阅读说明：本技术 一种含人端粒酶逆转录酶纳米透皮脂质体的制备方法及应用 (Preparation method and application of human telomerase reverse transcriptase-containing nano transdermal liposome ) 是由 南程成 于 2020-06-09 设计创作，主要内容包括：本发明公开一种含人端粒酶逆转录酶纳米透皮脂质体的制备方法,所述方法包括以下步骤：使用乙醇溶液将卵磷脂、胆固醇、PEG-2000在磁力搅拌的条件下混匀,获得混合液一；配置hTERT水溶液,在磁力搅拌的条件下混匀,获得混合液二；将所述混合液一与混合液二混合获得混合液三；将所述混合液三使用磁力搅拌器搅拌后进行超声处理；将经过超声后的混合液三继续使用磁力搅拌器搅拌；将获得的脂质体用无菌滤器进行过滤；将过滤后的溶液存于无菌离心管中。本发明有益效果在于,该纳米透皮脂质体具有良好的透皮性和生物活性,能够用作化妆品中面膜、爽肤水、冻干粉等的添加剂,具有良好的面部细胞抗衰老作用；同时该脂质体功效温和,不会给面部细胞增加额外的负担。(The invention discloses a preparation method of a human telomerase reverse transcriptase-containing nano transdermal liposome, which comprises the following steps: evenly mixing lecithin, cholesterol and PEG-2000 under the condition of magnetic stirring by using an ethanol solution to obtain a mixed solution I; preparing an hTERT aqueous solution, and uniformly mixing under the condition of magnetic stirring to obtain a mixed solution II; mixing the mixed solution I and the mixed solution II to obtain a mixed solution III; stirring the mixed solution III by using a magnetic stirrer and then carrying out ultrasonic treatment; continuing stirring the mixed solution III subjected to ultrasonic treatment by using a magnetic stirrer; filtering the obtained liposome with a sterile filter; the filtered solution was stored in a sterile centrifuge tube. The nano transdermal liposome has the beneficial effects that the nano transdermal liposome has good transdermal property and biological activity, can be used as additives of facial masks, toner, freeze-dried powder and the like in cosmetics, and has good anti-aging effect on facial cells; meanwhile, the liposome has mild efficacy, and does not increase additional burden on facial cells.)

1. A preparation method of a human telomerase reverse transcriptase-containing nano transdermal liposome is characterized by comprising the following steps:

s1, evenly mixing lecithin, cholesterol and PEG-2000 under the condition of magnetic stirring by using an ethanol solution to obtain a mixed solution I;

s2, preparing 0.1-0.5mM of hTERT aqueous solution, and uniformly mixing under the condition of magnetic stirring to obtain a mixed solution II;

s3, slowly dropping the mixed solution I into the mixed solution II to obtain a mixed solution III;

s4, stirring the mixed solution III for 2 hours by using a magnetic stirrer;

s5, putting the mixed solution III stirred in the S4 into an ultrasonic instrument, and carrying out ultrasonic treatment for 45 min;

s6, continuing stirring the mixed solution III subjected to the S5 ultrasonic treatment for 10 hours by using a magnetic stirrer to obtain a liposome;

s7 filtering the liposome obtained in S6 with a sterile filter;

s8, storing the solution filtered by the S7 in a sterile centrifuge tube, and storing the solution in an environment at 4 ℃ to obtain the human telomerase reverse transcriptase-containing nano transdermal liposome.

2. The method for preparing the human telomerase reverse transcriptase-containing nano transdermal liposome of claim 1, wherein the molar ratio of the lecithin, the cholesterol and the PEG-2000 is 15: 23: 6.

3. the method for preparing the human telomerase reverse transcriptase-containing nano transdermal liposome of claim 1, wherein the stirring speed of the magnetic stirrer of the steps S1, S4 and S6 is 1000-3000 rpm.

4. The method of claim 1, wherein the dripping mode of step S3 is to suck the first mixed solution with a 5ml sterile syringe and then to slowly push the syringe piston, so that the liquid in the syringe is slowly dripped into the second mixed solution at a speed of 1-5 drops per minute.

5. The method of preparing the human telomerase reverse transcriptase-containing nano transdermal liposome of claim 1, wherein in said step S7, the filtration is first performed three times using a 0.45um sterile filter, and then is performed three times using a 0.22um sterile filter.

6. The method for preparing the transdermal nanoliposome containing human telomerase reverse transcriptase of claim 1, wherein the size of the transdermal nanoliposome containing human telomerase reverse transcriptase is 30-50 nm.

7. The use of the human telomerase reverse transcriptase-containing nano transdermal liposome prepared according to claim 1, wherein the human telomerase reverse transcriptase-containing nano transdermal liposome is finally applied into basal cell nucleus by carrying hTERT across the dense stratum corneum of skin and the cell membrane structure of basal cells.

Technical Field

The invention belongs to the field of medical cosmetology, and particularly relates to a preparation method and application of a human telomerase reverse transcriptase-containing nano transdermal liposome.

Background

Aging is a process in which the function of body tissues and organs gradually declines and finally dies as the organism grows and matures. The aging of the facial skin is the visual expression of the aging of human body, and the aging of the skin is mainly expressed by the loose skin, the appearance of wrinkles, the pigmentation of pigment spots, dull color and luster and the like. Aging has been a major area of life science concern. Mechanisms by which cells undergo senescence include replicative senescence, DNA damage-induced senescence, proto-oncogene-induced senescence, and the like. Of which continuous cell replication leading to senescence is the most common.

In the normal human cell division process, with the increase of the cell division times, the telomeres of the cells are continuously shortened, and finally, the cells are aged and killed. The telomere is positioned at the tail end of the chromosome, and can protect the stability of the genetic information of the subtelomere region. As an important marker associated with biological aging, telomeres play a crucial role in the pathophysiological processes of cell replicative aging. If telomeres in chromosomes do not become short during cell replication, the cells will not age.

The relative molecular mass of telomerase is 200-500KD, and the telomerase is a ribonucleoprotein complex which is responsible for lengthening telomeres in cells, and the activity of the telomerase reverse transcriptase (hTERT) is closely related to the expression of human telomerase reverse transcriptase (hTERT). Research shows that exogenous hTERT can improve the activity of cell telomerase and endow cells with the capacity of delaying senility or immortalization.

Disclosure of Invention

Human telomerase reverse transcriptase needs to pass through the dense stratum corneum and cell membrane structures in order to reach the dermal layers of the facial skin to function. Meanwhile, human telomerase reverse transcriptase is protein in nature, is very sensitive to changes of light, temperature and Ph in the environment, and is easy to inactivate telomerase. Based on the defects, a nanoliposome with a phospholipid bilayer structure similar to a cell membrane structure is constructed. The nanoliposome wraps human telomerase reverse transcriptase, so that the activity of telomerase can be maintained for a long time, and the wrapped telomerase can smoothly reach cells in a dermis layer to play a role.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of a human telomerase reverse transcriptase-containing nano transdermal liposome comprises the following steps:

s1, evenly mixing lecithin, cholesterol and PEG-2000 under the condition of magnetic stirring by using an ethanol solution to obtain a mixed solution I;

s2, preparing 0.1-0.5mM of hTERT aqueous solution, and uniformly mixing under the condition of magnetic stirring to obtain a mixed solution II;

s3, slowly dropping the mixed solution I into the mixed solution II to obtain a mixed solution III;

s4, stirring the mixed solution III for 2 hours by using a magnetic stirrer;

s5, putting the mixed solution III stirred in the S4 into an ultrasonic instrument, and carrying out ultrasonic treatment for 45 min;

s6, continuing stirring the mixed solution III subjected to the S5 ultrasonic treatment for 10 hours by using a magnetic stirrer to obtain a liposome;

s7 filtering the liposome obtained in S6 with a sterile filter;

s8, storing the solution filtered by the S7 in a sterile centrifuge tube, and storing the solution in an environment at 4 ℃ to obtain the human telomerase reverse transcriptase-containing nano transdermal liposome.

Preferably, the molar ratio of the lecithin to the cholesterol to the PEG-2000 is 15: 23: 6.

preferably, the stirring speed of the magnetic stirrer in the steps S1, S4 and S6 is 1000-.

Preferably, the dropping manner in the step S3 is to suck the first mixed solution by using a 5ml sterile syringe, and then slowly push the syringe piston to slowly drop the liquid in the syringe into the second mixed solution, wherein the dropping speed is 1-5 drops per minute.

Preferably, in the step S7, the filter is first filtered three times using a 0.45um sterile filter, and then filtered three times using a 0.22um sterile filter.

Preferably, the prepared transdermal nano-liposome containing the human telomerase reverse transcriptase has the size of 30-50 nm.

The invention also provides application of the human telomerase reverse transcriptase-containing nano transdermal liposome prepared by the method, and the human telomerase reverse transcriptase-containing nano transdermal liposome finally acts in basal cell nucleus by carrying hTERT to penetrate through the compact cuticle of skin and the cell membrane structure of basal cells.

The nano transdermal liposome has the beneficial effects that the nano transdermal liposome has good transdermal property and biological activity, can be used as additives of facial masks, toner, freeze-dried powder and the like in cosmetics, and has good anti-aging effect on facial cells; meanwhile, the liposome has mild efficacy, and does not increase additional burden on facial cells.

Drawings

FIG. 1 is a schematic diagram of a human telomerase reverse transcriptase-containing nano transdermal liposome of the present invention;

FIG. 2 is a schematic representation of the appearance of senescence in cells in a control group of the invention;

FIG. 3 is a schematic diagram showing that the cells in the experimental group of the present invention did not undergo senescence.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.

The invention relates to a preparation method of a human telomerase reverse transcriptase-containing nano transdermal liposome, which comprises the following steps:

s1, evenly mixing lecithin, cholesterol and PEG-2000 under the condition of magnetic stirring by using an ethanol solution to obtain a mixed solution I;

s2, preparing 0.1-0.5mM of hTERT aqueous solution, and uniformly mixing under the condition of magnetic stirring to obtain a mixed solution II;

s3, slowly dropping the mixed solution I into the mixed solution II to obtain a mixed solution III;

s4, stirring the mixed solution III for 2 hours by using a magnetic stirrer;

s5, putting the mixed solution III stirred in the S4 into an ultrasonic instrument, and carrying out ultrasonic treatment for 45 min;

s6, continuing stirring the mixed solution III subjected to the S5 ultrasonic treatment for 10 hours by using a magnetic stirrer to obtain a liposome;

s7 filtering the liposome obtained in S6 with a sterile filter;

s8, storing the solution filtered by the S7 in a sterile centrifuge tube, and storing the solution in an environment at 4 ℃ to obtain the human telomerase reverse transcriptase-containing nano transdermal liposome.

Preferably, the molar ratio of the lecithin to the cholesterol to the PEG-2000 is 15: 23: 6.

preferably, the stirring speed of the magnetic stirrer in the steps S1, S4 and S6 is 1000-.

Preferably, the dropping manner in the step S3 is to suck the first mixed solution by using a 5ml sterile syringe, and then slowly push the syringe piston to slowly drop the liquid in the syringe into the second mixed solution, wherein the dropping speed is 1-5 drops per minute.

Preferably, in the step S7, the filter is first filtered three times using a 0.45um sterile filter, and then filtered three times using a 0.22um sterile filter.

Preferably, the prepared transdermal nano-liposome containing the human telomerase reverse transcriptase has the size of 30-50 nm.

The invention also provides application of the human telomerase reverse transcriptase-containing nano transdermal liposome prepared by the method, and the human telomerase reverse transcriptase-containing nano transdermal liposome finally acts in basal cell nucleus by carrying hTERT to penetrate through the compact cuticle of skin and the cell membrane structure of basal cells.