CN114470236B - Composite liposome for improving brain cognitive memory and neuroinflammation and preparation method thereof - Google Patents
Composite liposome for improving brain cognitive memory and neuroinflammation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a composite liposome for improving brain cognitive memory and neuroinflammation and a preparation method thereof, belonging to the technical field of liposome preparation. The liposome capable of simultaneously loading the hydrophobic substance curcumin and the hydrophilic substance epigallocatechin gallate (EGCG) is prepared, and on the basis, lactoferrin is utilized to carry out surface modification, and a hyaluronic acid further stabilizes a system through electrostatic interaction. The composite liposome obtained by the method has good stability and free radical scavenging efficiency, can obviously inhibit lipopolysaccharide-induced cognitive dysfunction, and is used for improving cerebral cognitive memory and neuroinflammation.
Description
Technical Field
The invention relates to the technical field of liposome preparation, in particular to a composite liposome for improving brain cognitive memory and neuroinflammation and a preparation method thereof.
Background
As human lifestyle changes, neurological diseases have become a threat to human health. Neuroinflammation is a critical pathological event that triggers and maintains neurodegenerative processes associated with many neurological diseases. Research has shown that natural ingredients in foods have great potential in alleviating neurological disorders.
Curcumin is hydrophobic polyphenol extracted from rhizome of Curcuma longa, has biological activity and pharmacological activity such as oxidation resistance and anti-inflammatory, and can reduce occurrence and development of Alzheimer disease by inhibiting NF- κB binding with amyloid plaque. Epigallocatechin gallate (EGCG) is a water-soluble polyphenol, has good antioxidant activity, and can scavenge free radicals in vivo. Both curcumin and EGCG show resistance to iron-induced toxicity, and have shown synergy in health promotion, but curcumin and EGCG have lower stability and higher pharmacokinetic differences, thereby impairing their efficacy.
Disclosure of Invention
In order to improve the bioavailability and the bioactivity of the polyphenol curcumin and the EGCG with different polarities, the invention uses lactoferrin and hyaluronic acid to modify liposome and carries out co-embedding on the curcumin and the EGCG. The liposome prepared by the invention has the characteristics of strong antioxidant capacity, can improve mitochondrial dysfunction of cells, reduce generation of ROS in cells, relieve synaptic dysfunction and neuronal damage, inhibit activation of microglial cells, and improve lipopolysaccharide-induced neuroinflammation and related cognitive dysfunction.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a composite liposome for improving brain cognitive memory and neuroinflammation, which comprises a drug-loaded liposome and a composite liposome with an outer layer modified lactoferrin and hyaluronic acid, wherein the drug-loaded liposome is a liposome in which curcumin and epigallocatechin gallate (EGCG) are co-embedded.
The invention provides a preparation method of a composite liposome for improving brain cognitive memory and neuroinflammation, which comprises the following steps:
dissolving phospholipid, cholesterol, tween 80 and curcumin in a mixed solvent, fully dissolving, transferring to a round bottom flask, evaporating the solvent by using a rotary evaporator, adding EGCG water solution into the sample, dispersing the system by using an ultrasonic cell disruption instrument after rotary evaporation to obtain a uniformly dispersed liposome solution, filtering, packaging by using nitrogen, sequentially adding lactoferrin solution and hyaluronic acid solution dropwise into the liposome solution, and stirring to fully react to obtain the composite liposome.
Further, the preparation method specifically comprises the following steps:
step 1, dissolving phospholipid (preferably yolk phospholipid), cholesterol, tween 80 and curcumin in a mixed solution of organic solvent chloroform and ethanol (volume ratio of the two is 2:1) by adopting a film ultrasonic method.
Step 2, transferring the mixed solution obtained in the step 1 into a round bottom flask, evaporating the solvent by using a rotary evaporator, placing the sample into a vacuum drying oven overnight, and thoroughly evaporating the organic reagent.
And 3, adding the EGCG aqueous solution into the sample obtained in the step 2, and steaming the hydrated sample.
And 4, placing the solution obtained in the step 3 in an ultrasonic cell disruption instrument to disperse the system, so as to obtain a uniformly dispersed liposome solution.
Step 5, filtering with a filter membrane (0.22 μm) to remove unencapsulated polyphenols (curcumin and EGCG), processing in dark place, and packaging with nitrogen.
And 6, weighing the lactoferrin solution, dissolving the lactoferrin solution in distilled water, and performing ultrasonic treatment to fully dissolve the lactoferrin to obtain the lactoferrin solution.
And 7, dropwise adding the lactoferrin solution obtained in the step 6 into the liposome solution, and stirring in a magnetic stirrer to fully react.
And 8, weighing the hyaluronic acid solution, dissolving the hyaluronic acid solution in distilled water, and completely dissolving the hyaluronic acid solution.
And 9, dropwise adding the hyaluronic acid solution obtained in the step 8 into the liposome solution modified by the lactoferrin obtained in the step 7, and stirring in a magnetic stirrer to fully react, wherein the finally prepared liposome is a composite liposome.
Further, the mass ratio of curcumin to ECGC in the liposome solution is 1:1, a step of; the mass ratio of the phospholipid to the cholesterol is 2:1-18:1, a step of; the mass ratio of the phospholipid to the tween 80 is 10:1-10:3, a step of; the mass ratio of phospholipids to the mixture of curcumin and ECGC was 14:1-6:1.
further, the concentration of the lactoferrin solution is 15mg/mL, the adding amount is 0-0.9mL/10mL of liposome, and the adding amount is not 0; the concentration of the hyaluronic acid is 1mg/mL, and the addition amount is 0.3-0.5mL/10mL of liposome.
Further, the ultrasonic time is 5-20min, preferably 15min.
Further, the mixed solvent is a mixed solvent of chloroform and ethanol, and the volume ratio of the chloroform to the ethanol is 2:1.
further, the spin steaming time is 30-50min.
Further, the whole process is protected from light.
In the preparation method of the invention, the preferable scheme is as follows: the mass ratio of the yolk phospholipids to the cholesterol is 6:1; the mass ratio of the yolk phospholipids to the tween 80 is 10:3; the mass ratio of yolk phospholipids and polyphenols (curcumin and EGCG) is 10:1; the hydration time of the preparation of the composite liposome is 40min; preparing liposome with the addition amount of lactoferrin of 0.3mL/10mL in the composite liposome; the added amount of hyaluronic acid in the preparation of the composite liposome is 0.5mL/10 mL.
The average particle size of the liposome prepared by the invention is 131.53 +/-0.258 nm, is in a uniform sphere shape, is uniformly dispersed, and has higher DPPH free radical clearance rate compared with free curcumin and EGCG suspension.
The invention also provides application of the composite liposome for improving the brain cognitive memory and the neuroinflammation in preparing a medicament for improving the brain cognitive memory and the neuroinflammation.
Further, the complex liposome is used for preparing a medicament for inhibiting lipopolysaccharide-induced cognitive dysfunction.
The invention discloses the following technical effects:
the invention prepares liposome capable of loading hydrophobic substance curcumin and hydrophilic substance EGCG simultaneously, and based on the liposome, lactoferrin is utilized to carry out surface modification, and hyaluronic acid is adopted to further stabilize a system through electrostatic interaction. The composite liposome obtained by the method has good free radical scavenging efficiency, can obviously inhibit lipopolysaccharide-induced cognitive dysfunction, and is used for improving brain cognitive memory and neuroinflammation.
Compared with the mixed suspension of curcumin and EGCG, the composite liposome loaded with the curcumin and the EGCG has stronger DPPH free radical scavenging capability, can improve cell mitochondrial dysfunction, reduce the generation of intracellular ROS, relieve synaptic dysfunction and neuronal damage of mice, inhibit activation of microglial cells, and improve Lipopolysaccharide (LPS) -induced neuroinflammation and related cognitive dysfunction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a morphology diagram of different liposomes under a transmission electron microscope, wherein a is a blank liposome, B is a liposome loaded with curcumin and EGCG, and C is a liposome modified with lactoferrin and loaded with curcumin and EGCG; d is a hyaluronic acid stabilized lactoferrin modified curcumin and EGCG loaded liposome.
FIG. 2 shows DPPH radical scavenging for various samples. Results are expressed as Mean ± standard deviation (Mean ± SD, n=3).
FIG. 3 is a Y maze test result, wherein A is the total arm advance number; b is the success rate.
FIG. 4 is a graph of open field test results, wherein A is a representative trace; b is the total distance.
FIG. 5 shows the effect of complex liposomes on Lipopolysaccharide (LPS) -induced mouse encephalitis factor, wherein A is IL-1. Beta. MRNA expression level, B is TNF-. Alpha.mRNA expression level, C is COX-2 mRNA expression level, and D is iNOS mRNA expression level.
FIG. 6 is the effect of different interventions on BV2 microglial morphology (×200), arrows representing cell side shoots.
FIG. 7 is the effect of complex liposomes on LPS-induced reactive oxygen species levels in BV2 microglia.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
Preparation method of liposome loaded with curcumin and EGCG
Dissolving yolk phospholipid (4 mg/mL), cholesterol, tween 80 and curcumin (0.2 mg/mL) in a mixed solvent of chloroform and ethanol (the volume ratio of the two is 2:1) by adopting a film ultrasonic method, wherein the mass ratio of the phospholipid to the cholesterol is 6:1; the mass ratio of the phospholipid to the tween 80 is 10:3. The resulting solution was transferred to a round bottom flask, the solvent was evaporated to dryness using a rotary evaporator, the sample was placed in a vacuum oven overnight, and the organic reagent was completely evaporated off. Adding EGCG water solution (0.2 mg/mL) into the obtained sample, steaming for 40min, and placing the obtained solution in an ultrasonic cell disruption instrument for 15min to disperse the system to obtain uniformly dispersed liposome solution. Filtering with 0.22 μm membrane, removing unencapsulated polyphenols (curcumin and EGCG), processing in dark, and packaging with nitrogen to obtain liposome loaded with curcumin and EGCG.
The preparation method of the lactoferrin modified curcumin and EGCG loaded liposome comprises the following steps: weighing lactoferrin to dissolve in distilled water, performing ultrasonic treatment to fully dissolve the lactoferrin to obtain 15mg/mL of lactoferrin solution, dropwise adding 0.3mL of lactoferrin solution into liposome solution loaded with curcumin and EGCG, and placing under a magnetic stirrer to stir so as to fully mix the liposome solution, thereby obtaining the lactoferrin modified liposome loaded with curcumin and EGCG.
The preparation method of hyaluronic acid-stabilized lactoferrin-modified curcumin and EGCG-loaded liposome comprises the following steps: weighing hyaluronic acid, dissolving in distilled water, performing ultrasonic treatment to completely dissolve the hyaluronic acid to obtain 1mg/mL hyaluronic acid solution, dropwise adding 0.5mL hyaluronic acid solution into the lactoferrin modified liposome solution loaded with curcumin and EGCG, placing under a magnetic stirrer, stirring to fully mix the hyaluronic acid and the hyaluronic acid, wherein the finally prepared liposome is the hyaluronic acid-stabilized lactoferrin modified liposome loaded with curcumin and EGCG (namely the composite liposome).
The preparation method of the blank liposome comprises the following steps: the preparation method of the blank liposome is the same as that of the liposome loaded with curcumin and EGCG, except that no EGCG and curcumin are added.
The morphology diagram of different liposomes under a transmission electron microscope is shown in figure 1, wherein A is blank liposome, B is liposome loaded with curcumin and EGCG, and C is liposome modified by lactoferrin and loaded with curcumin and EGCG; d is a hyaluronic acid stabilized lactoferrin modified curcumin and EGCG loaded liposome.
As shown in FIG. 1, the composite liposome prepared in example 1 had an average particle diameter of 131.53.+ -. 0.258nm, and was uniformly spherical and uniformly dispersed.
The DPPH radical scavenging rate of the different samples is shown in fig. 2, and the results are expressed as mean±standard deviation (mean±sd, n=3), and as can be seen from fig. 2, the complex liposome prepared in example 1 has higher DPPH radical scavenging rate.
The prepared composite liposome is used for feeding mice, and then the mice behavioural experiment is carried out.
The improvement effect of the composite liposome on brain cognitive memory and neuroinflammation was examined as follows:
1. grouping and treatment of test animals
The 60C 57BL/6J male mice were randomly grouped after 2 weeks of adaptive feeding, and 10 (n=10/group) of each group were normal control group, LPS model group, curcumin and EGCG suspension group, blank liposome group, curcumin and EGCG loaded liposome group, lactoferrin and hyaluronic acid modified curcumin and EGCG loaded complex liposome group, respectively (example 1). Wherein, normal control mice were intraperitoneally injected with an equal volume of physiological saline, and the other mice were treated with 0.25mg/kg/d LPS for 9d. All test mice were kept under 12h light and 12h dark conditions and given free diet and drinking water.
2.Y maze test
The Y maze is a classical behavioral test for testing the working memory of mice, consisting of three equiangular arms (length: 35cm, width: 5cm, height 15cm, included angle between arms of 120 °). After LPS was injected intraperitoneally for 4 hours, the mice were placed in the very centers of the three equiangular arms and allowed to freely explore for a fixed period of time (8 min), at the same time, the course of action, total arm-in times and continuous arm-in times of each test animal were automatically recorded by the video analysis system, and the alternating percentages were calculated according to the formula:
alternating percentage (%) = number of consecutive entries into three different arms/(total number of entries-2) ×100%
3. Barns labyrinth test
And (3) an adaptation stage: the day before the start of the test, the mice were individually placed in an avoidance box (escape chamber) communicating with the target hole for 3min, and the mice were placed in a black start box in the center of the maze to restrict their movement for 3min.
(1) Training phase: the mice were placed in a black starter box in the center of the maze, their activity was restricted for 5s, and then the starter box was removed to allow the mice to find the escape room. The experimenter views through the camera of another room. If all the four limbs of one mouse enter the escape chamber, the escape is calculated once, and the mouse stays in the chamber for 30 seconds. Each mouse was observed for a maximum of 3min. If a mouse still cannot find the escape chamber during this training period, the mouse should be guided into the escape chamber and stay there for 30s. After each test, the feces of the mice are cleaned, the disk surface is wiped by alcohol, the peculiar smell is removed, and the disk is randomly rotated. From the second training, the upper disc is randomly rotated before each training, but the escape chamber is always fixed in the same direction. The purpose is to prevent the mouse from relying on smell rather than memory to locate the escape chamber. Each mouse was trained once daily for 3 days.
(2) Testing: the escape chamber was taken out, the animal was placed in a black box in the center of the maze for 5s, and then it was allowed to move freely on the upper tray. The number of times it reaches the target area and the number of times the error area is explored are recorded. A camera is arranged above the maze, and the whole process is recorded.
4. Open field test
Mice were placed in the center of an open box (30 cm. Times.30 cm) with all four sides being white, and the box was divided into 25 small squares (5X 5) on average. The mice were first left free to explore for 5 minutes, and then the movement trajectories of the mice were recorded by a video tracking system for five minutes, each of which was repeated three times.
RNA extraction and reverse transcription
Accurately weighing 0.7g of brain tissue, adding 1mL of a Biozol RNA extraction reagent, and extracting RNA according to steps. The concentration was measured and the RNA of brain tissue was defined as a uniform concentration. According to the instruction of mRNA reverse transcription kit of century company, the reagents are uniformly mixed, the reaction condition is designed for reverse transcription, after the reverse transcription is finished, the sample is cooled to room temperature, the sample is diluted 5 times by using enzyme-free water, and the final sample is stored in a refrigerator at-80 ℃ for standby.
6. Real-time quantitative PCR assay
And carrying out fluorescent quantitative detection on the sample according to the operation sequence of the real-time fluorescent quantitative PCR kit of century company. The reaction system and the program were designed as follows:
TABLE 1 qPCR reaction System and Programming
The primer sequences of the target genes of mouse origin are shown in Table 2. The fluorescence signal of the reference gene GAPDH is used as an internal standard, and 2 is adopted -△△Ct The relative expression level of the genes was calculated by the method.
TABLE 2 qPCR primer information
The intervention of the complex liposomes on LPS-induced BV2 microglial inflammatory response was examined as follows:
7. cell morphology observations
Will be in logarithmic growthPhase BV2 microglial cells were uniformly seeded in 6-well cell culture plates containing serum medium (1X 10) 5 ～5×10 5 Individual/well), placed in a cell incubator for culturing. After the cells in the 6-hole cell culture plate are attached, the old culture medium is sucked and removed, a serum-free culture medium containing free drugs, blank liposome and drug-loaded liposome is added, and the mixture is placed at 37 ℃ and 5% CO 2 Incubating for 4 hours in a constant temperature and humidity incubator. After the incubation, 1mL of sterile phosphate buffer was added and washed once, and the mixture was treated with LPS (1. Mu.g/mL) in serum-free medium for 12 hours. After the treatment, 1mL of sterile phosphate buffer was added and washed once, and morphological changes of BV2 cells were observed under natural light using an optical microscope.
8. Detection of cellular Mitochondrial Membrane Potential (MMP)
MMP was measured as described in Solebao JC-1 kit, using the relative ratio of red/green fluorescence to measure the rate of mitochondrial depolarization.
BV2 cells were seeded simultaneously in 6-well cell culture plates and 96-well cell culture plates. BV2 cells are subjected to the same drug stimulation treatment. After 12h of LPS treatment, the medium was aspirated, washed once with sterile phosphate buffer and 1mL of cell culture medium was added. JC-1 staining working fluid (0.5 mL JC-1 staining working fluid is added to 50-100 ten thousand cells, and 1mL of working fluid is needed for a 6-well plate generally) is added to the 6-well cell culture plate. Incubation was carried out for a further 20min after thorough mixing. After the incubation, the supernatant was aspirated and washed 2 times with buffer. 2mL of cell culture medium was added and observed under a fluorescence microscope.
9. Intracellular ROS content assay
Intracellular ROS were detected using DCFH-DA probe. The specific method comprises the following steps:
(1) Cell pretreatment: BV2 cells were seeded in 75mm X75 mm cell culture dishes prior to the assay, and medium was aspirated off after 12h LPS treatment was completed.
(2) Probe addition and incubation: adding a specific fluorescent dye DCFH-DA probe into a serum-free culture medium under a dark condition: 1, the method comprises the following steps: 1000 ratio in serum free medium dilution to final concentration of 10uM DCFH-DA. The old medium was replaced with the probe-containing medium (typically a 6-well plate was added to 1mL of probe-containing medium). The cells were incubated at 37℃for half an hour to one hour, and in order to bring the probes into sufficient contact with the cells, they were gently mixed with shaking at intervals of 3 to 5 minutes, and a clear green fluorescence was observed at the end of the incubation.
(3) Collecting cells: after the incubation, the medium was aspirated off, washed twice with serum-free medium or sterile phosphate buffer, and photographed under a fluorescence microscope.
(4) Active oxygen measurement: after photographing, the cells are gently blown down until the bottom of the culture dish is changed from semitransparent to transparent, and almost all cell layers are blown into serum-free culture medium or sterile PBS. The cell suspension is rapidly spotted into a 96-well plate, fluorescence intensity measurement is carried out by using a fluorescence enzyme-labeled instrument, the excitation wavelength is set to be 500nm, and the emission wavelength is set to be 525nm.
FIG. 3 is a graph showing the results of a Y maze test, wherein A is the total arm advance number; b is the success rate. FIG. 4 is a graph showing the results of open field experiments, wherein A is a representative trace; b is the total distance. It can be found that: compared with the LPS model group and the curcumin and EGCG suspension group, the composite liposome obviously improves the times of mice on target holes and the success rate in Y maze test, which proves that the composite liposome obviously improves the cognitive dysfunction of mice induced by systemic inflammation.
The real-time quantitative PCR measurement result shows that the composite liposome can improve LPS-induced mouse neuron injury, improve mRNA expression of neurotrophic factor BDNF, and mRNA expression of postsynaptic compact PSD95, prevent injury of postsynaptic compact of brain of the mouse, and improve synaptic disturbance of the mouse. Meanwhile, the compound lipid can obviously inhibit the overactivation of microglia and reduce the expression of mRNA of IBA-1 in the brain of mice. FIG. 5 shows the effect of the complex liposomes on LPS-induced inflammatory factors in the brain of mice, indicating that the complex liposomes inhibit mRNA expression of inflammatory-related factors such as iNOS, COX-2, IL-1. Beta. And TNF-alpha.
The effect of different sample interventions on BV2 microglial morphology (x 200) is shown in fig. 6, the arrow indicates the cell side branch, the effect of the complex liposomes on LPS-induced levels of reactive oxygen species in BV2 microglial cells is shown in fig. 7, and the detection results indicate: the composite liposome can obviously improve the cell morphology change of BV2 microglial cells induced by LPS, and has better effect than curcumin+EGCG suspension treatment.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (2)
1. The composite liposome for improving brain cognitive memory and neuroinflammation is characterized by comprising a drug-loaded liposome and lactoferrin and hyaluronic acid modified by outer layers, wherein the drug-loaded liposome is a liposome in which curcumin and epigallocatechin gallate are co-embedded;
the composite liposome is used for preparing medicines for inhibiting lipopolysaccharide-induced cognitive dysfunction;
the preparation method of the composite liposome for improving the cognitive memory and the neuroinflammation of the brain comprises the following steps:
dissolving phospholipid, cholesterol, tween 80 and curcumin in a mixed solvent, evaporating the solvent, adding an epigallocatechin gallate aqueous solution, performing ultrasonic dispersion after rotary evaporation to obtain a uniformly dispersed liposome solution, filtering, packaging by using nitrogen, sequentially adding a lactoferrin solution and a hyaluronic acid solution dropwise into the liposome solution, and stirring to fully react to obtain a composite liposome;
the mass ratio of curcumin to epigallocatechin gallate in the liposome solution is 1:1; the mass ratio of the phospholipid to the cholesterol is 6:1; the mass ratio of the phospholipid to the tween 80 is 10:3; the mass ratio of the phospholipid to the mixture of curcumin and epigallocatechin gallate is 10:1;
the concentration of the lactoferrin solution is 15mg/mL, and the addition amount is 0.3mL/10mL liposome; the concentration of the hyaluronic acid is 1mg/mL, and the addition amount of the liposome is 0.5mL/10 mL;
the ultrasonic time is 15min;
the mixed solvent is a mixed solvent of chloroform and ethanol, and the volume ratio of the chloroform to the ethanol is 2:1;
the spin steaming time is 40min;
the whole process is protected from light.
2. The use of the composite liposome for improving brain cognitive memory and neuroinflammation according to claim 1 in the preparation of a medicament for improving brain cognitive memory and neuroinflammation.
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