CN116617166A - Polypeptide modified liposome for targeted treatment of glioblastoma and preparation and application thereof - Google Patents
Polypeptide modified liposome for targeted treatment of glioblastoma and preparation and application thereof Download PDFInfo
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- CN116617166A CN116617166A CN202310568754.6A CN202310568754A CN116617166A CN 116617166 A CN116617166 A CN 116617166A CN 202310568754 A CN202310568754 A CN 202310568754A CN 116617166 A CN116617166 A CN 116617166A
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- liposome
- polypeptide
- hlr
- dox
- lipo
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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Abstract
The invention discloses a polypeptide modified liposome for targeted treatment of glioblastoma, and preparation and application thereof. The liposome comprises a polypeptide of a targeting TREM2 protein for binding glioblastoma, a medicament with killing effect on glioblastoma cells and a liposome wrapping the medicament; the amino acid sequence of the polypeptide targeting TREM2 protein is as follows: HLRKLRKR; the polypeptide targeting TREM2 protein is modified on the liposome, and the liposome modified with the polypeptide encapsulates the drug. The polypeptide modified doxorubicin liposome prepared by the invention realizes active drug delivery by connecting the targeting polypeptide, and the doxorubicin with therapeutic effect on human glioblastoma is wrapped in the liposome, so that the acting time is prolonged, the cytotoxicity is reduced, and the overall stability of a targeting drug delivery system is improved.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a polypeptide modified liposome for targeted treatment of glioblastoma, and preparation and application thereof.
Background
Glioblastoma multiforme (Glioblastoma multiforme, GBM, WHO grade IV) is the most common and most invasive type of malignant brain tumor. Because of the characteristics of wettability, molecular heterogeneity and the like and the specificity of growth positions, the conventional treatment methods (surgery, external irradiation and chemotherapy) have relatively limited therapeutic effects of improving the overall survival of patients. At present, the main difficulty in brain tumor treatment is that the medicine cannot reach the tumor part or the accumulated amount of the medicine at the tumor part is small due to the existence of blood brain barriers (Blood Brain Barrier, BBB), so that the treatment effect cannot be achieved. Thus, improving drug targeting and delivery efficiency is an urgent problem to be solved in GBM therapeutic drug research.
In recent years, researchers have developed a drug delivery system that can effectively cross the blood-brain barrier using polypeptide-modified liposomes, improving the targeting and biocompatibility of the drug. The polypeptide and the liposome combine the advantages of the polypeptide and the liposome, and have wide application in drug treatment. With the development of biotechnology and molecular biology, this technology has gradually become a leading-edge technology for the treatment of malignant tumors.
The liposome is a microparticle composed of phospholipid, cholesterol and other components, and has good drug loading and metabolism capability. Researchers find that encapsulation of the drug in liposomes can improve the stability and bioavailability of the drug while reducing the toxic side effects of the drug on normal cells. The polypeptide with the capability of specifically binding to the tumor cell surface marker can enhance the binding capability of the liposome and the tumor cell surface receptor after being modified with the liposome, and the targeting effect of the liposome is effectively improved by modifying and changing the structure of the liposome and the configuration of the polypeptide, so that the accurate identification and targeting treatment of tumor cells are realized, the treatment effect is improved, and the toxic and side effects are reduced. Therefore, the polypeptide modified liposome drug delivery system with strong targeting specificity and small toxic and side effects is applied to GBM treatment, and has good research value and application prospect.
Disclosure of Invention
In order to solve the practical problem of glioblastoma treatment, the invention designs a polypeptide ligand with high affinity with the TREM2 protein based on the specific high-expression TREM2 protein of the glioblastoma, and provides a preparation method of a targeting delivery drug system capable of treating the glioblastoma. The drug delivery system can penetrate through the blood brain barrier, target U87-MG cells to transplant tumors in situ, and show a certain treatment effect, and has potential application prospects in treatment research of glioblastoma.
The invention aims to provide a liposome modified by polypeptide for targeted treatment of glioblastoma.
The polypeptide modified liposome for targeted treatment of glioblastoma provided by the invention comprises a polypeptide for combining targeting TREM2 protein of glioblastoma, a medicament with killing effect on glioblastoma cells and a liposome wrapping the medicament;
wherein, the amino acid sequence of the polypeptide targeting TREM2 protein is as follows: HLRKLRKR (histidine-leucine-arginine-lysine-arginine, his-Leu-Arg-Lys-Leu-Arg-Lys-Arg), abbreviated HLR (SEQ ID NO: 1);
The polypeptide of the targeting TREM2 protein is modified on the liposome, and the liposome modified with the polypeptide encapsulates the drug;
the medicine is doxorubicin;
the liposome is prepared from lecithin and DSPE-PEG2000 serving as raw materials.
The polypeptide modified liposome for targeted therapy of glioblastoma is prepared by a method comprising the following steps:
1) Reacting DSPE-PEG2000-Mal with polypeptide HLR to obtain DSPE-PEG2000-HLR;
2) Preparing liposome solution by using lecithin and DSPE-PEG2000-HLR as raw materials and adopting an ammonium sulfate gradient method;
3) Adding a medicament with killing effect on glioblastoma cells into the obtained liposome solution, and incubating to obtain the glioblastoma cell killing agent.
In the above method step 1), the molar ratio of DSPE-PEG2000-Mal to polypeptide HLR is 1:1.5-2, specifically 1:1.5;
the reaction is carried out in water, the reaction is carried out at 4 ℃ under the protection of inert gas, and the time of the reaction can be 24-48 hours, and can be particularly 24 hours;
the operation of the method step 2) is as follows: dissolving lecithin and DSPE-PEG2000-HLR in chloroform, respectively, steaming to form film, adding (NH) 4 ) 2 SO 4 Solution, hydration; preparing liposome solution after ultrasonic dispersion; dialysis to remove uncoated (NH) 4 ) 2 SO 4 Obtaining liposome solution;
wherein, the mass ratio of lecithin to DSPE-PEG2000-HLR can be 6-9:1, and can be 9:1 specifically;
said (NH) 4 ) 2 SO 4 The concentration of the solution may be 250mM,
lecithin and (NH) 4 ) 2 SO 4 The ratio of the solution can be 18mg:5mL;
the dialysis was dialysis overnight in 50mM ph=7.4 PBS;
in the step 3), the drug having killing effect on glioblastoma cells may specifically be doxorubicin;
the mass ratio of lecithin to the drug may be 18mg:1mg;
the incubation was at 50℃for 1 hour,
dialysis was also performed after incubation was completed to remove non-entrapped drug.
The invention also provides application of the polypeptide modified liposome for targeted treatment of glioblastoma in preparation of medicines for treating human brain glioma or human brain glioma cell transplantation tumor.
In the application, the human brain glioma cells can be specifically U87-MG cells.
According to an embodiment of the invention, the selected human brain glioma cells are U87-MG cells, and the selected in vivo brain glioma cell transplantation tumor is a BABL/c nude mouse in situ-bearing U87-MG cell transplantation tumor.
Compared with the prior art, the nano targeting material prepared by the invention has the beneficial effects that:
1) The target polypeptide designed by the invention can effectively identify and specifically bind with human brain glioma U87-MG cells, has a equilibrium dissociation constant KD of 8.17 mu M of interaction with TREM2 protein, and has strong binding capacity with TREM2 protein.
2) The liposome prepared by the invention can pass through a blood brain barrier, has certain active targeting property, enhances the targeting effect of the nano targeting material through synergistic effect after being combined with the targeting polypeptide, and can target glioblastoma cells.
3) The polypeptide modified doxorubicin liposome prepared by the invention realizes active drug delivery by connecting the targeting polypeptide, and the doxorubicin with therapeutic effect on human glioblastoma is wrapped in the liposome, so that the acting time is prolonged, the cytotoxicity is reduced, and the overall stability of a targeting drug delivery system is improved.
Drawings
FIG. 1 shows the structural formula of polypeptide HLR of the present invention.
Fig. 2 is a three-dimensional docking model of polypeptide HLR and TREM2 protein in example 1 of the present invention.
FIG. 3 is a synthetic route diagram of polypeptide HLR of example 2 of the present invention.
FIG. 4 shows the HPLC and MS identification results of the polypeptide HLR prepared in example 2 of the present invention.
Fig. 5 shows the results of the affinity test of HLR and TREM2 protein in example 2 of the present invention.
FIG. 6 shows the effect of HLR on the survival of U87-MG cells in example 3 of the present invention. * p < 0.05, ×p < 0.01.
Fig. 7 is a synthetic route diagram of FITC-HLR in example 4 of the present invention.
Fig. 8 is a structural formula of FITC-HLR in embodiment 4 of the present invention.
FIG. 9 shows the HPLC and MS identification results of FITC-HLR prepared in example 4 of the present invention.
FIG. 10 shows the binding of the TREM2 protein of U87-MG cells to HLR in example 4 of the present invention.
FIG. 11 shows uptake of HLR by flow cytometer comparison U87-MG cells in example 4 of the present invention.
Figure 12 is a synthetic route diagram of the cy5.5-HLR in example 5 of the present invention.
FIG. 13 shows the structural formula of Cy5.5-HLR prepared in example 5 of the present invention.
FIG. 14 shows the HPLC and MS identification results of the Cy5.5-HLR prepared according to example 5 of the present invention.
FIG. 15 shows the distribution and brain uptake of Cy5.5-HLR in U87-MG cells orthotopic tumor nude mice in example 5 of the present invention. # p < 0.001, fluorescence distribution at different time points after brain uptake of polypeptide by nude mice A; fluorescent intensity at various time points after uptake of polypeptide by brain of nude mice.
FIG. 16 shows the uptake of Cy5.5-HLR in U87-MG cells in situ in nude mice with tumors 24 hours after administration in example 5 of the present invention. # p < 0.001, fluorescence distribution of the viscera and brain of the nude mice A after taking up the polypeptide; fluorescence intensity of nude mice after uptake of the polypeptide by brain.
FIG. 17 is a nuclear magnetic resonance chart of DSPE-PEG2000-HLR according to example 6 of the present invention.
FIG. 18 shows the HPLC and MS identification of LRK in example 6 of the present invention.
FIG. 19 is a schematic diagram showing the synthesis of the liposome-encapsulated drug Dox, the surface-modified polypeptide and Cy5.5 of example 6 of the present invention.
FIG. 20 is a characterization of the properties of the polypeptide modified doxorubicin liposome in example 7 of the invention. Particle size A, B; c, D Zeta potential; E. f, analyzing the morphology by a transmission electron microscope; g, H encapsulation efficiency; i, J drug loading; k doxorubicin accumulation release rate.
FIG. 21 shows the effect of polypeptide-modified doxorubicin liposome in example 8 of the invention on U87-MG cell viability.
FIG. 22 shows the effect of polypeptide-modified doxorubicin liposome in example 8 of the invention on the apoptosis rate of U87-MG cells.
FIG. 23 shows the effect of polypeptide-modified doxorubicin liposome in example 8 of the invention on the invasive potential of U87-MG cells. * P < 0.01, # p﹤0.001。
FIG. 24 shows the effect of polypeptide-modified doxorubicin liposome in example 8 of the present invention on the migration ability of U87-MG cells. * p < 0.05, ×p < 0.01.
FIG. 25 shows the effect of polypeptide-modified doxorubicin liposomes of example 8 of the invention on the survival of U87-MG cell clone. * P < 0.01.
FIG. 26 shows the uptake of polypeptide-modified doxorubicin liposome by U87-MG cells in example 9 of the present invention.
FIG. 27 shows the passage of polypeptide-modified doxorubicin liposome in example 9 of the present invention across the blood brain barrier and brain uptake of nude mice with in situ transplantation tumors. Fluorescence distribution at different time points after taking up polypeptide modified doxorubicin liposome by brain of nude mice; fluorescence intensity at different time points after uptake of polypeptide modified doxorubicin liposomes by brain of nude mice.
FIG. 28 shows uptake of polypeptide-modified doxorubicin liposomes 24 hours after administration in example 9 of the present invention in the brain. A, taking up polypeptide modified doxorubicin liposome by viscera of nude mice and performing fluorescence imaging; fluorescence intensity of nude mice after uptake of polypeptide-modified doxorubicin liposomes.
FIG. 29 shows the case of the polypeptide modified doxorubicin liposome of example 10 of the present invention for treating nude mice in situ-transplanted tumor. A, comparing fluorescent signals of in-situ transplantation tumors of the brain of the nude mice in different time periods; b, fluorescence intensity of in-situ transplantation tumor of nude mice brain in different time periods; c comparison of nude mice body weight at different time periods.
FIG. 30 is an H & E staining observation of polypeptide modified doxorubicin liposome treated nude mice in situ engrafting tumor in example 10 of the present invention.
FIG. 31 is TUNEL staining of the polypeptide modified doxorubicin liposome treated nude mice in situ engrafted tumor in example 10 of the invention.
FIG. 32 is an expression profile of Ki67 after immunofluorescence detection of polypeptide-modified doxorubicin liposomes in example 10 of the invention in nude mice treated with in situ transplantation tumors.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 molecular docking of HLRKLRKR with TREM2 protein
1) TREM2 protein Structure determination
Human TREM2 protein, 230 amino acids in length, was subjected to butt scoring by using the Cluspro algorithm using the A chain of 5UD7 crystal structure.
2) Molecular binding pattern analysis
The binding pattern of the polypeptide with the highest score and TREM2 protein is analyzed, the polypeptide sequence is histidine-leucine-arginine-lysine-arginine (His-Leu-Arg-Lys-Leu-Arg-Lys-Arg, abbreviated HLRKLRKR, the structural formula is shown in figure 1. The result of the interaction position of the HLRKLRKR and TREM2 in butt joint is shown in figure 2, and the result shows that the HLRKLRKR can be effectively bound with TREM2 protein.
Example 2 affinity identification of HLR with TREM2 protein
1) Artificial HLR
The HLR synthesis route is shown in fig. 3, synthesis sequence: from the C-terminal to the N-terminal. (1) Swelling resin, placing 2-Chlorotrityl Chloride Resin into a reaction tube, adding DCM (dichloromethane) (15 mL/g), and oscillating for 30min; (2) the first amino acid was taken up, the solvent was filtered off with sand core suction, 3-fold molar excess of Fmoc-His (Trt) -OH amino acid was added, DMF (dimethylformamide) was added for dissolution, and 10-fold molar excess of DIEA (diisopropylethylamine) was added and shaking was carried out for 60min. Blocking with methanol; (3) deprotection, removal of DMF, addition of 20% piperidine DMF solution (15 mL/g), reaction for 5min, removal of 20% piperidine DMF solution (15 mL/g) and reaction for 15min; (4) detecting, namely pumping out piperidine solution, taking more than ten pieces of resin, washing with ethanol for three times, adding a detection reagent for detection, heating for 5min at 105-110 ℃, and turning deep blue into positive reaction; (5) washing with DMF (10 mL/g) twice, DCM (10 mL/g) twice, DMF (10 mL/g) twice; (6) condensing, namely protecting amino acid by three times and HBTU (O-benzotriazole-tetramethyl urea hexafluorophosphate) by three times, dissolving with DMF as little as possible, adding into a reaction tube, immediately adding DIEA by ten times and reacting for 30min; (7) detecting, namely taking more than ten pieces of resin, washing the resin with ethanol for three times, adding a detection reagent for detection, heating the resin for 5 minutes at the temperature of 105-110 ℃, and taking the resin as a colorless negative reaction. (8) Washing with DMF (10 mL/g) twice, DCM (10 mL/g) twice, DMF (10 mL/g) twice; (9) repeating the three to six steps, and sequentially connecting amino acids in the sequence from right to left; and (3) pumping, and washing: DMF (10 mL/g) was taken twice, methanol (10 mL/g) was taken twice, DMF (10 mL/g) was taken twice, DCM (10 mL/g) was taken twice, and the mixture was drained for 10min.11 Cleavage of the polypeptide from the resin to prepare a cleavage solution (10 mL/g) of TFA (trifluoroacetic acid) 95%; 1% of water; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120min; drying and washing, namely drying the pyrolysis liquid with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature; analyzing and purifying, and purifying the crude product by high performance liquid chromatography; freeze-drying, collecting target polypeptide solution, concentrating in freeze-dryer, and freeze-drying to obtain powder. The HPLC and MS identification results of HLR are shown in FIG. 4.
2) HLR and TREM2 protein affinity assay
TREM2 protein was diluted to 20. Mu.g/mL with PBS for chip immobilization and polypeptides were diluted to 100. Mu.M with PBST (pH 7.4). The TREM2 protein solution was dropped onto the NTA chip, 200. Mu.L of PBST (pH 7.4) was added to each well of the cured NTA chip as a control, and 200. Mu.L of 100. Mu.M polypeptide was added to the other wells. The sensor was injected with 250 μl of PBS buffer (pH 7.4), the buffer was run at maximum flow rate (150 μl/min) to reach the signal baseline, and the flow rate of buffer was reduced to 20 μl/min to obtain a more stable baseline. Signals of binding of HLR to TREM2 protein are shown in fig. 5. The results show that the HLR and TREM2 protein have strong binding effect, and the equilibrium dissociation constant KD of the interaction between the HLR and the TREM2 protein is 8.17 mu M.
EXAMPLE 3 Effect of HLR on U87-MG cell viability
Cell viability was determined using CCK-8 kit, human glioblastoma multiforme U87-MG cells were seeded in 96-well plates, and HLR at different concentrations, 37℃and 5% CO were added when the growth density reached 70% 2 Culturing for 24 hours under the condition. Then 10. Mu.L of CCK-8 reaction solution was added to each well at 37℃with 5% CO 2 The incubation was continued for 4 hours and the absorbance was measured at 450nm using a 96-well plate in an microplate reader. Cell viability formula [ A (sample well) -A (blank) ]/[ A (PBS) -A (blank)]X 100%. The results showed that concentrations of 5, 10, 20. Mu.g/mL were not significantly different from the toxicity of U87-MG cells, and therefore 20. Mu.g/mL was selected for the cell uptake experiments (FIG. 6).
EXAMPLE 4 uptake analysis of HLR by U87-MG cells
1) FITC-HLR preparation
The FITC-HLR synthesis route is shown in FIG. 7. Polypeptide synthesis is carried out in the same steps (1) to (8) in example 2, amino acids in the sequence are sequentially connected from right to left, 5-FTIC is linked, and the subsequent steps are the same as the steps in example 2. The structural formula of FITC-HLR is shown in FIG. 8, and the HPLC and MS identification results are shown in FIG. 9.
2) Qualitative analysis of HLR in combination with U87-MG cell TERM2
U87-MG cells were inoculated on a glass slide (placed on a phi 35 dish), after the cell density reached 60%, washed 3 times with precooled PBS, then fixed with 4% paraformaldehyde for 15min, naturally dried, added with 1 mug/mL of FITC-HLR, incubated overnight at 4 ℃, added with 5 mug/mL of DAPI to stain the nuclei, finally blocked with a quenching inhibitor, and photographed under a fluorescence microscope for observation. The results show that HLR is capable of binding to U87-MG cells (FIG. 10).
3) Quantitative analysis of uptake of U87-MG cells into HLR
Human umbilical vein endothelial cells HUVEC and U87-MG cells were plated in the upper and lower Transwell chambers, respectively, at a ratio of 1:5, cultured for 48 hours to simulate tumor microvascular environment, HUVEC cell culture broth in the upper Transwell chamber was aspirated, FITC-HLR with final concentration of 20. Mu.g/mL containing 10% FBS was added, incubated at 37℃and digested with 0.25% trypsin and washed 3 times with cold PBS for 2 and 4 hours, respectively, and then resuspended in 300. Mu.l of cold PBS for flow measurement of cell fluorescence intensity. The results show that the FITC fluorescence intensity of U87-MG cells at 4 hours was greater than 2 hours and PBS group, indicating that HLR was able to be taken up by U87-MG cells (FIG. 11).
Example 5 HLR crossing blood brain Barrier experiment
1) Cy5.5-HLR preparation
The Cy5.5-HLR synthesis route is shown in FIG. 12. Polypeptide synthesis is carried out in the same steps (1) to (8) in example 2, amino acids in the sequence are sequentially connected from right to left, cy5.5 is linked, and the subsequent steps are the same as the steps in example 2. Cy5.5-HLR has a structural formula shown in FIG. 13, and HPLC and MS identification results shown in FIG. 14.
2) U87-MG cell in situ tumor animal model establishment
And establishing an in-situ tumor model of the U87-MG cell mouse by using a brain stereotactic instrument. Taking U87-MG-luc cells in logarithmic growth phase, digesting with 0.25% pancreatin, and adjusting cell density to 1×10 5 5. Mu.L of the cell suspension was aspirated with a brain stereotactic apparatus and injected into the intracranial posterior ventricle (striatum, right bregma 1.8mm, depth 3 mm) of female BABL/c nude mice. The tumor formation was observed by photographing in vivo images of the animals 14 days after inoculation.
3) Tumor-bearing rat tail vein administration and HLR imaging observation
Tumor-bearing nude mice were injected with 100 μl (1 μg/μl) of cy5.5-HLR physiological saline injection of example 5 via tail vein, images were collected 1, 3, 6, 12, 24 hours after injection using a small animal fluorescence living imager (fig. 15), and control tumor-bearing mice were injected with PBS of the same volume as cy 5.5-HLR. The results show that the brain of the nude mice of the administration group can continuously display fluorescent signals. The mice viscera and brain were separated 24 hours after injection, and images were acquired using a small animal fluorescence living imager, which showed a significant increase in fluorescence signal of the nude mice brain in the dosing group compared to the PBS group (fig. 16), demonstrating that HLR was able to cross the blood brain barrier and had a sustained targeted binding effect.
EXAMPLE 6 preparation of polypeptide-modified Adriamycin Liposome
1) Synthesis of DSPE-PEG2000-HLR/LRK
Dissolving DSPE-PEG2000-Mal and polypeptide HLR/LRK in ultrapure water according to a molar ratio of 1:1.5, and carrying out nitrogen protection reaction for 24 hours to obtain a product DSPE-PEG2000-HLR/LRK. The yields were calculated by nuclear magnetic resonance imaging of polypeptide and DSPE-PEG2000-HLR (FIG. 17).
LRK is another polypeptide targeting TREM2 protein, the amino acid sequence of which is shown below: LRKLRLRL (leucine-arginine-lysine-leucine-arginine-leucine, leu-Arg-Lys-Leu-Arg-Leu), abbreviated LRK (sequence 2).
The preparation method comprises the following steps: artificial synthesis of LRK
Sequence of LRK synthesis: from the C-terminal to the N-terminal. (1) Swelling resin, placing 2-Chlorotrityl Chloride Resin into a reaction tube, adding DCM (dichloromethane) (15 ml/g), and oscillating for 30min; (2) the first amino acid was taken up, the solvent was filtered off with sand core suction, 3-fold molar excess of Fmoc-His (Trt) -OH amino acid was added, DMF (dimethylformamide) was added for dissolution, and 10-fold molar excess of DIEA (diisopropylethylamine) was added and shaking was carried out for 60min. Blocking with methanol; (3) deprotection, removal of DMF, addition of 20% piperidine DMF solution (15 mL/g), reaction for 5min, removal of 20% piperidine DMF solution (15 mL/g) and reaction for 15min; (4) detecting, namely pumping out piperidine solution, taking more than ten pieces of resin, washing with ethanol for three times, adding a detection reagent for detection, heating for 5min at 105-110 ℃, and turning deep blue into positive reaction; (5) washing with DMF (10 mL/g) twice, DCM (10 mL/g) twice, DMF (10 mL/g) twice; (6) condensing, namely protecting amino acid by three times and HBTU (O-benzotriazole-tetramethyl urea hexafluorophosphate) by three times, dissolving with DMF as little as possible, adding into a reaction tube, immediately adding DIEA by ten times and reacting for 30min; (7) detecting, namely taking more than ten pieces of resin, washing the resin with ethanol for three times, adding a detection reagent for detection, heating the resin for 5 minutes at the temperature of 105-110 ℃, and taking the resin as a colorless negative reaction. (8) Washing with DMF (10 mL/g) twice, DCM (10 mL/g) twice, DMF (10 mL/g) twice; (9) repeating the steps (3) to (6), and connecting the amino acids in the sequences from right to left; and (3) pumping, and washing: DMF (10 mL/g) was taken twice, methanol (10 mL/g) was taken twice, DMF (10 mL/g) was taken twice, DCM (10 mL/g) was taken twice, and the mixture was drained for 10min.11 Cleavage of the polypeptide from the resin to prepare a cleavage solution (10 mL/g) of TFA (trifluoroacetic acid) 95%; 1% of water; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120min; drying and washing, namely drying the pyrolysis liquid with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature; analyzing and purifying, and purifying the crude product by high performance liquid chromatography; freeze-drying, collecting target polypeptide solution, concentrating in freeze-dryer, and freeze-drying to obtain powder.
The HPLC and MS identification results are shown in FIG. 18.
2) Liposome-encapsulated drug Dox
Weighing 18mg of lecithin and 2mg of DSPE-PEG2000, respectively dissolving in 5mL of chloroform, and performing rotary evaporation at 40 ℃ to form a film; 5mL 250mM (NH) 4 ) 2 SO 4 Solution, hydration; after ultrasonic dispersion, liposome solution with proper particle size is obtained by adopting a liposome extruder; dialyzing in a nanodialysis apparatus at 50mM pH=7.4 PBS overnight to remove uncoated (NH) 4 ) 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Taking out liposome solution, adding 1mg of doxorubicin, and incubating at 50 ℃ for 1 hour; dialysis was performed for about 1 hour to remove non-entrapped doxorubicin; the absorbance of the solution was measured at 480nm and the concentration was determined from the doxorubicin standard absorption curve.
3) Liposome-encapsulated medicine Dox, surface modified HLR/LRK
Weighing 18mg of lecithin and 2mg of DSPE-PEG2000-HLR/LRK, respectively dissolving in 5mL of chloroform, and performing rotary evaporation at 40 ℃ to form a film; 5mL 250mM (NH) 4 ) 2 SO 4 Solution, hydration; after ultrasonic dispersion, liposome solution with proper particle size is obtained by adopting a liposome extruder; dialyzing in a nanodialysis apparatus at 50mM pH=7.4 PBS overnight to remove uncoated (NH) 4 ) 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Taking out liposome solution, adding 1mg of doxorubicin, and incubating at 50 ℃ for 1 hour; dialysis was performed for about 1 hour to remove non-entrapped doxorubicin; the absorbance of the solution was measured at 480nm and the concentration was determined from the doxorubicin standard absorption curve.
4) Liposome-entrapped drug Dox, surface-modified polypeptide and cy5.5
35mg lecithin and 1mg DSPE-PEG200-HLR were weighedLRK and 3.5mg DSPE-PEG2000-Cy5.5 are dissolved in 5mL chloroform respectively, and the mixture is steamed into a film at 40 ℃; 5mL 250mM (NH) 4 ) 2 SO 4 Solution, hydration; after ultrasonic dispersion, liposome solution with proper particle size is obtained by adopting a liposome extruder; dialyzing in 50mM pH=7.4 PBS overnight in a nanodialysis apparatus to remove uncoated (NH 4) 2 SO4; taking out liposome solution, adding 1mg of doxorubicin, and incubating at 50 ℃ for 1 hour; dialyzing for about 2 hours to remove the target product (the synthetic schematic diagram is shown in figure 19); the absorbance of the solution was measured at 480nm and the concentration was determined from the doxorubicin standard absorption curve.
EXAMPLE 7 characterization of polypeptide-modified Adriamycin Liposome Properties
1) Zeta potential and particle size detection
10 mu L of sample to be tested is dispersed in about 1.5mL of deionized water, and the mixture is uniformly mixed and placed into a sample tank of a nanometer particle size potentiometer, a DLS test mode is selected, and the particle size is tested (figure 20A, B). After the test, the electrodes were inserted into the sample cell and connected to the particle analyzer, and the PALS mode was selected to test the potential (fig. 20, C, D).
2) Electron microscope detection
The morphology of the samples was observed using a transmission electron microscope: and dispersing 10 mu L of a sample to be tested in 2mL of deionized water, uniformly mixing, dripping the sample to be tested on a copper mesh, dripping phosphotungstic acid to be enriched again after the copper mesh is dried, continuously drying, and placing the copper mesh in a transmission electron microscope instrument to shoot an electron microscope image after the copper mesh is completely dried (figure 20E, F).
3) Encapsulation efficiency
Preparing a Dox solution with known concentration, respectively diluting with deionized water to different multiples to obtain the Dox solutions with different known concentrations, and measuring the absorbance at 480nm by using an ultraviolet spectrophotometer to construct a Dox standard curve. Taking liposome drug sample, centrifuging at 10000rpm for 20min at 4deg.C, and collecting supernatant to obtain liposome without free Dox. 10. Mu.L of the sample to be tested was taken, 1% Triton X-100 was added to break the membrane, the absorbance was measured by an ultraviolet spectrophotometer, and the amount of the drug encapsulated in the liposome was calculated by substituting the standard curve (FIG. 20G, H). And directly demulsifying the same amount of sample to obtain a clear solution, wherein the same step can obtain the sum of the encapsulated drug quantity and the unencapsulated drug quantity of the liposome, namely the total drug quantity. The encapsulation efficiency calculation formula: encapsulation efficiency = encapsulation amount/total drug amount x 100%.
4) Drug loading rate
Taking liposome drug sample, centrifuging at 10000rpm for 20min at 4deg.C, and collecting supernatant to obtain liposome without free Dox. 10. Mu.L of a sample to be tested was taken, 1% TritonX-100 was added to break the membrane, the absorbance was measured by an ultraviolet spectrophotometer, and the standard curve was substituted to calculate the drug loading in the liposome (FIG. 20I, J). The total amount of Dox added in the preparation of doxorubicin liposomes was the total amount. The drug loading rate calculation formula: drug loading = drug loading/total drug loading x 100%.
5) Drug release profile
In vitro release of Dox from liposomes was detected by dialysis: preparing PBS buffer solution with PH=7.4, adding 2mL of sample solution into a nano dialysis device, dialyzing at room temperature and rotating speed of 1500rpm/min, taking 50 mu L of dialysate samples after 3, 20, 24, 44, 51, 68 and 72 hours respectively, adding 1% Triton x-100 membrane rupture, measuring absorbance at 480nm by an ultraviolet spectrophotometer, measuring the content of Dox in liposome, and plotting a cumulative release curve of Dox by taking time as an abscissa and cumulative release rate as an ordinate (figure 20K).
Example 8 cytological detection of drug delivery System
1) U87-MG cell culture and treatment
Taking out the U87-MG cells from liquid nitrogen, rapidly placing the U87-MG cells into a water bath kettle at 37 ℃, slightly shaking a freezing tube to dissolve the freezing solution, transferring the cells into a centrifuge tube containing 5mL of culture medium after dissolving, centrifuging to collect the cells, centrifuging at room temperature of 1000rmp for 5min, and discarding supernatant. Suspending cells in MEM complete medium containing 10% foetal calf serum, inoculating into culture dish, gently stirring, mixing at 37deg.C with 5% CO 2 Culturing under saturated humidity.
2) U87-MG cell viability assay
Cell viability experiments were performed using the CCK-8 method, with the Dox, liposome-Dox (Lipo@Dox), HLR modified liposome-Dox (HLR-Lipo@Dox) and LRK modified liposome-Dox set (LRK-Lipo@Dox) set with the effective concentration gradients of Dox set at 0.1, 0.5, 2, 10, 50 μg/mL. According to the experimental group, each group was set with 3 duplicate wells, each of which was seeded with 3000 cells in the logarithmic growth phase. Observing the cell state, when the confluence is 50-60%, diluting the medicine to be detected into different concentrations by using a culture medium in advance, absorbing the culture medium, adding 100 mu L of the culture medium containing different medicine concentrations into each hole, culturing for 48 hours, adding 10 mu L of CCK-8 solution into each hole, incubating for 4 hours, placing a 96-well plate into an enzyme-labeled instrument, and measuring the absorbance at the wavelength of 450 nm. Cell viability formula [ A (sample well) -A (blank) ]/[ A (PBS) -A (blank) ]. Times.100%. The results showed that U87-MG cells were dose-dependent in that U87-MG cells were treated with the drug for 24 hours, and that Dox, lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox inhibited U87-MG cell viability, indicating that liposome loading of Dox and polypeptide modification of liposome-Dox did not affect the inhibition of U87-MG cells by Dox (FIG. 21).
3) U87-MG apoptosis assay
Control, dox, liposome-Dox (Lipo@Dox), HLR modified Liposome-Dox (HLR-Lipo@Dox) and LRK modified Liposome-Dox group (LRK-Lipo@Dox) were set, with the effective concentration gradient of Dox set to 0.1, 0.5, 2, 10, 50 μg/mL. Washing U87-MG cells in logarithmic growth phase with PBS for 3 times, respectively digesting and collecting with EDTA-free 0.25% pancreatin, centrifuging at 1000rpm/min for 5min, removing supernatant, washing with PBS for two times, and washing off residual culture medium; apoptotic cells were detected using an annexin V/PI double-staining kit, 100. Mu.L of 1 Xbinding Buffer was added to resuspend cells, 5. Mu.L of Lannexin V-FITC and 5. Mu.L of PI were added respectively, gently mixed, reacted at room temperature for 10min in the absence of light, 400. Mu.L of Binding Buffer was added, and detection was performed on the cells. The results showed that U87-MG cells were dose-dependent in that U87-MG cells were treated with the drug for 24 hours, and that Dox, lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox induced apoptosis in U87-MG cells, indicating that loading of liposomes with Dox and modification of liposomes with polypeptide-Dox did not affect apoptosis in U87-MG cells induced by Dox (FIG. 22).
4) U87-MG cell invasion assay
Control, dox, liposome-Dox (Lipo@Dox), HLR modified Liposome-Dox (HLR-Lipo@Dox) and LRK modified Liposome-Dox group (LRK-Lipo@Dox) were set, where the effective concentration of Dox was set to 5 μg/mL. The position of the part Washing U87-MG cells in logarithmic growth phase with PBS for 3 times, respectively digesting and collecting with 0.25% pancreatin, centrifuging at 1000rpm/min for 5min, removing supernatant, washing with PBS for two times, and washing to remove residual serum; serum-free MEM medium was used to resuspend cells, and the cell counting plate was used to count the cells, and the serum-free MEM medium was used to dilute the cells to a concentration of 4X 10 5 cell/mL for use; mu.L of MEM medium (containing double antibody) of 10% FBS was previously added to a 24-well plate, and placed in a Transwell chamber, and 200. Mu.L of each cell suspension was added to the Transwell upper chamber after 1 hour, at 37℃and 5% CO 2 Culturing in an incubator for 24 hours; the Transwell was removed, the chamber was carefully washed once with PBS, and cells were fixed with 70% glacial ethanol solution for 1 hour; dyeing with 0.5% crystal violet dye solution, standing at room temperature for 20min, washing with PBS, and wiping off non-migrated cells at one side of the upper chamber with clean cotton ball; the Transwell cells were photographed under microscopic observation, 400 x magnification, and 3 fields of view were taken for each group. The results show that the cell invasion capacity of the Dox, lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox groups is obviously reduced compared with that of the control group after the U87-MG cells are treated by the medicaments, and the Dox group is more than the lipo@Dox group, the LRK-lipo@Dox group is more than the HLR-lipo@Dox group (figure 23), which shows that the cells can absorb more lipo@Dox due to the targeting combination effect of the LRK and the HLR; the targeting binding effect of HLR is superior to LRK, therefore, HLR-lipo@Dox group has the least cell invasiveness.
5) U87-MG cell migration assay
Control, dox, liposome-Dox (Lipo@Dox), HLR modified Liposome-Dox (HLR-Lipo@Dox) and LRK modified Liposome-Dox group (LRK-Lipo@Dox) were set, where the effective concentration of Dox was set to 5 μg/mL. U87-MG cells in logarithmic growth phase were washed 3 times with PBS, collected by digestion with 0.25% pancreatin, centrifuged at 1000rpm/min for 5min, the supernatant removed, washed twice with PBS, and residual serum was washed off. Serum-free MEM medium was used to resuspend cells, and the cell counting plate was used to count the cells, and the serum-free MEM medium was used to dilute the cells to a concentration of 3X 10 5 /mL, ready for use. Thawing Matrigel one day in advance at 4deg.C, pre-cooling the Transwell chamber, 24-well culture plate and gun head overnight at-20deg.C; diluting Matrigel with serum-free medium to a final concentration of 5mg/mL, and performing on ice; mu.L of 10% FBS MEM medium (containing double antibody) was pre-chilled at 4deg.C in 24 well plates) Placing into a Transwell chamber, vertically adding 100 μl of Matrigel with final concentration of 1mg/mL at the center of the bottom of the upper chamber of the Transwell chamber, incubating at 37deg.C for 4-5 hr to make Matrigel dry into gel, and respectively introducing 200 μl of cell suspensions of each group into the upper chamber of the Transwell chamber after Matrigel is dried into gel, 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours; the Transwell was removed, the chamber was carefully washed once with PBS, and cells were fixed with 70% glacial ethanol solution for 1 hour; dyeing with 0.5% crystal violet dye solution, standing at room temperature for 20min, washing with PBS once, and wiping off non-migrated cells at one side of the upper chamber with clean cotton balls; the Transwell cells were photographed under microscopic observation, 400 x magnification, and 3 fields of view were taken for each group. The results show that the cell migration capacity of the Dox, lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox groups is obviously reduced after the U87-MG cells are treated by the medicament, and the Dox group is more than the lipo@Dox group, the LRK-lipo@Dox group is more than the HLR-lipo@Dox group, which indicates that the cells can absorb more lipo@Dox due to the targeting combination effect of the LRK and the HLR; the targeting binding of HLR is superior to LRK, therefore, the cell migration capacity of HLR-lipo@dox group is the weakest (fig. 24).
6) Cloning experiments on U87-MG cells
Control, dox, liposome-Dox (Lipo@Dox), HLR modified Liposome-Dox (HLR-Lipo@Dox) and LRK modified Liposome-Dox group (LRK-Lipo@Dox) were set, with an effective concentration gradient of Dox set at 5 μg/mL. U87-MG cells in the logarithmic growth phase were washed 3 times with PBS, digested with 0.25% trypsin and blown into individual cells, and the cells were suspended in the medium for use. The cell suspension was diluted and seeded at 400 cell densities in 6-well plates. The cells were dispersed evenly by gentle rotation. The dishes were placed at 37℃with 5% CO 2 Culturing in an incubator for 3 weeks. When clones appeared, the supernatant was discarded, washed 2 times with PBS and fixed with 75% glacial ethanol for 15min. Removing the fixing solution, and adding a proper amount of 0.5% crystal violet for dyeing for 30min. Washed with PBS and counted. The calculation formula is as follows: clone formation rate = number of clones/number of inoculated cells x 100%. The results show that the cell clone survival of the Dox, lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox groups is significantly reduced after the U87-MG cells are treated by the drug, and that the Dox group > Lipo@Dox group > LRK-lipo@Dox group > HLR-lipo@DoGroup x, demonstrating that cells are able to ingest more lipo@dox due to the targeted binding of LRK and HLR; the targeted binding of HLR is superior to LRK, therefore, cell survival of HLR-lipo@dox group is minimal (fig. 25).
EXAMPLE 9 polypeptide modified Adriamycin Liposome crossing the blood brain Barrier experiment
1) U87-MG cell uptake polypeptide modified doxorubicin liposome
Control, dox, liposome-Dox (Lipo@Dox), HLR modified Liposome-Dox (HLR-Lipo@Dox) and LRK modified Liposome-Dox group (LRK-Lipo@Dox) were set, where the effective concentration of Dox was set to 5 μg/mL. Human umbilical vein endothelial cells HUVEC and U87-MG cells were spread on the upper and lower chambers of Transwell (coverslips) respectively at a ratio of 1:5, cultured for 48 hours to simulate tumor microvascular environment, HUVEC cell culture solution in the upper chamber of Transwell was aspirated, and coverslips were added to the upper chamber and incubated with 5. Mu.g/mL of Dox, lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox containing 10% FBS at an effective concentration of Dox, and incubated at 37℃for 8 hours, respectively, to collect coverslips of the lower chamber. Washing 3 times with precooled PBS, fixing with 4% paraformaldehyde for 15min, naturally airing, adding 5 mug/mL DAPI to dye the cell nuclei, sealing with a quenching inhibitor, scanning with a tissue section scanner and analyzing. The results show that compared with the control and Dox groups, the Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox groups can all show fluorescence signals of Cy5.5 after co-culture dosing treatment, which indicates that all three groups of drugs can pass through HUVEC cells and are taken up by U87-MG cells; in addition, the targeted binding of HLR is superior to LRK because of the targeted binding of LRK to HLR, which allows cells to take up more lipo@Dox, thus the fluorescent signal of HLR-lipo@Dox group is strongest (FIG. 26).
2) U87-MG cell in situ tumor animal model establishment
And establishing an in-situ tumor model of the U87-MG-luc cell mouse by using a brain stereotactic instrument. Taking U87-MG-luc cells in logarithmic growth phase, digesting with 0.25% pancreatin, and adjusting cell density to 1×10 5 5. Mu.L of the cell suspension was aspirated with a brain stereotactic apparatus and injected into the intracranial posterior ventricle (striatum, right bregma 1.8mm, depth 3 mm) of female BABL/c nude mice. 14 days after inoculation, the tumor formation is observed by imaging and photographing living bodies of small animalsThe condition is as follows.
3) Polypeptide modified doxorubicin liposomes across blood brain barrier experiments
Tumor-bearing nude mice were injected with Cy5.5 modified liposome-Dox of example 5 via tail vein respectively
(lipo @ Dox), cy5.5 and HLR modified Liposome-Dox (HLR-lipo @ Dox), cy5.5 and LRK modified Liposome-Dox group (LRK-lipo @ Dox) saline injection, dox effective concentration of 2.5MG/kg, 2, 4, 8, 24 hours post injection using small animal fluorescence biopsy imaging device to collect images (FIG. 27), nude mice not inoculated with U87-MG cells were intravenously injected with the same volume of saline as the drug. The results show that the nude mice brains of Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox groups can continuously display fluorescent signals, and the fluorescent signals at each time point are HLR-Lipo@Dox group & gt, LRK-Lipo@Dox group & gt, lipo@Dox group, which shows that the efficiency of taking medicines by the brains is higher due to the targeting combination effect of the LRK and the HLR. The mice organs and brains were separated 24 hours after injection, and images were acquired using a small animal fluorescence living imager, and the results showed that the tumor-bearing mice brain fluorescence signal intensity was HLR-lipo@Dox group & gt, LRK-lipo@Dox group & gt, lipo@Dox group (FIG. 28), indicating that lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox were all able to cross the blood brain barrier, and that the targeting effect of HLR was stronger and the order of LRK-lipo@Dox was superior to that of LRK.
Example 10 polypeptide-modified Adriamycin Liposome experiments for treating brain glioma
1) Administration and tumor observation
Experiments were divided into PBS group, dox group, cy5.5 modified liposome-Dox (Lipo@Dox), cy5.5 and HLR modified liposome-Dox (HLR-Lipo@Dox), cy5.5 and LRK modified liposome-Dox (LRK-Lipo@Dox) groups, administered intravenously at an effective concentration of 2.5mg/kg of Dox via the tail of the nude mice (days 0, 3, 6, 9, 12), once every 3 days with a weight change recorded (days 0, 3, 6, 9, 12), and tumor changes were observed with fluorescent biopsy (days 3, 6, 9, 12, 15), and nude mice were treated on day 15 of administration (FIG. 29A). The results showed that lipo@Dox, HLR-lipo@Dox and LRK-lipo@Dox can significantly inhibit tumor growth at various time points, and the inhibition effect of HLR-lipo@Dox is strongest (FIG. 29B), compared with PBS group and Dox group. HLR-lipo@Dox and LRK-lipo@Dox have no significant difference in the effect on the body weight of nude mice (FIG. 29C), indicating that the polypeptide-modified doxorubicin liposome has little toxic effect on the whole body of nude mice.
2) Paraffin section production
The brain of nude mice is taken after the treatment is finished on the 15 th day, and the nude mice are fixed for more than 24 hours by a fixing solution. (1) Dehydrating: the tissue is sequentially dehydrated by gradient alcohol. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, absolute alcohol I for 30 minutes, absolute alcohol II for 30 minutes, alcohol/xylene for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, wax III for 1 hour. (2) Embedding: and (3) putting melted wax into an embedding frame, taking out tissues from the dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20 deg.c, after solidification of the wax, the wax block is taken out of the embedding frame and trimmed. (3) Slicing: the trimmed wax block was sliced in a paraffin slicer to a thickness of 3 μm. Floating the slices on warm water at 40 ℃ of a slice spreading machine to spread tissues, spreading the tissues with a glass slide, and baking the slices in a baking oven at 60 ℃.
3) H & E staining
Sequentially placing the slices into xylene I for 20min, xylene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min, and washing with tap water. The slices are dyed by hematoxylin for 3 to 5 minutes, differentiated by hydrochloric acid aqueous solution, returned to blue by ammonia aqueous solution and washed by water; the slices are sequentially dehydrated by gradient alcohol of 85% and 95%, and finally are placed into 1% eosin dye solution for dyeing for 5min. And sequentially placing the slices into absolute ethyl alcohol I5 min, absolute ethyl alcohol II 5min, absolute ethyl alcohol III 5min, dimethylbenzene I5 min and dimethylbenzene II 5min to finish transparency, and finally sealing the slices with neutral resin, and adopting pannarac MIDI scanning slices to acquire images. The results showed that the nude mice brain grafts in the Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox groups all had reduced outlines compared to the control and Dox groups, indicating that Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox all inhibited the graft growth, and since the targeting binding effect of HLR was superior to LRK, the inhibition effect of HLR-Lipo@Dox group was most pronounced, and LRK-Lipo@Dox was inferior (FIG. 30).
3) TUNEL experiment
Apoptotic cells were detected using TUNEL assay as follows: (1) slice dewaxing: sequentially placing the slices into xylene I15 min, xylene II 15min, absolute ethanol I5 min, absolute ethanol II 5min, 85% ethanol 5min, 75% ethanol 5min, and washing with distilled water for 30min. (2) Rupture of membranes: proteinase K working solution (20. Mu.g/mL) was added dropwise to the sections to cover the tissue and incubated for 25min at 37 ℃. The slides were placed in PBS and washed 3 times with shaking on a decolorizing shaker for 5min each. (3) TUNEL reaction solution was added dropwise: appropriate amounts of reagent 1 (TdT) and reagent 2 (dUTP) in TUNEL kit were taken at 1:9, mixing, dripping the covered tissue, and incubating for 2 hours at the temperature of 37 ℃. (4) DAPI counterstaining nuclei: sections were washed 3 times with PBS for 5min each. DAPI dye (5. Mu.g/mL) was added dropwise after PBS was removed, and incubated at room temperature for 10min in the dark. (5) Sealing piece: the tablet is capped with an anti-fluorescence quench. (6) Scanning observation and result interpretation: and collecting images by adopting pannolic MIDI scanning slices, wherein nuclei dyed by DAPI are blue under the excitation of ultraviolet rays, and apoptotic nuclei are green. The results show that Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox can induce U87-MG cell apoptosis, and the number of apoptotic cells is HLR-Lipo@Dox group, LRK-Lipo@Dox group, indicating that the efficiency of inducing U87-MG cell apoptosis is higher due to the targeted binding effect of LRK and HLR (FIG. 31); and because the targeting binding effect of HLR is superior to LRK, the apoptosis of U87-MG cells of HLR-lipo@Dox group is most obvious.
3) Detection of Ki-67 expression
The expression of Ki67 protein is detected by immunofluorescence technique, and the steps are as follows: (1) slice dewaxing: sequentially placing slices into xylene I15 min, xylene II 15min, absolute ethanol I5 min, absolute ethanol II 5min-85% ethanol 5min, 75% ethanol 5min, and washing with distilled water for 30min. (2) Antigen retrieval: the sections were placed in EDTA-containing antigen retrieval buffer and subjected to antigen retrieval in a microwave oven. Stopping the fire for 8min at the middle fire and turning the fire for 8min to the middle fire and the low fire for 7min. (3) Closing: BSA was added dropwise and incubated for 30min. (4) Incubation resistance: the blocking solution was gently aspirated, ki67 primary antibody formulated in a proportion with PBS was added dropwise to the sections, and the sections were placed flat in a wet box for incubation overnight at 4 ℃. (5) Secondary antibody binding: and (3) dripping secondary antibody covering tissues of the corresponding species with the primary antibody, and incubating for 1 hour at room temperature in dark. (6) DAPI counterstaining nuclei: DAPI dye (5. Mu.g/mL) was added dropwise and incubated at room temperature for 10min in the dark. (7) Sealing piece: and (5) after the slices are slightly dried, sealing the slices by using an anti-fluorescence quenching sealing tablet. (8) Scanning observation and result interpretation: images were acquired using pannarac MIDI scan slices. Nuclei stained with DAPI are blue under uv excitation, and positive expression is red light of the corresponding fluorescein label. The results show that each of Lipo@Dox, HLR-Lipo@Dox and LRK-Lipo@Dox can inhibit the expression of U87-MG cells Ki67, and the number of cells of Ki67 is smaller than that of the HLR-Lipo@Dox group and smaller than that of the LRK-Lipo@Dox group, lipo@Dox group (FIG. 32), which shows that the efficiency of inhibiting the expression of U87-MG cells Ki67 is higher due to the targeting combination effect of LRK and HLR; and because the targeting binding effect of HLR is superior to LRK, the apoptosis of U87-MG cells of HLR-lipo@Dox group is most obvious.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (7)
1. A liposome modified by polypeptide for targeted treatment of glioblastoma, comprising polypeptide for binding to targeting TREM2 protein of glioblastoma, medicine with killing effect on glioblastoma cells and liposome wrapping the medicine;
the polypeptide of the targeting TREM2 protein is modified on the liposome, and the liposome modified with the polypeptide encapsulates the drug;
wherein, the amino acid sequence of the polypeptide targeting TREM2 protein is as follows: HLRKLRKR, histidine-leucine-arginine-lysine-arginine, his-Leu-Arg-Lys-Leu-Arg-Lys-Arg, abbreviated HLR.
2. The liposome according to claim 1, characterized in that: the medicine is doxorubicin;
the liposome is prepared from lecithin and DSPE-PEG2000 serving as raw materials.
3. A method of preparing the liposome of claim 1 or 2, comprising the steps of:
1) Reacting DSPE-PEG2000-Mal with polypeptide HLR to obtain DSPE-PEG2000-HLR;
2) Preparing liposome solution by using lecithin and DSPE-PEG2000-HLR as raw materials and adopting an ammonium sulfate gradient method;
3) Adding a medicament with killing effect on glioblastoma cells into the obtained liposome solution, and incubating to obtain the glioblastoma cell killing agent.
4. A method according to claim 3, characterized in that: in the step 1), the molar ratio of DSPE-PEG2000-Mal to polypeptide HLR is 1:1.5-2,
the reaction is carried out in water at 4 ℃ under the protection of inert gas, and the reaction time is 24-48 hours.
5. A method according to claim 3 or 4, characterized in that: the operation of the step 2) is as follows: dissolving lecithin and DSPE-PEG2000-HLR in chloroform, respectively, steaming to form film, adding (NH) 4 ) 2 SO 4 Solution, hydration; preparing liposome solution after ultrasonic dispersion; dialysis to remove uncoated (NH) 4 ) 2 SO 4 Obtaining liposome solution;
wherein the mass ratio of lecithin to DSPE-PEG2000-HLR is 6-9:1;
Said (NH) 4 ) 2 SO 4 The concentration of the solution was 250mM,
lecithin and (NH) 4 ) 2 SO 4 The proportion of the solution is 18mg:5mL;
the dialysis was performed overnight in 50 mmph=7.4 PBS.
6. A method according to claim 3 or 4, characterized in that: in the step 3), the drug with killing effect on glioblastoma cells is doxorubicin;
the mass ratio of lecithin to the drug was 18mg:1mg;
the incubation was at 50℃for 1 hour,
dialysis was also performed after incubation was completed to remove non-entrapped drug.
7. Use of a polypeptide-modified liposome for targeted therapy of glioblastoma according to claim 1 or 2 in the preparation of a medicament for the treatment of human glioma or human glioma cell transplantation tumor.
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