CN110693838A

CN110693838A - Preparation of folic acid modified tetrapeptide YGLF-loaded core-shell type nanoliposome

Info

Publication number: CN110693838A
Application number: CN201911173336.7A
Authority: CN
Inventors: 杨剑; 宋相容; 赵胜楠
Original assignee: Shenzhen Polytechnic
Current assignee: Shenzhen Polytechnic
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2020-01-17

Abstract

A preparation method of a folic acid modified core-shell nano-liposome carrying tetrapeptide YGLF relates to folic acid modified cholesterol carrying antihypertensive peptide YGLF. Dissolving YGLF in a buffer solution with the pH of 4-9 to serve as a water phase; the oil phase is a mixed phase of dichloromethane and methanol, YGLF is placed into the oil phase for ultrasonic treatment, and the water phase enters the oil phase in an ice bath to obtain a primary emulsion liquid; mixing emulsifier as external water phase solution with oil phase, stirring and ultrasonically treating to form emulsion, mixing the initial emulsion, and stirring to obtain milky emulsion; mixing phospholipid as lipid membrane material, cholesterol and FA-PEG cholesterol, dissolving in organic solvent chloroform, reducing pressure, and rotary evaporating to remove organic solvent to obtain yellowish membrane; and mixing the film with the milky emulsion, hydrating at high temperature for demoulding, and homogenizing or extruding after ultrasonic treatment to obtain the core-shell type nano liposome carrying the tetrapeptide YGLF modified by the folic acid.

Description

Preparation of folic acid modified tetrapeptide YGLF-loaded core-shell type nanoliposome

Technical Field

The invention relates to folic acid modified cholesterol loaded antihypertensive peptide YGLF, in particular to a preparation method of core-shell type nano-liposome taking folic acid as a target head and encapsulating tetrapeptide YGLF.

Background

In recent years, proteins and polypeptides have been successively found to have pharmacological activity and thus have received a great deal of attention from scientists, among which a number of fragments of peptides have been demonstrated to have hypotensive activity. The peptides are found from natural products and can also be prepared by recombinant DNA technology, so that the peptides are safer and open a new field for treating hypertension. Hypertension is most clinically administered orally to increase patient compliance. However, these active peptides capable of lowering blood pressure are easily degraded and inactivated by gastrointestinal enzymes when prepared into oral preparations; the permeability is poor, and the mucus layer on the inner wall of the small intestine cannot be smoothly and efficiently passed; can not be specifically identified and absorbed by the epithelial cells of the small intestine through the tight connection among the epithelial cells of the small intestine; and short half-life in blood circulation, etc. Therefore, there is an urgent need to develop a novel antihypertensive peptide preparation to solve the above problems and to improve bioavailability. The leaf modification modified core-shell nanoliposome (nanoliposome) is used for loading antihypertensive peptide YF4(FA-YF4-LNPs), can overcome the severe environment of gastrointestinal tracts and play a role in protecting the gastrointestinal tracts, can be specifically absorbed by folate receptors on small intestine epithelial cells, greatly maintains the physiological characteristics of YF4 by virtue of a slow release function, and improves the stability of the YF 4.

The document reports that the folic acid modified nano preparation loaded with insulin can increase the oral bioavailability of insulin and enhance the hypoglycemic effect of insulin. The folic acid ligand is modified on the surface of the liposome to prepare the folic acid modified nanoliposome, so that the absorption efficiency of small intestinal epithelial cells to the nanoliposome can be improved, and the polypeptide is effectively protected from the degradation of gastrointestinal tracts, thereby improving the blood concentration and bioavailability of the polypeptide.

Disclosure of Invention

The invention aims to provide a preparation method of a core-shell type nano liposome carrying tetrapeptide YGLF modified by folic acid.

The invention comprises the following steps:

1) dissolving YGLF in a buffer solution with the pH of 4-9 to serve as a water phase; the oil phase is a mixed phase of dichloromethane and methanol, YGLF is placed into the oil phase for ultrasonic treatment, and the water phase enters the oil phase in an ice bath to obtain a primary emulsion liquid;

2) mixing emulsifier as external water phase solution with the oil phase in the step 1), stirring and ultrasonically treating to form emulsion, mixing with the primary emulsion obtained in the step 1), and stirring to obtain milky emulsion;

3) mixing phospholipid as lipid membrane material, cholesterol and FA-PEG cholesterol, dissolving in organic solvent chloroform, reducing pressure, and rotary evaporating to remove organic solvent to obtain yellowish membrane;

4) mixing the film obtained in the step 3) with the milky white emulsion obtained in the step 2), hydrating at high temperature for demoulding, and homogenizing or extruding after ultrasonic treatment to obtain the folic acid modified tetrapeptide YGLF-loaded core-shell type nanoliposome.

In step 1), the time of the ultrasound may be 90 s; the obtained colostrum-like liquid can be water solution.

In the step 2), the emulsifier can adopt a PVA emulsifier, and the content of the emulsifier can be 1% by mass; the time of stirring and ultrasonic treatment can be 40-50 s; the obtained emulsion is W/O/W type emulsion, and the volume ratio of primary emulsion to external water phase in the emulsion can be 1: 3.

In step 3), the lipid membrane material may consist of cholesterol, soy lecithin, FA-pegylated cholesterol; the mass ratio of the cholesterol to the soybean lecithin can be 2: 1; the proportion of the added FA-PEGylated cholesterol can be 1% of the mixture of the cholesterol and the soybean lecithin; the reduced pressure conditions may be at 50rmp for 1 h.

In the step 4), the solution for hydration and demoulding can adopt the solution obtained in the step 2); the mass ratio of the milky white emulsion to the film can be 1.5: 1; the power for homogenizing or extruding may be 100W.

The invention adopts lipid membrane modified nano particles, and solves the complex problems of poor cell affinity of nano particles and low stability of liposome. Due to the excellent drug encapsulation efficiency and slow release behavior, the bioavailability can be effectively enhanced, and the half-life period of the peptide can be prolonged, so that the oral bioavailability of the polypeptide can be improved.

The invention takes PLGA as an inner core and soybean lecithin, cholesterol and FA cholesterol as wall materials, prepares a core-shell nano liposome which adopts folic acid as a target head and entraps tetrapeptide YGLF by adopting a thin film hydration method, and analyzes the morphological structure and stability of the nano liposome, the in vitro cell uptake, the in vivo blood pressure lowering condition and the in vivo pharmacokinetic experiment of SD rats, and the evaluation of the in vivo intestinal absorption kinetics and the safety.

Drawings

FIG. 1 is a particle size diagram of an example of the present invention.

FIG. 2 is a Zeta potential diagram of an embodiment of the present invention.

FIG. 3 is a Tyndall effect diagram of an embodiment of the present invention.

FIG. 4 is a transmission electron microscope image of an embodiment of the invention.

FIG. 5 is a stability diagram of an embodiment of the present invention.

FIG. 6 is a graph of in vitro release of an embodiment of the present invention.

FIG. 7 is a differential thermal scan of an embodiment of the present invention.

Fig. 8 is an uptake plot on Caco-2 cells for an example of the invention data are expressed as mean ± SD (n ═ 3) · ＊ P <0.05, P < 0.01, P < 0.001, and LNPS/NPS.

Fig. 9 is an uptake plot on HT-29 cells for an example of the invention data are expressed as mean ± SD (n ═ 3) · ＊ P <0.05, × P < 0.01, × P < 0.001 and LNPS/NPS.

FIG. 10 is a graph showing the drug duration of 0-72 h in SD rats according to the present invention.

FIG. 11 is a graph showing the drug duration of 0-12 h in SD rats according to the present invention.

FIG. 12 is a chart of the antihypertensive effect of the present invention on SHR rats.

FIG. 13 is a safety evaluation of examples of the present invention on SD rats.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings.

1. Preparation of folic acid ligand modified tetrapeptide YGLF-loaded core-shell type nanoliposome

Preparing YF4-NPs multiple emulsion by adopting an emulsifying solvent volatilization method: taking a proper volume of YF4 solution as an internal water phase (W1) and a proper concentration of a methylene dichloride methanol mixed solution of PLGA as an oil phase (O), and quickly adding the internal water phase into the PLGA oilIn phase, under ice-water bath, the cell disruptor probe is subjected to ultrasonic treatment to obtain W₁The first emulsion of/O; then W is₁The first emulsion is slowly added into the external water phase solution (W)₂) In, ultrasonic obtaining of W by probe under ice-water bath₁/O/W₂Compounding milk; and (3) carrying out reduced pressure rotary evaporation (75rpm) to remove the organic solvent under the condition of 37 ℃ water bath, thus obtaining a colloidal solution, namely YF 4-NPs.

Based on YF4-NPs emulsion, YF4-NPs are coated with lipid material by using a thin film hydration-ultrasonic dispersion method to prepare nanoliposomes (FA-FA-YF 4-LNPs). Firstly preparing a phospholipid membrane by a thin film method, then hydrating the phospholipid membrane by using YF4-NPs colloidal solution prepared by the optimal process, and finally crushing and dispersing by using ultrasound to obtain YF4 nanoliposome (FA-YF 4-LNPs). The specific operation is as follows: weighing soybean lecithin, cholesterol (Chol) and FA cholesterol in appropriate amount, adding chloroform to dissolve the materials completely, mixing, performing rotary evaporation at 37 deg.C under reduced pressure to form film (50rpm, 1h), and slowly removing organic solvent to obtain a uniform film; then 3ml of prepared YF4-NPs were added and hydrated at 60 deg.C (75rpm) for 0.5 h. And (4) carrying out ultrasonic treatment on the sample after hydration is finished under the condition of ice-water bath for 3s and stopping the ultrasonic treatment for 3 s. The obtained colloidal solution is FA-YF 4-LNPs.

2. Determination of embedding rate of folic acid modified YGLF nanoliposome

2.1 drawing of YGLF Standard Curve

Drawing a YF4 standard curve by using high performance liquid chromatography, which comprises the following steps: YF4 stock solution with the concentration of 1mg/mL is precisely prepared. The stock solutions were diluted with an appropriate amount of purified water to give a series of standard solutions of 1, 25, 50, 100, 200, 300, 400, and 500. mu.g/mL, and the peak areas were recorded by passing through a 0.22 μm filter and measuring as follows. And performing linear regression by taking the concentration as an abscissa and the peak area as an ordinate. The HPLC chromatographic conditions for YF4 detection were as follows: a chromatographic column: (

Symmetry C18 chromatography column, 250 × 4.6mm, 4 μm); mobile phase: 0.1% aqueous trifluoroacetic acid: acetonitrile (70:30, v/v); flow rate: 1 mL/min; detection wavelength: 210 nm; column temperature: 30 ℃; sample introduction amount: 20 μ L.

2.2 determination of the embedding rate and drug-loading rate of Folic acid modified YGLF nanoliposome

Separating the unencapsulated drug and the nanoliposome by adopting an ultrafiltration centrifugation method (the molecular weight cutoff is 30000Da) to determine the encapsulation rate of FA-YF 4-LNPs. The supernatant was centrifuged at 13000rpm for 10min, 200. mu.L of the separated supernatant was measured by the method under "2.2 chromatographic conditions", the amount of unencapsulated YF4 was measured, and the encapsulation efficiency and drug loading of FA-YF4-LNPs were calculated according to the following formula.

3. Characterization of Nanoliposomes

3.1 measurement of Liposomal particle size and potentiometric assay

The particle size and potential of YF 4-loaded nano preparation were determined by Malvern laser particle sizer. A certain volume of colloidal solution is measured, the colloidal solution is diluted by 10 times by purified water and then transferred to a particle size cup for particle size measurement, and the potential is directly measured without dilution. The measurement temperature was set at 25 ℃ and the instrument was equilibrated for 2min before measurement, 3 replicates per sample.

3.2 appearance morphology of FA-YF4-LNPs

And (3) placing YF4-NPs and FA-YF4-LNPs colloidal solution prepared by proper purified water and an optimal process in a penicillin bottle, observing the appearance of the solution by naked eyes, and irradiating by using a laser pen to observe whether the Tyndall phenomenon exists.

3.3 microscopic morphology Transmission Electron microscopy of FA-YF4-LNPs

Diluting colloidal solution of FA-YF4-LNPs, YF4-NPs and common LPs (the preparation process is the same as that of LNPs) prepared by an optimal process by 1 time, dripping the colloidal solution onto a copper net coated with a Formavar membrane, settling at room temperature for 45s, dyeing with 2% phosphotungstic acid dye solution for 35s, carefully sucking the dye solution on the edge of the copper net by using filter paper, naturally drying at room temperature, placing the copper net on an observation handle of a transmission electron microscope instrument, and observing the shape of the copper net.

3.4 FA-YF4-LNPs stability Studies

It was stored in a refrigerator at 4 ℃ in the dark. And (3) taking the particle size and the encapsulation rate as comprehensive evaluation indexes, and inspecting the stability of the FA-YF 4-LNPs.

3.5 FA-YF4-LNPs in vitro Release study

The in vitro release profile of FA-YF4-LNPs was examined using a dynamic dialysis method. 2mL of free YF4, YF4-NPs and FA-YF4-LNPs were taken and placed in dialysis bags (Mw is 3500Da), and then the dialysis bags were placed in 40mL of buffer solutions (pH1.0, pH4.5, pH6.8 and pH7.4) with different pH, shaken in a constant temperature shaker at 37 ℃ at a rotation speed of 100rpm, and 1mL was sampled at predetermined time points (0, 0.5, 1, 2, 4, 6, 8, 12, 14, 16, 18h) while adding equal amounts of isothermal fresh release medium. After centrifuging the sample at 13000rpm for 10min, the supernatant was measured according to the method under the item "2.2 chromatographic conditions" to calculate the concentration of YF4, and the cumulative release percentage was calculated according to the following formula,

Release％＝Q_n/W×100％

in the formula Q_n: cumulative release at each time point; w: the total drug amount; % Release: cumulative percent release at each time point; c_n: actual drug concentration measured at the nth sampling time point; c_i: the actual drug concentration measured at the ith sampling time point; v₀: total dissolution medium volume; v_i: the volume was sampled.

In vitro release profiles of FA-YF4-LNPs were plotted using time as the abscissa and cumulative release rate as the ordinate.

3.6 Differential Scanning Calorimetry (DSC)

And (4) carrying out freeze-drying treatment on the prepared blank nano preparation and the prepared drug-loaded nano preparation, and preparing for differential scanning calorimetry. And (3) taking a proper amount of the mechanical mixture of the YF4, the blank folic acid modified nanoliposomes (B-LNPs), the YF4 nanoliposomes (FA-YF4-LNPs) and the YF4 and blank modified nanoliposomes (the proportion of the two is the same as that of the FA-YF4-LNPs) after freeze-drying, placing the mixture in an aluminum crucible, covering the crucible, placing the crucible in a differential scanning calorimetry sample cell, and carrying out thermal analysis and determination. And observing the existence state of YF4 in FA-YF4-LNPs, and verifying whether YF4 is successfully encapsulated in the FA-YF 4-LNPs. The condition parameters are set as follows: the nitrogen is used as a protective gas, the flow rate is 20mL/min, the temperature rise speed is 10 ℃/min, and the temperature range is 50-250 ℃.

3.7 in vitro cell assay

3.7.1 cells

Human colon cancer cells Caco-2 and HT-29 were obtained from Sichuan biotherapy national focus laboratories and cultured in DMEM containing 10% fetal calf serum and 1640 containing 10% fetal calf serum, respectively.

3.7.2 cellular uptake protocol

Caco-2 cells and HT29 cells were inoculated into a culture dish at a certain density, and placed at 37 ℃ in 5% CO₂Culturing in incubator, and changing liquid once every other day. When the degree of cell confluence reached about 80% to 90%, cell passaging was performed. Sucking out original cell culture solution, washing for 2 times by using serum-free culture medium, adding 2mL of 0.25% trypsin-0.02% EDTA mixed digestive solution, gently swirling to ensure that cells are fully digested, then adding 4mL of culture medium to stop digestion, gently blowing, sucking cell suspension into a centrifuge tube, centrifuging at 1000rpm for 3min, discarding supernatant, adding 2mL of culture medium into the centrifuge tube, blowing, suspending, and sucking cell suspension according to a certain proportion into a flat dish for passage.

Cou-6 preparation of labeled NPs: cou-6 stock solution and 20mg PLGA are dissolved in 1.6mL dichloromethane to be used as an organic phase (O), 33 muL pure water is used as an internal aqueous phase (W1), and after the internal aqueous phase is rapidly added into the organic phase, the ice bath is carried out for 90s by 100W probe ultrasound to form colostrum; slowly dripping the prepared primary emulsion into 3mL of 1% PVA, and carrying out ultrasonic treatment for 45s by a 100W probe in an ice bath to form multiple emulsion. And transferring the multiple emulsion into a round-bottom flask, and performing reduced pressure rotary evaporation to remove the organic solvent under the condition of 37 ℃ water bath to obtain a colloidal solution, namely Cou-6-labeled NPs.

Cou-6 preparation of labeled FA-LNPs: firstly, preparing a folic acid modified phospholipid membrane, then hydrating the phospholipid membrane by using the Cou-6 labeled NPs colloidal solution, and finally homogenizing by using a homogenizer to obtain folic acid modified Cou-6 nanoliposome (FA-LNPs). The specific operation is as follows: weighing soybean phospholipid, Chol and appropriate amount of folic acid cholesterol, placing in 250mL eggplant-shaped bottle, adding chloroform to dissolve the materials completely, mixing, performing rotary evaporation at 37 deg.C under reduced pressure to form film (50rpm, 2h), removing organic solvent to obtain uniform film; then 3mL of prepared NPs were added and hydrated at 60 deg.C (100rpm) for 1 h. Homogenizing the hydrated sample in a homogenizer for 3s, stopping 3s, and homogenizing for 2 min. The obtained colloidal solution is LNPs.

Collecting pancreatin-digested Caco-2 cells at a cell concentration of 2 × 10⁴Density per well, inoculated into 24-well plates (sterile cell slide placed in advance), placed at 37 ℃ with 5% CO₂Culturing in an incubator. Changing the culture medium every other day, changing the culture medium every day after 1 week, culturing for 10-15 days, and changing fresh culture medium before 2h of ingestion test. Cou-6 labeled preparation was added at predetermined time points (0.5, 1, 2, 3h), and the plates were placed at 37 ℃ in 5% CO₂The incubator continues to culture. When the preset time point is reached, the following processing is carried out: sucking out the culture medium containing the medicine, and gently washing the culture medium once by PBS; adding 4% paraformaldehyde, fixing at room temperature for 15min, and washing with PBS; adding Hoechst 33258 (final concentration is 1ug/mL) to stain cell nucleus for 10min, and washing with PBS once; add 200. mu.L PBS. 5 mu L of fluorescent anti-quenching agent is dripped on the glass slide, and then the cell slide in the 24-hole plate is picked up by a needle head to cover the cell surface on the glass slide. The observation was performed under an upright fluorescence microscope. Meanwhile, another 24-well plate (without adding a slide) is operated in parallel, and after the cell uptake is finished, the cells are collected and detected by a flow cytometer.

HT-29 was cultured for 2 days and the procedure was the same.

3.8 in vivo pharmacokinetic experiments in SD rats

3.8.1 animal

SPF grade Sprague-Dawley rats (SD rats), 8-12 weeks old, body weight 200 + -20 g, total 18, male and female halves. Purchased from Duodda animal experiments Co., Ltd, and raised in animal center of tumor biotherapy laboratory of Sichuan university.

3.8.2 animal grouping and dosing regimens

Sprague-Dawley rats (SD rats, purchased from Dawley, Sichuan) were 24 in total, 8-12 weeks old, 200. + -.20 g in body weight, and male and female halves. The groups are randomly divided into Free YF4 group, YF4-NPs group, YF4-LNPs group and FA-YF4-LNPs group, wherein each group comprises six animals and half animals. SD rats are fasted for at least 12h before administration without water deprivation, and are fed 2h after administration. The dosage of the drug for intragastric administration is 1.2 mg/kg.

Healthy SD rats were taken and randomly divided into 4 groups of 6 rats each with half of males and females. Fasted the day before the experiment (about 12h), water was freely available. After weighing, Free YF4, YF4-NPs, YF4-LNPs and FA-YF4-LNPs are respectively administered by intragastric administration, the administration dosage is 1.2mg/kg calculated by YF4, the whole blood is collected in the orbit about 0.4mL after the administration and is placed in an EP tube containing a proper amount of heparin sodium, and the anesthesia is carried out in ether for 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h. The collected blood sample is immediately centrifuged at 4000rpm at 4 ℃ for 10min, and the supernatant plasma is taken out and placed in a 1.5mL EP tube to be stored at-20 ℃ for detection.

And (3) performing data statistical treatment by using GraphPad Prism 5 software, comparing the change before and after administration among groups by using repeated measurement variance analysis, and performing pairing t test on the change before and after administration in the groups, wherein the difference has statistical significance when P is less than 0.05.

3.9 in vivo antihypertensive drug effect test in SHR rats

3.9.1 animal

SPF grade SHR rats, 8-12 weeks old, 200 + -20 g weight, 24 in total, half male and female. Purchased from Duodda animal experiments Co., Ltd, and raised in animal center of tumor biotherapy laboratory of Sichuan university.

3.9.2 animal grouping and dosing regimens

The SHR rats are 24 in total, 8-12 weeks old, 200 +/-20 g in body weight and half male and female. The groups are randomly divided into Free YF4 group, YF4-NPs group, YF4-LNPs group and FA-YF4-LNPs group, wherein each group comprises six animals and half animals. The dosage of the gavage administration is 0.8 mg/kg.

Healthy SHR rats were selected and randomized into 3 groups of 6 rats each with half of males and females. After weighing, FreeYF4, YF4-NPs, YF4-LNPs and FA-YF4-LNPs are respectively intragastrically administered, the administration dosage is 0.8mg/kg calculated by YF4, and the blood pressure change is respectively measured and recorded at 0, 2, 4, 8, 12, 24, 48 and 72 hours after administration.

3.10 evaluation of in vivo safety in SD rats

3.10.1 animal

SPF grade Sprague-Dawley rats (SD rats), 8-12 weeks old, body weight 200 + -20 g, 24 total, male and female halves. Purchased from Duodda animal experiments Co., Ltd, and raised in animal center of tumor biotherapy laboratory of Sichuan university.

3.10.2 animal grouping and dosing regimens

Sprague-Dawley rats (SD rats, purchased from Dawley, Sichuan) were 24 in total, 8-12 weeks old, 200. + -.20 g in body weight, and male and female halves. The groups are randomly divided into Free YF4 group, YF4-NPs group, YF4-LNPs group and FA-YF4-LNPs group, wherein each group comprises six animals and half animals.

The low, medium and high doses (1, 10, 20mg/kg) were administered separately for 14 days. After the experiment, the patient is sacrificed and stained to observe pathological conditions.

4. Statistical analysis

Each test was repeated 3 times, with the results given

And (4) showing. Performing statistical analysis by using Graphpad Prism 5 and SPSS data processing software, when P is<The difference was considered significant at 0.05.

5. Characterization of core-shell type nanoliposome modified by folic acid ligand and loaded with tetrapeptide YGLF

5.1 measurement of Liposomal particle size and potentiometric assay

The results are shown in FIGS. 1 and 2. The most preferred FA-YF4-LNPs have an average particle size of 227.3 + -2.8 nm and a narrow size distribution (PDI ═ 0.09 + -0.01). The Zeta potential of FA-YF4-LNPs is-2.7 +/-0.1 mV.

5.2 appearance morphology of FA-YF4-LNPs

The results are shown in FIG. 3. It can be seen that, when observed under white light, the prepared nano preparation samples are clear and transparent, have light blue opalescence, have no visible foreign matters, and have good appearance. Under dark field, the laser irradiation has obvious Tyndall phenomenon.

5.3 microscopic morphology Transmission Electron microscopy of FA-YF4-LNPs

As a result, as shown in FIG. 4, the shape of the nanoparticle was spherical, while the liposome had a distinct lipid membrane structure due to the difference in the preparation process. As the results show, the size of the LNPs was 30 nm larger than the nanoparticles, and the shape of the LNPs appeared to be irregularly elliptical with significant shading.

5.4 FA-YF4-LNPs stability Studies

It was stored in a refrigerator at 4 ℃ in the dark. And (3) taking the particle size and the encapsulation rate as comprehensive evaluation indexes, and inspecting the stability of the FA-YF 4-LNPs. As shown in FIG. 5, after FA-YF4-LNPs are stored at 4 ℃ for 7 days, the encapsulation efficiency and the particle size of the FA-YF4-LNPs are not obviously changed; after being placed for 14 days, the encapsulation efficiency is obviously reduced, and the particle size is increased sharply. The above results suggest that FA-YF4-LNPs are stable at 4 ℃ for at least 7 days.

5.5 FA-YF4-LNPs in vitro Release study

The results are shown in FIG. 6. In release media of different pH, FA-YF4-LNPs did not show the burst release effect of free YF 4. The release behavior of FA-YF4-LNPs has a certain pH dependence, and the results show that almost all free YF4 is released at pH7.4 within 12 hours, more than half of YF4 is released from YF4-NPs, and only 42% of YF4 is released from FA-YF4-LNPs, indicating that the phospholipid layer can effectively slow down the drug release profile. The result indicates that YF4 is encapsulated in PLGA nanoparticles, YF4 can be effectively protected from being damaged by acid in the gastrointestinal tract, and release is delayed; after the liposome is further encapsulated in the lipid membrane, the release of the drug can be effectively retarded due to the protective effect of the lipid membrane, the drug is protected from being degraded, and the drug release time is obviously prolonged.

5.6 Differential Scanning Calorimetry (DSC)

From the DSC results FIG. 7, DSC curves of free YF4, blank LNPs, FA-YF4-LNPs and a mixture of free YF4 and blank LNPs were studied. Free YF4 exhibited a strong endothermic peak at 225.6 ℃, probably due to its degradation. However, FA-YF4-LNPs showed no endothermic peak at 225.6 ℃ and two broad endothermic peaks at 118.2 and 193.6 ℃ and the analysis should be the glass transition temperatures of both materials. Interestingly, the physical mixture showed three endothermic peaks, free YF4 and material, respectively. The results show that YF4 was successfully encapsulated in FA-YF 4-LNPs.

5.7 in vitro cell assay

As shown in fig. 8 and 9, all of free Cou-6, NPs, and LNPs exhibited a time-dependent behavior with progressively increasing absorption over 2 hours. The effect of increased cellular uptake of FA-LNPs was confirmed by quantitative analysis. The fluorescence intensity of the cells of the FA-LNPs group was stronger than that of the other groups. All data indicate that in both cell lines, FA-LNPs entered more cells than NPs, indicating that FA-LNPs had better small intestine targeting properties.

5.8 in vivo pharmacokinetics assay in SD rats

The pharmacokinetic profile of YF4 following oral administration to SD rats is shown in figures 10 and 11. The results show that YF4 exhibits a short half-life behavior, absorbing rapidly in the early phase. C after treatment of SD rats with FA-YF4-LNPs_{Maximum of}、AUC_0-72hAnd t_1/2The z value is respectively improved by more than 2.46 times with YF4-NPs treatment group rats. These results indicate that the bioavailability of FA-YF4-LNPs is improved after oral dosing in SD rats.

5.9 in vivo antihypertensive drug effect experiment of SHR rats

As shown in FIG. 12, after oral administration, the results showed that the antihypertensive effect was significantly enhanced and the drug effect was prolonged to 144 h.

5.10 in vivo safety evaluation experiment in SD rats

As shown in figure 13, after 14 days of continuous administration, the heart, liver, spleen, lung and kidney of the rat have no obvious lesion and are safer.

Claims

1. The preparation method of the core-shell type nanoliposome modified by folic acid and loaded with tetrapeptide YGLF is characterized by comprising the following steps:

2. The method for preparing the folic acid modified core-shell type nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in the step 1), the time of the ultrasound is 90 s; the obtained colostrum-like liquid is water solution.

3. The preparation method of the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in the step 2), the emulsifier is PVA emulsifier, and the content of the emulsifier is 1% by weight.

4. The preparation method of the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in the step 2), the time of the stirring ultrasound is 40-50 s.

5. The method for preparing the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in the step 2), the prepared emulsion is W/O/W type emulsion, and the volume ratio of colostrum volume to external water phase in the emulsion is 1: 3.

6. The method for preparing the folic acid modified core-shell type nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in step 3), the lipid membrane material is composed of cholesterol, soybean lecithin, FA-PEGylated cholesterol.

7. The preparation of the folate-modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 6, wherein the mass ratio of cholesterol to soybean lecithin is 2: 1; the proportion of FA-pegylated cholesterol added was 1% of the mixture of cholesterol and soya lecithin.

8. The method for preparing the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in step 3), the reduced pressure is applied by rotating at 50rmp for 1 h.

9. The method for preparing the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in step 4), the solution for hydration and membrane removal is the solution obtained in step 2).

10. The preparation of the folic acid modified core-shell nanoliposome carrying tetrapeptide YGLF of claim 1, wherein in step 4), the mass ratio of the milky white emulsion to the thin film is 1.5: 1; the power for homogenizing or extruding may be 100W.