to 8 - 9 to obtain an aqueous phase; then, an oil phase matrix and a surfactant at a first temperature are mixed to obtain a first mixture, and the first mixture is subjected to a first stirring treatment to make the oil phase matrix and the surfactant in the first mixture uniformly mixed to obtain an oil phase; the aqueous phase is added to the oil phase to obtain a second mixture, and the second mixture is subjected to emulsification treatment and cooled to a second temperature to obtain an emulsion. The emulsification treatment is to achieve the uniform dispersion of the aqueous phase and the oil phase to form a stable water-in-oil emulsion system, laying a foundation for the formation of the subsequent porous structure. The purpose of cooling to the second temperature is to provide a suitable environment for subsequent crosslinking and curing; a curing agent is added to the emulsion for crosslinking and curing to make the curing agent combine with PDRN in the emulsion to obtain an initially crosslinked emulsion; a calcium chloride solution is added to the initially crosslinked emulsion, and metal ion crosslinking is carried out under stirring to obtain a product after metal ion crosslinking; the product after metal ion crosslinking is subjected to precipitation treatment to precipitate the precipitate, and PDRN porous microspheres with controllable size, excellent anti-disintegration performance and excellent anti-swelling property are prepared, and it has strong tolerance to moist heat sterilization and has the advantage of high strength.

[0073] In a specific embodiment, the concentration of PDRN in the above aqueous phase is 10 - 50 mg / mL.

[0074] In a specific embodiment, the above metal salt solution is prepared by mixing a metal salt with water, and the concentration of the metal salt in the metal salt solution is 5 - 15 mg / mL.

[0075] In a specific embodiment, the above first temperature is 40 - 50 °C.

[0076] When the parameter of the first temperature is within the above range, the viscosity of the oil phase matrix can be reduced, and the uniform mixing of the oil phase matrix and the surfactant (such as Span - 80) can be promoted, thereby obtaining an oil phase.

[0077] Exemplarily, the first temperature can be any one of 40 °C, 42 °C, 44 °C, 46 °C, 48 °C, 50 °C or the range composed of any two of them.

[0078] In a specific embodiment, during the above stirring treatment, the rotation speed is 300 - 600 r / min and the time is 10 - 30 min.

[0079] The present invention does not particularly limit the temperature during the stirring treatment. For example, the stirring treatment of the present invention can be carried out at room temperature.

[0080] During the stirring treatment, when the parameters of the rotation speed and time are within the above ranges, it is possible to prevent the oil phase from agglomerating, which is beneficial to uniformly mixing the oil phase matrix and the surfactant.

[0081] In a specific embodiment, the time of the above emulsification treatment is 30 - 60 min.

[0082] When the time of the emulsification treatment is within the above range, it is possible to achieve uniform dispersion of the water phase and the oil phase, forming a stable water-in-oil emulsion system, which lays a foundation for the formation of the subsequent porous structure.

[0083] In a specific embodiment, the above second temperature is a temperature less than or equal to 4°C, and can preferably be 0 - 4°C.

[0084] When the parameter of the second temperature is within the above range, it provides a suitable environment for subsequent crosslinking and curing, and protects the integrity of the PDRN molecular chain.

[0085] In a specific embodiment, the time of the above crosslinking and curing is 6 - 12 h.

[0086] When the time of the crosslinking and curing is within the above range, it is possible to enable the curing agent to fully combine with the PDRN in the emulsion, which is beneficial to enhancing the stability of the PDRN porous microspheres.

[0087] In a specific embodiment, the time of the above metal ion crosslinking is 12 - 24 h.

[0088] When the time of the second stirring treatment is within the above range, it is possible to enable the metal ions to fully carry out metal ion crosslinking with the initially crosslinked emulsion, and finally prepare PDRN porous microspheres with controllable size, excellent anti-disintegration performance, excellent anti-swelling property, and strong tolerance to moist heat sterilization (high strength).

[0089] In a specific embodiment, the volume-mass ratio of the above oil phase matrix to the surfactant is (100 - 400 mL) : (0.5 - 5 g), and can preferably be 200 mL : 1 g; the surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

[0090] When the parameter of the mass ratio of the oil phase matrix to the surfactant is within the above range, it is possible to make the oil phase matrix and the surfactant more matched, and obtain a stable oil phase.

[0091] In a specific embodiment, it further includes washing treatment, centrifugation treatment and drying treatment of the PDRN porous microspheres.

[0092] The present invention does not particularly limit the specific manner of the above drying treatment. For example, the above drying treatment is vacuum drying treatment.

[0093] Specifically, the precipitate after precipitation treatment is washed to remove the residual unreacted curing agent, free PDRN, surfactant and oil phase impurities in the precipitate, and the washed precipitate is obtained. Finally, the washed precipitate is centrifuged and dried to prepare PDRN porous microspheres.

[0094] The third aspect of the present invention provides an application of the above PDRN porous microspheres or PDRN porous microspheres prepared by the preparation method of the above PDRN porous microspheres in the preparation of aesthetic medical filling materials or tissue engineering scaffolds. Through the research of the present inventors, it is shown that the PDRN porous microspheres have the advantages of good dispersibility, controllable size, excellent anti-disintegration performance and strong tolerance to moist heat sterilization. Therefore, they can be used as ideal candidate materials for aesthetic medical filling materials or tissue engineering scaffolds.

[0095] The embodiments of the present invention will be described in detail below through the following examples.

[0096] Example 1

[0097] The PDRN porous microspheres provided in this example are obtained by a preparation method including the following process:

[0098] (1) Dissolve polydeoxyribonucleotide (PDRN) in water, and adjust the pH to 8 with sodium hydroxide solution to obtain a PDRN solution with pH = 8 and a concentration of 50 mg / mL, and use the PDRN solution as the aqueous phase;

[0099] (2) Heat 200 mL of liquid paraffin to 50 °C, then mix the liquid paraffin at 50 °C with 1 g of Span-80 to obtain a first mixture, and mechanically stir the first mixture at a speed of 300 r / min for 30 min to obtain the oil phase;

[0100] (3) Add 10 mL of the aqueous phase to the oil phase to obtain a second mixture, and perform emulsification treatment on the second mixture for 30 min, and cool to 4 °C to obtain an emulsion;

[0101] (4) Add 1 g of poly(ethylene glycol) acrylate with a molecular weight of 575 to the emulsion for crosslinking and curing for 6 h to obtain a preliminarily crosslinked emulsion;

[0102] (5) Dissolve calcium chloride in water to prepare a calcium chloride solution with a concentration of 10 mg / mL. Then add 5 mL of the calcium chloride solution to the initially cross-linked emulsion, and carry out metal ion cross-linking for 12 h under stirring to obtain the product after metal ion cross-linking. Then place the product after metal ion cross-linking in 1 L of ethanol for precipitation treatment, let it stand to precipitate the precipitate, then wash the precipitate with deionized water to obtain the washed precipitate, and finally carry out centrifugation treatment and vacuum drying treatment on the washed precipitate to obtain PDRN porous microspheres.

[0103] Example 2

[0104] The preparation of the PDRN porous microspheres provided in this example is basically the same as that in Example 1, except that:

[0105] Replace 1 g of polyethylene glycol acrylate with a molecular weight of 575 with 1 g of polylysine with a molecular weight of 3500.

[0106] Example 3

[0107] The preparation of the PDRN porous microspheres provided in this example is basically the same as that in Example 1, except that:

[0108] Replace calcium chloride with zinc chloride.

[0109] Example 4

[0110] The preparation of the PDRN porous microspheres provided in this example is basically the same as that in Example 1, except that:

[0111] Replace 1 g of polyethylene glycol acrylate with a molecular weight of 575 with 0.5 g of polyethylene glycol acrylate with a molecular weight of 575.

[0112] Example 5

[0113] The preparation of the PDRN porous microspheres provided in this example is basically the same as that in Example 1, except that:

[0114] Replace 1 g of polyethylene glycol acrylate with a molecular weight of 575 with 2 g of polyethylene glycol acrylate with a molecular weight of 575.

[0115] Comparative Example 1 (without metal ion cross-linking)

[0116] The PDRN porous microspheres provided in this comparative example are obtained by a preparation method including the following process:

[0117] (1) Dissolve polydeoxyribonucleotide (PDRN) in water, adjust the pH to 8 with sodium hydroxide solution to obtain a PDRN solution with pH = 8 and a concentration of 50 mg / mL, and use the PDRN solution as the aqueous phase;

[0118] (2) Heat 200 mL of liquid paraffin to 50 °C, then mix the liquid paraffin at 50 °C with 1 g of Span-80 to obtain a first mixture, and mechanically stir the first mixture at a speed of 300 r / min for 30 min to obtain an oil phase;

[0119] (3) Add 10 mL of the aqueous phase to the oil phase to obtain a second mixture, and perform emulsification treatment on the second mixture for 30 min, and cool to 4 °C to obtain an emulsion;

[0120] (4) Add 1 g of poly(ethylene glycol) acrylate with a molecular weight of 575 to the emulsion for crosslinking and curing for 6 h to obtain a crosslinked and cured product, then place the crosslinked and cured product in 1 L of ethanol for precipitation treatment, let it stand for the precipitate to precipitate, then wash the precipitate with deionized water to obtain a washed precipitate, and finally perform centrifugation treatment and vacuum drying treatment on the washed precipitate to obtain PDRN porous microspheres.

[0121] Performance Test

[0122] 1. Scanning Electron Microscope Test

[0123] The PDRN porous microspheres in Examples 1-5 and Comparative Example 1 were respectively tested by scanning electron microscope (SEM), and the results are as Figures 1 - 6 shown; Figure 1 This is the SEM image of the PDRN porous microspheres in Example 1 of the present invention; Figure 2 This is the SEM image of the PDRN porous microspheres in Example 2 of the present invention; Figure 3 This is the SEM image of the PDRN porous microspheres in Example 3 of the present invention; Figure 4 This is the SEM image of the PDRN porous microspheres in Example 4 of the present invention; Figure 5 This is the SEM image of the PDRN porous microspheres in Example 5 of the present invention; Figure 6 This is the SEM image of the PDRN porous microspheres in Comparative Example 1 of the present invention.

[0124] It can be seen from Figure 1 that the PDRN porous microspheres in Example 1 are spherical, have a honeycomb-like pore structure, good monodispersity, and the particle size distribution is between 20-60 μm.

[0125] It can be seen from Figure 2 that the PDRN porous microspheres in Example 2 are nearly spherical, with slight deformation at the edges, rough surfaces, visible pore structures, and the particle size distribution is between 20-60 μm.

[0126] It can be seen from Figure 3It can be seen that the PDRN porous microspheres in Example 3 are nearly spherical, with smooth edges, a pore structure visible on the surface, consistent sphericity, good monodispersity, a narrow size distribution, and a particle size distribution between 40 - 50 μm.

[0127] From Figure 4 It can be seen that the PDRN porous microspheres in Example 4 are nearly spherical, with a visible pore structure, poor dispersibility, aggregation occurring, significant size differences, and a particle size distribution between 2 - 40 μm.

[0128] From Figure 5 It can be seen that the PDRN porous microspheres in Example 5 are nearly spherical, with a significantly reduced visible pore structure on the surface, being denser, and a particle size distribution between 50 - 60 μm.

[0129] From Figure 6 It can be seen that the PDRN porous microspheres in Comparative Example 1 have a nearly spherical profile, smooth edges, a pore structure visible on the surface, consistent sphericity, good monodispersity, and a particle size concentrated distribution between 20 - 40 μm.

[0130] 2. Anti - degradation performance test

[0131] The anti - degradation performance of the PDRN porous microspheres in Examples 1 - 5 and Comparative Example 1 was tested respectively. The specific method is as follows: 0.5 g of PDRN porous microspheres, 1 mL of deoxyribonuclease I buffer, and 0.25 mL of deoxyribonuclease I (1000 U / mL) were mixed evenly and placed in a 37 °C water bath for 2 hours. The enzyme was removed by boiling, the supernatant was taken by centrifugation, and the absorbance of the PDRN porous microspheres at 595 was detected according to the diphenylamine method to determine the initial nucleic acid content, and the absorbance of the supernatant at 595 was detected according to the diphenylamine method to determine the degraded nucleic acid content. The enzymatic hydrolysis rate was calculated (enzymatic hydrolysis rate = (degraded nucleic acid content / initial nucleic acid content) × 100%). The lower the enzymatic hydrolysis rate, the better the anti - degradation performance of the PDRN porous microspheres. The specific calculation results are shown in Table 1.

[0132] Table 1 Results of the enzymatic hydrolysis rate of PDRN porous microspheres

[0133] Item Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Enzymolysis rate 50％ 60％ 9％ 10％ 25％ 90％

[0134] As can be seen from Table 1, the enzymatic hydrolysis rates of the PDRN porous microspheres in Examples 1 - 5 of the present invention are all lower than those of the PDRN porous microspheres in Comparative Example 1, with the lowest being 9%, indicating that the PDRN porous microspheres in the examples of the present invention have excellent anti - degradation performance.

[0135] 3. Anti - swelling test

[0136] The swelling ratio tests were respectively carried out on the PDRN porous microspheres in Examples 1-5 and Comparative Example 1. The specific method is as follows: Take 0.05 g of PDRN porous microspheres, place them in a vacuum drying oven at 60 °C for 6 h, weigh them after cooling to room temperature, obtain the dried PDRN porous microspheres and weigh them with an analytical balance, and record the initial mass W0; Immerse the dried PDRN porous microspheres in 10 mL of physiological saline, place them in a constant temperature shaking water bath at 25 °C, sample at the time points of 10 min and 30 min after immersion, and quickly separate the PDRN porous microspheres from the solvent with filter paper; Then spread the separated PDRN porous microspheres on the filter paper, gently touch to absorb the free liquid on the surface, weigh with an analytical balance, record the mass W1, calculate the swelling ratio according to the following formula, and the calculation results are shown in Table 2. The lower the swelling ratio, the better the anti-swelling property of the PDRN porous microspheres;

[0137] Swelling ratio = (W1 - W0) / W0 × 100%.

[0138] Table 2 Swelling ratio results of PDRN porous microspheres

[0139] Item Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Swelling rate (10 min) 108％ 230％ 48％ 88％ 60％ 410％ Swelling rate (30 min) 190％ 350％ 85％ 117％ 98％ 600％

[0140] As can be seen from Table 2, the swelling ratios of the PDRN porous microspheres in Examples 1-5 of the present invention are all lower than those of the PDRN porous microspheres in Comparative Example 1, indicating that the PDRN porous microspheres in Examples 1-5 of the present invention have excellent anti-swelling properties, and among them, the PDRN porous microspheres in Example 3 have the most excellent anti-swelling properties.

[0141] 4. Strength test

[0142] The strength tests were respectively carried out on the PDRN porous microspheres in Examples 1-5 and Comparative Example 1 by the method of moist heat sterilization. The specific method is as follows:

[0143] Mix the PDRN porous microspheres with water to prepare a PDRN porous microsphere solution with a concentration of 20 mg / mL (the PDRN porous microsphere solution before sterilization);

[0144] Carry out moist heat sterilization on the PDRN porous microsphere solution at 121 °C for 20 min to obtain the sterilized PDRN porous microsphere solution;

[0145] Respectively place the PDRN porous microsphere solutions before and after sterilization under an optical microscope to observe the structure of the PDRN porous microspheres.

[0146] Figure 7 For the strength test of the PDRN porous microspheres in Example 1 of the present invention, where Figure 7 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 1, Figure 7b is the optical microscope image of the sterilized PDRN porous microsphere solution of Example 1;

[0147] Figure 8 This is the strength test of the PDRN porous microspheres in Example 2 of the present invention, where Figure 8 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 2, Figure 8 b is the optical microscope image of the sterilized PDRN porous microsphere solution in Example 2;

[0148] Figure 9 This is the strength test of the PDRN porous microspheres in Example 3 of the present invention, where Figure 9 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 3, Figure 9 b is the optical microscope image of the sterilized PDRN porous microsphere solution in Example 3;

[0149] Figure 10 This is the strength test of the PDRN porous microspheres in Example 4 of the present invention, where Figure 10 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 4, Figure 10 b is the optical microscope image of the sterilized PDRN porous microsphere solution in Example 4;

[0150] Figure 11 This is the strength test of the PDRN porous microspheres in Example 5 of the present invention, where Figure 11 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 5, Figure 11 b is the optical microscope image of the sterilized PDRN porous microsphere solution in Example 5;

[0151] Figure 12 This is the strength test of the PDRN porous microspheres in Comparative Example 1 of the present invention, where Figure 12 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Comparative Example 1, Figure 12 b is the optical microscope image of the sterilized PDRN porous microsphere solution in Comparative Example 1.

[0152] Consisting of Figures 7 - 12It can be seen that before sterilization, the PDRN porous microspheres of Comparative Example 1 would rapidly swell in water, while the morphologies of the PDRN porous microspheres of Examples 1-5 basically did not swell; after sterilization, the PDRN porous microspheres of Comparative Example 1 no longer existed and were completely dissolved in water. The PDRN porous microspheres of Example 1 retained a partial intact spherical structure, the PDRN porous microspheres of Example 2 were broken into fragments, the PDRN porous microspheres of Example 3 had a complete structure, the PDRN porous microspheres of Example 4 underwent severe aggregation to form lamellae, and the dispersibility of the microspheres was poor. The edge contours of the PDRN porous microspheres of Example 5 were blurred and deformed. The above results illustrate that the PDRN porous microspheres provided in Examples 1-5 of the present invention have stronger tolerance to moist heat sterilization than the PDRN porous microspheres of Comparative Example 1 and have the advantage of high strength. Among them, the PDRN porous microspheres of Example 3 have the highest tolerance to moist heat sterilization.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PDRN porous microsphere, characterized in that, The raw materials for preparing the PDRN porous microspheres include: PDRN, an oil-phase matrix, a curing agent, and a metal salt; The curing agent is at least one of polyethylene glycol acrylate and polylysine; The metal salt is at least one of calcium chloride, zinc chloride, magnesium chloride, and iron chloride.

2. The PDRN porous microspheres according to claim 1, characterized in that, The particle size of the PDRN porous microspheres is 20 - 60 μm.

3. The PDRN porous microspheres according to claim 1, characterized in that, The volume-mass ratio of the PDRN, the oil-phase matrix, the curing agent, and the metal salt is (0.1 - 4 g) : (100 - 400 mL) : (0.5 - 5 g) : (0.05 - 2 g).

4. The PDRN porous microspheres according to claim 3, characterized in that, The oil-phase matrix is any one of liquid paraffin, coconut oil, silicone oil, oleic acid, and rapeseed oil.

5. The PDRN porous microspheres according to claim 1, characterized in that, The PDRN porous microspheres are obtained by a preparation method including the following process: Dissolve PDRN in water to obtain an aqueous phase, and mix the oil-phase matrix with a surfactant to obtain an oil phase; add the aqueous phase to the oil phase for emulsification treatment, then cool the emulsified product and add a curing agent for crosslinking and curing, then add a metal salt for metal ion crosslinking, and then perform precipitation treatment on the product after metal ion crosslinking to obtain the precipitate, which is the PDRN porous microspheres; The surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

6. A method for preparing the PDRN porous microspheres according to any one of claims 1-5, characterized in that, It includes the following steps: Dissolve PDRN in water and adjust the pH to 8 - 9 to obtain an aqueous phase; Mix the oil-phase matrix at a first temperature with a surfactant and then perform stirring treatment to obtain an oil phase; Add the aqueous phase to the oil phase for emulsification treatment, and cool to a second temperature to obtain an emulsion; Add a curing agent to the emulsion for crosslinking and curing to obtain a preliminarily crosslinked emulsion; Add a metal salt solution to the preliminarily crosslinked emulsion, perform metal ion crosslinking under stirring, and obtain PDRN porous microspheres after precipitation treatment.

7. The preparation method of the PDRN porous microspheres according to claim 6, characterized in that, The concentration of PDRN in the aqueous phase is 10 - 50 mg / mL; The metal salt solution is prepared by mixing a metal salt with water, and the concentration of the metal salt in the metal salt solution is 5 - 15 mg / mL.

8. The preparation method of the PDRN porous microspheres according to claim 6, characterized in that, The first temperature is 40 - 50 °C; And / or, in the stirring treatment, the rotation speed is 300 - 600 r / min, and the time is 10 - 30 min; And / or, the time of the emulsification treatment is 30 - 60 min; And / or, the second temperature is a temperature less than or equal to 4 °C; And / or, the time of the crosslinking and curing is 6 - 12 h; And / or, the time of the metal ion crosslinking is 12 - 24 h.

9. The preparation method of the PDRN porous microspheres according to claim 6, wherein, The volume-mass ratio of the oil-phase matrix to the surfactant is (100 - 400 mL) : (0.5 - 5 g); The surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

10. Use of the PDRN porous microspheres according to any one of claims 1 - 5 or the PDRN porous microspheres prepared by the preparation method of the PDRN porous microspheres according to any one of claims 6 - 9 in the preparation of a filling material for medical aesthetics or a tissue engineering scaffold.

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