Nanocomposition with tissue repair and anti-inflammatory effects, its preparation method and application

Abstract

The present invention belongs to the technical field of medical materials, and discloses a nano-composition with tissue repair and anti-inflammatory effects, its preparation method and application. The nano-composition of the present invention is formed by encapsulating collagen and lysozyme with a cationic polymer. The cationic polymer has amphiphilic characteristics, and the molecular chain segment of the cationic polymer contains a hydrophilic block and a hydrophobic block. The amphiphilic cationic polymer prepared in the present invention encapsulates collagen and lysozyme to obtain a nano-composition with tissue repair and anti-inflammatory effects. Compared with collagen and lysozyme, the nano-composition of the present invention exhibits better repair (promoting cell proliferation and cell migration) and anti-inflammatory effects. It can be used for preparing cosmetics and drugs with tissue repair and anti-inflammatory effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a nano - composition with tissue - repairing and anti - inflammatory effects, a preparation method thereof, and an application thereof. Background Art

[0002] The skin is the largest organ of the human body and plays a very important role in resisting foreign chemical substances and pathogenic microorganisms, regulating body temperature, maintaining the stability of the internal environment, and preventing the loss of body moisture. However, skin traumas can be caused by surgery, infection, radiation, external pressure, burns, scalds, and pathological factors (such as diabetes or other vascular diseases). Skin trauma is the defect or injury of the skin caused by external injury factors or internal factors of the human body, accompanied by the destruction of the skin barrier integrity and functional impairment, which is prone to cause various skin diseases, such as eczema, psoriasis, contact dermatitis, and atopic dermatitis.

[0003] Collagen is the main component of the extracellular matrix and is widely used in the field of biomaterials due to its good physical and chemical properties. In addition, collagen also has good coagulation effects and significantly promotes the repair, regeneration, and reconstruction of defective tissues. Collagen has low immunogenicity, good biocompatibility, and is easy to process and form. Currently, its application in tissue repair mainly uses collagen as the matrix raw material to make various different types and shapes of materials. For example, using the electrospinning technique, collagen is prepared into a medical dressing for skin tissue repair; or a protein sponge is prepared based on collagen as a dressing for mild skin wound repair. However, the above - mentioned applications of collagen in skin repair mainly act on the outer layer of the skin and it is difficult to penetrate the skin cutin layer. Summary of the Invention

[0004] In order to overcome the above - mentioned disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a nano - composition with tissue - repairing and anti - inflammatory effects.

[0005] Another object of the present invention is to provide a preparation method of the above - mentioned nano - composition with tissue - repairing and anti - inflammatory effects.

[0006] Still another object of the present invention is to provide an application of the above - mentioned nano - composition with tissue - repairing and anti - inflammatory effects.

[0007] The objects of the present invention are achieved by the following solutions:

[0008] A nano - composition with tissue - repairing and anti - inflammatory effects, wherein the nano - composition is formed by a cationic polymer encapsulating collagen and lysozyme, and the molecular chain segment of the cationic polymer contains a hydrophilic block and a hydrophobic block.

[0009] In some embodiments, the molecular weight of the cationic polymer is Mn = 17820 - 29631, preferably 23726.

[0010] In some embodiments, the hydrophobic block of the cationic polymer is a polymethacrylate, which includes one or more of dimethylaminoethyl methacrylate (PDMAEMA), diethylaminoethyl methacrylate (PDEAEMA), 2-(diisopropylamino)ethyl methacrylate (PDIPAEMA), and 2-(tert-butylamino)ethyl methacrylate (PTBAEMA).

[0011] Preferably, the hydrophobic block of the cationic polymer includes dimethylaminoethyl methacrylate (PDMAEMA), which is obtained by atom transfer radical polymerization (ATRP) of dimethylaminoethyl methacrylate (DMAEMA), with a degree of polymerization of 20 - 40, and the molecular weight of the dimethylaminoethyl methacrylate is 3100 - 6300.

[0012] Preferably, the hydrophobic block of the cationic polymer includes diethylaminoethyl methacrylate, which is obtained by ATRP of diethylaminoethyl methacrylate (DEAEMA), with a degree of polymerization of 20 - 40, and the molecular weight of the diethylaminoethyl methacrylate is 3700 - 9300.

[0013] Preferably, the hydrophobic block of the cationic polymer includes 2-(diisopropylamino)ethyl methacrylate, which is obtained by ATRP of 2-(diisopropylamino)ethyl methacrylate (DIPAEMA), with a degree of polymerization of 20 - 40, and the molecular weight of the 2-(diisopropylamino)ethyl methacrylate is 4200 - 8600.

[0014] Preferably, the hydrophobic block of the cationic polymer includes 2-(tert-butylamino)ethyl methacrylate (PTBAEMA), which is obtained by ATRP of 2-(tert-butylamino)ethyl methacrylate (TBAEMA), with a degree of polymerization of 20 - 40, and the molecular weight of the 2-(tert-butylamino)ethyl methacrylate is 3700 - 7500.

[0015] In some embodiments, the hydrophilic block of the polymer is poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).

[0016] Preferably, the poly(2-methacryloyloxyethyl phosphorylcholine) is obtained by ATRP of 2-methacryloyloxyethyl phosphorylcholine (MPC), with a degree of polymerization of 20 - 40, and the molecular weight of the poly(2-methacryloyloxyethyl phosphorylcholine) is 11800 - 23600.

[0017] In some embodiments, the method for preparing the cationic polymer comprises the following steps:

[0018] Dissolve the polymerization monomers corresponding to the hydrophobic block of the cationic polymer, i.e., methacrylates, bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide, and N,N,N',N',N''-pentamethyldiethylenetriamine, in an organic solvent. Add a catalyst under the protection of an inert gas and react at 40 - 80 °C for 12 - 48 h; then add the polymerization monomer (MPC) of the hydrophilic block of the cationic polymer and continue to react at 40 - 80 °C for 12 - 48 h; thus obtaining the cationic polymer.

[0019] In some embodiments, the organic solvent includes at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; preferably tetrahydrofuran.

[0020] In some embodiments, the catalyst includes at least one of CuBr, CuCl, and CuI; preferably CuBr.

[0021] In some embodiments, the molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the polymerization monomer methacrylates of the hydrophobic block is 1:10 - 50; the molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the polymerization monomer MPC of the hydrophilic block is 1:40 - 80.

[0022] In some embodiments, the molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to N,N,N',N',N''-pentamethyldiethylenetriamine is 1:4 - 8; the molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the catalyst is 1:4 - 8.

[0023] In some embodiments, after the ATRP reaction is completed, a purification step is further included:

[0024] Elute the reaction mixture through a neutral alumina column to remove the catalyst. After the eluate is concentrated by rotary evaporation, drop the concentrated solution into n-hexane for precipitation, collect the solid product, and dry it at 40 - 60 °C for 12 - 48 h to obtain the cationic polymer.

[0025] Preferably, in the step of passing the reaction product through the neutral alumina column, the eluate is a tetrahydrofuran solution.

[0026] Preferably, the temperature of the rotary evaporation concentration is 30 - 60 °C and the rotation speed is 100 - 450 rpm.

[0027] A method for preparing the above-mentioned nano-composition with tissue repair and anti-inflammatory effects, comprising the following steps:

[0028] (1) Dissolve the cationic polymer in a solvent, heat and stir. After the polymer is dissolved, a polymer solution is obtained;

[0029] (2) Add collagen and lysozyme to water, heat and stir. After collagen and lysozyme are dissolved, an aqueous solution is obtained;

[0030] (3) Add the polymer solution to the aqueous solution and stir to obtain a mixed solution;

[0031] (4) The mixed solution is subjected to high-speed shear dispersion treatment and high-pressure microfluidic homogenization treatment to obtain a nano-composition with tissue repair and anti-inflammatory effects.

[0032] In some embodiments, the mass percentage of the cationic polymer in step (1) in the nano-composition is 1-7%, preferably 3-7%;

[0033] In some embodiments, the solvent in step (1) includes at least one of dichloromethane, acetone, tetrahydrofuran, N,N'-dimethylformamide, dimethyl sulfoxide, propylene glycol, glycerol, butylene glycol, pentylene glycol, hexylene glycol, ethylene glycol, pentaerythritol, dipropylene glycol, and diglycol; the amount of the solvent used satisfies that the mass percentage of the solvent in the nano-composition is 20-40%;

[0034] In some embodiments, the heating temperatures in steps (1) and (2) are independently 40-65°C; the stirring speeds are independently 30-300 rpm; the stirring times are independently 10-60 min;

[0035] In some embodiments, the mass percentage of the collagen in step (2) in the nano-composition is 1-10%, preferably 1-7%;

[0036] In some embodiments, the mass percentage of the lysozyme in step (2) in the nano-composition is 0.1-2%;

[0037] In some embodiments, the mass ratio of the collagen to the lysozyme in step (2) is 1:0.05-0.2;

[0038] In some embodiments, the amount of water used in step (2) satisfies that its mass percentage in the nano-composition is 41.0-77.9%;

[0039] In some embodiments, the feeding rate of the polymer solution in step (3) is 0.5 - 20 mL / min, preferably 0.5 - 3.0 mL / min.

[0040] In some embodiments, in step (3), the stirring speed is 30 - 300 rpm; the stirring time is 10 - 60 min;

[0041] In some embodiments, the high-speed shear dispersion treatment in step (4) is carried out by using a high-shear homogenizing emulsifier for shear homogenization, where the rotation speed of the high-shear homogenizing emulsifier is 5000 - 9000 rpm and the shear dispersion time is 3 - 15 min.

[0042] In some embodiments, the high-pressure microfluidic homogenization treatment in step (4) is carried out by using a microfluidic homogenizer, where the homogenization pressure is 50 - 120 Mpa and the number of homogenization cycles is 1 - 6 times.

[0043] Optionally, in some embodiments of the present invention, the stirring method in step (1), step (2) and step (3) is magnetic stirring.

[0044] The application of the above-mentioned nano-composition with tissue repair and anti-inflammatory effects in the cosmetic field and the pharmaceutical field, especially in the preparation of cosmetics and the preparation of drugs with tissue repair and anti-inflammatory effects.

[0045] The present invention has the following advantages and beneficial effects compared with the prior art:

[0046] The amphiphilic cationic polymer prepared by the present invention encapsulates collagen and lysozyme to obtain a nano-composition with tissue repair and anti-inflammatory effects. Compared with collagen and lysozyme, the nano-composition of the present invention shows better repair (promoting cell proliferation and cell migration) and anti-inflammatory effects. Description of the Drawings

[0047] Figure 1 is the content of TNFα after using Examples 1 - 4, Comparative Example 6, Comparative Example 7, collagen (5% solution), lysozyme (0.5% solution), lysozyme + collagen (5% + 0.5% solution).

[0048] Figure 2 is the inhibition rate of TNFα after using Examples 1 - 4, Comparative Example 6, Comparative Example 7, collagen (5% solution), lysozyme (0.5% solution), lysozyme + collagen (5% + 0.5% solution). Detailed Embodiments

[0049] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0050] In the embodiments of the present invention, the ATRP reaction polymerization monomers DMAEMA, DEAEMA, DIPAEMA, DTBAEMA, and MPC can all be directly purchased from the market.

[0051] The administration concentration in the embodiments refers to the concentration after diluting the nano - composition prepared in the embodiments or comparative examples with deionized water, which is the mass - percentage concentration. Throughout the application document, unless otherwise specified, it refers to the mass - percentage.

[0052] Example 1

[0053] (1) Preparation of cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 (taking the preparation of 1.0 mmol as an example): Dissolve bis[2 - (2’ - bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (5.558 g, 30.0 mmol) and N,N,N’,N’,N”-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Pass the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution) to remove CuBr. After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n - hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 (the number - average molecular weight Mn is 23726).

[0054] (2) The prepared cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30(3%) was dissolved in a mixed solution of 20% glycerol, 5% 1,3-propanediol, and 5% 1,4-butanediol by mass percentage. Stirring was maintained at 60 °C (rotation speed of 450 rpm) for 2 h. After the polymer was fully dissolved, it was cooled to room temperature to obtain a polymer solution; 5% collagen and 0.5% lysozyme by mass percentage were added to 61.5% deionized water by mass percentage. Stirring was maintained at 40 °C (rotation speed of 450 rpm) for 1 h. After the collagen and lysozyme were fully dissolved, an aqueous solution was obtained; under the conditions of 40 °C and stirring (rotation speed of 300 rpm), the aqueous phase was uniformly added dropwise to the polymer solution at a dropping rate of 1 ml / min. After the dropping was completed, stirring (450 rpm) was continued for 1 h; then it was dispersed by high-speed shearing (8000 rpm) for 10 min; and then it was treated by high-pressure microfluidization. The homogenization pressure was 80 MPa, and the number of homogenization times was 3 times to obtain a nano-composition with tissue repair and anti-inflammatory effects. The percentages in this step all refer to the mass percentages of each component in the nano-composition.

[0055] Examples 2-4 (different types of polyacrylates)

[0056] Example 2

[0057] (1) Preparation of cationic polymer PMPC 30 -PDMAEMA 30 -PMPC 30 (taking the preparation of 1.0 mmol as an example): Bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DMAEMA (4.716 g, 30.0 mmol), and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) were dissolved in tetrahydrofuran (50 mL). Under the protection of inert gas, CuBr (0.84 g, 6.0 mmol) was quickly added to the reaction system. The reaction was carried out at 60 °C for 24 h. Then MPC (17.7 g, 60.0 mmol) was added to the reaction system, and the reaction was continued at 60 °C for 24 h. The reaction mixture was passed through a neutral alumina column (the eluent was a tetrahydrofuran solution) to remove CuBr. After the eluent was concentrated by rotary evaporation, it was dropped into an excess of n-hexane for precipitation. The solid product was collected and dried to obtain the cationic polymer PMPC 30 -PDMAEMA 30 -PMPC 30 (number average molecular weight Mn is 22884).

[0058] (2) 3% cationic polymer PMPC in Example 1 30 -PDEAEMA 30 -PMPC30 Replace it with cationic polymer PMPC with a mass percentage of 3% 30 -PDMAEMA 30 -PMPC 30 , and keep the rest unchanged to prepare a nano - composition with tissue repair and anti - inflammatory effects.

[0059] Example 3

[0060] (1) Preparation of cationic polymer PMPC 30 -PDIPAEMA 30 -PMPC 30 (Taking the preparation of 1.0 mmol as an example): Dissolve bis[2 - (2’ - bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DIPAEMA (6.400 g, 30.0 mmol) and N,N,N’,N’,N”-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n - hexane for precipitation. The collected solid product is dried to obtain the cationic polymer PMPC 30 -PDIPAEMA 30 -PMPC 30 (The number - average molecular weight Mn is 24568).

[0061] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a mass percentage of 3% in Example 1 with cationic polymer PMPC 30 -PDIPAEMA 30 -PMPC 30 , and keep the rest unchanged to prepare a nano - composition with tissue repair and anti - inflammatory effects.

[0062] Example 4

[0063] (1) Cationic polymer PMPC 30 -PTBAEMA 30 -PMPC 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), TBAEMA (5.558 g, 30.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The collected solid product is dried to obtain the cationic polymer PMPC 30 -PTBAEMA 30 -PMPC 30 (The number-average molecular weight Mn is 23726).

[0064] (2) Replace the cationic polymer PMPC with a mass percentage of 3% in Example 1 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC with a mass percentage of 3% 30 -PTBAEMA 30 -PMPC 30 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0065] Examples 5-6 (different degrees of polymerization of the hydrophobic block PDEAEMA)

[0066] Example 5

[0067] (1) The cationic polymer PMPC 30 -PDEAEMA 20 -PMPC 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (3.705 g, 20.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The collected solid product is dried to obtain the cationic polymer PMPC 30 -PDEAEMA 20 -PMPC 30 (The number-average molecular weight Mn is 21873).

[0068] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC 30 -PDEAEMA 20 -PMPC 30 with a mass percentage of 3%, and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0069] Example 6

[0070] (1) Cationic polymer PMPC 30 -PDEAEMA 40 -PMPC 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (7.410 g, 40.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 30 -PDEAEMA 40 -PMPC 30 (The number-average molecular weight Mn is 25578).

[0071] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a 3% (by mass) cationic polymer PMPC 30 -PDEAEMA 40 -PMPC 30 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0072] Examples 7 - 8 (different degrees of polymerization of the hydrophilic block PMPC)

[0073] Example 7

[0074] (1) The cationic polymer PMPC 20 -PDEAEMA 30 -PMPC 20Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (5.558 g, 30.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (0.70 g, 4.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.56 g, 4.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (11.8 g, 40.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 20 -PDEAEMA 30 -PMPC 20 (The number-average molecular weight Mn is 17820).

[0075] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC 20 -PDEAEMA 30 -PMPC 20 with a mass percentage of 3%, and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0076] Example 8

[0077] (1) Cationic polymer PMPC 40 -PDEAEMA 30 -PMPC 40Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (5.558 g, 30.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.40 g, 8.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (1.12 g, 8.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (23.6 g, 80.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, drop it into an excess of n-hexane for precipitation. Collect the solid product and dry it to obtain the cationic polymer PMPC 40 -PDEAEMA 30 -PMPC 40 (The number-average molecular weight Mn is 29631).

[0078] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a 3% (by mass) cationic polymer PMPC 40 -PDEAEMA 30 -PMPC 40 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0079] Examples 9 - 10 (Polymer feeding ratio)

[0080] Example 9

[0081] Replace the 3% (by mass) cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 in Example 1 with a 5% (by mass) cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 , and appropriately adjust the mass percentage of deionized water to 59.5% so that the total mass percentage is 100%, and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0082] Example 10

[0083] Replace the 3% (by mass) cationic polymer PMPC 30 -PDEAEMA30 -PMPC 30 Change to cationic polymer PMPC with a mass percentage of 7% 30 -PDEAEMA 30 -PMPC 30 , appropriately adjust the mass percentage of deionized water to 57.5% so that the total mass percentage is 100%, and the rest remains unchanged, to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0084] Examples 11 - 13 (Collagen feeding ratio)

[0085] Example 11

[0086] Change the collagen with a mass percentage of 5% in Example 1 to collagen with a mass percentage of 1%, appropriately adjust the mass percentage of deionized water to 65.5% so that the total mass percentage is 100%, and the rest remains unchanged, to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0087] Example 12

[0088] Change the collagen with a mass percentage of 5% in Example 1 to collagen with a mass percentage of 3%, appropriately adjust the mass percentage of deionized water to 63.5% so that the total mass percentage is 100%, and the rest remains unchanged, to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0089] Example 13

[0090] Change the collagen with a mass percentage of 5% in Example 1 to collagen with a mass percentage of 7%, appropriately adjust the mass percentage of deionized water to 59.5% so that the total mass percentage is 100%, and the rest remains unchanged, to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0091] Example 14 (Process)

[0092] PMPC 30 -PDEAEMA 30 -PMPC 30 The preparation steps are the same as those in Example 1.

[0093] The prepared cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30(3%) was dissolved in a mixed solution containing 20% glycerol, 5% 1,3-propanediol, and 5% 1,4-butanediol by mass percentage. Stirring was maintained at 60 °C (rotation speed: 450 rpm) for 2 h. After the polymer was fully dissolved, it was cooled to room temperature to obtain a polymer solution. 5% collagen and 0.5% lysozyme by mass percentage were added to 61.5% deionized water by mass percentage. Stirring was maintained at 40 °C (rotation speed: 450 rpm) for 1 h. After the collagen and lysozyme were fully dissolved, an aqueous solution was obtained. Under the conditions of 40 °C and stirring (rotation speed: 300 rpm), the aqueous phase was uniformly added dropwise to the polymer solution at a dropping rate of 1 ml / min. After the dropping was completed, stirring was continued (450 rpm) for 1 h. Then, it was dispersed by high-speed shearing (5000 rpm) for 5 min. Subsequently, it was treated by high-pressure microfluidization. The homogenization pressure was 80 MPa, and the number of homogenization times was 1 time to obtain a nano-composition with tissue repair and anti-inflammatory effects. The percentages in this step all refer to the mass percentages of the respective components in the nano-composition.

[0094] Example 15

[0095] PMPC 30 -PDEAEMA 30 -PMPC 30 The preparation steps were the same as those in Example 1.

[0096] The prepared cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 (3%) was dissolved in a mixed solution containing 20% glycerol, 5% 1,3-propanediol, and 5% 1,4-butanediol by mass percentage. Stirring was maintained at 60 °C (rotation speed: 450 rpm) for 2 h. After the polymer was fully dissolved, it was cooled to room temperature to obtain a polymer solution. 5% collagen and 0.5% lysozyme by mass percentage were added to 61.5% deionized water by mass percentage. Stirring was maintained at 40 °C (rotation speed: 450 rpm) for 1 h. After the collagen and lysozyme were fully dissolved, an aqueous solution was obtained. Under the conditions of 60 °C and stirring (rotation speed: 300 rpm), the aqueous phase was uniformly added dropwise to the polymer solution at a dropping rate of 5 mL / min. After the dropping was completed, stirring was continued (450 rpm) for 1 h. Then, it was dispersed by high-speed shearing (5000 rpm) for 5 min. Subsequently, it was treated by high-pressure microfluidization. The homogenization pressure was 80 MPa, and the number of homogenization times was 1 time to obtain a nano-composition with tissue repair and anti-inflammatory effects. The percentages in this step all refer to the mass percentages of the respective components in the nano-composition.

[0097] Examples 16 - 19 (types and ratios of solvents when preparing nano-compositions)

[0098] Example 16

[0099] In Example 1, change “20% glycerol, 5% 1,3 - propanediol, and 5% 1,4 - butanediol” to 30% glycerol, and keep the rest unchanged to prepare a nano - composition with tissue - repairing and anti - inflammatory effects.

[0100] Example 17

[0101] In Example 1, change “20% glycerol, 5% 1,3 - propanediol, and 5% 1,4 - butanediol” to 30% 1,3 - propanediol, and keep the rest unchanged to prepare a nano - composition with tissue - repairing and anti - inflammatory effects.

[0102] Example 18

[0103] In Example 1, change “20% glycerol, 5% 1,3 - propanediol, and 5% 1,4 - butanediol” to 30% 1,4 - butanediol, and keep the rest unchanged to prepare a nano - composition with tissue - repairing and anti - inflammatory effects.

[0104] Example 19

[0105] In Example 1, change “20% glycerol, 5% 1,3 - propanediol, and 5% 1,4 - butanediol” to 10% glycerol, 10% 1,3 - propanediol, 10% 1,4 - butanediol, and keep the rest unchanged to prepare a nano - composition with tissue - repairing and anti - inflammatory effects.

[0106] Comparative Example 1 (uncoated)

[0107] Mix 20% glycerol, 5% 1,3 - propanediol, and 5% 1,4 - butanediol by mass percentage evenly to obtain a mixed alcohol solution. Add 5% collagen and 0.5% lysozyme by mass percentage to 64.5% deionized water, keep stirring (at a rotation speed of 450 rpm) at 40 °C for 1 h. Wait until the collagen and lysozyme are fully dissolved to obtain an aqueous solution. Under the conditions of 40 °C and stirring (at a rotation speed of 300 rpm), add the aqueous solution to the mixed alcohol solution at a dropping rate of 1 ml / min evenly. After the dropping is completed, continue to stir (450 rpm) for 1 h; then disperse by high - speed shearing (8000 rpm) for 10 min; then perform high - pressure micro - jet treatment with a homogenization pressure of 80 MPa and a homogenization number of 3 times to obtain a tissue - repairing and anti - inflammatory composition. The percentages in this step all refer to the mass percentages of each component in the nano - composition.

[0108] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, cationic polymer is not used to coat collagen and lysozyme.

[0109] Comparative Example 2 (the degree of polymerization of PDEAEMA is 10)

[0110] (1) Cationic polymer PMPC 30 -PDEAEMA 10 -PMPC 30 Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (1.853 g, 10.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Pass the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution) to remove CuBr. After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 30 -PDEAEMA 10 -PMPC 30 (The number average molecular weight Mn is 19568).

[0111] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a 3% (by mass) cationic polymer PMPC 30 -PDEAEMA 10 -PMPC 30 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0112] Comparative Example 3 (the degree of polymerization of PDEAEMA is 50)

[0113] (1) Cationic polymer PMPC 30 -PDEAEMA 50 -PMPC 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (9.265 g, 50.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (17.7 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, drop it into an excess of n-hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 30 -PDEAEMA 50 -PMPC 30 (The number-average molecular weight Mn is 27431).

[0114] (2) Replace the cationic polymer PMPC with a mass percentage of 3% in Example 1 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC with a mass percentage of 3% 30 -PDEAEMA 50 -PMPC 30 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0115] Comparative Example 4 (the degree of polymerization of PMPC is 10)

[0116] (1) Cationic polymer PMPC 10 -PDEAEMA 30 -PMPC 10Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (5.558 g, 30.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (0.52 g, 3.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.42 g, 3.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (5.9 g, 20.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The solid product is collected and dried to obtain the cationic polymer PMPC 10 -PDEAEMA 30 -PMPC 10 (The number-average molecular weight Mn is 11915).

[0117] (2) Replace the cationic polymer PMPC with a mass percentage of 3% in Example 1 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC with a mass percentage of 3% 10 -PDEAEMA 30 -PMPC 10 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0118] Comparative Example 5 (the degree of polymerization of PMPC is 50)

[0119] (1) Cationic polymer PMPC 50 -PDEAEMA 30 -PMPC 50Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (5.558 g, 30.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (0.52 g, 3.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.42 g, 3.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPC (5.9 g, 20.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Remove CuBr from the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution). After the eluent is concentrated by rotary evaporation, drop it into an excess of n-hexane for precipitation. Collect the solid product and dry it to obtain the cationic polymer PMPC 50 -PDEAEMA 30 -PMPC 50 (The number-average molecular weight Mn is 35537).

[0120] (2) Replace the cationic polymer PMPC with a mass percentage of 3% in Example 1 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC with a mass percentage of 3% 50 -PDEAEMA 30 -PMPC 50 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0121] Comparative Example 6 (HO-PLA-HO replaces PDEAEMA)

[0122] (1) Cationic polymer PMPC 30 -PCL 52 -PMPC 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve polyester PCL52 (5.93 g, 1.0 mmol) in dichloromethane (50 mL) to obtain a polyester solution; under the protection of inert gas, add 2-bromo-2-methylpropionyl bromide (0.9 g, 4.0 mmol) to the polyester solution, react at 0 °C for 2 h, and then continue to react at room temperature for 48 h. The reaction mixture is washed with dilute hydrochloric acid solution (1.0 mol / L), saturated NaHCO3 solution and deionized water respectively. The organic phase is dried with anhydrous MgSO4 overnight. Take the upper clear liquid, concentrate it by rotary evaporation, precipitate the product with an excessive amount of n-hexane solution, and the macromolecular initiator can be obtained after drying; Dissolve the macromolecular initiator (5.93 g, 1.0 mmol), MPC (17.7 g, 60 mmol) and N,N,N’,N’,N”-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.85 g, 6.0 mmol) to the reaction system and react at 60 °C for 24 h. Pass the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution) to remove CuBr. After the eluent is concentrated by rotary evaporation, it is dropped into an excessive amount of n-hexane to precipitate. The solid product is collected and dried to obtain the cationic polymer PMPC 30 -PCL 52 -PMPC 30 (The number-average molecular weight Mn is 23644).

[0123] (2) Replace the cationic polymer PMPC with a mass percentage of 3% in Example 1 30 -PDEAEMA 30 -PMPC 30 with a cationic polymer PMPC with a mass percentage of 3% 30 -PCL 52 -PMPC 30 , and the rest remains unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0124] Comparative Example 7 (MPEG replaces MPC)

[0125] (1) Cationic polymer PMPEG 30 -PDEAEMA 30 -PMPEG 30Preparation (taking the preparation of 1.0 mmol as an example): Dissolve bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (0.452 g, 1.0 mmol), DEAEMA (9.265 g, 50.0 mmol) and N,N,N',N',N''-pentamethyldiethylenetriamine (1.05 g, 6.0 mmol) in tetrahydrofuran (50 mL). Under the protection of inert gas, quickly add CuBr (0.84 g, 6.0 mmol) to the reaction system. React at 60 °C for 24 h, then add MPEG (18.0 g, 60.0 mmol) to the reaction system and continue to react at 60 °C for 24 h. Pass the reaction mixture through a neutral alumina column (the eluent is a tetrahydrofuran solution) to remove CuBr. After the eluent is concentrated by rotary evaporation, it is dropped into an excess of n-hexane for precipitation. The solid product collected is dried to obtain the cationic polymer PMPEG 30 -PDEAEMA 30 -PMPEG 30 (The number average molecular weight Mn is 24010).

[0126] (2) Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 with a 3% by mass of the cationic polymer PMPEG 30 -PDEAEMA 30 -PMPEG 30 , and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0127] Comparative Example 8 (polymer 1%)

[0128] Replace the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 in Example 1 with a 1% by mass of the cationic polymer PMPC 30 -PDEAEMA 30 -PMPC 30 , and appropriately adjust the mass percentage of deionized water to 63.5% so that the total mass percentage is 100%, and keep the rest unchanged to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0129] Comparative Example 9 (collagen 10%)

[0130] Change the collagen with a mass percentage of 5% in Example 1 to collagen with a mass percentage of 10%, and appropriately adjust the mass percentage of deionized water to 56.5% so that the total mass percentage is 100%, and the rest remains unchanged, to prepare a nano-composition with tissue repair and anti-inflammatory effects.

[0131] Test Example 1 - Particle Size Measurement

[0132] Characterize the particle size and particle size distribution coefficient of the samples of Examples 1 - 19 and Comparative Examples 1 - 9 above using a Malvern Nano-ZS90 dynamic light scattering particle size analyzer. The test angle is 90°, the test temperature is 25°C, and three parallel experiments are carried out for each group of experiments, and the arithmetic mean of the experimental results is taken.

[0133] Test Example 2 - Encapsulation Efficiency Measurement

[0134] Measure the encapsulation efficiency of collagen in the samples of Examples 1 - 19 and Comparative Examples 1 - 9: Take 200 μL of the nano-composition with tissue repair and anti-inflammatory effects, and after ultrafiltration centrifugation (9000 rpm, 30 min), take 5 μL of the filtrate and determine the content of collagen in the filtrate using a high-performance liquid chromatograph (HPLC, Shimadzu, Japan), that is, obtain the content of unencapsulated collagen in the nano-composition with tissue repair and anti-inflammatory effects. Take another portion of the above nano-composition with tissue repair and anti-inflammatory effects, add deionized water, and perform ultrasonic demulsification for 30 min according to sample:deionized water = 1:9 (v / v). After filtering through a 0.45 μm organic filter membrane, take 5 μL of the sample solution and determine the content of collagen using a high-performance liquid chromatograph (HPLC, Shimadzu, Japan), that is, obtain the total content of collagen in the nano-composition with tissue repair and anti-inflammatory effects. Calculate the encapsulation efficiency (EE) of collagen in the nano-composition with tissue repair and anti-inflammatory effects according to formula (1). The analytical column used in the HPLC system is a non-polar C18 column, the mobile phase is acetonitrile, the flow rate is 1.0 mL / min, the column temperature of the chromatographic column is 30°C, and three parallel experiments are carried out for each group of experiments, and the arithmetic mean of the experimental results is taken.

[0135]

[0136] C1 represents the concentration of unencapsulated collagen in the sample; C0 represents the total concentration of collagen in the sample after ultrasonic demulsification with deionized water.

[0137] The particle size characterization and encapsulation efficiency test results of Examples 1 - 19 and Comparative Examples 1 - 9 are shown in Table 1.

[0138] Table 1 Particle Size and Encapsulation Efficiency Results of Samples of Examples 1 - 19 and Comparative Examples 1 - 9

[0139]

[0140]

[0141] Comparing Comparative Examples 1-4, it can be seen that among cationic polymers, different types of hydrophobic block polyacrylates result in different particle sizes and encapsulation efficiencies of the finally prepared nano-compositions with tissue repair and anti-inflammatory effects. In Examples 1-4, the hydrophobic block polyacrylates are PDEAEMA, PDMAEMA, PDIPAEMA, and PDTBAEMA respectively. The results show that the sample of Example 1 has the smallest particle size, which is 82.6 nm, and the highest encapsulation efficiency, which is 77.8%. In the present invention, the polymer hydrophobic block is selected as polyacrylate PDEAEMA, and the polymer has the best encapsulation effect on collagen.

[0142] Comparing Example 1, Example 5, Example 6, Comparative Example 2, and Comparative Example 3, it can be seen that the degree of polymerization of the hydrophobic block PDEAEMA affects the particle size and encapsulation efficiency of the final product. As the degree of polymerization of the polyacrylate block increases, the particle size of the product gradually increases. When the degree of polymerization of the polyacrylate block reaches 50, the particle size of the product suddenly increases to 121.3 nm. As the degree of polymerization of the polyacrylate block increases, the encapsulation efficiency of the product first increases and then decreases. When the degree of polymerization of the polyacrylate block is 10 and 50, the encapsulation efficiencies of the products are both low. Therefore, in this application, the degree of polymerization of the polyacrylate block is selected to be 20-40, preferably 30 (Example 1).

[0143] Comparing Example 1, Example 7, Example 8, Comparative Example 4, and Comparative Example 5, it can be seen that the degree of polymerization of the cationic block PMPC ultimately affects the particle size and encapsulation efficiency of the product. As the degree of polymerization of PMPC increases, the particle size of the product first decreases and then increases, and the encapsulation efficiency first increases and then decreases. When the degree of polymerization of the cationic block PMPC is 10 and 50, the encapsulation efficiencies of the products are both low. Therefore, in this application, the degree of polymerization of the cationic block PMPC is selected to be 20-40, preferably 30 (Example 1).

[0144] Comparing Example 1, Comparative Examples 6 and 7, it can be seen that the types of hydrophilic and hydrophobic blocks in the cationic polymer ultimately affect the particle size and encapsulation efficiency of the product. Compared with polyester (PCL) and polyethylene glycol ester (MPEG), polyacrylate (PDEAEMA) is used as the hydrophobic block; cationic PMPC is used as the hydrophilic block. The synthesized polymer encapsulates collagen and lysozyme, and the finally obtained product has a smaller particle size and a higher encapsulation efficiency.

[0145] Comparing Comparative Example 1, Example 9, Example 10 and Comparative Example 8, it can be seen that as the mass percentage of the cationic polymer increases, the particle size of the sample gradually increases; the encapsulation efficiency first increases and then decreases. When the mass percentage of the cationic polymer is 3%, the encapsulation efficiency of the sample is the highest, which is 77.8%, and the particle size of the sample is 82.6 nm at this time.

[0146] Comparing Comparative Example 1, Example 11-13 and Comparative Example 9, it can be seen that as the mass percentage of collagen increases, the particle size of the sample gradually increases, and the encapsulation efficiency first increases and then decreases. When the mass percentage of collagen is 5%, the encapsulation efficiency of the sample is the highest, which is 77.8%, and the particle size of the sample is 82.6 nm at this time.

[0147] Comparing Example 1, Examples 16-19, it can be seen that the type and ratio of the solvent for preparing the nano-composition affect the particle size and encapsulation efficiency of the final product. In the present invention, the nano-composition prepared by using a mixed solvent of glycerol, 1,3-propanediol, and 1,4-butanediol has a smaller particle size and a higher encapsulation efficiency; the optimal ratio of glycerol, 1,3-propanediol, and 1,4-butanediol is 20:5:5.

[0148] Test Example 3 - Transdermal Absorption (Penetration Enhancement) Test

[0149] The transdermal absorption (penetration enhancement) test was used to evaluate the effects of Examples 1-8 and Comparative Examples 1-9 on the transdermal absorption (penetration enhancement) ability of collagen. The specific method is as follows:

[0150] For the in vitro transdermal experiment, a vertical diffusion cell was selected, and the skin of nude mice was used as a model (abdominal skin, removing the subcutaneous fat layer and blood vessels). The receiving solution was PBS solution. The skin slice was fixed between the supply pool and the receiving pool, with the skin layer facing up, and equilibrated for 20 min. The liquid of each sample was added to the supply pool, and the receiving solution was taken at 1 h, 2 h, 6 h, 8 h, and 24 h. The receiving solution was ultrasonically demulsified with deionized water, and then the content of collagen was determined by high performance liquid chromatography (HPLC, Shimadzu, Japan), and the cumulative permeation amount per unit area was calculated therefrom. Each group of experiments was carried out in three parallel experiments, and the experimental results were taken as the arithmetic mean. The calculation formula for the cumulative permeation amount per unit area on the skin slice is as shown in formula (2):

[0151]

[0152] Among them, Q n is the cumulative permeation rate per unit area of the sample at time t (μg / cm 2 ), A is the permeation area, C n is the concentration measurement value at time t, C i is the concentration measurement value before time t, V is the total volume of the receiving solution, and V0 is the sampling volume.

[0153] Table 2 Transdermal permeation amount of collagen per unit area in samples of Examples 1-8 and Comparative Examples 1-9

[0154]

[0155] The results of the transdermal absorption characterization of the samples of Examples 1-8 and Comparative Examples 1-9 are shown in Table 2. It can be seen from the results in the table that the type of hydrophobic block polyacrylate, the degree of polymerization of polyacrylate, and the degree of polymerization of the hydrophilic block cationic polymer PMPC all affect the transdermal absorption effect of collagen.

[0156] The transdermal absorption effects of Example 1 at 1 h, 4 h, 8 h, and 24 h are better than those of Examples 2-4, indicating that the type of hydrophobic block polyacrylate affects the transdermal absorption effect of collagen. In the present invention, when the polyacrylate is PDEAEMA, the polymer obtained after graft polymerization with cationic MPC wraps collagen, and the resulting product has a better transdermal absorption effect.

[0157] The transdermal absorption effects of Example 1 at 1 h, 4 h, 8 h, and 24 h are better than those of Examples 5-6 and Comparative Examples 2-3, indicating that the degree of polymerization of the hydrophobic block polyacrylate affects the transdermal absorption effect of collagen. In the present invention, when the degree of polymerization of polyacrylate is 30, the final product has a better transdermal absorption effect.

[0158] The transdermal absorption effects of Example 1 at 1 h, 4 h, 8 h, and 24 h are better than those of Examples 7-8 and Comparative Examples 4-5, indicating that the degree of polymerization of the hydrophilic block PMPC affects the transdermal absorption effect of collagen. In the present invention, when the degree of polymerization of the hydrophilic block PMPC is 30, the final product has a better transdermal absorption effect.

[0159] The transdermal absorption effects of Example 1 at 1 h, 4 h, 8 h, and 24 h are better than those of Comparative Example 6 and Comparative Example 7, indicating that compared with polyester (PCL) and polyethylene glycol ester (MPEG), using polyacrylate (PDEAEMA) as the hydrophobic block; using cationic PMPC as the hydrophilic block, the polymer synthesized to wrap collagen has a better transdermal absorption effect.

[0160] Test Example 4 - Anti-inflammatory test - Determination of TNFα content

[0161] The objects of this anti-inflammatory test are Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, collagen (5% aqueous solution), lysozyme (0.5% aqueous solution), and a mixed aqueous solution of collagen and lysozyme (the concentrations of collagen and lysozyme are 5% and 0.5 respectively). The anti-inflammatory test is divided into two steps: cytotoxicity test and determination of TNFα content.

[0162] Test Step 1: Cytotoxicity test

[0163] Test materials

[0164] Cell line: Mouse macrophage Raw 264.7. Culture medium: High-glucose DMEM medium containing 10% fetal bovine serum. Culture conditions: Incubate at 37 °C, 5% CO2, and saturated humidity. Solution and control: The control group is the culture medium. Thiazolyl blue (MTT) solution: 5 mg / mL.

[0165] Test procedures

[0166] Cell culture: Prepare a cell suspension 24 h before the test. Inoculate the cell suspension into a 96-well cell culture plate, 100 μL per well, and the number of cells per well is 3×10 4 cells, and culture for 24 h.

[0167] Exposure: Discard the original culture medium in the wells. Add 100 μL of test samples with different concentrations to each well (the administration concentrations of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen aqueous solution, 0.5% lysozyme aqueous solution, and the mixed sample of 5% collagen + 0.5% lysozyme aqueous solution are 1.25 wt%, 2.5 wt%, 5 wt%, 10 wt% in sequence), and negative control (the negative control is to add 100 μL of the culture medium, that is, the cells cultured under normal conditions). Incubate in an incubator for 24 h. Observe the cell morphology and characteristics under an inverted microscope.

[0168] MTT test

[0169] Add 100 μL of 0.1 mg / mL MTT solution to each well, and incubate in an incubator for 4 h. Remove the liquid in the wells. Add 150 μL of DMSO to each well, place it on an oscillator and shake for 15 min, and then measure the absorbance at a wavelength of 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0170] Data analysis

[0171] The data of each group are expressed as mean ± standard deviation. Taking the cell viability of the negative control group as 100%, calculate the relative cell viability (Viability) of each group. In this anti-inflammatory test, select the concentration with cell viability ≥ 90% as the highest safe concentration of macrophages, and the selection range of the anti-inflammatory test concentration should not exceed this highest safe concentration.

[0172] Table 3 Cytotoxicity test results of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen aqueous solution, 0.5% lysozyme aqueous solution, and the mixed sample of 5% collagen + 0.5% lysozyme aqueous solution

[0173]

[0174]

[0175] The measurement results of the cytotoxicity of the mixed samples of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen aqueous solution, 0.5% lysozyme aqueous solution, and 5% collagen + 0.5% lysozyme aqueous solution are shown in Table 3. It can be seen from the results in the table that for the above-mentioned Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen aqueous solution, 0.5% lysozyme aqueous solution, and the mixed sample of 5% collagen + 0.5% lysozyme aqueous solution within the concentration range of 5% (administration concentration), the cell survival rate is greater than 90%, and there is no obvious cytotoxicity in the cells. Therefore, the sample concentration is selected as 3%, and the above test samples are formulated into a 3% solution for the determination of TNFα content.

[0176] Test Step 2: Determination of TNFα Content

[0177] Experimental principle: Macrophages are immune effector cells with various immunomodulatory functions, so they are often used as an ideal model to evaluate the immunomodulatory properties of some bioactive substances. When there is an excess of bacterial lipopolysaccharide (LPS) in the body, it will induce macrophages to release inflammatory mediators such as TNFα, NO, and ILs, thereby triggering an inflammatory response in the body. Among them, TNFα can promote T cells to produce various inflammatory factors to promote the inflammatory response and is one of the important inflammatory factors. Establishing an inflammatory model by stimulating macrophages with LPS in vitro is a classic cell model for studying the anti-inflammatory efficacy of active components.

[0178] The determination of TNFα content uses the enzyme-linked immunosorbent assay (ELISA). The specific principle is as follows: After TNFα specifically binds to the TNFα antibody coated on the enzyme-labeled plate, it binds to the anti-TNFα antibody with a substrate label. After the substrate is catalyzed by the enzyme, a colored product is generated, and the TNFα content is positively correlated with the depth of the color of the colored product. The optical density value (OD) is measured at a wavelength of 450 nm with an enzyme-labeled instrument, and the TNFα content is calculated.

[0179] (2) Test Procedures

[0180] Cell culture: Inoculate the cells into a 96-well cell culture plate, with 3×10 4 cells per well, and culture for 24 h.

[0181] Exposure: Discard the original culture medium in the wells. Add the 3% sample solution to each well in the sample group, and supplement the complete culture medium to the negative control group and the modeling group. Pretreat for 2 h. Add 10 ng / mL lipopolysaccharide to the modeling group and the sample group, and culture for 24 h. Only 100 μL of 10 ng / mL lipopolysaccharide is added to the modeling group, aiming to stimulate macrophages to produce TNFα, thereby simulating the "inflammatory" reaction. The negative control group is the cells cultured normally.

[0182] ELISA test: Absorb the culture medium, centrifuge to obtain the supernatant, and test the content of TNFα in the supernatant.

[0183] The anti-inflammatory test results of the samples of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen solution, 0.5% lysozyme solution, and the mixed solution of collagen and lysozyme (the content of collagen in the mixed solution is 5%, and the lysozyme is 0.5%) are as Figure 1 and Figure 2 shown. After using the samples of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen solution, 0.5% lysozyme solution, and the mixed solution of collagen and lysozyme, the contents of TNFα are 9.841 ng / mL (Example 1), 12.647 ng / mL (Example 2), 13.154 ng / mL (Example 3), 13.664 ng / mL (Example 4), 14.754 ng / mL (Comparative Example 1), 17.110 ng / mL (Comparative Example 6), 16.684 ng / mL (Example 7), 26.654 ng / mL (5% collagen solution), 15.648 ng / mL (0.5% lysozyme solution), and 15.101 ng / mL (the mixed solution of collagen and lysozyme), respectively. Compared with the LPS group (i.e., the modeling group, 35.516 ng / mL), the inhibition rates of the samples of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, 5% collagen solution, 0.5% lysozyme solution, and the mixed solution of collagen and lysozyme on the production of the inflammatory factor TNFα are 72.3%, 64.4%, 62.9%, 61.5%, 58.4%, 51.8%, 53.0%, 24.9%, 55.9%, and 57.5%, respectively. The above results can show that the nano-composition with tissue repair and anti-inflammatory effects in Example 1 has the best anti-inflammatory effect, which is better than the samples of Examples 2-4, Comparative Example 6, Comparative Example 7, 5% collagen solution, 0.5% lysozyme solution, and the mixed solution of collagen and lysozyme.

[0184] Test Example 5 - Tissue repair

[0185] Cell growth promotion experiment

[0186] Make the revived human fibroblasts or epidermal cells into a single-cell suspension and count the number to make the cell density 10 4Cells were inoculated at a density of [number] cells / mL into a 96-well plate, with 200 μL of cell suspension added to each well. After the cells adhered to the wall, samples of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, and collagen (5% solution) were added. Five replicates were set for each sample, and blank wells and control wells were also set (the blank wells were normal cultured cells without adding anything; the control wells were wells without adding cell suspension but adding an equal volume of sterile water). The plate was continuously cultured in an incubator at 37 °C and 5% CO₂ for 72 h. 20 μL of MTT solution (prepared with PBS at pH = 7.4) was added to the corresponding wells, and the plate was further cultured in an incubator at 37 °C for 4 h. Then, the supernatant was discarded, and 150 μL of DMSO was added to each well and shaken for 10 min to dissolve the MTT crystals. The absorbance of each well at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0187] (2) Cell migration experiment

[0188] ① Cell seeding: A 6-well plate was evenly divided with a marker pen, with an interval of 0.5 - 1 cm in the middle. Each well was crossed by at least 3 lines, and approximately [number] cells / mL were added to each well. After overnight incubation, the wells could be filled with cells. 5 Cells were added at a density of [number] cells / mL, and the wells could be filled after overnight incubation.

[0189] ② Scratch assay: On the second day, a pipette tip was used to make scratches perpendicular to the horizontal lines. The cells were washed 3 times with PBS to elute the scratched cells, which were then added to a serum-free medium containing RHC as the control group; the cells were added to a medium containing the test sample as the experimental group, with a final concentration of 3% (the dosing concentration). The cells were cultured in an incubator at 37 °C and 5% CO₂, and the sampling time was 24 h.

[0190] ③ Data processing: The overall migration distance of the cells between different time periods was calculated according to formula (2).

[0191] S i = S0 - S t Formula (2)

[0192] where S i represents the overall migration distance of the cells for each time period; S0 represents the distance length at time 0; S t represents the distance length at each time point.

[0193] Table 4 Effects of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, collagen aqueous solution, lysozyme aqueous solution, and the mixed solution of collagen and lysozyme on cell proliferation

[0194]

[0195]

[0196] Table 5 Results of cell migration experiments of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, aqueous collagen solution, aqueous lysozyme solution, and mixed solution of collagen and lysozyme

[0197]

[0198] The results of the proliferation experiments of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, aqueous collagen solution, aqueous lysozyme solution, and mixed solution of collagen and lysozyme on fibroblasts and epidermal cells are shown in Table 4. It can be seen from the results in the table that the results of the lysozyme solution are similar to those of the blank group, and the lysozyme solution cannot promote the proliferation of fibroblasts and epidermal cells. Compared with the blank group, the above-mentioned Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, aqueous collagen solution, and mixed solution of collagen + lysozyme can all promote the proliferation of fibroblasts and epidermal cells, and the nano-composition with tissue repair and anti-inflammatory effects in Example 1 has the best effect on promoting the proliferation of fibroblasts and epidermal cells, which is better than Examples 2-4, Comparative Example 6, Comparative Example 7, aqueous collagen solution, and mixed solution of collagen + lysozyme.

[0199] The results of the cell migration experiments of Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, aqueous collagen solution, aqueous lysozyme solution, and mixed solution of collagen + lysozyme on fibroblasts and epidermal cells are shown in Table 5. It can be seen from the results in the table that the lysozyme solution cannot promote the cell migration of fibroblasts and epidermal cells, and it has no ability to promote tissue repair. Compared with the blank group, the above-mentioned Examples 1-4, Comparative Example 1, Comparative Example 6, Comparative Example 7, aqueous collagen solution, aqueous lysozyme solution, and mixed solution of collagen + lysozyme can all promote the cell migration of fibroblasts and epidermal cells, and the nano-composition with tissue repair and anti-inflammatory effects in Example 1 has the best effect on promoting the cell migration of fibroblasts and epidermal cells, which is better than Examples 2-4, Comparative Example 6, Comparative Example 7, aqueous collagen solution, aqueous lysozyme solution, and mixed solution of collagen + lysozyme.

[0200] The above examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A nano-composition with the efficacy of tissue repair and anti-inflammation, characterized in that, The nano-composition is formed by encapsulating collagen and lysozyme with a cationic polymer, and the molecular chain of the cationic polymer includes a hydrophilic block and a hydrophobic block; The hydrophilic block includes poly(2-methacryloyloxyethyl phosphorylcholine); The hydrophobic block includes at least one of poly(dimethylaminoethyl methacrylate), poly(diethylaminoethyl methacrylate), poly(2-(diisopropylamino)ethyl methacrylate), and poly(2-tert-butylaminoethyl methacrylate); The molecular weight of the cationic polymer is Mn = 17820 - 29631; The degree of polymerization of the cationic polymer is 20 - 40.

2. The nano-composition with tissue repair and anti-inflammatory effects according to claim 1, characterized in that: The preparation method of the cationic polymer includes the following steps: Dissolve the polymerization monomers of the hydrophobic block of the cationic polymer, such as methacrylate, bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide, and N,N,N',N',N''-pentamethyldiethylenetriamine in an organic solvent. Under the protection of an inert gas, add a catalyst and react at 40 - 80 °C for 12 - 48 h; then add the polymerization monomer of the hydrophilic block of the cationic polymer, 2-methacryloyloxyethyl phosphorylcholine, and continue to react at 40 - 80 °C for 12 - 48 h to prepare the cationic polymer.

3. The nano-composition with tissue repair and anti-inflammatory effects according to claim 2, characterized in that: The organic solvent includes at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; The catalyst includes at least one of CuBr, CuCl, and CuI; The molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the polymerization monomer of the hydrophobic block, methacrylate, is 1:10 - 50; The molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the polymerization monomer of the hydrophilic block is 1:40 - 80; The molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to N,N,N',N',N''-pentamethyldiethylenetriamine is 1:4 - 8; The molar ratio of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide to the catalyst is 1:4 - 8.

4. A method for preparing a nano-composition with tissue repair and anti-inflammatory effects according to any one of claims 1-3, characterized in that, Including the following steps: (1) Dissolve the cationic polymer in a solvent, heat and stir. After the cationic polymer is dissolved, a polymer solution is obtained; (2) Add collagen and lysozyme to water, heat and stir. After collagen and lysozyme are dissolved, an aqueous solution is obtained; (3) Add the polymer solution to the aqueous solution and stir to obtain a mixed solution; (4) The mixed solution is subjected to high-speed shear dispersion treatment and high-pressure microfluidic homogenization treatment to obtain a nano-composition with tissue repair and anti-inflammatory effects.

5. The preparation method of the nano-composition with tissue repair and anti-inflammatory effects according to claim 4, characterized in that: The mass percentage of the cationic polymer in the nano-composition in step (1) is 1 - 7%; The dosage of the solvent described in step (1) is 20 - 40% by mass percentage in the nano-composition; The mass percentage of the collagen described in step (2) in the nano-composition is 1 - 10%; The mass percentage of the lysozyme described in step (2) in the nano-composition is 0.1 - 2%; The dosage of water described in step (2) is 41.0 - 77.9% by mass percentage in the nano-composition.

6. The preparation method of the nano-composition with tissue repair and anti-inflammatory effects according to claim 4, characterized in that: The solvent described in step (1) includes at least one of dichloromethane, acetone, tetrahydrofuran, N,N'-dimethylformamide, dimethyl sulfoxide, propylene glycol, glycerol, butylene glycol, pentylene glycol, hexylene glycol, ethylene glycol, pentaerythritol, dipropylene glycol, and diglycol; The heating temperatures described in step (1) and step (2) are independently 40 - 65 °C; the stirring speeds are independently 30 - 300 rpm; the stirring times are independently 10 - 60 min; In step (3), the stirring speed is 30 - 300 rpm; the stirring time is 10 - 60 min.

7. The preparation method of the nano-composition with tissue repair and anti-inflammatory effects according to claim 4, characterized in that: The high-speed shear dispersion treatment described in step (4) is carried out by using a high-shear homogenizing emulsifier for shear homogenization, wherein the rotation speed of the high-shear homogenizing emulsifier is 5000 - 9000 rpm, and the shear dispersion time is 3 - 15 min; The high-pressure microfluidic homogenization treatment described in step (4) is carried out by using a microfluidic homogenizer, wherein the homogenization pressure is 50 - 120 Mpa, and the number of homogenization cycles is 1 - 6 times.

8. The application of the nano-composition with tissue repair and anti-inflammatory effects according to any one of claims 1 - 3 in the preparation of cosmetics and the preparation of drugs with tissue repair and anti-inflammatory effects.

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