Liposome as well as preparation method and application thereof

Abstract

The invention discloses a liposome as well as a preparation method and application thereof. The molecule of the lipid comprises a lipid chain and a head group part connected with the lipid chain, wherein the head group part is a hot alcohol head group. The head group part of the liposome provided by the invention is not phospholipid or sugar any more, but is replaced by a hot alcohol head group with acid resistance. The existence of the hot alcohol head group has an excellent resisting effect on an acid environment. The liposome provided by the invention has very strong stability, especially in an acid environment.

Description

Technical Field

[0001] The present invention relates to the technical field of liposome preparation, and in particular to a liposome and a preparation method and application thereof. Background Art

[0002] Liposomes are single or multiple concentric lipid bilayer vesicles that were discovered in the 1960s and can encapsulate substances. These approximately spherical lipid vesicles can vary in size from a few nanometers to several micrometers. Lipids are naturally occurring molecules that contain hydrophilic (water-loving) and hydrophobic (water-repelling) components. These structures can be formed by the self-assembly of lipids in an aqueous environment, forming a closed bilayer structure. Liposomes are often used in the field of drug delivery because they are able to encapsulate both hydrophilic and hydrophobic drugs. The lipid bilayer structure of liposomes allows hydrophobic drugs to be incorporated into the lipid bilayer, while hydrophilic drugs can be loaded into the inner aqueous compartment. This versatility makes liposomes promising vehicles for the delivery of various types of drugs.

[0003] All liposome preparations currently on the market use phospholipids to encapsulate drugs to achieve drug delivery. However, due to the easy oxidation and hydrolysis of phospholipids themselves, the stability of phospholipid liposomes still needs to be improved.

[0004] Therefore, the prior art needs to be further improved and enhanced. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a liposome and a preparation method and application thereof, aiming to solve the problems of the prior art liposome being easy to oxidize, easy to hydrolyze and having poor stability.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a liposome, the molecule of which comprises: a lipid chain and a head group portion connected to the lipid chain, wherein the head group portion is a thermophilic alcohol head group.

[0008] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. Through the following preferred technical solutions, the objectives and beneficial effects of the present invention can be better achieved and realized.

[0009] As a preferred technical solution, the liposome, wherein the lipid chain is modified with N five-membered rings, wherein N=1, 2, 3...8.

[0010] In a second aspect, a liposome, the molecule of which comprises: a lipid chain, and a head group portion connected to the lipid chain, wherein the lipid chain is modified with N five-membered rings, wherein N=1, 2, 3...8.

[0011] In a third aspect, a method for preparing the liposome described above, comprising:

[0012] Sulfolobus was used as the original strain for gene editing to construct knockout plasmids;

[0013] Transforming the knockout plasmid into a Sulfolobus strain with uracil auxotrophy to obtain a first strain;

[0014] Cultivating the first strain, extracting lipids therefrom, and obtaining membrane lipids;

[0015] The membrane lipid and phospholipid are mixed, and the liposome is prepared by a thin film hydration method.

[0016] As a preferred technical solution, the method for preparing liposomes, wherein the knockout plasmid includes: pSVA407-saci_1585UD plasmid, pSVA407-saci_0240UD plasmid; the construction of the knockout plasmid specifically includes:

[0017] The upstream and downstream sequences of saci_1585 and saci_0240 of 700 bp each were inserted into the pSVA407 plasmid using the SLIC technique to form the knockout plasmid;

[0018] The saci_1585 and saci_0240 are two genes related to the two phenotypes of Sulfolobus.

[0019] As a preferred technical solution, the method for preparing liposomes, wherein the method for preparing liposomes further comprises:

[0020] The 700 bp SSO2881 was inserted into the pSVA1561 plasmid using the SLIC method to construct the pSVA1561-SSO2881 expression plasmid;

[0021] The pSVA1561-SSO2881 expression plasmid is transformed into the first strain to obtain a second strain; the first strain is a strain transformed with the pSVA407-saci_0240UD plasmid;

[0022] Cultivating the second strain and extracting lipids therefrom to obtain membrane lipids;

[0023] The membrane lipid and phospholipid are mixed, and the liposome is prepared by a thin film hydration method.

[0024] As a preferred technical solution, in the method for preparing liposomes, the mixing ratio of the membrane lipid to the phospholipid is 20%.

[0025] In a fourth aspect, a method for preparing the liposome according to the first aspect or the second aspect, comprising:

[0026] Constructing an engineered strain of a model strain for synthesizing liposomes, and using the engineered strain to synthesize the liposomes; or

[0027] A thermoalcohol head group is provided, and the thermoalcohol head group is linked to a tetraether lipid or a phospholipid in vitro to obtain the liposome.

[0028] In a fifth aspect, the liposome described above is used to prepare a drug delivery vehicle.

[0029] Beneficial effects: Compared with the prior art, the liposome provided by the present invention has a head group part that is no longer a phospholipid or a sugar, but is replaced by a thermoalcohol head group that has acid resistance. The presence of the thermoalcohol head group has an excellent resistance to an acidic environment. The liposome provided by the present invention has a strong stability, especially in an acidic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the synthesis and main modified configuration of archaeal GDGTs in the present invention; wherein (a) represents the synthesis of tetraether GDGTs from diether archaeal alcohols; (b) represents the synthesis of thermoalcohol head groups from hexoses; (c) represents the intrachain cyclization of GDGTs;

[0031] Figure 2 Schematic diagram of leakage of liposomes encapsulating fluorescein calcein in the present invention; wherein the green circle represents calcein; the black bilayer represents the phospholipid bilayer; and the red monolayer represents the tetraether lipid GDGTs;

[0032] Figure 3 is the leakage curve of five liposomes in the present invention at pH = 4.6 over time;

[0033] Figure 4 The calcein residues of the five liposomes of the present invention at pH = 4.6 after 60 minutes and the significance test. NS (no significance): no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001;

[0034] Figure 5 The residual calcein of the five liposomes of the present invention at pH = 3.6 after 60 minutes and the significance test; NS (no significance): no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001;

[0035] Figure 6The figure shows the residual calcein in the five liposomes of the present invention at pH = 2.6 after 60 minutes and the significance test. NS (no significance): no significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION

[0036] The present invention provides a liposome and a preparation method and application thereof. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementation methods. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and do not mean that they are necessary sequences, unless otherwise specified that a certain sequence must be followed. The serial numbers themselves, such as "first", "second", etc., assigned to the components herein are only used to distinguish the objects described and do not have any order or technical meaning. The instruments used are all commercially available products.

[0038] like Figure 1 As shown, in view of the poor stability of existing liposomes, the inventors of the present invention provide the following technical solutions:

[0039] The polar head group calditol is used to modify the liposomes, and the head group of the liposome molecule is replaced with the acid-resistant calditol head group, thereby improving the stability of the liposome in an acidic environment.

[0040] In one implementation of the present invention, the lipid chains of the liposomes can also be modified, that is, the lipid chains are cyclized, such as the lipid chains are modified with 1 five-membered ring, 2 five-membered rings, 3 five-membered rings, 4 five-membered rings, 5 five-membered rings, 6 five-membered rings, 7 five-membered rings or 8 five-membered rings. As the number of rings in the lipid chain increases, its resistance to acidic environments also increases.

[0041] It is easy to understand that in one implementation of the present invention, in addition to the above-mentioned single hot alcohol head group modification and then cyclization modification, a single cyclization modification can also be used.

[0042] Based on the same inventive concept, the present invention also provides a method for preparing the above liposome, comprising the following steps:

[0043] S100, using Sulfolobus solvatis as the original strain for gene editing, the knockout plasmid was constructed.

[0044] Specifically, for Sulfolobus acidocaldarius, the editing methods for this gene are relatively mature and easy to operate. It is easy to understand that in addition to Sulfolobus acidocaldarius, other commonly used strains can also be used as original strains for gene editing. The genes related to the two phenotypes of Sulfolobus acidocaldarius were named cds: saci_1489; grsA: saci_1585; grsB: saci_0240, SSO2881, which were used to construct knockout plasmids with the plasmid of pSVA407. The construction of the plasmid can adopt SLIC (Sequence and Ligation Independent Cloning) technology, that is, sequence and ligase independent cloning technology. 700bp of saci_1489, saci_1585 and saci_0240 upstream and downstream sequences are inserted into the pSVA407 plasmid.

[0045] S200, transferring the knockout plasmid into a Sulfolobus strain with uracil auxotrophy to obtain a first strain.

[0046] Specifically, pSVA407-saci_1489UD and pSVA407-saci_0240UD in step S100 were transferred into the uracil auxotrophic strain Sulfolobus acidocaldarius MW001, and 5-FOA was used to induce the construction of MW001Δsaci_1489 and MW001Δsaci_0240 strains (ie, the first strain).

[0047] S300, culturing the first strain, extracting lipids therefrom, and obtaining membrane lipids.

[0048] Specifically, Brock medium can be used, cultured at 75°C and 200rpm until the stable phase, and then the cells are collected and extracted to obtain membrane lipids. The collection, extraction and other operations involved are common techniques in the art, which will be described in the examples below and will not be repeated here.

[0049] S400, mixing the membrane lipid and phospholipid, and preparing the liposome by thin film hydration method.

[0050] Specifically, the thin-film hydration method is a commonly used method for preparing liposomes, which includes the following main steps:

[0051] Mixing lipids: First, the desired membrane lipids (such as archaeal membrane lipids GDGTs) and phospholipids are mixed together in a specific ratio. This mixture can be a mixture of solid powders or by dissolving them in an appropriate solvent (usually an organic solvent, such as chloroform).

[0052] Evaporation of solvent: Pour the mixed lipid solution into a container and evaporate the solvent using a rotary evaporator or heating under reduced pressure, leaving a thin film of dry lipids at the bottom of the container.

[0053] Hydration: Add water or buffer to the dried lipid film. Water molecules will penetrate into the lipid film, and because lipid molecules have hydrophilic and hydrophobic ends, they will spontaneously form a double-layer structure, encapsulating water molecules in the middle to form multi-layer liposome vesicles.

[0054] Sonication: To reduce the size of liposomes and increase their homogeneity, sonication is often used. During sonication, the liposome vesicles are ruptured and reformed, resulting in unilamellar liposomes of the desired size.

[0055] Filtration or Extrusion: Finally, the size and distribution of the liposomes can be further adjusted by filtration or extrusion techniques to obtain a liposome product with specific properties.

[0056] The advantages of the thin film hydration method are that it is simple, low cost, and can be used to prepare a variety of liposomes. This method is particularly suitable for laboratory-scale liposome preparation because it can adjust the ratio of lipids and water as needed and control the size of liposomes by means such as ultrasound.

[0057] Based on the same inventive concept, the present invention also provides another method for preparing liposomes, such as using molecular biological methods to construct engineered strains of model strains such as yeast and Escherichia coli to obtain thermophilic head groups or cyclization modifications; or purifying thermophilic head groups and connecting the thermophilic head groups with lipids such as tetraether lipids and phospholipids in vitro.

[0058] Based on the same inventive concept, the present invention also provides liposomes for use as drug delivery carriers. Since liposomes have excellent acid resistance and stability, they can resist the destruction of digestive juices such as gastric acid and pancreatic enzymes and protect drugs from metabolism by enzymes in the body during oral drug delivery, thereby protecting the precise release of drugs. During targeted delivery, the high stability of liposomes can ensure that they are not destroyed while circulating in the body but before reaching the target, thereby improving the therapeutic effect.

[0059] The technical solution provided by the present invention is further explained below through specific preparation examples.

[0060] Example 1

[0061] The engineering strains for GDGTs thermoalcohol head group and intrachain cyclization are constructed as shown in Table 1. Sulfolobus acidocaldarius, which has relatively mature gene editing methods, was selected as the original strain for gene editing. The three genes related to the two phenotypes are cds: saci_1489; grsA: saci_1585; grsB: saci_0240, SSO2881. First, we selected pSVA407 as the knockout plasmid, and used the SLIC technology to insert 700 bp of saci_1489, saci_1585 and saci_0240 upstream and downstream sequences into the pSVA407 plasmid, forming the knockout plasmids pSVA407-saci_1489UD, pSVA407-saci_1585UD, and pSVA407-saci_0240UD. The expression plasmid pSVA1561 was selected and inserted into SSO2881 using the SLIC method to construct the pSVA1561-SSO2881 expression plasmid. We transferred pSVA407-saci_1489UD and pSVA407-saci_0240UD into the uracil auxotrophic strain Sulfolobus acidocaldarius MW001 and used 5-FOA induction to construct the MW001Δsaci_1489 and MW001Δsaci_0240 strains. pSVA407-saci_1585UD was added to Δsaci_0240, and 5-FOA was used to induce the MW001Δsaci_1585Δsaci_0240 strain. The pSVA1561-SSO881 plasmid was added to the Δsaci_0240 strain to construct the grsB complemented strain MW001Δsaci_0240+pSVA1561-SSO2881.

[0062] Table 1 Selected strains and their lipid compositions

[0063]

[0064] The gene-edited strain was cultured in Brock medium at 75°C and 200rpm until the stationary phase, and the cells were collected by centrifugation at 10,000×g for 5 minutes and frozen at -20°C. The core lipids were obtained by resuspending the centrifuged cells with a small amount of pure water and acid hydrolyzing them overnight in a 40mL capped glass bottle containing 7mL of 10% HCl (hydrochloric acid) in MeOH (methanol) (vol / vol) solution at 70°C. 10mL DCM (dichloromethane) and 10mL pure water were added to the acid-treated mixture, and the lipids in the mixture were extracted 3 times with DCM in a 40mL polytetrafluoroethylene tube. The sample was centrifuged for 10 minutes to separate the aqueous and organic phases, and the bottom organic phase was transferred to a collection glass bottle. The organic phases were combined and filtered through a 0.22-μm PTFE filter and dried under nitrogen. Polar lipids were extracted using a modified Bligh / Dyer method. First, resuspend the cells with 2 mL of phosphate buffer or trichloroacetic acid solution, transfer to a PTFE centrifuge tube, add 2.5 mL of DCM and 5 mL of MeOH to the tube, vortex for 1 min, then ultrasonicate in a water bath for 10 min, and centrifuge at 2800×g for 10 min. Transfer the supernatant to a 500 mL flask. Extract twice with phosphate buffer and TCA buffer respectively. Add 40 mL of pure water and 40 mL of DCM, transfer to a separatory funnel to collect the lower organic phase and transfer it to a collection flask. The combined organic phases are concentrated using a rotary evaporator, filtered through a 0.22-μm PTFE filter, and dried under nitrogen.

[0065] Take GDGTs and add DCM: MeOH (2: 1) and mix well to make a stock solution (10 mg / ml). Use a vacuum pump to evacuate until the pressure reaches 0 mbar, set the water temperature to 35°C, and 120 rpm for 60 min of rotary evaporation to form a uniform lipid film on the bottle wall. Add 3 mL of PBS buffered saline solution containing calcein (80 mM) to the pear-shaped bottle for hydration, and ultrasonicate in an ultrasonic cleaner to control the ultrasonic temperature to never exceed 35°C. The prepared liposomes are stored at 4°C.

[0066] like Figures 2 to 6As shown, in vitro leakage: using the self-quenching property of calcein, the tetraether liposomes with different lipid compositions encapsulating calcein will have different degrees of leakage at a certain pH, and calcein will be released from the liposomes into the external system. Trace amounts of calcein leak into the external system, causing its concentration to decrease. At this time, the quenching phenomenon disappears, and the corresponding fluorescence intensity can be detected by a fluorescence spectrophotometer. After the reaction is completed, 100 microliters of 10% (W / V) TritonX-100 is added to the reaction tube and incubated at 37°C for 30 minutes to completely lyse the liposomes. The fluorescence intensity in the system is measured, which is the fluorescence of all calcein initially encapsulated inside the liposomes. Finally, the percentage of calcein released at each time point is calculated. Because calcein is in a positive linear relationship within a low concentration range, the strength of the fluorescence signal reflects the degree of calcein release by the liposomes under different conditions. The tetraether liposomes and phospholipid liposomes prepared by the thin film dispersion method were placed at pH 2.6, 3.6, and 4.6 to investigate the stability of liposomes with four different tetraether lipid compositions. The results are as follows Figures 3 to 6 As shown, it can be seen that the prepared liposomes have strong acid resistance and stability.

[0067] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A liposome, the molecules of which include: A lipid chain and a head group portion connected to the lipid chain, characterized in that the head group portion is a thermoalcohol head group.

2. The liposome according to claim 1, characterized in that The lipid chain is modified with N five-membered rings, wherein N=1, 2, 3...

8.

3. A liposome, the molecules of which include: A lipid chain and a head group portion connected to the lipid chain, characterized in that the lipid chain is modified with N five-membered rings, wherein N=1, 2, 3...

8.

4. A method for preparing liposomes according to claim 1 or 2, characterized in that: include: Sulfolobus was used as the original strain for gene editing to construct knockout plasmids; Transforming the knockout plasmid into a Sulfolobus strain with uracil auxotrophy to obtain a first strain; Cultivating the first strain, extracting lipids therefrom, and obtaining membrane lipids; The membrane lipid and phospholipid are mixed, and the liposome is prepared by a thin film hydration method.

5. The method for preparing liposomes according to claim 4, characterized in that: The knockout plasmids include: pSVA407-saci_1585UD plasmid, pSVA407-saci_0240UD plasmid; the construction of the knockout plasmid specifically includes: Using SL IC technology, 700 bp of saci_1585 and saci_0240 upstream and downstream sequences were inserted into the pSVA407 plasmid to form the knockout plasmid; The saci_1585 and saci_0240 are two genes related to the two phenotypes of Sulfolobus.

6. The method for preparing liposomes according to claim 4, characterized in that: The preparation method further comprises: The 700 bp SSO2881 was inserted into the pSVA1561 plasmid using the SLIC method to construct the pSVA1561-SSO2881 expression plasmid; The pSVA1561-SSO2881 expression plasmid is transformed into the first strain to obtain a second strain; the first strain is a strain transformed with the pSVA407-saci_0240UD plasmid; Cultivating the second strain and extracting lipids therefrom to obtain membrane lipids; The membrane lipid and phospholipid are mixed, and the liposome is prepared by a thin film hydration method.

7. The method for preparing liposomes according to claim 6, characterized in that: The mixing ratio of the membrane lipid to the phospholipid is 20%.

8. A method for preparing the liposome according to claim 1 or 3, characterized in that: include: Constructing an engineered strain of a model strain for synthesizing liposomes, and synthesizing the liposomes using the engineered strain; or A thermoalcohol head group is provided, and the thermoalcohol head group is linked to a tetraether lipid or a phospholipid in vitro to obtain the liposome.

9. A liposome according to any one of claims 1 to 2 or claim 3 for use in preparing a drug delivery vehicle.

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