CN115531554B - Synthetic lethal nano-drug combination carrier and application thereof in preparation of GBM targeted drugs - Google Patents

Synthetic lethal nano-drug combination carrier and application thereof in preparation of GBM targeted drugs Download PDF

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CN115531554B
CN115531554B CN202211270462.6A CN202211270462A CN115531554B CN 115531554 B CN115531554 B CN 115531554B CN 202211270462 A CN202211270462 A CN 202211270462A CN 115531554 B CN115531554 B CN 115531554B
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CN115531554A (en
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吴海刚
师冰洋
刘媛媛
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Henan University
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Abstract

The invention belongs to the technical field of nano-drug preparation, and discloses a nano-drug combination carrier, which comprises polypeptide, polyhydroxy-containing amide compounds, bipyridyl group-containing amide compounds, boron-hydroxyl-containing proteasome chymotrypsin-like activity inhibition drugs, histone deacetylase inhibition drugs and disulfide bond-containing compounds; the polyhydroxy-containing amide compound and the proteasome chymotrypsin-like activity inhibiting drug containing boron hydroxyl generate a boron ester bond through reaction, and the bipyridyl-containing amide compound and the histone deacetylase inhibiting drug are chelated through zinc ions. The combined carrier has the capability of responding to the reduction, acid and chelation competition to release the medicines, can simultaneously load two medicines, has high release capability and controllable time and space, and plays a synergistic effect.

Description

Synthetic lethal nano-drug combination carrier and application thereof in preparation of GBM targeted drugs
Technical Field
The invention belongs to the technical field of nano-drug preparation, and relates to a synthetic lethal nano-drug combination carrier and application thereof in preparing a GBM targeted drug.
Background
Glioblastoma (GBM) is the most common primary in-furnace malignancy that originates from glial cells. The annual incidence of GBM is 3-8/10 ten thousand, accounting for about 27% of all tumors of the central nervous system, 80% of malignant tumors, and GBM is also increasing year by year at an annual growth rate of 1-2%. The world health organization characterizes GBM malignancy as grade IV (highest) based on whether tumor cells have the characteristics of multiple nuclear divisions, polymorphism, vascular endothelial cell proliferation, and necrosis. Currently, no effective GBM treatment means exists clinically, the survival rate of GBM patients in1 year and 5 years is only 30% and 4%, respectively, and the average median survival time is only 12-15 months.
The p53 gene encodes a protein with a molecular weight of 43.7 kDa. Inactivation of the p53 Gene against swellingNeoplasia plays an important role. In all malignant tumors, mutations in this gene occur in more than 50%. Mutation of p53 (TP 53) mut ) Highly involved in the development and progression of various tumors including glioblastoma multiforme (GBM), and directly targets TP53 mut Drug resistance is always induced and treatment is limited. Synthetic Lethality (SL) is a strategy to block the compensatory pathway of oncogene mutations by pharmaceutical intervention to induce cell death, with great potential for the treatment of GBM.
For therapeutic approaches using combinatorial chemicals, reasonable drug ratios, simultaneous delivery, effective drug concentration, tissue targeting and accurate therapeutic window remain unresolved issues, which limit further application of chemicals in tumor therapy. Random encapsulation of the formed nano-drug makes it difficult to precisely adjust the ratio of drug combinations, and pre-mixing the nano-drug alone to encapsulate the drug may overcome this disadvantage, but the uniformity of the properties of the nano-particles prepared when different drugs are prepared is difficult to control. Thus, rational assignment of co-delivery nanoplatforms may be an ideal strategy for synthetic lethality, with significantly reduced toxicity to normal cells and reduced potential damage to non-target organs or tissues by precisely controlled drug administration.
Disclosure of Invention
The invention aims to provide a synthetic lethal nano-drug combination carrier which has the capacity of responding to drug release by reduction, acid and chelation competition, can be used for simultaneously loading two drugs, has high drug release capacity and controllable space and time, and plays a synergistic effect.
The second object of the invention is to provide the application of the synthetic lethal nano-drug combination carrier in preparing the GBM targeted drug, which has good biological safety, and compared with the free drug, the nano-drug enhances the synthetic lethal effect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a synthetic lethal nano-drug combination carrier, which comprises polypeptide, an amide compound containing polyhydroxy, an amide compound containing bipyridyl groups, a proteasome chymotrypsin-like activity inhibiting drug containing boron hydroxy groups, a histone deacetylase inhibiting drug and a compound containing disulfide bonds; the polyhydroxy-containing amide compound and the proteasome chymotrypsin-like activity inhibiting drug containing boron hydroxyl generate a boron ester bond through reaction, and the bipyridyl-containing amide compound and the histone deacetylase inhibiting drug are chelated through zinc ions.
In one embodiment, the proteasome chymotrypsin-like activity inhibiting drug containing a boron hydroxyl group is bortezomib.
In one embodiment, the histone deacetylase inhibitor is panobinostat.
In one embodiment, the polypeptide is a sulfhydryl modified polypeptide.
In one embodiment, the polyhydroxy containing amide compound is selected from the group consisting of N-acryloylglucosamine.
In one embodiment, the amide containing a bipyridyl group is selected from the group consisting of N- (2-acrylamidoethyl) - [2,2' -bipyridyl ] -5-carboxamide, N- (3-acrylamidopropyl) - [2,2' -bipyridyl ] -5-carboxamide, and N- (4-acrylamidobutyl) - [2,2' -bipyridyl ] -5-carboxamide.
In one embodiment, the composite carrier has a spherical structure.
The invention also provides application of the nano-drug combination carrier in preparing a GBM targeted drug.
Compared with the prior art, the invention has the beneficial effects that:
the nano-drug combination carrier prepared by the invention has the capability of responding to drug release by reduction, acid and chelation competition, can simultaneously load two drugs, has high drug loading efficiency, has high drug release capability and controllable time and space, and plays a synergistic effect.
Compared with the nano-drug without the polypeptide, the nano-drug combination carrier prepared by the invention comprises the polypeptide modified by sulfhydryl, and has the capability of actively targeting GBM cells (SNB 19 and LN 229) with high expression of LRP1 protein on the surface of cell membrane.
The invention uses nano drug combination carrier to prepare nano drug, in vitro cell experiment and in situ TP53 mut In GBM mouse model, nano-drugs enhance synthetic lethal effects compared to free drugs; has good biological safety, and has no side effects on blood routine, blood biochemical, organs of mice and normal brain tissues.
Drawings
FIG. 1 shows the synthetic route of AGA according to the invention.
FIG. 2 shows the results of nuclear magnetic characterization of AGA according to the invention.
FIG. 3 shows the synthetic route of AGA-BTZ according to the invention.
FIG. 4 shows the results of nuclear magnetic characterization of AGA-BTZ of the present invention.
FIG. 5 is a synthetic route for AABC according to the invention.
FIG. 6 is a graph showing the results of nuclear magnetic characterization of AABC in accordance with the present invention.
FIG. 7 shows the synthetic route of AABC-Zn-LBH589 of the present invention.
FIG. 8 is an ultraviolet absorption spectrum of AABC-Zn-LBH589 of the present invention.
FIG. 9 shows a process for the preparation of ApoE-NM@ (BTZ/LBH 589) according to the invention.
FIG. 10 shows TEM, size, dispersion coefficient and surface potential of ApoE-NM@ (BTZ/LBH 589), NM@ (BTZ/LBH 589), NM@BTZ, NM@LBH589 and ApoE-NM of the present invention.
FIG. 11 is a graph of the ability of the nanomedicine of the present invention to responsively release a drug, wherein FIG. 11A is a graph of drug release over time; fig. 11B is a change in nanoparticle morphology (TEM characterization) after drug release.
FIG. 12 shows the characterization of receptor expression of polypeptide ApoE, including LRP1, LDLR and LRP2, on the surface of the cell membrane of GBM (SNB 19 and LN 229) and normal astrocytes (HA 1800) by immunoblotting.
FIG. 13 shows the targeting ability of ApoE of the invention to SNB19 and LN229 cells.
FIG. 14 is an in vitro BBB model construction and nanomaterials' ability to cross the blood brain barrier assessment of the present invention.
FIG. 15 is an evaluation of the effect of the nanomedicine of the present invention on cell survival inhibition.
FIG. 16 is a graph showing the effect of free drug and nanomedicine of the present invention on SNB19 and LN229 endoplasmic reticulum stress.
FIG. 17 shows the degree of apoptosis of SNB19 and LN229 cells induced by free drug and nano-drug of the invention.
FIG. 18 is an in vivo distribution of a nano-drug according to the present invention, wherein FIG. 18A is an IVIS Lumina III imaging system for monitoring in real time the in vivo distribution of a nano-drug in an in situ tumor-bearing mouse; 18B is that after the nano-drug is injected for 4 hours, the IVIS Lumina III imaging system detects the distribution condition of the nano-drug in each organ of the mice; the 18C is obtained after the nano-drug is injected for 4 hours, and the distribution condition of the nano-drug is detected by a fluorescence microscope after the frozen section treatment; and 18D is the distribution of the nano-drugs quantitatively counted after taking normal brain tissues, tumor tissues and organs of the mice and homogenizing the normal brain tissues, tumor tissues and organs of the mice after the nano-drugs are injected for 4 hours.
FIG. 19 is an in situ TP53 of a nano-drug according to the present invention mut GBM treatment experimental procedures and results, wherein fig. 19A is a flow chart of an animal experiment; FIGS. 19B and 19C show the weight change and survival period of mice in SNB19 in situ tumor-bearing model; fig. 19D and 19E show the change in weight and survival period of mice in LN229 in situ tumor-bearing model.
FIG. 20 is H & E staining results for each treatment group according to the invention, wherein FIG. 20A is tumor size; FIG. 20B shows the presence or absence of lesions in normal brain tissue and organs of mice.
FIG. 21 shows TUNEL positive signal distribution and statistical data for each treatment group according to the invention.
FIG. 22 shows the results of immunohistochemical analysis of each treatment group according to the present invention, and FIG. 22A shows the distribution of Ki67 and CC3 positive signals in each treatment group by immunohistochemical characterization; fig. 22B and 22C show the distribution of CC3 in normal and tumor tissues of the brain and statistical data.
FIG. 23 shows the nano-drug pair in situ TP53 in the present invention mut Apoptosis-related protein regulation results in GBM tumors.
FIG. 24 shows the effect of the nano-drug of the present invention on the routine and biochemical results of mouse blood, wherein FIG. 24A shows the routine results of mouse blood, and FIG. 24B shows the biochemical results of mouse blood.
FIG. 25 is a graph showing the effect of the nano-drug of the present invention on inflammatory factor expression in the liver and kidney of mice.
Detailed Description
The following examples are illustrative of the present invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified.
Example 1
1. Preparation of prodrug AGA-BTZ
1.1 preparation of AGA
N-acryloylglucosamine (AGA) was prepared using D- (+) -galactosamine hydrochloride and acryloyl chloride (Aladine, china), the specific synthetic route is shown in FIG. 1. 20mL of 1mol/L K containing 0.02mol of D- (+) -galactosamine hydrochloride 2 CO 3 Put into a reaction bottle and put into an ice bath for cooling. To the solution was slowly added dropwise 0.024mol of acryloyl chloride with vigorous stirring. The reaction was maintained at 0-4 ℃ for 4 hours and then slowly returned to room temperature for 24 hours to recover unreacted acryloyl chloride. Subsequently, the crude product was freeze-dried and the product AGA was purified by column chromatography on a silica gel column with a methanol/ethyl acetate (20/80 v/v) mixture as eluent. Using 1 AGA was detected by H NMR (300 MHz) using DMSO-d6 as a solvent, and the results are shown in FIG. 2. Nuclear magnetism @ 1 HNMR) results showed that AGA was successfully prepared and nearly 100% pure.
1.2 preparation of AGA-BTZ
Bortezomib (BTZ) is a reversible inhibitor of 26S proteasome chymotrypsin-like activity in mammalian cells. AGA and BTZ react to generate AGA-BTZ, the boron ester bond in the AGA-BTZ has acid response capability, controllable release of the medicine can be realized, and the synthetic route of the AGA-BTZ is shown in figure 3.
AGA and BTZ (Nanjing Kang Manlin) of the same molar mass were dissolved in ddH respectively 2 O and methanol, and added to the reaction flask. The pH of the reaction solution was adjusted to 8.5 to 9.0 with NaOH, and the reaction was continued for 48 hours. By first rotatingThe methanol was removed by evaporator, the sample was lyophilized with a freeze dryer, and finally the product AGA-BTZ was purified on a silica gel column using column chromatography. Using 1 H NMR (300 MHz) with CD 3 OD was used as a solvent to measure the purity of AGA-BTZ, and the results are shown in FIG. 4. Nuclear magnetism @ 1 HNMR) results showed that AGA-BTZ was successfully prepared and had a purity approaching 100%.
2. Preparation of prodrug AABC-Zn-LBH589
2.1 preparation of AABC
The AABC synthesis route is shown in figure 5. Will [2,2' -bipyridine]-5-Carboxylic acid (0.6 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 1.2 mmol) and N-hydroxysuccinimide (NHS, 0.8 mmol) were dispersed in Tetrahydrofuran (THF), respectively, and added to a reaction flask, reacted for 2 hours to activate carboxyl groups, and then N- (2-aminoethyl) acrylamide (0.8 mmol) was dispersed in ddH 2 O was added to the flask, and the reaction was continued for 24 hours. After removal of THF by rotary evaporator, extraction with ethyl acetate gave the product N- (2-acrylamidoethyl) - [2,2' -bipyridine]-5-carboxamide (AABC). And is used in combination 1 H NMR detection. Using 1 H NMR (400 MHz) in DMSO-d 6 The results of the purity test of AABC as a solvent are shown in fig. 6. Nuclear magnetism @ 1 HNMR) results showed that AABC was successfully prepared and nearly 100% pure.
Similarly, the present invention can also replace N- (2-aminoethyl) acrylamide with N- (3-aminopropyl) acrylamide, N- (4-aminobutyl) acrylamide according to the synthetic route shown in FIG. 6 to give N- (3-acrylamidopropyl) - [2,2 '-bipyridine ] -5-carboxamide (AABC-1) and N- (4-acrylamidobutyl) - [2,2' -bipyridine ] -5-carboxamide (AABC-2), respectively.
2.2 preparation of AABC-Zn-LBH589
Panobinostat (LBH 589) is a potent oral Histone Deacetylase (HDAC) inhibitor. The prodrug AABC-Zn-LBH589 was prepared by zinc ion chelation between hydroxamic acid of LBH589 and bipyridine of AABC, the synthetic route is shown in FIG. 7.
First, equimolar AABC is slowly added dropwise to ZnSO 4 ·7H 2 In the O solution, after chelation for 15 minutes,equimolar LBH589 (south tokyo Kang Manlin) was added to the reaction solution and stirred overnight. Finally, whether AABC-Zn-LBH589 was successfully prepared or not was determined by ultraviolet absorption spectroscopy, and the result is shown in FIG. 8. The detection result of the ultraviolet absorption spectrometer shows that AABC and LBH589 are successfully connected through zinc ion chelation, and AABC-Zn-LBH589 is obtained.
3. Preparation of ApoE-PEG
The polypeptide ApoE sequence is LRKLRKRLLLRKLRKRLLC, and is an amino acid complex with higher brain targeting efficiency. Thiol-modified polypeptides ApoE (ApoE-SH, jiang Yao organism), acrylic ester PEG2000 maleimide (Acrylamide-PEG 2000-Mal, abbreviated as APEG-Mal, 5. Mu. Mol) and triethylamine (15. Mu. Mol) were dispersed in dimethyl sulfoxide (DMSO) which had previously been deoxygenated, respectively, and after mixing, reacted at 37℃for 24 hours under nitrogen-protected anaerobic conditions in the absence of light. To remove unreacted ApoE-SH, APEG-Mal and triethylamine, the reaction solution was placed in a dialysis bag of 3.4kDa cut-off, at ddH 2 Dialysis in O for 2 days (6 times change of dialysate) followed by lyophilization afforded the product ApoE-PEG. Finally, the purity of APEG was detected by BCA protein quantification. The BCA protein quantification demonstrated that the ratio of ApoE to PEG was approximately 100%.
4. Preparation and characterization of nanomedicines
4.1 preparation of nano-drug
To prepare the nano-drug ApoE-NM@ (BTZ/LBH 589), the flask was first purged with nitrogen for 30min to remove oxygen, followed by 0.60. Mu. Mol of AABC-Zn-LBH589 (dispersed in ethanol/ddH) 2 O (3/1), 0.18. Mu. Mol AGA-BTZ, 0.045. Mu. Mol ApoE-PEG, 0.135. Mu. Mol APEG and 0.18. Mu. Mol N, N' -bis (acryl) cystamine (BACA) were mixed under nitrogen protection and added to a reaction flask placed in an ice bath, and stirred and mixed for 15 minutes. Subsequently, tetramethyl ethylenediamine (TEMED, 0.88 nmol) and ammonium persulfate (APS, 0.88 nmol) were added to the reaction flask under nitrogen protection, the flask was closed, and the polymerization was performed in an ice bath for 2 hours. After the reaction is terminated, the polymer solution is dropwise added into 1 XPBS buffer solution to form nano-drugs. Finally, to purify the nanomaterials, excess components were removed using ultrafiltration centrifuge tubes with a molecular weight cut-off of 30kDaAnd washing the nano-drug 3 times by using 1 XPBS to finally obtain the nano-drug ApoE-NM@ (BTZ/LBH 589). The specific route is shown in fig. 9.
In the preparation of nano-drug NM@ (BTZ/LBH 589), no ApoE-PEG is required, the molar amount of APEG added is 0.18 mu mol, and other components and steps are the same as those in the preparation of ApoE-NM@ (BTZ/LBH 589).
In the preparation of nano-drug NM@BTZ, AGA-BTZ is replaced by equimolar AGA, and other components and steps are the same as those in the preparation of ApoE-NM@ (BTZ/LBH 589).
In the preparation of nano-drug NM@BTZ, AABC-Zn-LBH589 is replaced by equimolar AABC, and other components and steps are the same as those in the preparation of ApoE-NM@ (BTZ/LBH 589).
In the preparation of drug-free nanoparticles ApoE-NM, AABC-Zn-LBH589 was replaced with equimolar AABC, AGA-BTZ with equimolar AGA, and the other components and steps were the same as in the preparation of ApoE-NM@ (BTZ/LBH 589).
In addition, to prepare a Cy 5-loaded nano-drug, N- (2-aminoethyl) acrylamide (0.09. Mu. Mol) and NHS-Cy5 (0.09. Mu. Mol) were added during polymerization, with the other steps unchanged.
4.2 drug Loading efficiency
And taking an external liquid in the ultrafiltration centrifuge tube, detecting the contents of BTZ and LBH589 in ApoE-NM@ (BTZ/LBH 589) and NM@ (BTZ/LBH 589) by using a High Performance Liquid Chromatograph (HPLC), namely, redundant free drugs which do not form nano drugs, and then calculating to obtain the Drug Loading Efficiency (DLE). The results are shown in Table 1. Results and analysis: the loading efficiencies of NM@BTZ/LBH589 for BTZ and LBH589 were about 68.7% and 75%, respectively; the loading efficiencies of ApoE-NM@BTZ/LBH589 for BTZ and LBH589 were about 65.3% and 79.5%, respectively. The loading efficiency of NM@BTZ to BTZ is about 65.3%; the loading efficiency of NM@LBH589 to LBH589 was about 71.6%.
TABLE 1 Loading efficiency of nanomedicine on BTZ and LBH589
Nanometer medicine BTZ-DLE(％) LBH589-DLE(％)
ApoE-NM@(BTZ/LBH589) 68.7±0.9 75.0±5.0
NM@(BTZ/LBH589) 65.3±6.0 79.5±5.6
NM@BTZ 65.3±1.9 /
NM@LBH589 / 71.6±0.5
4.3 characterization of the nano-drug
In order to detect whether various nano-drugs are successfully prepared, a dynamic light scattering instrument (DLS) and a Transmission Electron Microscope (TEM) are adopted to respectively detect hydrodynamic size, dispersion coefficient, surface potential, morphology and the like of the nano-drugs. The results are shown in FIG. 10. Results and analysis: the nano-drug has a size smaller than 200nm and uniform size distribution (PDI < 0.3), a spherical structure and a negative charge on the surface.
5. Responsive drug release properties
Since the disulfide bonds in BACA have Glutathione (GSH) or Dithiothreitol (DTT) response capability, the boron ester bonds in AGA-BTZ have acid response capability, and AABC-Zn-LBH589 has chelating competition capability, the invention simulates the stimulus response capability of the chemical bonds and the conditions of drug release and nanoparticle structural disruption caused by the response in vitro.
The prepared nano-drug ApoE-NM@ (BTZ/LBH 589) was placed in dialysis bags and dispersed in1 XPBS buffer solution at pH7.4, pH5.0, glycine (Gly, n (Gly)/n (AABC-Zn-LBH 589) =10/1), pH5.0/DTT (10 mM) and Gly (n (Gly)/n (AABC-Zn-LBH 589) =10/1)/DTT (10 mM), respectively, and the outer solution was taken at different time points (2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours) in a shaking table at 37℃and rotational speed of 100 rpm. Finally, the content of released BTZ and LBH589 in the external liquid is detected by a high performance liquid chromatograph. At 24h of mode drug release, the "nano-drug" in the dialysis bag was removed and its particle size and morphology were detected with a dynamic light scatterometer and transmission electron microscope, respectively, to characterize the effect of drug release on the nanostructure after stimulus response. The results are shown in FIG. 11. The results show that: under responsive stimulation, the drug is slowly released over time. At 24 hours, there was little drug release at ph 7.4; at ph5.0, about 44% btz is released from the nanoparticle; about 77% of the BTZ is released from the nanoparticle at pH5.0/DTT (10 mM); about 53% of LBH589 was released from the nanoparticle under Gly (n (Gly)/n (AABC-Zn-LBH 589) =10/1); about 75% of LBH589 was released from the nanoparticle under Gly (n (Gly)/n (AABC-Zn-LBH 589) =10/1)/DTT (10 mM). TEM characterization showed that at 24 hours of responsive drug release, the nanoparticle structure was destroyed, exhibiting an irregular structure.
6. Receptor expression and ApoE targeting ability assays
6.1 receptor expression of ApoE on GBM cell membranes
Expression of LRP1, LRP2 and LDLR on SNB19 and LN229 cell membranes was studied with normal astrocyte HA1800 as a control.
The cell membrane proteins were first extracted according to the instructions of the cell membrane and cytoplasmic protein extraction kit, then the protein concentration was determined with the BCA protein quantification kit, and the protein solution (20 μg/sample) was mixed with 1 x SDS-PAGE loading buffer and denatured at 95 ℃ for 10 minutes. Protein samples were then added to a 7.5% SDS-polyacrylamide gel well and electrophoresed at constant pressure 80v until the protein markers were completely separated, and the samples were transferred to polyvinylidene fluoride (PVDF) membranes (300 mA,1.5 hours). After the PVDF membrane is blocked by 5% skimmed milk for 1 hour, the PVDF membrane is cut according to the molecular weight and the position of a protein marker, and is respectively incubated with a primary antibody anti-LRP1 rabit (1:1000 dilution), anti-LRP2 rabit (1:1000 dilution), anti-LDLR rabit (1:1000 dilution) and anti-beta-actin rabit (1:1000 dilution) at 4 ℃ for overnight. The primary antibody was collected, the membrane was washed 3 times with 1×tbst for 10 minutes each, and then PVDF membrane was incubated with the secondary antibody at room temperature for 1 hour. After the secondary antibody was collected, the membrane was washed 3 times with 1×tbst, ECL super-sensitized chemiluminescent developer was added, and luminescent imaging was performed using an Amersham Imager 680RGB instrument. The results are shown in FIG. 12. The results show that: compared to normal astrocytes HA1800, LRP1 protein was highly expressed on SNB19 and LN229 cell membranes.
6.2 detection of targeting ability of ApoE modified nano-drug to GBM cells
To examine whether ApoE modified nanomedicines had the ability to target SNB19 and LN229 cells, the prepared nanomedicines NM@ (BTZ/LBH 589/Cy 5) and ApoE-NM@ (BTZ/LBH 589/Cy 5) were incubated with SNB19 and LN229 cells, respectively, for 4 hours at the same Cy5 content (microplate reader assay). Removing the culture solution, washing the cells 3 times by using 1 XPBS, digesting, centrifuging and collecting the cells, collecting and detecting Cy5 fluorescent signals in the cells by using a flow cytometer, and then analyzing the results by using FlowJo_V10 software; alternatively, 4% paraformaldehyde was added to the cells, after 10 minutes, the cells were washed 2 times with 1 XPBS, then nuclei were stained with DAPI (10. Mu.g/mL) for 10 minutes, the cells were washed 3 times with 1 XPBS, and fluorescence signals were collected and imaged by laser confocal microscopy (parameters were kept consistent when Cy5 fluorescence was collected in each sample). The results are shown in FIG. 13. The results show that: compared with NM@BTZ/LBH589/Cy5, the nano-drug ApoE-NM@BTZ/LBH589 with the surface modified ApoE can enter SNB19 and LN229 cells more, which indicates that the ApoE can be combined with LRP1 specifically, and the capacity of actively targeting SNB19 and LN229 cells is exerted.
7. Assessment of the ability of a nano-drug to cross the blood brain barrier
In order to examine the ability of nanomaterials to cross the BBB, the present invention establishes a blood brain barrier model in vitro. Bond.3 (50,000 cells/well) was inoculated into a transwell chamber and 800. Mu.L of cell culture broth was added to the lower well plate (24 well plate). At 37℃with 5% CO 2 Is cultured in a sterile cell incubator when the trans-endothelial cell electronic impedance (TEER) value is higher than 200 Ω cm 2 At this time, nano-drug NM@ (BTZ/LBH 589/Cy 5) or ApoE-NM@ (BTZ/LBH 589/Cy 5) with the same Cy5 content was added to the chamber, and then 50. Mu.L of the culture solution was withdrawn from the well plate at 4 hours, 12 hours, 24 hours, respectively. Finally, the Cy5 fluorescence value in the culture solution is detected by an enzyme-labeled instrument. The results are shown in FIG. 14. The results show that: at 24 hours, the number of surface modified ApoE nano-drugs ApoE-NM@BTZ/LBH589 crossing the BBB was about 1.76 times that of NM@BTZ/LBH589/Cy 5.
8. MTT method for detecting influence of nano-drug on GBM cell survival
Each GBM cell was seeded in 96-well plates (5,000 cells/well) at 37℃with 5% CO 2 After 24 hours of culture in a sterile cell incubator, the drug carrier ApoE-NM, free drugs with different drug concentrations BTZ/LBH589 or nano drugs NM@BTZ+NM@LBH589NM@ (BTZ/LBH 589) and ApoE-NM@ (BTZ/LBH 589) are respectively added into the culture solution for 48 hours of incubation. Then, after adding a 10. Mu. LMTT (5 mg/mL) solution to the culture broth and incubating for 4 hours, the culture broth was removed and 150. Mu.L of dimethyl sulfoxide was added to the sample well, and the incubation was performed with shaking at room temperature for 15 minutes, and finally, the absorbance at a wavelength of 490nm was measured using an enzyme-labeled instrument. Each sample had three duplicate wells. The results are shown in FIG. 15. The results show that: the nanoparticles without drug loading have little cytotoxicity and exhibit good biocompatibility; drug-loaded nanoparticles affect cell survival, and the anti-cell proliferation effect of the BTZ and LBH589 co-loaded nanomaterials NM@ (BTZ/LBH 589) is stronger than that of ApoE-NM@ (BTZ/LBH 589), and due to modification of ApoE, apoE-nm@btz/LBH589 is able to enter more cells, exhibiting better cell survival inhibition effect than nm@btz/LBH 589.
9. ER-tracker characterizes endoplasmic reticulum stress caused by nano-drugs
GBM cells were seeded in 6-well plates (150,000 cells/well) at 37℃with 5% CO 2 After 24 hours of incubation in a sterile cell incubator of (a), apoE-NM, free drug BTZ/LBH589 or nano drug NM@BTZ+NM@LBH589, NM@ (BTZ/LBH 589), apoE-NM@ (BTZ/LBH 589) were added to the culture medium and incubated at a concentration of 4.0nM/8.0nM (BTZ/LBH 589), respectively, for 48 hours. Next, the culture broth was removed and the cells were washed 2 times with 1×pbs, ER-tracker Red working solution previously preheated at 37 ℃ was added to the cells, and incubated at 37 ℃ for 20 minutes. ER-Tracker Red staining working solution was removed and cells were washed 2 times with cell culture solution. Cells were digested, centrifuged and collected, and ER-Tracker Red fluorescent signals were collected and detected in the cells using a flow cytometer, and then analyzed for results using FlowJo_V10 software. The results are shown in FIG. 16. The results show that: at the same drug concentration, the free drug did not induce endoplasmic reticulum stress, whereas the nanomedicine induced endoplasmic reticulum stress, and ApoE-NM@ (BTZ/LBH 589) induced endoplasmic reticulum stress to a degree of 2.5 and 1.3 times that of NM@ (BTZ/LBH 589) in SNB19 and LN229 cells, respectively; NM@ (BTZ/LBH 589) induced endoplasmic reticulum stress to a degree of 2.3 and 1.2 times that of NM@BTZ+NM@LBH589 in SNB19 and LN229 cells, respectively.
10. Flow cytometry detection of influence of nano-drugs on GBM apoptosis
Various GBM cells were seeded in 12-well plates (70,000 cells/well) at 37℃with 5% CO 2 After 24 hours of incubation in a sterile cell incubator, the free drug BTZ/LBH589 or the nano drug NM@ (BTZ/LBH 589), apoE-NM@ (BTZ/LBH 589), apoE-NM were added to the culture medium at a concentration of 4.0nM/8.0nM (BTZ/LBH 589), respectively, and incubated for 48 hours. All cells in the well plate were collected separately, centrifuged (400×g,3 min) and washed 2 times with 1×pbs, the cells were dispersed in1×annexin V-FITC conjugate, 5 μ LAnnexin V-FITC was added and gently mixed at room temperature, 10 μ L propidium iodide staining (PI) was added and gently mixed after 5 min, and after 15 min, the cells were placed on ice, fluorescent signals in the cells were collected and detected with a flow cytometer, and the results were analyzed with flowjo_v10 software. The results are shown in FIG. 17. The results show that: at the same drug concentration, the free drug does not cause apoptosis, but is nano-sizedThe drugs ApoE-NM@ (BTZ/LBH 589) and NM@ (BTZ/LBH 589) clearly caused apoptosis of SNB19 and LN229 cells.
11. Establishment of in situ TP53 mut GBM model
SNB19 or LN229 cells were seeded in 15cm cell culture dishes at 37℃with 5% CO 2 When cultured to logarithmic growth phase, the culture medium was removed, the cells were washed 2 times with 1X PBS, digested with 1X trpysin for 3 minutes, collected by centrifugation, and concentrated at a density of 5X 10 5 Individual cells/5. Mu.L were dispersed in1 XPBS buffer and placed on ice for use. After anesthetizing 6-8 week old female Balb/c nude mice with chloral solution (8 mg/20 g), the skin of the scalp was scratched with a special blade for animals, and the left upper part of the brain of the mice was perforated with skull drill to a depth of about 3.5mm, and then 5X 10 with a microneedle type syringe 5 The SNB19 or LN229 cells were injected into the wells, the wells were closed with melted bone wax, the skin was cut before gluing with tissue seal glue, and the mice were placed in an electric blanket until awakened and returned to the cages for feeding. All animal experiments were processed according to the protocol approved by the university of henna, meeting the requirements of the laboratory animal center and the committee for animal care and use.
12. In vivo distribution experiment of nano-drug
The nanomedicine NM@ (BTZ/LBH 589/Cy 5), apoE-NM@ (BTZ/LBH 589/Cy 5) or free Cy5, respectively, was injected into the Balb/c nude mice already bearing tumors (Cy 5:0.1 mg/kg) via the tail vein, and the distribution of these nanomedicine in the mice was monitored at different time points (1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours) using the lumine IVIS III near infrared fluorescence imaging system (ex=620 nm, em=710 nm). In addition, in order to more clearly understand the distribution of the nano-drugs in each organ and brain of the tumor-bearing Balb/c nude mice, when the various nano-drugs are injected into the tail vein for 4 hours, the mice are sacrificed according to the cervical dislocation method, the heart, liver, spleen, lung, kidney and brain of the mice are taken out, and the Cy5 fluorescence distribution in each organ and brain of the mice is detected through the Lumina IVIS III system of a small animal imager.
In order to further observe the distribution and penetration depth of nano-drugs in brain and in situ tumors, the sameAfter nano-drug injection for 4 hours, tumor-bearing mice are sacrificed by cervical dislocation, and the brains of the mice are taken out and placed in neutral universal tissue fixing solution for light-shielding preservation for 48 hours. The mouse brain was removed and placed in 20% w / v Until it has settled completely to the bottom of the solution (about 12 hours), and then the mouse brain is placed in 30% w / v For about 12 hours. Then, the mouse brain placed in the sucrose solution was taken out, placed in the center of the embedding well, and OCT embedding medium was added to the embedding well, which was then frozen in liquid nitrogen to fix the position of the mouse brain. The specimen was placed on a holding table, sliced with a constant temperature cryomicrotome (generally at a slice temperature of-22 ℃ C.) and a slice thickness of 15 μm, attached to a pre-chilled slide, and stored in a-20 ℃ refrigerator. Sections were fixed with acetone pre-chilled at 4 ℃ for 15 min, washed 3 times with 0.3% Triton X-100, stained nuclei with DAPI for 10 min, removed and washed for DAPI dye, subjected to a plate-sealing treatment, and scanned with a pannarac MIDI instrument to capture images.
Furthermore, in order to quantify the accumulation of nanomaterials in each organ, mice were sacrificed according to cervical dislocation method at 4 hours of injection of nanomaterials, tumors in heart, liver, spleen, lung, kidney, brain and brain of mice were taken out, they were weighed and placed in EP tubes containing 0.6ml of 1% triton X-100 solution, two round magnetic beads having a diameter of about 0.3mm were added to the tubes, each tissue was homogenized for 4 minutes (power: 70 Hz) with a homogenizer (JXFSTPRP-48), left standing overnight at room temperature, and then supernatant was collected by centrifugation (14,000×g,30 minutes), and then the fluorescence intensity of Cy5 (ex=630 nm, em=670 nm) in the samples was measured with an enzyme-labeled instrument, and the Cy5 content was calculated according to a standard curve, and the final value was displayed in units of percent of tissue injection per gram (% ID/g) (3 mice per group).
The results of fig. 18 show: the amount of nanomedicine enriched in the mouse brain is significantly increased compared to free Cy5, and is maximal in the mouse tumor sites due to the ability of ApoE-NM@ (BTZ/LBH 589/Cy 5) to cross the BBB and actively target SNB19 and LN229 cells.
13. In situ TP53 of nano-drug mut GBM therapy experiments
At 10 days tumor-bearing, mice were randomized for treatment evaluation. ApoE-NM@ (BTZ/LBH 589), NM@ (BTZ/LBH 589), free BTZ/LBH589, apoE-NM or 1 XPBS were injected into mice by tail vein (drug dose 2 mg/kg), respectively, 1 time every 2 days for 5 times. Mice were monitored for changes in body weight during treatment, and survival time of mice was monitored for a long period of time to day 90 when in situ GBM models were established. The results are shown in FIG. 19. The results show that: in the SNB19 and LN229 models, apoE-NM@ (BTZ/LBH 589) treated mice remained essentially stable in weight during the treatment period, with other treated mice continuing to lose weight; in the SNB19 model, NM@ (BTZ/LBH 589) and ApoE-NM@ (BTZ/LBH 589) prolonged the total survival of mice to 48 days and 70 days, respectively, in contrast to PBS, apoE-NM and free BTZ/LBH589 treated mice with shorter than 32 days. Likewise, in LN229 model, the total survival time of mice in the NM@ (BTZ/LBH 589) and ApoE-NM@ (BTZ/LBH 589) treatment groups was prolonged to 51 days and over 90 days, respectively.
14. In situ TP53 mut Histological analysis after GBM treatment
At the end of the animal level treatment (5 dosing treatments were completed), the mouse brain tissue or organ was placed in a neutral universal tissue fixative solution, after 2 days, the tissue was removed and placed in a dedicated card tank for running water to flush overnight (for brain tissue, olfactory bulb and cerebellum could be excised), followed by dehydration treatment, i.e., the tissue was placed in sequence in 70% ethanol for 1 hour, 80% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol for 1 hour, 50% ethanol and 50% xylene mixed solution for 1 hour, 100% xylene (I) for 1 hour, 100% xylene (II) for 1 hour. The tissue was then placed in melted paraffin (65 ℃) for a 3 hour wax dipping treatment, and finally the tissue was placed in an embedding tank and melted paraffin was added dropwise to the tank with an embedding machine until the tissue was completely submerged, followed by cooling on a cold table for at least 2 hours. Next, the tissue was taken out of the embedding bath and sliced to a thickness of 4. Mu.m, the slices were spread in a water bath at 40℃for about 30 seconds (completely spread but without rotting), the slices were fished with an adhesive glass slide, and the slices were baked in an oven at 60℃for 2 hours. Before any histological staining, paraffin sections are dewaxed, and the specific steps are sequentially placed in xylene (I) for 5 minutes, xylene (II) for 5 minutes, xylene (III) for 5 minutes, 100% ethanol (I) for 5 minutes, 100% ethanol (II) for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, distilled water (I) for 5 minutes, distilled water (II) for 5 minutes, distilled water (III) for 5 minutes, and distilled water for a long time.
14.1 hematoxylin & eosin (H & E) staining
Firstly, placing a tissue slice into a hematoxylin (Hemagtoxy in, stained cell nuclei, purple) solution for staining for 6 minutes, and then washing the slice with running water for 10 minutes to change the purple color into blue (if the hematoxylin is stained too deeply, alcoholic hydrochloric acid or a commercial hematoxylin differentiation reagent is required for differentiation, the differentiation time is generally 1-2 seconds, and if the differentiation degree is too large, counterstaining can be performed). Then the flakes were put into Eosin (stained cytoplasma, orange red) solution for 3 minutes, washed 2 times, and then directly put into the step of removing water flakes (excessive color separation with hydrochloric acid is not required because Eosin masks the color of hematoxylin in cytoplasm). Sequentially placing the slices in 80% ethanol for 5 seconds, 90% ethanol for 5 seconds, 95% ethanol for 5 seconds, 100% ethanol (I) for 5 seconds, and 100% ethanol (II) for 5 seconds (the placing time in 90% ethanol can be longer, so as to avoid excessive eosin concentration). Finally, the flakes were placed in xylene (which may be long-standing in xylene), followed by a volume ratio of 1:1 neutral gum: sealing the sheet with xylene, removing bubbles, and air-drying in a fume hoodThe 40 instrument performs scan shots and analyzes the data using the quaath software. The results are shown in fig. 20, in the SNB19 and LN229 in situ tumor-bearing models, apoE-NM@ (BTZ/LBH 589) treated group significantly inhibited tumor growth with minimal tumor size compared to the other treated groups; the normal brain tissues and organs of mice in each treatment group are found pathological changes and side effects, which indicates that the nano-drug has good biocompatibility.
14.2 use of TUNEL for apoptosis detection
To a sample of the tissue section, 20. Mu.g/mL (diluted with 10mM Tris-HCl pH 7.4-7.8) of proteinase K free of DNase was added dropwise, and the reaction was carried out at 20-37℃for 25 minutes, followed by removing the excess proteinase K, and washing with 1 XPBS 3 times for 5 minutes each. 50. Mu.L TUNEL assay solution was added dropwise to the sample and placed in a water-filled, light-resistant wet box to keep it moist and minimize evaporation of TUNEL assay solution, incubated at 37℃for 60 minutes in the dark, then excess TUNEL assay solution was removed, and washed 3 times with 1 XPBS for 2 minutes each. Nuclei were stained with DAPI (10. Mu.g/mL) for 10 min, and the dishes were washed with 1 XPBST 2 min 3 times. After sealing the tablet with the anti-fluorescence quenching sealing tablet, a laser confocal microscope is used for fluorescence signal acquisition and picture shooting. The results are shown in FIG. 21. The results show that: in SNB19 and LN229 in situ tumor-bearing models, the TUNEL positive signal was significantly increased in the ApoE-NM@ (BTZ/LBH 589) treatment group compared to the other treatment groups, resulting in tumor cell apoptosis.
14.3 immunohistochemistry
Drawing a small circle (for isolating samples and saving reagents such as antibodies) as possible around the samples on the sections by using an immunohistochemical pen, leaving a corner in the circle, conveniently sucking the sample, placing the chip in a wet box, and placing 3% H 2 O 2 After 20 minutes of dripping onto the sectioned tissue, the endogenous peroxidase was removed and excess H was washed off with 1 XPBST 2 O 2 3 times per 2 minutes. The slices are soaked in 0.01M citrate buffer solution, then put into a microwave oven for heating until boiling, the sub-boiling temperature (95 ℃ -98 ℃) is kept for 10 minutes, antigen thermal restoration is carried out, and finally the slices are cooled on an experiment table for 30 minutes. The pieces were soaked in1 XPBST for 2-3 min, wiped dry, blocked with 3% BSA for 30min, washed 3 times with PBST for 2 min each. An anti-Ki67, anti-clean caspase-3 and other primary antibodies (diluted in 1% BSA, the dilution ratio is referred to the antibody instruction) were added to the samples (in the assembled circles) in an amount to cover the samples, and the samples were placed in a wet box at 4℃overnight (water was added to the wet box), and then the primary antibodies were removed and the 1 XPBST chip was washed for 2 minutes 3 times. The sample was incubated with biotin-labeled goat anti-rabbit IgG for 30 minutes at 37℃and the pieces were washed with 1 XPBST for 2 minutes 3 times. SABC was added dropwise to the sample, incubated at 37℃for 30 minutes, and the shoots were washed with 1 XPBST 2 minutes 3 times. By usingDAB is used as a color reagent, is dripped on a sample for 5-10 minutes (not more than 10 minutes, or nonspecific dyeing exists), is washed by running water for 10 minutes, dyeing is stopped, microscopic examination is carried out, and if the effect is poor, the dyeing can be continued. Finally, the cell nucleus is stained with hematoxylin solution for 2-3 min, and the washing is carried out for 5-10 min, and the steps of dehydration and sealing the sample are carried out with H&The subsequent steps of E dyeing are consistent. By usingThe 40 instrument performs scan shots and analyzes the data using the quaath software. The results are shown in FIG. 22. The results show that: in SNB19 and LN229 in situ tumor-bearing models, ki67 positive signals were significantly reduced in tumor tissue of the ApoE-NM@ (BTZ/LBH 589) treated group compared to the other treated group, CC3 positive signals were significantly increased, and CC3 positive signals were distributed mainly in tumor tissue but not normal brain tissue, indicating that ApoE-NM@ (BTZ/LBH 589) was able to inhibit tumor cell proliferation, promote apoptosis, and not affect normal brain tissue cells.
14.4 detection of in situ TP53 by immunoblotting of nanomedicine pairs mut Apoptosis-related protein modulation in GBM tumors
After 5 treatments were completed, tumor tissues in the brain of the mice were removed after dislocation and death of cervical vertebrae of the mice, placed in a high-efficiency RIPA lysate containing 1mM PMSF, and two round magnetic beads having a diameter of about 0.3mM were added to the tube, each tissue was homogenized for 4 minutes (power: 70 Hz) with a homogenizer (JXFSTPRP-48), left to crack on ice for 30 minutes, and after centrifugation (15,000 rpm,10 minutes), the supernatant was taken, and after protein concentration was measured with BCA protein quantification kit, protein solution (10 μg/sample) was mixed with 1 x SDS-PAGE loading buffer and denatured at 95 ℃ for 10 minutes. Protein samples were then added to a 7.5% SDS-polyacrylamide gel well and electrophoresed at constant pressure 80v until the protein markers were completely separated, and the samples were transferred to polyvinylidene fluoride (PVDF) membranes (300 mA,1 hour). After the PVDF membrane is blocked by 5% milk for 1 hour, the PVDF membrane is cut according to the molecular weight and the position of a protein marker, and incubated with a primary antibody anti-Bip rabit pAb (1:1000 dilution), anti-caspase-12 rabit (1:1000 dilution), anti-caspase-3 rabit (1:1000 dilution), anti-clean caspase-3 rabit (1:1000 dilution), anti-calpain1 rabit pAb (1:1000 dilution) and anti-beta-actin rabit pAb (1:1000 dilution) at 4 ℃ for overnight respectively. The primary antibody was collected, the membrane was washed 3 times with 1×tbst for 10 minutes each, and then PVDF membrane was incubated with the secondary antibody at room temperature for 1 hour. After the secondary antibody was collected, the membrane was washed 3 times with 1×tbst, ECL super-sensitized chemiluminescent developer was added, and luminescent imaging was performed using an Amersham Imager 680RGB instrument. The results are shown in FIG. 23. The results show that the amount of Bip/CAPN1/cCasp12/cCasp3 protein expression in tumor tissue of the ApoE-NM@ (BTZ/LBH 589) treated group was significantly increased compared to the other treated groups.
15. Influence of nano-drug on routine and biochemical blood of mouse
First, healthy Balb/c mice (6-8 weeks old) were randomly divided into two groups (4 mice per group). Mice were injected with different doses of ApoE-NM@ (BTZ/LBH 589) (1 mg/kg,2mg/kg,4 mg/kg) or 1×pbs via the tail vein, respectively. About 200 μl of blood was taken from the mouse canthus at days 2,7 and 14, respectively. A portion of the collected whole blood is subjected to conventional blood analysis including Red Blood Cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean hematocrit (MCV), mean red blood cell hemoglobin (MCH), mean red blood cell hemoglobin concentration (MCHC), white Blood Cell (WBC), and Platelets (PLT) index measurements. After another portion of the blood was centrifuged (800×g,10 minutes), serum was collected and assayed for alanine Aminotransferase (ALT), alkaline phosphatase level (ALP), serum Albumin (ALB), aspartic Aminotransferase (AST), UREA (ura), creatinine (CREA), and Uric Acid (UA). The results are shown in FIG. 24. The results show that: after the apoE-NM@BTZ/LBH589 with different drug doses is injected, the values of the conventional blood parameters and the biochemical blood parameters of the mice are similar to those of a control group on days 2,7 and 14, and no obvious difference exists, so that the apoE-NM@BTZ/LBH589 has good biocompatibility and no side effect.
16. Influence of nano-drug on mouse liver and kidney
To examine whether the nanomedicine caused inflammation, we examined IL-1β, IL-6 and TNF-. Alpha.mRNA expression in the liver and kidney of mice after the nanomedicine injection.Healthy Balb/c white rats (6-8 weeks old) were selected and randomly divided into 2 groups of 3 animals each. The nano-drug ApoE-NM@ (BTZ/LBH 589) was injected into mice via the tail vein (2 mg/kg). After 2 days, cervical dislocation of mice was fatal and kidneys and livers were removed, they were placed in an EP tube containing an RNA extraction solution, and two round magnetic beads having a diameter of about 0.3mm were added to the tube, each tissue was homogenized for 4 minutes (power: 70 Hz) with a homogenizer (JXFSPRP-48), and total RNA in the tissue was extracted according to the total RNA extraction kit standard procedure. By Thermo Scientific TM After the NanoDrop instrument detects the RNA concentration, the RNA was reverse transcribed to form cDNA according to the experimental instructions of the RT reagent Kit (Perfect Real Time), and then qRT-PCR experiments were performed according to the standard procedure of the SYBR Premix Ex Taq II Kit and using a real-time fluorescent quantitative PCR instrument (lightcyler 480 II). Experimental data using 2 -ΔΔCt The formula performs further calculations. The results are shown in FIG. 25. The results show that: after 2 days of injection of ApoE-NM@BTZ/LBH589 into mice, qRT-PCR characterization shows that mRNA expression levels of inflammatory factors (IL-1 beta, IL-6 and TNF-alpha) in livers and kidneys of the mice are not significantly different from those of a control group, which indicates that ApoE-NM@BTZ/LBH589 does not produce side effects on livers and kidneys of the mice, does not induce inflammatory responses, and has good biocompatibility.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.

Claims (2)

1. The composite carrier is characterized by comprising polypeptide, N-acryl glucosamine, amide compounds containing bipyridyl groups, bortezomib, panobinostat and N, N' -bis (acryl) cystamine;
the N-acryl-glucosamine and bortezomib generate a boron ester bond compound through reaction, and the structure is as follows:
；
chelating the amide compound containing the bipyridyl group and panobinostat through zinc ions;
the amide compound containing bipyridyl group is selected from N- (2-acrylamide ethyl) - [2,2' -bipyridine ] -5-formamide, N- (3-acrylamide propyl) - [2,2' -bipyridine ] -5-formamide and N- (4-acrylamide butyl) - [2,2' -bipyridine ] -5-formamide;
the combined carrier is of a spherical structure;
the polypeptide is sulfhydryl modified polypeptide, and has the structure ofWherein the sequence of the polypeptide ApoE is LRKLRKRLLLRKLRKRLLC.
2. The use of a synthetic lethal nano-drug combination carrier of claim 1 in the preparation of a targeted drug for the treatment of GBM.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105348157A (en) * 2015-12-18 2016-02-24 苏州大学 Cystamine diisocyanate monomer, cystamine diisocyanate monomer based polymers as well as preparation method and application of cystamine diisocyanate monomer
CN110290781A (en) * 2017-01-26 2019-09-27 南京三迭纪医药科技有限公司 The dosage form of stomach and intestine privileged site control release
CN111437258A (en) * 2020-03-11 2020-07-24 苏州大学 Anti-tumor nano adjuvant based on cross-linked biodegradable polymer vesicles and preparation method and application thereof
CN115054704A (en) * 2022-07-18 2022-09-16 河南大学 Nano composition, preparation method and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105348157A (en) * 2015-12-18 2016-02-24 苏州大学 Cystamine diisocyanate monomer, cystamine diisocyanate monomer based polymers as well as preparation method and application of cystamine diisocyanate monomer
CN110290781A (en) * 2017-01-26 2019-09-27 南京三迭纪医药科技有限公司 The dosage form of stomach and intestine privileged site control release
CN111437258A (en) * 2020-03-11 2020-07-24 苏州大学 Anti-tumor nano adjuvant based on cross-linked biodegradable polymer vesicles and preparation method and application thereof
CN115054704A (en) * 2022-07-18 2022-09-16 河南大学 Nano composition, preparation method and application

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