Encapsulates of a complex molecular spike and methods of making same

Abstract

The application provides an encapsulating object of a composite molecule spike and a preparation method thereof, relates to the technical field of biological medical materials, and the encapsulating object comprises an inner core and an outer layer. The inner core is a molecule spike liposome. Active ingredients of the molecule spike liposome comprise 2-5 wt% of a molecule spike NP, 1-2 wt% of a molecule spike AP and 0.05-0.15 wt% of a plant sphingosine. The outer layer is an extracellular vesicle of long bifidobacterium wrapped outside the inner core. The encapsulating object can effectively improve the problem that the molecule spike is not suitable for common formula systems, significantly enhances the stability of the molecule spike system, and significantly enhances water solubility. A new composite delivery system is constructed by utilizing the natural carrier characteristics of the extracellular vesicle of long bifidobacterium and the biomimetic structure advantages of the molecule spike liposome, and the technical problems of poor solubility, low transdermal absorption rate and insufficient stability of the molecule spike delivery system in the prior art are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medical materials, and in particular to a complex molecular spike encapsulant and a preparation method thereof. BACKGROUND

[0002] The skin barrier is mainly composed of corneocyte and intercellular lipid, among which the lipid bilayer structure formed by molecular spike, cholesterol and free fatty acid in a specific ratio is crucial for preventing water loss and resisting external stimuli. When this structure is damaged, it will lead to problems such as dry, sensitive and even inflammatory skin. Traditional moisturizing products often focus on the use of single moisturizing agent or occlusive agent, but it is difficult to achieve real barrier repair function. With the in-depth research of skin science, people gradually realize that it is necessary to supplement all kinds of lipid components naturally existing in the stratum corneum and ensure that the ratio is close to the physiological state, so as to effectively rebuild a healthy skin barrier.

[0003] Molecular spike, also known as ceramide, is the main lipid component between corneocytes in the skin stratum corneum, accounting for 50% of the stratum corneum lipid. It forms a protective film on the surface of the skin, resists external stimuli, locks in moisture and keeps the skin hydrated. Topical molecular spike can play a role through the construction of liquid crystal and / or gel structure, repair of lamellar bilayer structure, as a precursor of endogenous molecular spike synthesis, etc. Phytosphingosine is a precursor of molecular spike, which can promote the synthesis of molecular spike, thereby repairing and enhancing the barrier function of the skin. However, the traditional molecular spike delivery system faces technical bottlenecks such as poor solubility, low transdermal absorption rate and insufficient stability, which seriously limits its application effect.

[0004] Liposome is a spherical vesicle composed of phospholipid bilayer, which can encapsulate hydrophilic and hydrophobic compounds, avoid their decomposition and improve the penetration of encapsulated contents. Liposome has a wide application prospect in drug delivery and skincare products, but there are some problems, such as hydrolysis or oxidative degradation during non-frozen storage, and sedimentation, aggregation or fusion with other liposomes in the dispersion system.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a complex molecular spike encapsulant to solve the technical problems of poor solubility, low transdermal absorption rate and insufficient stability of the molecular spike delivery system in the prior art.

[0007] The second purpose of the present application is to provide a preparation method of the above-mentioned encapsulant.

[0008] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides an encapsulate of a composite molecular spike, the encapsulate comprising:

[0010] a core, the core being a molecular spike liposome, active ingredients of the molecular spike liposome comprising molecular spike NP 2-5 wt%, molecular spike AP 1-2 wt% and phytosphingosine 0.05-0.15 wt%;

[0011] an outer layer, the outer layer being an extracellular vesicle of long bifidobacterium wrapped outside the core.

[0012] Further, the average particle size of the encapsulate is 135-155 nm.

[0013] Further, the molecular spike liposome further comprises glycerin 40-55 wt%, hexyldecanol 8-20 wt%, hydrogenated lecithin 2-5 wt% and cholesterin 1-2 wt%, the rest being water.

[0014] Further, the hydrogenated lecithin is hydrogenated lecithin with hydrogenated phosphatidylcholine content ≥75%, preferably hydrogenated lecithin with hydrogenated phosphatidylcholine content ≥90%.

[0015] In a second aspect, the present application provides a preparation method of the above-mentioned encapsulate, comprising:

[0016] adding the extracellular vesicle of long bifidobacterium into the molecular spike liposome, ultrasonic treatment, stirring and incubation to obtain the encapsulate;

[0017] The mass ratio of the extracellular vesicle of long bifidobacterium to the molecular spike liposome is 1:4-1:9, preferably 1:8.

[0018] Further, the ultrasonic treatment time is 20-60 min, preferably 30 min;

[0019] Preferably, the ultrasonic power is 90-360 W, preferably 200 W;

[0020] Preferably, the incubation time is 0.5-2 hours, preferably 1 hour;

[0021] Further, the preparation method of the molecular spike liposome comprises:

[0022] mixing and stirring hydrogenated lecithin and glycerin to obtain a uniform viscous liquid; stirring molecular spike NP, molecular spike AP, phytosphingosine, cholesterin and hexyldecanol until completely dissolved to obtain a transparent solution; pouring the transparent solution into the uniform viscous liquid, slowly pouring into water after uniform stirring, homogenizing to obtain the molecular spike liposome.

[0023] Further, the mixing and stirring the hydrogenated lecithin and glycerol to obtain a uniform viscous liquid comprises: after mixing the hydrogenated lecithin and glycerol, preheating, and then stirring under high temperature and vacuum conditions.

[0024] Preferably, the preheating comprises a water bath at 55-85°C.

[0025] Preferably, the high temperature comprises 55-85°C.

[0026] Preferably, the vacuum condition comprises -0.01 to -0.03 MPa.

[0027] Preferably, the stirring speed is 300-500 rpm.

[0028] Preferably, the stirring time is 30 min.

[0029] Preferably, the pouring the transparent solution into the uniform viscous liquid further comprises pouring at 55-85°C and a stirring speed of 300-500 rpm.

[0030] Preferably, the continuous stirring time is 5-10 min.

[0031] Preferably, before slowly pouring into the water, the water is preheated to 55-85°C.

[0032] Preferably, the homogenization is performed using a high-pressure microfluidization technique.

[0033] Preferably, the homogenization pressure is controlled to be 5000-15000 psi, and the number of cycles is 2-6.

[0034] Preferably, the discharge temperature is 10-30°C.

[0035] Further, the preparation method of the B. longum extracellular vesicles comprises culturing B. longum, centrifuging to collect supernatant, filtering to remove the supernatant, and obtaining B. longum extracellular vesicles.

[0036] Further, the centrifuging to collect the supernatant comprises first centrifuging to collect the supernatant, and second centrifuging to collect the supernatant.

[0037] Preferably, the first centrifuging condition is 400-1000 xg, 4°C, 15-30 min.

[0038] Preferably, the second centrifuging condition is 10,000-20,000 xg, 4°C, 15-30 min.

[0039] Preferably, the filtering to remove the supernatant comprises filtering through a 0.22 μm filter, centrifuging at 4°C, 100,000-200,000 xg, 60-90 min, and removing the supernatant.

[0040] The present application provides a composite molecular spike encapsulant, which effectively improves the problem of incompatibility of the molecular spike with common formula systems, significantly enhances the stability of the molecular spike system, and significantly enhances the water solubility. A new composite delivery system is constructed by utilizing the natural carrier characteristics of the extracellular vesicles (EVs) of Bifidobacterium longum and the biomimetic structure advantages of the molecular spike liposome. The liposome technology and the microbial nanocarrier technology are organically combined to improve the stability of the molecular spike liposome, enhance the penetration of the molecular spike on the skin surface, and enable the molecular spike to better play a role in the epidermis and dermis of the skin. At the same time, no chemical penetration enhancer such as azone is introduced, and the safe and effective application of the molecular spike NP is realized. The technical problems of poor solubility, low transdermal absorption rate, and insufficient stability of the molecular spike delivery system in the prior art are solved. DETAILED DESCRIPTION

[0041] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by a person of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, the definitions provided herein take precedence. In this application, the use of "or" means "and / or" unless otherwise stated. Moreover, the use of the term "including" as well as other forms such as "include", "includes" or "including" is not limiting.

[0042] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0043] The technical solutions of the present application will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0044] In one aspect, the present application provides a composite molecular spike encapsulant, which comprises:

[0045] The inner core is a molecular spike liposome, and active ingredients of the molecular spike liposome include 2-5 wt% of molecular spike NP, 1-2 wt% of molecular spike AP, and 0.05-0.15 wt% of phytosphingosine.

[0046] The outer layer is an outer layer of Bifidobacterium longum extracellular vesicles.

[0047] The encapsulant effectively improves the problem that the molecular spike is not suitable for common formula systems, significantly enhances the stability of the molecular spike system, and significantly enhances the water solubility. A new type of composite delivery system is constructed by taking advantage of the natural carrier characteristics of Bifidobacterium longum extracellular vesicles (EVs) and the biomimetic structure of the molecular spike liposome. The liposome technology and the microbial nanocarrier technology are organically combined to improve the stability of the molecular spike liposome, enhance the penetration of the molecular spike on the skin surface, and enable the molecular spike to better play a role in the epidermis and dermis of the skin. At the same time, no chemical penetration enhancer such as azone is introduced, and the safe and effective application of the molecular spike NP is realized. The technical problems of poor solubility, low transdermal absorption rate, and insufficient stability of the molecular spike delivery system in the prior art are solved.

[0048] In some specific embodiments, the average particle size of the encapsulant is 135-155 nm.

[0049] The average particle size of the encapsulant can be, but is not limited to, 135 nm, 137 nm, 139 nm, 141 nm, 143 nm, 145 nm, 147 nm, 149 nm, 151 nm, 153 nm, or 155 nm, or any value between 135 nm and 155 nm.

[0050] In some specific embodiments, the molecular spike liposome further includes 40-55 wt% of glycerin, 8-20 wt% of hexyldecanol, 2-5 wt% of hydrogenated lecithin, and 1-2 wt% of cholesterols, and the balance is water.

[0051] Hydrogenated lecithin, as the main membrane material, cooperates with cholesterols to form a stable lipid bilayer structure, and the molecular spike and phytosphingosine repair active substances are embedded in the lipid bilayer. This design not only solves the dispersion problem of liposoluble components such as the molecular spike, but also significantly improves the skin permeability of active substances through a biomimetic mechanism.

[0052] The fatty acid chain is hydrogenated to improve stability. In some specific embodiments, the hydrogenated lecithin is hydrogenated lecithin with a hydrogenated phosphatidylcholine content of ≥75%, preferably hydrogenated lecithin with a hydrogenated phosphatidylcholine content of ≥90%. This is conducive to the stability of the system.

[0053] According to another aspect of the present application, there is further provided a preparation method of the encapsulate, comprising: adding the B. longum extracellular vesicles into the molecular spike liposome, ultrasonic treatment, stirring and incubation to obtain the encapsulate; the mass ratio of the B. longum extracellular vesicles to the molecular spike liposome is 1:4-1:9, preferably 1:8.

[0054] The natural carrier property of the B. longum extracellular vesicles and the biomimetic structure advantage of the molecular spike liposome are utilized to construct a new composite delivery system by assembly. The liposome is a nanoscale carrier composed of a phospholipid bilayer, and its structure has high similarity with the skin keratin layer lipid, which provides an ideal delivery platform for the functional ingredients in the formula. Not only can the problems such as low efficiency and insufficient stability of the molecular spike delivery in the prior art be overcome, but also the liposome technology and the microbial nanocarrier technology are organically combined, breaking through the limitations of traditional formulations, and having a broad application prospect in the fields of dermatology and cosmetics.

[0055] In some specific embodiments, the ultrasonic treatment time is 20-60 min, preferably 30 min; in some specific embodiments, the ultrasonic power is 90-360 W, preferably 200 W; in some specific embodiments, the incubation time is 0.5-2 hours, preferably 1 hour.

[0056] In some specific embodiments, the preparation method of the molecular spike liposome comprises: mixing and stirring hydrogenated lecithin and glycerol to obtain a uniform viscous liquid; stirring molecular spike NP, molecular spike AP, phytosphingosine, cholestanol and hexyl decanol until completely dissolved to obtain a transparent solution; pouring the transparent solution into the uniform viscous liquid, stirring uniformly, then slowly pouring into water, homogenizing to obtain the molecular spike liposome.

[0057] In some specific embodiments, the mixing and stirring of hydrogenated lecithin and glycerol to obtain a uniform viscous liquid comprises mixing and preheating the hydrogenated lecithin and glycerol, then stirring under high temperature and vacuum conditions; in some specific embodiments, the preheating comprises water bath at 55-85°C; in some specific embodiments, the high temperature comprises 55-85°C; in some specific embodiments, the vacuum condition comprises -0.01 to -0.03 MPa; in some specific embodiments, the stirring speed is 300-500 rpm; in some specific embodiments, the stirring time is 30 min.

[0058] In some specific embodiments, pouring the transparent solution into the uniform viscous liquid further comprises pouring at a temperature of 55-85℃ and a rotation speed of 300-500 rpm; in some specific embodiments, the duration of the stirring is 5-10 min; in some specific embodiments, the method further comprises preheating the water to a temperature of 55-85℃ before slowly pouring into the water.

[0059] In some specific embodiments, the homogenization is performed by high pressure microfluidization; in some specific embodiments, the homogenization pressure is controlled to be 5000-15000 psi and the number of cycles is 2-6.

[0060] In some specific embodiments, the discharge temperature is 10-30℃.

[0061] In some specific embodiments, the method for preparing the long Bifidobacterium extracellular vesicles comprises culturing the long Bifidobacterium, centrifuging to collect the supernatant, filtering to remove the supernatant, and obtaining the long Bifidobacterium extracellular vesicles.

[0062] In some specific embodiments, the long Bifidobacterium strain can be selected from, but not limited to, Bifidobacterium longum or Bifidobacterium longum subsp. infantis.

[0063] In some specific embodiments, the centrifuging to collect the supernatant comprises centrifuging to collect the supernatant for the first time and centrifuging to collect the supernatant for the second time; in some specific embodiments, the centrifugation for the first time is performed at 400-1000 x g at 4℃ for 15-30 min; in some specific embodiments, the centrifugation for the second time is performed at 10,000-20,000 x g at 4℃ for 15-30 min.

[0064] In some specific embodiments, the filtering to remove the supernatant comprises filtering through a 0.22 μm filter, centrifuging at 100,000-200,000 x g at 4℃ for 60-90 min, and removing the supernatant.

[0065] The encapsulating material of the long Bifidobacterium extracellular vesicle-encapsulated molecule nail liposome provided by the present application can be added to cosmetics such as water, emulsion, cream, serum, and mask liquid, and the addition ratio can be preferably 0.5-10%, which can maintain stability under various conditions.

[0066] The present application is further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0067] Example 1

[0068] An encapsulating object of Bifidobacterium longum extracellular vesicles encapsulating molecular spike liposomes, comprising an inner core and an outer layer, wherein the inner core is the molecular spike liposomes; the outer layer is the Bifidobacterium longum extracellular vesicles encapsulating the inner core; wherein the components of the molecular spike liposomes are shown in Table 1.

[0069] Table 1

[0070]

[0071] The encapsulating object is prepared according to the following steps:

[0072] 1. Preparation of molecular spike liposomes:

[0073] (1) Hydrogenated lecithin (hydrogenated phosphatidylcholine content 90%, purchased from LIPOID, Germany) and glycerol were weighed according to the above mass ratio and placed in a clean container. After preheating in a 75℃ water bath, the mixture was continuously stirred at 500 rpm, 75℃ and-0.03Mpa vacuum condition for 30 min to obtain a uniform viscous liquid.

[0074] (2) Molecular spike NP, molecular spike AP, phytosphingosine, cholesterol, and hexyl decanol were weighed according to the above mass ratio and dissolved in a transparent solution under the condition of 75℃ and stirring.

[0075] (3) The transparent solution obtained in step (2) was poured into the viscous liquid obtained in step (1) at a speed of 500 rpm and 75℃, and continuously stirred for 10 min to obtain a phospholipid phase.

[0076] (4) Water was weighed according to the above mass ratio and preheated to 75℃. The phospholipid phase obtained in step 3 was slowly poured into the water to hydrate the phospholipid phase, and a lipid body initial milk was obtained. The lipid body initial milk was homogenized using high-pressure microjet technology, with a homogenization pressure of 12000 psi, a cycle number of 4 times, and an outlet temperature of 30℃. A light yellow translucent liquid was obtained as the molecular spike liposomes.

[0077] 2. Preparation of Bifidobacterium longum extracellular vesicles:

[0078] (1) Bifidobacterium longum (Bifidobacterium longum CICC®6207, purchased from China Industrial Microbial Culture Collection Center, strain number: CICC 6068) was resuspended with 0.5 mL sterile water and inoculated into 10 mL BSM broth (purchased from Merck) under sterile conditions. The culture was incubated at 37℃ in an anaerobic box for 48 h. The recovered strain was inoculated into BSM broth at a ratio of 0.5% under sterile conditions and incubated at 37℃ in an anaerobic condition for 4 days with shaking (60 rpm).

[0079] (2) 400xg, 4℃ centrifugation for 15 min to precipitate B. longum, collect the supernatant; 12,000xg, 4℃ centrifugation for 20 min for the second ultracentrifugation to remove dead bacterial debris, collect the supernatant; the supernatant is filtered through a 0.22 μm filter, and then centrifuged at 200,000xg for 60 min at 4℃ to remove the supernatant and collect the precipitate, which is the B. longum extracellular vesicle.

[0080] 3. Preparation of the encapsulate:

[0081] The B. longum extracellular vesicle is added to the aqueous solution of the molecular spike liposome at a mass ratio of 1:8; the mixture is treated with an ultrasonic instrument for 30 min (200 w, 3 s on 3 s off); stirring and incubation at room temperature for 1 hour, which is the encapsulate of the B. longum extracellular vesicle wrapping the molecular spike liposome.

[0082] Example 2

[0083] An encapsulate of the B. longum extracellular vesicle wrapping the molecular spike liposome, comprising an inner core and an outer layer, the inner core is the molecular spike liposome; the outer layer is the B. longum extracellular vesicle wrapped outside the inner core; wherein the composition of the molecular spike liposome is shown in Table 2.

[0084] Table 2

[0085]

[0086] The encapsulate is prepared according to the following steps:

[0087] 1. Preparation of the molecular spike liposome:

[0088] (1) The hydrogenated lecithin (hydrogenated phosphatidylcholine content 75%, purchased from LIPOID, Germany) and glycerol are weighed according to the above mass ratio, and then placed in a clean container. After preheating in a 70℃ water bath, 400 rpm stirring is carried out at 70℃ and-0.03 Mpa vacuum condition for 30 min to obtain a uniform viscous liquid;

[0089] (2) The molecular spike NP, the molecular spike AP, the phytosphingosine, the cholesterols, and the hexyldecanol are weighed according to the above mass ratio, and then stirred at 70℃ until completely dissolved to obtain a transparent solution;

[0090] (3) The transparent solution obtained in step (2) is poured into the viscous liquid obtained in step (1) at 70℃ and 400 rpm, and stirring is continued for 10 min to obtain a phospholipid phase;

[0091] (4) Weigh water according to the above mass ratio, preheat to 70°C, then slowly pour the phospholipid phase obtained in step 3 into the water to hydrate the phospholipid phase, and obtain the liposome initial milk. The liposome initial milk is homogenized using a high-pressure microjet technique, the homogenization pressure is controlled at 10000 psi, the cycle number is 6 times, and the discharge temperature is 20°C, to obtain a light yellow translucent liquid as the molecular nail liposome.

[0092] 2. Preparation of B. longum extracellular vesicles:

[0093] (1) Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. infantis CICC® 6069, purchased from China Industrial Microbial Culture Collection Center, strain number: CICC 6069) was dissolved in 0.5 mL sterile water, then inoculated into 10 mL BSM broth (purchased from Merck) under sterile conditions, and cultured at 37°C in an anaerobic box for 48 h; the recovered strain was inoculated into BSM broth at a proportion of 0.5% under sterile conditions, and cultured at 37°C in an anaerobic condition with shaking (60 rpm) for 4 days.

[0094] (2) The Bifidobacterium longum cells were precipitated by centrifugation at 1000×g and 4°C for 15 min, and the supernatant was collected; the second ultracentrifugation was performed at 15,000×g and 4°C for 30 min, and the supernatant was collected; the supernatant was filtered through a 0.22 μm filter, then centrifuged at 100,000×g and 4°C for 90 min, the supernatant was removed, and the precipitate was retained, which was the Bifidobacterium longum extracellular vesicles.

[0095] 3. Preparation of the encapsulating material:

[0096] The Bifidobacterium longum extracellular vesicles were added to the molecular nail liposome aqueous solution at a mass ratio of 1:9; the mixture was treated with an ultrasonic instrument for 20 min (90w, 3s on 3s off); and the mixture was stirred and incubated at room temperature for 0.5 h, to obtain the encapsulating material of the Bifidobacterium longum extracellular vesicles wrapped molecular nail liposome.

[0097] Example 3

[0098] An encapsulating material of Bifidobacterium longum extracellular vesicles wrapped molecular nail liposome, comprising an inner core and an outer layer, wherein the inner core is a molecular nail liposome, and the outer layer is Bifidobacterium longum extracellular vesicles wrapped outside the inner core; and the components of the molecular nail liposome are shown in Table 3.

[0099] Table 3

[0100]

[0101] The encapsulating material was prepared according to the following steps:

[0102] 1. Preparation of molecular spike liposome:

[0103] (1) Hydrogenated lecithin (hydrogenated phosphatidylcholine content 90%, purchased from LIPOID, Germany) and glycerol were weighed according to the above mass ratio and placed in a clean container. After preheating at 70°C, the mixture was continuously stirred at 500 rpm, 70°C and -0.03 MPa vacuum for 30 min to obtain a uniform viscous liquid.

[0104] (2) Molecular spike NP, molecular spike AP, phytosphingosine, cholesterol, and hexyl decanol were weighed according to the above mass ratio and dissolved in a transparent solution under stirring at 70°C.

[0105] (3) The transparent solution obtained in step (2) was poured into the viscous liquid obtained in step (1) at 70°C and 500 rpm, and continuously stirred for 10 min to obtain a phospholipid phase.

[0106] (4) Water was weighed according to the above mass ratio and preheated to 70°C. The phospholipid phase obtained in step 3 was slowly poured into the water to hydrate the phospholipid phase, and a liposome primary emulsion was obtained. The liposome primary emulsion was homogenized using high-pressure microjet technology, with a homogenization pressure of 15000 psi, 4 cycles, and an outlet temperature of 30°C. A light yellow translucent liquid was obtained as the molecular spike liposome.

[0107] 2. Preparation of Bifidobacterium longum extracellular vesicles:

[0108] (1) Bifidobacterium longum (Bifidobacterium longum CICC®6207, purchased from China Industrial Microbial Culture Collection Center, strain number: CICC 6068) was reconstituted with 0.5 mL sterile water and inoculated into 10 mL BSM Broth (purchased from Merck) under sterile conditions. The culture was incubated at 37°C in an anaerobic chamber for 48 h. The recovered strain was inoculated into BSM Broth at a ratio of 0.5% under sterile conditions and incubated at 37°C in an anaerobic condition with shaking (60 rpm) for 4 days.

[0109] (2) The Bifidobacterium longum was precipitated by centrifugation at 500 x g and 4°C for 15 min, and the supernatant was collected. The supernatant was subjected to a second ultracentrifugation at 12,000 x g and 4°C for 20 min to remove dead bacterial debris, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter and centrifuged at 150,000 x g and 4°C for 75 min to remove the supernatant and collect the precipitate, which was the Bifidobacterium longum extracellular vesicles.

[0110] 3. Preparation of encapsulated material:

[0111] B. frigidus extracellular vesicles were added into the aqueous solution of molecular spike liposomes at a ratio of 1:4 (molecular spike liposomes:B. frigidus extracellular vesicles); the mixture was treated by ultrasonic instrument for 60 min (90 w, 3 s on 3 s off); stirring and incubation at room temperature for 0.5 h, which was the encapsulated product of B. frigidus extracellular vesicles encapsulating molecular spike liposomes.

[0112] Comparative Example 1

[0113] Different from Example 1, no B. frigidus extracellular vesicles were used for encapsulation, and the molecular spike liposomes were prepared according to Step 1.

[0114] Comparative Example 2

[0115] Different from Example 1, soybean lecithin (phosphatidylcholine content 90%, purchased from LIPOID, Germany) was used instead of hydrogenated lecithin to prepare the molecular spike liposomes, and no B. frigidus extracellular vesicles were used for encapsulation, and the rest of the process and ingredients were prepared according to the method of Example 1.

[0116] Comparative Example 3

[0117] Different from Example 1, polyglyceryl-10 stearate was used instead of hydrogenated lecithin, and no B. frigidus extracellular vesicles were used for encapsulation, and the rest of the process and ingredients were prepared according to the method of Example 1.

[0118] Comparative Example 4

[0119] Different from Example 1, polyglyceryl-10 stearate was used instead of hydrogenated lecithin to prepare the molecular spike emulsion system, and the rest of the process and ingredients were prepared according to the method of Example 1.

[0120] Comparative Example 5

[0121] Different from Example 1, soybean lecithin (phosphatidylcholine content 90%, purchased from LIPOID, Germany) was used instead of hydrogenated lecithin to prepare the molecular spike liposomes, and the rest of the process and ingredients were prepared according to the method of Example 1.

[0122] Comparative Example 6

[0123] This comparative example is a deuterated molecular spike NP-butanediol solution without process treatment, containing 5.0% deuterated molecular spike NPs.

[0124] Comparative Example 7

[0125] The difference between Example 1 is that the amount of hydrogenated lecithin is 1.5%, the ratio of molecular spike NP is adjusted to 1.0%, the ratio of molecular spike AP is adjusted to 5.0%, the ratio of phytosphingosine is adjusted to 1.0%, and the rest of the process and ingredients are prepared according to the method of Example 1.

[0126] Comparative Example 8

[0127] The difference between Example 1 is that the amount of hydrogenated lecithin is 5.5%, the ratio of molecular spike NP is adjusted to 6.0%, the ratio of molecular spike AP is adjusted to 0.5%, the ratio of phytosphingosine is adjusted to 0.5%, and the rest of the process and ingredients are prepared according to the method of Example 1.

[0128] Comparative Example 9

[0129] The difference between Example 1 is that the amount of hydrogenated lecithin is 5.5%, the ratio of molecular spike NP is adjusted to 6.0%, the ratio of molecular spike AP is adjusted to 0.5%, the ratio of phytosphingosine is adjusted to 0.5%, and the rest of the process and ingredients are prepared according to the method of Example 1.

[0130] Comparative Example 10

[0131] The difference between Example 1 is that the amount of hydrogenated lecithin is 5.5%, the ratio of molecular spike NP is adjusted to 6.0%, the ratio of molecular spike AP is adjusted to 0.5%, the ratio of phytosphingosine is adjusted to 0.5%, and the rest of the process and ingredients are prepared according to the method of Example 1.

[0132] Test 1 Particle size test

[0133] Take Example 1~3 and Comparative Example 1-5, 7-10 with laboratory pure water dispersion, get 1% pure water dispersion, then use BeNano 180 zeta Pro particle size tester for particle size and PDI test, test conditions: 25℃, equilibrium time 120s, test angle 173°. The specific data are shown in Table 4.

[0134] Table 4 Initial appearance and particle size characterization

[0135]

[0136] The particle size of Example 1~3 is in the range of 135~155nm, which is larger than that of Comparative Example 1 (97.3 nm), indicating that the particle size of the encapsulated body is increased after EVs encapsulation, but still maintains nanoscale size. Comparative Example 7 shows delamination phenomenon just after preparation, indicating that the combination of this molecular spike ratio has poor stability.

[0137] Test 2 Stability test

[0138] Take Example 1~3 and Comparative Example 1-5, 8-10 in sample bottles, respectively, under room temperature conditions for 90 days, under room temperature and non-sunlight direct observation of the appearance change of the product and test 1% water dispersion particle size change, 1% water dispersion change same as test 1. The specific data are shown in Table 5.

[0139] Table 5 Appearance and particle size characterization for 90 days

[0140]

[0141] Appearance stability: The samples were observed at room temperature for 90 days. The samples of Examples 1-3 remained light yellow with a bluish translucent appearance, without precipitation or stratification. The samples of Comparative Examples 1 and 2 showed a decrease in transparency or the precipitation of flocculation, indicating that the liposomes without the protection of B. longum EVs were more prone to aggregation, and the encapsulation by EVs could significantly improve the physical stability of the system. The emulsion systems of Comparative Examples 3 and 4 were stratified, indicating that the stability of the emulsion system was less than that of the liposome system. The sample of Comparative Example 9 showed a decrease in transparency, indicating that a high proportion of B. longum EVs was not conducive to the physical stability of the system.

[0142] Particle size change: The particle size of the samples of Examples 1-3 increased by less than 5 nm, indicating that the system was stable. The particle size of the sample of Comparative Example 1 increased by more than 40 nm. The samples of Comparative Examples 2-4 and 8 were stratified into non-uniform systems after 90 days, and the particle size was not tested again. PDI change: The PDI of the samples of Examples 1-3 remained less than 0.2, indicating that the system was uniform. The PDI of the sample of Comparative Example 5 increased, indicating that the stability of the soybean lecithin system was weaker than that of the hydrogenated lecithin system. The sample of Comparative Example 8 was stratified, indicating that the stability of the molecular spike with different proportions was different from that of the sample of Example. The particle size and PDI of the sample of Comparative Example 9 increased significantly, indicating that a high proportion of EVs was not conducive to the stability of the system.

[0143] It can be seen that the stability of the molecular spike system prepared by the emulsion system and the soybean lecithin system is lower than that of the hydrogenated lecithin system. The introduction of EVs at a suitable proportion can enhance the stability of the system and delay the stratification time, but the proportion should not be too high.

[0144] Test 3: Penetration amount test

[0145] (1) Sample preparation: The samples of Examples and Comparative Examples (all using deuterated molecular spike NPs instead of the original molecular spike NPs to exclude the interference of the original molecular spike in the skin) were added to the serum gel base at a mass fraction of 1%, stirred uniformly, and obtained as the samples to be tested. All the samples to be tested were prepared using a unified serum gel base, and the formula was as follows: 3% propylene glycol, 3% glycerol, 0.8% thickening agent SIMULGEL FL (hydroxyethyl acrylate & dimethyl sodium acrylate copolymer & isohexadecane & polysorbate 60), and the rest was water.

[0146] (2) Test procedure: The transdermal validation was performed according to the in vitro transdermal test guideline of OECD / OCDE, and the specific operation method was as follows: during the experiment, the bama pigskin was first placed in phosphate buffer (pH 7.4) for hydration for 30 min, after hydration, the surface water of the bama pigskin was wiped dry and the bama pigskin was placed between the receiving pool and the supply pool of the Franz diffusion cell, the epidermis of the bama pigskin was towards the diffusion chamber, and the dermis layer was towards the receiving chamber. The receiving pool was phosphate buffer (pH 7.4); 0.1 g of the sample was added to the supply pool; the assembled Franz diffusion cell was placed in the transdermal diffusion instrument, and the experimental conditions were set as follows: the temperature was 37°C, the stirring speed was 600 rpm, and the penetration time was 12 h. The receiving liquid after sample penetration was taken, in order to exclude the interference of the originally existing molecular nail NP in the skin, the skin residence amount of deuterated molecular nail NP was quantified by LC-MS, so as to extrapolate the skin residence amount of molecular nail NP in the sample, and the specific data were shown in Table 6.

[0147] Table 6 Molecular nail NP penetration data

[0148]

[0149] From the test data, the residence amount (1.34 µg / cm 2 ) of Example 1 was 2.0 times that of Comparative Example 1 and 3.9 times that of Comparative Example 6, indicating that the EVs further enhanced the penetration of the molecular nail on the basis of the liposome improving the transdermal penetration and residence of the molecular nail. Example 3 was further improved to 1.74 µg / cm 2 , suggesting that the high load formula could continue to improve the effect. Comparative Example 2 and Comparative Example 5 showed that by changing to a soy lecithin system and introducing EVs, the penetration could also be further improved.

[0150] Comparative Example 3 and Comparative Example 4 showed that whether the polyglycerol-10 stearate system was wrapped with EVs or not, the residence amount was significantly lower than that of the liposome system, indicating that the liposome structure itself played a decisive role in the delivery of the molecular nail.

[0151] Although the particle size of Example was larger than that of Comparative Example 1, the residence amount was significantly higher than that of Comparative Example 1 with smaller particle size, indicating that the contribution of EVs wrapping and phosphatidylcholine content to the penetration was much higher than the influence of particle size.

[0152] In summary, the combination of hydrogenated lecithin with high phosphatidylcholine content + long bifidobacterium extracellular vesicles could significantly improve the skin residence amount of the molecular nail under the premise of small particle size stability, and provided an efficient and stable solution for barrier repair products.

[0153] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An encapsulate of a complex molecular spike, characterized in that, The encapsulate comprises an inner core, the inner core being a molecular spike liposome, an active ingredient of the molecular spike liposome comprising molecular spike NP 2~5wt%, molecular spike AP 1~2wt% and phytosphingosine 0.05~0.15wt%; an outer layer, the outer layer being long-bifidobacterium extracellular vesicles wrapped outside the inner core; the molecular spike liposome further comprises glycerin 40~55wt%, hexyldecanol 8~20wt%, hydrogenated lecithin 2~5wt% and cholesterin 1~2wt%, the rest being water; a mass ratio of the long-bifidobacterium extracellular vesicles to the molecular spike liposome is 1:4~1:

9.

2. The enclosure of claim 1, wherein, The average particle size of the encapsulate is 135~155nm.

3. The enclosure of claim 1, wherein, The hydrogenated lecithin is hydrogenated lecithin with hydrogenated phosphatidylcholine content ≥75%.

4. The enclosure of claim 1, wherein, The hydrogenated lecithin is hydrogenated lecithin with hydrogenated phosphatidylcholine content ≥90%.

5. A process for the preparation of the encapsulates according to any one of claims 1 to 4, characterized in that, comprising: adding long-bifidobacterium extracellular vesicles into the molecular spike liposome, ultrasonic treatment, stirring and incubation to obtain an encapsulate; a mass ratio of the long-bifidobacterium extracellular vesicles to the molecular spike liposome is 1:4~1:

9.

6. The production method according to claim 5, wherein a mass ratio of the long-bifidobacterium extracellular vesicles to the molecular spike liposome is 1:

8.

7. The preparation method according to claim 5, characterized in that, The ultrasonic treatment time is 20~60min.

8. The preparation method according to claim 7, characterized in that, The ultrasonic treatment time is 30min.

9. The preparation method according to claim 5, characterized in that, The ultrasonic power is 90~360W.

10. The method of claim 9, wherein, The ultrasonic power is 200W.

11. The preparation method according to claim 5, characterized in that, The incubation time is 0.5~2 hours.

12. The method of claim 11, wherein, The incubation time is 1 hour.

13. The preparation method according to claim 5, characterized in that, The preparation method of the molecular spike liposome comprises: mixing and stirring hydrogenated lecithin and glycerin to obtain a uniform viscous liquid; stirring molecular spike NP, molecular spike AP, phytosphingosine, cholesterin and hexyldecanol until completely dissolved to obtain a transparent solution; pouring the transparent solution into the uniform viscous liquid, slowly pouring into water after uniform stirring, homogenizing to obtain the molecular spike liposome.

14. The method of claim 13, wherein, The mixing and stirring of hydrogenated lecithin and glycerin to obtain a uniform viscous liquid comprises mixing hydrogenated lecithin and glycerin, preheating, high temperature and vacuum stirring.

15. The method of claim 14, wherein, The preheating comprises 55~85℃ water bath.

16. The method of claim 14, wherein, The high temperature comprises 55~85℃.

17. The method of claim 14, wherein, The vacuum condition comprises -0.01~ -0.03MPa.

18. The method of claim 14, wherein, The stirring speed is 300~500rpm.

19. The method of claim 14, wherein, The stirring time is 30min.

20. The method of claim 13, wherein, Pouring the transparent solution into the uniform viscous liquid further comprises pouring at 55~85℃, 300~500rpm.

21. The method of claim 13, wherein, The uniform stirring time is 5~10min.

22. The method of claim 13, wherein, Preheating the water to 55~85℃ before slowly pouring into water.

23. The method of claim 13, wherein, The homogenization is carried out by high pressure microjet technology.

24. The method of claim 23, wherein, The homogenization pressure is controlled to be 5000~15000psi, and the cycle number is 2~6 times.

25. The method of claim 13, wherein, The discharge temperature is 10~30℃.

26. The method of claim 5, wherein, The preparation method of the long-bifidobacterium extracellular vesicles comprises culturing long-bifidobacterium, centrifuging to collect supernatant, filtering to remove supernatant, and obtaining long-bifidobacterium extracellular vesicles.

27. The method of claim 26, wherein, The centrifuging to collect supernatant comprises first centrifuging to collect supernatant, and second centrifuging to collect supernatant.

28. The method of claim 27, wherein, The first centrifuging condition is 400~1000×g, 4℃ centrifuging for 15~30min.

29. The preparation method according to claim 27, characterized in that, The second centrifugation is performed at 10,000-20,000 x g at 4°C for 15-30 min.

30. The method of claim 26, wherein, The filtration to remove the supernatant includes filtration through 0.22 μm, centrifugation at 4°C at 100,000-200,000 x g for 60-90 min, and removal of the supernatant.

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