A composite protein gel and a method for preparing the same

Abstract

The application discloses a kind of composite protein gels and preparation method thereof, its preparation method includes the following steps: with the blood collection tube with anticoagulant is collected blood;The above-mentioned blood is centrifuged at 300-700g centrifugal acceleration at room temperature 10-20min;After centrifugation, blood is transferred to biological safety cabinet, and the upper plasma layer is extracted, and then it is placed in-80 ℃ ultra-low temperature refrigerator and rapidly frozen, and the freezing time is 24-72h;Then, the frozen plasma is taken out, and freeze-drying treatment is carried out under the condition that the temperature is-55-65 ℃ and the pressure is 50-100Pa, and the freeze-dried plasma is prepared;Calcium chloride injection is added to the freeze-dried plasma at a volume ratio of 1:0.5-2, then reacted in a water bath at 60-90 ℃ for 5-15min, and removed to prepare.The protein gel can effectively solve the problem of easy skin stiffening, collapse, high in vivo absorption rate and poor filler stability caused by hyaluronic acid and autologous fat.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetic products, and particularly relates to a composite protein gel and a preparation method thereof. BACKGROUND

[0002] In recent years, the fillers applied to the skin are mainly the following two kinds: hyaluronic acid and fat.

[0003] Hyaluronic acid (HA) as a dermal layer filler has been widely used in the field of skin treatment. Hylauronic acid has the effects of enhancing the elasticity of soft tissue, promoting the proliferation and differentiation of epidermal cells, and high biocompatibility. However, hyaluronic acid has the disadvantages of causing skin stiffening and collapse after injection, high absorption rate in the body, and no anti-aging and tissue regeneration promoting ability.

[0004] Autologous fat filling is also a technique that is more clinically applied. Autologous fat produces a vascular stromal tissue (SVF) containing a large amount of cell growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor (TGF-β), after preliminary processing in vitro. Among them, the pro-angiogenic effect of VEGF can provide energy supply for the proliferation of stem cells and fibroblasts. However, autologous fat filling has a large trauma to the body and a poor experience; and many clinical data show that the absorption rate of autologous fat filling within 3-6 months is more than 50%, and the stability of the filling material is poor. SUMMARY

[0005] In view of the above problems existing in the prior art, the present application provides a composite protein gel and a preparation method thereof. The protein gel can effectively solve the problems of easy stiffening and collapse of the skin, high absorption rate in the body, and poor stability of the filling material when hyaluronic acid or autologous fat is filled.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is:

[0007] A preparation method of a composite protein gel, comprising the following steps:

[0008] (1) collecting blood with a blood collection tube with an anticoagulant;

[0009] (2) centrifuging the blood in step (1) at room temperature at a centrifugal acceleration of 300-700g for 10-20min;

[0010] (3) aspirating the upper plasma layer in the centrifuged blood in step (2), and placing it at-80℃ for 24-72h of freezing time;

[0011] (4) taking out the frozen blood plasma in step (3), and performing freeze-drying treatment on the blood plasma at a temperature of -55 to -65 DEG C and a pressure of 50 to 100 Pa to obtain freeze-dried blood plasma;

[0012] (5) mixing the freeze-dried blood plasma obtained in step (4) with calcium chloride injection with a concentration of 20 to 50 μmol / L at a volume ratio of 1:0.5 to 2, and then reacting in a water bath at 60 to 90 DEG C for 5 to 15 min to obtain the protein gel.

[0013] Further, the blood in step (1) is umbilical cord blood or autologous venous blood.

[0014] Further, the anticoagulant in step (1) is 3.8% sodium citrate.

[0015] Further, the centrifugal acceleration in step (2) is 600 to 700 g, and the centrifugal time is 10 to 15 min.

[0016] Further, the upper blood plasma layer in step (3) is frozen at -80 DEG C for 36 h.

[0017] Further, the freeze-drying temperature in step (4) is -58 DEG C, and the pressure is 80 Pa.

[0018] Further, the concentration of the calcium chloride injection in step (5) is 30 to 40 μmol / L.

[0019] Further, the freeze-dried blood plasma and the calcium chloride injection are mixed at a volume ratio of 1:0.5 in step (5).

[0020] Further, the water bath temperature in step (5) is 75 to 78 DEG C, and the reaction time is 5 to 7 min.

[0021] A composite protein gel is prepared by the above preparation method.

[0022] The present application has the following advantages:

[0023] 1. The umbilical cord blood is used as the raw material for preparing the protein gel, and the umbilical cord blood contains a large amount of undifferentiated stem cells and growth factors beneficial to cell proliferation and tissue repair. The immune cells in the umbilical cord blood are not mature, the NK and T cells are in the juvenile stage, the antigen expression and immune function activity are low, the immunoglobulin is less, the stem cells have strong regenerative capacity, the immune rejection after filling is small, and the protein gel prepared by treating the umbilical cord blood has low immunogenicity, natural shaping effect and filling effect, no stiffening and collapse phenomenon, long maintenance time after filling, etc.

[0024] 2. In this invention, the freeze-dried plasma undergoes conformational changes under heat treatment and the action of calcium ions, forming a protein gel. This protein gel has a three-dimensional network structure, which is mainly maintained by physical interactions such as hydrophobic interactions and hydrogen bonding. After heat treatment, the interactions between proteins are enhanced, and the hydrophobic groups inside the proteins are exposed, promoting the further formation of disulfide bonds. The presence of a large number of disulfide bonds strengthens the intermolecular cross-linking, forming a stable network gel structure. This gel structure is not easily decomposed or absorbed. When this gel is filled into the skin, it acts as a scaffold, providing a three-dimensional growth environment for the growth of subcutaneous tissue and cells. Calcium ions can also activate platelet-derived growth factor, promote cell differentiation and regeneration, improve cell regeneration capacity, and promote microcirculation in the tissues surrounding the network structure. After a period of growth, the subcutaneous tissue and cells fill the interior of the three-dimensional network structure, thus achieving the purpose of improving the filling effect and prolonging the maintenance time. Because the protein gel provides energy for cell growth at the filling site, cells proliferate in large numbers at this site, and skin tissue is regenerated. Therefore, there is no phenomenon of collapse, absorption, or stiffening after filling, resulting in a good filling effect.

[0025] 3. In this invention, freeze-drying technology is used to prepare freeze-dried plasma. The freeze-dried plasma can be stored for a long time after being sealed. The freeze-dried plasma is convenient to use and transport, and is easy to produce and sell on a large scale. When in use, it is dissolved in a suitable concentration of calcium chloride solvent, and the protein structure inside the freeze-dried plasma is quickly restored. The proteins and growth factors are basically not lost. The protein gel can be prepared by continuing to heat it in a water bath at a suitable temperature. It can be used for filling without the need for on-site preparation, thus improving surgical efficiency. Detailed Implementation

[0026] Example 1

[0027] A composite protein gel, the preparation method of which includes the following steps:

[0028] (1) Umbilical cord blood was collected using a blood collection tube containing 3.8% sodium citrate anticoagulant, with a volume ratio of sodium citrate to umbilical cord blood of 1:13.

[0029] (2) Centrifuge the umbilical cord blood from step (1) at room temperature with a centrifugal acceleration of 300g for 10 minutes;

[0030] (3) Take the upper plasma layer from the blood after centrifugation in step (2) and place it in an ultra-low temperature freezer at -80℃ for 24 hours to freeze rapidly.

[0031] (4) Take out the frozen plasma from step (3) and freeze-dry it at a temperature of -55℃ and a pressure of 50Pa to obtain freeze-dried plasma;

[0032] (5) The freeze-dried plasma obtained in step (4) is mixed with calcium chloride injection solution with a concentration of 20 μmol / L at a volume ratio of 1:0.5, and then reacted in a water bath at 60°C for 5 min. The mixture is then removed to obtain the final product.

[0033] Example 2

[0034] A composite protein gel, the preparation method of which includes the following steps:

[0035] (1) Collect autologous venous blood using blood collection tubes containing 3.8% sodium citrate anticoagulant, with a volume ratio of sodium citrate to autologous venous blood of 1:13;

[0036] (2) Centrifuge the autologous venous blood from step (1) at room temperature with a centrifugal acceleration of 700g for 20 minutes;

[0037] (3) Take the upper plasma layer from the blood after centrifugation in step (2) and place it in an ultra-low temperature freezer at -80℃ for 72 hours to freeze rapidly.

[0038] (4) Take out the frozen plasma from step (3) and freeze-dry it at a temperature of -65℃ and a pressure of 100Pa to obtain freeze-dried plasma;

[0039] (5) The freeze-dried plasma obtained in step (4) is mixed with calcium chloride injection solution with a concentration of 50 μmol / L at a volume ratio of 1:2, and then reacted in a water bath at 90°C for 15 min. The mixture is then removed and the product is obtained.

[0040] Example 3

[0041] A method for preparing a composite protein gel includes the following steps:

[0042] (1) Umbilical cord blood was collected using a blood collection tube containing 3.8% sodium citrate anticoagulant, with a volume ratio of sodium citrate to umbilical cord blood of 1:13.

[0043] (2) Centrifuge the umbilical cord blood from step (1) at room temperature with a centrifugal acceleration of 500g for 15 minutes;

[0044] (3) Take the upper plasma layer from the blood after centrifugation in step (2) and place it in an ultra-low temperature freezer at -80℃ for 48 hours to freeze rapidly.

[0045] (4) Take out the frozen plasma from step (3) and freeze-dry it at a temperature of -65℃ and a pressure of 70Pa to obtain freeze-dried plasma;

[0046] (5) The freeze-dried blood plasma prepared in step (4) is mixed with calcium chloride injection with a concentration of 30 μmol / L at a volume ratio of 1:1.5, and then reacted in a water bath at 80°C for 10 min, and taken out to prepare.

[0047] Example 4

[0048] A composite protein gel, a preparation method thereof, comprising the following steps:

[0049] (1) Collecting umbilical cord blood with blood collection tubes with 3.8% sodium citrate anticoagulant, the volume ratio of sodium citrate to umbilical cord blood being 1:13;

[0050] (2) Centrifuging the umbilical cord blood in step (1) at a centrifugal acceleration of 600g at room temperature for 12 min;

[0051] (3) Absorbing the upper plasma layer in the centrifuged blood in step (2), and rapidly freezing it in a ultra-low temperature refrigerator at -80°C for 36 h;

[0052] (4) Taking out the frozen blood plasma in step (3), and freeze-drying it at a temperature of -58°C and a pressure of 80 Pa to prepare freeze-dried blood plasma;

[0053] (5) Mixing the freeze-dried blood plasma prepared in step (4) with calcium chloride injection with a concentration of 35 μmol / L at a volume ratio of 1:1, and then reacting in a water bath at 76°C for 6 min, and taken out to prepare.

[0054] Comparative Example 1

[0055] A composite protein gel, a preparation method thereof, comprising the following steps:

[0056] (1) Collecting umbilical cord blood with blood collection tubes with 3.8% sodium citrate anticoagulant, the volume ratio of sodium citrate to umbilical cord blood being 1:13;

[0057] (2) Centrifuging the umbilical cord blood in step (1) at a centrifugal acceleration of 900g at room temperature for 5 min;

[0058] (3) Absorbing the upper plasma layer in the centrifuged blood in step (2), and rapidly freezing it in a ultra-low temperature refrigerator at -80°C for 36 h;

[0059] (4) Taking out the frozen blood plasma in step (3), and freeze-drying it at a temperature of -38°C and a pressure of 110 Pa to prepare freeze-dried blood plasma;

[0060] (5) Mixing the freeze-dried blood plasma prepared in step (4) with calcium chloride injection with a concentration of 35 μmol / L at a volume ratio of 1:1, and then reacting in a water bath at 76°C for 6 min, and taken out to prepare.

[0061] Comparative Example 2

[0062] A composite protein gel, a preparation method thereof, comprising the following steps:

[0063] (1) Collecting blood with blood collection tube with 3.8% sodium citrate anticoagulant, the volume ratio of sodium citrate to umbilical cord blood is 1:13;

[0064] (2) Centrifuging the blood in step (1) at a centrifugal acceleration of 600g at room temperature for 12min;

[0065] (3) Absorbing the upper plasma layer in the blood after centrifugation in step (2), and rapidly freezing it in an ultra-low temperature refrigerator at-80℃ for 36h;

[0066] (4) Taking out the frozen plasma in step (3), and performing freeze-drying treatment at a temperature of-58℃ and a pressure of 80Pa to obtain freeze-dried plasma;

[0067] (5) Mixing the freeze-dried plasma prepared in step (4) with calcium chloride injection with a concentration of 15μmol / L at a volume ratio of 1:3, and then reacting in a water bath at 55℃ for 17min, and taking out to obtain.

[0068] Test Example

[0069] I. Randomly selecting 90 female subjects aged 30-50 years old, with an average age of 40 years old, and dividing them into 6 groups, 15 people in each group, according to the blood type matching principle, respectively injecting the composite protein gels prepared by Examples 1-4 and Comparative Examples 1-2 into the 6 groups of subjects, the injection method is point injection, and the maximum injection amount of each part is 2ml, and the shape of the injection site of the subjects is observed and recorded after 7 days, 15 days, 30 days, 90 days, half a year, one year and two years after injection, and the specific results are shown in Table 1.

[0070]

[0071] It can be seen from Table 1 that the protein gels prepared by Examples 1-4 and Comparative Examples 1-2 can be used for facial filling, and have little immune rejection reaction, which disappears after 15 days. The overall effect of the face is good after filling with the protein gels prepared by Examples 1-4 and Comparative Examples 1-2 for a certain period of time, and there is no phenomenon of collapse and rigidity, and the stability is good. The protein gels prepared by Examples 1-4 are gradually decomposed and absorbed after being injected for one year, and the saturation of the injection site decreases more after being injected for about two years. Among them, the protein gel in Example 4 maintains the longest time and has the best filling effect. The protein gels prepared by Comparative Examples 1-2 begin to be decomposed and absorbed after being injected for half a year, the face shows a decrease in three-dimensionality, and there is a slight collapse. After being injected for one year, the face basically returns to the original state, the saturation disappears, and there is no three-dimensionality.

[0072] II. After the freeze-dried plasma prepared in step (4) in Examples 1-4 and Comparative Examples 1-2 is sealed and stored for one year, it is taken out and the protein gel is prepared according to the operation of step (5). The filling effect of the protein gel used in the face of the user according to the above method is basically consistent with direct injection, the maintenance time is basically unchanged, which proves that the protein and growth factors in the freeze-dried plasma prepared according to the operation of step (4) do not disappear or decrease after being sealed and stored for one year. Moreover, the umbilical cord blood is treated and stored in the form of freeze-drying, which is convenient for subsequent use and storage.

Claims

1. A method for preparing a composite protein gel, characterized by, It is prepared by the following steps: (1) collecting blood with a blood collection tube with an anticoagulant, wherein the blood is umbilical cord blood, and the anticoagulant is 3.8% sodium citrate; (2) centrifuging the blood in step (1) at a centrifugal acceleration of 600-700 g for 10-15 min at room temperature; (3) sucking the upper plasma layer in the blood after centrifugation in step (2), and placing it at-80℃, with a freezing time of 36 h; (4) taking out the frozen plasma in step (3), and performing freeze-drying treatment at a temperature of-58℃ and a pressure of 80 Pa to prepare freeze-dried plasma; (5) mixing the freeze-dried plasma prepared in step (4) with calcium chloride injection with a concentration of 30-40 µmol / L at a volume ratio of 1:1, then reacting in a water bath at 75-78℃ for 5-7 min, and taking out to prepare.

2. A composite protein gel, characterized in that, It is prepared by the method of claim 1.

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