CN114904008A - Mulberry leaf liposome extraction preparation method, mulberry leaf liposome product and application of mulberry leaf liposome product in nucleic acid delivery - Google Patents
Mulberry leaf liposome extraction preparation method, mulberry leaf liposome product and application of mulberry leaf liposome product in nucleic acid delivery Download PDFInfo
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- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The invention relates to a mulberry leaf liposome extraction preparation method, a mulberry leaf liposome product and application of the mulberry leaf liposome product in nucleic acid delivery, wherein the method comprises the following steps: preparing a mulberry leaf extraction aqueous solution, dissolving the mulberry leaf extraction aqueous solution in an organic solvent, uniformly mixing to obtain an organic phase, adding the mixed solution, uniformly mixing to obtain a mixed system, adding a set amount of KCl solution into the lower organic phase after centrifugal treatment, uniformly mixing, freezing and centrifuging, adding a set amount of PF127 into the lower organic phase of the solution, uniformly mixing, placing the organic phase into a container, performing rotary evaporation for 5-10min at a set temperature, dissolving the non-evaporated part in a set amount of HEPES buffer solution to obtain liposome; the mulberry leaf liposome provided by the invention has uniform particle size, good biocompatibility, degradability and mucosa penetrating capability, and can improve the stability of nano-drugs in gastrointestinal tracts and the aggregation of nano-drugs at focus parts after oral administration, thereby improving the treatment effect of the nano-drugs on inflammatory bowel diseases and cancer parts.
Description
Technical Field
The invention relates to the technical field of nano drug carriers and tumor targeting, in particular to a mulberry leaf liposome extraction preparation method, a mulberry leaf liposome product and application of the mulberry leaf liposome product in nucleic acid delivery.
Background
The liposome is a bilayer vesicle constructed by amphiphilic molecules such as phospholipid and the like, and becomes one of promising nano-carriers for local drug delivery; the lipid nanoparticle carrier is a novel nano-drug delivery system which takes a nanoparticle material as a drug carrier and dissolves or wraps drug molecules in a phospholipid bilayer of the lipid molecules or in an interlayer, or adsorbs and attaches the drug molecules to the surface of the nanoparticles. The lipid nano-drug can not only improve the absorption of the organism to the drug and change the in vivo process of the drug, but also has the advantages of sustained release and controlled release of the drug, improvement of the stability of the drug, enhancement of the curative effect, reduction of toxic and side effects and the like. The carrier system is widely applied to genetic drugs, antitumor drugs, proteins, polypeptides and other drugs. The existing systems such as liposome, lipid nano-drug, micelle and the like are used clinically;
nanostructures and nanoparticles of plant origin find applications in many disciplines such as health care, food, feed, cosmetics, biomedicine, energy science, drug gene delivery, environmental health, etc. Plants contain a variety of vesicles, and studies have found that they all have the following characteristics: (1) the biocompatibility is good; (2) has wide stress on the carried medicine. Hydrophilic and hydrophobic drugs can be simultaneously encapsulated in the same liposome; (3) the safety is high. The phospholipid is a cell membrane component, so that the nano liposome is nontoxic when injected into a body, has high bioavailability and does not cause immune reaction; (4) can protect the carried medicine, prevent the dilution of the medicine by body fluid and the decomposition and damage of enzyme in the body; (5) has extremely strong targeting property. Therefore, the plant nano-liposome is a nano material with great development prospect. These edible plant-derived vesicles are in daily contact with the human intestinal tract. Moreover, these plant vesicles are also involved in the intestinal tissue turnover process in healthy subjects, regulate intestinal microbiota, and have important biological functions in inflammatory diseases (e.g. colitis, hepatic steatosis). In addition, research data indicate that the nano-vesicle of plant origin is an excellent carrier for delivering therapeutic agents (such as siRNA, anticancer drugs) or insoluble natural compounds (such as curcumin and camptothecin), for example, it can be used as a carrier for drugs for anti-tumor, antibacterial, anticoagulant drugs, rheumatoid arthritis treatment drugs and transdermal and mucosal drug delivery systems. The anti-tumor drug carrier is used as the most researched and valuable application carrier, can slowly release the anti-tumor drug, prolongs the retention time of the drug in the tumor, slows down the growth of the tumor, improves the curative effect, and reduces the administration dosage and the toxic reaction. For example, the existing research shows that the nano liposome can effectively transfer the p53 gene into nasopharyngeal carcinoma cells, inhibit the growth of the tumor cells and induce apoptosis. The mannan-modified targeted nanoliposome has a certain curative effect on mouse lung cancer; the podophyllotoxin nano liposome has better anti-liver cancer effect than podophyllotoxin suspension. Edible plant-derived nanoparticles were isolated and identified from ginger, carrot, grape, grapefruit, lemon, apple, broccoli, and the like, and were similar in size and structure to mammalian-derived exosomes. They contain protein, lipid and miRNA, can be absorbed by intestinal macrophages and stem cells, and can achieve the treatment of corresponding diseases;
the natural plant lipid contains natural targeting molecules such as galactose, phosphatidylserine and the like, so that natural targeting to cells can be realized, and the lipid serving as a drug carrier has good biomedical performances such as biocompatibility, biodegradability and the like, and is approved by FDA (food and drug administration) to be applied to clinical medicine; at present, the natural plant lipid products are still to be developed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a mulberry leaf liposome extraction preparation method aiming at the defects in the prior art, and provide a mulberry leaf liposome product and application of the mulberry leaf liposome in nucleic acid delivery.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a mulberry leaf liposome extraction preparation method is constructed, and comprises the following steps:
the first step is as follows: preparing or taking mulberry leaf extract water solution;
the second step is that: dissolving the mulberry leaf extraction aqueous solution obtained in the first step in an organic solvent, and uniformly mixing to obtain an organic phase;
the third step: adding the organic phase obtained in the second step into the mixed solution, and uniformly mixing to obtain a mixed system;
the fourth step: centrifuging the mixed system obtained in the third step;
the fifth step: adding a certain amount of KCl solution into the lower organic phase of the solution obtained in the fourth step, and uniformly mixing to obtain a mixed system;
and a sixth step: freezing and centrifuging the mixed system obtained in the fifth step;
the seventh step: adding the lower organic phase of the solution obtained in the sixth step into PF127 with a set amount, mixing uniformly, placing the organic phase into a container, performing rotary evaporation at a set temperature for 5-10min, and dissolving the non-evaporated part in HEPES buffer solution with a set amount to obtain liposome;
or adding the lower layer organic phase of the solution in the sixth step into a container, rotary evaporating at set temperature for 5-10min, adding the unevaporated part into a mixed solution of PF127 and HEPES buffer solution in a set amount, and dissolving lipid components by ultrasound to obtain liposome.
The mulberry leaf liposome extraction preparation method of the invention, wherein in the seventh step, the amount of added PF127 has a relationship with the protein concentration in the mulberry leaf extraction aqueous solution:
the protein concentration of the mulberry leaf extraction aqueous solution is equal to the volume of the mulberry leaf extraction aqueous solution/PF 127 (80-120): 5.
The mulberry leaf liposome extraction preparation method provided by the invention comprises the following steps of:
dissolving a certain amount of mulberry leaves in a certain amount of PBS buffer solution, soaking for a certain time, putting into a crusher for juicing and crushing, taking supernate, centrifuging, and taking supernate as mulberry leaf extraction aqueous solution.
The mulberry leaf liposome extraction preparation method provided by the invention is characterized in that the organic solvent in the second step is a mixed solution of methanol and dichloromethane with a set volume.
The mulberry leaf liposome extraction preparation method provided by the invention is characterized in that the mixed solution in the third step is prepared by uniformly mixing dichloromethane and deionized water with set volumes.
A mulberry leaf liposome product is prepared according to the extraction preparation method of the mulberry leaf liposome.
An application of folium Mori liposome in nucleic acid delivery is provided.
The invention has the beneficial effects that: the mulberry leaf liposome provided by the invention has uniform particle size, good biocompatibility, degradability and mucosa penetrating capability, and can improve the stability of nano-drugs in gastrointestinal tracts and the aggregation of nano-drugs at focus parts after oral administration, thereby improving the treatment effect of the nano-drugs on inflammatory bowel diseases and cancer parts.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described with reference to the accompanying drawings and embodiments, wherein the drawings in the following description are only part of the embodiments of the present invention, and for those skilled in the art, other drawings can be obtained without inventive efforts according to the accompanying drawings:
FIG. 1 is a flow chart of the method for preparing liposome of mulberry leaf according to the preferred embodiment of the present invention;
FIG. 2 is a schematic of a screening and stability analysis of five different Pluronic surfactants;
FIG. 3 is a graph showing the results of dynamic light scattering particle size, Zeta potential and PDI distribution tests on liposomes of mulberry leaves successfully modified with Pluronic F127;
FIG. 4 is a schematic diagram of the compositional analysis of successfully prepared liposomes of mulberry leaf without Pluronic F127 modification;
FIG. 5 is a schematic representation of the biocompatibility evaluation of liposomes of mulberry leaves modified by Pluronic F127 prepared successfully;
FIG. 6 is a graph showing the transfection efficiency of successfully prepared Pluronic F127 modified liposomes of mulberry leaves compared with Lipo 6000.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of the present invention.
The method for extracting and preparing the mulberry leaf liposome of the preferred embodiment of the invention, as shown in figure 1, comprises the following steps:
s01: preparing or taking mulberry leaf extract water solution;
s02: dissolving the mulberry leaf extraction aqueous solution obtained in the first step in an organic solvent, and uniformly mixing to obtain an organic phase;
s03: adding the organic phase obtained in the second step into the mixed solution, and uniformly mixing to obtain a mixed system;
s04: centrifuging the mixed system obtained in the third step;
s05: adding a certain amount of KCl solution into the lower organic phase of the solution obtained in the fourth step, and uniformly mixing to obtain a mixed system;
s06: freezing and centrifuging the mixed system obtained in the fifth step;
s07: adding the lower organic phase of the solution obtained in the sixth step into PF127 with a set amount, mixing uniformly, placing the organic phase into a container, performing rotary evaporation at a set temperature for 5-10min, and dissolving the non-evaporated part in HEPES buffer solution with a set amount to obtain liposome;
or adding the lower organic phase of the solution in the sixth step into a container, performing rotary evaporation at a set temperature for 5-10min, adding the non-evaporated part into a mixed solution of a set amount of PF127 and HEPES buffer solution, and dissolving lipid components by ultrasound to obtain liposome;
the mulberry leaf liposome provided by the invention has uniform particle size, good biocompatibility, degradability and mucosa penetrating capability, and can improve the stability of nano-drugs in gastrointestinal tracts and the aggregation of nano-drugs at focus parts after oral administration, thereby improving the treatment effect of the nano-drugs on inflammatory bowel diseases and cancer parts;
the research utilizes lipid extracted from mulberry leaves as a carrier, and the liposome from the mulberry leaves is placed in a refrigerator (3-5 ℃) for refrigeration, has small state change, low permeability and strong stability, and is suitable for long-term storage. The transmission electron microscope picture shows that the liposome is the vesicle with uniform size and regular round shape. The particle size measurement data show that the average particle size is about 230.5nm, which meets the requirements of the pharmaceutical injection preparation; the dispersion coefficient was 0.231, indicating a small particle size distribution range. The potential measurement result is averaged to be zeta-potential value-15.7, and the stability is better;
the invention has the outstanding advantages that nucleic acid materials can be delivered, the successfully prepared mulberry leaf liposome is used for transfecting GFP plasmids, and the transfection efficiency is better compared with the marketed Lipofectamin 6000.
In the method of the present application, there are two different embodiments in the seventh step, but both are based on the same concept, except that the adding time node of the PF127 is slightly changed.
One specific implementation is:
(1) providing 3-4mL of mulberry leaf extraction aqueous solution;
(2) dissolving the mulberry leaf water solution obtained in the step (1) in an organic solvent, and uniformly mixing by swirling for 1-2min to obtain an organic phase;
(3) and (3) adding the mixed solution into the organic phase obtained in the step (2), and uniformly mixing by vortex for 1-2min to obtain a mixed system.
(4) Centrifuging the mixed system obtained in the step (3) for 5-10min at 4000g/min by a high-speed centrifuge;
(5) and (4) adding 2-3mL of 1mol/L KCl solution into the lower-layer organic phase obtained in the step (4), and uniformly mixing by swirling for 1-2min to obtain a mixed system.
(6) Centrifuging the mixed system in the step (5) for 5-10min by a high-speed refrigerated centrifuge at 2000 g/min;
(7) respectively continuing the experiment of the lower organic phase obtained in the step (6) by two methods to finally obtain the liposome, and storing the liposome in a refrigerator below-20 ℃ for later use;
the method comprises the following steps: adding 3mg of PF127 into the lower organic phase obtained in the step (6), and mixing by vortex. And adding the organic phase into a 50ml round-bottom flask, placing the flask into a rotary evaporator at 38-40 ℃ for rotary evaporation for 5-10min, taking the part without evaporation, and finally taking 1 ml HEPES water bath for ultrasonic resuspension to obtain the final liposome.
The second method comprises the following steps: adding the lower organic phase obtained in the step (6) into a 50ml round-bottom flask, placing the flask into a 38 ℃ rotary evaporator for rotary evaporation for 5-10min, taking the part without evaporation, adding 2ml of solution prepared by PF127 (3mg/ml, unfreezing 24mg of PF127 in advance and adding 8ml of HEPES buffer solution) (using an ultrasonic instrument probe for ultrasonic power of 100-;
preferably, in the seventh step, the amount of PF127 added is related to the protein concentration of the mulberry leaf extract aqueous solution:
the protein concentration (mg/ml) of the mulberry leaf extraction aqueous solution, the volume (ml) of the mulberry leaf extraction aqueous solution/PF 127 (mg) ═ 80-120): 5; preferably, a ratio of 100:5 is used;
the proportion is an optimal proportion value, and of course, adaptive adjustment can be performed, and simple changes based on the principle also belong to the protection scope of the application.
Preferably, in the first step, the preparation of the aqueous solution of mulberry leaf extract comprises the following steps:
dissolving a set amount of mulberry leaves in a set amount of PBS buffer solution, soaking for a set time, putting the mulberry leaves into a crusher for juicing and crushing, taking supernate, centrifuging, and taking supernate as mulberry leaf extraction aqueous solution;
one preferred method is: selecting 25g of dry mulberry leaves, dissolving the dry mulberry leaves in 50ml of PBS (pH is 7.4, the mass-volume ratio of the mulberry leaves to a buffer is 1: 2), soaking the dry mulberry leaves overnight, putting the dry mulberry leaves into a crusher, juicing and crushing the mixture, taking supernate, centrifuging the supernate for 1.5 to 2 hours at the room temperature of 10000g/min by using a high-speed centrifuge, and taking supernate as a mulberry leaf extraction aqueous solution; of course, parameters can also be adjusted on the basis of this principle, and simple changes based on this principle also belong to the scope of protection of the present application.
Preferably, the organic solvent in the second step is a mixed solution of methanol and dichloromethane with a set volume;
preferably, the organic solvent is obtained by uniformly mixing 8mL of methanol and 4mL of dichloromethane in a vortex manner for 1-2 min; of course, parameters can also be adjusted on the basis of this principle, and simple changes based on this principle also belong to the scope of protection of the present application.
Preferably, the mixed solution in the third step is prepared by uniformly mixing dichloromethane and deionized water with set volumes;
preferably, the mixed solution is obtained by uniformly mixing 4mL of dichloromethane and 2mL of deionized water through vortex for 1-2 min; of course, parameters can also be adjusted on the basis of this principle, and simple changes based on this principle also belong to the scope of protection of the present application.
Preferably, the solution of the upper layer and the solution of the lower layer in each step are sucked by a medical needle;
a folium Mori liposome product is prepared according to the above folium Mori liposome extraction method.
An application of folium Mori liposome in nucleic acid delivery is provided.
Experimental mode:
screening and stability analysis of one, five different Pluronic surfactants:
the stability of nanoparticles composed of different materials can be evaluated through the changes of particle size, potential and polydispersity index under different pH conditions for a certain time. In the preparation process of the mulberry leaf liposome, the stability difference of five pluronics of PF127, PF108, P123, PF68 and L65 is compared, namely, different types of surfactants are added before rotary evaporation in the preparation process of the liposome, wherein the protein concentration (mg/ml) of mulberry leaf extraction aqueous solution is about 100:5 of the mulberry leaf extraction aqueous solution volume/Pluronic (mg). Re-suspending in buffer solution with pH6.8, incubating at 37 deg.C for 72h, and sucking partial suspension at certain time point to determine the particle size distribution of NPs by Dynamic Light Scattering (DLS);
in FIG. 2, (a) is the basic information for five different Pluronic surfactants; (b) stability evaluation of mulberry leaf liposome nanoparticles modified with five different Pluronic surfactants; (c) and (d) are dispersivity comparisons of Pluronic F127 and L65 of similar stability;
in the mulberry leaf nano-liposome prepared by a film hydration method, Pluronic F127 (PF127) is selected to modify NPs, and the PF127 mainly plays roles in stabilizing, uniformly dispersing and solubilizing as a surfactant. Before this, we also screened different classes of pluronic surfactants, including five types PF127, PF108, P123, PF68, L65, in order to determine the best modification effect. Through preliminary comparison, the particle sizes of PF127 and L65 are relatively stable compared with the other three, the hydrodynamic particle size is controlled to be 156.8-276.4nm (FIG. 2(b)), and secondly, PF127 is used as a surfactant (FIG. 2(c)) in successfully prepared mulberry leaf liposome, and the dispersibility of the mulberry leaf liposome nanoparticles using L65 as a surfactant (FIG. 2(d)) is better, so that PF127 is used as a surfactant of the nanoparticles in the following nanoparticle preparation process, and the comparison shows that the hydrodynamic particle size, the polydispersity index (PDI) and the Zeta potential of blank liposome nanoparticles containing PF127 surfactant are all more stable than NPs without PF127 (FIG. 3). Stability testing of the folium mori liposome nanoparticles: the results show that the particle size distribution of the mulberry leaf liposome nanoparticles in 72h is 220.5-264.3 nm, and the fluctuation within a certain range is acceptable.
Secondly, analyzing the components of the mulberry leaf liposome:
the successfully prepared liposomes of mulberry leaves were subjected to lipid component analysis by Shanghai Cluster company (Wealso thane Shanghai Sensiticipip Infotech Co. Ltd, Shanghai, People's Republic of China.), briefly, lipid composition and major molecular species analysis were performed by LC-MS/MS (Thermo, ultra 3000LC, Q active), reversed phase high performance liquid chromatography (UPLC) BEH C18 (100X 2.1mm, 1.7 μm, Waters), and the measurement of the instrumental parameters was performed by LC-MS/MS method. Data results are presented as percent molecules;
stability testing of the folium mori liposome nanoparticles: in the application of the delivery carrier in drug delivery, certain requirements are placed on the entrapment and targeted delivery of the drug, as shown in fig. 4, natural mulberry leaf liposome contains about 24.9% of TG, 16.9% of PE, 9.9% of MG, 9.2% of hexlcar, 9.2% of LdMePE, 8.1% of DG, 6.3% of dmpe, 4.0% of LPG, 3.0% of PI and 8.4% of other substances, wherein TG and PE not only serve as main structural support of liposome, but also help absorption of nano-drug in vivo, and the absorption rate of the delivery system can be about 25%, which is very high for an oral drug.
Thirdly, cell compatibility experiment of the mulberry leaf liposome nano particles:
the toxicity of the mulberry leaf liposome nano-particles to cells is detected by using an MTT method, and the succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into insoluble purple crystals. RAW 264.7 cells were first seeded in 96-well plates at a cell density of 1.0X 10 per well 4 And (4) respectively. After overnight culture, the cell state at the bottom of the well plate was observed to be good, and then the culture solution was discarded, followed by washing three times with PBS containing calcium and magnesium ions pre-cooled at 4 ℃. The corresponding serum-free medium containing the mulberry leaf liposome nanoparticles was then added to the corresponding experimental wells, supplemented with complete medium after 6 hours, and then co-incubation continued for 24 or 48 hours. After the incubation was completed, the co-incubation solution was discarded, and washed three times with PBS containing calcium and magnesium ions to completely remove the remaining NPs. Then 100. mu.L of MTT solution (0.5mg/mL) was added per well and incubated with the cells for 4 hours, with the MTT being plated out in phenol red free medium. After the incubation is finished, the incubation solution is discarded, then 100 μ L DMSO is added into each well, and the well plate is placed in a horizontal shaking table to shake for 10-15 minutes at 150rpm and 100-. The optical density of each well was then measured using a microplate reader. Wherein untreated cells were used as negative controls and cells treated with Triton X-100 (5%, w/v) were used as positive controls, with 6 replicates per group;
as shown in fig. 5, evaluation of in vitro biocompatibility of the mulberry leaf liposome nanoparticles: in vitro biocompatibility evaluation is one of important parameters, and results show that even higher NPs concentration in mouse peritoneal macrophages shows less cytotoxicity, the survival rate of cells at 24 hours is more than 83%, and the survival rate of cells at 48 hours is more than 75%, which indicates that healthy macrophages show good tolerance to NPs. In addition, the survival rate of the cells is reduced to a certain extent along with the increase of the concentration, which is probably because the increase of the concentration of the nano-drug influences the gas exchange, osmotic pressure and pH maintenance of the cells in the culture solution, thereby causing 'passive' toxicity to the cells. When plasmid with the administration concentration of 1 mu g/ml is transfected, a purchased conventional transfection reagent lipo6000 is used as a control, the cytotoxicity of lipo6000 transfection is far greater than that of mulberry leaf liposome, and obvious significant difference exists; using GFP as a plasmid control, GFP showed good cellular compatibility, indicating that the biocompatibility effect on tumor-associated cells was carried by NPs. The results show that the present study indeed developed a and almost negligible effect on normal cells, and that for commercial liposomes on the market, natural plant liposomes showed good cell compatibility, reflecting the superiority of natural liposomes as a material for delivering nucleic acids.
Fourthly, evaluating the transfection efficiency of the mulberry leaf liposome nano particles: respectively transfecting GFP plasmids in macrophage RAW 264.7 by using extracted mulberry leaf liposome nanoparticles and a commercial transfection reagent Lipo6000, and observing the fluorescence expression condition of GPF by a confocal microscope after 24 hours to evaluate the transfection efficiency of the mulberry leaf liposome nanoparticles on the GFP plasmids;
as shown in FIG. 6, the fluorescence expression of GFP plasmid delivered by using mulberry leaf liposome nanoparticle as delivery vector is significantly higher than that of GFP plasmid with the same quality transfected by using Lipo6000 as transfection reagent after 24 hours, which shows that the preparation of successful Pluronic F127 modified mulberry leaf liposome has the effect of transfecting nucleic acid and the transfection efficiency is higher than that of commercial transfection reagent Lipo6000 which is already on the market.
It will be appreciated that modifications and variations are possible to those skilled in the art in light of the above teachings, and it is intended to cover all such modifications and variations as fall within the scope of the appended claims.
Claims (7)
1. The extraction and preparation method of the mulberry leaf liposome is characterized by comprising the following steps:
the first step is as follows: preparing or taking mulberry leaf extract water solution;
the second step is that: dissolving the mulberry leaf extraction aqueous solution obtained in the first step in an organic solvent, and uniformly mixing to obtain an organic phase;
the third step: adding the organic phase obtained in the second step into the mixed solution, and uniformly mixing to obtain a mixed system;
the fourth step: centrifuging the mixed system obtained in the third step;
the fifth step: adding a certain amount of KCl solution into the lower organic phase of the solution obtained in the fourth step, and uniformly mixing to obtain a mixed system;
and a sixth step: freezing and centrifuging the mixed system obtained in the fifth step;
the seventh step: adding the lower organic phase of the solution obtained in the sixth step into PF127 with a set amount, mixing uniformly, placing the organic phase into a container, performing rotary evaporation at a set temperature for 5-10min, and dissolving the non-evaporated part in HEPES buffer solution with a set amount to obtain liposome;
or adding the lower layer organic phase of the solution in the sixth step into a container, rotary evaporating at a set temperature for 5-10min, adding the non-evaporated part into a mixed solution of PF127 and HEPES buffer solution in a set amount, and dissolving lipid components by ultrasound to obtain liposome.
2. The method for extracting and preparing the mulberry leaf liposome according to claim 1, wherein in the seventh step, the amount of added PF127 has a relationship with the protein concentration in the aqueous solution of mulberry leaf extract:
the protein concentration of the mulberry leaf extraction aqueous solution is equal to the volume of the mulberry leaf extraction aqueous solution/PF 127 (80-120): 5.
3. The method for preparing the liposome of folium mori as claimed in claim 1, wherein the preparation of the aqueous solution of folium mori extract in the first step comprises the steps of:
dissolving a certain amount of mulberry leaves in a certain amount of PBS buffer solution, soaking for a certain time, putting into a crusher for juicing and crushing, taking supernate, centrifuging, and taking supernate as mulberry leaf extraction aqueous solution.
4. The method for preparing the liposome of folium mori as claimed in claim 1, wherein the organic solvent in the second step is a mixed solution of methanol and dichloromethane with a predetermined volume.
5. The method for extracting and preparing the liposome of mulberry leaf according to claim 1, wherein the mixed solution in the third step is prepared by mixing dichloromethane and deionized water with a predetermined volume.
6. A mulberry leaf liposome product, which is characterized in that the mulberry leaf liposome product is prepared according to the extraction preparation method of the mulberry leaf liposome of any one of claims 1 to 5.
7. An application of folium Mori liposome in nucleic acid delivery is provided.
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| CN109846825A (en) * | 2019-01-16 | 2019-06-07 | 江西科技师范大学 | A kind of preparation method of folic acid-Pluronic F127 modification curcumin nano-lipid body |
| CN113384705A (en) * | 2021-06-28 | 2021-09-14 | 西南大学 | Preparation of poloxamer modified liposome and application of poloxamer modified liposome in oral drug delivery |
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| CN109846825A (en) * | 2019-01-16 | 2019-06-07 | 江西科技师范大学 | A kind of preparation method of folic acid-Pluronic F127 modification curcumin nano-lipid body |
| CN113384705A (en) * | 2021-06-28 | 2021-09-14 | 西南大学 | Preparation of poloxamer modified liposome and application of poloxamer modified liposome in oral drug delivery |
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| MINGZHEN ZHANG等: "Edible Ginger-derived Nano-lipids Loaded with Doxorubicin as a Novel Drug-delivery Approach for Colon Cancer Therapy" * |
| NANXI CHEN等: "Edible plant-derived nanotherapeutics and nanocarriers: recent progress and future directions" * |
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