CN111329837B

CN111329837B - Melatonin flexible liposome and preparation method and application thereof

Info

Publication number: CN111329837B
Application number: CN201911254151.9A
Authority: CN
Inventors: 侯雪芹; 张萌; 张汉霆
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2019-08-01
Filing date: 2019-12-10
Publication date: 2023-03-28
Anticipated expiration: 2039-12-10
Also published as: CN111329837A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a melatonin flexible liposome, and a preparation method and application thereof. The melatonin flexible liposome is prepared from the following raw materials in parts by weight: 10-30 parts of lecithin, 70.00-90.00 parts of cholesterol and 10-30 parts of sodium deoxycholate. The melatonin flexible liposome has better percutaneous permeation effect. The results of pharmacokinetic experiments in rats show that the AUC of the flexible liposome is improved by 1.82 times compared with that of the common liposome, which indicates that the flexible liposome can improve the bioavailability of melatonin absorbed through skin. The pharmacodynamic experiment result of mice shows that the external flexible liposome has the function of resisting photoaging. The flexible liposome enters into the body through the skin, changes the structures of stratum corneum lipid and keratin, and can resist skin damage caused by ultraviolet sunburn.

Description

Melatonin flexible liposome and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a melatonin flexible liposome as well as a preparation method and application thereof.

Background

Melatonin (Meldonin, MT) is also called as pineal gland hormone, commonly called as human body 'brain platinum', has a chemical structural formula of N-acetyl-5-methoxytryptamine and a molecular formula of C13N2H16O2, and is a hormone with antioxidant activity secreted by animals and plants. In human body, melatonin is a compound secreted by pineal gland in human brain, can resist oxidation, regulate sleep and waking rhythm, help to improve sleep, enhance human immunity, keep the balance of internal environment of organism and improve the defense ability of organism; and can act on secretion system to delay senility, maintain the zinc content in blood, and resist tumor and cardiac and cerebral vascular diseases. But the content of the melatonin in the body is very small, and the exogenous melatonin is usually supplemented to maintain the normal physiological function of the body.

Melatonin is usually supplemented by oral administration, but most of the drugs are degraded by the liver and are rarely available to the human body due to the liver first-pass effect. In addition, the melatonin has short half-life period in vivo, low and unstable solubility, so that the absorption of the melatonin in vivo is limited, and the curative effect of a common melatonin preparation is influenced. In addition, long-term oral melatonin can cause emotional anxiety, uneasiness, dysphoria and suspiciousness. Therefore, the problem that the oral administration mode of melatonin is changed by developing a new melatonin dosage form, so that the problems of strong liver first pass effect, low absorption availability and great side effect of oral melatonin are solved, and the problem needs to be solved at present.

Disclosure of Invention

The invention aims to provide a melatonin flexible liposome.

The invention also aims to provide a preparation method of the melatonin flexible liposome.

The invention also aims to provide application of the melatonin flexible liposome in preparing medicines or skin care products for resisting skin photoaging.

The invention also aims to provide application of the melatonin flexible liposome in preparing a medicament for treating skin lesions caused by resisting ultraviolet sunburn and/or application of the melatonin flexible liposome in preparing a skin care product for resisting skin lesions caused by ultraviolet sunburn.

The invention solves the technical problems through the following technical scheme:

a melatonin flexible liposome is prepared from the following raw materials in parts by weight: 10.00-30.00 parts, 70.00-90.00 parts of lecithin, 10.00-30.00 parts of cholesterol and 10.00-30.00 parts of sodium deoxycholate.

Preferably, the melatonin flexible liposome is prepared from the following raw materials in parts by weight: 15.00-25.00 parts, 75.00-85.00 parts of lecithin, 15.00-25.00 parts of cholesterol and 15.00-25.00 parts of sodium deoxycholate.

More preferably, the melatonin flexible liposome is prepared from the following raw materials in parts by weight:

18.90 parts, 80.00 parts of lecithin, 20.00 parts of cholesterol and 18.91 parts of sodium deoxycholate.

The preparation method of the melatonin flexible liposome comprises the following steps:

precisely weighing melatonin, lecithin, cholesterol and sodium deoxycholate in the formula amount, placing the mixture into a reactor, adding chloroform and absolute ethyl alcohol for water bath ultrasonic dissolution, placing the reactor on a rotary evaporator for rotary evaporation under reduced pressure at 40 ℃ until an organic solvent is volatilized to form a layer of film on the wall of the reactor, then adding distilled water for continuous rotary evaporation under reduced pressure for 1h to fully hydrate the film, and finally carrying out ultrasonic treatment for 15min by using an ultrasonic cell crusher to obtain the melatonin flexible liposome.

The ratio of the chloroform dosage to the total dosage of lecithin and cholesterol is 1: and (5) mg. The total dosage of the lecithin and the cholesterol is the sum of the dosages of the lecithin and the cholesterol.

The ratio of the dosage of the absolute ethyl alcohol to the total dosage of the cholesterol, the lecithin and the cholesterol is 1: and (5) mg.

The ratio of the amount of the distilled water to the total amount of the lecithin and the cholesterol is 1: and (5) mg.

The melatonin flexible liposome is applied to the preparation of anti-skin photoaging medicines or skin care products.

The melatonin flexible liposome is applied to the preparation of the medicine for treating skin lesion caused by resisting ultraviolet sunburn.

The melatonin flexible liposome is applied to the preparation of skin care products for resisting skin lesions caused by ultraviolet sunburn.

The melatonin flexible liposome is applied to the preparation of skin care products for resisting the damage of ultraviolet rays to skin elastic fibers.

The melatonin flexible liposome is applied to the preparation of skin care products for resisting the reduction of collagen content in skin tissues caused by photoaging.

The melatonin flexible liposome is applied to the preparation of skin care products for resisting skin relaxation and wrinkles caused by photoaging.

The invention has the beneficial effects that:

(1) The optimal prescription after being optimized by Design-Expert software is as follows: 18.90mg of melatonin, 80.00mg of lecithin, 20.00mg of cholesterol and 18.91mg of sodium deoxycholate, wherein the particle diameter of the flexible liposome prepared by the method is 49.47nm, the entrapment rate is 73.91 percent, and the drug loading rate is 9.92 percent. FTIR, DSC and XRD analysis results prove that the chemical structure of the melatonin prepared into the flexible liposome is not changed, and the medicine is successfully encapsulated in the flexible liposome membrane material. The mouse in vitro skin permeation experiment result shows that the unit area accumulated permeation amount of the flexible liposome is 1.5 times higher than that of the common liposome, and the flexible liposome is more easily permeated into the body. The results of pharmacokinetic experiments in rats show that the AUC of the flexible liposome is improved by 1.82 times compared with that of the common liposome, which indicates that the flexible liposome can improve the bioavailability of melatonin absorbed through skin. The pharmacodynamic experiment result of mice shows that the external melatonin preparation has the function of resisting photoaging. Experimental results of a melatonin percutaneous permeation mechanism show that the flexible liposome enters a body through skin to change the structures of stratum corneum lipid and keratin, and the quantity of the flexible liposome phagocytosed by Langerhans cells is different in different administration time.

(2) The melatonin flexible liposome prepared by the film dispersion method has smaller particle size and PDI, higher entrapment rate and drug-loading rate, and good physical and chemical property representation of the melatonin flexible liposome. The results of in vitro percutaneous permeation experiments indicate that the melatonin flexible liposome has better percutaneous permeation effect. The research on a transdermal mechanism proves that the flexible liposome can change the structure of the skin stratum corneum lipid and keratin through transdermal penetration, and can be phagocytized and presented by Langerhans cells into the body to play a role. As proved by pharmacokinetic and pharmacodynamic experiments, compared with the common liposome, the melatonin flexible liposome is easier to permeate into the body through the skin to exert the drug effect, the bioavailability is improved, the melatonin flexible liposome has the effect of resisting skin photoaging, the biocompatibility is good, and a certain theoretical basis is provided for preventing and treating skin lesion caused by ultraviolet sunburn resistance by the melatonin.

(3) The common oral dosage of the melatonin is 1-3 mg, and the melatonin flexible liposome is adopted for administration, so that the dosage of the melatonin can be greatly reduced through external application to the skin, and the side effect of the melatonin is reduced.

(4) The nano delivery system is utilized to change the dosage form and the administration mode of the medicament, the medicament is wrapped in the flexible liposome for transdermal administration, the particle size of the medicament can be reduced, the solubility of the medicament is increased, the first-pass effect is avoided, in addition, more medicaments can be encapsulated in the flexible liposome due to the existence of the phospholipid bilayer and the membrane softener, the medicament can be extruded into the deeper layer of the skin through the self deformability, the absorption condition of the melatonin is improved, and the bioavailability is improved. As a novel skin drug delivery system, the flexible liposome has the advantages of common liposome as skin drug delivery, high flexibility and permeability, can carry more drugs to permeate into subcutaneous tissues through the barrier effect of the skin stratum corneum, and can restore the deformation to prevent the drugs from leaking.

(5) Examination of melatonin flexible liposome in vitro in mice by using improved Franz diffusion cellThe result shows that compared with the common liposome, the flexible liposome prepared from the medicament can further improve the transdermal absorption effect of the medicament, does not pass through the liver and intestine circulation, and reduces the adverse reaction. The average transdermal speed of the melatonin flexible liposome prepared in the experiment is 0.0176 mg-cm ^-2 ·h ^-1 2 times higher than common liposomes; the cumulative permeation amount is 0.2514mg cm ^-2 Compared with the common liposome, the melatonin flexible liposome is 1.5 times higher, which shows that the melatonin flexible liposome has good transdermal effect and is an ideal administration form applied to clinical treatment.

(6) The pharmacokinetic characteristics of the melatonin liposome in a rat body are measured in a transdermal administration mode, and the experimental results show that the area under the curve and the blood concentration of the melatonin flexible liposome are larger than those of the common liposome, so that the content of the melatonin entering the body through the skin in the flexible liposome is higher, and the bioavailability is improved. But the clearance rate is reduced compared with the common liposome, which reflects that the retention time of the drug in the body is prolonged, the drug absorption is increased, and the treatment effect is enhanced.

(7) The experimental result of the effect of the external melatonin preparation on treating skin photoaging caused by ultraviolet radiation shows that compared with a mouse irradiated under an ultraviolet lamp, the elasticity and the water content of the skin of the mouse applied with the melatonin liposome are increased after the skin is irradiated by the ultraviolet lamp, collagen fiber bundles are arranged regularly and increased, the skin structure is complete, and the preparation has the advantages of no influence on the normal skin structure, good biocompatibility and reference and basis for the development of melatonin cosmetics.

(8) The mechanism by which flexible liposomes enhance skin penetration was examined by SEM, DSC and FTIR analysis. The results indicate that flexible liposomes can alter the structure of stratum corneum lipids and keratin, thereby overcoming the barrier effect of the stratum corneum to more effectively penetrate into the skin. Finally, from the immunological perspective, the preliminary research on how the liposome penetrates into the body through the skin to play a role shows that the liposome is coated on the skin as an antigen which can be taken up and treated by LC on the skin epidermis and finally presented to T cells to play a role, and the LC intake changes along with the change of time, which has a positive effect on the deep research of LC in the skin immunological role in the later period.

(9) The experimental result of the influence of melatonin on skin elastic fibers shows that the melatonin flexible liposome is found to resist the damage of UV on the skin elastic fibers, and the effect of the melatonin flexible liposome is close to the normal form and physiological structure of the melatonin flexible liposome. The experimental result of the influence of the melatonin flexible liposome on the collagen content in the skin tissue of the photoaging mouse shows that the collagen content of the skin of the MC group is remarkably reduced (P is less than 0.05) compared with that of the SC group, and the success of model building of the photoaging model of the skin of the mouse is prompted. Compared with the MC group, the skin collagen content of the mice in the VC group has no significant difference (P > 0.05), and compared with the VC group, the collagen content of the skin of the mice is greatly increased (P < 0.05) after the melatonin liposome, especially the flexible liposome, is administered to the skin of the mice, and is close to the normal level. The experimental result of the influence of the melatonin liposome on the MMPs level in the skin tissue of the photoaging mouse shows that the skin of the mouse is relaxed and wrinkled, the expressions of MMP-1 and MMP-3 in the skin tissue are abnormally increased, and the contents of MMP-1 and MMP-3 in the skin tissue of the mouse given to the melatonin liposome are obviously reduced; the melatonin flexible liposome can obviously inhibit the increase of MMP-1 and MMP-3 secretion in the skin of a mouse caused by UV, and the effect of the melatonin flexible liposome is superior to that of the common melatonin liposome.

Drawings

Fig. 1 is a three-dimensional effect surface trend graph of the relationship between different factors and response values of the melatonin flexible liposome prescription optimization experiment of the invention.

Fig. 2 is a graph in which the cumulative release amount of melatonin flexibel lipid is plotted according to the cumulative release amount and sampling time points.

Fig. 3 shows HPLC chromatograms of blank plasma (a), blank liposomes (b) and melatonin-containing plasma (c).

Fig. 4 is a graph of two different melatonin liposome drugs.

FIG. 5 is a graph showing the change in body weight of mice in each group.

FIG. 6 shows the skin appearance evaluation of each group of mice (a: appearance of mice irradiated with UV rays, b: evaluation of skin of the back of mice after UV irradiation, P <0.05, P <0.01, P < 0.001)

FIG. 7 shows the evaluation of the skin elasticity of the mice in each group (a: skin test method and results, b: recovery time after stretching statistical chart, P <0.05, P <0.01, P < 0.001)

Figure 8 is a statistical plot of the percent skin hydration of each group of mice (P <0.05, P <0.01, P < 0.001).

FIG. 9 shows the results of histopathological examination of mouse skin HE staining NC (100X), SC (200X), MC (100X), VC (100X), EL (100X), CL (100X) (ED: epidermis; DR: dermis; ST: subcutaneous tissue; HF: hair follicle; SG: sebaceous gland; DEG: dermal-epidermal junction; DAC: densely arranged collagen fibers; and DC: degenerated, randomly arranged collagen fibers).

FIG. 10 is a scanning electron micrograph of mouse skin (a: control group, b: melatonin flexible liposome group).

FIG. 11 is mouse skin DSC (a) and FTIR spectrum (B) (A: v asCH2, B: v sCH2, C: v sC = O, D: amide I, E: amide II).

FIG. 12 is a schematic diagram of a Langerhans cell flow analysis strategy.

FIG. 13 is a flow chart of the dynamic changes in phagocytosis of FITC fluorescent liposomes at different time points (a: FITC-EL, b: FITC-CL).

FIG. 14 is a graph showing the dynamic change of the proportion of cells phagocytosing FITC fluorescent liposomes at different time points.

FIG. 15 is a graph showing the results of pathological changes in the elastic fibers of mouse skin tissues.

Fig. 16 is a graph showing the effect of melatonin flexible liposomes on collagen content in skin tissues of photoaged mice.

Fig. 17 is a graph of the effect of melatonin liposomes on the levels of MMPs in skin tissue of photoaged mice.

Detailed Description

The invention will be further described with reference to the accompanying drawings and specific embodiments so that those skilled in the art may better understand the invention, but the invention is not limited thereto.

Example 1

A melatonin flexible liposome, prescription:

18.90mg, lecithin 80.00mg, cholesterol 20.00mg and sodium deoxycholate 18.91mg.

The preparation method comprises the following steps:

precisely weighing melatonin, lecithin, cholesterol and sodium deoxycholate in the formula amount, placing the mixture into a round-bottom flask, adding 10mL chloroform and 10mL absolute ethyl alcohol for water bath ultrasonic dissolution, placing the flask on a rotary evaporator at 40 ℃, performing reduced pressure rotary evaporation until an organic solvent is volatilized to form a layer of thin film on the wall of the flask, then adding 10mL distilled water, continuing to perform reduced pressure rotary evaporation for 1h to fully hydrate the thin film, and finally performing ultrasonic treatment for 15min by using an ultrasonic cell crusher to obtain the melatonin flexible liposome.

Example 2

A melatonin flexible liposome, prescription:

15.00mg, lecithin 75.00mg, cholesterol 25.00mg, and sodium deoxycholate 15.00mg.

The preparation method comprises the following steps:

Example 2

A melatonin flexible liposome, which is prepared by the following steps:

25.00mg, lecithin 85.00mg, cholesterol 15mg, and sodium deoxycholate 15.00mg.

The preparation method comprises the following steps:

Example 4

A melatonin flexible liposome, which is prepared by the following steps:

10.00mg, lecithin 70.00mg, cholesterol 10.00mg, and sodium deoxycholate 30.00mg.

The preparation method is the same as example 1.

Example 5

A melatonin flexible liposome, which is prepared by the following steps:

30.00mg, lecithin 90.00mg, cholesterol 10.00mg, sodium deoxycholate 10.00mg.

The preparation method is the same as example 1.

A series of experiments were carried out on the melatonin flexible liposomes of the present invention, the details of which are as follows:

1. preparation and prescription optimization of melatonin flexible liposome

(I) Experimental method

1. Preparation of melatonin flexible liposome

The melatonin flexible liposome is prepared by a film dispersion method. Precisely weighing melatonin, lecithin, cholesterol and sodium deoxycholate in the formula amount, placing the mixture into a round-bottom flask, adding 10mL chloroform and 10mL absolute ethyl alcohol for water bath ultrasonic dissolution, placing the flask on a rotary evaporator at 40 ℃, performing reduced pressure rotary evaporation until an organic solvent is volatilized to form a layer of thin film on the wall of the flask, then adding 10mL distilled water, continuing to perform reduced pressure rotary evaporation for 1h to fully hydrate the thin film, and finally performing ultrasonic treatment for 15min by using an ultrasonic cell crusher to obtain the melatonin flexible liposome. Sodium deoxycholate is not added when a film forming material of the flexible liposome is prepared, and other conditions are not changed, so that the melatonin ordinary liposome can be prepared.

2. Melatonin flexible liposome particle size determination

And (3) taking 100 mu L of the prepared melatonin flexible liposome, diluting the melatonin flexible liposome to 1mL by ultrapure water, and measuring the particle size by using a Malvern laser particle size analyzer.

3. Melatonin flexible liposome encapsulation efficiency and drug loading rate determination

The entrapment rate and the drug-loading rate of the melatonin flexible liposome are measured by adopting an ultrafiltration centrifugation method. Respectively and precisely measuring 200 mu L of the two melatonin liposome solutions and placing the two melatonin liposome solutions in an ultrafiltration centrifugal tube. The tube was centrifuged at 8000rpm for 20min at 4 ℃ in a centrifuge, 50. Mu.L of the solutions containing free melatonin in the two outer tubes were measured precisely, diluted with 4mL of methanol and shaken up, respectively, and then the content of free melatonin was measured by HPLC. The entrapment efficiency (EE%) and drug loading (DL%) of the liposomes were calculated according to the following formulas.

Encapsulation efficiency

Drug loading capacity->

4. Melatonin flexible liposome formulation optimization

4.1 prescription design

On the basis of a preliminary experiment, a melatonin flexible liposome prescription is optimized by using a central combined response surface optimization method in Design-Expert software. The prescription is optimized by taking the content of sodium deoxycholate (X1, mg), the ratio of the drug to lecithin and cholesterol, namely the drug-to-lipid ratio (X2,%), as dependent variables and the particle size (Y1, d/nm), the entrapment rate (Y2, EE/%), the drug loading rate (Y3, DL/%) of the prepared liposome as response values, and the experimental factor levels are shown in Table 1.

TABLE 1 experiment factor level table

4.2 data processing

And designing a prescription according to the table 1, preparing melatonin flexible liposomes, respectively measuring the response values, performing regression analysis on each index data, and fitting the index data into a binomial exponential equation model.

4.3 response surface analysis and prediction

Preparing melatonin flexible liposome with different factor levels according to the requirements of a design prescription, and measuring the particle size, the entrapment rate and the drug-loading rate. And drawing a three-dimensional effect surface trend graph of the influence of the factors on the response value by using Design-Expert software to investigate the influence of the prescription factors on the response value.

4.4 analysis of actual and predicted values of prescription after optimization

The prepared flexible liposome is expected to have smaller particle size and larger entrapment rate and drug-loading capacity, so that the flexible liposome is optimized by Design-Expert software according to limited conditions, then 3 batches of flexible liposomes are prepared according to the optimized prescription, and the particle size, the entrapment rate and the drug-loading capacity are measured and analyzed.

(II) results of the experiment

1. The design results of the melatonin flexible liposome prescription optimization experiments are shown in table 2.

Table 2 melatonin flexible liposome prescription optimization experimental design table

2. Center combined response surface optimization analysis result

From the trend graph (fig. 1) of the three-dimensional effect surface, when the drug-lipid ratio is increased, the change of the particle size is not large, but the encapsulation efficiency is reduced along with the increase of the drug loading; when the content of sodium deoxycholate is increased, the encapsulation rate is increased and then decreased, and the particle size is decreased and then increased, but the drug loading amount is not changed greatly.

The three response values were subjected to regression analysis according to the experimental data of table 2 to obtain a binomial equation model:

Y ₁ ＝75.569-4.587X ₁ -2.168X ₂ +5.000X ₁ X ₂ +0.291X ₁ ² +0.076X ₂ ² ，Y ₂ ＝68.846-1.665X ₁ +0.469X ₂ +0.023X ₁ X ₂ -0.571X ₁ ² -0.011X ₂ ² ，Y ₃ ＝3.201+1.485X ₁ -7.700X ₂ +0.025X ₁ X ₂ -0.032X ₁ ² -2.672X ₂ ² the fitting equation is tested by F to obtain P < 0.0001, which shows that the equation model has significance and good fitting degreeTherefore, the prepared melatonin flexible liposome can be optimized by the prescription of the model.

3. Optimized result analysis of actual value and predicted value of prescription

TABLE 3 analysis table of results of optimized prescription actual value and predicted value

The Design-Expert software is used for optimizing to obtain the optimal prescription X ₁ ＝18.91mg，X ₂ =3.78%. The actual values of particle size, encapsulation efficiency and drug loading of the flexible liposomes prepared by the optimal formulation are shown in table 3. The results in the table show that the actual measured values and the model predicted values are within the error allowable range, which indicates that the software has good prediction on the designed model, so the model prediction results are selected as the optimal prescription of the experiment for carrying out the experiment.

(III) discussion

The ability of flexible liposomes to readily deform to penetrate the skin barrier is of great interest for improving transdermal drug delivery. The experimental result of melatonin lipid-water partition coefficient proves that melatonin is an amphiphilic molecule, but lipophilicity is higher than hydrophilicity, the flexible melatonin liposome is prepared by adopting a film dispersion method and adding sodium deoxycholate into a film forming material in the research, medicine powder is directly added into the film forming material to be subjected to reduced pressure rotary evaporation to form film hydration so as to be wrapped in a phospholipid bilayer, and the obtained liposome encapsulation rate reaches (73.91 +/-0.13)%, so that the flexible melatonin liposome is higher than other documents. In addition, as melatonin can be better dissolved in organic solvents such as chloroform and the like, and the solubility in water is lower, the medicine and the film-forming material are dissolved in the organic solvents together to be rotated, evaporated and hydrated to form a film, more medicines can be wrapped in a phospholipid bilayer, and the experimental result proves that the flexible liposome prepared by the method has high encapsulation efficiency and good stability.

The experimental result of prescription optimization of the melatonin flexible liposome shows that the increase of the drug-lipid ratio has little influence on the particle size, but the entrapment rate is reduced, the drug loading capacity is increased, probably because the drug encapsulated in the flexible liposome membrane has certain saturation, and the drug loading capacity exceeds the phospholipid loading capacity due to excessive drug, so that the drug amount encapsulated in the flexible liposome vesicle is reduced, the free drug amount is increased, and the entrapment rate of the flexible liposome is reduced, and the drug loading capacity is increased. The encapsulation rate is increased and then decreased along with the increase of the content of the sodium deoxycholate, the particle size tends to increase and decrease, and the influence on the drug loading is small, probably because a small amount of a proper amount of a surfactant is inserted into a phospholipid bilayer to increase the intermolecular distance of phospholipid, interfere the sequence of the acyl chains of the phospholipid, increase the fluidity of the phospholipid, correspondingly reduce the content of free drugs, reduce the particle size and increase the encapsulation rate, but when the added amount is excessive, the phospholipid bilayer membrane structure formed by a lipid material is damaged, so that aggregated drug leakage occurs to increase the particle size and decrease the encapsulation rate. Therefore, the film softener is used as little as possible while ensuring the encapsulation efficiency and the drug loading.

It should be noted that after the flexible liposome is formed into a film, the organic solvent must be completely volatilized and then hydrated, the residual organic solvent may affect the aggregation state of the phospholipid, and emulsion droplets may exist in the obtained product. The phase transition temperature of the phospholipid is noticed during hydration so as to prevent phase separation when the phospholipid undergoes phase transition, so that the fluidity of the membrane is increased, the leakage of contents is caused, and the encapsulation efficiency is reduced; and must be completely hydrated, otherwise the resulting liposomes may be non-uniform in size.

2. Melatonin flexible liposome in vitro percutaneous permeation experiment

After transdermal administration, the medicine can be directly absorbed through skin and enter blood circulation, and the first pass effect of liver can be avoided, so that the bioavailability and the treatment effect of the medicine are improved, and the irritation of the medicine to the gastrointestinal tract is reduced. In order to evaluate the transdermal permeation effect of the drug, the summary adopts an improved Franz diffusion cell to research the in-vitro transdermal permeation accumulated release amount, transdermal rate constant, permeation coefficient, retention time and other permeation kinetics parameters of the melatonin flexible liposome.

(I) experimental animal Kunming female mouse 8, weight 35g + -5 g, provided by pharmacological laboratory of Taishan medical college.

(II) Experimental methods

1. Drawing of melatonin standard curve

Accurately weighing 4.0mg of melatonin, placing the melatonin in a 50mL volumetric flask, adding a methanol solution, shaking up to fully dissolve the melatonin, and fixing the volume to a scale to obtain a stock solution. The prepared stock solutions are respectively diluted into series of standard solutions with the concentrations of 0.010, 0.015, 0.020, 0.030, 0.035 and 0.040mg/mL by methanol. Taking a proper amount of the melatonin solution with different concentration gradients, and measuring the absorbance of the melatonin solution at 223nm by using an ultraviolet spectrophotometer. And (3) drawing a standard curve by taking the absorbance (A) as a vertical coordinate and the concentration (C) as a horizontal coordinate, and establishing a regression equation.

2. In vitro percutaneous permeation test

2.1 preparation of skin in vitro

Taking 8 Kunming mice with the weight of 30-35g, depilating the backs of the 8 Kunming mice by using depilatory cream, cleaning the backs of the 8 Kunming mice by using normal saline, feeding the mice for 12 hours, dislocating the cervical vertebrae, killing the hairless backs of the mice, removing subcutaneous fat by using a blade, and cleaning the mice by using the normal saline for later use.

2.2 Experimental facility and method

The transdermal absorption experiment was performed in a modified Franz diffusion cell, which was placed in a drug transdermal diffusion tester under a constant temperature circulating water bath set at 37 ℃. Spreading the prepared skin of mouse between the supply tank and the receiving tank, fixing with a special clamp, with the skin surface facing the supply tank, and the effective permeation area of the diffusion tank being 3.5cm ₂ The receiving tank has a volume of 6.5mL, and the receiving solution is a phosphate buffer solution with a pH value of 7.2-7.4. The skin of 8 mice was randomly divided into 2 groups, and 1.0mL of melatonin flexible liposome and 1.0mL of melatonin ordinary liposome were added to the feeding wells of the two groups, respectively. The receiving solution was taken out 3.0mL at 0.5, 1, 2, 3, 4, 6, 8, 10, 24h, placed in a test tube, and supplemented with an equal amount of phosphate buffer immediately after each sampling. The obtained sample was measured for absorbance at 223nm with an ultraviolet spectrophotometer.

2.3 Experimental data processing

After the absorbance is measured, the concentration corresponding to each time point can be solved by using a melatonin standard curve equation, and then the accumulated permeation quantity and the permeation parameters are solved. The cumulative permeation per unit area can be obtained by the following equation:

in the formula, S-effective diffusion area (cm) ² )

V-receiving liquid volume in receiving chamber (mL)

C _i Concentration of drug in the receiving fluid (mg/mL) from the first time to the last sampling

C _n The concentration of drug in the receiving fluid (mg/mL) at this time

V _i Volume per sample (mL)

J-transdermal Rate constant

(III) results of the experiment

1. The melatonin standard curve is drawn by taking the concentration as an abscissa and the absorbance as an ordinate, and linear regression is carried out. The resulting linear regression equation was a =23.76C-0.075 ₂ =0.9994, and the result shows that the melatonin has a good linear relation in the concentration range of 0.01-0.04 mg/mL.

2. Results of in vitro percutaneous permeation in mice

TABLE 4 melatonin liposome mouse in vitro transdermal penetration kinetics parameter table

The cumulative release is plotted against the cumulative release and the sampling time points as shown in FIG. 2, and the calculated osmotic kinetic parameters are shown in Table 4. Table 4 results show that the melatonin flexible liposomes and normal liposomes had 24h cumulative permeabilities of 0.2514 and 0, respectively.1655mg·cm ^-2 The permeation rates are respectively 0.0176 and 0.0087 mg-cm ^-2 ·h ^-1 . The results show that the unit area cumulative permeation amount and the permeation rate of the melatonin flexible liposome are respectively improved by 1.5 times and 2 times compared with the common liposome, and the figure 2 can visually show that the unit area cumulative permeation amounts of the two liposomes are not obviously different in 0.5h, and the unit area cumulative permeation amount of the flexible liposome is larger and larger than that of the common liposome along with the time. The flexible liposome prepared from melatonin is shown to further improve the transdermal permeation efficiency.

(IV) discussion

It can be seen from table 4 that the cumulative permeation amount per unit area of the flexible liposome 24h is significantly higher than that of the common liposome, which may be due to the fact that sodium deoxycholate is inserted into the middle of the phospholipid bilayer, increasing the distance between phospholipid molecules, changing the sequence of the acyl chains of the phospholipids, increasing the fluidity of the phospholipid, so that the flexible liposome has high deformability, can efficiently enter subcutaneous tissues through the skin cuticle through self deformation, and does not cause drug leakage due to the change of the self structure, while the common liposome is easy to be damaged and retained in the cuticle when passing through the narrow channel of the skin cuticle due to the absence of a membrane softener, and is difficult to enter the subcutaneous tissues to play a role. On the other hand, the phospholipids forming the liposome membrane material can be fused with lipid layers of the skin stratum corneum structure, so that the stratum corneum lipid composition and structure are changed; meanwhile, the liposome can also increase the humidifying and hydrating action of the stratum corneum to change the structure among corneocytes, thereby causing the barrier action of the skin stratum corneum to be reversed, and the flexible liposome containing sodium deoxycholate can keep the original shape after entering subcutaneous tissues through the skin stratum corneum so as to ensure that more medicaments can enter the body to exert the curative effect

3. In vivo pharmacokinetics study of melatonin flexible liposome transdermal administration rat

(I) Experimental animals

Sprague-Dawley (SD) female rats 8 with body weights of 220g + -20 g purchased from Jinanpunyue laboratory animal Breeding Co., ltd.

(II) Experimental method

1. Establishment of method for analyzing melatonin in biological sample

1.1 chromatographic conditions

C18 column (4.6 mm. Times.200mm, 5 μm); mobile phase: methanol: water =1:1; detection wavelength: 223nm; flow rate: 1mL/min; sample introduction volume: 20 μ L.

1.2 sample Pre-treatment

About 0.2mL of blood is taken from rat tail breakage, the rat tail breakage blood is put into a centrifuge tube containing heparin, the centrifuge tube is centrifuged for 10min by a low-speed centrifuge at 3000r/min, 50 mu L of supernatant is taken, 3 times of methanol is added, vortex mixing is carried out for 2min, water bath ultrasound is carried out for 5min, centrifugation is carried out for 10min at 12000r/min under the condition of 4 ℃, and 20 mu L of supernatant is taken for HPLC determination.

1.3 specialization examination

The HPLC chromatograms of the blank plasma (a), blank liposomes (b) and plasma containing melatonin (c) were recorded at 1.1, after taking the blank plasma from rats and adding melatonin, and processing the plasma samples at 1.2.

1.4 drawing of Standard Curve

Respectively taking 6 parts of 100 mu L of blank plasma, adding 20 mu L of melatonin reference substance solutions with different concentrations to prepare plasma containing reference substances with concentrations of 0.05, 1.00, 2.50, 5.00 and 10.00 mu g/mL, operating and processing samples according to 1.2 items, feeding samples for five times for each sample, drawing a standard curve by taking a peak area A as a vertical coordinate Y and taking a blood concentration C as an abscissa X, and establishing a linear regression equation.

1.5 precision test

Preparing samples with low, medium and high concentrations of 0.5, 1.5 and 8.0 mu g/mL of rat blank plasma, preparing 5 parts of samples with each concentration, operating and processing the samples according to 1.2 items, detecting by HPLC, continuously measuring the samples for 3 days, continuously measuring each sample for 5 times in one day, recording peak areas, calculating actually measured concentrations according to a standard curve, and calculating the precision of the plasma samples with each concentration in the day and the day.

1.6 stability test

Rat blank plasma was prepared into samples with low, medium and high concentrations of 0.5, 1.5 and 8.0. Mu.g/mL, respectively, and the samples were processed as described under item 1.2, and then stability of the plasma samples was measured by HPLC after being left at room temperature for 2 hours (A) and 24 hours (B), respectively, and each concentration was measured 3 times in succession to examine its stability.

1.7 recovery test

Taking rat blank plasma, preparing three sample solutions with different concentrations of low, medium and high, wherein the concentrations are respectively 0.5, 1.5 and 8.0 mu g/mL, operating and processing the samples according to item 1.2, detecting peak areas of the samples with the three concentrations by adopting HPLC, substituting the peak areas into a standard curve to calculate the concentration, taking the three standard solutions with the different concentrations to directly measure by using HPLC, recording the peak areas, and respectively calculating the relative recovery rate and the absolute recovery rate according to the following formula.

Relative recovery rate R _R = measured/theoretical concentration × 100%

Absolute recovery rate R _A ＝A _Yuan /A _{Straight bar} ×100％

(A _Yuan : the peak area, A, is determined after the plasma sample has been treated _{Straight bar} : direct sample introduction to measure peak area)

2. Pharmacokinetic experiment of melatonin flexible liposome transdermal administration rat

2.1 grouping and administration of Experimental animals

8 SD female rats with the body weight of 220g +20g are randomly divided into two groups, and the two groups are both administered in a skin administration mode, and the administration dosage is 43 mu g/mL, wherein one group is provided with melatonin flexible liposomes, and the other group is provided with melatonin ordinary liposomes as a control group.

2.2 blood sampling time points and blood sample processing

At 0h before administration and 0.2, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10h after administration, respectively, 500. Mu.L of blood was collected from rat tail-broken ends and placed in an EP tube containing heparin, centrifuged at 3000r/min for 10min, 50. Mu.L of plasma was collected, and then plasma was treated according to 1.2 procedures and measured by HPLC.

2.3 sample measurement and data processing

Substituting peak area measured by HPLC under item 2.2 into standard curve to calculate blood concentration at each time point, and drawing pharmaceutical time curve. The pharmacokinetic parameters were calculated by fitting analysis of the drug-time data using DAS pharmacokinetic processing software without a compartmental model.

(III) results of the experiment

1. Results of the specificity investigation

As can be seen from the HPLC chromatogram 3, the retention time of the melatonin absorption peak is about 6min, and the separation effect of the chromatographic peak is good without interference of other substances, which indicates that the melatonin absorption peak has good specificity under the chromatographic condition.

2. Drawing of standard curve

Taking the blood concentration as abscissa and the peak area as ordinate, performing linear regression to obtain linear regression equation Y =2887.5X-168, R ₂ =0.9993, which shows that the melatonin concentration in the plasma of rats is in a good linear relation within the concentration range of 0.05-10.00 mu g/mL.

3. Results of precision

TABLE 5 melatonin precision results in rat plasma samples

As can be seen from the results in Table 5, the daily and daytime accuracies RSD of the melatonin in the rat plasma sample are less than 5%, which shows that the measurement method meets the requirements of methodology.

4. Stability results

Table 6 melatonin stability results in rat plasma samples

As can be seen from table 6, the RSD of melatonin in the plasma samples was <5% at different concentrations and under different conditions, indicating that the plasma samples containing melatonin had good stability at room temperature.

5. Recovery results

Table 7 melatonin recovery results in rat plasma samples

According to the table 7, the relative recovery rate and the absolute recovery rate value of the plasma samples with different concentrations are both more than 80%, and the RSD is less than 10%, which shows that the recovery rates are not greatly different and the recovery rates are higher, thereby proving that the method is reliable in operation and can be used for determining the content of the plasma samples containing the melatonin.

6. Pharmacokinetic experimental results in rats

TABLE 8 melatonin Flexible liposomes and common liposomes pharmacokinetic parameters in rats

As can be seen from the curve of fig. 4, after the rats were administered two different melatonin liposomes transdermally, the pharmacokinetic characteristics in the rats were substantially the same, and the maximum blood concentration occurred at 30min, but from the pharmacokinetic parameters tables 5-4, it was found that the area under the curve of the flexible liposomes was 21.389, which is 1.82 times higher than that of the common liposomes, i.e., bioavailability F =1.82, and the peak blood concentration of the flexible liposomes was 2.35 times higher than that of the common liposomes, suggesting that the bioavailability of the flexible liposomes was higher than that of the common liposomes. Meanwhile, the clearance rate CL shows that the flexible liposome is slower than the common liposome to eliminate in vivo, and the result shows that the bioavailability of the melatonin prepared into the flexible liposome is higher than that of the melatonin prepared into the common liposome.

(IV) discussion of

Before rat pharmacokinetics research, firstly, an HPLC method for measuring the melatonin is established to measure the content of the melatonin in rat plasma, and meanwhile, the linear range of the content, the specificity of the method, the day-to-day precision, the stability and the recovery rate are examined to prove whether the measured result under the method is credible. The result shows that the melatonin has a good linear relation in the concentration range of 0.05-10.00 mu g/mL, the daily precision, the stability and the recovery rate all meet the methodological requirements, and the method can be used for researching the pharmacokinetic rule of the melatonin flexible liposome in a rat body.

Since the liposome is rapidly eliminated from the blood circulation after intravenous injection, and the drug is first taken up by tissues rich in reticuloendothelial cells such as liver, spleen and the like when the drug enters the body through intravenous injection, the experiment is carried out by adopting a transdermal administration mode (namely, cotton balls containing equal amounts of melatonin flexible liposome and common liposome are respectively fixed on the back of a depilated rat by using adhesive tapes and then are taken down). As can be seen from the time curve and the pharmacokinetic parameter table, the bioavailability of the melatonin flexible liposome is improved by 1.82 times compared with that of the common liposome; the peak reaching concentration of the blood is 6.442mg/L, which is 2.35 times higher than that of the common liposome; the clearance rate of blood plasma is lower than that of common liposome, thus prolonging the metabolism time of the medicine, and improving the absorption of the medicine and the curative effect of the medicine acting in vivo.

4. In vivo pharmacodynamic evaluation of melatonin flexible liposome after transdermal administration in mice

(I) laboratory animals

36 Kunming female mice, weighing 35g + -5 g, were provided by the pharmacological laboratory of Taishan medical college.

(II) Experimental method

1. Grouping and administration irradiation mode for experimental animals

Experimental mice were randomly divided into 6 groups of 6 mice each. Irradiating with ultraviolet lamp for 2 hr every time in the same time period, and irradiating every other day for 10 weeks.

A normal control group (NC): normally feeding in the whole experiment period without any treatment;

b shaving control group (SC): the electric shaver is used together with other groups of mice to gently shave off the back hairs of the mice in the whole experimental period, and the shaving area is about 2.5 multiplied by 3cm ₂ ；

Model C control (MC): the electric shaver is used together with other groups of mice to gently shave off the back hairs of the mice in the whole experimental period, and the shaving area is about 2.5 multiplied by 3cm ₂ (ii) a After shaving, giving ultraviolet irradiation for 2 h;

vehicle control group (VC): with other groups of miceThe electric shaver is used for gently shaving the hair on the back of the mouse, and the shaving area is about 2.5 multiplied by 3cm ₂ (ii) a After shaving, ultraviolet irradiation is given for 2h, and after irradiation, ethanol-propylene glycol (7, vol/vol) is applied to the skin at a dose of 300 μ l/skin for treatment;

e melatonin flexible liposome group (EL): the electric shaver is used together with other groups of mice to gently shave off the back hairs of the mice in the whole experimental period, and the shaving area is about 2.5 multiplied by 3cm ₂ (ii) a After shaving, giving 2h ultraviolet irradiation, and after irradiation, smearing melatonin flexible liposome according to 300 mul/skin for treatment;

f melatonin common liposome group (CL): the electric shaver is used together with other groups of mice to gently shave off the back hairs of the mice in the whole experimental period, and the shaving area is about 2.5 multiplied by 3cm ₂ (ii) a After shaving, ultraviolet irradiation is given for 2h, and after irradiation, melatonin ordinary liposome is applied to the skin according to the dosage of 300 mul/skin for treatment.

2. Body weight changes in mice

The weights of the groups of mice were weighed on a weekly basis throughout the experimental period, and the weight change curves of the mice were plotted.

3. Skin appearance evaluation

After the experiment period was completed, the skin exposed to ultraviolet rays was observed on the back of each mouse after anesthetizing the mouse, and the damage of the skin was determined with reference to the score in Table 9 _[26] . Skin photoaging grades were divided into 7 grades, 0 for normal skin and 6 for severely damaged skin.

TABLE 9 skin photoaging score Standard Table

4. Skin elasticity evaluation (skin lifting test)

Skin elasticity evaluation method established by reference to Kazue et al _[27] After the experimental period ended, each group of mice was anesthetized, and then the thumb and forefinger were gently applied along both sides of the center of the skin on the back of the miceLifting the mouse, taking the mouse out of the air as a standard, timing for 1s, putting down the mouse, immediately taking a picture, and recording the time for recovering to the original state.

5. Skin moisture determination

After the experimental period is finished, the mouse is killed by dislocation of cervical vertebrae, the exposed skin on the back of the mouse to be depilated is taken down by about 0.2g, if subcutaneous fat exists, the skin needs to be scraped off by a blade, the wet weight is precisely weighed, the mouse is immediately placed into a drying oven, the skin is dried to constant weight at 70 ℃, then the weight of the dry skin is weighed, and the water content of the skin of each group of mice is calculated according to the following formula:

percent skin moisture = (M) _{(Wet-Dry weight)} /M _{Wet weight} )×100％。

6. Examination of skin histopathology

After the experimental period was over, the mice were sacrificed by cervical dislocation and the exposed skin on the backs of the depilated mice was removed by about 1.0X 1.0cm ₂ Size, fixing in paraformaldehyde for about 24h, dehydrating, embedding in paraffin, slicing, dewaxing, and finally staining with hematoxylin-eosin (HE) and observing structural change under an optical microscope.

(III) results of the experiment

1. Evaluation results of mouse body weight and appearance

The weight change curve of the mouse is shown in fig. 5, and the result shows that the whole weight of the mouse does not change greatly in the whole experimental period, which indicates that the ultraviolet irradiation has little influence on the life activity of the mouse and does not influence the normal vital signs of the mouse.

As can be seen from the appearance of the mice after uv irradiation in fig. 6 (a), no significant difference was observed between the NC and SC groups on the back skin surface, indicating that the skin appearance was not significantly affected by shaving without uv irradiation during the entire experimental period. At the end of the experimental period, some skin lesions and lesions were observed in the MC and VC groups of mice. However, no macroscopic lesions appeared in both EL and CL groups, but the skin was rough compared to NC and SC groups, suggesting that melatonin had some protective effect on the skin by uv radiation.

The scoring results for each mouse are shown in fig. 6 (b), and the scores of the two groups of MC and VC are not significantly different, but both groups are higher than those of SC and NC. However, the scores were reduced for both EL and CL groups compared to the VC group. These results indicate that the entire period of uv irradiation causes damage to the skin of mice, but melatonin can effectively prevent these macroscopic damages.

2. Results of measuring skin elasticity and water content of mice

The elasticity of the skin of the back of the mouse after irradiation with ultraviolet rays was tested, and a photograph after stretching and setting down was taken as shown in fig. 7 (a). The recovery time after stretching is shown in FIG. 7 (b), and the skin recovery time of the mice in the MC and VC groups is significantly longer than that of the SC and NC groups. Although there was no significant difference between the EL and CL groups, there was a shortening compared to the VC group. These results indicate that melatonin can improve skin elasticity problems caused by ultraviolet irradiation.

The skin moisture content results of fig. 8 show that there was no significant difference in skin moisture content between the NC and SC groups, indicating that depilation alone had no effect on the skin moisture content of the mice; compared with the SC group, the skin moisture percentage of the MC group and the VC group is obviously reduced and has obvious difference, which shows that the moisture content of the skin of a mouse can be reduced by ultraviolet irradiation, but the MC group and the VC group have no obvious difference, which shows that the moisture content of the skin irradiated by ultraviolet can not be improved by coating a solvent; the percent of water content of the skin is reduced in the two groups of EL and CL compared with the SC group, but is increased compared with the two groups of MC and VC, which shows that the water content of the skin is improved after the melatonin liposome is applied to the skin irradiated by ultraviolet rays, the effect is better than that of the melatonin liposome applied by a solvent, but no significant difference exists between the two liposome preparations.

3. Mouse skin histopathology section results

The pathological changes of the skin tissues of the mice induced by the ultraviolet rays are shown in fig. 9, the skin structures of the mice in the NC group and the SC group are relatively similar, the structures of all layers are complete and normal, and the subcutaneous hair follicles and the sebaceous glands are plump and complete; specifically, the epidermis tissue structures of the two groups of tested mice are complete, the cell layers are clearly divided, the epidermis is thin and uniform in thickness, the epidermis is tightly connected with the dermis, the boundary line is clear, and wavy connecting lines, epidermal bulges and dermal papillae are obviously seen; the thickness of the dermis is normal, the wavy collagen fiber bundles which are orderly arranged and uniformly distributed can be seen, and the cell components and the number are moderate.

The skin structures of mice in the MC group and the VC group are basically similar, the epidermis of the skin is irregularly thickened, the structure is incomplete and unclear, the junction between the epidermis and the dermis is linear, and the epidermis protrusion and the dermal papilla disappear; the dermis is shrunk and thinned, the cells are disorganized and even disintegrated, the collagen fiber bundles are disorganized and loosened and are mostly broken and even broken, the inflammatory cell infiltration is accompanied, and meanwhile, the solvent has no obvious influence on the histopathology of the photoaging skin. The skin conditions of the CL group and the EL group are recovered to a certain degree compared with those of the VC group and the MC group, the thick epidermis and dermis layers of the CL group are irregular, hair follicles and sebaceous glands with complete structures can be seen, collagen fiber bundles are scattered and tend to be orderly arranged, and inflammatory infiltration can be seen; the skin structure of the EL group is more complete, the epidermis is slightly thickened but more uniform, the boundary line with the dermis is clear and tends to be wavy, the collagen fiber bundles of the dermis are increased and thickened, and the arrangement is compact and more uniform. The result shows that the melatonin liposome preparation has better biocompatibility and basically does not influence the normal shape of the skin.

(IV) discussion

In the whole experiment period, the change situation of the body weight of each group of mice every week is weighed, and the self development situation of the mice may be influenced by the conditions such as the change of the surrounding environment, ultraviolet irradiation and the like in the experiment process, so that the experiment result is influenced to a certain extent. The change in the ambient environment or the ultraviolet irradiation was judged by weighing them and comparing them with mice raised in the same environment without any treatment. From the experimental results, the body weight of each group of mice has no obvious difference compared with the control group and has a trend of continuous fluctuation, but the body weight of the control group or the experimental group has no obvious increase in the irradiation process of 10 weeks, which is probably caused by individual difference or artificial measurement deviation between the mice of each group, and further indicates that the normal vital signs of the mice are not influenced.

The effect of the melatonin on resisting photoaging after transdermal administration is further evaluated through evaluating the appearance of the skin of a mouse, testing the elasticity and measuring the water content of the skin of the mouse, and experimental results show that compared with a control group, the elasticity and the water content of the skin of the mouse coated with the melatonin liposome under the condition of ultraviolet irradiation are higher than those of a solvent-coated group and a group only irradiated by ultraviolet rays, and the result preliminarily shows that the melatonin can resist the body aging caused by the ultraviolet irradiation after transdermal administration.

Since the decrease in collagen content and the change in collagen fiber bundle structure are important causes of skin aging, the effect of continuous ultraviolet irradiation on the arrangement and structure of collagen fibers in mouse skin was further studied in this experiment. The experimental results show that compared with the normal control group, the collagen fiber bundles in the ultraviolet irradiation group and the solvent group are disorderly and loose in arrangement and are broken or even broken frequently; the melatonin liposome-treated group maintained the integrity of the collagen structure in the skin of the irradiated mice and was able to promote an increase in collagen fiber bundles. These results indicate that melatonin may be useful in preventing skin aging by ultraviolet radiation, and that subsequent determination of collagen content in the skin is necessary in order to confirm the reliability of the results, in order to lay the foundation for the application of melatonin in the treatment of skin aging by ultraviolet radiation.

5. Melatonin flexible liposome transdermal mechanism research

(I) Experimental animals

33C 57BL/6 female mice with weight of 25g +/-5 g and IVC grade are purchased from Beijing Huafukang Biotechnology Limited company and are bred in the experimental animal center of Taishan medical college.

(II) Experimental method

1. Scanning Electron Microscope (SEM) analysis of skin surface

And observing the influence of the physiological saline group and the melatonin flexible liposome group on the skin surface structure by using SEM. Taking 3 mice of each group of 6C 57BL/6 mice, respectively coating a proper amount of physiological saline and melatonin flexible liposome on the back of each depilated mouse, taking a next small piece of back skin after 12 hours, placing the small piece of back skin in glutaraldehyde for fixation for 24 hours, then dehydrating and drying the treated skin sample by using graded ethanol, and then placing the dried skin sample on an SEM for observation by spraying a gold coating film.

2. Differential Scanning Calorimetry (DSC) analysis of skin structure

DSC was used to analyze the changes in the texture of the stratum as a function of temperature under temperature program control. The back skin of the mouse treated by the physiological saline and the melatonin flexible liposome is taken down after 12 hours, is immediately frozen by liquid nitrogen, is ground by a mortar (the liquid nitrogen is continuously added into the mortar in the grinding process so as to prevent the skin from softening and adhering), and is placed in a freeze dryer to remove the moisture in the skin to obtain the skin freeze-dried powder. The testing temperature is set to be 20-250 ℃, the heating rate is 10 ℃/min, and the measurement is carried out under the protection of nitrogen.

3. Fourier transform Infrared Spectroscopy (FTIR) analysis of skin Components

FTIR was used to further study the effect of melatonin flexible liposomes on stratum corneum lipids and keratin. Tabletting the skin freeze-dried powder with KBr respectively at 400-4000 cm _-1 And performing spectrum scanning by using an infrared spectrometer in the scanning range.

4. Dynamic change study of fluorescent liposome uptake by skin Langerhans Cells (LC)

4.1 preparation of Fluorescein Isothiocyanate (FITC) liposomes

Dissolving lecithin, cholesterol and sodium deoxycholate in a prescription amount by using a mixed solvent of chloroform and ethanol, carrying out reduced pressure rotary evaporation to form a membrane, then hydrating by using an aqueous solution containing a proper amount of FITC to obtain the FITC flexible liposome (FITC-EL), and obtaining the FITC common liposome (FITC-CL) without adding sodium deoxycholate in a membrane forming material. The prepared fluorescent liposomes were then centrifuged through an ultrafiltration tube to remove free FITC.

4.2 grouping and administration

C57 mice are randomly divided into 9 groups, each group comprises 3 mice, one group is a blank control group, the other 8 groups are respectively coated with FITC flexible liposome and common liposome with the same dose on the ears of the mice, the administration is carried out once a day, the ears of the mice are respectively taken down after 12 hours, 24 hours, 48 hours and 72 hours after the administration, and single cells are prepared for detection.

4.3 preparation of mouse ear epidermal unicell

Killing the mouse by dislocation of cervical vertebra, taking down the ear of the mouse, tearing the ear from the middle by a forceps, dividing into two pieces, firstly putting the two pieces into 1640 cell culture medium, then uniformly removing cartilage, some blood vessels and adipose tissues, putting the 24-pore plate containing dispaseII type enzyme with the epidermis facing upwards, and putting the 24-pore plate into a 37 ℃ incubator for incubation for 50min. Separating epidermis and dermis layers, cutting the epidermis layer, adding the cut epidermis layer into 1% type IV collagenase, incubating for 50min in an incubator at 37 ℃, filtering into a 50mLEP tube by using a 40-micron filter screen, slightly grinding by using a grinding rod, removing cell aggregates and residual tissues, preparing into single cell suspension, centrifuging, discarding supernatant, finally utilizing residual liquid in the tube to resuspend cells, filtering into a 96-pore plate by using a 40-micron filter screen again, centrifuging, detecting by using a flow cytometer after dyeing is finished.

4.4 detection of Single cells in Tabs by flow cytometry

Detection was performed by flow cytometry according to the analytical strategy shown in FIG. 12. SSC-A and FSC-A were used first to remove impurities and se:Sub>A portion of the red blood cells, FSC-H and FSC-A were used to remove adherent cells, A430 dye was used to remove dead cells, and the live cells of the subpopulation were analyzed next. Non-mononuclear and neutrophils were analyzed with CD11b and Ly6c antibodies, followed by double positive analysis of Dendritic Cells (DCs) with CD11c and IA/IE, and finally analysis of langerhans cells containing FITC (LC) with EPCAM. Experimental results were processed and statistically analyzed using Flowjo software and graphpadprism6.0 software.

(III) results of the experiment

1. Mouse skin SEM, DSC and FTIR analysis results

SEM figure 10 shows that the skin surface was rough and stratum corneum cells shrunk in the control group, while the skin surface was smooth and stratum corneum cells expanded in the group treated with flexible liposomes, resulting in decreased intercellular spaces, which initially indicates that the skin surface texture was altered by the penetration of flexible liposomes into the body.

The characteristic peak of the skin sample in the DSC curve is a keratin denaturation peak and is in the temperature range of 110-120 ℃. As can be seen from fig. 11 (a), characteristic peaks appeared in both the control group and the experimental group at 110 to 120 ℃, but the melting point of the flexible liposome group was significantly increased and the skin peak height was also changed compared to the control group, indicating that the keratin structure was changed.

The infrared characteristic absorption peak of the skin sample is the horny layer lipid peak (v) _as CH ₂ ，ν _s CH ₂ ，ν _s C = O) and keratin peaks (amide I, amide II). From the results of fig. 11 (b), it can be seen that the peak of stratum corneum lipid in the infrared spectrum of the flexible liposome group is shifted to the right compared to the control group, indicating that the flexible liposome changes the stratum corneum structure by penetrating into the skin through skin.

2. Results of changes in LC uptake into FITC liposomes at different time points

Flow chart 13 and cell number percentage dynamics figure 14 show that LC shows an increasing trend in phagocytosis of both fluorescent liposomes and reaches a maximum at 48h, followed by a decrease. The phagocytosis amount of the flexible liposome is larger than that of the common liposome, which shows that the liposome is smeared on the surface of the skin, when the liposome overcomes the barrier effect of the stratum corneum of the skin and permeates into the skin, the liposome can be absorbed into the body by Langerhans cells on the epidermis of the skin, and the change of the absorption amount along with time has a dynamic change process, and further proves that compared with the common liposome, the flexible liposome can better permeate into the skin through the skin.

(IV) discussion of

In this experiment, the change of the skin surface structure was first detected by SEM analysis. The SEM result shows that the skin surface of the mice treated by the melatonin flexible liposome is smoother, the cells of the horny layer are expanded and arranged orderly, and the intercellular spaces are reduced compared with the control group. The results preliminarily show that the melatonin flexible liposome overcomes the skin barrier effect by changing the structure of the skin surface.

DSC was then applied to investigate the mechanism by which flexible liposomes enhance permeation by analyzing the thermodynamic properties of the stratum corneum. Keratin is a temperature sensitive substance and the conformation of the protein is easily altered by external heat energy, which is related to the barrier function of the stratum corneum. From the DSC graph results, the skin melting point of the mice treated with the flexible liposomes was increased and the peak height of the skin was changed to directly change the keratin structure, compared to the blank control group, indicating that the lipid nanocarriers can reduce the barrier function of the stratum corneum by changing the helix structure of keratin.

The mechanism of penetration of the liposomes is then explained by the structural change of the stratum corneum lipid peak and keratin peak by FTIR, since FTIR can provide information on the vibration of keratin and stratum corneum lipids of the stratum corneum _[29] . FTIR patterns showed that the right shift of the stratum corneum lipid peak compared to the blank control indicated a change in the keratin structure. Indicating that the transdermal permeability of liposomes depends on changes in the stratum corneum lipid and keratin structures.

Finally, the transdermal penetration mechanism of the liposome is studied from the immunological perspective. As is well known, LCs are a part of DCs, playing an important role in the immune response of the skin, and mainly have the role of taking up, processing, presenting antigens and inducing T cell responses as antigen-presenting cells of the skin. The experimental results show that the change of the antigen uptake amount by LC is a dynamic change process, the antigen uptake is correspondingly increased along with the prolonging of the stimulation time, but the highest point exists, namely when the uptake amount reaches a certain limit, the antigen continues to be stimulated, the antigen uptake is reduced, and the skin epidermal LC plays an important role in the transdermal delivery process of the liposome.

6. Effect of melatonin on skin elastic fibers

Once the elastic fiber is damaged or broken, the reticular structure of the dermis layer can be directly damaged, so that the reticular structure is loosened and sunken, and the phenomenon of macroscopic wrinkles and laxity of the skin is caused. The method utilizes a Victoria blue dyeing method to compare and observe KM mouse skin elastic fibers which are normal and irradiated by ultraviolet lamps, and inspects whether the melatonin flexible liposome has a protective effect on the skin elastic fibers. The results are shown in fig. 15, the elastic fiber structures in the skin of the mice in the NC group and the sc group are complete (the elastic fiber net with clear structures can be seen), and the arrangement is orderly; after irradiation by an ultraviolet lamp (M groups), the elastic fibers of the skin of the mice are thickened, broken and twisted, and part of the elastic fibers are gathered, tangled and disorganized; the VC group is similar to the MC group in the structure and distribution of elastic fibers in the skin of mice. The results show that corresponding to the macroscopic characterization of wrinkles and looseness of the skin of the tested mouse, the chronic ultraviolet irradiation can damage the elastic fibers of the skin of the tested mouse and destroy the net physiological structure of the elastic fibers, and the experiment successfully establishes a mouse SP model, and the melatonin liposome and the melatonin flexible liposome administration group have different degrees of recovery effects on the elastic fibers in the skin of the mouse, are orderly arranged, and occasionally have aggregation, fracture and entanglement phenomena. The comparison shows that the melatonin flexible liposome can resist the damage of UV to the skin elastic fibers, and the effect is close to the normal form and physiological structure.

7. Effect of melatonin Flexible liposomes on collagen content in skin tissues of photoaged mice

The results are shown in fig. 16, and the NC group and the SC group showed no significant difference in collagen content in the skin tissue of the test mice, indicating that the depilatory operation had no significant effect on the collagen content in the skin of the mice. Compared with the SC group, the MC group has extremely obviously reduced skin collagen content (P < 0.05), which indicates that the mouse skin photoaging model is successfully modeled. Compared with the MC group, the skin collagen content of the mice in the VC group has no significant difference (P > 0.05), and compared with the VC group, the collagen content of the skin of the mice is greatly increased (P < 0.05) after the melatonin liposome, especially the flexible liposome, is administered to the skin of the mice, and is close to the normal level.

8. Effect of melatonin liposomes on MMPs levels in skin tissue of photoaged mice

The expression level of MMPs in the skin tissue of the mouse is detected by adopting enzyme-linked immunosorbent assay in the experiment. The results are shown in FIG. 17, the levels of MMP-1 and MMP-3 in the skin tissues of mice in the NC group and the SC group are close, and no significant difference exists (all P > 0.05); compared with the SC group, the skin of the mice in the MC group has obviously increased levels of MMP-1 and MMP-3; the levels of MMP-1 and MMP-3 in the skin of mice in the NC group and the VC group are also close to each other, and no significant difference exists (P is more than 0.05); the results show that the skin of the mice is loose and wrinkled, the expressions of MMP-1 and MMP-3 in the skin tissues of the mice are abnormally increased, and the contents of MMP-1 and MMP-3 in the skin tissues of the mice given melatonin liposome are obviously reduced; the melatonin flexible liposome can obviously inhibit the increase of MMP-1 and MMP-3 secretion in the skin of a mouse caused by UV, and the effect of the melatonin flexible liposome is superior to that of the common melatonin liposome.

Claims

1. The application of the melatonin flexible liposome in preparing a skin care product for resisting the reduction of collagen content in skin tissues caused by photoaging is characterized in that the melatonin flexible liposome is prepared from the following raw materials in parts by weight:

18.90 parts of melatonin, 80.00 parts of lecithin, 20.00 parts of cholesterol and 18.91 parts of sodium deoxycholate;

precisely weighing melatonin, lecithin, cholesterol and sodium deoxycholate in a formula amount, placing the mixture into a reactor, adding chloroform and absolute ethyl alcohol to carry out water bath ultrasonic dissolution, placing the reactor on a rotary evaporator to carry out rotary evaporation under reduced pressure at 40 ℃ until an organic solvent is volatilized to form a layer of film on the wall of the reactor, then adding distilled water to continue rotary evaporation under reduced pressure for 1h to fully hydrate the film, and finally carrying out ultrasonic treatment for 15min by using an ultrasonic cell crusher to obtain the melatonin flexible liposome; the ratio of the chloroform dosage to the total dosage of lecithin and cholesterol is 1: mg; the ratio of the dosage of the absolute ethyl alcohol to the total dosage of the lecithin and the cholesterol is 1: mg; the ratio of the amount of the distilled water to the total amount of the lecithin and the cholesterol is 1: and (5) mg.