Intestinal targeted biological membrane liposome nano preparation as well as preparation method and application thereof
By constructing intestinal-targeted biomembrane liposome nanoformulations, the problem of liposome instability in the gastrointestinal environment is solved, and efficient targeted delivery and therapeutic effects of drugs in intestinal diseases are achieved.
Patent Information
- Application Number
- CN202410291589.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-14
- Publication Date
- 2025-09-23
AI Technical Summary
Existing liposomes are unstable in the gastrointestinal environment, resulting in low drug bioavailability and difficulty in achieving effective oral targeted drug delivery, especially the lack of active targeting in non-tumor intestinal diseases such as inflammatory bowel disease.
A phospholipid bilayer biomimetic nanoplatform is used to construct an intestinal-targeted biomembrane liposome nanoformulation. By wrapping the probiotic Escherichia coli or macrophage membrane on the liposome surface and coating it with the intestinal-specific degradation material chitosan, the stability and targeting in the gastrointestinal tract are enhanced.
It improves the delivery efficiency of drugs in the intestine, enhances the active targeting and biocompatibility of intestinal lesions, reduces the toxic and side effects of drugs, and achieves effective treatment of intestinal diseases.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an intestinal tract-targeted biomembrane liposome nanoformulation, a preparation method thereof, and an application thereof. Background Art
[0002] Oral administration is the main route of administration. Compared with parenteral administration, oral administration is a simple and painless method of administration with higher compliance and convenience, and is therefore more popular with patients. Oral targeted drug delivery systems are considered to be an effective drug delivery system that can significantly improve patient compliance and also improve the local therapeutic effect of drugs in the gastrointestinal tract. However, most of the small molecule drugs currently used have poor water solubility, and drugs with low solubility are difficult to be absorbed by the gastrointestinal tract, resulting in low bioavailability. In addition, the harsh conditions in the digestive tract and the complex absorption and digestion mechanisms are huge obstacles to the oral administration of many active pharmaceutical ingredients.
[0003] Liposomes are one of the most successful carriers in the field of drug delivery systems. Recent research on oral targeted drug delivery systems has demonstrated their potential to enhance the bioavailability of encapsulated materials and improve their intestinal absorption. Major obstacles to efficient oral drug delivery using liposomes are the high acidity of the gastric environment, the presence of enzymes, the mucosal barrier lining much of the digestive tract, and the tight junctions of epithelial cells. Traditional liposomes are composed of phospholipids self-assembled into concentric bilayers. Unfortunately, these liposomes have been found to be unstable after oral administration in the presence of bile salts, digestive tract enzymes, and acidic conditions such as the stomach, leading to dissolution of the lipid bilayer. This ultimately reduces the bioavailability of oral delivery materials.
[0004] In order to better utilize liposomes for oral targeted drug delivery systems, modifying and modifying liposomes to enhance their stability during digestive tract transport and introduce new functions is a good approach. In addition, although liposomes can be passively enriched at tumor sites based on the enhanced permeability and retention effect (EPR effect) of tumors, passive targeting lacks high efficiency, and for other non-tumor intestinal diseases such as inflammatory bowel disease, the EPR effect does not exist. Therefore, further modification is usually required to enhance the ability of liposomes to actively target tumors / other intestinal cells.
[0005] Regarding liposome modification and modification, we first selected a blend of natural and synthetic phospholipids as raw materials for liposome preparation, replacing the traditional phospholipids that form the basis of liposomes. For intestinal diseases without granulomatous tissue, we chose DSPE-PEG to enhance its circulation time. For diseases with granulomatous lesions, such as Crohn's disease, we chose DSPE-PEG-PLGA, which exhibits targeted accumulation in granulomatous tissue. Furthermore, Pluronic F127 was incorporated into the liposome preparation, which loosens the tight junctions of intestinal epithelial cells, improving drug solubility and enhancing liposome penetration into the intestinal mucosa. Liposome modification typically involves multiple synthetic steps, making the process complex and challenging. However, biomimetic techniques, which utilize cell membranes to coat nanoparticles, camouflaging them as autologous components to evade immune recognition and clearance, have attracted considerable attention. This top-down approach bypasses laborious modification engineering and preserves the antigen and cell membrane structure, resulting in excellent targeting. Therefore, the membranes of macrophages, which are abundant in inflammatory sites, and the membrane of the probiotic Escherichia coli Nissle 1917, a normal component of the intestinal microenvironment, are used to coat the liposomes. Finally, the negatively charged cell membrane is coated with a gut-specific degradable material, such as chitosan, to improve its stability in the digestive tract environment, enhance its adhesion, and cellular absorption, thereby preventing leakage or premature release of the encapsulated drug.
[0006] In summary, we introduced Pluronic F127 into the liposome preparation raw materials, coated the liposome surface with a biofilm, and finally adsorbed a layer of intestinal-specific degradation material on the biofilm surface through electrostatic interaction. This is used for single / combination loading of water-soluble / lipid-soluble drugs, effectively preventing phospholipids from being exposed to the acidic gastrointestinal environment, thereby preventing drug leakage from the liposomes, improving the intestinal targeted delivery efficiency of drugs and the therapeutic effect of intestinal diseases. Summary of the Invention
[0007] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide an intestinal-targeted biomembrane liposome nanoformulation and its preparation method and application, which is mainly used to improve the delivery efficiency of drugs in the treatment of intestinal diseases. It has a simple preparation method, a wide drug loading range, and excellent active targeting, biocompatibility and gastrointestinal environmental stability.
[0008] In order to achieve the above object, the present invention adopts the following technical solutions:
[0009] In one aspect, the present invention provides an intestinal-targeted biomembrane liposome nanoformulation, characterized in that it comprises a liposome nanocore and a biomembrane wrapped on its surface, and an intestinal-specific degradation material coated on the surface of the biomembrane;
[0010] The lipid nanocore comprises at least one phospholipid liposome and a single / combined hydrophilic / lipophilic drug loaded in its aqueous phase / lipid phase;
[0011] The biofilm includes a probiotic Escherichia coli Nissle 1917 derived membrane or a macrophage membrane;
[0012] The surface of the biofilm is coated with an intestinal specific degradation material.
[0013] The raw materials for preparing the liposome nanocore include at least one phospholipid, which can be one or more of natural phospholipids such as soy lecithin and egg yolk lecithin, synthetic phospholipids such as DSPE, DPPC, PSPC, DPPE, and DSPC, and functionalized phospholipid derivatives such as DSPE-PEG and DSPE-PEG-PLGA. Liposomes primarily targeting inflammatory lesions in the intestine are preferably a combination of soy lecithin and DSPE-PEG, while liposomes primarily targeting granulomatous lesions in the intestine are preferably a combination of soy lecithin and DSPE-PEG-PLGA.
[0014] Meanwhile, the raw materials for preparing the liposome also include steroids, wherein the steroid is preferably cholesterol.
[0015] In addition, we also added Pluronic F127 to the raw materials for the preparation of liposomes to stabilize the liposome structure and enhance the intestinal epithelial penetration of liposomes.
[0016] The intestinal specific degradation material coated on the surface of the biofilm may be chitosan, pectin, sodium alginate, etc., preferably chitosan.
[0017] The intestinal tract-targeted biomembrane liposome nanoformulation can achieve single or combined loading of water-soluble / lipid-soluble drugs in the aqueous phase / lipid phase of the liposome according to the medication requirements of different disease courses.
[0018] Preferably, in the raw materials for preparing liposomes, the mass ratio of phospholipids to cholesterol is 10:1 to 2:1; the mass ratio of phospholipids to the total drug amount is 10:1 to 40:1, the mass ratio of natural phospholipids to synthetic phospholipids is 1:2 to 2:1; the mass ratio of the liposome nanocore to the biological membrane is 1:10 to 10:1, and the mass ratio of the water-soluble and fat-soluble drugs loaded on the liposomes is 1:5 to 5:1.
[0019] Based on a phospholipid bilayer biomimetic nanoplatform, the present invention constructs an intestinal-targeted biomembrane liposome nanoformulation with a three-layer structure of lipid nanocore + biomembrane wrapping + intestinal-specific degradation material. Pluronic F127 is also introduced into the preparation of the lipid nanocore to achieve the dual purposes of stabilizing the liposome structure and enhancing the intestinal epithelial penetration of the liposome. This specific structure allows the chitosan adsorbed on the outermost layer of the delivery system to remain wrinkled in the low pH environment of the stomach when the delivery system passes through the upper digestive tract, thereby protecting the structural stability of the delivery system. When it reaches the lower digestive tract with a neutral pH, the structure of the chitosan responds to the change in pH and becomes loose, which is beneficial for the biofilm to achieve targeted adhesion and accumulation at the lesion site, and then expose the internal liposome nanocore. At this time, the Pluronic F127 in the liposome nanocore can allow the liposome to penetrate the intestinal epithelium in a relatively complete form, enter the cell and release the encapsulated drug. The DSPE-PEG and DSPE-PEG-PLGA used in it both have excellent long-circulation effects, and the latter also has granulation tissue targeted distribution characteristics, so it can greatly improve the targeted drug delivery efficiency of the delivery system and achieve drug efficacy enhancement and toxicity reduction.
[0020] In another aspect, the present invention provides a method for preparing an intestinal tract-targeted biomembrane liposome nanoformulation, the preparation method comprising:
[0021] (1) dissolving the liposome raw material and the liposoluble drug in an organic solvent, removing the organic solvent by rotary evaporation to form a phospholipid bilayer, adding an aqueous solution containing a water-soluble drug according to the delivery requirements, hydrating and ultrasonically dispersing, and filtering the whole particles to obtain drug-loaded liposome nanoparticles;
[0022] (2) Extraction of Escherichia coli Nissle 1917 cell membrane: After lysing the Escherichia coli cells, the cell membrane was extracted using extraction buffer, and the purified Escherichia coli cell membrane was obtained after centrifugation; for the extraction of macrophage membrane, the macrophage membrane was broken by multiple extrusions, and the obtained cell homogenate was mixed with sucrose solution, centrifuged, and the supernatant was collected and centrifuged again. The collected precipitate was washed with buffer to obtain the purified macrophage membrane.
[0023] (3) The lipid nanocores were mixed with Escherichia coli Nissle 1917 cell membranes or macrophage cell membranes with ultrasound assistance and then co-incubated.
[0024] This invention, based on a phospholipid bilayer biomimetic nanoplatform, constructs an intestinal-targeted biomembrane liposome nanoformulation. First, a drug-loaded liposome nanocore is prepared using a thin-film dispersion method. Escherichia coli Nissle 1917 cell membranes and macrophage membranes are purified by lysis extraction and extrusion centrifugation, respectively. Finally, a nanoscale drug delivery system is obtained by co-incubating the lipid nanocore and cell membranes. This preparation method is simple, has a wide drug loading range, and exhibits excellent active targeting and biocompatibility, making it suitable for improving the efficiency of intestinal-targeted drug delivery.
[0025] In step (1), the rotary evaporation is carried out at 45-65°C for 40-120 minutes; and the hydration is carried out at 20-30°C.
[0026] The pH value of the E. coli extraction buffer in step (2) is in the range of 7.0 to 8.0, preferably 7.4; the number of macrophage extrusions is 10 to 50 times, preferably 20 to 30 times.
[0027] The ultrasound-assisted mixing and co-incubation in step (3) is carried out at a temperature of 30-40°C, preferably 37°C.
[0028] In the present invention, other specific point values in all the numerical ranges mentioned above can be selected. Due to space limitations and for the sake of brevity, they will not be described in detail here.
[0029] Thirdly, the intestinal targeted biomembrane liposome nanoformulation described in the present invention is mainly used for targeted intestinal delivery of drugs to treat intestinal-related diseases, including primary intestinal diseases such as inflammatory bowel disease and intestinal tumors, as well as diseases mediated by the intestine or intestinal microenvironment, such as non-alcoholic fatty liver disease and metabolic syndrome.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The present invention is based on a phospholipid bilayer biomimetic nano-platform and has constructed a kind of intestinal targeted biomembrane liposome nano-formulation.This biomimetic nano-liposome has a lipid nano-core+biomembrane wrap+intestinal specific degradation material three-layer structure, has improved gastrointestinal stability and improved intestinal targeted distribution characteristics, utilizes the delivery system of biomembrane wrapped drug-loaded liposome, can not only realize active targeting of lesion site, reduce the toxic and side effects of drugs, and uses it as the method for loading drugs on the platform, is simple and easy, has excellent biocompatibility.In addition, certain diseases such as intestinal Behcet's disease often need to use fat-soluble immunosuppressants such as thalidomide and water-soluble hormones such as dexamethasone simultaneously when the disease acute phase is excessive to the remission phase.This delivery system proposes that the purpose of delivering drugs simultaneously can be achieved by the combined drug loading of liposome aqueous phase and lipid phase.In short, this biomembrane liposome nano-formulation provides an ideal biomimetic carrier for the effective intestinal targeted delivery of drugs, has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 Flow chart for the preparation of an intestinal-targeted biomembrane liposome nanoformulation.
[0033] Figure 2 This is the particle size distribution diagram of the macrophage membrane-encapsulated liposome nanoformulation prepared in Example 1.
[0034] Figure 3 This is a transmission electron micrograph of the macrophage membrane-encapsulated liposome nanoformulation prepared in Example 1.
[0035] Figure 4 This is a graph showing the particle size stability of the macrophage membrane-encapsulated liposome nanoformulation prepared in Example 1 after incubation in PBS for 7 days.
[0036] Figure 5 1 is a graph showing the particle size distribution of the liposome nanoformulation coated with the Nissle 1917-derived membrane of Escherichia coli prepared in Example 2.
[0037] Figure 6 This is a transmission electron micrograph of the Escherichia coli Nissle 1917-derived membrane-encapsulated liposome nanoformulation prepared in Example 2.
[0038] Figure 7 This is a graph showing the particle size stability of the E. coli Nissle 1917-derived membrane-encapsulated liposome nanoformulation prepared in Example 2 after incubation in PBS for 7 days. DETAILED DESCRIPTION
[0039] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as limiting the present invention. It should be understood by those skilled in the art that any modification or replacement of the method, steps or conditions of the present invention, without departing from the spirit and essence of the present invention, falls within the scope of the present invention.
[0040] Example 1
[0041] (1) Soy lecithin, cholesterol, and DEPS-PEG were dissolved in chloroform at a mass ratio of 5:1:5 to form an 11 mg lipid solution. 0.1 M Pluronic F127 and an appropriate amount of colchicine were then added. The lipid solution was mixed in a round-bottom flask and rotary evaporated. After forming a thin layer on the flask wall, the film was further dried in a vacuum chamber for 2–4 hours to remove the remaining organic solvent.
[0042] (2) Then, an appropriate amount of PBS solution containing dexamethasone was added to the lipid membrane and hydrated at room temperature to obtain the liposome nanocore.
[0043] (3) Extraction of macrophage membrane: RAW264.7 cells were suspended in pre-chilled buffer (pH 7.4; 10 mM Tris + 1 mM MgCl2) and then disrupted by 30 extrusions through a 400 nm microextruder without a polycarbonate membrane. The resulting cell homogenate was then mixed with 1 M sucrose to a final concentration of 0.25 M sucrose, and the mixture was centrifuged at 2000 × g and 4°C for approximately 15 minutes. The resulting supernatant was centrifuged again at 3000 × g for 20–30 minutes to remove remaining organelles. Cell membranes were then collected and washed twice with pre-chilled buffer in the presence of 0.25 M sucrose for purification.
[0044] (4) The obtained lipid nanocores and the extracted macrophage membranes were co-incubated at 37°C for 10 to 30 minutes, and ultrasonically disrupted to obtain nano-modulators with uniform particle size.
[0045] (5) The particle size of the macrophage membrane-encapsulated liposome nanoformulation was determined by DLS and characterized by transmission electron microscopy.
[0046] (6) The prepared macrophage membrane-encapsulated liposome nanoformulation was placed at room temperature for 7 days, and the particle size of the nanoformulation was measured every day to characterize its stability.
[0047] The particle size test results of macrophage membrane-encapsulated liposome nanoformulations are as follows Figure 2 As shown, the average particle size of the sample is 236.1±3.9 nm.
[0048] Transmission electron microscopy characterization results of macrophage membrane-encapsulated liposome nanoformulations are shown in Figure 2. Figure 3 As shown, the sample exhibits an obvious core-shell structure.
[0049] The particle size test results of the macrophage membrane-wrapped liposome nanoformulation after 7 days of storage are as follows Figure 4 As shown in the figure, the hydrodynamic diameter of the nano-regulator remained basically unchanged for seven days, indicating that the particle size of this nano-preparation was stable and uniform and had good biological stability.
[0050] Example 2
[0051] (1) Soy lecithin, cholesterol, and DEPS-PEG were dissolved in chloroform at a mass ratio of 5:1:5 to form an 11 mg lipid solution. 0.1 M Pluronic F127 and an appropriate amount of colchicine were then added. The lipid solution was mixed in a round-bottom flask and rotary evaporated. After forming a thin layer on the flask wall, the film was further dried in a vacuum chamber for 2–4 hours to remove the remaining organic solvent.
[0052] (2) Then, an appropriate amount of PBS solution containing dexamethasone was added to the lipid membrane and hydrated at room temperature to obtain the liposome nanocore.
[0053] (3) Extraction of E. coli Nissle 1917-derived membranes: After culturing E. coli, the E. coli cells were harvested and resuspended in 0.2M Tris-HCl buffer (pH 7.4-8.0). They were lysed with 200 μg / mL lysozyme, 20 mM sucrose, and 0.2 mM EDTA at room temperature for 10-20 minutes. Apronin (20 μg / mL) and PMSF (1 mM) were then added. Subsequently, an equal volume of extraction buffer (2% Triton X-100, 50 mM 7.4-8.0 Tris-HCl, 10 mM MgCl2) containing DNAse (10 μg / mL) was added to isolate the cell membranes of the E. coli cells. Finally, after incubation on ice for 30 minutes, the lysate was centrifuged at 4000-6000 rpm for 5 minutes. The collected precipitate was the E. coli Nissle 1917-derived membrane.
[0054] (4) The obtained lipid nanocore and the extracted Escherichia coli Nissle 1917 derived membrane were co-incubated at 37°C for 10 to 30 minutes, and ultrasonically disrupted to obtain a nanomodulator with uniform particle size.
[0055] (5) The particle size of the obtained Escherichia coli Nissle 1917-derived membrane-encapsulated liposome nanoformulation was determined by DLS and characterized by transmission electron microscopy.
[0056] (6) The prepared E. coli Nissle 1917-derived membrane-encapsulated liposome nanoformulation was placed at room temperature for 7 days, and the particle size of the nanoformulation was measured every day to characterize its stability.
[0057] The particle size test results of the liposome nanoformulation encapsulated by the Escherichia coli Nissle 1917 derived membrane are as follows Figure 5 As shown, the average particle size of the sample is 257.4±5.1 nm.
[0058] Transmission electron microscopy characterization results of liposome nanoformulations encapsulated by Escherichia coli Nissle 1917 derived membranes are shown in Figure 2. Figure 6 As shown, the sample exhibits an obvious core-shell structure.
[0059] The particle size test results of the liposome nanoformulation coated with the Escherichia coli Nissle 1917 derivative membrane after 7 days are as follows Figure 7 As shown in the figure, the hydrodynamic diameter of the nano-regulator remained basically unchanged for seven days, indicating that the particle size of this nano-preparation was stable and uniform and had good biological stability.
Claims
1. An intestinal targeted biomembrane liposome nanoformulation, characterized in that: It includes a liposome nano core and a biofilm wrapped on its surface, and an intestinal specific degradation material coated on the surface of the biofilm; The lipid nanocore comprises: at least one phospholipid liposome and a single / combined hydrophilic / lipophilic drug loaded in its aqueous phase / lipid phase; The biofilm includes a probiotic Escherichia coli Nissle 1917 derived membrane or a macrophage membrane; The surface of the biofilm is coated with an intestinal specific degradation material.
2. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 1, characterized in that: The raw materials for preparing the liposomes contain at least one phospholipid, wherein the phospholipid can be one or more of natural phospholipids such as soybean lecithin, egg yolk lecithin, etc., synthetic phospholipids such as DSPE, DPPC, PSPC, DPPE, DSPC, and functionalized phospholipid derivatives such as DSPE-PEG and DSPE-PEG-PLGA.
3. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 2, characterized in that: The raw materials for preparing the liposomes are preferably a combination of soybean lecithin and DSPE-PEG for liposomes targeting mainly inflammatory lesions in the intestine, and preferably a combination of soybean lecithin and DSPE-PEG-PLGA for liposomes targeting mainly granulomatous lesions in the intestine.
4. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 2, characterized in that: The raw materials for preparing the liposomes also include steroids, wherein the steroid is preferably cholesterol.
5. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 2, characterized in that: The raw materials for preparing the liposomes also include Pluronic F127 adsorbed on the surface of the liposomes to stabilize the liposome structure and enhance the intestinal epithelial penetration of the liposomes.
6. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 1, wherein the intestinal tract-specific degradation material coated on the biomembrane surface can be chitosan, pectin, sodium alginate, etc., preferably chitosan.
7. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 1, characterized in that: The intestinal tract-targeted biomembrane liposome nanoformulation can achieve single or combined loading of water-soluble / lipid-soluble drugs in the aqueous phase / lipid phase of the liposome according to the medication requirements of different disease courses.
8. The intestinal tract-targeted biomembrane liposome nanoformulation according to claim 1, characterized in that: The mass ratio of phospholipid to cholesterol is 10:1 to 2:1; the mass ratio of phospholipid to total drug amount is 10:1 to 40:1, the mass ratio of natural phospholipid to synthetic phospholipid is 1:2 to 2:1; the mass ratio of liposome nanocore to biological membrane is 1:10 to 10:1, and the mass ratio of water-soluble and fat-soluble drugs loaded on the liposome is 1:5 to 5:
1.
9. The method for preparing the intestinal tract-targeted biomembrane liposome nanoformulation according to claims 1-8, characterized in that: The preparation method comprises: (1) dissolving the liposome raw material and the liposoluble drug in an organic solvent, removing the organic solvent by rotary evaporation to form a phospholipid bilayer, adding an aqueous solution containing a water-soluble drug according to the delivery requirements, hydrating and ultrasonically dispersing, and filtering the whole particles to obtain drug-loaded liposome nanoparticles; (2) Extraction of Escherichia coli Nissle 1917 cell membrane: After lysing the Escherichia coli cells, the cell membrane was extracted using extraction buffer, and the purified Escherichia coli cell membrane was obtained after centrifugation; for the extraction of macrophage membrane, the macrophage membrane was broken by multiple extrusions, and the obtained cell homogenate was mixed with sucrose solution, centrifuged, and the supernatant was collected and centrifuged again. The collected precipitate was washed with buffer to obtain the purified macrophage membrane. (3) The lipid nanocores were mixed with Escherichia coli Nissle 1917 cell membranes or macrophage cell membranes with ultrasound assistance and then co-incubated.
10. The preparation method according to claim 9, wherein the rotary evaporation in step (1) is carried out at 45-65°C for 40-120 minutes; and the hydration is carried out at 20-30°C.
11. The preparation method according to claim 9, wherein the pH value of the E. coli extraction buffer in step (2) is in the range of 7.0 to 8.0, preferably 7.4; and the macrophage extrusion times are 10 to 50 times, preferably 20 to 30 times.
12. The preparation method according to claim 9, wherein the ultrasound-assisted mixing and co-incubation in step (3) is carried out at a temperature of 30-40°C, preferably 37°C.
13. An intestinal targeted biomembrane liposome nanoformulation for use in the preparation of intestinal related targeted drug delivery systems.