CD47 modified pH sensitive liposome delivery system and preparation method and application thereof
By using CD47-modified pH-sensitive liposomes to bind CD47 mimic peptides to macrophage SIRPα receptors, the circulating half-life is extended and the drug is released in an acidic environment, solving the problems of difficulty in brain accumulation and low targeting of existing antiepileptic drugs, and achieving efficient drug delivery and therapeutic effects.
Patent Information
- Application Number
- CN202510851183.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-11-07
AI Technical Summary
Existing antiepileptic drugs are ineffective in 30% of patients. Most drugs do not address the pathological mechanisms such as neuroinflammation and oxidative stress. Liposomes are difficult to accumulate in the brain, have short circulating half-lives, and have low targeted release efficiency.
The pH-sensitive liposomes modified with CD47 contain soybean lecithin, cholesterol, dioleoylphosphatidylethanolamine, distearate phosphatidylethanolamine-polyethylene glycol 2000, icariin, and CD47 mimic peptides. By binding to the SIRPα receptor on macrophages through CD47 mimic peptides, the circulating half-life is prolonged, and the drug is released by pH-sensitive protonation to target the epileptogenic focus.
It improves the bioavailability and targeting of drugs in the brain, enhances the drug release efficiency at the epileptogenic focus, reduces side effects, and improves treatment efficacy.
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Figure CN120899641A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a CD47 modified pH-sensitive liposome delivery system and a preparation method and application thereof. BACKGROUND
[0002] At present, the treatment of epilepsy mainly depends on anti-seizure drugs, but about 30% of patients have no response to existing drugs, and most drugs only alleviate symptoms by inhibiting neuronal excitability, without intervention on pathological mechanisms such as neural inflammation and oxidative stress. Icaritin has anti-inflammatory, antioxidant and neuroprotective potential, but its bioavailability is low (only 12% after gavage) and its blood-brain barrier (BBB) penetration ability is limited (the apparent permeability coefficient is 1.5 x 10⁻ 6 cm / s), making it difficult to reach an effective concentration in the epileptogenic focus.
[0003] As a kind of nanocarrier, liposomes have been widely used in drug delivery systems due to their low toxicity, good biocompatibility and biodegradability, but the monocyte-macrophage system can still recognize the liposomes in the circulation and be quickly cleared by the reticuloendothelial system, resulting in a shortened circulation half-life of the liposomes and difficulty in accumulating in the brain. In addition, the liposomes also have the problem of low targeted release efficiency. SUMMARY
[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide a CD47 modified pH-sensitive liposome delivery system that can have long circulation, targeted controlled release and high blood-brain barrier penetration efficiency, as well as a preparation method and application thereof.
[0005] To solve the above technical problems, the technical solution provided by the present application is as follows: a CD47 modified pH-sensitive liposome delivery system, comprising: soybean lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icaritin and CD47 mimetic peptide.
[0006] In an embodiment, the mass ratio of the soybean lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icaritin and CD47 mimetic peptide is 4:6:8:5:4.5:2.
[0007] Based on the same inventive concept, a preparation method of the CD47 modified pH-sensitive liposome delivery system is also provided, comprising: dissolving soybean lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icariin and CD47 mimetic peptide in methanol according to a mass ratio of 4:6:8:5:4.5:2, fully dissolving and mixing, then performing rotary evaporation under reduced pressure to remove the organic solvent to form a lipid film, performing hydration reaction of the lipid film with a phosphate buffer, then performing ultrasonic treatment in an ice bath, and performing extrusion with a micro-extruder to obtain the product.
[0008] In an embodiment, the rotary evaporation under reduced pressure is performed at 40-50 DEG C for 10-15 min.
[0009] In an embodiment, the hydration reaction is performed at a temperature of 25-37 DEG C for 15-30 min.
[0010] Based on the same inventive concept, the CD47 modified pH-sensitive liposome delivery system is also provided for use in epilepsy.
[0011] Compared with the prior art, in the CD47 modified pH-sensitive liposome delivery system, the CD47 mimetic peptide is combined with the SIRP alpha receptor of macrophages to activate the "don't eat me" signal, reduce the recognition of the liposome by the monocyte-macrophage system (MPS), prolong the ICA circulation half-life, improve the bioavailability of ICA, and solve the problems of low ICA bioavailability (only 12% after gavage) and poor blood-brain barrier penetration ability (apparent permeability coefficient 1.5 x 10-7 cm / s), so that the ICA can reach an effective concentration in the epileptogenic focus to exert its anti-inflammatory and antioxidant potential. 6 The phosphatidyl ethanolamine head of DOPE is protonated in an acidic environment, leading to phase transition of the liposome membrane layer, triggering the disintegration of the liposome and the release of the drug, ensuring the specific release of the drug in the acidic microenvironment of the epileptogenic focus, and enhancing the targeting and release efficiency of the liposome. BRIEF DESCRIPTION OF DRAWINGS
[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.
[0013] Figure 1 It is an embodiment of the synthesis process of the CD47 modified pH-sensitive liposome delivery system; Figure 2Transmission electron microscope (TEM) image of ICA@LipD-CD47 of an embodiment; Figure 3 Particle size distribution diagram of ICA@LipD-CD47 of an embodiment; Figure 4 Zeta potential of LipD, ICA@LipD, ICA@LipD-CD47 at pH 7.4 of an embodiment; Figure 5 Fourier transform infrared spectroscopy (FTIR) of LipD, ICA, ICA@LipD-CD47 of an embodiment; Figure 6 Ultraviolet-visible spectroscopy (UV-Vis) of ICA, ICA@LipD-CD47 of an embodiment; Figure 7 Hydrodynamic diameter of ICA@LipD-CD47 on day 1, 3, 7 of an embodiment; Figure 8 Diagram showing the total amount of ICA leakage outside the dialysis bag on day 1, 3, 5, 7 and 14, respectively, measured by UV-Vis at 271 nm of an embodiment; Figure 9 ICA release amount of ICA@LipD-CD47 in PBS at pH 5.0, 6.5 and 7.4 of an embodiment; Figure 10 Diagram showing the amount of ICA, Lip, ICA@Lip, ICA@LipD and ICA@LipD-CD47 transferred from the upper chamber to the lower chamber of the BBB in vitro model constructed by the Transwell model; Figure 11 Fluorescence images of nude mice at 6h, 12h, 24h after administration of DiR-labeled liposomes of each formulation; Figure 12 Representative fluorescence images of main organs ex vivo and quantitative diagram of fluorescence intensity 24h after administration of each formulation; Figure 13 Fluorescence images of the brain of epileptic mice and quantitative fluorescence intensity diagram 24h after administration of PBS, ICA@Lip-CD47, ICA@LipD-CD47 of each group, wherein DiR-labeled liposomes were used; Figure 14 Representative immunofluorescence images of the hippocampus of epileptic mice 24h after administration; Figure 15 Percentage of M1 phenotype and M2 phenotype microglial cells in total microglial cells; Figure 16The schematic diagram of serum liver and kidney function indexes of the mice in each group after the mice were treated with PBS, Lip, ICA, ICA@Lip, ICA@LipD and ICA@LipD-CD47, respectively. DETAILED DESCRIPTION
[0014] In order to facilitate the understanding of the present application, the present application will be described in more detail and in a more complete, specific manner below in conjunction with the preferred embodiments and the accompanying drawings of the specification, but the scope of protection of the present application is not limited to the following specific embodiments.
[0015] Unless otherwise defined, all the professional terms used herein have the same meanings as generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing the specific embodiments and are not intended to limit the scope of protection of the present application.
[0016] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.
[0017] Please refer to Figures 1-16 The CD47 modified pH-sensitive liposome delivery system (ICA@LipD-CD47) of an embodiment comprises soybean phosphatidylcholine (SPC), cholesterol, dioleoyl phosphatidyl ethanolamine (DOPE), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), Icaritin (ICA) and CD47 mimic peptide (DSPE-PEG2000-CERVIGTGWVRC), and the mass ratio is 4:6:8:5:4.5:2.
[0018] Based on the same inventive concept, a preparation method of the CD47 modified pH-sensitive liposome delivery system is also provided, comprising: dissolving soybean phosphatidylcholine, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, Icaritin and CD47 mimic peptide in methanol according to a mass ratio of 4:6:8:5:4.5:2, fully dissolving and mixing, then performing rotary evaporation under reduced pressure to remove the organic solvent to form a lipid film, performing hydration reaction of the lipid film with a phosphate buffer, then performing ultrasonic treatment in an ice bath, and using a micro-extruder to extrude the product.
[0019] Specifically, in an embodiment, the rotary evaporation under reduced pressure is rotary evaporation under reduced pressure at 50°C for 15 min.
[0020] Specifically, in an embodiment, the preparation method of the CD47 modified pH-sensitive liposome delivery system, the hydration reaction is performed at a temperature of 37°C for 30 min.
[0021] Based on the same inventive concept, the application also provides a use of the CD47 modified pH-sensitive liposome delivery system in epilepsy.
[0022] Example 1: Preparation of ICA@LipD-CD47 ICA@LipD-CD47 was prepared by typical thin film dispersion method. SPC, cholesterol, DOPE, DSPE-MPEG2000, ICA and DSPE-PEG2000-CERVIGTGWVRC (CD47 mimetic peptide) were dissolved in 10 mL methanol, and the mass ratio of each component was 4:6:8:5:4.5:2. After thorough mixing, the organic solvent was removed by rotary evaporation at 40-50°C under reduced pressure for 10-15 min to form a thin film. The lipid film was hydrated with PBS at 25-37°C for 15-30 min, and then ultrasonicated in an ice bath for 10 min. Finally, the product was extruded by a micro-extruder and stored at 4°C. The synthesis process is shown in Figure 1
[0023] Comparative Example 1: Preparation of ICA@Lip ICA@Lip was prepared by typical thin film dispersion method. SPC, cholesterol, DSPE-MPEG2000, ICA were dissolved in 10 mL methanol, and the mass ratio of each component was 4:6:5:4.5. After thorough mixing, the organic solvent was removed by rotary evaporation at 40-50°C under reduced pressure for 10-15 min to form a thin film. The lipid film was hydrated with PBS at 25-37°C for 15-30 min, and then ultrasonicated in an ice bath for 10 min. Finally, the product was extruded by a micro-extruder and stored at 4°C.
[0024] Comparative Example 2: Preparation of ICA@LipD ICA@LipD was prepared by typical thin film dispersion method. SPC, cholesterol, DOPE, DSPE-MPEG2000, ICA were dissolved in 10 mL methanol, and the mass ratio of each component was 4:6:8:5:4.5. After thorough mixing, the organic solvent was removed by rotary evaporation at 40-50°C under reduced pressure for 10-15 min to form a thin film. The lipid film was hydrated with PBS at 25-37°C for 15-30 min, and then ultrasonicated in an ice bath for 10 min. Finally, the product was extruded by a micro-extruder and stored at 4°C.
[0025] Example 2: Characterization of ICA@LipD-CD47 The hydrodynamic diameter and Zeta potential of ICA@LipD-CD47 were measured by dynamic light scattering (DLS) using a Malvern nanoparticle size analyzer (Model: C-W10X10XB22) at room temperature. To further obtain the microscopic morphological characteristics, transmission electron microscopy (Talos F200X, Czech) was used to image and observe the samples. FTIR spectra were collected by a Fourier transform infrared spectrometer (Nicolet 6700). TEM and DLS analysis showed that ICA@LipD-CD47 presented a uniform spherical structure, with an average particle size of 134 ± 0.75 nm ( Figure 2 , 3 ), a Zeta potential of -38.3 ± 0.40 mV ( Figure 4 ), and a drug loading rate and encapsulation efficiency of 6.1% and 52%, respectively. FTIR spectral detection showed that ICA@LipD-CD47 exhibited characteristic C=C double bond stretching vibration peaks at 1650 cm⁻¹ and C-O bond stretching vibration peaks at 1260 cm⁻¹, indicating that ICA was successfully loaded into the liposome system ( Figure 5 ). To further verify whether ICA was loaded in ICA@LipD-CD47, 2 mg of ICA and 2 mg of ICA@LipD-CD47 were dissolved in 2 mL of deionized water and diluted to 10 μg / mL and 20 μg / mL, respectively, and then the absorption curves were determined by UV-Vis. The results showed that the ultraviolet characteristic peak of ICA appeared at 271 nm ( Figure 6 ), indicating that ICA was successfully loaded. 1 mg of ICA@LipD-CD47 was dispersed in 10 mL of PBS (pH 7.4), and the Malvern nanoparticle instrument was used to detect the hydrodynamic diameter distribution of ICA@LipD-CD47 at different times (1, 3, 7 days). At the same time, 1 mL (1 mg / mL) of ICA@LipD-CD47 was added to a dialysis bag (500D) and placed in 3 mL of PBS (pH 7.4). On the 1st, 3rd, 5th, 7th, and 14th days, the total amount of ICA leakage outside the dialysis bag was determined by UV-Vis at 271 nm. Figure 7 and Figure 8The displayed particle size and ICA drug leakage rate changed little, indicating that they were reliable nanocarriers for in vivo application. To verify that ICA@LipD-CD47 released ICA through acid response, the cumulative release amount of ICA during the degradation of ICA@LipD-CD47 was detected by UV-Vis. 1 mL of ICA@LipD-CD47 (1 mg / mL) was placed in 3 mL of buffer solution with different pH conditions (500D) (pH 5.0, 6.5, 7.4) for dialysis. At each set time point (1, 2, 4, 8, 12, 24, 48 h), 2 mL of supernatant was collected and supplemented with 2 mL of fresh buffer solution. Finally, the cumulative release amount was calculated by measuring the absorption peak of ICA at 271 nm by UV-Vis. At pH 7.4, ICA@LipD-CD47 showed slow release of ICA, with only about 7% of the drug released within 12 hours, indicating that ICA@LipD-CD47 has good biological stability and is suitable for in vivo application; while in a slightly acidic environment at pH 6.5, ICA was rapidly released from ICA@LipD-CD47, reaching 18% within 12 hours Figure 9 , indicating that ICA@LipD-CD47 has good epileptogenic focus targeting and controlled release performance.
[0026] Example 3: Blood-brain barrier permeability, long circulation and epileptogenic focus targeting of ICA@LipD-CD47 After constructing the liposome delivery of ICA, the blood-brain barrier permeability of ICA monomers and different liposome preparations such as ICA@LipD and ICA@LipD-CD47 was evaluated by the Transwell experiment, and it was found that various types of liposomes successfully delivered ICA to permeate the blood-brain barrier, among which the blood-brain barrier permeability of ICA@LipD-CD47 was more than 4 times that of ICA monomers Figure 10 .
[0027] To verify whether the coating of CD47 mimetic peptide can effectively prolong the circulation half-life of liposomes and improve the bioavailability, the distribution of ICA@LipD-CD47 and ICA@LipD in vivo was studied. The liposomes were labeled with DiR fluorescence, and the IVIS Lumina III imaging system was used for observation. In nude mice, the fluorescence signal of the ICA@LipD-CD47 group at 6 h, 12 h and 24 h after drug injection was higher than that of the ICA@LipD group, confirming the long circulation effect of the ICA@LipD-CD47 preparation Figure 11 . To further confirm the immune escape effect of CD47 mimetic peptide, the mice were euthanized and the main organs were collected for in vitro observation. The research results found that the fluorescence signal of the liver and spleen of the ICA@LipD-CD47 group of mice was significantly lower than that of the ICA@LipD group Figure 12), which proved that the use of CD47 mimetic peptide coating on the surface of liposomes could reduce the phagocytosis of nanoparticles by macrophages in the reticuloendothelial system. Due to the significant reduction in the accumulation of liposomes in the liver and spleen, it is expected to reduce the side effects of liposomes on the liver and spleen.
[0028] To verify the targeting of the pH-sensitive liposome, the liposomes (ICA@Lip-CD47 and ICA@LipD-CD47) were labeled with DiR (red), and after intravenous injection into the epileptic mice for 24 h, the ex vivo mouse brain was observed using the IVIS Lumina III imaging system. It was found that the fluorescence intensity of the ICA@LipD-CD47 group was significantly higher than that of the control group and the ICA@Lip-CD47 group ( Figure 13 ), and the quantitative results showed that the fluorescence intensity of the ICA@LipD-CD47 group was more than 4 times that of the ICA@Lip-CD47 group, which proved the improvement of the brain targeting of the pH-sensitive liposome. In addition, further DAPI immunofluorescence staining of the brain tissue of the epileptic mice found that in the epileptogenic focus of the epileptic mice, i.e. the hippocampal region, the liposomes labeled with DiR red fluorescence in the ICA@LipD-CD47 group were significantly more than those in the ICA@Lip-CD47 group ( Figure 14 ), which indicated that the pH-sensitive liposome significantly enhanced the epileptogenic focus targeting of the liposome-delivered ICA, and the targeting of the epileptogenic focus had the advantages of improving the efficacy of ICA, reducing side effects, and reducing drug resistance.
[0029] Example 4: Evaluation of the efficacy of ICA@LipD-CD47 on the epileptic mouse model After evaluating the brain targeting performance, the in vivo efficacy of ICA@LipD-CD47 was further evaluated using a mouse epilepsy model. Taking the PBS group as a control, the efficacy of ICA, Lip, ICA@Lip, ICA@LipD, and ICA@LipD-CD47 on epileptic mice was evaluated, respectively. First, the hippocampal tissue of the epileptic mice in each group was taken to prepare a single cell suspension, and the M1 microglial cell marker CD86 and the M2 microglial cell marker CD163 were immunofluorescently labeled to clarify the changes in microglial cell phenotype. Flow cytometry results showed that compared with the PBS group, ICA@LipD and ICA@LipD-CD47 treatment both increased the percentage of M2 microglial cells, while reducing the proportion of M1 microglial cells, indicating that the microglial cells appeared to be transformed into an anti-inflammatory phenotype, proving that ICA@LipD and ICA@LipD-CD47 groups could significantly improve the neuroinflammation of epileptic mice, with ICA@LipD-CD47 being the best ( Figure 15 ).
[0030] Example 5: In vivo safety evaluation To determine the biological safety of ICA@LipD-CD47 in vivo application, a comprehensive evaluation was carried out. After 7 days of treatment, there was no significant difference in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), creatinine (CRE) and urea (UREA) in epileptic mice compared with the PBS group, which proved that there was no obvious abnormality in the function of kidney and liver Figure 16
[0031] The above is only a preferred embodiment of the present application, it should be noted that the present application is not limited to the above-mentioned embodiments, for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be considered as the protection scope of the present application.
Claims
1. A CD47-modified pH-sensitive liposome delivery system, characterized in that, It comprises: soy lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icariin and CD47 mimetic peptide.
2. The CD47-modified, pH-sensitive liposome delivery system of claim 1, wherein, The mass ratio of the soy lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icariin and CD47 mimetic peptide is 4:6:8:5:4.5:
2.
3. A method of preparing the CD47-modified pH-sensitive liposome delivery system of claim 1, wherein, It comprises: The soy lecithin, cholesterol, dioleoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, icariin and CD47 mimetic peptide are dissolved in methanol according to the mass ratio of 4:6:8:5:4.5:2, and after being fully dissolved and mixed, organic solvent is removed by rotary evaporation under reduced pressure to form a lipid film, the lipid film is subjected to hydration reaction with a phosphate buffer, then is ultrasonicated in an ice bath, and the product is obtained by extrusion using a micro-extruder.
4. The method of claim 3, wherein the CD47-modified pH-sensitive liposome delivery system is prepared by the steps of: The rotary evaporation under reduced pressure is rotary evaporation under reduced pressure at 40-50℃ for 10-15min.
5. The method of claim 3, wherein the CD47-modified pH-sensitive liposome delivery system is prepared by the steps of: The hydration reaction is reacted at a temperature of 25-37℃ for 15-30min.
6. The use of the CD47 modified pH-sensitive liposome delivery system according to any one of claims 1-2 in epilepsy.