CN113521035A - Preparation method and application of nano-drug for chemo-immune combination therapy - Google Patents

Preparation method and application of nano-drug for chemo-immune combination therapy Download PDF

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CN113521035A
CN113521035A CN202110900098.6A CN202110900098A CN113521035A CN 113521035 A CN113521035 A CN 113521035A CN 202110900098 A CN202110900098 A CN 202110900098A CN 113521035 A CN113521035 A CN 113521035A
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drug
tmz
otx
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tumor
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CN113521035B (en
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邹艳
刘艳杰
师冰洋
郑蒙
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Henan University
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses a preparation method and application of a chemo-immune combination therapy nano-drug, and relates to the technical field of medicines. The invention discloses a red blood cell membrane camouflage bionic nano-drug co-loaded with a TMZ (Tetramethylbenzidine) and BET (BET bromodomain inhibitor). The bionic nano-drug serving as a multifunctional nano-platform can enhance the blood circulation time and the permeability of BBB, and the co-delivered BET bromodomain inhibitor can reduce the proliferation of cancer cells by improving the TMZ sensitivity. The BET bromodomain inhibitor can inhibit the DNA damage repair of GBM tumor cells, thereby enhancing the drug effect of TMZ. TMZ chemotherapy and OTX can stimulate the immune system in vivo to be activated, and the BET bromodomain inhibitor can destroy the axis of immunosuppressive PD-L1/PD-1, recruit T cells to attack tumors, induce anti-tumor immune stress response, and achieve the effect of chemotherapy and immune combined treatment.

Description

Preparation method and application of nano-drug for chemo-immune combination therapy
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of a nano-drug for chemo-immune combination therapy.
Background
Temozolomide (TMZ) is currently the first-line chemotherapeutic drug for the treatment of glioblastoma multiforme (GBM). Although conventional TMZ chemotherapy can improve the efficacy to some extent, GBM patients are very resistant to TMZ due to the existence of GBM tumor cell DNA self-repair mechanism, which significantly compromises the efficacy of TMZ. Therefore, by inhibiting the DNA repair of tumor cells, the drug resistance of GBM to TMZ is reduced, and the effect of improving the chemotherapeutic effect of GBM is played a great importance.
Recently, the use of immunomodulation to treat tumors has attracted considerable attention by researchers. Immunotherapy, such as tumor vaccines, immune checkpoint blockade therapy and CAR-T, show encouraging clinical outcomes in the treatment of different types of cancer, including melanoma, leukemia and GBM. In the immunotherapy strategy, blocking therapy based on PD1/PD-L1 immune checkpoint inhibitors brings new hopes for the treatment of cancer. However, commercial PD1 antibodies (e.g., nivolumab, pembrolizumab) have limited therapeutic efficacy in GBM patients, with less than 10% of patients responding chronically. The main reason is attributed to the high immunosuppressive cytokine levels in the tumor microenvironment of GBM and the poor T cell infiltration, and thus, the conversion of "cold" immunosuppressive GBM into T cell inflammatory "hot" tumors plays a key role in the reversal of immunosuppression and GBM immunotherapy.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preparation method and application of a chemo-immune combination therapy nano-drug to solve the technical problems.
Bromodomain protein 4(Brd4) is the most important functional protein in the bromodomain and superterminal family of proteins. It has been found that Brd4 is closely related to DNA repair, and that inhibition of Brd4 can reduce the response of various cells to DNA damage repair. In addition, Brd4 was able to reduce the expression of PD-L1 on the surface of tumor cells. Various Brd4 inhibitors (e.g., JQ1, OTX015, etc.) have been used in GBM clinical studies because they exhibit good anti-tumor effects at the cellular level. However, the clinical experiment effect is not ideal due to the short pharmacokinetics and strong hydrophobicity of the naked Brd4 inhibitor.
Based on the Brd4 inhibitor, the inventor utilizes nano-drug loaded BET bromodomain inhibitor to inhibit GBM tumor cell DNA damage repair, thereby enhancing the drug effect of TMZ. We developed a red blood cell membrane camouflage biomimetic nano-drug (ABNM @ TMZ/OTX) co-loaded with TMZ and BET bromodomain inhibitors. The bionic nano-drug serving as a multifunctional nano-platform can enhance the blood circulation time and the permeability of BBB, and the co-delivered BET bromodomain inhibitor can reduce the proliferation of cancer cells by improving the sensitivity of TMZ. In addition, TMZ chemotherapy and BET bromodomain inhibitors can stimulate the immune system in vivo to be activated, and the BET bromodomain inhibitor can destroy the immunosuppressive PD-L1/PD-1 axis, recruit T cells to attack tumors, induce anti-tumor immune stress response, and achieve the effect of chemotherapy and immune combined treatment. In mice bearing in situ GL261 brain tumors, treatment with ABNM @ TMZ/OTX significantly inhibited tumor growth and significantly prolonged survival of the mice with reduced side effects.
Specifically, the present invention is realized by:
the invention provides a bionic nano-drug, which comprises an inner core and a shell coated outside the inner core, wherein the inner core comprises a first component, a second component and a carrier, the first component comprises temozolomide, the second component comprises a BET bromodomain inhibitor, and the carrier is pH sensitive nano-particles;
the shell comprises an erythrocyte membrane, a cancer cell membrane, an immune cell membrane or a blood platelet, and a targeting agent is modified on the biomembrane so that the bionic nano-medicament can target cells.
Chemotherapy with the first component (TMZ) activates the immune response in vivo, and the second component (BET bromodomain inhibitor) not only interferes with cell proliferation, but also prevents DNA repair to increase tumor sensitivity to TMZ and enhance the efficacy of TMZ. In addition, the BET bromodomain inhibitor can inhibit the expression of PD-L1, recruit T cells to attack tumors, induce anti-tumor immune stress response and achieve the effect of chemotherapy and immune combined treatment.
The carrier can meet the requirement that the bionic nano-drug can realize the release of the drug under the specific pH environment.
The shell adopts erythrocyte membranes, has the characteristic of no immunogenicity, and the target cells can be targeted by modifying a targeting agent on the biomembrane, so that the accurate targeting of the medicament is realized. In other embodiments, the biological membrane may be selected from one or more of a cell membrane, a bacterial membrane, and a viral membrane.
The bionic nano-drug provided by the invention can improve BBB permeability, increase tumor accumulation and blood retention, realize drug release of tumor microenvironment response, and reduce cancer cell proliferation by improving TMZ sensitivity through the co-delivered BET bromodomain inhibitor.
In a preferred embodiment of the present invention, the BET bromodomain inhibitor is an inhibitor of at least one protein selected from the group consisting of: BRD2, BRD3, BRD4, and BRDT;
preferably, the BET bromodomain inhibitor is an inhibitor of BRD 4;
preferably, the inhibitor of BRD4 is selected from an inhibitor of at least one of the following, or a pharmaceutically acceptable salt or solvate of any of the following: derivatives of plant polyphenol-resveratrol, isoxazole structure BRD4 inhibitors, benzodiazepine BRD inhibitors, pyridone BRD4 targeted inhibitors, JQ1, CeMMEC2, PF-1, bromosporine, OTX-015, TEN-010, BI2536, TG101348 and LY 294002.
Preferably, the isoxazole structure BRD4 inhibitor is selected from CPI-203 or CPI-0610; the benzodiazepine BRD inhibitor is selected from I-BET 151 or I-BET 762; the plant polyphenol-resveratrol derivative is selected from RVX-208; the pyridone BRD4 targeted inhibitor is selected from ABBV-075.
In addition, in other embodiments, the BET bromodomain inhibitor may be selected as long as it can achieve expression deregulation of BET family member proteins within the scope of the inventive concept of the present invention, and is not limited to the above-listed several inhibitors.
The scope of the present invention includes all pharmaceutically acceptable salt forms of the compounds used according to the invention, which can be formed, for example, by protonation of an atom bearing a lone electron pair susceptible to protonation, such as amino groups, with inorganic or organic acids, or as salts of acid groups (such as carboxylic acid groups) with physiologically acceptable cations. Exemplary base addition salts include, for example: alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; a zinc salt; an ammonium salt; fatty amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as ethylenediamine N, N-dibenzyl salt, benzathine salt, benzphetamine salt; heterocyclic aromatic amine salts such as pyridinium, picolinate, quinolinate or isoquinolinium salts; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt or tetrabutylammonium salt; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts include, for example: inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate (e.g., sulfate or bisulfate), nitrate, phosphate (e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate), carbonate, hydrogencarbonate, perchlorate, borate, or thiocyanate; organic acid salts such as acetate, propionate, butyrate, valerate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate (camphorate), glucoheptanoate (glucoheptonate), or pivalate; sulfonates such as methanesulfonate (methanesulfonate), ethanesulfonate (ethanesulfonate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (benzenesulfonate), p-toluenesulfonate (toluenesulfonate), 2-naphthalenesulfonate (naphthalenesulfonate), 3-benzenesulfonate, or camphorsulfonate; a glycerophosphate salt; and acidic amino acid salts such as aspartate or glutamate.
Furthermore, the scope of the present invention includes any solvate form of a compound used according to the present invention, including, for example, solvates with water (i.e. as hydrates) or with organic solvents, such as, for example, methanol, ethanol or acetonitrile (i.e. as methanolate, ethanolate or acetonitrilate), or any crystalline form (i.e. as any polymorph) or amorphous form of a compound used according to the present invention. It will be understood that these solvates of the compounds for use according to the invention also include solvates of the pharmaceutically acceptable salts of the respective compounds.
Furthermore, the compounds used according to the invention may exist in the form of different isomers, in particular stereoisomers (including, for example, geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers. All such isomers of the compounds mentioned in the present specification are considered to be part of the present invention, as mixtures, in pure or substantially pure form. With respect to stereoisomers, the present invention includes isolated optical isomers of the compounds used according to the invention and any mixtures thereof (including, in particular, racemic mixtures/racemates). The racemates can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The respective optical isomers can also be obtained from the racemates by forming a salt with an optically active acid and then crystallizing. The invention also includes any tautomer of the compounds provided herein.
The scope of the present invention also includes compounds for use according to the present invention wherein one or more atoms are replaced by a particular isotope of the corresponding atom. For example, the present invention includes the ones mentioned in the present specificationUse of a compound wherein one or more hydrogen atoms (or for example all hydrogen atoms) are replaced by deuterium atoms (i.e. 2H; also referred to as "D"). Thus, the invention also includes deuterium enriched compounds for use according to the invention. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol% hydrogen-1 (1H) and about 0.0156 mol% deuterium (2H or D). Deuterium content in one or more hydrogen positions in the compounds used according to the invention can be increased using deuteration techniques known in the art. For example, the compounds mentioned in the present specification or the reactants or precursors for synthesizing the corresponding compounds may use, for example, heavy water (D)2O) carrying out H/D exchange reaction. Other suitable deuteration techniques are described in: atzrodt J et al, Bioorg Med Chem,20(18),5658-5667, 2012; william JS et al, Journal of laboratory Compounds and Radiopharmaceuticals,53(11-12),635-644, 2010; modvig A et al, J Org Chem,79, 5861-. Deuterium content can be determined, for example, using mass spectrometry or NMR spectroscopy. Unless otherwise specifically indicated, it is preferred that the compounds used according to the invention are not deuterium-enriched. It is therefore preferred that naturally occurring hydrogen atoms or 1H hydrogen atoms are present in the compounds used according to the invention.
In a preferred embodiment of the present invention, the target cell is a glioblastoma cell.
In one embodiment, the targeting agent is DSPE-PEG-ApoE, the targeting agent is covalently linked to the biological membrane via DSPE, and the DSPE-PEG-ApoE is prepared by reacting DSPE-PEG-Mal with an apolipoprotein E polypeptide.
DSPE-PEG-Mal (phosphatidylethanolamine-polyethylene glycol-maleimide) and apolipoprotein E polypeptide are selected from commercially available products. In other embodiments, the molecular weight of PEG can be adaptively adjusted as needed, and can be, for example, 2000, 5000, 10000, etc.
In a preferred embodiment of the present invention, the theoretical drug loading of the first component in the biomimetic nano-drug is 1-40 wt.%, and the theoretical drug loading of the second component in the biomimetic nano-drug is 1-40 wt.%. Optionally, the theoretical drug-loading rate of the first component is 5-30 wt.%, and the theoretical drug-loading rate of the second component in the biomimetic nano-drug is 5-30 wt.%.
In a preferred embodiment of the present invention, the pH sensitive nanoparticle is a-glucan, alkali lignin or a lignin macromolecular derivative;
preferably, the lignin macromolecule derivatives are one or more of lignin macromolecule derivatives obtained by introducing macromolecules into lignin macromolecules by a physicochemical method.
In one embodiment, the polymer introduced into the lignin macromolecule by a physicochemical method or the like includes one or more of polyethylene glycol, polyglycerol, polyvinyl alcohol, polyacrylic acid, polylactic acid, polyhydroxylactic acid, polycaprolactone, lactic acid-glycolic acid copolymer, polyhydroxybutyrate.
In one embodiment, the pH-sensitive nanoparticle may also be a pH-sensitive nanocomposite hydrogel, which is prepared by preparing nanoparticles from alkali lignin or a derivative thereof, and then mixing the nanoparticles with a conventional hydrogel material to obtain the pH-sensitive nanocomposite hydrogel.
The invention provides a preparation method of a bionic nano-drug, which comprises the step of coating a biological membrane outside an inner core to prepare the bionic nano-drug.
In a preferred embodiment of the present invention, the preparation method comprises preparing an inner core and coating a biological membrane on the outer periphery of the inner core to form a shell;
the preparation of the inner core is to mix the first component, the second component and the carrier to prepare the inner core; the modification of the biological membrane refers to that the biological membrane is mixed with a targeting agent to prepare the biological membrane modified with the targeting agent;
preferably, when the inner core is prepared, after the first component, the second component and the carrier are mixed, the solvent of the carrier is evaporated, and the first component and the second component which are not carried are removed by dialysis;
the modification of the biological membrane is that DSPE-PEG-Mal reacts with apolipoprotein E polypeptide to prepare a targeting agent DSPE-PEG-ApoE, and then the targeting agent DSPE-PEG-ApoE is mixed with the biological membrane for incubation.
In a preferred embodiment of the present invention, the inner core is mixed with a biological membrane with a modifier, and the mixture is extruded with a filter membrane;
preferably, a filter extrusion of 100-200nm is used.
The invention provides application of a bionic nano-drug in at least one of the following situations:
(1) the application in preparing tumor cell proliferation inhibitor; (2) the application in preparing the medicine for preventing and/or treating the tumor;
preferably, the application is the application of the bionic nano-drug in preparing a tumor multidrug resistance reversal agent, preparing an anti-tumor drug sensitizer or preparing a recurrent tumor therapeutic agent;
preferably, the application is the application of the bionic nano-drug as a tumor multidrug resistance reversal agent or an anti-tumor drug sensitizer in the preparation of drugs for combined therapy of drug-resistant tumors.
Preferably, the tumor cell is a human brain glioma.
The invention provides a chemo-immune combined treatment preparation, which comprises a bionic nano-medicament.
The invention has the following beneficial effects:
the invention provides a hemoglobin cell membrane camouflage bionic nano-drug co-loaded with a TMZ (Tetramethylbenzidine) and BET (BET bromodomain inhibitor). The bionic nano-drug serving as a multifunctional nano-platform can enhance the blood circulation time and the permeability of BBB, and the co-delivered BET bromodomain inhibitor can reduce the proliferation of cancer cells by improving the sensitivity of TMZ. The BET bromodomain inhibitor can inhibit the DNA damage repair of GBM tumor cells, thereby enhancing the drug effect of TMZ.
In addition, TMZ chemotherapy and OTX can stimulate the immune system in vivo to be activated, and the BET bromodomain inhibitor can destroy the axis of immunosuppressive PD-L1/PD-1, recruit T cells to attack tumors, induce anti-tumor immune stress response, and achieve the effect of chemotherapy and immune combined treatment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic of ABNM @ TMZ/OTX versus GL261 induced anti-tumor immune response and enhanced TMZ chemotherapy;
FIG. 2 is a representation of a biomimetic nanoparticle;
FIG. 3 shows the measurement of the protein expression amounts of LRP1 and LDLR in GL261 and normal HA1800 astrocytes by WB;
FIG. 4 is a graph of the cumulative release ratios of (a) TMZ and (b) OTX in ABNM @ TMZ/OTX in either pH 5.0 and pH 6.5 acetate buffer or pH 7.4 phosphate buffer at 37 ℃;
FIG. 5 is a graph of immunofluorescence images of γ H2AX DNA lesion foci in cells and results of synergistic effect validation experiments;
FIG. 6 flow cytometry analysis ABNM @ OTX down-regulated the expression of PD-L1 on GL261 cell membranes (OTX:400 nM);
FIG. 7CLSM analyzes TMZ-induced translocation of calreticulin in GL261 cells (OTX:400nM, TMZ: 150. mu.M), scale 10 μ M;
FIG. 8 is a diagram showing the results of flow cytometry and the localization of nanoparticles in cells;
FIG. 9 is a graph of in vivo fluorescence imaging of GL261-Luc tumor C57BL/6 in situ-loaded mice at various time points after injection of ABNM @ DiR, BNM @ DiR and NM @ DiR (DiR dose: 0.2mg/kg) and of the fluorescence imaging of each organ;
FIG. 10 concentrations of (a) IFN-. gamma., (b) TNF-. alpha., (c) and IL-6 in mouse sera following single dose ABNM @ TMZ/OTX injection;
FIG. 11 Single injection of ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ/OTX, free TMZ (TMZ:5mg/kg, OTX:5mg/kg) and PBS to in situ tumor-bearing GL261-Luc mice the immune cell content (a) mature DC cells (CD11c + CD80+ CD86 +). (b) Tumor and (c) the level of T cells (CD3+ CD4+ CD8+) in the blood;
FIG. 12 is a graph showing the results of the anti-tumor efficacy study time presentation intent and the characterization of bioluminescence;
FIG. 13 shows the quantitative results of bioluminescence in each group of mice after treatment and the body weight change and survival data of the mice;
FIG. 14 Whole brain H & sections of bioluminescence from groups of mice after treatment;
figure 15TUNEL assay and immunohistochemical staining for analysis of γ H2AX, apoptosis (CC3), proliferation (Ki67), PD-L1, CD4+ and CD8+ content in tumor sections of treated mice on a scale of 200 μm;
FIG. 16ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ/OTX, free TMZ or day 22 after PBS treatment, H & E stained pictures of sections of GL261 mice were taken, ruler: 200 mu m;
FIG. 17 schematic representation of the establishment and treatment of a recurrent GL261-Luc tumor-bearing mouse model and bioluminescence imaging of mice bearing in situ GL261-Luc tumors following various treatments;
figure 18(a) quantitative luminescence levels of GL261-Luc tumor burden in situ in mice, (b) body weight change (n-8, data expressed as mean standard deviation, × p <0.01), (c) survival rate;
FIG. 19 is a graph of (a) flow analysis and (b) ratiometric analysis of memory-producing T cells in the spleen of a mouse, (c) and (d) ratiometric analysis of T cells in blood, and (e) and (f) ratiometric analysis of T cells in tumors;
FIG. 20 in vivo biocompatibility assessment of ABNM @ TMZ/OTX. (a-i) blood Biochemical blood routine examination. Expression of pro-inflammatory factors Il-1 β, Il-6 and TNF- α in liver (j-l) and kidney (m-o). Data are expressed as mean standard deviation (n-3).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
The experimental materials used in the examples and experimental examples of the present invention are as follows:
apolipoprotein E polypeptide [ ApoE, (LRKLRKRLL)2C]Purchased from peptide stocks, inc (suzhou, china). OTX015 was purchased from APExBIO (Houston, USA). RIPAy lysate, protease inhibitors, and red blood cell lysate were purchased from petunia. FITC-dextran was purchased from sigma. Flow antibodies anti-CD11c-FITC, anti-CD80-PE, anti-CD86-APC, anti-CD3-PerCP-Cy5.5, anti-CD4-FITC, anti-CD8-PE, anti-CD3-FITC, anti-CD8-PerCP-Cy5.5, anti-CD62L-APC, and anti-CD44-PE were all purchased from eBioscience. LRP1, LDLR, PD-L1, isotype control IgG and β -actin antibodies were purchased from Abcam, and γ H2AX antibody was purchased from EMD Millipore. Calreticulin antibodies, Alexafluor secondary antibodies and anti-quencher were purchased from sermer fly. GL261-luc cells were given by Zhang Lei professor at university of Shanxi. BALB/C, C57BL/6 animal mice were purchased from Spbefu. The content of TMZ and OTX is detected by high performance liquid chromatography (Agilent, 1260), and a nuclear magnetic resonance spectrometer (400MHz) of Bruker, Switzerland is used for performing nuclear magnetic resonance hydrogen spectrum on the compound (A), (B), (C), (1H NMR), the morphology of the nanoparticles was determined by Transmission Electron Microscopy (TEM) at an accelerating voltage of 120 kV. And (3) dripping 10 mu L of nano-drug solution on a copper net, adding a drop of 1% uranyl acetate coloring agent into the copper net after 10min, washing with water for 5 times after dyeing, and testing a sample by using a TEM after drying. High speed flow cytometers (FACS), both purchased from semer fly, usa, observe fluorescent pictures inside cells, mainly by laser scanning confocal microscopy (CLSM, German Zeiss).
Example 1
The embodiment provides a bionic nano-drug, which consists of an inner core and an outer shell, wherein the inner core comprises a first component, a second component and a carrier, the first component is temozolomide, the second component is an inhibitor (OTX-015 in the embodiment) of BRD4 protein, the carrier is a-glucan, the outer shell is an erythrocyte membrane, and a targeting agent is modified on the erythrocyte membrane so that the bionic nano-drug targets a target cell. The targeting agent is DSPE-PEG-ApoE.
The preparation method of the bionic nano-drug comprises the following steps:
(1) preparation of core (NM):
1mg of a-dextran (i.e., the acid-dextran) was dissolved in 200 μ L of THF, and OTX015 and TMZ were added, respectively, at 10% and 20% of the theoretical drug loading (DLC (wt.%) x 100 (total amount of drug/drug and polymer). And (3) dropwise adding the mixture into a pH 8 aqueous solution after uniformly mixing, stirring the solution at room temperature for 3 hours to completely volatilize THF, and dialyzing to remove the OTX and TMZ which are not loaded, thus obtaining the acid-sensitive drug-loaded nano-drug NM @ TMZ/OTX.
The method for quantifying DLC for TMZ and OTX is as follows: the drug-loaded nanoparticles are lyophilized or directly dissolved in DMSO, and then measured by high performance liquid chromatography (absorption at 260 nm). DLC for TMZ and OTX can be calculated based on standard curves drawn for known concentrations of TMZ/DMSO, OTX/DMSO.
The reaction of the a-dextran needs to be carried out under anhydrous and oxygen-free conditions, and the specific process is as follows: 1g of dextran was added to 10mL of DMSO and stirred until it was completely dissolved, 37mmol of diethoxypropylene was added thereto and mixed well, and then 15.6mg of PPTS was added to catalyze the reaction. After reacting at room temperature for 30min, 1mL of triethylamine was added to terminate the chemical reaction, the solution was added dropwise to cold deionized water (pH 8) to perform precipitation reaction, and the precipitate was collected by centrifugation (8000rpm, 10min), washed 3 times repeatedly, and then freeze-dried.
(2) The preparation method of DSPE-PEG-ApoE comprises the following steps: stirring DSPE-PEG-Mal and sulfhydryl ApoE (molar ratio 1:3) in PBS buffer solution for 24h, dialyzing to remove unreacted free polypeptide, and freeze drying to obtain product DSPE-PEG-ApoE.
(3) Referring to Yan Zou, Effective and Targeted Human ortho lipids therapeutic Materials,2018,30, e1803717, the extracted erythrocyte membrane (100 μ L of cell membrane obtained from blood) and the targeting agent DSPE-PEG-ApoE (40 μ g) are mixed uniformly, incubated in a shaker (200rpm) for 30min, and then subjected to water bath ultrasound for 5min to obtain ApoE Targeted modified RBCm (AB) through 400nm and 200nm filters respectively.
(4) Mixing NM @ TMZ/OTX (1mg) with ApoE targeted modified RBCm (AB), repeatedly extruding for 7 times under a 200NM filter membrane to finally obtain the bionic nano-drug ABNM @ TMZ/OTX, wherein the particle size and the morphology of the bionic nano-drug can be determined by DLS and TEM. The prepared bionic nano-drug ABNM @ TMZ/OTX is shown in figure 1.
Experimental example 1
In this experimental example, DLS measurement and TEM observation were performed on the biomimetic nanoparticles prepared in example 1.
The alpha-dextran loaded TMZ and OTX are self-assembled to obtain the acid-sensitive nano-drug core (NM @ TMZ/OTX). TMZ and OTX were tested for drug loading capacity by HPLC. The actual Drug Loading (DLC) of TMZ and OTX in ABNM @ TMZ/OTX was 6.7% and 7.6%, respectively.
And modifying the RBCm vesicle by introducing ApoE as a targeting ligand to obtain the functionalized ApoE-RBCm. ApoE polypeptides can specifically bind to LDLR and LRP1 present on tumor cells, thereby enhancing tumor uptake. Coating ApoE-RBCm on the surface of NM @ TMZ/OTX by adopting an ultrasonic method to obtain the final bionic nano-drug ABNM @ TMZ/OTX. The size of the biomimetic nanomedicine was 186NM (fig. 2(a)), 18NM larger than the unencapsulated nanomedicine (168NM), consistent with reported cell membrane thickness, and further the integrity of ABNM @ TMZ/OTX nucleocapsid structure was observed using transmission electron microscopy (fig. 2(b)), NM (left) and ABNM (right), scale: 50 nm.
Experimental example 2
To investigate whether the low density lipoprotein receptor family (i.e. Low Density Lipoprotein Receptor (LDLR) and LRP1) is overexpressed in GL261 cells, we performed WB analyses in GL261 tumor cells and normal HA1800 astrocytes. Specifically, the present experimental example carried out the detection of GL261 cell surface receptors.
The expression levels of LRP1 and LDLR on GL261 glioma cells and normal astrocytes (HA1800) were determined by Western blotting (Western blot, WB). After the GL261 and HA1800 cell lysates were centrifuged, the supernatants were harvested and protein quantification was performed by BCA. Equal amounts of total protein samples from different cells were then mixed with sample loading buffer (5 ×), denatured at 100 ℃ for 5min, and subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis. After electrophoresis was complete, the gel was transferred to polyethylene film by electrotransfer for 1h, then blocked with 5% skim milk, and then incubated overnight with the primary antibody (LRP1 or LDLR) followed by incubation of the secondary antibody, each of which was washed three times with TBST containing tween-20. ECL luminophores were used on a Bio-Rad ChemiDoc MP system to obtain signals of the bands of interest.
The results indicated that GL261 tumor cells over-expressed LRP1 and LDLR, while normal astrocytes (HA1800) were relatively weakly expressed (fig. 3). ApoE has been reported in the literature to specifically recognize LDLR and LRP1 proteins. Therefore, the ApoE modified nano-drug can be specifically recognized by GL261 cells highly expressed by LDLR and LRP1, and has high-efficiency active targeting capability on GL 261.
Experimental example 3
In this example, ABNM @ TMZ/OTX in vitro drug release and cytotoxicity experiments were performed.
The ABNM @ TMZ/OTX in vitro release assay was performed in a 37 ℃ shaker, protected from light, by adding 600. mu.L of ABNM @ TMZ/OTX to a dialysis bag (Spectra/Pore, MWCO 12000), then placing the dialysis bag in a 50mL round bottom centrifuge tube, followed by the addition of equal volumes (25mL) of PBS buffer (pH 7.4) and sodium acetate buffer (pH 5.0 and 6.5) to the tube, respectively, and simulating an in vivo physiological environment in a 37 ℃, 200rpm/min shaker. At the specified time point, 5mL of the solution was removed from the centrifuge tube and supplemented with the same volume of fresh buffer.
The amount of TMZ and OTX in the release medium was determined by high performance liquid chromatography. The release results are the average of three replicates.
In cytotoxicity experiments, GL261 was plated in 96-well plates (5X 10)3Cells/well) for 24h, then DMEM (100 μ L) solution containing 10 μ L of empty vector AB @ NM was added and after 48h incubation, cell viability was determined.
To evaluate the synergistic effect of TMZ and OTX, we added different concentrations of TMZ (0-160. mu.M) and OTX (0-1.6. mu.M) nanoparticles and free drug to GL261 cells at 5% CO2After incubation for 72h at 37 ℃, MTT was added and cell viability was measured.
ABNM @ TMZ/OTX in vitro drug release results show that TMZ and OTX release is pH responsive with 67% and 72% drug release over 24h at pH 5.0 (TMZ and OTX correspond to figures 4(a) and (b), respectively), and the results are expressed as the mean standard deviation (n ═ 3). However, at pH 7.4, the ABNM @ TMZ/OTX drug release rate (less than 20%) is significantly inhibited, indicating the pH responsiveness of the nano drug release, and also indicating that the modification of RBCm has little influence on TMZ and OTX release.
Experimental example 4
In this example, a cell synergy experiment was performed, and the current gold standard for GBM chemotherapy is the use of the alkylating agent TMZ, which induces DNA damage. Brd4 inhibitors have been found to modulate DNA damage responses in a variety of cancer cell lines at the cellular level. Since gliomas are highly resistant to TMZ, we hypothesized that the use of Brd4 inhibitors interferes with self-repair following DNA damage, thereby increasing the sensitivity of gliomas to TMZ. Sensitivity of the GL261 glioma cell line to Brd4 inhibitors was demonstrated by the following experiment.
GL261 cells were plated in 6-well plates (1X 10)6Cells/well) for 24h, 100 μ L of FITC-labeled AB @ NM, B @ NM, and NM (FITC concentration: 0.5. mu.g/mL) in PBS for 4h, the cells were trypsinized after washing with aspirates, and then GL261 cells were dispersed in 500. mu.L PBS for flow cytometry analysis and data were analyzed using FlowJo software.
To study the cellular localization of AB @ NM, GL261 cells were plated on confocal dishes (1X 10)5Cells/well) for 24h, 50 μ L of AB @ NM, B @ NM and NM (FITC concentration: 0.5. mu.g/mL), after 4h incubation. The medium was removed and washed twice with PBS. After 15min fixation with 4% paraformaldehyde, washing was performed 2 times, followed by 10min DAPI staining of nuclei and two additional washes. The fluorescence pictures were taken by a confocal laser microscope.
We observed the formation of γ H2AX DNA lesions in cells after treating GL261 cells with 400nM free OTX for 72H (fig. 5 (a)). FIG. 5a is an immunofluorescence image (OTX:400nM, TMZ: 150. mu.M) of γ H2AX DNA lesion foci in cells treated with GL261 for 72H with ABNM @ TMZ/OTX, ABNM @ TMZ, AB @ NM-OTX, free TMZ/OTX, free OTX and free TMZ, at a scale bar of 10 μ M. The results show that: DNA damage foci were also observed in cells treated with 150 μ M free TMZ, and when cells were treated with free OTX and TMZ, a significant increase in DNA damage in the cells was observed (fig. 5 (a)). The GL261 cell DNA damage effect treated by TMZ and OTX co-loaded nano-drug ABNM @ TMZ/OTX is far stronger than that of single-loaded ABNM @ TMZ, ABNM @ OTX and free drugs, and the fact that ABNM @ TMZ/OTX bionic nano-drug co-delivery causes stronger DNA damage is confirmed, and the TMZ drug effect is improved.
To further explore the synergistic effect of TMZ and OTX co-loading, we performed cytotoxicity assays in GL261 cells. FIG. 5b shows the survival of GL261 treated 72h cells with ABNM @ TMZ/OTX, ABNM @ TMZ, AB @ NM-OTX, TMZ concentration 0-160. mu.M, OTX concentration: 0-1.6. mu.M. FIG. 5c is a Fa-CI plot of cells after 72h treatment with ABNM @ TMZ/OTX, ABNM @ TMZ, AB @ NM-OTX. FIG. 5d is the viability of the GL261 cells after treatment for 72h with free TMZ/OTX, free TMZ and free OTX, TMZ concentration 0-160. mu.M, OTX concentration: 0-1.6. mu.M. FIG. 5e is a graph of Fa-CI of cells after treatment of GL261 with free TMZ/OTX, free TMZ and free OTX for 72 h.
The results show that ABNM @ TMZ/OTX showed enhanced cytotoxicity in GL261 cells (fig. 5(b)) compared to the drug-loaded nanopharmaceutical alone (fig. 5(d)), and that using Chou-Talalay method to calculate synergy index (CI) after ABNM @ TMZ/OTX, ABNM @ TMZ, AB @ NM-OTX treatment, CI values were between 0.2-0.8 at different inhibition rates (Fa, Fa ═ 1-cell viability) (fig. 5(c)), indicating that TMZ and OTX combination of ABNM @ TMZ/OTX achieved enhanced antitumor activity (CI < I synergy, CI ═ I additive, CI > I antagonism). Since the synergistic effect of the free drug TMZ and OTX physical mixed combination is obvious when Fa is lower, the synergistic effect of the two free drugs is gradually weakened along with the increase of Fa, and slight synergistic effect is shown (figure 5 (e)). The above data indicate that single drug OTX or TMZ treatment can inhibit cell proliferation in glioma cells, but the extent of inhibition is limited, but co-delivery of OTX and TMZ can improve the cell injury effect, and the synergistic effect is enhanced after nano-drug loading, which may be related to the property of slow release of nano-drug. This data is consistent with the results of figure 5(a), i.e., the combination of OTX and TMZ was significantly more effective than the use of a single drug, primarily due to the co-delivered OTX's ability to inhibit DNA repair, thereby enhancing the TMZ efficacy.
Experimental example 5
In this example, a PD-L1 inhibition test and calreticulin assay were carried out.
To investigate the OTX-induced expression of PD-L1 on the cell membrane of GL261, GL261 cells were seeded in 6-well plates (1X 10)6Cells/well) for 24h, add ABNM @ TMZ/OTX or free OTX at 400nM concentration for 72 h. The cells were collected and incubated with PD-L1 antibody and isotype control IgG antibody, washed after 30min, and then assayed for expression on GL261 cells on the surface of PD-L1 cells by flow cytometry.
The results show that the biomimetic nano-drug ABNM @ OTX completely inhibits PD-L1 expression in GL261 cells by delivering Brd4 inhibitor OTX at an OTX concentration of 400nM (FIG. 6), and the effect is similar to that of free OTX, indicating that ABNM @ TMZ/OTX has a high PD-L1 inhibition effect, which lays a solid foundation for in vivo immunotherapy.
The cellular immunofluorescence technique was used to study the calreticulin translocation induced by TMZ and OTX. The method comprises the following steps: GL261 was plated on CLSM dishes (1X 10)5Cells/well) and then ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ, free OTX and free TMZ and OTX (concentrations of TMZ and OTX are 150 μ M and 400nM, respectively). After 72h incubation, the medium was removed, the cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100 for 10min, followed by blocking with goat serum for 1h, followed by calreticulin-primary antibody incubation overnight at 4 ℃, followed by Alexa 488-secondary antibody and nuclear dye incubation for 1h at room temperature. All above procedures were washed 3 times with PBS. Add 5. mu.L of anti-quencher to the slide, cover slip was mounted on the slide and images were taken with CLSM.
Calreticulin is an important signal of in vivo immune reaction, and the results of detection by a cellular immunofluorescence technique show that compared with free drugs and nano-drugs with single-loaded TMZ and OTX, ABNM @ TMZ/OTX has the highest degree of calreticulin translocation (figure 7), which indicates that TMZ and OTX have the potential of activating in vivo immune reaction and treating glioma.
Experimental example 6
ABNM @ TMZ/OTX-induced DNA damage experiment
We used CLSM to observe the damage of ABNM @ TMZ/OTX to GL261 cellular DNA. GL261 cells were plated on CLSM dishes (1X 10)5Cells/well), cultured for 24 h. 100 μ L of ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ, free OTX and free TMZ/OTX were added and incubated for 72h, at concentrations of TMZ and OTX of 150 μ M and 400nM, respectively. After 72H incubation, the medium was removed and immunofluorescence was performed as in example 5, except that the primary antibody was replaced with γ H2AX antibody.
M-dextran labeled with FITC was used to study ABNM uptake and intracellular drug release in GL261 cells. Flow cytometry results demonstrated that AB @ NM was efficiently taken up by cells with 2.3 and 2.8 times higher fluorescence intensity than the non-targeted group B @ NM and bare nanoparticle NM, respectively (fig. 8 (a)). FIG. 8a shows that GL261 cells were detected by flow cytometry after incubation with ABNM, BNM, NM for 4h (FITC concentration: 0.5. mu.g/mL).
In addition, after 4h incubation with ABNM, significant FITC fluorescence was observed in the cytoplasm of GL261 cells with much stronger intensity than BN or NM (fig. 8(b)), confirming that after the biomimetic nanomedicine was modified with ApoE polypeptide, GL261 cells had receptor-mediated high efficiency endocytosis and active targeting ability. Fig. 8b shows the intracellular localization of nanoparticles in 4h GL261 cells treated with ABNM, BNM, NM, scale 10 μm.
Experimental example 7
In order to evaluate the in vivo tumor targeting ability of the biomimetic nano-drugs, the near infrared dye DiR was loaded into the nano-drugs to monitor the in vivo distribution of the nano-drugs.
Loading the near-infrared dye DiR into the nano-drug to obtain ABNM @ DiR. 200 μ L of ABNM @ DiR, no-targeting control group BNM @ DiR or no-membrane modification group NM @ DiR (DiR doses are all 0.2mg/kg) nanoparticles were injected tail vein into GL261-Luc tumor-bearing mice. The distribution of DiR in vivo at different time points (excitation 747nm, emission 774nm) was followed by a small animal imager. In order to explore the in vitro imaging effect of the mice, after 4h of the nano-drug, a part of the mice are dissected and the aggregation in the main organs is observed.
200 μ L of ABNM @ DiR, BNM @ DiR and NM @ DiR (DiR dose: 0.2mg/kg) were intravenously injected into GL261-Luc tumor-bearing C57BL/6 mice in situ. After 4h injection, strong DiR fluorescence was observed in the brain in ABNM @ DiR group, with fluorescence intensity reaching a maximum at 8h and strong fluorescence continuing to be maintained for 24h (fig. 9 (a)). Importantly, the co-localization degree of the DiR fluorescence and the tumor bioluminescence is high, which proves that the nano-drug can specifically target tumor parts and has small accumulation amount in normal brain. In contrast, the non-targeting group BNM @ DiR showed less tumor accumulation, suggesting an important role for ApoE in promoting BBB crossing, active targeting and tumor accumulation, while the naked NM @ DiR without cell membrane modification accumulated less tumor in the brain because of shorter blood circulation time.
To further confirm the targeting effect of ApoE, mice bearing in situ GL261-Luc tumors were dissected 8h after injection of ABNM @ DiR, BNM @ DiR and NM @ DiR (DiR dose: 0.2mg/kg), and the mice were imaged for DiR fluorescence in the major organs (heart, liver, spleen, lung, kidney and brain). Imaging results showed (figure 9(b)) that mice injected with ABNM @ DiR had significantly enhanced DiR fluorescence in situ tumors, while weaker DiR fluorescence was detected in tumors of mice treated with red blood cell membrane camouflaged non-targeted BNM @ DiR and naked NM @ DiR. Furthermore, brain accumulation of BNM @ DiR was higher than NM @ DiR, suggesting that camouflaging of the red blood cell membrane is beneficial to prolong circulation time in vivo to increase tumor accumulation of the nano-drug (fig. 9 (b)). The results all show that the ApoE modified bionic nano-drug can increase the BBB crossing of the nano-drug, realize the specific targeting of the tumor, and accumulate and stay in the tumor for a long time.
Experimental example 8
ABNM @ TMZ/OTX immunoreaction detection was performed. GL261(GL261-Luc) stably expressed by luciferase is used for detecting the size and the position of a mouse tumor. 200 μ L of ABNM @ TMZ/OTX or PBS (TMZ:5mg/kg, OTX:5mg/kg) was injected intravenously into C57BL/6 tumor-bearing (GL261-Luc) mice (n-3). Blood was collected from mice using 1, 3 or 7 days after the injection of the nano-drug. The blood was centrifuged at 1000rpm/min for 10min to collect serum. According to the product specification of an enzyme linked immunosorbent assay kit (ELISA), the mouse serum is subjected to an ELISA experiment to detect tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin-6 (IL-6).
After a single injection of ABNM @ TMZ/OTX nano-drug for 72h, high levels of INF-gamma, TNF-alpha and IL-6 were detected in peripheral serum of mice, which were 3.2 times, 2.1 times and 2.8 times respectively as high as those of PBS group (FIG. 10), demonstrating that the biomimetic nano-drug can effectively stimulate immune system in a short period.
To further investigate whether ABNM @ TMZ/OTX could accelerate Dendritic Cell (DC) maturation, we injected ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ, a mixture of free TMZ and OTX (TMZ:5mg/kg, OTX:5mg/kg) or PBS intravenously into GL261-Luc tumor-bearing mice. 3 days after injection, mice were sacrificed and lymph nodes were removed, gently homogenized into single cell suspensions, corresponding amounts of CD11c-FITC, CD80-PE and CD86-APC antibodies were added according to the recommended amounts of the product instructions, stained at 4 ℃ in the dark for 30min (during staining in the immunoassay, the volume of the solution cannot exceed 100. mu.L), and after PBS washing, cells were resuspended in PBS buffer for flow cytometry assay.
Lymph nodes from each group of mice were collected following a single injection of ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ/OTX mixture or free TMZ72 h. We then used flow cytometry to quantify the expression of CD80 and CD86 to study the immunomodulatory effects of ABNM @ TMZ/OTX on DC cells following injection. The results showed that CD80 and CD86 expression was highest in the lymph nodes of mice injected with ABNM @ TMZ/OTX nano-drug (25.40%) and similar to ABNM @ TMZ (21.09%), significantly higher than ABNM @ OTX (13.13%) control group (fig. 11 (a)). FIG. 11a is the content of mature DC cells (CD11c + CD80+ CD86+) in mice.
Interestingly, free TMZ/OTX mixture or free TMZ hardly induced DC maturation, probably due to the fact that free drug was rapidly metabolized in vivo and was difficult to work with. The mouse DC maturation level after ABNM @ TMZ/OTX bionic nano-drug treatment is higher, which indicates that strong immune response is induced, and the mouse DC maturation level has the potential of immunotherapy.
To investigate whether the biomimetic nano-drug ABNM @ (TMZ/OTX) could induce immune responses in vivo, blood was collected from mice 3 days after injection, serum was removed after centrifugation (1000rpm,10min), and blood cells were mixed with erythrocyte lysis buffer at a volume ratio of 1:3, incubation at 4 ℃ for 3min, and centrifugation (1000rpm,10min) to collect lymphocytes. After washing 2 times with PBS and adding the corresponding amounts of CD3-PerCP-Cy5.5, CD4-FITC and CD8-PE antibodies according to the recommended amount of the product instructions, staining for 30min at 4 ℃ in the dark, after PBS washing, the cells were resuspended in PBS buffer solution for flow cytometry.
To examine whether the nano-drug ABNM @ (TMZ/OTX) could activate anti-tumor immune response in vivo, the brain tumors of the mice were collected 3 days after injection, and the tumors of the mice in each group were collected 3 days after injection of the nano-drug, homogenized into a single cell suspension, and centrifuged (1000rpm,3 min). Corresponding amounts of CD3-PerCP-Cy5.5, CD4-FITC and CD8-PE antibodies were added according to the recommended amounts of the product instructions, stained at 4 ℃ in the dark for 30min, and after PBS washing, the cells were resuspended in PBS buffer for flow cytometry assay.
Flow cytometry assay results showed: cytotoxic CD8+ T cells and CD4+ helper T cells play a key role in the immune process, and the increase in CD8+ and CD4+ T cells promotes an anti-tumor immune response. We observed that the total percentage of CD8+ and CD4+ T cells in the tumors of mice treated with ABNM @ TMZ/OTX was 25.36% (fig. 11(b)) compared to the other dosing groups, which was 2.1-fold higher than mice treated with free TMZ/OTX. The quantification results further showed that ABNM @ TMZ/OTX treatment induced the highest levels of CD8+ and CD4+ in the blood of the dosed groups, consistent with the results in tumors (fig. 11 (c)). These results indicate that our nano-drug delivery strategy successfully elicits an anti-tumor immune response in vivo.
Experimental example 9
After successful construction of the in situ loaded GL261 tumor C57BL/6 mouse model, 200. mu.L of ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ and OTX mixture, free TMZ (TMZ:5mg/kg, OTX:5mg/kg) and PBS were injected every two days of tail vein. Tumor size and body weight of the mice were recorded with a small animal imager and a weight scale, respectively. After treatment is finished, one mouse is randomly selected from each group, brain and other main organs are taken for H & E histological analysis, CD4+, CD8+, PD-L1, Ki-67 and Caspase 3 immunohistochemical staining is carried out, a TUNEL experiment is operated according to the TUNEL product instruction, and then the system toxicity, the anti-tumor activity and the anti-tumor immune activation effect of the bionic nano-drug on the GL261-Luc mouse in the treatment process are analyzed. The remaining mice were used to observe the life cycle.
Evaluation of the antitumor Effect of ABNM @ TMZ/OTX was carried out in C57BL/6 mice bearing GL261-Luc brain glioma in situ. On day 12 after successful tumor implantation, treatment was performed by intravenous injection of 200 μ L of ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free drug cocktail TMZ/OTX, free TMZ (TMZ:5mg/kg, OTX:5mg/kg) and PBS, administered once every two days for a total of 5 times (FIG. 12 (a)). Qualitative and quantitative results of bioluminescence indicate that the tumor growth was effectively delayed after ABNM @ TMZ/OTX treatment, and the tumor was minimal in the six treatment groups (FIG. 12(b), FIG. 13 (a)). In addition, the treatment effect of the nano-drug is superior to that of the free drug, which shows the superiority of the nano-drug in the aspect of tumor treatment.
Although the free drug mixture TMZ/OTX treated group delayed glioma growth to some extent, the body weight loss was dramatic, indicating the presence of systemic toxicity (fig. 13(b), (n ═ 8, data expressed as mean standard deviation,. p < 0.05)). Mice treated with PBS also showed significant weight loss, reflecting increased brain damage as the disease progressed. In contrast, there was little weight loss in mice following ABNM @ TMZ/OTX treatment (fig. 13(b)), indicating that this treatment effectively inhibited brain tumor growth without causing side effects. Interestingly, ABNM @ TMZ/OTX significantly prolonged the survival time of GL261 mice compared to the control group with the free drug or the single drug-loaded nano-drug (fig. 13 (c)). Median survival value 45d of mice after ABNM @ TMZ/OTX treatment was significantly longer than ABNM @ TMZ (33 days), ABNM @ OTX (28 days), free TMZ/OTX (25 days), free TMZ (24 days) or PBS (22 days).
Whole brain H & sections of bioluminescence from each group of mice after treatment are shown in FIG. 14.
Immunofluorescence and immunohistochemistry imaging results showed that ABNM @ TMZ/OTX exhibited the highest levels of DNA damage (γ H2AX) and tumor cell apoptosis (TUNEL, CC3) signals compared to single-loaded nanopharmaceutical groups ABNM @ TMZ, ABNM @ OTX (fig. 15). Interestingly, the apoptosis signal of the nano-drug was more pronounced compared to the free drug mixture TMZ/OTX and free drug TMZ groups (fig. 15). Meanwhile, the tumor cell proliferation marker (Ki67) showed the lowest signal in mice treated with the nano-drug ABNM @ TMZ/OTX (fig. 15). Importantly, a significant reduction in PD-L1 expression was observed in tumor sections of biomimetic nanomedicine-treated mice, demonstrating that OTX co-delivered by nanomedicine is effective in inhibiting PD-L1 expression. In addition, the number of CD8+ T cells and CD4+ T auxiliary cells in mouse tumors after ABNM @ TMZ/OTX treatment is remarkably increased compared with other treatments, and the remarkable effect of the bionic nano-drug ABNM @ TMZ/OTX chemotherapy immune combination treatment is further proved, so that the excellent immune activation capability of the nano-drug is demonstrated.
On the 22 nd day after ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ/OTX, free TMZ or PBS treatment, one mouse is taken from each group, and H & E staining is carried out on heart, liver, spleen, lung and kidney slices of the mouse, so that the result shows that after ABNM @ TMZ/OTX treatment, the mouse has no toxic or side effect on main organs of the mouse, and the free drug mixture TMZ/OTX and free drug TMZ groups can cause renal toxicity (figure 16), thereby indicating that the bionic nano-drug ABNM @ TMZ/OTX has good biological safety.
Experimental example 10
The tumor of GBM patients is easy to relapse after operation and is the main reason for causing GBM difficult to cure, and the experimental example further evaluates the chemo-immune combined treatment effect of the bionic nano-drug ABNM @ TMZ/OTX in a relapse tumor model. This example was conducted for the combination therapy and biosafety assessment of ABNM @ (TMZ/OTX) in a model of recurrent brain tumors.
To simulate clinical recurrence of glioma, we constructed a GL261-Luc glioma recurrence model (see FIG. 17 a). The method comprises the following specific steps: on day 7 after successful GL261-Luc implantation, mice were surgically resected for tumors and the procedure was performed under 10-fold microscope. The operation time is about 20min, and the overall death rate is lower than 5 percent. After surgery, mice were weighed and randomized into 6 groups (n-8). On days 10 and 13 post-implantation, ABNM @ TMZ/OTX, ABNM @ TMZ, ABNM @ OTX, free TMZ and OTX mixture, free TMZ (TMZ:5mg/kg, OTX:5mg/kg) and PBS were injected tail vein. And a living body bioluminescence Lumina IVIS III system is used for optical imaging to record the proliferation condition of the tumor. To investigate its anti-tumor mechanism, we analyzed immune cells in recurrent tumors. After the tumor-bearing mice were treated, tumors and blood were collected according to the method of Experimental example 8 to obtain single cell suspensions, which were then stained with antibodies against CD3-FITC, CD4-PE, and CD 8-APC. After flow cytometry analysis, analysis was performed using FlowJo software. For analysis of memory T cells, spleens were harvested from surviving mice on day 18 and stained with CD3-FITC, CD8-PerCP-Cy5.5, CD62L-APC, and CD44-PE antibodies. The detection of mouse lymph node DC cells was in accordance with the method of Experimental example 8. After testing, data analysis was performed using FlowJo software.
Compared to mice treated with free drug (free TMZ/OTX mixture or free TMZ), single drug-loaded nano-drugs ABNM @ TMZ and ABNM @ OTX may partially delay tumor recurrence, while ABNM @ TMZ/OTX showed significant therapeutic effect (fig. 17(b), fig. 18 (a)). The change of the body weight of the mouse after the ABNM @ TMZ/OTX nano-drug treatment is found to be small by tracking the body weight of the mouse, so that the mouse is proved to have good anti-tumor activity and less toxicity. In contrast, mice treated with free drug or single drug encapsulated nano-drug significantly lost body weight (fig. 18(b)), which is likely due to the deterioration of mouse status due to the continuous growth of GL261 tumor. Median survival following treatment with ABNM @ TMZ/OTX was extended to 52 days, significantly longer than mice treated with ABNM @ TMZ (38 days), ABNM @ OTX (32 days), free TMZ and OTX mixed (25 days), free TMZ (25 days), or PBS (20 days), respectively (fig. 18 (c)).
Since immunological memory plays an important role in anti-tumor immune responses, we investigated the content of memory T cells in the spleen of mice at day 18 post-treatment. The results show that the number of memory T cells (CD3+ CD8a + CD44+ CD62L +) in the spleen of mice receiving ABNM @ TMZ/OTX was significantly increased compared to mice receiving the single drug (fig. 19(a) and (b)), indicating that ABNM @ TMZ/OTX elicited a strong immunological memory effect, which explains its good antitumor activity in the relapse model. Fig. 19(c) and (d) further confirm that the ABNM @ TMZ/OTX nano-drug significantly increases the percentage of activated CD8+ and CD4+ T cells (CD3+ CD4+ CD8+) in blood, and the content of CD8+ and CD4+ T cells in tumor-infiltrating lymphocytes (fig. 20(e) and (f)) is also significantly increased, indicating that the biomimetic nano-drug ABNM @ TMZ/OTX can achieve immunotherapy of in-situ brain tumors by activating anti-tumor response, and has strong immune activation and memory capacity.
To evaluate the biological safety of ABNM @ TMZ/OTX, a single dose thereof was injected into healthy mice, and blood was taken from the mice at the prescribed time points, and blood routine and blood biochemistry were examined (fig. 20 (a-i)). Healthy Balb/c mice were selected and randomized into two groups (n ═ 6). ABNM @ (TMZ/OTX) (TMZ:5mg/kg, OTX:5mg/kg) or PBS was injected into the mice via tail vein. Mice were subjected to eye bleeds on days 0, 2, 4, 7 and 14. Serum was obtained by centrifuging 800g of whole blood for 5min, and ALT, AST, ALP, plasma urea (BUN), Uric Acid (UA), and Creatinine (CR) in the plasma were detected by Dri-Chem 7000IZ of Fuji film company, Tokyo, Japan. Carrying out blood routine detection on whole blood, wherein the detection items comprise: platelets (PLT), Red Blood Cells (RBC), White Blood Cells (WBC).
To evaluate the induction of the potential inflammatory process by nano-drug treatment, mice were sacrificed on days 0, 2, organs (kidneys and liver) were collected and tested for changes in inflammatory factors (interleukin-1 β (Il-1 β), Il-6 and TNF- α) (fig. 20 (j-o)). The results show that there was no significant difference between the indices of the PBS and ABNM @ TMZ/OTX groups over a period of two weeks after injection, indicating that they had low systemic toxicity and good biocompatibility.
In conclusion, the invention provides a bionic nano-drug carried by TMZ and Brd4 inhibitor OTX together, so as to promote the combined administration of TMZ and OTX and realize the combined treatment of brain tumor by chemotherapy and immunotherapy. ABNM @ TMZ/OTX can improve BBB permeability, increase tumor accumulation and retention, and realize drug release of tumor microenvironment response. Chemotherapy induced by TMZ and OTX release can activate immune responses in vivo, whereas OTX not only interferes with cell proliferation, but also prevents DNA repair thereby increasing the sensitivity of the tumor to TMZ and enhancing the efficacy of TMZ. In addition, OTX can also inhibit the expression of PD-L1 to generate a potent anti-tumor immune response. ABNM @ TMZ/OTX induced high levels of cytokines in serum, promoted maturation of DCs, increased expression of CD4+ CD8+ T cells in tumors and blood to further enhance the effect of brain tumor immunotherapy. ABNM @ TMZ/OTX demonstrated that chemotherapeutic-immune combination therapy, in both primary and recurrent orthotopic glioma mouse models, could provide more effective glioma treatment than monotherapy. It is worth noting that these biomimetic nano-drugs have little damage to normal tissues and high safety in vivo. The intelligent bionic nano-drug provides a multifunctional platform for regulating and controlling an immunosuppressive microenvironment and treating tumors which are easy to generate drug resistance.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The bionic nano-drug is characterized by comprising an inner core and an outer shell coated outside the inner core, wherein the inner core comprises a first component, a second component and a carrier, the first component comprises temozolomide, the second component comprises a BET bromodomain inhibitor, and the carrier is a pH sensitive nano-particle;
the shell comprises a biological membrane, the biological membrane is selected from erythrocyte membrane, cancer cell membrane, immune cell membrane or blood platelet, and a targeting agent is modified on the biological membrane so that the bionic nano-drug can target cells.
2. The biomimetic nanomedicine according to claim 1, wherein the BET bromodomain inhibitor is an inhibitor of at least one protein selected from the group consisting of: BRD2, BRD3, BRD4, and BRDT;
preferably, the BET bromodomain inhibitor is an inhibitor of BRD 4;
preferably, the inhibitor of BRD4 is selected from an inhibitor of at least one of the following, or a pharmaceutically acceptable salt or solvate of any one of the following: derivatives of plant polyphenol-resveratrol, isoxazole structure BRD4 inhibitors, benzodiazepine BRD inhibitors, pyridone BRD4 targeted inhibitors, JQ1, CeMMEC2, PF-1, bromoshorine, OTX-015, TEN-010, BI2536, TG101348 and LY 294002;
preferably, the isoxazole structure BRD4 inhibitor is selected from CPI-203 or CPI-0610; the benzodiazepine BRD inhibitor is selected from I-BET 151 or I-BET 762; the plant polyphenol-resveratrol derivative is selected from RVX-208; the pyridone BRD4 targeted inhibitor is selected from ABBV-075.
3. The biomimetic nano-drug according to claim 1, wherein the target cell is a glioblastoma cell;
preferably, the targeting agent is DSPE-PEG-ApoE, which is covalently linked to the biofilm via DSPE, and the DSPE-PEG-ApoE is prepared by reacting DSPE-PEG-Mal with an apolipoprotein E polypeptide.
4. The biomimetic nano-drug according to claim 1, wherein the theoretical drug loading of the first component in the biomimetic nano-drug is 1-40 wt.%, and the theoretical drug loading of the second component in the biomimetic nano-drug is 1-40 wt.%.
5. The biomimetic nano-drug according to claim 1, wherein the pH sensitive nanoparticle is a-glucan, alkali lignin or a lignin macromolecular derivative;
preferably, the lignin macromolecule derivatives are one or more of lignin macromolecule derivatives obtained by introducing macromolecules into lignin macromolecules by a physicochemical method.
6. A method of preparing a biomimetic nano-drug according to any of claims 1-5, comprising coating a biological membrane outside the inner core to prepare the biomimetic nano-drug.
7. The preparation method of the biomimetic nano-drug according to claim 6, wherein the preparation method comprises the steps of preparing an inner core and coating a biological membrane on the periphery of the inner core to form an outer shell;
the preparation of the inner core is to mix the first component, the second component and the carrier to prepare the inner core; the inner core is coated with the biological membrane and also comprises the modification of the biological membrane: mixing the biological membrane with a targeting agent to prepare the biological membrane modified with the targeting agent;
preferably, when the inner core is prepared, after the first component, the second component and the carrier are mixed, the solvent of the carrier is evaporated, and the first component and the second component which are not carried are removed by dialysis;
the modification of the biological membrane is that DSPE-PEG-Mal reacts with apolipoprotein E polypeptide to prepare a targeting agent DSPE-PEG-ApoE, and then the targeting agent DSPE-PEG-ApoE is mixed with the biological membrane for incubation.
8. The method for preparing a biomimetic nano-drug according to claim 7, wherein the inner core is mixed with a biological membrane with a modifier, and extruded with a filter membrane;
preferably, a filter extrusion of 100-200nm is used.
9. Use of a biomimetic nano-drug as in any of claims 1-5 in at least one of the following situations:
(1) the application in preparing tumor cell proliferation inhibitor; (2) the application in preparing the medicine for preventing and/or treating the tumor;
preferably, the application is the application of the bionic nano-drug in preparing a tumor multi-drug resistance reversal agent, preparing an anti-tumor drug sensitizer or preparing a recurrent tumor therapeutic agent;
preferably, the application is the application of the bionic nano-drug as a tumor multidrug resistance reversal agent or an anti-tumor drug sensitizer in the preparation of drugs for combined therapy of drug-resistant tumors;
preferably, the tumor cell is a human brain glioma.
10. A chemo-immune combination therapeutic formulation, comprising a biomimetic nano-drug according to any of claims 1-5.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115068622A (en) * 2022-05-30 2022-09-20 江苏省中医院 Preparation method of macroporous polydopamine nano material of cross-linked small molecule inhibitor OTX015

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016123054A2 (en) * 2015-01-26 2016-08-04 The University Of North Carolina At Chapel Hill Kinase drug combinations and methods of use thereof
CN106029076A (en) * 2013-11-18 2016-10-12 福马疗法公司 Benzopiperazine compositions as BET bromodomain inhibitors
CN106893118A (en) * 2017-03-24 2017-06-27 东华大学 A kind of pH and redox double-response polysaccharide hydrogel and its preparation and application
CN108355139A (en) * 2018-03-28 2018-08-03 河南大学 A kind of bionical acid-sensitive Nano medication and its preparation and application method
CN108601746A (en) * 2016-01-08 2018-09-28 加利福尼亚大学董事会 The mesoporous silica nano particle with double-layer of lipoid coating for load delivering
CN109893660A (en) * 2019-03-25 2019-06-18 河南大学 A kind of bionic nano carrier and preparation method thereof for glioma treatment
CN110101685A (en) * 2019-05-21 2019-08-09 河南大学 A kind of bionic nano drug, preparation method and application
CN111956618A (en) * 2020-08-31 2020-11-20 河南中医药大学 Preparation method and application of lignin-based antitumor drug carrier
CN112569207A (en) * 2019-09-30 2021-03-30 复旦大学 Apolipoprotein-modified bionic nano tumor vaccine and preparation method and application thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106029076A (en) * 2013-11-18 2016-10-12 福马疗法公司 Benzopiperazine compositions as BET bromodomain inhibitors
US20180312524A1 (en) * 2013-11-18 2018-11-01 Forma Therapeutics, Inc. Benzopiperazine compositions as bet bromodomain inhibitors
WO2016123054A2 (en) * 2015-01-26 2016-08-04 The University Of North Carolina At Chapel Hill Kinase drug combinations and methods of use thereof
CN108601746A (en) * 2016-01-08 2018-09-28 加利福尼亚大学董事会 The mesoporous silica nano particle with double-layer of lipoid coating for load delivering
CN106893118A (en) * 2017-03-24 2017-06-27 东华大学 A kind of pH and redox double-response polysaccharide hydrogel and its preparation and application
CN108355139A (en) * 2018-03-28 2018-08-03 河南大学 A kind of bionical acid-sensitive Nano medication and its preparation and application method
CN109893660A (en) * 2019-03-25 2019-06-18 河南大学 A kind of bionic nano carrier and preparation method thereof for glioma treatment
CN110101685A (en) * 2019-05-21 2019-08-09 河南大学 A kind of bionic nano drug, preparation method and application
CN112569207A (en) * 2019-09-30 2021-03-30 复旦大学 Apolipoprotein-modified bionic nano tumor vaccine and preparation method and application thereof
CN111956618A (en) * 2020-08-31 2020-11-20 河南中医药大学 Preparation method and application of lignin-based antitumor drug carrier

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
YAN ZOU ET AL: "Effective and Targeted Human Orthotopic Glioblastoma Xenograft Therapy via a Multifunctional Biomimetic Nanomedicine", 《ADVANCED MATERIALS》, 16 October 2018 (2018-10-16) *
YANJIE LIU ET AL.: "Brain co-delivery of irst-line chemotherapy drug and epigenetic bromodomain inhibitor for multidimensional enhanced synergistic glioblastoma therapy", vol. 2, no. 4 *
闻乃妍: "溴结构域抑制剂JQ1通过VEGF/PI3K/AKT信号通路促进脑胶质瘤干细胞周期阻滞和凋亡", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》, 15 February 2020 (2020-02-15), pages 57 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115068622A (en) * 2022-05-30 2022-09-20 江苏省中医院 Preparation method of macroporous polydopamine nano material of cross-linked small molecule inhibitor OTX015
CN115068622B (en) * 2022-05-30 2024-03-19 江苏省中医院 Preparation method of macroporous polydopamine nano material of cross-linked small molecule inhibitor OTX015

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