Pdrn porous microspheres, preparation method and application thereof

Abstract

The present application relates to the technical field of biological materials, and particularly relates to a PDRN porous microsphere, a preparation method and application thereof. The preparation raw material of the PDRN porous microsphere comprises 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. The PDRN porous microsphere has the advantages of controllable size, excellent anti-disintegration performance and excellent anti-swelling performance. In addition, the PDRN porous microsphere has strong resistance to moist heat sterilization and high strength, and can be used as an ideal candidate material for medical and cosmetic filling materials or tissue engineering scaffolds, and has potential application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a PDRN porous microsphere and a preparation method and application thereof. BACKGROUND

[0002] Polydeoxyribonucleotide (PDRN) is a kind of bioactive polymer composed of deoxyribonucleotides, which has the characteristics of promoting tissue regeneration, reducing inflammatory response, enhancing angiogenesis, moisturizing and antioxidant, and has been widely concerned in the fields of tissue engineering and medical beauty.

[0003] The porous microsphere has a larger specific surface area and space capacity due to its porous structure. The increase of the specific surface area will attach more tissue cells, which is conducive to the growth and migration of tissue cells. At the same time, the porous structure is also conducive to the adsorption of proteins, the removal of metabolic waste, the transport of nutrients and the transmission of biological factors, so it has attracted much attention in the fields of medicine and material science. For example, the porous microsphere can be made into subcutaneous filling, wrinkle removal and other products by compounding different suspension stabilizers, and it can also be obtained by 3D printing technology to obtain medical products for tissue defect repair, which has important application value in the fields of tissue engineering and medical beauty.

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

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

[0006] The present application 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 in the preparation process, has the advantages of controllable size, excellent anti-disintegration performance and excellent anti-swelling performance; and it has strong resistance to moist heat sterilization and high strength, which can be used as an ideal candidate material for medical beauty filling materials or tissue engineering scaffolds, and has potential application value.

[0007] The application further provides a preparation method of the PDRN porous microspheres, which does not need to rely on traditional cross-linking agents such as glutaraldehyde or 1,4-butanediol diglycidyl ether, and can prepare the PDRN porous microspheres with controllable size, excellent anti-disintegration performance and excellent anti-swelling performance, and the PDRN porous microspheres have strong resistance to moist heat sterilization and good thermal stability; the preparation method is convenient to operate and can be widely applied.

[0008] The application further provides application of the PDRN porous microspheres in preparation of medical and cosmetic filling materials or tissue engineering scaffolds.

[0009] The first aspect of the application provides a PDRN porous microsphere, and preparation raw materials of the PDRN porous microsphere include PDRN, an oil phase matrix, a curing agent and a 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] The PDRN porous microsphere has a particle size of 20-60 μm.

[0013] The PDRN, the oil phase matrix, the curing agent and the metal salt have a volume mass ratio of (0.1-4 g):(100-400 mL):(0.5-5 g):(0.05-2 g).

[0014] The oil phase matrix is any one of liquid paraffin, coconut oil, silicone oil, oleic acid and rapeseed oil.

[0015] The PDRN porous microsphere is obtained by a preparation method including the following process:

[0016] The PDRN is dissolved in water to obtain an aqueous phase, and the oil phase matrix is mixed with a surfactant to obtain an oil phase; the aqueous phase is added to the oil phase for emulsification treatment, and then the product after the emulsification treatment is cooled and added with the curing agent for cross-linking and curing, and then the metal salt is added for metal ion cross-linking, and then the product after the metal ion cross-linking is subjected to precipitation treatment, and the obtained precipitate is the PDRN porous microsphere.

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

[0018] The second aspect of the present application provides a preparation method of the PDRN porous microspheres, 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 then cool to a second temperature to obtain an emulsion;

[0022] Add a solidifying agent to the emulsion for cross-linking solidification to obtain a primary cross-linked emulsion;

[0023] Add a metal salt solution to the primary cross-linked emulsion, perform metal ion cross-linking under stirring, and then perform precipitation treatment to obtain PDRN porous microspheres.

[0024] The preparation method of the PDRN porous microspheres as described above, wherein 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] The preparation method of the PDRN porous microspheres as described above, wherein the first temperature is 40-50℃;

[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 emulsification treatment time is 30-60 min;

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

[0030] And / or, the cross-linking solidification time is 6-12 h;

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

[0032] The preparation method of the PDRN porous microspheres as described above, wherein the volume-to-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, Tween-80.

[0034] The third aspect of the present application provides an application of the PDRN porous microsphere or the PDRN porous microsphere prepared by the preparation method of the PDRN porous microsphere in preparing a medical filling material or a tissue engineering scaffold.

[0035] The scheme of the present application has at least the following effects:

[0036] The PDRN porous microsphere provided by the present application has the following advantages: the preparation raw material of the PDRN porous microsphere comprises 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; the polyethylene glycol acrylate and / or polylysine are used as the curing agent to combine with the PDRN to enhance the stability (anti-disintegration performance and anti-swelling performance) of the porous microsphere, and then the metal ions are crosslinked by the metal salt to further enhance the strength of the porous microsphere, so that the finally prepared PDRN porous microsphere has the advantages of controllable size, excellent anti-disintegration performance and excellent anti-swelling performance; in addition, the PDRN porous microsphere has the advantages of strong resistance to moist heat sterilization and high strength; and the PDRN porous microsphere provided by the present application does not need to rely on traditional crosslinking agents such as glutaraldehyde and 1,4-butanediol diglycidyl ether, and overcomes the problems of inflammatory reactions and tissue damage caused by the residues of traditional crosslinking agents. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0038] Figure 1 SEM image of the PDRN porous microsphere in Example 1 of the present application;

[0039] Figure 2 SEM image of the PDRN porous microsphere in Example 2 of the present application;

[0040] Figure 3 SEM image of the PDRN porous microsphere in Example 3 of the present application;

[0041] Figure 4 SEM image of the PDRN porous microsphere in Example 4 of the present application;

[0042] Figure 5 SEM image of the PDRN porous microsphere in Example 5 of the present application;

[0043] Figure 6 SEM image of PDRN porous microspheres in Inventive Example 1;

[0044] Figure 7 Strength test of PDRN porous microspheres in Inventive Example 1, wherein Figure 7 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 1, Figure 7 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 1;

[0045] Figure 8 Strength test of PDRN porous microspheres in Inventive Example 2, wherein Figure 8 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 2, Figure 8 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 2;

[0046] Figure 9 Strength test of PDRN porous microspheres in Inventive Example 3, wherein Figure 9 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 3, Figure 9 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 3;

[0047] Figure 10 Strength test of PDRN porous microspheres in Inventive Example 4, wherein Figure 10 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 4, Figure 10 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 4;

[0048] Figure 11 Strength test of PDRN porous microspheres in Inventive Example 5, wherein Figure 11 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 5, Figure 11 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 5;

[0049] Figure 12 Strength test of PDRN porous microspheres in Inventive Example 1, wherein Figure 12 a is an optical microscope image of PDRN porous microspheres solution before sterilization of Example 1, Figure 12 b is an optical microscope image of PDRN porous microspheres solution after sterilization of Example 1. DETAILED DESCRIPTION

[0050] In order to make the objects, technical solutions and advantages of the present application clearer, the following will combine the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0051] Unless otherwise specified, the technical or conditions not specified in the embodiments of the present application are carried out according to the technical or conditions described in the literature in the art or according to the product manual. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by purchase.

[0052] It should be noted that the descriptions of "first", "second" and the like in the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence, and therefore should not be understood as a limitation on the present application.

[0053] The first aspect of the present application provides a PDRN porous microsphere, and the preparation raw materials of the PDRN porous microsphere comprise: 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.

[0054] The present application does not particularly limit the source of the above-mentioned raw materials, which can be purchased through commercial channels or prepared by known methods in the art.

[0055] In the present application, the curing agent is used to combine with PDRN to enhance the stability (anti-degradation performance and anti-swelling performance) of the porous microsphere, and the metal salt is used for metal ion crosslinking to enhance the strength of the porous microsphere, so that the finally prepared PDRN porous microsphere has the advantages of controllable size, excellent anti-disintegration performance and excellent anti-swelling performance; and the PDRN porous microsphere has strong resistance to moist heat sterilization and has the advantage of high strength.

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

[0057] When the particle size of the PDRN porous microsphere is within the above-mentioned range, the surface of the PDRN porous microsphere has visible pore structure, consistent sphericity, good monodispersity and narrow size distribution.

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

[0059] When the mass ratio of the PDRN, the oil phase base, the curing agent and the metal salt is within the above range, the PDRN porous microspheres with controllable size, excellent anti-disintegration performance, excellent anti-swelling performance and strong resistance to moist heat sterilization can be prepared.

[0060] In one embodiment, the oil phase base is any one of liquid paraffin, coconut oil, silicone oil, oleic acid and rapeseed oil, which can be preferably liquid paraffin.

[0061] When the above-mentioned substance is used as the oil phase base, the oil phase base can be mixed with the surfactant to prepare the oil phase, which can form a water-in-oil system with the water phase (PDRN solution) smoothly.

[0062] In one embodiment, the PDRN porous microspheres are prepared by a preparation method comprising the following steps:

[0063] After dissolving the PDRN in water, a water phase is obtained, and the oil phase base is mixed with the surfactant to obtain an oil phase; the water phase is added to the oil phase for emulsification treatment, and then the product after the emulsification treatment is cooled and added with the curing agent for cross-linking and curing, followed by adding the metal salt for metal ion cross-linking, and then the product after the metal ion cross-linking is subjected to precipitation treatment, and the obtained precipitate is the PDRN porous microspheres;

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

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

[0066] The PDRN is dissolved in water and the pH is adjusted to 8-9 to obtain a water phase;

[0067] The oil phase base at the first temperature is mixed with the surfactant and subjected to stirring treatment to obtain an oil phase;

[0068] The water phase is added to the oil phase for emulsification treatment, and the emulsion is obtained after being cooled to the second temperature;

[0069] The curing agent is added to the emulsion for cross-linking and curing to obtain a primary cross-linked emulsion;

[0070] The metal salt solution is added to the initial cross-linked emulsion, and metal ion cross-linking is carried out under stirring, and PDRN porous microspheres are obtained after precipitation treatment.

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

[0072] The object of the present application is to prepare PDRN porous microspheres. Specifically, first, PDRN is dissolved in water and the pH is adjusted to 8-9 to obtain an aqueous phase; then the oil phase matrix at the first temperature is mixed with the surfactant to obtain a first mixture, and the first mixture is subjected to a first stirring treatment to uniformly mix the oil phase matrix and the surfactant in the first mixture 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 an emulsification treatment to obtain an emulsion after cooling to a second temperature, wherein the emulsification treatment is to realize the uniform dispersion of the aqueous phase and the oil phase, form a stable water-in-oil emulsion system, and lay the foundation for the formation of the subsequent porous structure, and the purpose of cooling to the second temperature is to provide a suitable environment for subsequent cross-linking and solidification; a solidifying agent is added to the emulsion for cross-linking and solidification, so that the solidifying agent combines with PDRN in the emulsion to obtain an initial cross-linked emulsion; a calcium chloride solution is added to the initial cross-linked emulsion, and metal ion cross-linking is carried out under stirring to obtain a product after metal ion cross-linking; the product after metal ion cross-linking is subjected to precipitation treatment to precipitate the precipitate, and PDRN porous microspheres with controllable size, excellent anti-disintegration performance, and excellent anti-swelling performance are prepared, and they have strong resistance to moist heat sterilization and high strength.

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

[0074] In a specific embodiment, the above-mentioned 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 first temperature is 40-50°C.

[0076] When the first temperature parameter is within the above-mentioned 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) is promoted, thereby obtaining the oil phase.

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

[0078] In one embodiment, the stirring treatment is performed at a rotation speed of 300-600 r / min for 10-30 min.

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

[0080] When the rotation speed and the time during the stirring treatment are within the above ranges, the oil phase does not agglomerate, and the oil phase matrix and the surfactant can be uniformly mixed.

[0081] In one embodiment, the emulsification treatment is performed for 30-60 min.

[0082] When the time of the emulsification treatment is within the above range, the water phase and the oil phase can be uniformly dispersed, and a stable water-in-oil emulsion system is formed, which lays a foundation for the formation of a porous structure.

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

[0084] When the parameter of the second temperature is within the above range, a suitable environment is provided for subsequent cross-linking and curing, and the integrity of the PDRN molecular chain is protected.

[0085] In one embodiment, the cross-linking and curing is performed for 6-12 h.

[0086] When the time of the cross-linking and curing is within the above range, the curing agent can be fully combined with the PDRN in the emulsion, which is beneficial to enhancing the stability of the PDRN porous microspheres.

[0087] In one embodiment, the metal ion cross-linking is performed for 12-24 h.

[0088] When the time of the second stirring treatment is within the above range, the metal ions can be fully cross-linked with the initially cross-linked emulsion, and finally, the PDRN porous microspheres with controllable size, excellent anti-disintegration performance, excellent anti-swelling performance, and strong resistance to moist heat sterilization (high strength) are prepared.

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

[0090] When the mass ratio of the oil phase matrix to the surfactant is within the above range, the oil phase matrix and the surfactant can be more matched, and a stable oil phase is obtained.

[0091] In a specific embodiment, the PDRN porous microspheres are further subjected to a washing treatment, a centrifugal treatment and a drying treatment.

[0092] The drying treatment is not particularly limited in the present application, and for example, the drying treatment is a vacuum drying treatment.

[0093] Specifically, the precipitate after the precipitation treatment is subjected to a washing treatment to remove unreacted curing agent, free PDRN, surfactant and oil phase impurities remaining in the precipitate, to obtain a washed precipitate, and finally the washed precipitate is subjected to a centrifugal treatment and a drying treatment, thereby obtaining the PDRN porous microspheres.

[0094] The third aspect of the present application provides a use of the PDRN porous microspheres or the PDRN porous microspheres prepared by the preparation method of the PDRN porous microspheres in preparing a medical and aesthetic filling material or a tissue engineering scaffold. The PDRN porous microspheres have good dispersibility, controllable size, excellent anti-disintegration performance and strong resistance to moist heat sterilization, and thus can be used as an ideal candidate material for medical and aesthetic filling materials or tissue engineering scaffolds.

[0095] The embodiments of the present application are described in detail by the following examples.

[0096] Example 1

[0097] The PDRN porous microspheres provided in the present embodiment are prepared by a preparation method comprising the following processes:

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

[0099] (2) Heat 200 mL of liquid paraffin to 50℃, then mix 1 g of Span-80 with the liquid paraffin at 50℃ 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;

[0100] (3) Add 10 mL of the water phase to the oil phase to obtain a second mixture, and emulsify the second mixture for 30 min to obtain an emulsion, and cool the emulsion to 4℃;

[0101] (4) Add 1 g of polyethylene glycol acrylate with a molecular weight of 575 to the emulsion for cross-linking and curing for 6 h to obtain a primary cross-linked emulsion;

[0102] (5) dissolving calcium chloride in water to prepare a calcium chloride solution with a concentration of 10 mg / mL, then adding 5 mL of the calcium chloride solution into the primary crosslinked emulsion, and carrying out metal ion crosslinking under stirring for 12 h to obtain a metal ion crosslinked product, then placing the metal ion crosslinked product in 1 L of ethanol for precipitation treatment, standing to allow the precipitate to precipitate, then washing the precipitate with deionized water to obtain a washed precipitate, and finally carrying out centrifugal 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] 1 g of polyethylene glycol acrylate with a molecular weight of 575 is replaced by 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] Calcium chloride is replaced by 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] 1 g of polyethylene glycol acrylate with a molecular weight of 575 is replaced by 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] 1 g of polyethylene glycol acrylate with a molecular weight of 575 is replaced by 2 g of polyethylene glycol acrylate with a molecular weight of 575.

[0115] Comparative Example 1 (without metal ion crosslinking)

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

[0117] (1) dissolving polydeoxyribonucleotides (PDRN) in water, adjusting the pH to 8 using a sodium hydroxide solution to obtain a PDRN solution with a pH of 8 and a concentration of 50 mg / mL, and using the PDRN solution as the water phase;

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

[0119] (3) 10 mL of the water phase was added to the oil phase to obtain a second mixture, and the second mixture was emulsified for 30 min to obtain an emulsion after cooling to 4℃;

[0120] (4) 1 g of polyethylene glycol acrylate with a molecular weight of 575 was added to the emulsion for cross-linking and curing for 6 h to obtain a cross-linked and cured product, then the cross-linked and cured product was placed in 1 L of ethanol for precipitation treatment, and the precipitate was allowed to settle, then the precipitate was washed with deionized water to obtain a washed precipitate, and finally the washed precipitate was subjected to centrifugal treatment and vacuum drying treatment 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 subjected to scanning electron microscope (SEM) tests, and the results are shown in Figures 1-6 ; Figure 1 is an SEM image of the PDRN porous microspheres in Example 1 of the application; Figure 2 is an SEM image of the PDRN porous microspheres in Example 2 of the application; Figure 3 is an SEM image of the PDRN porous microspheres in Example 3 of the application; Figure 4 is an SEM image of the PDRN porous microspheres in Example 4 of the application; Figure 5 is an SEM image of the PDRN porous microspheres in Example 5 of the application; Figure 6 is an SEM image of the PDRN porous microspheres in Comparative Example 1 of the application.

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

[0125] As can be seen from Figure 2 , the PDRN porous microspheres in Example 2 are nearly spherical, have slight deformation at the edges, a rough surface, visible pore structure, and a particle size distribution of 20-60 μm.

[0126] As can be seen from Figure 3It can be seen that the PDRN porous microspheres in Example 3 are near-spherical, the edges are smooth, the surface visible pore structure is uniform, the monodispersity is good, the size distribution is narrow, and the particle size distribution is between 40-50 μm.

[0127] By Figure 4 It can be seen that the PDRN porous microspheres in Example 4 are near-spherical, the visible pore structure is poor, the aggregation occurs, the size difference is significant, and the particle size distribution is between 2-40 μm.

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

[0129] By Figure 6 It can be seen that the PDRN porous microspheres in Comparative Example 1 are near-spherical, the edges are smooth, the surface visible pore structure is uniform, the monodispersity is good, and the particle size is concentrated between 20-40 μm.

[0130] 2. Anti-degradation performance test

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

[0132] Table 1 Enzymolysis rate of PDRN porous microspheres

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

[0134] As shown in Table 1, the enzymolysis rate of the PDRN porous microspheres in Examples 1-5 of the present application is lower than that of the PDRN porous microspheres in Comparative Example 1, and the lowest is 9%, which indicates that the PDRN porous microspheres in the examples of the present application have excellent anti-degradation performance.

[0135] 3. Anti-swelling test

[0136] The PDRN porous microspheres in Examples 1-5 and Comparative Example 1 were respectively subjected to a swelling rate test, and the specific method was as follows: 0.05 g of the PDRN porous microspheres was placed in a 60℃ vacuum drying oven for drying for 6 h, and after cooling to room temperature, the dried PDRN porous microspheres were weighed to obtain the dried PDRN porous microspheres and the initial mass W0 was recorded; the dried PDRN porous microspheres were immersed in 10 mL of normal saline, and placed in a 25℃ constant-temperature oscillation water bath, and samples were taken at 10 min and 30 min, respectively, and the PDRN porous microspheres were quickly separated from the solvent by using filter paper; then the separated PDRN porous microspheres were laid on the filter paper, the surface free liquid was absorbed by gently touching, and the mass W1 was weighed by using an analytical balance, and the swelling rate was calculated according to the following formula, and the calculation results were shown in Table 2, and the lower the swelling rate, the better the anti-swelling property of the PDRN porous microspheres.

[0137] Swelling rate = (W1-W0) / W0 x 100%.

[0138] Table 2: Swelling rate 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 shown in Table 2, the swelling rates of the PDRN porous microspheres in Examples 1-5 were lower than that of the PDRN porous microspheres in Comparative Example 1, which indicated that the PDRN porous microspheres in Examples 1-5 had excellent anti-swelling property, and the PDRN porous microspheres in Example 3 had the most excellent anti-swelling property.

[0141] 4. Strength test

[0142] The PDRN porous microspheres in Examples 1-5 and Comparative Example 1 were respectively subjected to a strength test by using a wet heat sterilization method, and the specific method was as follows:

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

[0144] The PDRN porous microsphere solution was subjected to wet heat sterilization at 121℃ for 20 min to obtain a PDRN porous microsphere solution after sterilization;

[0145] The PDRN porous microsphere solutions before and after sterilization were respectively placed 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, wherein Figure 7 a is the optical microscope image of the PDRN porous microsphere solution before sterilization in Example 1, Figure 7b is an optical microscope image of the sterilized PDRN porous microsphere solution of Example 1;

[0147] Figure 8 is a strength test of PDRN porous microspheres in the present invention Example 2, wherein Figure 8 a is an optical microscope image of the non-sterilized PDRN porous microsphere solution of Example 2, Figure 8 b is an optical microscope image of the sterilized PDRN porous microsphere solution of Example 2;

[0148] Figure 9 is a strength test of PDRN porous microspheres in the present invention Example 3, wherein Figure 9 a is an optical microscope image of the non-sterilized PDRN porous microsphere solution of Example 3, Figure 9 b is an optical microscope image of the sterilized PDRN porous microsphere solution of Example 3;

[0149] Figure 10 is a strength test of PDRN porous microspheres in the present invention Example 4, wherein Figure 10 a is an optical microscope image of the non-sterilized PDRN porous microsphere solution of Example 4, Figure 10 b is an optical microscope image of the sterilized PDRN porous microsphere solution of Example 4;

[0150] Figure 11 is a strength test of PDRN porous microspheres in the present invention Example 5, wherein Figure 11 a is an optical microscope image of the non-sterilized PDRN porous microsphere solution of Example 5, Figure 11 b is an optical microscope image of the sterilized PDRN porous microsphere solution of Example 5;

[0151] Figure 12 is a strength test of PDRN porous microspheres in the present invention Comparative Example 1, wherein Figure 12 a is an optical microscope image of the non-sterilized PDRN porous microsphere solution of Comparative Example 1, Figure 12 b is an optical microscope image of the sterilized PDRN porous microsphere solution of Comparative Example 1.

[0152] from Figures 7-12It can be seen that before sterilization, the PDRN porous microspheres of Comparative Example 1 will quickly swell in water, and the PDRN porous microspheres of Example 1-Example 5 have no swelling in appearance; after sterilization, the PDRN porous microspheres of Comparative Example 1 no longer exist and are completely dissolved in water, the PDRN porous microspheres of Example 1 retain part of the complete spherical structure, the PDRN porous microspheres of Example 2 are broken into fragments, the PDRN porous microspheres of Example 3 are complete in structure, the PDRN porous microspheres of Example 4 are severely aggregated to form lamellas, and the PDRN porous microspheres of Example 5 have fuzzy edge contours and are deformed. The above results show that the PDRN porous microspheres of Examples 1-5 have stronger resistance to moist heat sterilization than the PDRN porous microspheres of Comparative Example 1, and have the advantage of high strength, and the PDRN porous microspheres of Example 3 have the highest resistance to moist heat sterilization.

[0153] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A PDRN porous microsphere, characterized in that, The preparation raw material of the PDRN porous microspheres comprises 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; The PDRN porous microspheres are obtained by a preparation method comprising the following steps: After dissolving PDRN in water, a water phase is obtained, and an oil phase is obtained by mixing the oil phase matrix and a surfactant; the water phase is added to the oil phase for emulsification treatment, and then the product after the emulsification treatment is cooled and added with the curing agent for cross-linking and curing, followed by adding the metal salt for metal ion cross-linking, and then the product after the metal ion cross-linking is subjected to precipitation treatment to obtain the PDRN porous microspheres; The surfactant is any one of Span-60, Span-80, Tween-60, and Tween-80.

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-to-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. A method of preparing the PDRN porous microspheres according to any one of claims 1-4, characterized in that, The method comprises the following steps: PDRN is dissolved in water and the pH is adjusted to 8-9 to obtain a water phase; The oil phase matrix at a first temperature is mixed with a surfactant and then subjected to stirring treatment to obtain an oil phase; The water phase is added to the oil phase for emulsification treatment, and an emulsion is obtained after cooling to a second temperature; A curing agent is added to the emulsion for cross-linking and curing to obtain a primary cross-linked emulsion; A metal salt solution is added to the primary cross-linked emulsion, and metal ion cross-linking is performed under stirring, and PDRN porous microspheres are obtained after precipitation treatment.

6. The method for preparing PDRN porous microspheres according to claim 5, characterized in that, The concentration of PDRN in the water 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.

7. The method of claim 5, wherein the PDRN porous microspheres are prepared by the steps of: 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 emulsification treatment is performed for 30-60 min; And / or, the second temperature is a temperature less than or equal to 4 °C; And / or, the cross-linking and curing are performed for 6-12 h; And / or, the metal ion cross-linking is performed for 12-24 h.

8. The method of claim 5, wherein the PDRN porous microspheres are prepared by the steps of: The volume-to-mass ratio of the oil phase matrix and 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.

9. Use of the PDRN porous microspheres of any one of claims 1-4 or the PDRN porous microspheres prepared by the preparation method of any one of claims 5-8 in the preparation of a medical and cosmetic filling material or a tissue engineering scaffold.

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