FRET fluorescent probe entrapped liposome and application thereof

Abstract

The invention provides a lipidosome entrapped with an FRET fluorescent probe and application of the lipidosome, the FRET fluorescent probe is DiO and / or DiI, and the molar ratio of the FRET fluorescent probe to soybean phospholipid in the lipidosome is 1: 200. According to the invention, the FRET technology which is widely used for intermolecular distance determination is ingeniously applied to liposome drug targeting ability evaluation, and the liposome cell permeability and the cell model integrity are comprehensively evaluated by encapsulating FRET fluorescent probes (DiO, DiI and DiO + DiI) in a liposome drug and combining with a constructed in-vitro cell model.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to liposomes encapsulating FRET fluorescent probes and applications thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is a tight structure composed of brain microvascular endothelial cells (BMVEC) and their intercellular tight junctions (TJ), capillary basement membranes, and pericytes, astrocytes, and narrow extracellular spaces embedded therein. As a physiological barrier, the BBB is selective for the entry of substances, can prevent most harmful substances from entering the brain, and maintain the stability of the brain's internal environment. But at the same time, the BBB also limits the entry of drugs into the brain to play a therapeutic role in brain diseases. CN113880913A discloses two dual-mediated brain-targeted liposomes PTB-SSL (the present invention uses RVGPR9-SSL to represent) and RVGP-R9-SSL co-modified by RVGP and R9. The structure of the PTB-modified liposomes obtained by molecular docking and coarse-grained computational simulation is studied at the atomic and molecular scale level. This technology has the advantages of high efficiency, low cost, and high accuracy. It can analyze the molecular interactions in the drug delivery system and provide key physicochemical information. However, whether PTB-SSL and RVGP-R9-SSL can effectively penetrate the BBB in actual application, and the integrity of the liposomes after crossing the BBB, will directly affect their concentration entering the brain and the form of the preparation that reaches the lesion site. Therefore, investigating the BBB permeability and integrity of PTB-SSL and RVGP-R9-SSL across the BBB is of great significance for evaluating their brain drug delivery effects.

[0003] The in vitro BBB models currently established mainly include the following four types: 1) BMVEC monolayer cell model; 2) BMVEC and astrocyte co-culture model; 3) BMVEC, astrocyte and pericyte co-culture model; 4) primary cultured brain microvascular endothelial cells and pericyte co-culture model. Since the barrier function of the BBB mainly comes from BMVEC cells, the monolayer BMVEC cell model has the barrier characteristics of the BBB and can meet the evaluation of the ability of brain-targeted drug delivery systems to cross the BBB. The first in vitro BBB model is a general model. The model has a simple structure and is easily interfered by external factors. The use of this model to evaluate the targeting ability of brain-targeted liposomes cannot truly simulate the in vivo situation and may produce a large deviation from the in vivo results. The second and third in vitro BBB models, although they are closer to the in vivo BBB in structure, are complex to construct in vitro, difficult to operate and costly. In addition, the trophic factors secreted by astrocytes in vitro can easily cause the BMVEC cells to grow in clusters, making it difficult to maintain a monolayer barrier structure. Therefore, they are not suitable for the evaluation of the ability of drug delivery systems to cross the BBB. The fourth in vitro BBB model requires co-culture of primary BMVEC cells and pericytes, but primary cells are difficult to separate and culture in vitro, so the use of this model is also limited. Based on this, how to combine these existing in vitro BBB models to design an accurate evaluation method for the brain targeting ability of these two dual-mediated brain-targeted liposomes RVGPR9-SSL and RVGP-R9-SSL is a difficulty that needs to be solved urgently. Summary of the invention

[0004] In response to the difficult problems pointed out in the background technology, the present invention provides liposomes encapsulating FRET fluorescent probes and applications thereof. By encapsulating the FRET pair (DiO+DiI) in the dual-mediated brain-targeted liposomes and utilizing the FRET effect, the BBB permeability and trans-BBB integrity of the brain-targeted liposomes can be accurately evaluated. This method provides a new idea for the subsequent evaluation of the brain drug delivery ability of brain-targeted liposome preparations.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0006] In a first aspect, the present invention provides liposomes encapsulating FRET fluorescent probes, wherein the FRET fluorescent probes are DiO and / or DiI, and the molar ratio of the FRET fluorescent probes to soybean lecithin in the liposomes is 1:200.

[0007] Preferably, the liposome is a brain-targeted liposome, preferably RVGPR9-SSL or RVGP-R9-SSL.

[0008] In a second aspect, the present invention provides a method for preparing the liposomes described in the first aspect, the preparation method comprising: mixing the substrate, the FRET fluorescent probe and a solvent according to the substrate ingredients of the liposomes, and then removing the solvent until the liposomes are formed.

[0009] Preferably, the solvent is selected from at least one of chloroform, acetone, methanol and ethanol.

[0010] Preferably, the liposome is RVGPR9-SSL encapsulating a FRET fluorescent probe, and the preparation method comprises: mixing soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGPR9 and the FRET fluorescent probe, and then removing the solvent until the liposome is formed; further preferably, the molar ratio of soybean lecithin, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-RVGPR9 is 100:50:6:2.

[0011] Preferably, the preparation method of the DSPE-PEG2000-RVGPR9 comprises:

[0012] Step 1, RVGPR9 polypeptide and DSPE-PEG2000-NHS are prepared into solutions respectively, and the DSPE-PEG2000-NHS solution is added to the RVGPR9 polypeptide solution, and DMAP is further added after the addition, and the reaction is performed under light shielding at room temperature;

[0013] Step 2, after the reaction is completed, dialysis purification is performed, the purified product is pre-frozen, and then freeze-dried to obtain the DSPE-PEG2000-RVGPR9;

[0014] Further preferably, in step 1, the molar ratio of DSPE-PEG2000-NHS to RVGPR9 polypeptide is 1:2; and / or, the reaction is carried out in the dark for 30 to 40 hours.

[0015] Preferably, the liposome is RVGP-R9-SSL encapsulating a FRET fluorescent probe, and the preparation method comprises: mixing soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGP, DSPE-PEG1000-R9 and the FRET fluorescent probe, and then removing the solvent until the liposome is formed; preferably, the molar ratio of soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGP and DSPE-PEG1000-R9 is: 95-105:45-55:1-3:1-3:3-5.

[0016] Preferably, the preparation method of the DSPE-PEG2000-RVGP comprises:

[0017] Step 1, RVGP polypeptide and DSPE-PEG2000-NHS are prepared into solutions respectively, and the pH of the RVGP polypeptide solution is adjusted to 7.4, and then the DSPE-PEG2000-NHS solution is added to the pH-adjusted RVGP polypeptide solution, and DMAP is further added after the addition, and the reaction is performed under light shielding at room temperature; preferably, the molar ratio of DSPE-PEG2000-NHS to RVGP polypeptide is 1:2; and / or, the reaction is performed under light shielding for 30 to 40 hours;

[0018] Step 2, after the reaction is completed, dialysis purification is performed, the purified product is pre-frozen, and then freeze-dried to obtain the DSPE-PEG2000-RVGP;

[0019] And / or, the preparation method of the DSPE-PEG1000-R9 comprises:

[0020] Step 1, prepare R9 polypeptide and DSPE-PEG1000-NHS into solutions respectively, and adjust the pH of the R9 polypeptide solution to 7.4, then add the DSPE-PEG1000-NHS solution to the R9 polypeptide solution after adjusting the pH, and then further add DMAP after the addition, and perform light-shielding reaction at room temperature; preferably, the molar ratio of DSPE-PEG1000-NHS to R9 polypeptide is 1:2; and / or, the reaction is performed in the dark for 30 to 40 hours;

[0021] Step 2: After the reaction is completed, dialysis purification is performed, and the purified product is pre-frozen and then freeze-dried to obtain the DSPE-PEG1000-R9.

[0022] In a third aspect, the present invention provides use of the liposome described in the first aspect in preparing a kit for evaluating drug targeting ability, wherein the kit is based on FRET technology for evaluating drug targeting ability.

[0023] In a fourth aspect, the present invention provides a drug targeting capability evaluation kit, comprising: the liposome and cell model described in the first aspect.

[0024] In a fifth aspect, the present invention provides a method for evaluating drug targeting ability, which uses the kit described in the fourth aspect, and the evaluation method comprises:

[0025] Constructing cell models;

[0026] administering the liposome to the cell model;

[0027] FRET technology was used to detect the liposome permeability and integrity of the cell model after drug administration.

[0028] Preferably, the liposome is RVGPR9-SSL or RVGP-R9-SSL encapsulating a FRET fluorescent probe.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention cleverly applies the FRET technology widely used in intermolecular distance determination to the evaluation of liposome drug targeting ability. By encapsulating FRET fluorescent probes (DiO, DiI, DiO+DiI) in liposome drugs and combining the constructed in vitro cell model, the liposome cell permeability and cell model integrity are comprehensively evaluated. Taking RVGPR9-SSL and RVGP-R9-SSL, two new brain-targeted liposomes, as examples, the present invention combines the monolayer BMVEC cell model and uses FRET technology to accurately evaluate the BBB permeability and integrity of these two brain-targeted liposomes across the BBB, providing a new idea for the evaluation of the targeted drug delivery ability of subsequent liposome drug preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0032] in:

[0033] Figure 1 The tight junctions between BMVEC cells observed by SEM in Example 1 of the present invention (×1500).

[0034] Figure 2 The SEM images before and after liposome incubation in Example 3 of the present invention, wherein, Figure a shows the BBB morphology before liposome incubation; Figure b shows the BBB morphology after RVGPR9-SSL liposome incubation for 4 hours; Figure c shows the BBB morphology after RVGP-R9-SSL liposome incubation for 4 hours. The red arrows point to the tight junctions between BMVEC cells (×1500).

[0035] Figure 3 This is the standard curve of RVGP-R9-SSL (DiO) in Example 4 of the present invention.

[0036] Figure 4 This is the standard curve of RVGP-R9-SSL (DiI) in Example 4 of the present invention.

[0037] Figure 5 This is the standard curve of RVGP-R9-SSL (DiO+DiI) in Example 4 of the present invention.

[0038] Figure 6 This is the standard curve of RVGPR9-SSL (DiO) in Example 4 of the present invention.

[0039] Figure 7 This is the standard curve of RVGPR9-SSL (DiI) in Example 4 of the present invention.

[0040] Figure 8 This is the standard curve of RVGPR9-SSL((DiO+DiI) in Example 4 of the present invention.

[0041] Fig. 9 This is the cumulative permeability curve of the liposomes across the in vitro BBB model in Example 4 of the present invention.

[0042] Fig.10 This is a laser confocal image of the liposome stock solution encapsulating the fluorescent probe in Example 5 of the present invention.

[0043] Fig.11 This is a laser confocal image of the liposomes encapsulating the fluorescent probe after transport across the BBB in Example 5 of the present invention.

[0044] Fig.12 These are the fluorescence intensity values ​​of the original solution dilution and transwell receptor liquid of the fluorescent probe-encapsulated liposome in Example 5 of the present invention after uptake in the cells, wherein Figure a corresponds to RVGPR9-SSL; Figure b corresponds to RVGP-R9-SSL.

[0045] Fig.13 These are the fluorescence spectra of the two dual-mediated liposomes crossing the BBB after the receptor pool samples in Example 5 of the present invention, wherein Figure a corresponds to RVGP-R9-SSL; Figure b corresponds to RVGPR9-SSL. DETAILED DESCRIPTION

[0046] In the embodiment of the present invention, DiO is 3,3′-DiOctadecyloxacarbocyanine perchlorate)(Anaspec inc., USA); DiI is 1,1′-DiOctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)(Anaspec inc., USA).

[0047] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0048] In the embodiment of the present invention, the BBB permeability and BBB integrity of brain-targeted liposomes are evaluated by fluorescence resonance energy transfer (FRET) technology. FRET refers to when the emission spectrum of a fluorescent molecule (fluorescence donor) overlaps with the absorption spectrum of another fluorescent molecule (fluorescence acceptor), and the distance between the two fluorescent molecules is less than 10nm, then when the donor is excited by the incident light, its energy can be transferred to the acceptor molecule in a non-radiative manner through dipole-dipole coupling, and the donor molecule decays to the ground state without emitting fluorescence, and the acceptor molecule transitions from the ground state to the excited state, and then decays to the ground state while emitting fluorescence, and the donor fluorescence disappears and the acceptor fluorescence is enhanced. Its energy transfer efficiency is closely related to the distance between fluorescent molecules. In the embodiment, fluorescent probes DiO (fluorescence donor, λex428 nm, λem504 nm) and DiI (fluorescence acceptor, λex549 nm, λem565 nm) are selected as FRET pairs, DiO and DiI are co-encapsulated in liposomes, and the integrity of the liposomes after crossing the BBB barrier is investigated by the FRET effect. If the distance between DiO and DiI encapsulated in the liposomes is still less than 10 nm after the liposomes cross the blood-brain barrier in a complete form, then the FRET effect still exists; if the liposomes rupture during the process of crossing the blood-brain barrier, the fluorescent probes DiO and DiI encapsulated in the liposomes leak out of the liposomes, so that the distance between the two is greater than 10 nm, and the FRET phenomenon disappears. The present invention evaluates the integrity of the liposomes and the ligand during the trans-BBB transport of the liposomes by measuring the BBB permeability of the two probes and the FRET pair, and uses fluorescence spectrum scanning and laser confocal microscopy to observe and evaluate the trans-BBB transport form of the liposomes.

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0050] In the following examples, the preparation methods of DSPE-PEG2000-RVGPR9, DSPE-PEG2000-RVGP, and DSPE-PEG2000-R9 are as follows:

[0051] (1) Preparation process of DSPE-PEG2000-RVGPR9: RVGPR9 peptide and DSPE-PEG2000-NHS were dissolved in anhydrous dimethyl sulfoxide (DMSO) respectively. 200 μL of triethylamine reagent was added to the RVGP peptide solution to adjust the pH of the reaction solution to 7.4, and the DSPE-PEG2000-NHS solution was slowly and evenly added to the RVGPR9 peptide solution (DSPE-PEG2000-NHS:RVGPR9) = 1:2, mol / mol). Then, an appropriate amount of catalyst DMAP was added, and the reaction mixture was placed on a magnetic stirrer and reacted at room temperature in the dark for 36 hours. After the reaction was completed, the reaction mixture was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in 1000mL deionized water for 36 hours, and the dialysis external solution was changed every 6 hours. In order to remove the reactants and solvents that were not completely reacted. After the dialysis, the dialyzate was pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample was freeze-dried at -40°C and 0.22 mbar for 48 hours to obtain a white loose solid sample DSPE-PEG2000-RVGPR9.

[0052] (2) Preparation process of DSPE-PEG2000-RVGP: RVGP polypeptide and DSPE-PEG2000-NHS were dissolved in anhydrous dimethyl sulfoxide (DMSO) respectively. 200 μL of triethylamine reagent was added to the RVGP polypeptide solution to adjust the pH of the reaction solution to 7.4, and then the DSPE-PEG2000-NHS solution was slowly and evenly added to the RVGP polypeptide solution (DSPE-PEG2000-NHS:RVGP=1:2, mol / mol). Then, an appropriate amount of catalyst DMAP was added, and the reaction mixture was placed on a magnetic stirrer and reacted at room temperature for 36 hours in a dark environment. After the reaction was completed, the reaction mixture was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in 1000mL deionized water for 36 hours, and the dialysis external solution was changed every 6 hours. In order to remove the reactants, triethylamine and solvent that were not completely reacted. After the dialysis is completed, the dialyzate is pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample is placed at -40°C and 0.22 mbar for freeze-drying for 48 hours to obtain a white loose solid sample.

[0053] (3) Preparation process of DSPE-PEG1000-R9: R9 polypeptide and PEGylated active phospholipid (DSPE-PEG1000-NHS) were dissolved in anhydrous dimethylformamide (DMF) respectively, and 200 μL of triethylamine reagent was added to the above R9 polypeptide solution to adjust the pH of the reaction solution to 7.4. Then, the DSPE-PEG1000-NHS solution was slowly and evenly added to the R9 solution (DSPE-PEG1000-NHS:R9=1:2, mol / mol), and then an appropriate amount of catalyst DMAP was added. The reaction mixture was placed on a magnetic stirrer and reacted at room temperature in the dark for 36 hours. After the reaction was completed, the reaction mixture was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in 1000mL deionized water for 36 hours, and the dialysis external solution was changed every 6 hours. In order to remove the reactants, triethylamine and solvent that were not completely reacted. After the dialysis is completed, the dialyzate is pre-frozen in a -20°C refrigerator for 12 hours, and then the pre-frozen sample is placed at -40°C and 0.22 mbar for freeze-drying for 48 hours to obtain a white loose solid sample.

[0054] Example 1

[0055] The establishment of the in vitro BBB model is as follows:

[0056] The BMVEC in the culture flask that was fused to 90% were digested and evenly perfused with BMVEC-specific culture medium to form a single cell suspension, and the cells were counted. Then, the BMVEC cells were plated at 1×10 5 The density of cells / well was inoculated into the Transwell donor pool, and the volume of the cell suspension added to the donor pool was 1.5mL. Then 2mL of BMVEC-specific culture medium was added to the Transwell recipient pool, and then the Transwell culture plate was transferred to a 5% CO2, 37°C incubator for culture. Culture in the incubator for 3-7 days until the BBB model was established. During the culture process, the culture medium was replaced every 2 days to obtain an in vitro BBB model.

[0057] Evaluation of in vitro BBB models:

[0058] 1) 4h leakage test: The Transwell culture plate cultured for 3-7 days was placed under a microscope for observation. After the cells were fused, the BMVEC-specific culture medium was added to the donor pool of the Transwell culture plate, so that the liquid level of the donor pool of the Transwell culture plate was 5mm higher than the liquid level of the receiving pool. The Transwell culture plate was then placed in a 5% CO2, 37°C incubator for 4h, after which the Transwell culture plate was taken out to observe whether the liquid level difference between the donor pool and the receiving pool could be maintained. The test results showed that the liquid level difference between the donor pool and the receiving pool could be maintained at about 0.5cm, and three groups of parallel tests were performed.

[0059] 2) TEER measurement: Place the Transwell culture plate cultured for 3-7 days under a microscope for observation. After cell fusion, use an EVOM resistance meter to measure the resistance between the Transwell donor pool and the receiver pool, i.e., TEER. If the TEER is greater than 200Ω·cm 2 , indicating that the BBB model was successfully established. The test results showed that the TEER was greater than 200Ω·cm 2 , three groups of parallel experiments were conducted.

[0060] 3) SEM observation of tight junctions between cells: After 4h leakage test and TEER identification, the Transwell culture plate was taken out, the culture medium of the donor pool was discarded, and it was washed 3 times with sterile PBS buffer, and then 2% glutaraldehyde fixative was added to fix at 4℃ for 2h, 2% glutaraldehyde was discarded, and sterile PBS was added to wash 3 times, and then 1% kaolin fixative was added to fix at 4℃ for 2h, 1% kaolin was discarded, and sterile PBS buffer was added to wash 3 times, 50% ethanol was added to dehydrate for 10min, and then 70% ethanol was added to dehydrate for 10min, and then gold was sprayed, and the tight junctions between cells on the polycarbonate membrane of the Transwell donor pool were observed by SEM to further determine the fusion state of BMVEC cells and evaluate whether the in vitro BBB model was successfully established. The results are shown in Figure 1 As shown, by observing the cell state through SEM, it can be seen that the BMVEC cells are densely arranged and there are obvious tight junctions between cells, indicating that the in vitro BBB model was successfully established and can be used to determine the BBB permeability of each group of liposomes.

[0061] Example 2

[0062] The preparation and encapsulation efficiency of brain-targeted liposomes encapsulating FRET fluorescent pairs are as follows:

[0063] 2.1 Preparation of liposomes loaded with fluorescent probes, including:

[0064] 1) RVGPR9-SSL liposomes loaded with fluorescent probe: accurately weigh a certain amount of soybean phospholipids (SPC), cholesterol (Chol), DSPE-PEG2000, DSPE-PEG2000-RVGPR9 and an appropriate amount of DiO (or DiI or DiO+DiI (1:1)) and add them to an eggplant-shaped bottle in a certain proportion, SPC:Chol:DSPE-PEG2000:DSPE-PEG2000-RVGPR9=100:50:6:2 mol / mol; DiO (DiI, DiO+DiI):SPC=1:200 mol / mol, then add an appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, spin dry the organic solvent in a constant temperature water bath at 37°C until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, continue to spin dry for 30 minutes, remove the residual organic solvent, and obtain RVGPR9-SSL liposomes loaded with fluorescent probe. When determining the encapsulation efficiency, the liposomes encapsulating the fluorescent probe RVGPR9-SSL are re-dissolved. The solvent for the re-dissolution may be physiological saline, deionized water or other solvents. After the re-dissolution, the final concentration of SPC is 2 mg / mL.

[0065] 2) Encapsulation of fluorescent probe RVGP-R9-SSL liposomes: Accurately weigh a certain amount of SPC, Chol, DSPE-PEG2000, DSPE-PEG2000-RVGP, DSPE-PEG1000-R9 and an appropriate amount of DiO (DiI or DiO+DiI (1:1)) and add them into an eggplant-shaped bottle in a certain ratio, SPC:Chol:DSPE-PEG2000:DSPE-PEG2000-RVGPR9:DSPE-PE G1000-R9=100:50:2:2:4mol / mol;DiO(DiI, DiO+DiI):SPC=1:200mol / mol, then add appropriate amount of chloroform to dissolve, place on a vortex oscillator to oscillate and mix evenly, place on a rotary evaporator, spin dry the organic solvent in a 37°C constant temperature water bath until a uniform lipid film is formed at the bottom of the eggplant-shaped bottle, continue to spin dry for 30 minutes, remove the residual organic solvent, and obtain the RVGPR9-SSL liposomes containing the fluorescent probe. When determining the encapsulation efficiency, the RVGP-R9-SSL liposomes containing the fluorescent probe are re-dissolved, and the re-dissolving solvent can be selected from physiological saline, deionized water or other solvents, and the final concentration of SPC after re-dissolution is 2mg / mL.

[0066] 2.2 Investigation of the encapsulation efficiency of liposomes encapsulating fluorescent probes: Sephadex G-50 gel columns (2cm×22cm) were used to separate liposomes from free DiO or DiI, and then the encapsulation efficiency of the above-mentioned RVGPR9-SSL and RVGP-R9-SSL was determined. Take 0.5mL of the above-mentioned reconstituted sample of liposomes encapsulating fluorescent probes (deionized water as solvent) and load it onto a Sephadex G-50 gel column. The fluorescence intensity of the probe in the liposome part and the original preparation was measured at the corresponding wavelengths. DiO excitation wavelength (λex) 482nm, emission wavelength (λem) 504nm; DiI excitation wavelength (λex) 549nm, emission wavelength (λem) 565nm; DiO+DiI excitation wavelength (λex) 482nm, emission wavelength (λem) 565nm. The encapsulation efficiency calculation formula is:

[0067]

[0068] Among them, C liposomes and C0 are the concentration of the fluorescent probe encapsulated in the liposomes collected by column separation and the total concentration of the fluorescent probe in the liposome stock solution. The encapsulation efficiency results are shown in Table 1.

[0069] Table 1 Encapsulation efficiency of liposomes carrying fluorescent probes (n=3)

[0070]

[0071]

[0072] The results in Table 1 show that the liposome encapsulation efficiency is higher than 95%. According to the t-test, there is no significant difference in the encapsulation efficiency among the groups, indicating that the content of the fluorescent probe encapsulated in each liposome preparation is consistent, which can be used for comparative study of the trans-BBB permeability of each group of liposomes.

[0073] Example 3

[0074] The integrity of the BBB was investigated before and after liposome translocation across the BBB, as follows:

[0075] The in vitro BBB model was constructed by the method of Example 1, the culture medium in each well was discarded, and RVGPR9-SSL (DiO), RVGPR9-SSL (DiI), RVGPR9-SSL (DiO + DiI), RVGP-R9-SSL (DiO), RVGP-R9-SSL (DiI), RVGP-R9-SSL (DiO + DiI) diluted ten times with phenol red-free DMEM medium were added, and the drug concentration was set according to SPC of 0.2 mg / mL, and placed in a 5% CO2, 37°C incubator for 4 hours. The Transwell culture plate was taken out, the drug solution in the supply pool was discarded, and it was washed 3 times with sterile PBS buffer. The TEER value of each well was measured again according to the method in Example 1, and the tight junctions between cells were observed by SEM to determine the integrity of the in vitro BBB after 4 hours of incubation and drug administration.

[0076] SEM images of liposomes in each group before and after transport across the BBB Figure 2 As shown in the figure, the BBB structure was intact before liposome incubation, BMVEC cells were densely arranged, and there were obvious tight junctions between cells. After 4 hours of incubation with RVGPR9-SSL and RVGP-R9-SSL, the in vitro BBB model still maintained good integrity, and there were obvious tight junctions between cells, indicating that the 4-hour incubation of the two targeted liposomes did not destroy the integrity of the BBB model.

[0077] The TEER values ​​of the BBB model before and after incubation with liposomes loaded with fluorescent probes are shown in Table 2:

[0078] Table 2 TEER values ​​of BBB before and after liposome incubation (n=3)

[0079]

[0080]

[0081] The data in Table 2 show that the TEER values ​​of each well of the Transwell culture plate were greater than 200 Ω·cm before incubation with the liposome preparation. 2 This indicates that the in vitro BBB model was successfully established and can be used to investigate BBB permeability. After 4 h of incubation with the liposome preparation, the TEER value of each well was still greater than 200 Ω·cm 2 , indicating that 4 h incubation did not destroy the structure of the BBB, and the in vitro BBB model always maintained a relatively intact structure during the incubation process.

[0082] Example 4

[0083] BBB permeability determination, as follows:

[0084] 4.1 Preparation of liposome standard curve: Take appropriate amount of liposomes prepared in Example 2 and prepare standard solutions with fluorescent probe concentrations of 0, 0.875, 1.75, 3.5, 7, 14, 28, 56, and 112 ng / mL respectively with deionized water. Use a fluorescence spectrophotometer to measure the fluorescence intensity at: DiO excitation wavelength (λex) 482nm, emission wavelength (λem) 504nm; DiI excitation wavelength (λex) 549nm, emission wavelength (λem) 565nm, DiO+DiI excitation wavelength (λex) 482nm, emission wavelength (λem) 565nm, each concentration is measured in parallel 3 times, and the average value is calculated. Linear regression is performed on the concentration C of the liposome solution using the fluorescence intensity (I) of DiO, DiI, and DiO+DiI to draw a standard curve, as shown in FIG. Figures 3 to 8 As shown in the figure, after linear fitting, the liposome preparations encapsulating the fluorescent probe have good linearity in the range of 0.875-112 ng / mL and can be used for subsequent quantitative experiments. For different liposomes encapsulating the same probe, there is no obvious difference in the molar absorption coefficient (proportional coefficient of the linear equation). Since there is no significant difference in the encapsulation rate of each liposome, it can be seen that the fluorescence intensity of the probe is independent of the liposome. From the molar absorption coefficients of the preparations encapsulating different probes, it can be seen that: DiO>DiO+DiI>DiI.

[0085] 4.2 BBB permeability determination: An in vitro BBB model was established according to the method of Example 1. Appropriate amounts of RVGPR9-SSL and RVGP-R9-SSL encapsulated with DiO, DiI and co-encapsulated with DiO+DiI were sterilized by membrane sterilization, and then diluted tenfold with DMEM basal medium without phenol red to an SPC concentration of 0.2 mg / mL. The original culture medium in the donor pool of the BBB model was discarded, and then 1.5 mL of the above-prepared liposome solution was added to the donor pool, and 2 mL of basal medium without phenol red was added to the recipient pool, so that the liquid surface of the Transwell donor pool and the recipient pool were level, eliminating the effect of the static pressure caused by the liquid surface difference on the permeability. The Transwell culture plate was placed in a 5% CO2, 37°C constant temperature incubator for culture, and 2 mL of samples were taken from the recipient pool at 0, 0.5, 1, 2, and 4 h after administration, and 2 mL of DMEM medium without phenol red was added to the recipient pool. The fluorescence intensity (I) of each sample was measured using a fluorescence spectrophotometer. According to the standard curve prepared in 4.1, the concentration was calculated. The cumulative permeability Pe calculation formula is as follows:

[0086]

[0087] Among them, C tis the concentration of fluorescent probes such as DiO in the receiving pool at time t; C0 is the initial concentration of fluorescent probes such as DiO in the dosing pool; Vdonor is the sample volume in the dosing pool, i.e. 1.5 mL; V is the sample volume in the receiving pool, i.e. 2 mL; Ci represents the sample concentration measured at time point i, i is calculated in hours.

[0088] The BBB permeability curves of RVGPR9-SSL and RVGP-R9-SSL loaded with different fluorescent probes are shown in Figure 2. Fig. 9 As shown. It can be seen from the figure: whether in the RVGPR9-SSL group or the RVGP-R9-SSL group, the BBB permeability of the FRET pair DiO+DiI encapsulated liposomes is consistent with that of the liposomes encapsulating single probe DiO or DiI, indicating that the transport of fluorescent probes across the BBB is mainly completed by liposomes. During the trans-BBB transport, the liposomes did not rupture and crossed the BBB in a complete form. The 4h cumulative BBB permeability of RVGP-R9-SSL was 7.9%, while the 4h cumulative BBB permeability of RVGPR9-SSL was 10.3%, which was 1.3 times higher than that of the RVGP-R9-SSL group. This shows that the ligand modification method will affect the efficiency of liposomes crossing the BBB. For the dual-mediated liposomes with two different modification methods, the single-ligand liposome RVGPR9-SSL has a stronger ability to transport across the BBB.

[0089] Example 5

[0090] The integrity of RVGPR9-SSL and RVGP-R9-SSL across the BBB was evaluated as follows:

[0091] 5.1 Observation of FRET effect by laser confocal microscopy: After completing the BBB permeability experiment, collect 2 mL of each Transwell receptor pool sample (i.e., the sample obtained in the Transwell receptor pool) after incubation for 4 hours for later use. Take another 2 mL of the liposome stock solution containing each fluorescent probe. Dilute the Transwell receptor pool sample and the liposome stock solution with phenol red-free DMEM culture medium to the same fluorescence intensity for later use. Digest the BMVEC cells that have been fused to 90% into a single cell suspension, and count the cells. 1×10 4 / well density was inoculated in a laser confocal dish and placed in a 5% CO2, 37℃ incubator for 24h. After the cells adhered to the wall and grew stably, they were taken out. The original culture medium was discarded, and sterile PBS buffer was added to wash 3 times. The above-mentioned pool sample diluted to the same fluorescence intensity with phenol red-free DMEM culture medium and 1mL of liposome stock solution were added respectively, and placed in a 5% CO2, 37℃ incubator for 2h. After the culture was completed, the culture medium was discarded, and sterile PBS buffer was added to wash three times, and tissue cell fixative was added to fix for 15min, and then washed 3 times with sterile PBS buffer, Hoechst33342 was added to label the cell nucleus for 10min, and sterile PBS was washed 3 times, and glycerol: PBS (9:1) mounting medium was added to mount the slices. The FRET phenomenon was observed using a laser confocal microscope. Wavelength selection: DiO excitation wavelength (λex) 482nm, emission wavelength (λem) 504nm; DiI excitation wavelength (λex) 549nm, emission wavelength (λem) 565nm. The excitation wavelength (λex) of DiO+DiI is 482nm, and the emission wavelength (λem) is 565nm.

[0092] After the original solution of RVGPR9-SSL and RVGP-R9-SSL containing fluorescent probes was diluted to an appropriate concentration, the results of laser confocal microscopy observation of BMVEC cell uptake were as follows: Fig.10 As shown in the figure, it can be seen that the two liposomes encapsulating DiO show clear green fluorescence after being taken into BMVEC cells, the liposomes encapsulating DiI show red fluorescence after being taken into cells, and the liposomes mixed with two fluorescent probes show enhanced red fluorescence and almost disappearance of green fluorescence after being taken into cells, which is the FRET phenomenon. This shows that when the two fluorescent probes are co-encapsulated in the liposomes, the distance between the fluorescent probes is less than 10nm, and there is a FRET effect. After the BBB permeability experiment was completed, the Transwell receptor pool samples were collected after incubation for 4 hours, diluted to an appropriate concentration (consistent with the fluorescence intensity of the above-mentioned liposome preparation stock solution), and the BMVEC cell uptake experiment was performed. The results of laser confocal microscopy observation are shown as follows Fig.11 As shown, the liposome group mixed with two fluorescent probes still has the FRET effect, indicating that the transport across the BBB model for 4 h did not destroy the structure of the liposome.

[0093] Will Fig.10 and Fig.11 After the laser confocal images were processed by LASX software (version 3.2, Leica, Germany), the intracellular fluorescence intensity values ​​of each group were as follows: Fig.12As shown in the figure: (1) Whether before or after crossing the BBB, the fluorescence intensity of the carrier containing dual probes is significantly higher than that of the liposome containing single probes, and the fluorescence intensity is the same, indicating that there is no leakage of fluorescent probes after the two dual-mediated liposomes cross the BBB, and the integrity of the liposomes is still maintained. (2) Regardless of whether they encapsulate a single fluorescent probe or dual fluorescent probes, the fluorescence intensity of the two dual-mediated liposomes (RVGPR9-SSL and RVGP-R9-SSL) in the receptor fluid after crossing the BBB is consistent with the original solution dilution, indicating that the ligands in the two dual-mediated liposomes still maintain integrity after crossing the BBB.

[0094] 5.2 Fluorescence spectrum scanning experiment: After completing the BBB permeability experiment, collect the Transwell pool samples after incubation for 4 hours, and perform a fluorescence spectrum scanning experiment using a fluorescence photometer to observe whether the liposomes mixed with two fluorescent probes still have the FRET phenomenon, so as to examine the integrity of the liposomes after crossing the BBB.

[0095] After the BBB permeability experiment was completed, the Transwell receptor pool samples were collected and the fluorescence spectrum was scanned at the corresponding excitation wavelength using a fluorescence spectrophotometer. The results are as follows: Fig.13 As shown: Compared with the liposomes encapsulating a single probe, the two dual-mediated liposomes co-encapsulating DiO+DiI have a FRET effect, that is, the fluorescence of the donor DiO almost disappears, and the fluorescence of the acceptor DiI is enhanced, showing the same properties as the original solution of the liposome preparation. This shows that during the trans-BBB model transport of the two dual-mediated liposomes, the liposome structure remains intact, and no leakage of the fluorescent probe occurs. Both RVGPR9-SSL and RVGP-R9-SSL achieve trans-BBB transport with intact structures.

[0096] In summary, the present invention cleverly applies the technical advantages of FRET to evaluate the brain targeting ability of the dual-mediated liposome drug delivery system, provides a clear formulation form for the subsequent entry of the two liposomes, RVGPR9-SSL and RVGP-R9-SSL, into the glioma area to exert a therapeutic effect, and provides guidance for further application of dual-mediated liposomes in the treatment of brain gliomas.

[0097] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A liposome encapsulating a FRET fluorescent probe, characterized in that: The FRET fluorescent probe is DiO and / or DiI, and the molar ratio of the FRET fluorescent probe to the soybean lecithin in the liposome is 1:

200.

2. The liposome according to claim 1, characterized in that The liposome is a brain-targeted liposome, preferably RVGPR9-SSL or RVGP-R9-SSL.

3. The method for preparing liposomes according to claim 1 or 2, characterized in that: The preparation method comprises: mixing the substrate, the FRET fluorescent probe and a solvent according to the substrate ingredients of the liposome, and then removing the solvent until the liposome is formed.

4. The preparation method according to claim 3, characterized in that: The solvent is selected from at least one of chloroform, acetone, methanol and ethanol.

5. The preparation method according to claim 3 or 4, characterized in that: The liposome is RVGPR9-SSL encapsulating a FRET fluorescent probe, and the preparation method comprises: mixing soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGPR9 and the FRET fluorescent probe, and then removing the solvent until the liposome is formed; preferably, the molar ratio of soybean lecithin, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-RVGPR9 is 100:50:6:

2.

6. The preparation method according to claim 5, characterized in that: The preparation method of the DSPE-PEG2000-RVGPR9 comprises: Step 1, RVGPR9 polypeptide and DSPE-PEG2000-NHS are prepared into solutions respectively, and the DSPE-PEG2000-NHS solution is added to the RVGPR9 polypeptide solution, and DMAP is further added after the addition, and the reaction is performed under light shielding at room temperature; Step 2, after the reaction is completed, dialysis purification is performed, the purified product is pre-frozen, and then freeze-dried to obtain the DSPE-PEG2000-RVGPR9; Preferably, in step 1, the molar ratio of DSPE-PEG2000-NHS to RVGPR9 polypeptide is 1:2; and / or, the reaction is carried out in the dark for 30 to 40 hours.

7. The preparation method according to claim 3 or 4, characterized in that: The liposome is RVGP-R9-SSL encapsulating a FRET fluorescent probe, and the preparation method comprises: mixing soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGP, DSPE-PEG1000-R9 and the FRET fluorescent probe, and then removing the solvent until the liposome is formed; preferably, the molar ratio of soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVGP and DSPE-PEG1000-R9 is: 95-105: 45-55: 1-3: 1-3: 3-5. .

8. The preparation method according to claim 7, characterized in that: The preparation method of the DSPE-PEG2000-RVGP comprises: Step 1, RVGP polypeptide and DSPE-PEG2000-NHS are prepared into solutions respectively, and the pH of the RVGP polypeptide solution is adjusted to 7.4, and then the DSPE-PEG2000-NHS solution is added to the pH-adjusted RVGP polypeptide solution, and DMAP is further added after the addition, and the reaction is performed under light shielding at room temperature; preferably, the molar ratio of DSPE-PEG2000-NHS to RVGP polypeptide is 1:2; and / or, the reaction is performed under light shielding for 30 to 40 hours; Step 2, after the reaction is completed, dialysis purification is performed, the purified product is pre-frozen, and then freeze-dried to obtain the DSPE-PEG2000-RVGP; And / or, the preparation method of the DSPE-PEG1000-R9 comprises: Step 1, prepare R9 polypeptide and DSPE-PEG1000-NHS into solutions respectively, and adjust the pH of the R9 polypeptide solution to 7.4, then add the DSPE-PEG1000-NHS solution to the R9 polypeptide solution after adjusting the pH, and then further add DMAP after the addition, and perform light-shielding reaction at room temperature; preferably, the molar ratio of DSPE-PEG1000-NHS to R9 polypeptide is 1:2; and / or, the reaction is performed in the dark for 30 to 40 hours; Step 2: After the reaction is completed, dialysis purification is performed, and the purified product is pre-frozen and then freeze-dried to obtain the DSPE-PEG1000-R9.

9. Use of the liposome according to claim 1 or 2 in preparing a kit for evaluating drug targeting ability, wherein the kit is based on FRET technology for evaluating drug targeting ability.

10. A drug targeting capability evaluation kit, characterized in that: include: The liposome and cell model according to claim 1 or 2.

11. A method for evaluating drug targeting ability, characterized in that: The kit according to claim 10 is used, wherein the evaluation method comprises: Constructing cell models; administering the liposome to the cell model; FRET technology was used to detect the liposome permeability and integrity of the cell model after drug administration.

12. The evaluation method according to claim 11, characterized in that: The liposome is RVGPR9-SSL or RVGP-R9-SSL encapsulating a FRET fluorescent probe.

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