CN115531554A - Synthetic lethal nano-drug combined carrier and application thereof in preparation of GBM targeted drugs - Google Patents

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

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CN115531554A
CN115531554A CN202211270462.6A CN202211270462A CN115531554A CN 115531554 A CN115531554 A CN 115531554A CN 202211270462 A CN202211270462 A CN 202211270462A CN 115531554 A CN115531554 A CN 115531554A
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drug
btz
drugs
apoe
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CN115531554B (en
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吴海刚
师冰洋
刘媛媛
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Henan University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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Abstract

The invention belongs to the technical field of nano-drug preparation, and discloses a nano-drug combined carrier, which comprises polypeptide, an amide compound containing polyhydroxy, an amide compound containing bipyridyl groups, a proteasome chymotrypsin-like activity inhibition drug containing boron hydroxyl, a histone deacetylase inhibition drug and a compound containing disulfide bonds; the amide compound containing polyhydroxy and proteasome chymotrypsin-like activity inhibition drugs containing boron hydroxyl react to generate boron ester bonds, and the amide compound containing bipyridyl groups and the histone deacetylase inhibition drugs are chelated through zinc ions. The combined carrier has the capability of responding to the reduction, acid and chelation competition to release the drugs, can load two drugs simultaneously, has high release capability and space-time controllability of the two drugs, and plays a role in synergy.

Description

Synthetic lethal nano-drug combined 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 combined carrier and application thereof in preparation of a GBM targeted drug.
Background
Glioblastoma (GBM) is a primary, intraoven malignancy originating from glial cells, the most common. The annual incidence of GBM is 3-8/10 ten thousand, accounting for about 27% of all tumors in the central nervous system and 80% of malignant tumors, and GBM also increases year by year at an annual rate of 1-2%. The world health organization characterizes the malignancy of GBM as grade IV (highest grade) depending on whether the tumor cells are characterized by multiple mitosis, polymorphism, proliferation of vascular endothelial cells, and necrosis. At present, no effective GBM treatment means exists clinically, the survival rates of GBM patients in1 year and 5 years are only 30 percent and 4 percent 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 plays an important role in tumor formation. In all malignant tumors, more than 50% of the tumors show mutations in the gene. p53 mutation (TP 53) mut ) Is highly involved in the development and progression of various tumors including glioblastoma multiforme (GBM), and directly targets TP53 mut Resistance is always induced and thus treatment is limited. Synthetic Lethality (SL) is a strategy to induce cell death by blocking the compensatory pathway of oncogene mutations through pharmaceutical intervention, with great potential for treating GBM.
For therapeutic approaches using combinatorial chemicals, reasonable drug ratios, synchronized delivery, effective drug concentrations, tissue targeting, and accurate therapeutic windows remain unsolved issues that limit the further application of chemical drugs in tumor therapy. The random encapsulation of nano-drugs makes it difficult to precisely adjust the combination ratio of the drugs, and the pre-mixing of nano-drugs to individually encapsulate the drugs may overcome this disadvantage, but the uniformity of the nano-particles prepared when preparing different drugs is difficult to control. Thus, rational assignment of co-delivery nano-platforms may be an ideal strategy for synthetic lethality, with significantly reduced toxicity to normal cells and potential damage to non-target organs or tissues through precisely controlled drug application.
Disclosure of Invention
One of the purposes of the invention is to provide a synthetic lethal nano-drug combined carrier, which has the capability of responding to drug release by reduction, acid and chelation competition, can load two drugs simultaneously, has high release capability and space-time controllability of the two drugs, and plays a role in synergy.
The second purpose of the invention is to provide the application of the synthetic lethal nano-drug combined carrier in preparing the GBM targeted drug, the biological safety is good, and compared with free drugs, the nano-drug enhances the synthetic lethal effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a synthetic lethal nano-drug combined carrier, which comprises polypeptide, polyhydroxy amide compound, bipyridyl group-containing amide compound, borohydroxyl-containing proteasome chymotrypsin-like activity inhibiting drug, histone deacetylase inhibiting drug and disulfide bond-containing compound; the amide compound containing polyhydroxy and the proteasome chymotrypsin-like activity inhibition drug containing boron hydroxyl react to generate a boron ester bond, and the amide compound containing bipyridyl groups and the histone deacetylase inhibition drug are chelated by zinc ions.
In one embodiment, the proteasome chymotrypsin-like activity inhibiting drug comprising a boronic hydroxyl group is bortezomib.
In one technical scheme, the histone deacetylase inhibitory drug is panobinostat.
In one embodiment, the polypeptide is a thiol-modified polypeptide.
In one technical scheme, the amide compound containing the polyhydroxy group is selected from N-acryloyl glucosamine.
In one embodiment, the amide compound containing a bipyridyl group is selected from the group consisting of N- (2-acrylamidoethyl) - [2,2' -bipyridine ] -5-carboxamide, N- (3-acrylamidopropyl) - [2,2' -bipyridine ] -5-carboxamide, and N- (4-acrylamidobutyl) - [2,2' -bipyridine ] -5-carboxamide.
In one embodiment, the composite carrier has a spherical structure.
The invention also provides application of the nano-drug combination carrier in preparation of 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 reduction, acid and chelation competition to release drugs, can load two drugs simultaneously, has high drug loading efficiency, high release capability of the two drugs and space-time controllability, and plays a role in synergy.
The nano-drug combination carrier prepared by the invention comprises the sulfhydryl modified polypeptide, and has the capability of actively targeting GBM cells (SNB 19 and LN 229) with high LRP1 protein expression on the surface of cell membranes compared with nano-drugs without the polypeptide.
The invention utilizes the nano-drug prepared by the nano-drug combination carrier to carry out in-vitro cell experiments and in-situ TP53 mut In the GBM mouse model, compared with free drugs, the nano-drugs enhance the synthetic lethal effect; has good biological safety and no side effect on blood routine, blood biochemistry, organs of mice and normal brain tissue.
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FIG. 1 shows a scheme for the synthesis of AGA according to the invention.
FIG. 2 shows the nuclear magnetic characterization of AGA of the present invention.
FIG. 3 shows a synthetic route of AGA-BTZ of the present invention.
FIG. 4 shows the nuclear magnetic characterization of AGA-BTZ of the present invention.
FIG. 5 is a scheme of synthesis of AABC of the present invention.
FIG. 6 shows the nuclear magnetic characterization results of AABC of the present invention.
FIG. 7 shows the synthesis scheme of AABC-Zn-LBH589 of the present invention.
FIG. 8 shows an ultraviolet absorption spectrum of AABC-Zn-LBH589 according to the present invention.
FIG. 9 shows the preparation method of ApoE-NM @ (BTZ/LBH 589) according to the present invention.
FIG. 10 shows TEM, size, dispersion coefficient and surface potential of ApoE-NM @ (BTZ/LBH 589), NM @BTZ, NM @LBH589 and ApoE-NM according to the present invention.
FIG. 11 is a graph of the ability of the nano-drug of the present invention to responsively release the drug, wherein FIG. 11A is a graph of the drug release over time; fig. 11B is the nanoparticle morphology change after drug release (TEM characterization).
FIG. 12 shows the immunoblotting of the invention for characterizing the receptor expression of the polypeptide ApoE, including LRP1, LDLR and LRP2, on the cell membrane surface of GBM (SNB 19 and LN 229) and normal astrocyte (HA 1800).
FIG. 13 is a graph of the ability of ApoE of the invention to target SNB19 and LN229 cells.
FIG. 14 is the construction of the BBB model in vitro and the evaluation of the ability of the nano-drug to cross the blood brain barrier.
FIG. 15 is an evaluation of the effect of the nano-drugs of the present invention on inhibiting cell survival.
FIG. 16 is a graph of the effect of free drug and nano-drug of the present invention on SNB19 and LN229 endoplasmic reticulum stress in cells.
FIG. 17 is a graph of the degree of SNB19 and LN229 cell apoptosis induced by free drug and nano-drug of the present invention.
FIG. 18 is the in vivo distribution of the nano-drug of the present invention, wherein FIG. 18A is the in vivo distribution of the nano-drug in tumor-bearing mice monitored in real time by the IVIS Lumina III imaging system; 18B, after the nano-drug is injected for 4 hours, detecting the distribution condition of the nano-drug in each organ of the mouse by an IVIS Lumina III imaging system; 18C, obtaining mouse brain after injecting the nano-drug for 4 hours, and detecting the distribution condition of the nano-drug by a fluorescence microscope after freezing and slicing; and 18D is the distribution condition of the nano-drugs quantitatively counted after 4 hours of nano-drug injection by taking normal brain tissues, tumor tissues and organs of the mice and carrying out homogenate treatment.
FIG. 19 shows in situ TP53 of a Nanoparticulate drug of the present invention mut GBM treatment course and results, wherein fig. 19A is a flow chart of animal experiments; FIGS. 19B and 19C show the change in mouse body weight and mouse survival in the SNB19 orthotopic tumor-bearing modelA period; FIGS. 19D and 19E show the change in body weight and the survival period of mice in LN229 in situ tumor-bearing model.
FIG. 20 is the H & E staining results for treatment groups of the invention, where FIG. 20A is the tumor size; FIG. 20B shows the presence or absence of lesions in normal brain tissue and mouse organs.
FIG. 21 shows the distribution and statistics of TUNEL positive signals in the treatment groups of the present invention.
FIG. 22 is the immunohistochemical analysis results of each treatment group of the present invention, and FIG. 22A is the immunohistochemical characterization of the distribution of Ki67 and CC3 positive signals in each treatment group; fig. 22B and 22C show the distribution and statistics of CC3 in normal and tumor brain tissues.
FIG. 23 shows the in situ TP53 of the present invention with nano-drug pairs mut Results of apoptosis-related protein regulation in GBM tumors.
FIG. 24 shows the effect of the nano-drug of the present invention on the blood regulation and blood biochemistry of mice, wherein FIG. 24A shows the blood regulation results of mice, and FIG. 24B shows the blood biochemistry results of mice.
FIG. 25 shows the effect of the nano-drug of the present invention on the expression of inflammatory factors in the liver and kidney of mice.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.
Example one
1. Preparation of prodrug AGA-BTZ
1.1 preparation of AGA
The specific synthetic route of preparing N-Acryloyl Glucosamine (AGA) by using D- (+) -galactosamine hydrochloride and acryloyl chloride (Aladdin, china) is shown in figure 1. 20mL of 1mol/L K containing 0.02mol of D- (+) -galactosamine hydrochloride 2 CO 3 Put into a reaction flask and placed in an ice bath for cooling. 0.024mol of acryloyl chloride was slowly added dropwise to the solution with vigorous stirring. The reaction was maintained at 0-4 ℃ for 4 hours and then slowly resumedThe reaction mixture was cooled to room temperature for 24 hours to recover the 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. Use of 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 the purity was close to 100%.
1.2 preparation of AGA-BTZ
Bortezomib (BTZ) is a reversible inhibitor of 26S proteasome chymotrypsin-like activity in mammalian cells. AGA reacts with BTZ to generate AGA-BTZ, a boron ester bond in the AGA-BTZ has acid response capability, controllable release of the drug can be realized, and a synthetic route of the AGA-BTZ is shown in figure 3.
Respectively dissolving AGA and BTZ (Nanjing Congralin) with the same molar mass in ddH 2 O and methanol, and added to the reaction flask. The pH of the reaction solution was adjusted to 8.5-9.0 with NaOH and the reaction was continued for 48 hours. Methanol is removed by a rotary evaporator, the sample is freeze-dried by a freeze dryer, and finally the product AGA-BTZ is purified on a silica gel column by column chromatography. Use of 1 H NMR (300 MHz) in CD 3 OD was used as a solvent to examine 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 the purity was close to 100%.
2. Preparation of prodrug AABC-Zn-LBH589
2.1 preparation of AABC
The AABC synthetic route is shown in fig. 5. Reacting [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 2h to activate the carboxyl group, and then N- (2-aminoethyl) acrylamide (0.8 mmol) was dispersed in ddH 2 O, and adding into a reaction flask, and continuing the reaction for 24 hours. After THF was removed by rotary evaporator, the product N- (2-acrylamidoethyl) - [2,2' -bipyridine ] was obtained by extraction with ethyl acetate]-5-carboxamide (AABC). In combination with 1 And H NMR detection. Use of 1 H NMR (400 MHz) in DMSO-d 6 The purity of AABC as a solvent was checked and the results are shown in FIG. 6. Nuclear magnetism 1 HNMR) results showed that AABC was successfully prepared and the purity was close to 100%.
Similarly, the present invention can also replace N- (2-aminoethyl) acrylamide with N- (3-aminopropyl) acrylamide and N- (4-aminobutyl) acrylamide according to the synthetic route shown in FIG. 6 to obtain 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 is prepared by zinc ion chelation between hydroxamic acid of LBH589 and bipyridyl of AABC, and the synthetic route is shown in figure 7.
Firstly, slowly dripping equimolar AABC into ZnSO 4 ·7H 2 After chelating in O solution for 15 minutes, equimolar LBH589 (nanjing compactin) was added to the reaction solution and stirred overnight. Finally, whether the preparation of AABC-Zn-LBH589 was successful or not is determined by ultraviolet absorption spectroscopy, and the result is shown in FIG. 8. The detection result of an 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 compound with higher brain targeting efficiency. Sulfhydryl modified polypeptide ApoE (ApoE-SH, qiaozhaoya), acrylate PEG2000 maleimide (Acrylamide-PEG 2000-Mal, APEG-Mal for short, 5 mu mol) and triethylamine (15 mu mol) are respectively dispersed in dimethyl sulfoxide (DMSO) which is deoxidized in advance, and after mixing, the mixture reacts for 24 hours at 37 ℃ in the absence of light under the anaerobic condition of nitrogen protection. To remove unreacted ApoE-SH, APEG-Mal and triethylamine, the reaction solution was placed in a 3.4kDa cut-off dialysis bag in ddH 2 Dialysis in O for 2 days (6 changes of dialysate) followed by lyophilization gave the product ApoE-PEG. Finally, the purity of APEG is detected by a BCA protein quantification method. BCA protein quantificationThe results confirmed that the ratio of ApoE to PEG attachment was close to 100%.
4. Preparation and characterization of Nanoparticulates
4.1 preparation of Nano medicine
To prepare the Nanoparticulate ApoE-NM @ (BTZ/LBH 589), the reaction 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) mixed solution), 0.18 μmol AGA-BTZ, 0.045 μmol apoe-PEG, 0.135 μmol apeg and 0.18 μmol N, N' -bis (acryloyl) cystamine (BACA) were mixed and added to a reaction flask placed in an ice bath under nitrogen protection, and stirred and mixed for 15 minutes. Subsequently, tetramethylethylenediamine (TEMED, 0.88 nmol) and ammonium persulfate (APS, 0.88 nmol) were added to the reaction flask under nitrogen, the reaction flask was closed, and polymerization was carried out in an ice bath for 2 hours. After the reaction is terminated, the polymer solution is added dropwise into 1 XPBS buffer solution to form the nano-drug. Finally, in order to purify the nano-drug, an ultrafiltration centrifugal tube with a molecular weight cutoff of 30kDa was used to remove excess components and the nano-drug was washed 3 times with 1 XPBS, finally obtaining 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), apoE-PEG is not needed to be added, the molar weight of APEG is 0.18 mu mol, and other components and steps are the same as those of 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 preparation of ApoE-NM @ (BTZ/LBH 589).
During 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 for preparing ApoE-NM @ (BTZ/LBH 589).
In the preparation of ApoE-NM nanoparticle without drug loading, AABC-Zn-LBH589 is replaced by equimolar AABC, 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 addition, in order 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, and the other steps were not changed.
4.2 drug Loading efficiency
Taking the external liquid in the ultrafiltration centrifugal 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 the content of the surplus free medicine without the nano-medicine, and then calculating to obtain the medicine loading efficiency (DLE). The results are shown in Table 1. Results and analysis: loading efficiencies of NM @ BTZ/LBH589 for BTZ and LBH589 were approximately 68.7% and 75%, respectively; the loading efficiency of ApoE-NM @ BTZ/LBH589 on BTZ and LBH589 was about 65.3% and 79.5%, respectively. The loading efficiency of NM @ BTZ on BTZ is about 65.3%; the loading efficiency of NM @ LBH589 to LBH589 was about 71.6%.
Table 1 loading efficiency of nano-drug on BTZ and LBH589
Nano 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 Nanoparticulates
In order to detect whether the preparation of various nano-drugs is successful, a dynamic light scattering instrument (DLS) and a Transmission Electron Microscope (TEM) are adopted to respectively detect the hydrodynamic size, the dispersion coefficient, the surface potential, the morphology and the like of the nano-drugs. The results are shown in FIG. 10. Results and analysis: the size of the nano-drug is less than 200nm, the size distribution is uniform (PDI < 0.3), the structure is spherical, and the surface presents negative charges.
5. Responsive drug release properties
Since disulfide bonds in BACA have Glutathione (GSH) or Dithiothreitol (DTT) response capability, boron ester bonds in AGA-BTZ have acid response capability, and AABC-Zn-LBH589 has chelating competition capability, the invention simulates the stimulation response capability of the chemical bonds and drug release and nanoparticle structure damage caused by response in vitro.
The prepared nano-drug ApoE-NM @ (BTZ/LBH 589) is filled in a dialysis bag, and is respectively dispersed in1 XPBS buffer solution with the conditions of 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), the dialysis external liquid is taken at different time points (2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours) in a shaking table with the temperature of 37 ℃ and the rotation speed of 100 rpm. And finally detecting the content of the released BTZ and LBH589 in the external liquid by using 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 examined by dynamic light scattering and transmission electron microscopy, respectively, to characterize the effect of drug release on the nanostructure after the 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, almost no drug was released at pH 7.4; about 44% of BTZ released from the nanoparticles under pH5.0; about 77% of the BTZ was released from the nanoparticles at pH5.0/DTT (10 mM); about 53% of LBH589 is released from the nanoparticles under Gly (n (Gly)/n (AABC-Zn-LBH 589) = 10/1) conditions; about 75% of LBH589 was released from the nanoparticles under Gly (n (Gly)/n (AABC-Zn-LBH 589) = 10/1)/DTT (10 mM). The TEM characterization structure shows that the nanoparticle structure is destroyed and shows an irregular structure when the responsive drug is released for 24 hours.
6. Receptor expression and ApoE targeting ability assays
6.1 ApoE receptor expression on GBM cell membranes
Expression of LRP1, LRP2 and LDLR on SNB19 and LN229 cell membranes was studied with normal astrocyte HA1800 as control.
Cell membrane proteins were first extracted according to the instructions of the cell membrane and cytosolic protein extraction kit, then protein concentration was determined using the BCA protein quantification kit, and the protein solution (20. Mu.g/sample) was mixed with 1 XSDS-PAGE loading buffer and denatured at 95 ℃ for 10 min. The protein samples were then added to the channels of a 7.5% SDS-polyacrylamide gel, electrophoresed at constant pressure 80v until the protein markers were completely separated, and transferred to polyvinylidene fluoride (PVDF) membranes (300 mA,1.5 hours). After 1 hour blocking of PVDF membranes with 5% skim milk, PVDF membranes were trimmed according to protein marker molecular weight and position and incubated with primary anti-LRP1 rabbitt (1. The primary antibody was collected, the membrane was washed 3 times with 1 × TBST for 10 minutes each, and then the PVDF membrane was incubated with the secondary antibody at room temperature for 1 hour. Secondary antibodies were collected, membranes were washed 3 times with 1 × TBST, and ECL hypersensitive chemiluminescent developer was added and imaged for luminescence using an Amersham Imager 680RGB instrument. The results are shown in FIG. 12. The results show that: LRP1 protein is highly expressed on SNB19 and LN229 cell membranes compared to normal astrocyte HA 1800.
6.2 detection of targeting ability of ApoE modified Nanoparticulate drug to GBM cells
To test whether the ApoE modified nano-drug has the ability to target SNB19 and LN229 cells, the prepared nano-drugs NM @ (BTZ/LBH 589/Cy 5) and ApoE-NM @ (BTZ/LBH 589/Cy 5) were co-incubated with SNB19 and LN229 cells, respectively, for 4 hours at the same Cy5 content (as measured by a microplate reader). Removing the culture solution, washing the cells for 3 times by using 1 XPBS (phosphate buffer solution), digesting, centrifuging and collecting the cells, collecting and detecting Cy5 fluorescent signals in the cells by using a flow cytometer, and then performing result analysis by using FlowJo _ V10 software; alternatively, 4% paraformaldehyde was added to the cells, and after 10 minutes, the cells were washed with 1 × PBS 2 times, followed by DAPI (10 μ g/mL) to stain the nuclei for 10 minutes, and the cells were washed with 1 × PBS 3 times, and then fluorescence signals were collected and imaged by a laser confocal microscope (in each sample, parameters were kept consistent when Cy5 fluorescence was collected). 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 surface modified ApoE can enter SNB19 and LN229 cells more, which indicates that ApoE can be specifically combined with LRP1 and exerts the capability of actively targeting SNB19 and LN229 cells.
7. Evaluation of capability of nano-drug to cross blood brain barrier
In order to detect the capacity of the nano-drug to cross BBB, the invention establishes a blood brain barrier model in vitro. Bend.3 (50,000 cells/well) was seeded in a transwell chamber and 800. Mu.L of cell culture broth was added to the lower well plate (24 well plate). At 37 deg.C, 5% CO 2 When the trans-endothelial cell electronic impedance (TEER) value is higher than 200 omega cm 2 In this case, the nano-drugs NM @ (BTZ/LBH 589/Cy 5) or ApoE-NM @ (BTZ/LBH 589/Cy 5) having the same Cy5 content were added to the chamber, and then 50. Mu.L of the culture solution was removed from the well plate at 4 hours, 12 hours, and 24 hours, respectively. And finally, detecting the fluorescence value of Cy5 in the culture solution by a microplate reader. The results are shown in FIG. 14. The results show that: at 24 hours, the nano-drug ApoE-NM @ BTZ/LBH589 with the surface modified ApoE crossed the BBB by about 1.76 times of NM @ BTZ/LBH589/Cy 5.
8. MTT method for detecting influence of nano-drug on GBM cell survival
Various GBM cells were seeded in 96-well plates (5,000 cells/well) at 37 ℃ with 5% CO 2 After culturing for 24 hours in a sterile cell culture box, adding the drug carrier ApoE-NM, free drug BTZ/LBH589 or nano drug NM @ BTZ + NM @ LBH589NM @ (BTZ/LBH 589), apoE-NM @ (BTZ/LBH 589) of different drug concentrations were added to the culture medium and incubated for 48 hours, respectively. Then, after adding 10. Mu. LMTT (5 mg/mL) solution to the culture solution and incubating for 4 hours, the culture solution was removed and 150. Mu.L of dimethyl sulfoxide was added to the sample well, incubated at room temperature for 15 minutes with shaking, and finally, absorbance at a wavelength of 490nm was measured using a microplate reader. Each sample had three replicate wells. The results are shown in FIG. 15. The results show that: the nano-particles without drug loading almost have no cytotoxicity, and show good biocompatibility; the nanoparticle loaded with drug affects cell survival, and the nano-drug NM @ (BTZ/LBH 589) co-loaded with BTZ and LBH589 has stronger anti-cell proliferation effect than ApoE-NM @ (BTZ/LBH 589), and due to modification of ApoE, apoE-NM @BTZ/LBH589 is capable of entering cells more, and exhibits better effect of inhibiting cell survival compared to 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 culturing in the sterile cell incubator of (1) for 24 hours, apoE-NM, free drug BTZ/LBH589 or nano drug NM @BTZ + NM @ LBH589, NM @ (BTZ/LBH 589), apoE-NM @ (BTZ/LBH 589), at a concentration of 4.0nM/8.0nM (BTZ/LBH 589), were added to the culture medium, respectively, and incubated for 48 hours. Next, the culture solution was removed, and the cells were washed 2 times with 1 XPBS, and ER-tracker Red working solution pre-warmed at 37 ℃ was added to the cells, and co-incubated at 37 ℃ for 20 minutes. The ER-Tracker Red staining solution was removed and the cells were washed 2 times with cell culture medium. Cells were digested, centrifuged and collected, and ER-Tracker Red fluorescent signals were collected and detected in the cells using a flow cytometer, followed by analysis of the results using FlowJo _ V10 software. The results are shown in FIG. 16. The results show that: at the same drug concentration, the free drug cannot cause endoplasmic reticulum stress, while the nano-drug causes endoplasmic reticulum stress, and ApoE-nm @ (BTZ/LBH 589) causes endoplasmic reticulum stress to a degree 2.5 and 1.3 times that of nm @ (BTZ/LBH 589), respectively, in SNB19 and LN229 cells; the degree of endoplasmic reticulum stress caused by NM @ (BTZ/LBH 589) is NM @ BTZ + NM @ LBH58 in SNB19 and LN229 cells, respectively2.3 and 1.2 times of 9.
10. Flow cytometry for detecting influence of nano-drug on GBM apoptosis
Various GBM cells were seeded in 12-well plates (70,000 cells/well) at 37 ℃ with 5% CO 2 After culturing in the sterile cell incubator of (1) for 24 hours, free drugs BTZ/LBH589 or nano drugs NM @ (BTZ/LBH 589), apoE-NM were added to the culture solution 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 Xg, 3 min) and washed 2 times with 1 XPBS, then the cells were dispersed in1 XPannexin V-FITC binding solution, 5 μ Lannexin V-FITC was added and mixed gently at room temperature, after 5 min, 10 μ L of propidium iodide staining solution (PI) was added and mixed gently, after 15 min, the cells were placed on ice, fluorescence signals in the cells were collected and detected by flow cytometry, and the results were analyzed by FlowJo _ V10 software. The results are shown in FIG. 17. The results show that: at the same drug concentration, the free drug did not cause apoptosis, while the nano-drugs ApoE-NM @ (BTZ/LBH 589) and NM @ (BTZ/LBH 589) apparently caused apoptosis of SNB19 and LN229 cells.
11. Establishment of in situ TP53 mut GBM model
The SNB19 or LN229 cells were seeded in 15cm cell culture dishes at 37 ℃ with 5% CO 2 When cultured under the conditions of (1) to a logarithmic growth phase, the culture solution was removed, the cells were washed 2 times with 1 XPBS, the cells were digested with 1 XPrysin for 3 minutes, the cells were collected by centrifugation, and the cells were cultured at a density of 5X 10 5 The individual cells/5. Mu.L were dispersed in1 XPBS buffer and placed on ice until use. Anaesthetizing 6-8 week-old female Balb/c nude mice with chloral hydrate solution (8 mg/20 g), cutting open scalp with special blade, perforating at the left upper part of mouse brain with skull drill to about 3.5mm depth, and injecting 5 × 10 with micro-needle injector 5 Injecting SNB19 or LN229 cells into the holes, sealing the holes with melted bone wax, adhering the cut skin with tissue sealant, and placing the mice in an electric blanket to recover and then feeding. All animal experiments were processed according to protocols approved by the university of Henan, and the animal center and animals were satisfiedRequirements of the nursing and use committee.
12. In vivo distribution experiment of nano-drug
The nano-drugs NM @ (BTZ/LBH 589/Cy 5), apoE-NM @ (BTZ/LBH 589/Cy 5) or free Cy5 were injected into the tumor-bearing Balb/c nude mice via caudal vein (Cy 5:0.1 mg/kg), respectively, and the distribution of these nano-drugs in the mice was monitored at different time points (1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours) using Lumina IVIS III near infrared fluorescence imaging system (Ex =620nm Em = 710nm). In addition, in order to more clearly understand the distribution of the nano-drugs in each organ and brain of a tumor-bearing Balb/c nude mouse, the mouse is killed according to a cervical dislocation method after 4 hours of tail vein injection of each nano-drug, the heart, liver, spleen, lung, kidney and brain of the mouse are taken out, and the Cy5 fluorescence distribution in each organ and brain of the mouse is detected by a Lumina IVIS III system of a small animal imager.
In order to further observe the distribution and penetration depth of the nano-drug in brain and in situ tumor, similarly, 4 hours after nano-drug injection, tumor-bearing mice were sacrificed by cervical dislocation, and the mouse brains were taken out and placed in neutral general tissue fixative to be protected from light for 48 hours. Taking out mouse brain and placing in 20% w / v Until it completely sinks to the bottom of the solution (about 12 hours), and then the mouse brain is placed at 30% w / v For about 12 hours in sucrose solution. Then, the mouse brain placed in the sucrose solution is taken out and placed in the center of the embedding groove, the OCT embedding medium is added into the embedding groove, and then the embedding groove is placed in liquid nitrogen to freeze the embedding medium so as to fix the position of the mouse brain. The specimen was placed on a holder, sliced with a constant temperature microtome (generally at a slicing temperature of minus 22 ℃) to a thickness of 15 μm, attached to a pre-cooled slide glass, and stored in a refrigerator at-20 ℃. Sections were fixed for 15 minutes in 4 ℃ pre-cooled acetone, washed 3 times with 0.3% Triton X-100, stained for 10 minutes for nuclei with DAPI, removed and washed for DAPI staining, mounted for mounting, and images were taken by scanning with a Pannoramic MIDI instrument.
Furthermore, in order to quantify the accumulation amount of the nano-drug in each organ, the mouse was sacrificed according to the cervical dislocation method at 4 hours after the nano-drug injection, the heart, liver, spleen, lung, kidney, brain and brain tumors of the mouse were removed, they were weighed and placed in an EP tube containing 0.6ml of a 1% triton X-100 solution, and two circular 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 overnight at room temperature, and then centrifuged (14,000 × g,30 minutes) to take the supernatant, followed by detecting the fluorescence intensity of Cy5 (Ex =630nm, em =670 nm) in the sample with a microplate reader, and calculating the Cy5 content according to a standard curve, and the final value was displayed in units of injection percentage of ID/g per gram of tissue (3 mice per group).
The results in FIG. 18 show that: the amount of nanomedicines enriched in mouse brain compared to free Cy5 was significantly increased and was the greatest at the tumor site in mice 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 Nanoparticulate drugs mut Experiment of GBM treatment
At 10 days tumor bearing, mice were randomized into groups for treatment evaluation. ApoE-NM @ (BTZ/LBH 589), free BTZ/LBH589, apoE-NM or 1 XPBS were injected into mice by tail vein injection (drug dose 2 mg/kg) 1 time every 2 days for 5 times, respectively. The change in body weight of the mice was monitored during the treatment period, and the survival time of the mice was monitored for a long period until day 90 of the establishment of the in situ GBM model. The results are shown in FIG. 19. The results show that: in the SNB19 and LN229 models, the body weight of mice in the ApoE-NM @ (BTZ/LBH 589) treated group remained essentially stable during the treatment period, and the body weight of mice in the other treated groups continued to decrease; in the SNB19 model, NM @ (BTZ/LBH 589) and ApoE-NM @ (BTZ/LBH 589) extended the overall survival of the mice to 48 days and 70 days, respectively, unlike the total survival of the PBS, apoE-NM and free BTZ/LBH589 treated groups of mice, which was less than 32 days. Similarly, in the LN229 model, the total survival time of mice in the NM @ (BTZ/LBH 589) and ApoE-NM @ (BTZ/LBH 589) treatment groups was extended to over 51 days and 90 days, respectively.
14. In situ TP53 mut Histological analysis after GBM treatment
At the end of the horizontal treatment of the animals (5 treatments with drug administration were completed), the brain tissue or organ of the mice was placed in a neutral general-purpose tissue fixing solution, and after 2 days, the tissue was taken out and placed in a dedicated card slot to be flushed overnight with running water (for the brain tissue, the olfactory bulb and cerebellum may be excised), and then dehydrated, that is, the tissue was placed in a mixed solution of 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 for 1 hour, 100% xylene (I) for 1 hour, and 100% xylene (II) for 1 hour in this order. The tissue was then placed in melted paraffin (65 ℃) for 3 hours of waxing, and finally placed in an embedding tank and melted paraffin was added dropwise to the tank with an embedding machine until completely submerged in the tissue, followed by cooling on a cold stage for at least 2 hours. The tissue was then removed from the embedding tank and sliced to a thickness of 4 μm, spread in a 40 ℃ water bath for about 30 seconds (fully spread but not broken), scooped with an adhesive slide, and then placed in a 60 ℃ oven for 2 hours for baking. Before any histological staining is carried out, paraffin sections are required to be dewaxed, and the specific steps are that the paraffin sections are placed in the xylene (I) for 5 minutes, the xylene (II) for 5 minutes, the xylene (III) for 5 minutes, the 100% ethanol (I) for 5 minutes, the 100% ethanol (II) for 5 minutes, the 90% ethanol for 5 minutes, the 80% ethanol for 5 minutes, the 70% ethanol for 5 minutes, the distilled water (I) for 5 minutes, the distilled water (II) for 5 minutes and the distilled water (III) for 5 minutes in sequence, and the paraffin sections can be placed in the distilled water for a long time.
14.1 hematoxylin & eosin (H & E) staining
Firstly, placing the tissue section into hematoxylin (hematoxylin, staining cell nucleus and purple) solution for staining for 6 minutes, then flushing the tissue section with running water for 10 minutes to change the purple color into blue color (if the hematoxylin is too deeply stained, alcohol hydrochloric acid or a commercial hematoxylin differentiation reagent is needed for differentiation, the differentiation time is generally 1-2 seconds, and if the differentiation degree is too large, counterstaining can be carried out). The slide was then stained in Eosin (Eosin, stained cytoplasm, orange red) solution for 3 minutes, washed 2 times and then directly submitted to the dehydration mounting step (no excessive hydrochloric acid color separation was needed because Eosin masks the hematoxylin color in the cytoplasm). Placing the slices in sequenceIn 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 standing time in 90% ethanol can be longer to avoid over-high concentration of eosin). Finally, the flakes are placed in xylene (which can be kept in xylene for a long time) and subsequently are mixed in a volume ratio of 1:1 neutral gum: sealing the slices with xylene, removing bubbles, and air drying in a fume hood
Figure BDA0003894963610000161
The 40 instruments take scan shots and analyze the data using QuPath 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 the mice in each treatment group are all used for discovering pathological changes and side effects, which indicates that the nano-medicament has good biocompatibility.
14.2 use of TUNEL for apoptosis detection
20. Mu.g/mL (10 mM Tris-HCl pH7.4-7.8 diluted) DNase-free proteinase K was added dropwise to the tissue section samples, allowed to act at 20-37 ℃ for 25 minutes, and then excess proteinase K was removed and washed 3 times with 1 XPBS for 5 minutes each. mu.L of TUNEL assay was added drop wise to the sample and placed in a humidified dark box containing water to keep moist to minimize evaporation of the TUNEL assay, incubated at 37 ℃ for 60 minutes in the dark, then excess TUNEL assay 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 slides were washed with 1 XPBST for 2 min, 3 times. After the mounting is carried out by using the anti-fluorescence quenching mounting agent, fluorescence signal acquisition and picture shooting are carried out by using a laser confocal microscope. 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, causing tumor cell apoptosis.
14.3 immunohistochemistry
Drawing a circle as small as possible around the sample on the section by using an immunohistochemical pen (aiming at isolating the sample and saving reagents such as antibody and the like), leaving a corner in the circle for convenient suction, placing the section in a wet box, and placing the section 3％ H 2 O 2 After 20 minutes of application to the sliced tissue, for removal of endogenous peroxidase, excess H was washed away with 1 XPBST 2 O 2 2 minutes each time, 3 times. Soaking the slices in 0.01M citrate buffer solution, then putting the slices into a microwave oven to be heated until the slices are boiled, continuously keeping the sub-boiling temperature (95-98 ℃) for 10 minutes, carrying out antigen thermal restoration, and finally cooling the slices on a laboratory bench for 30 minutes. The pieces were soaked in1 XPBST for 2-3 minutes, wiped dry, blocked with 3% BSA for 30 minutes, and washed 3 times with PBST for 2 minutes each. Primary antibodies such as anti-Ki67, anti-cleared caspase-3, etc. (diluted in 1% BSA at the dilution ratio referred to the antibody specification) were added to the samples (within the assembly circle) in an amount to cover the samples, the primary antibodies were removed after overnight incubation at 4 ℃ in a wet box (water was added to the wet box) and the plates were washed with 1 XPBST for 2 minutes 3 times. Biotin-labeled goat anti-rabbit IgG was added dropwise to the samples, incubated at 37 ℃ for 30 minutes, and the slides were washed with 1 XPBST for 2 minutes, 3 times in total. SABC was added dropwise to the samples, incubated at 37 ℃ for 30 minutes, and the slides were washed with 1 XPBST for 2 minutes, 3 times total. DAB is used as a color developing agent and is dripped on a sample for 5-10 minutes (no more than 10 minutes, otherwise, nonspecific dyeing exists), the sample is washed for 10 minutes by running water, the dyeing is stopped, microscopic examination is carried out, and if the effect is not good, the dyeing can be continued. Finally, staining the cell nucleus with hematoxylin solution for 2-3 min, flushing with running water for 5-10 min, dehydrating and mounting the sample, and&e, the subsequent steps of dyeing are consistent. By using
Figure BDA0003894963610000171
The 40 instruments take scan shots and analyze the data using QuPath software. The results are shown in FIG. 22. The results show that: in SNB19 and LN229 in situ tumor-bearing models, compared with other treatment groups, the tumor tissue of the ApoE-NM @ (BTZ/LBH 589) treatment group has obviously reduced Ki67 positive signals and obviously increased CC3 positive signals, and the CC3 positive signals are mainly distributed in the tumor tissue but not in normal brain tissue, which indicates that ApoE-NM @ (BTZ/LBH 589) can inhibit tumor cell proliferation and promote apoptosis without affecting normal brain tissue cells.
14.4 detection of in situ TP53 by nano-drug by immunoblotting mut Apoptosis-related protein regulation in GBM tumors
After completion of 5 treatments, tumor tissues in mouse brain were taken out after the mouse was sacrificed by dislocation of cervical vertebrae, placed in a high performance RIPA lysate containing 1mM PMSF, and two circular 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 (jxstprp-48), placed on ice for lysis for 30 minutes, centrifuged (15,000rpm, 10 minutes) to take supernatant, and after protein concentration was measured with a BCA protein quantification kit, a protein solution (10 μ g/sample) was mixed with a1 x SDS-PAGE loading buffer and denatured at 95 ℃ for 10 minutes. The protein samples were then added to 7.5% SDS-polyacrylamide gel channels, electrophoresed at constant pressure 80v until the protein markers were completely separated, and the samples were transferred to polyvinylidene difluoride (PVDF) membranes (300mA, 1 hour). After 1 hour of 5% milk blocking of the PVDF membrane, the PVDF membrane was cut according to protein marker molecular weight and position, and incubated overnight at 4 ℃ with primary anti-Bip rabbitflag pAb (1. The primary antibody was collected, the membrane was washed 3 times with 1 × TBST for 10 minutes each, and then the PVDF membrane was incubated with the secondary antibody at room temperature for 1 hour. Secondary antibodies were collected, and after washing the membrane 3 times with 1 × TBST, ECL hypersensitive chemiluminescent developer was added and luminescent imaging was performed using an Amersham Imager 680RGB instrument. The results are shown in FIG. 23. The results showed that the expression level of Bip/CAPN1/cCasp12/cCasp3 protein was significantly increased in the tumor tissues of the ApoE-NM @ (BTZ/LBH 589) treated group compared to the other treated groups.
15. Effect of nano-drugs on blood convention and blood biochemistry of mice
First, healthy Balb/c mice (6-8 weeks old) were randomly divided into two groups (4 mice per group). Mice were injected with different drug doses of ApoE-NM @ (BTZ/LBH 589) (1 mg/kg,2mg/kg,4 mg/kg) or 1 XPBS, respectively, via the tail vein. Approximately 200 μ L of blood was drawn from the mouse canthus on days 2,7 and 14, respectively. A portion of the collected whole blood is subjected to routine blood analysis including measurement of indicators such as Red Blood Cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean Corpuscular Volume (MCV), mean Corpuscular Hemoglobin (MCH), mean Corpuscular Hemoglobin Concentration (MCHC), white Blood Cells (WBC), and Platelets (PLT). The other part of the blood was centrifuged (800 Xg, 10 minutes), and serum was collected and examined for alanine Aminotransferase (ALT), alkaline phosphatase level (ALP), serum Albumin (ALB), aspartate Aminotransferase (AST), UREA (UREA), creatinine (CREA) and Uric Acid (UA). The results are shown in FIG. 24. The results show that: after injection of ApoE-NM @ BTZ/LBH589 with different medicine doses, the values of the blood routine and blood biochemical parameters of the mice are similar to those of the control group on the 2,7 and 14 days, and no significant difference exists, which indicates that ApoE-NM @ BTZ/LBH589 has good biocompatibility and no side effect.
16. Effect of nano-drugs on liver and kidney of mice
To determine whether the nano-drug caused inflammation, we examined the expression of IL-1 β, IL-6 and TNF- α mRNA in the liver and kidney of mice after nano-drug injection. Healthy Balb/c white mice (6-8 weeks old) were selected and randomly divided into 2 groups of 3 mice each. The nano-drug ApoE-NM @ (BTZ/LBH 589) was injected into mice via tail vein (2 mg/kg). After 2 days, the cervical vertebrae of the mouse were dislocated to death and the kidney and liver were removed, they were placed in an EP tube containing an RNA extract, two circular 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), and total RNA in the tissue was extracted according to the standard procedure of the total RNA extraction kit. Using Thermo Scientific TM After the NanoDrop instrument detects the RNA concentration, the RNA is reverse transcribed to form cDNA according to the instructions of RT reagent Kit (Perfect Real Time), and then qRT-PCR experiments are performed according to the standard procedures of SYBR Premix Ex Taq II Kit and using a Real-Time fluorescent quantitative PCR instrument (Lightcycler 480 II). Experimental data 2 -ΔΔCt The formula is further calculated. The results are shown in FIG. 25. The results show that: after 2 days of ApoE-NM @ BTZ/LBH589 injection into mice, mRNA expression levels of inflammatory factors (IL-1 beta, IL-6 and TNF-alpha) in liver and kidney of the mice were found to be not significantly different from those of the control group by qRT-PCR characterization, which indicates that ApoE-NM @ BTZ/LBH589Has no side effect on the liver and the kidney of the mouse, does not induce inflammatory reaction, and has good biocompatibility.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

Claims (8)

1. A synthetic lethal nano-drug combined carrier is characterized in that the combined carrier comprises polypeptide, amide compounds containing polyhydroxy, amide compounds containing bipyridyl groups, proteasome chymotrypsin-like activity inhibition drugs containing boron hydroxyl, histone deacetylase inhibition drugs and compounds containing disulfide bonds; the amide compound containing polyhydroxy and proteasome chymotrypsin-like activity inhibition drugs containing boron hydroxyl react to generate boron ester bonds, and the amide compound containing bipyridyl groups and the histone deacetylase inhibition drugs are chelated through zinc ions.
2. The synthetic lethal nano-drug combination carrier according to claim 1, wherein the proteasome chymotrypsin-like activity inhibition drug containing a boron hydroxyl group is bortezomib.
3. The synthetic lethal nano-drug combination carrier of claim 1, wherein the histone deacetylase inhibitory drug is panobinostat.
4. The synthetic lethal nano-drug combination carrier of claim 1, wherein the polypeptide is a sulfhydryl-modified polypeptide.
5. The synthetic lethal nano-drug combination carrier according to claim 1, wherein said amide compound containing polyhydroxy is selected from N-acryloyl glucosamine.
6. The synthetic lethal nano-drug combination carrier according to claim 1, wherein the amide compound containing a bipyridyl group is selected from the group consisting of N- (2-acrylamidoethyl) - [2,2' -bipyridine ] -5-carboxamide, N- (3-acrylamidopropyl) - [2,2' -bipyridine ] -5-carboxamide and N- (4-acrylamidobutyl) - [2,2' -bipyridine ] -5-carboxamide.
7. The synthetic lethal nano drug combination carrier according to claim 1, wherein the combination carrier has a spherical structure.
8. Use of the synthetic lethal nano-drug combination carrier of any one of claims 1 to 7 in the preparation of a targeted drug for treating GBM.
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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
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