Preparation method and application of ApoE peptide modified composite drug-loaded micelle

Abstract

The invention discloses a preparation method and application of an ApoE peptide modified composite drug-loaded micelle, and belongs to the technical field of nano biomedicine. The medicine provided by the invention comprises ApoE-UPGs-DHA (docosahexaenoic acid) capable of targeting and carrying medicine, the ApoE peptide modified composite drug-loaded micelle can induce ferroptosis and immunogenic death of brain glioma cells, efficiently kills tumor cells, almost has no toxic effect on normal brain tissue cells, and provides a new thought and direction for clinical treatment of brain glioma.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomedicine, and specifically relates to a preparation method of ApoE peptide-modified composite drug-loaded micelles and application thereof. Background Art

[0002] Glioma is the most common malignant tumor in the brain. The overall survival rate of patients is low, and most patients cannot be cured. Due to the complex anatomical structure of the brain and the existence of the blood-brain barrier, there are many limitations in its treatment, such as: low radical surgical resection rate, chemotherapy drugs are difficult to pass through the blood-brain barrier, radiotherapy causes systemic toxic side effects, and low tumor sensitivity. Even with the use of comprehensive treatments such as surgery, radiotherapy and chemotherapy, the patient's survival time is still limited and the prognosis is poor. In view of the high incidence and high mortality rate of gliomas, finding suitable therapeutic targets and exploring new methods for treating brain gliomas are current research hotspots. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a method for preparing an ApoE peptide-modified composite drug-loaded micelle and its application.

[0004] Currently, inducing ferroptosis is becoming an effective method to eliminate tumor cells. Dihydroartemisinin can induce ferroptosis and is one of the few inducers of immunogenic cell death. Malignant cells can change iron metabolism to increase uptake and reduce efflux, resulting in an increase in the unstable iron pool in the cell. Therefore, dihydroartemisinin may have selective toxicity to cancer cells. Upconversion nanomaterials overcome the obstacle of limited penetration depth and can convert near-infrared light into ultraviolet light, making Fe 3+ Reduction to Fe 2+ , providing more endogenous Fe for ferroptosis of tumor cells 2+ . In recent years, researchers have found that polymer micelles show great advantages as drug delivery systems, thanks to their good biocompatibility, high drug loading, and biodegradability. Apolipoprotein E peptide ApoE can bind to low-density lipoprotein receptor-related protein 1, which is highly expressed in both the blood-brain barrier and glioma cells, and achieve dual targeting of the blood-brain barrier and glioma.

[0005] A drug for treating brain glioma, including composite drug-loaded micelles modified with ApoE peptide;

[0006] The composite drug-loaded micelle comprises a core-shell structure, dihydroartemisinin and ApoE peptide; the dihydroartemisinin is arranged inside the core-shell structure, and the ApoE peptide is arranged on the surface of the core-shell structure.

[0007] A method for preparing ApoE peptide-modified composite drug-loaded micelles comprises the following steps:

[0008] The upconversion nanoparticles are suspended in chloroform, dispersed, and a solution is obtained and stored;

[0009] Distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylethanolamine-polyethylene glycol-maleimide are added to chloroform, ultrasonicated, the above solution and dihydroartemisinin are added, ultra-dispersed, and deionized water is added and ultra-dispersed again;

[0010] The chloroform was removed by evaporation, and the evaporation was stopped when there were no bubbles in the reaction. After cooling and standing, the supernatant was taken, ultrasonicated, balanced and centrifuged, and dispersed in deionized water to obtain UPGs-DHA;

[0011] ApoE peptide and UPGs-DHA were stirred and co-incubated;

[0012] The liquid was centrifuged to remove the uncoupled polypeptide, the supernatant was discarded, and the mixture was dispersed with deionized water to obtain ApoE peptide-modified composite drug-loaded micelles ApoE-UPGs-DHA.

[0013] Preferably, the sequence of the ApoE peptide is SEQ ID NO:1.

[0014] Preferably, the molar ratio of the distearoylphosphatidylethanolamine-polyethylene glycol to the distearoylphosphatidylethanolamine-polyethylene glycol-maleimide is 4:1.

[0015] Preferably, the evaporation temperature is 65°C to 68°C.

[0016] Preferably, the co-incubation time is 8 h.

[0017] Preferably, the mass ratio of the upconversion nanoparticles to dihydroartemisinin is 10:1.

[0018] Preferably, the molar ratio of the ApoE peptide to distearoylphosphatidylethanolamine-polyethylene glycol-maleimide is 1:1.2.

[0019] The use of the ApoE peptide modified composite drug-loaded micelles prepared by the above preparation method or the ApoE peptide modified composite drug-loaded micelles according to claim 1 in the preparation of drugs for treating brain gliomas.

[0020] Beneficial effects of the present invention:

[0021] In this experiment, a new composite nanosystem ApoE-UPGs-DHA for targeted treatment of brain glioma was designed. The composite nanosystem was observed to be round under an electron microscope, with uniform shape and good dispersion, and an average particle size of about 82nm, which is consistent with the application of subsequent experiments. The encapsulation rate of the composite nanosystem is about 57%, and the drug loading is about 12%, which provides the possibility of efficient killing of tumor cells in vitro. The composite nanosystem has good targeting, can cause ferroptosis and immunogenic cell death in tumor cells, and has almost no toxic effect on normal brain tissue cells. The present invention provides new ideas and methods for the clinical treatment of brain glioma, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The schematic diagram of the synthesis of the composite nanosystem includes: (1), OA-UCNPs, (2), mal-PEG-DSPE, (3), MPEG-2000-DSPE, (4), DHA, (5), ApoE-SH, (6), ApoE-UPGs-DHA;

[0024] Figure 2 It is the transmission electron microscope image of the composite nanosystem;

[0025] Figure 3 is the hydrated particle size diagram of the composite nanosystem;

[0026] Figure 4 is the drug release rate of the composite nanosystem;

[0027] Figure 5 This is a picture of the composite nanosystem killing mouse brain glioma cells in vitro;

[0028] Figure 6 This is a picture of the composite nanosystem targeting mouse brain glioma cells;

[0029] Figure 7 Western blotting was used to detect the expression of GPX4, an indicator related to ferroptosis, in mouse brain glioma cells after treatment with the composite nanosystem;

[0030] Figure 8 To observe the morphological changes of mitochondria in mouse brain glioma cells after treatment with the composite nanosystem under a projection electron microscope;

[0031] Fig. 9To use fluorescence microscopy to detect the expression of ICD-related index CRT induced in mouse brain glioma cells after treatment with the composite nanosystem;

[0032] Fig.10 To use the ATP kit to detect the expression of ATP, an ICD-related indicator induced in mouse brain glioma cells after treatment with the composite nanosystem;

[0033] Fig.11 To use Elisa kit to detect the expression of HMGB1, an ICD-related indicator induced in mouse brain glioma cells after treatment with the composite nanosystem. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] Preparation of upconversion nanomaterials:

[0037] Upconversion nanoparticles were prepared by thermal decomposition method;

[0038] First, the core NaYF4:Yb,Tm was prepared. YCl3·6H2O (0.64mmol), YbCl3·6H2O (0.17mmol), and TmCl3·6H2O (0.032mmol) were accurately weighed with a precision balance. 6mL of oleic acid and 14mL of octadecene were taken and placed in a 100mL three-necked round-bottom flask. Sufficient Ar2 was introduced into the reaction system for magnetic stirring, and the mixture was heated to 150℃ and maintained for 30min. The color of the solution changed from colorless to light yellow.

[0039] NaOH (2.5 mmol) and NH4F (3.9 mmol) were accurately weighed with a precision balance and ultrasonically dispersed in 10 mL of methanol. When the reaction system cooled to 50°C, Ar2 was turned off, and the mixture was added to a 10 mL syringe and the solution was slowly dripped into a three-necked round-bottom flask, and then stirred in a sealed container at 50°C for 30 min.

[0040] Continue to pass Ar2 and heat to 100°C, maintain for 30 minutes to completely remove impurities such as methanol; add a condenser, continue to pass sufficient Ar2, quickly heat to 160°C, maintain for 30 minutes, then heat to 300°C, maintain for 1 hour, and cool to room temperature;

[0041] 30 mL of anhydrous ethanol was added and stirred thoroughly, centrifuged at 12000 rpm for 10 min, the material was washed 3 times, and finally the material precipitate was ultrasonically dispersed evenly in 10 mL of chloroform.

[0042] Preparation of core-shell structured UCNPs: YCl3·6H2O (0.64mmol), YbCl3·6H2O (0.17mmol), and NdCl3·6H2O (0.24mmol, 0.06g) were accurately weighed with a precision balance, and 6mL of oleic acid and 14mL of octadecene were taken and placed in a 100mL three-necked round-bottom flask; sufficient Ar2 was introduced into the reaction system for magnetic stirring, and heated to 150°C for 30min, and the color of the solution changed from colorless to light yellow;

[0043] NaOH (2.5 mmol) and NH4F (3.9 mmol) were accurately weighed with a precision balance and ultrasonically dispersed in 10 mL of methanol. When the reaction system cooled to 50°C, Ar2 was turned off, and the mixture was added to a 10 mL syringe and the solution was slowly dripped into a three-necked round-bottom flask, and then stirred in a sealed container at 50°C for 30 min.

[0044] Continue to pass Ar2 and heat to 100°C, maintain for 30 minutes to completely remove impurities such as methanol; add a condenser, continue to pass sufficient Ar2, quickly heat to 160°C, maintain for 30 minutes, then heat to 300°C, maintain for 1 hour, and cool to room temperature;

[0045] 30 mL of anhydrous ethanol was added and stirred thoroughly, centrifuged at 12000 rpm for 10 min, the material was washed 3 times, and finally the material precipitate was ultrasonically dispersed evenly in 10 mL of chloroform.

[0046] The preparation method of the upconversion nanoparticles adopted in the present invention can also be obtained by other preparation methods. The upconversion nanoparticles have the advantages of low toxicity, good stability, deeper tissue penetration, etc. Therefore, the preparation method and steps thereof do not constitute a limitation on the protection scope of the present invention.

[0047] Preparation of composite nanosystem ApoE-UPGs-DHA:

[0048] ApoE-UPGs-DHA was prepared by rotary evaporation;

[0049] 5.6 mg of DSPE-PEG-2000 and 1.4 mg of Mal-PEG-DSPE were accurately weighed at a molar ratio of 4:1, 4 mL of chloroform was added and ultrasonically mixed, 0.5 mL of chloroform solution of OA-UCNPs and 0.35 mg of dihydroartemisinin were added, wherein OA-UCNPs was 3.5 mg, 4 mL of deionized water was added and ultrasonically dispersed and mixed again; the speed of the rotary evaporator was adjusted to 30 rpm, and the chloroform was evaporated at 65 ° C. When there were basically no bubbles in the reaction system, the evaporator could be stopped; the supernatant was taken, centrifuged at 6000g for 20 min, and dispersed in 0.5 mL of deionized water to obtain UPGs-DHA; ApoE peptide was added according to the molar ratio of ApoE peptide to Mal-PEG-DSPE of 1:1.2, and the sequence of ApoE peptide was SEQ ID NO: 1: LRKLRKRLLLRKLRKRLLC, incubate at room temperature with low speed stirring for 8 hours; centrifuge at 6000g for 20 minutes, discard the supernatant, disperse in 0.5mL deionized water to obtain ApoE-UPGs-DHA.

[0050] Example 2 The killing effect of the composite nanosystem on different cells

[0051] After ApoE-UPGs-DHA was co-incubated with mouse brain glioma cells for 24 hours, 100 μL CCK-8 solution was added to each well to detect cell activity, and the absorbance (OD) at 450 nm was measured using an ELISA reader. The killing effect of ApoE-UPGs-DHA on different cells was detected by calculating the cell survival rate.

[0052] Comparative Example 2

[0053] A monoclonal antibody against ACTIN was used as a protein internal control. Equal amounts of protein were loaded onto SDS-PAGE gels, separated proteins were transferred to PVDF membranes, and probed with antibodies against the target protein (GPX4). The experimental results are shown in Figure 2. Figure 8 shown.

[0054] Example 3 Study on the effect of ApoE-UPGs-DHA on ferroptosis induced by tumor cells

[0055] After the composite nanosystem was co-incubated with mouse brain glioma cells for 24 hours, the changes in mitochondrial morphology in the cells were observed under an electron microscope. Equal amounts of protein were loaded onto SDS-PAGE gels, the separated proteins were transferred to PVDF membranes, and probed with antibodies against the target protein (GPX4). The experimental results are shown in Figure 2. Figure 8 As shown in the figure, it can be seen that after ApoE-UPGs-DHA intervenes in brain glial cells, the expression level of GPX4 protein in the cells can be reduced, thereby inducing ferroptosis of tumor cells.

[0056] Comparative Example 3

[0057] After incubation for 24 h in simple culture medium, culture medium mixed with UPGs, culture medium mixed with UPGs-DHA, and culture medium mixed with ApoE-UPGs-DHA, the release of ATP and HMGB1 in the supernatant was detected, and the externalization of CRT was examined by fluorescence microscopy. The experimental results are shown in Figure 2. Fig. 9 .

[0058] Example 4 Study on the effect of ApoE-UPGs-DHA on inducing ICD in tumor cells

[0059] After the composite nanosystem was co-incubated with mouse glioma cells for 24 h, the release of ATP and HMGB1 in the supernatant was detected, and the externalization of CRT was examined by fluorescence microscopy; the experimental results are shown in Figure 2. Fig. 9 ,From the figure, we can see the expression of ICD-related index CRT induced in mouse glioma cells after ApoE-UPGs-DHA treatment. The results show the translocation of CRT biomarker to the surface of tumor cells, thereby inducing immunogenic cell death of tumor cells.

[0060] Comparative Example 1

[0061] After ApoE-UPGs-DHA was co-incubated with mouse astrocytes and mouse brain microvascular endothelial cells for 24 hours, the CCK8 method was used to detect its killing effect on different cells; the experimental results obtained in Example 2 and Comparative Example 1 are as follows Figure 5 As shown, from Figure 5 It can be seen that ApoE-UPGs-DHA can effectively kill brain glioma cells and has less cytotoxicity to normal brain tissue cells.

[0062] Figure 1 Schematic diagram of the synthesis of the composite nanosystem ApoE-UPGs-DHA

[0063] Figure 2 The prepared composite nanosystem ApoE-UPGs-DHA was placed on a double copper grid for 5 minutes to dry, and then negatively stained with 2% phosphotungstic acid staining solution. The morphology under a transmission electron microscope (TEM) was spherical particles with an average particle size of 82.3±5.8nm.

[0064] Figure 3 The composite nanosystem ApoE-UPGs-DHA was diluted and placed in a nanoparticle size and potential analyzer for detection. The hydrated particle size of the composite nanosystem ApoE-UPGs-DHA was about 136.56±4.32nm;

[0065] The composite nanosystem ApoE-UPGs-DHA was placed in a dialysis bag (3500Da), and PBS with pH 5.2, pH 6.8 and pH 7.2 was added respectively, and then dialyzed on a shaker (200rpm, 37°C). The solution was taken out at different time points to detect the drug release amount and then put back. The results showed that the drug release rate gradually increased with the extension of time; the results are as follows Figure 4 ;

[0066] Figure 5 The composite nanosystem ApoE-UPGs-DHA can effectively kill brain glioma cells and has less cytotoxicity to normal brain tissue cells;

[0067] The composite nanosystem UPGs / ApoE-UPGs-DHA was combined with rhodamine and FITC respectively and incubated with G422 cells for 4 hours, and then 4% paraformaldehyde was added and incubated in the dark for 15 minutes. Under the fluorescence microscope, the fluorescence signal of the targeted group was strong, indicating good in vitro targeting. After the composite nanosystem UPGs / ApoE-UPGs-DHA was combined with DIR and injected into tumor-bearing mice through the tail vein, the in vivo near-infrared light imaging showed that the fluorescence signal of the targeted group was strong. The mice were euthanized 48 hours after the injection, and the brain, heart, liver, spleen, lung, and kidney were taken for in vitro near-infrared light imaging. The fluorescence signal of the tumor tissue was strong, indicating good in vivo targeting. The results are as follows Figure 6 ;

[0068] After the composite nanosystem ApoE-UPGs-DHA intervened in brain glial cells, they were fixed with 2.5% glutaraldehyde, and then dehydrated, infiltrated, embedded, sliced, uranium-lead double-stained, and dried at room temperature overnight. Under the transmission electron microscope, the mitochondrial morphology in the cells was observed to be smaller, and the cristae in the cells were reduced or disappeared; the results are as follows Figure 7 ;

[0069] Figure 8 After the composite nanosystem ApoE-UPGs-DHA intervened in brain glial cells, the expression level of GPX4 protein in the cells was reduced;

[0070] Fig. 9 To detect the expression of ICD-related index CRT in mouse glioma cells induced by the composite nanosystem ApoE-UPGs-DHA after treatment using fluorescence microscopy, the results showed the translocation of CRT biomarker to the surface of tumor cells;

[0071] Fig.10 The ATP kit was used to detect the expression of ATP, an ICD-related indicator, in mouse glioma cells after treatment with the composite nanosystem ApoE-UPGs-DHA. The results showed that the release of ATP from tumor cells was further significantly increased.

[0072] Fig.11The Elisa kit was used to detect the expression of HMGB1, an ICD-related indicator induced in mouse glioma cells after treatment with the composite nanosystem ApoE-UPGs-DHA. The results showed that the release of HMGB1 in tumor cells was further significantly increased.

[0073] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0074] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A drug for treating brain glioma, characterized in that: Including ApoE peptide modified composite drug-loaded micelles; The composite drug-loaded micelle comprises a core-shell structure, dihydroartemisinin and ApoE peptide; the dihydroartemisinin is arranged inside the core-shell structure, and the ApoE peptide is arranged on the surface of the core-shell structure.

2. A method for preparing ApoE peptide-modified composite drug-loaded micelles, characterized in that: The following steps are involved: The upconversion nanoparticles are suspended in chloroform, dispersed, and a solution is obtained and stored; Distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylethanolamine-polyethylene glycol-maleimide are added to chloroform, ultrasonicated, the above solution and dihydroartemisinin are added, ultra-dispersed, and deionized water is added and ultra-dispersed again; The chloroform was removed by evaporation, and the evaporation was stopped when there were no bubbles in the reaction. After cooling and standing, the supernatant was taken, ultrasonicated, balanced and centrifuged, and dispersed in deionized water to obtain UPGs-DHA; ApoE peptide and UPGs-DHA were stirred and co-incubated; The liquid was centrifuged to remove the uncoupled polypeptide, the supernatant was discarded, and the mixture was dispersed with deionized water to obtain ApoE peptide-modified composite drug-loaded micelles ApoE-UPGs-DHA.

3. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The sequence of the ApoE peptide is SEQ ID NO:

1.

4. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The molar ratio of the distearoyl phosphatidylethanolamine-polyethylene glycol to the distearoyl phosphatidylethanolamine-polyethylene glycol-maleimide is 4:

1.

5. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The evaporation temperature is 65°C to 68°C.

6. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The co-incubation time is 8 h.

7. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The mass ratio of the upconversion nanoparticles to dihydroartemisinin is 10:

1.

8. The method for preparing ApoE peptide-modified composite drug-loaded micelles according to claim 2, characterized in that: The molar ratio of the ApoE peptide to distearoyl phosphatidylethanolamine-polyethylene glycol-maleimide is 1:1.

2.

9. Use of the ApoE peptide-modified composite drug-loaded micelles prepared by the preparation method of any one of claims 2 to 8 or the ApoE peptide-modified composite drug-loaded micelles according to claim 1 in the preparation of drugs for treating brain gliomas.

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