CN113521035B - Preparation method and application of chemical immune combined therapeutic nano-drug - Google Patents

Abstract

The invention discloses a preparation method and application of a chemical immune combined therapy nano-drug, and relates to the technical field of medicines. The invention disguises the bionic nano-drug from the red blood cell membrane co-loaded with TMZ and BET bromodomain inhibitors. As a multifunctional nano platform, the bionic nano drug can not only enhance the blood circulation time and the permeability of BBB, but also reduce the proliferation of cancer cells by improving TMZ sensitivity through co-delivered BET bromodomain inhibitors. BET bromodomain inhibitors can inhibit GBM tumor cell DNA damage repair, thereby enhancing the efficacy of TMZ. TMZ chemotherapy and OTX can both 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 immunity stress reaction, and achieve the effect of combined treatment of chemotherapy and immunity.

Description

Preparation method and application of chemical immune combined therapeutic nano-drug

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method and application of a chemical immune combined treatment nano-drug.

Background

Temozolomide (TMZ) is currently the first-line chemotherapeutic drug for the treatment of glioblastoma multiforme (GBM). Although conventional chemotherapy of TMZ can improve the curative effect to a certain extent, GBM patients are extremely easy to generate drug resistance to TMZ due to the existence of a DNA self-repair mechanism of GBM tumor cells, so that the drug effect of TMZ is greatly reduced. Therefore, the drug resistance of GBM to TMZ is reduced by inhibiting DNA repair of tumor cells, and plays a role in improving the chemotherapy curative effect of GBM.

Recently, the use of immunomodulation to treat tumors has attracted considerable attention by researchers. Immunotherapy, such as tumor vaccine, immune checkpoint blocking therapy and CAR-T, has shown encouraging clinical outcome in the treatment of different types of cancers, including melanoma, leukemia and GBM. In the immunotherapy strategy, the blocking therapy based on PD1/PD-L1 immune checkpoint inhibitors brings new hopes for the treatment of cancer therapy. However, commercial PD1 antibodies (e.g., nawuzumab, pembrolizumab) have limited therapeutic efficacy in GBM patients, with less than 10% of patients responding for a long period of time. The main reasons are attributed to the higher levels of immunosuppressive cytokines in the tumor microenvironment of GBM and poor T cell infiltration, so that the conversion of "cold" immunosuppressive GBM to T cell inflammatory "hot" tumors plays a key role in reversing immunosuppression and GBM immunotherapy.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a preparation method and application of a chemical immune combined therapeutic nano-drug to solve the technical problems.

Bromodomain protein 4 (Brd 4) is the most important functional protein in the bromodomain and superferminal family of proteins. It has been found that Brd4 is closely related to DNA repair and that inhibition of Brd4 reduces the response of various cells to DNA damage repair. In addition, brd4 is able to reduce 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 antitumor effects at the cellular level. However, the clinical test effect is not ideal due to the short pharmacokinetics and strong hydrophobicity of the naked Brd4 inhibitor.

Based on Brd4 inhibitor, the inventor loads BET bromodomain inhibitor by using nano-drug to inhibit GBM tumor cell DNA damage repair, thereby enhancing the drug effect of TMZ. We developed a biomimetic nano-drug (ABNM@TMZ/OTX) for camouflaging the red blood cell membrane co-loaded with TMZ and BET bromodomain inhibitors. As a multifunctional nano platform, the bionic nano drug not only can enhance the blood circulation time and the permeability of BBB, but also can reduce the proliferation of cancer cells by improving TMZ sensitivity through co-delivered BET bromodomain inhibitors. In addition, TMZ chemotherapy and a BET bromodomain inhibitor can both 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 immunity stress reaction and achieve the effect of combined chemotherapy and immunity 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 minimal side effects.

Specifically, the invention is realized as follows:

the invention provides a bionic nano-drug, which comprises 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 pH sensitive nano-particles;

The shell comprises erythrocyte membrane, cancer cell membrane, immune cell membrane or blood platelet, and the biological membrane is modified with a targeting agent so that the bionic nano-drug can target cells.

Chemotherapy with a first component (TMZ) can activate an immune response in vivo, and a second component (BET bromodomain inhibitor) not only interferes with cell proliferation, but also can prevent DNA repair, thereby increasing the sensitivity of the tumor to TMZ and enhancing 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 immunity stress reaction and achieve the effect of chemotherapy and immunity combined treatment.

The carrier can realize the release of the bionic nano-drug in a specific pH environment.

The shell adopts erythrocyte membranes, has the characteristic of no immunogenicity, and the targeting agent can target cells by modifying the biomembrane, thereby realizing the accurate targeting of the medicine. In other embodiments, the biofilm may be selected from more than one 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 the co-delivered BET bromodomain inhibitor can reduce proliferation of cancer cells by improving TMZ sensitivity.

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 at least one of the following inhibitors or a pharmaceutically acceptable salt or solvate of any of the following inhibitors: derivatives of plant polyphenol-resveratrol, isoxazole structure BRD4 inhibitors, benzoazepine BRD inhibitors, pyridone BRD4 targeted inhibitors, JQ1, ceMMEC2, PF-1, bromosporine, OTX-015, TEN-010, BI2536, TG101348 and LY294002.

Preferably, the inhibitor of the isoxazole structure BRD4 is selected from CPI-203 or CPI-0610; the benzoazepine BRD inhibitor is selected from I-BET 151 or I-BET 762; the derivative of plant polyphenol-resveratrol is selected from RVX-208; the pyridone BRD4 targeting inhibitor is selected from ABBV-075.

In other embodiments, the BET bromodomain inhibitors may be selected as the BET bromodomain inhibitors described above, without being limited to the several inhibitors listed above, as long as deregulation of the expression of BET family member proteins is achieved within the scope of the inventive concept of the present invention.

The scope of the present invention includes all pharmaceutically acceptable salt forms of the compounds used according to the invention, which may be formed, for example, by protonation of an atom bearing a lone pair of electrons which is prone to protonation, such as an amino group with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. 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; zinc salts; an ammonium salt; fatty amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkylamine salts such as N, N-dibenzylethylenediamine salt, benzathine salt, phenethylbenzylamine salt; heterocyclic aromatic amine salts such as pyridinium, picolinium, quinolinium 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, lysine 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, hydrogen phosphate, or dihydrogen phosphate), carbonate, bicarbonate, perchlorate, borate, or thiocyanate; organic acid salts such as acetate, propionate, butyrate, valerate, caproate, heptanoate, caprylate, cyclopentanepropionate, caprate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (emponate), camphorite (camphorite), glucoheptonate (glucoheptanoate) or pivalate; sulfonates such as methanesulfonate (methanesulfonate), ethanesulfonate (ethanesulfonate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (benzenesulfonate), p-toluenesulfonate (toluenesulfonate), 2-naphthalenesulfonate (naphthalenesulfonate), 3-benzenesulfonate, or camphorsulfonate; glycerophosphate; and acidic amino acid salts such as aspartate or glutamate.

Furthermore, the scope of the present invention includes any solvate form of a compound for use according to the present invention, including, for example, a solvate with water (i.e., as a hydrate) or a solvate with an organic solvent, such as, for example, methanol, ethanol or acetonitrile (i.e., as a methanolate, ethanolate or acetonitrile), or any crystalline form (i.e., as any polymorph) or amorphous form of a compound for use according to the present invention. It is to be understood that these solvates of the compounds used according to the invention also include solvates of 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 of these isomers of the compounds mentioned in this specification are considered to be part of the present invention, which may be in mixtures, or in pure or substantially pure form. With respect to stereoisomers, the 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 may be resolved by physical means, such as, for example, fractional crystallization, separation or crystallization of diastereoisomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates by salt formation with an optically active acid followed by crystallization. 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 in which one or more atoms are replaced with a specific isotope of the corresponding atom. For example, the invention includes the use of the compounds mentioned in this specification wherein one or more hydrogen atoms (or e.g., all hydrogen atoms) are replaced with deuterium atoms (i.e., 2H; also referred to as "D"). Thus, the present invention also includes deuterium enriched compounds for use according to the present invention. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98mol% hydrogen-1 (1H) and about 0.0156mol% deuterium (2H or D). Deuterium content in one or more hydrogen positions in the compounds used according to the present invention may 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 be prepared using, for example, heavy water (D ₂ O) H/D exchange reaction. Other suitable deuteration techniques are described in: atzrodt J et al, biorg Med Chem,20 (18), 5658-5667,2012; william JS et al, journal of Labelled Compounds and Radiopharmaceuticals,53 (11-12), 635-644,2010; modvig A et al, J Org Chem,79,5861-5868,2014. Can be measured, for example, using mass spectrometry or NMR spectrometry Deuterium content. The compounds used according to the invention are preferably not enriched in deuterium, unless specifically indicated otherwise. Thus, it is 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, which is prepared by reacting DSPE-PEG-Mal with an apolipoprotein E polypeptide, by covalently linking DSPE to a biological membrane.

DSPE-PEG-Mal (phosphatidylethanolamine-polyethylene glycol-maleimide), apolipoprotein E polypeptides are all selected from commercially available products. In other embodiments, the molecular weight of PEG may be adaptively adjusted as desired, e.g., 2000, 5000, 10000, etc.

In a preferred embodiment of the present invention, the theoretical drug loading of the first component in the bionic nano-drug is 1-40wt.%, and the theoretical drug loading of the second component in the bionic nano-drug is 1-40wt.%. Optionally, the theoretical drug loading of the first component is 5-30wt.%, and the theoretical drug loading of the second component in the biomimetic nano-drug is 5-30wt.%.

In a preferred embodiment of the present invention, the pH-sensitive nanoparticle is a-glucan, alkali lignin or lignin macromolecular derivative;

preferably, the lignin macromolecule derivatives are one or more of lignin macromolecule derivatives obtained by introducing macromolecules into lignin macromolecules by physicochemical methods.

In one embodiment, the macromolecule incorporated into the lignin macromolecule by physicochemical methods or the like comprises one or more of polyethylene glycol, polyglycerol, polyvinyl alcohol, polyacrylic acid, polylactic acid, polyhydroxylactic acid, polypropylene caproate, copolymers of lactic acid and glycolic acid, polyhydroxybutyrate.

In one embodiment, the pH-sensitive nanoparticle may be a pH-sensitive nanocomposite hydrogel, which is prepared by using alkali lignin or a derivative thereof as a raw material to prepare the nanoparticle, and then mixing the nanoparticle with a conventional hydrogel material.

The invention provides a preparation method of a bionic nano-drug, which comprises the step of coating a biomembrane outside a core to prepare the bionic nano-drug.

In a preferred embodiment of the present invention, the above preparation method comprises preparing a core and coating a biofilm around the core to form a shell;

The preparation of the kernel is to mix the first component, the second component and the carrier to prepare the kernel; modifying the biological film refers to mixing the biological film with a targeting agent to prepare the biological film modified with the targeting agent;

preferably, when the inner core is prepared, after the first component, the second component and the carrier are mixed, evaporating the solvent of the carrier, and dialyzing to remove the first component and the second component which are not encapsulated;

the modification of the biological membrane is to react DSPE-PEG-Mal with apolipoprotein E polypeptide to prepare targeting agent DSPE-PEG-ApoE, and then mix and incubate the targeting agent DSPE-PEG-ApoE with the biological membrane.

In a preferred embodiment of the invention, the inner core is mixed with a biological film with a modifier and extruded by a filter film;

preferably, a 100-200nm filter membrane extrusion 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) Application in preparing medicines for preventing and/or treating tumor;

preferably, the application is the application of bionic nano-drug in preparing tumor multi-drug resistance reversal agent, antitumor drug sensitizer or recurrent tumor therapeutic agent;

preferably, the application is the application of bionic nano-drug as tumor multi-drug resistance reversal agent or anti-tumor drug sensitizer in preparing drugs for combined treatment of drug-resistant tumors.

Preferably, the tumor cells are human brain gliomas.

The invention provides a chemical immune combined therapeutic preparation which comprises bionic nano-drugs.

The invention has the following beneficial effects:

the invention provides a red blood cell membrane camouflage bionic nano-drug co-loaded with TMZ and a BET bromodomain inhibitor. As a multifunctional nano platform, the bionic nano drug not only can enhance the blood circulation time and the permeability of BBB, but also can reduce the proliferation of cancer cells by improving TMZ sensitivity through co-delivered BET bromodomain inhibitors. BET bromodomain inhibitors can inhibit GBM tumor cell DNA damage repair, thereby enhancing the efficacy of TMZ.

In addition, TMZ chemotherapy and OTX can both 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 combined chemotherapy and immune treatment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic representation of ABNM@TMZ/OTX versus GL261 induced anti-tumor immune response and enhanced TMZ chemotherapy;

FIG. 2 is a representation of biomimetic nanoparticles;

FIG. 3 shows protein expression levels of LRP1 and LDLR in GL261 and normal HA1800 astrocytes measured by WB;

FIG. 4 is a graph showing the ratio of cumulative release of (a) TMZ and (b) OTX in acetate buffer at pH 5.0 and pH 6.5 or phosphate buffer at pH 7.4 at 37℃for ABNM@TMZ/OTX;

FIG. 5 is a diagram showing immunofluorescence images of γH2AX DNA damage lesions in cells and experimental results of synergistic effect verification;

FIG. 6 flow cytometry analysis of ABNM@OTX down-regulates 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 μm;

FIG. 8 is a graph of flow cytometry detection results and nanoparticle localization in cells;

FIG. 9 is an in vivo fluorescence imaging of GL261-Luc tumor-bearing C57BL/6 mice at various time points after injection of ABNM@DiR, BNM@DiR and NM@DiR (DiR dose: 0.2 mg/kg) and fluorescence imaging of each organ;

FIG. 10 concentration of (a) IFN-. Gamma., (b) TNF-. Alpha., (c) and IL-6 in serum of mice after single dose injection of ABNM@TMZ/OTX;

FIG. 11 content of immune cells (a) mature DC cells (CD11c+CD80+CD86+) in one injection of ABNM@TMZ/OTX, ABNM@TMZ, ABNM@OTX, free TMZ/OTX, free TMZ (TMZ: 5mg/kg, OTX:5 mg/kg) and PBS to in situ tumor-bearing GL261-Luc mice. (b) Tumor and (c) T cell (cd3+cd4+cd8+) content in blood;

FIG. 12 is a graph showing the results of intent and bioluminescence characterization over time of an anti-tumor efficacy study;

FIG. 13 is a graph showing the quantitative results of bioluminescence and weight change in mice and survival data of mice for each group of mice after treatment;

FIG. 14 bioluminescence whole brain H & sections of mice of each group after treatment;

fig. 15TUNEL assay and immunohistochemical staining for analysis of content of γh2ax, apoptosis (CC 3), proliferation (Ki 67), PD-L1, cd4+ and cd8+ in tumor sections of treated mice, scale = 200 μm;

FIG. 16ABNM@TMZ/OTX, ABNM@TMZ, ABNM@OTX, free TMZ/OTX, free TMZ or PBS treatment day 22 post-treatment, sections of GL261 mice were H & E stained with the pictures, scale: 200 μm;

FIG. 17 establishment and treatment schematic of recurrent GL261-Luc tumor-bearing mice models and bioluminescence imaging of mice bearing in situ GL261-Luc tumors after different treatments;

fig. 18 (a) quantitative luminescence levels of in situ GL261-Luc tumor burden in mice, (b) body weight change (n=8, data expressed as mean standard deviation, ×p < 0.01), (c) survival;

FIG. 19 flow charts (a) and (b) ratio statistics of memory T cells, flow charts (c) and (d) ratio statistics of T cells in blood, and flow charts (e) and (f) ratio statistics of T cells in tumors in the spleen of mice;

FIG. 20ABNM@TMZ/OTX in vivo biocompatibility evaluation. (a-i) blood biochemical blood routine examination. Expression of pro-inflammatory factors Il-1 beta, il-6 and TNF-alpha in the 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

The experimental materials used in the examples and experimental examples of the present invention are as follows:

apolipoprotein E polypeptide [ ApoE, (LRKLRKRLL) ₂ C]Purchased from chinese peptide stock limited (su state, china). OTX015 was purchased from apex bio (houston, usa). RIPAy lysate, protease inhibitor, erythrocyte lysate were purchased from bi yun. FITC-dextran was purchased from sigma. The streaming 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 corporation and γh2ax antibodies were purchased from EMD Millipore corporation. Calreticulin antibodies, alexafluor secondary antibodies and anti-quenchers were purchased from zemoeid. GL261-luc cells were given away by the university of Shaanxi, university Zhang Lei. BALB/C, C57BL/6 animal mice were purchased from St Bei Fu. The TMZ and OTX contents are detected by high performance liquid chromatography (Agilent, 1260) and selected from Bruce Switzerland Nuclear magnetic resonance spectrometer (400 MHz) for nuclear magnetic resonance hydrogen spectrum of compound ¹ H NMR), the morphology of the nanoparticles was measured by Transmission Electron Microscopy (TEM) at an accelerating voltage of 120 kV. And (3) dropwise adding 10 mu L of nano-drug solution onto the copper mesh, adding one drop of 1% uranyl acetate coloring agent into the copper mesh after 10min, washing with water for 5 times after dyeing, and testing by using a TEM after drying the sample. Fluorescence images of cells purchased from the sammer fly, usa, and observed by high-speed flow cytometry (FACS) were taken mainly by a laser scanning confocal microscope (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 of BRD4 protein (OTX-015 in the embodiment), the carrier is a-glucan, the outer shell is erythrocyte membranes, and a targeting agent is modified on the erythrocyte membranes so as to target the bionic nano-drug to target cells. The targeting agent is DSPE-PEG-ApoE.

The preparation method of the bionic nano-drug comprises the following steps:

(1) Preparation of kernel (NM):

1mg of a-dextran, i.e. acetate-dextran, was dissolved in 200. Mu.L THF and OTX015 and TMZ were added respectively according to a theoretical drug loading (DLC (wt.%) = (drug/total amount of drug and polymer) ×100) of 10%, 20%, respectively. And (3) dropwise adding the mixture into an aqueous solution with the pH value of 8 after uniformly mixing, stirring the solution at room temperature for 3 hours to completely volatilize THF, and dialyzing to remove the unsupported OTX and TMZ to obtain the acid-sensitive drug-loaded nano drug NM@TMZ/OTX.

The method for quantifying DLC for TMZ and OTX is as follows: drug-loaded nanoparticles were either lyophilized or directly dissolved in DMSO and then assayed by high performance liquid chromatography (absorption 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 a-dextran needs to be carried out under anhydrous and anaerobic conditions, and the specific process is as follows: 1g of dextran was added to 10mL of DMSO and stirred until it was completely dissolved, and after 37mmol of diethoxypropene was added and thoroughly mixed, 15.6mg of PPTS was added to catalyze the reaction. After reaction 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 carry out precipitation, the precipitate was collected by centrifugation (8000 rpm,10 min), repeatedly washed 3 times, and freeze-dried.

(2) The preparation method of DSPE-PEG-ApoE comprises the following steps: the DSPE-PEG-Mal and sulfhydryl ApoE (molar ratio 1:3) are stirred in PBS buffer solution for 24h, unreacted free polypeptide is removed by dialysis and freeze drying is carried out to obtain the product DSPE-PEG-ApoE.

(3) Method of erythrocyte membrane extraction referring to (Yan Zou, effective and Targeted Human Orthotopic Glioblastoma Xenografts Therapy via a Multifunctional Biomimetic nanomedicine. Advanced Materials,2018,30, e 1803717.), we mixed the extracted erythrocyte membrane (100 μl blood cell membrane) with the targeting agent DSPE-PEG-ApoE (40 μg), incubated in a shaker (200 rpm) for 30min, and then sonicated in a water bath for 5min, and filtered through 400nm and 200nm filters, respectively, to obtain ApoE-targeted modified RBCm (AB).

(4) Mixing NM@TMZ/OTX (1 mg) and 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 ABNM@TMZ/OTX can be determined by DLS and TEM. The prepared bionic nano-drug ABNM@TMZ/OTX is shown by referring to 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 loads TMZ and OTX, and acid sensitive nano-drug core (NM@TMZ/OTX) is obtained through self-assembly. TMZ and OTX were assayed 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.

Modification of rbc vesicles by introducing ApoE as targeting ligand yields functionalized ApoE-rbcs. ApoE polypeptides can specifically bind to LDLR and LRP1 present on tumor cells, thereby enhancing tumor uptake. And 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 biomimetic nanomedicine was 186NM (fig. 2 (a)) 18NM larger than the nanomedicine (168 NM) before encapsulation, consistent with the reported thickness of the cell membrane, and further the integrity of abnm@tmz/OTX core-shell structure was observed using transmission electron microscopy (fig. 2 (b)), NM (left) and ABNM (right), scale: 50nm.

Experimental example 2

To investigate whether the low density lipoprotein receptor family, i.e. Low Density Lipoprotein Receptor (LDLR) and LRP1, was overexpressed in GL261 cells, we performed WB analysis in GL261 tumor cells and normal HA1800 astrocytes. Specifically, the present experimental example detects the GL261 cell surface receptor.

The expression levels of LRP1 and LDLR on GL261 glioma cells and normal astrocytes (HA 1800) were determined by Western Blot (WB). After GL261 and HA1800 cell lysates were centrifuged, the supernatant was taken and protein quantification was performed by BCA. Then, an equal amount of total protein samples of different cells was mixed with a sample loading buffer (5×), denatured at 100℃for 5min, and subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis. At the end of electrophoresis, the gel was transferred onto polyethylene membrane by electrotransfer for 1h, followed by blocking with 5% skim milk, then incubation with primary antibody overnight (LRP 1 or LDLR), followed by incubation of 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 the signal of the band of interest.

The results indicate that GL261 tumor cells overexpressed LRP1 and LDLR, whereas normal astrocytes (HA 1800) were expressed relatively poorly (FIG. 3). It is reported in the literature that ApoE can specifically recognize LDLR and LRP1 proteins. Therefore, the ApoE modified nano-drug can be specifically identified with GL261 cells highly expressed by LDLR and LRP1, and has high-efficiency active targeting capability on GL 261.

Experimental example 3

In vitro drug release and cytotoxicity experiments of ABNM@TMZ/OTX were performed in this experimental example.

The abnm@tmz/OTX in vitro release experiments were performed in a shaking table at 37 ℃ under light-protected conditions, 600 μl of abnm@tmz/OTX was added to a dialysis bag (Spectra/Pore, MWCO 12000), then the dialysis bag was placed in a 50mL round bottom centrifuge tube, then the same volumes (25 mL) of PBS buffer (pH 7.4) and sodium acetate buffer (pH 5.0 and 6.5) were added to the tube, respectively, and the in vivo biological environment was simulated in a shaking table at 200rpm/min at 37 ℃. At the prescribed time point, 5mL of the solution was removed from the centrifuge tube and the same volume of fresh buffer was replenished.

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) ³ Cells/well) for 24h, then DMEM (100 μl) solution containing 10 μl of empty vector ab@nm was added, and after 48h incubation, the cell viability was measured.

To evaluate the synergistic effect of TMZ and OTX, we added nanoparticles and free drug containing different concentrations of TMZ (0-160. Mu.M) and OTX (0-1.6. Mu.M) to GL261 cells at 5% CO ₂ After incubation at 37℃for 72h, MTT was added and the cell viability was measured.

abnm@tmz/OTX in vitro drug release results showed that release of TMZ and OTX was pH responsive with 67% and 72% drug release, respectively, over 24h at pH 5.0 (TMZ and OTX correspond to fig. 4 (a) and (b), respectively), and the results were expressed as mean standard deviation (n=3). However, under the condition of pH 7.4, the release rate (less than 20%) of the ABNM@TMZ/OTX drug is obviously inhibited, which shows the pH responsiveness of the nano drug release, and also shows that the modification of RBCM has little influence on TMZ and OTX release.

Experimental example 4

The experimental example is used for cell synergic experiments, and the gold standard of GBM chemotherapy is that an alkylating agent TMZ capable of inducing DNA damage is used at present. It was found that Brd4 inhibitors can regulate DNA damage response at the cellular level in a variety of cancer cell lines. Since gliomas are extremely resistant to TMZ, we hypothesize that the use of Brd4 inhibitors would interfere with self-repair after DNA damage, thereby increasing glioma sensitivity 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) ⁶ Cells/well) for 24h, 100 μl of FITC-labeled ab@nm, b@nm, and NM (FITC concentration:0.5 μg/mL) in PBS for 4h, the cells were digested with pancreatin after washing the samples with aspiration, and then GL261 cells were dispersed in 500 μl PBS for flow cytometry detection and data analysis using FlowJo software.

To study the cell localization of AB@NM, GL261 cells were plated in confocal dishes (1X 10) ⁵ Cell/well) for 24h, 50 μl 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 fixation with 4% paraformaldehyde for 15min, the nuclei were washed 2 times, then stained with DAPI for 10min and washed twice. Fluorescence pictures were taken with a laser confocal microscope.

We observed formation of γh2ax DNA lesions in cells after 72H treatment of GL261 cells with 400nM free OTX (fig. 5 (a)). FIG. 5a is an immunofluorescence image of γH2AX DNA damage lesions in cells after 72H treatment of GL261 with ABNM@TMZ/OTX, ABNM@TMZ, AB@NM-OTX, free TMZ/OTX, free OTX and free TMZ (OTX: 400nM, TMZ:150 μm), scale = 10 μm. The results show that: DNA damage lesions were also observed in cells treated with 150 μm free TMZ, and a significant increase in DNA damage in cells was observed when cells were treated with free OTX and TMZ (fig. 5 (a)). The GL261 cell DNA injury effect of the nano-drug ABNM@TMZ/OTX co-carried by TMZ and OTX is far stronger than that of the single-carried ABNM@TMZ, ABNM@OTX and free drug, and the fact that the nano-drug ABNM@TMZ/OTX bionic nano-drug co-carried by ABNM@TMZ/OTX causes stronger DNA injury is proved, and the efficacy of the TMZ is improved.

To further explore the synergistic effect of co-loading of TMZ and OTX, we performed cytotoxicity assays in GL261 cells. FIG. 5b shows the survival rate of GL261 treated 72h with ABNM@TMZ/OTX, ABNM@TMZ, AB@NM-OTX, TMZ concentrations of 0-160. Mu.M, OTX concentrations: 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 shows the viability of GL261 treated with free TMZ/OTX, free TMZ and free OTX for 72h, TMZ concentrations 0-160. Mu.M, OTX concentrations: 0-1.6. Mu.M. FIG. 5e is a Fa-CI plot of cells after 72h treatment of GL261 with free TMZ/OTX, free TMZ and free OTX.

The results showed that abnm@tmz/OTX showed enhanced cytotoxicity in GL261 cells (fig. 5 (b)) compared to drug-alone nanomedicines, and that synergy index (CI) after treatment of abnm@tmz/OTX, abnm@tmz, ab@nm-OTX was calculated using Chou-talay method, with CI values between 0.2 and 0.8 at different inhibition rates (Fa, fa=1-cell survival) (fig. 5 (c)), indicating that the combination of TMZ and OTX administration of abnm@tmz/OTX achieved enhancement of antitumor activity (CI < I synergy, ci=i addition, CI > I antagonism). Since the synergy of the free drug TMZ and OTX physical mixture combinations is evident at lower Fa, the synergy of the two free drugs gradually diminishes as Fa increases, exhibiting a slight synergy (FIG. 5 (e)). The above data indicate that single drug OTX or TMZ treatment can inhibit cell proliferation in glioma cells, but to a limited extent, but co-delivery of OTX and TMZ can enhance the cell damaging effect, and synergistic effect is enhanced after nano drug loading, which may be related to the slow release properties of nano drug. This data is consistent with the results of fig. 5 (a), i.e., the combination of OTX and TMZ is significantly more effective than the single drug, which is largely attributed to the co-delivered OTX inhibiting DNA repair and thus enhancing TMZ efficacy.

Experimental example 5

The experimental example carries out PD-L1 inhibition experiment and calreticulin detection.

To investigate the OTX-induced expression of PD-L1 on GL261 cell membranes, GL261 cells were seeded in 6-well plates (1X 10 ⁶ Cells/well) for 24h, abnm@tmz/OTX or free OTX was added and incubated at 400nM concentration for 72h. The cells were collected for incubation of PD-L1 antibodies and antibodies to isotype control IgG, washed after 30min, and then flow cytometry was used to detect the expression of PD-L1 cell surfaces on GL261 cells.

The results show that when the OTX concentration is 400nM, the bionic nano-drug ABNM@OTX completely inhibits the PD-L1 expression in GL261 cells by delivering the Brd4 inhibitor OTX (figure 6), and the effect is similar to that of free OTX, so that the ABNM@TMZ/OTX has a high-efficiency PD-L1 inhibition effect, and a solid foundation is laid for in-vivo immunotherapy.

The TMZ and OTX induced calreticulin translocation was studied using a cellular immunofluorescence technique. The method comprises the following steps: GL261 was plated in CLSM dishes (1×10 ⁵ Cells/well), then abnm@tmz/OTX, ABNM@TMZ, ABNM@OTX, free TMZ, free OTX and free TMZ and OTX (TMZ and OTX concentrations of 150 μm and 400nM, respectively) were added. After 72h incubation, the medium was removed, cells were fixed by adding 4% paraformaldehyde, then permeabilized with Triton X-100 for 10min, then blocked with goat serum for 1h, then incubated overnight at 4 ℃ with calreticulin primary antibody, then Alexa 488 secondary antibody and nuclear dye were incubated for 1h at room temperature. The above procedure was all washed 3 times with PBS. 5. Mu.L of anti-quencher was added to the slide, the coverslip was snapped onto the slide and the image was taken with CLSM.

Calreticulin is an important signal of immune response in vivo, and the results of detection by the cytoimmunofluorescence technology show that compared with free drugs and nano-drugs of TMZ and OTX which are singly carried, ABNM@TMZ/OTX causes the translocation of calreticulin to the highest degree (figure 7), which shows that TMZ and OTX have the potential of activating immune response in vivo and carrying out glioma treatment.

Experimental example 6

ABNM@TMZ/OTX induced DNA damage experiment

We used CLSM to observe the damage of abnm@tmz/OTX to GL261 cell DNA. GL261 cells were plated in CLSM dishes (1X 10) ⁵ Cells/well), for 24h. 100. Mu.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 150. Mu.M and 400nM, respectively. After 72H incubation, the medium was removed and immunofluorescence was performed as in experimental example 5, except that the primary antibody was replaced with γh2ax antibody.

FITC-labeled m-dextran was used to study ABNM uptake and intracellular drug release in GL261 cells. Flow cytometry experiments demonstrated that ab@nm was able to be efficiently taken up by cells with fluorescence intensities 2.3 and 2.8 times that of the non-targeted group b@nm and bare nanoparticle NM, respectively (fig. 8 (a)). FIG. 8a shows flow cytometry detection of GL261 cells after 4h incubation with ABNM, BNM, NM (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 the receptor-mediated efficient endocytosis and active targeting ability of GL261 cells after modification of ApoE polypeptide with biomimetic nanomedicine. Fig. 8b is the intracellular localization of nanoparticles in GL261 cells treated with ABNM, BNM, NM for 4h, scale = 10 μm.

Experimental example 7

To evaluate the in vivo tumor targeting ability of biomimetic nanomaterials, near infrared dye DiR was loaded into the nanomaterials to monitor the in vivo distribution of the nanomaterials.

And loading the near infrared dye DiR into the nano-drug to obtain the ABNM@DiR. 200 mu L of ABNM@DiR, BNM@DiR of a non-targeting control group or NM@DiR of a non-membrane modification group (DiR doses are all 0.2 mg/kg) nanoparticle tail is intravenously injected into GL261-Luc tumor-bearing mice. The DiR distribution at different time points in the body (excitation 747nm, emission 774 nm) was tracked with a small animal imager. To explore the in vitro imaging effect of mice, after 4h of the nanomedicine, a portion of the mice were dissected and observed for aggregation in the major organs.

200. Mu.L of ABNM@DiR, BNM@DiR and NM@DiR (DiR dose: 0.2 mg/kg) were intravenously injected into C57BL/6 mice bearing GL261-Luc tumors in situ. After injection for 4h, strong DiR fluorescence was observed in the brain by abnm@dir group, with fluorescence intensity reaching maximum at 8h, and strong fluorescence was sustained for 24h (fig. 9 (a)). Importantly, the co-localization degree of DiR fluorescence and tumor bioluminescence is high, which proves that the nano-drug can specifically target the tumor part and has small accumulation in the normal brain. In contrast, the non-targeted group bnm@dir showed less tumor accumulation, indicating the important role of ApoE in promoting BBB crossing, active targeting and tumor accumulation, whereas the naked nm@dir without cell membrane modification had less accumulation in brain tumors due to shorter blood circulation time.

To further confirm the targeting effect of ApoE, mice were dissected and DiR imaged for fluorescence in the major organs (heart, liver, spleen, lung, kidney and brain) of the mice after injection of abnm@dir, bnm@dir and nm@dir (DiR dose: 0.2 mg/kg) for 8h into in situ GL261-Luc tumor mice. Imaging results showed (fig. 9 (b)) that the mice after abnm@dir injection had significantly enhanced DiR fluorescence in the in situ tumors, whereas weaker DiR fluorescence was detected in the tumors of the mice treated with red cell membrane camouflage without targeting bnm@dir and naked nm@dir. Furthermore, the brain accumulation of bnm@dir is higher than nm@dir, suggesting that camouflage of the red blood cell membrane is beneficial for prolonging the in vivo circulation time to increase tumor accumulation of the nanomedicine (fig. 9 (b)). The results show that the ApoE modified bionic nano-drug can increase BBB crossing of the nano-drug, realize tumor specific targeting, and accumulate and stay in tumors for a long time.

Experimental example 8

ABNM@TMZ/OTX immunoreaction detection is carried out. Luciferase-stably expressed GL261 (GL 261-Luc) was used to detect the size and location of mouse tumors. 200. Mu.L of ABNM@TMZ/OTX or PBS (TMZ: 5mg/kg, OTX:5 mg/kg) was intravenously injected into C57BL/6 tumor-bearing (GL 261-Luc) mice (n=3). Mouse blood was collected 1, 3, or 7 days after the administration of the nano-drug. Blood was centrifuged at 1000rpm/min for 10min to collect serum. Mouse serum was subjected to ELISA experiments according to the ELISA kit (ELISA) product instructions 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 nanomaterials for 72h, high levels of INF- γ, TNF- α and IL-6 were detected in the mouse peripheral serum, 3.2-fold, 2.1-fold and 2.8-fold, respectively, of the PBS group (fig. 10), demonstrating that the biomimetic nanomaterials can effectively stimulate the immune system in a short period of time.

To further investigate whether ABNM@TMZ/OTX could accelerate Dendritic Cell (DC) maturation, we intravenous injected GL261-Luc tumor-bearing mice with ABNM@TMZ/OTX, ABNM@TMZ, ABNM@OTX, free TMZ and OTX mixtures (TMZ: 5mg/kg, OTX:5 mg/kg) or PBS. 3 days after injection, mice were sacrificed for lymph node removal, gently homogenized into single cell suspensions, corresponding amounts of CD11c-FITC, CD80-PE and CD86-APC antibodies were added according to the amounts recommended in the product specifications, stained at 4℃for 30min in the dark (the solution volume during the staining procedure of the immunoassay cannot exceed 100. Mu.L), washed with PBS, and the cells were resuspended in PBS buffer for flow cytometry determination.

Lymph nodes from each group of mice were collected after a single injection of abnm@tmz/OTX, ABNM@TMZ, ABNM@OTX, free TMZ/OTX mixture or free TMZ for 72 h. We then used flow cytometry to quantify CD80 and CD86 expression to investigate the immunomodulatory effects of abnm@tmz/OTX injection on DC cells. The results showed that the highest expression of CD80 and CD86 (25.40%) in lymph nodes of abnm@tmz/OTX nano-drug injected mice was similar to abnm@tmz (21.09%) and 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 mixtures or free TMZ hardly induced maturation of DCs, probably due to rapid metabolism of free drugs in vivo, which was difficult to work with. The DC maturation level of the mice treated by the ABNM@TMZ/OTX bionic nano-drug is higher, which indicates that strong immune response is induced and the mice have the potential of immunotherapy.

In order to investigate whether the bionic nano-drug abnm@(TMZ/OTX) can induce an immune response in vivo, mouse blood was collected 3 days after injection, serum was removed after centrifugation (1000 rpm,10 min), and blood cells and erythrocyte lysis buffer were mixed according to a volume ratio of 1:3, incubating for 3min at 4 ℃, and centrifuging (1000 rpm,10 min) to collect lymphocytes. Washing with PBS for 2 times, adding corresponding amounts of CD3-PerCP-Cy5.5, CD4-FITC and CD8-PE antibodies according to the recommended dosage of the product specification, dyeing at 4 ℃ in a dark place for 30min, and re-suspending the cells in PBS buffer solution for flow cytometry measurement after washing with PBS.

To examine whether the nano-drug abnm@(TMZ/OTX) could activate an anti-tumor immune response in vivo, brain tumors of mice were collected 3 days after injection, tumors of mice in each group were collected 3 days after injection of the nano-drug, homogenized into a single cell suspension, and centrifuged (1000 rpm,3 min). Corresponding amounts of CD3-PerCP-Cy5.5, CD4-FITC and CD8-PE antibodies were added according to the amounts recommended in the product specifications, stained at 4deg.C for 30min in the absence of light, washed with PBS, and the cells were resuspended in PBS buffer for flow cytometry assays.

Flow cytometry assay results showed: cytotoxic cd8+ T cells and cd4+ helper T cells play a key role in the immune process, and increases in cd8+ and cd4+ T cells promote anti-tumor immune responses. We observed that the total percentage of cd8+ and cd4+ T cells in the tumor of mice treated with abnm@tmz/OTX was 25.36% (fig. 11 (b)) compared to the other dosing groups, 2.1 times that of mice treated with free TMZ/OTX. The quantitative results further showed that abnm@tmz/OTX treatment induced the highest levels of cd8+ and cd4+ in the blood of the dosing group, which is consistent with the results in the tumor (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-charged 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:5 mg/kg) and PBS were injected intravenously every two days. Tumor size and body weight of mice were recorded with a small animal imager and a body weight scale, respectively. After treatment, each group randomly selects a main organ such as brain from a mouse for H & E histological analysis, performs immunohistochemical staining of CD4+, CD8+, PD-L1, ki-67 and Caspase 3, and operates a TUNEL experiment according to a TUNEL product instruction book so as to analyze the systemic toxicity, anti-tumor activity and anti-tumor immune activation effect of the bionic nano-drug on GL261-Luc mice in the treatment process. The remaining mice were used to observe the life cycle.

The antitumor effect evaluation of ABNM@TMZ/OTX was performed in situ in C57BL/6 mice bearing GL261-Luc brain glioma. On day 12 after successful tumor implantation, treatment was performed by intravenous injection of 200. Mu.L of ABNM@TMZ/OTX, ABNM@TMZ, ABNM@OTX, free drug mixture TMZ/OTX, free TMZ (TMZ: 5mg/kg, OTX:5 mg/kg) and PBS, administered once every two days for a total of 5 times (FIG. 12 (a)). The results of bioluminescence characterization and quantification showed that the abnm@tmz/OTX treatment was effective in delaying tumor growth, and was the smallest tumor in the six treatment groups (fig. 12 (b), fig. 13 (a)). In addition, the therapeutic effect of the nano-drug is better than that of the free drug, which indicates the superiority of the nano-drug in tumor treatment.

Although the free drug mixture TMZ/OTX treatment 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, mice after abnm@tmz/OTX treatment had little decrease in body weight (fig. 13 (b)), indicating that this treatment effectively inhibited brain tumor growth without causing side effects. Interestingly, abnm@tmz/OTX significantly prolonged survival of GL261 mice compared to the nanopharmaceutical control group loaded with free drug or single drug (fig. 13 (c)). The median survival 45d of the abnm@tmz/OTX treated mice 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).

The bioluminescence whole brain H & sections of each group of mice after treatment are shown with reference to fig. 14.

Immunofluorescence and immunohistochemical image results showed that abnm@tmz/OTX exhibited the highest levels of DNA damage (γh2ax) and tumor cell apoptosis (TUNEL, CC 3) signals compared to the single loaded nano-drug groups abnm@tmz, abnm@otx (fig. 15). Interestingly, the apoptosis signal of the nanomedicine was more pronounced compared to the free drug mixture TMZ/OTX and free drug TMZ group (fig. 15). Meanwhile, in mice treated with the nano-drug abnm@tmz/OTX, the signal of the tumor cell proliferation marker (Ki 67) was lowest (fig. 15). Importantly, a significant decrease in PD-L1 expression was observed in tumor sections of biomimetic nanomedicine treated mice, demonstrating that OTX co-delivery by nanomedicine is effective in inhibiting PD-L1 expression. In addition, the number of CD8+ T cells and CD4+ T helper cells in the tumor of the mice after ABNM@TMZ/OTX treatment is obviously increased compared with other treatments, and further the effects of the bionic nano-drug ABNM@TMZ/OTX chemotherapy and immune combined treatment are obvious, and the excellent immune activation capability of the nano-drug is demonstrated.

On day 22 after abnm@tmz/OTX, abnm@tmz, abnm@otx, free TMZ/OTX, free TMZ or PBS treatment, one mouse was taken from each group and H & E staining was performed on its heart, liver, spleen, lung, kidney sections, which indicated that no toxic side effects were observed on the major organs of the mice after abnm@tmz/OTX treatment was given, whereas the free drug mixture TMZ/OTX and free drug TMZ group caused renal toxicity (fig. 16), demonstrating good biosafety of the biomimetic nano drug abnm@tmz/OTX.

Experimental example 10

The easy recurrence of the tumor of the GBM patient after operation is a main cause of difficult cure of the GBM, and the experimental example further evaluates the chemical immune combined treatment effect of the bionic nano-drug ABNM@TMZ/OTX in a recurrent tumor model. The present experimental example performs a combination therapy and biosafety assessment of abnm@ (TMZ/OTX) in a recurrent brain tumor model.

To simulate the clinical recurrence of glioma, we constructed a GL261-Luc glioma recurrence model (see FIG. 17 a). The method comprises the following steps: on day 7 after successful GL261-Luc implantation, the mice were surgically resected for tumor, and the procedure was performed under a 10-fold microscope. The operation time is about 20min, and the overall mortality is lower than 5%. Mice were weighed and randomized into 6 groups (n=8) after surgery. On days 10 and 13 post-implantation, the tail vein was injected with ABNM@TMZ/OTX, ABNM@TMZ, ABNM@OTX, free TMZ and OTX mixtures, free TMZ (TMZ: 5mg/kg, OTX:5 mg/kg) and PBS. And optical imaging is carried out by using a living organism bioluminescence Lumina IVIS III system to record the proliferation of the tumor. To investigate the anti-tumor mechanism, we analyzed immune cells in recurrent tumors. Following treatment of tumor-bearing mice, tumors and blood were collected according to the procedure of Experimental example 8 to obtain single cell suspensions, which were then stained with CD3-FITC, CD4-PE and CD8-APC antibodies. After measurement by flow cytometry, it was analyzed by FlowJo software. To analyze 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 consistent 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 nanomaterials abnm@tmz and abnm@otx can partially delay tumor recurrence, while abnm@tmz/OTX shows significant therapeutic effect (fig. 17 (b), fig. 18 (a)). The mice are found to have little weight change after being treated by the ABNM@TMZ/OTX nano drug by tracking the weight of the mice, so that the mice are proved to have good anti-tumor activity and smaller toxicity. In contrast, mice treated with free drug or single drug encapsulated nanomedicine had significantly reduced body weight (fig. 18 (b)), which may be due to the worsening of the mice status caused by the sustained growth of GL261 tumors. Median survival in mice treated with abnm@tmz/OTX was prolonged 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 immune memory plays an important role in anti-tumor immune response, we studied the content of memory T cells in the spleen of mice at day 18 post-treatment. The results show a significant increase in the number of memory T cells (cd3+cd8a+cd44+cd62l+) in the spleen of mice receiving abnm@tmz/OTX compared to mice receiving the single drug (fig. 19 (a) and (b)), indicating that abnm@tmz/OTX elicits a strong immune memory effect, which explains its good antitumor activity in the relapse model. Fig. 19 (c) and (d) further demonstrate that abnm@tmz/OTX nanomaterials significantly increase 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)) also significantly increases, indicating that biomimetic nanomaterials abnm@tmz/OTX can achieve immunotherapy of in situ brain tumors by activating anti-tumor responses, with stronger immune activation and memory capabilities.

To evaluate the biosafety of abnm@tmz/OTX, a single dose thereof was injected into healthy mice, blood from the mice was taken at a prescribed time point, and blood routine and blood biochemistry were examined (fig. 20 (a-i)). Healthy Balb/c mice were selected and randomly divided into two groups (n=6). ABNM@ (TMZ/OTX) (TMZ: 5mg/kg, OTX:5 mg/kg) or PBS was injected into mice via tail vein. Mice were subjected to eyeball blood collection on days 0, 2, 4, 7 and 14. Whole blood was centrifuged at 800g for 5min to obtain serum, and ALT, AST, ALP, plasma urea (BUN), uric Acid (UA), and Creatinine (CR) in the plasma were detected by Dri-Chem 7000IZ of tokyo fuji film company, japan. Whole blood is subjected to blood routine detection, and detection items comprise: platelets (PLT), red Blood Cells (RBC), white Blood Cells (WBC).

To evaluate the induction of potential inflammatory processes by nanomedicine treatment, mice were sacrificed on days 0, 2, and organs (kidneys and livers) were collected to detect changes in inflammatory factors (interleukin-1β (Il-1β), il-6, and TNF- α) (fig. 20 (j-o)). The results show that in the two weeks after injection, there is no significant difference between the various indexes of PBS and ABNM@TMZ/OTX groups, indicating that the PBS and ABNM@TMZ/OTX groups have lower systemic toxicity and good biocompatibility.

In conclusion, the invention provides a TMZ and Brd4 inhibitor OTX co-carried bionic nano-drug 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 caused by TMZ and OTX release can activate immune response in vivo, and OTX not only interferes with cell proliferation, but also can prevent DNA repair so as to increase sensitivity of tumor to TMZ and enhance TMZ drug effect. In addition, OTX can also inhibit the expression of PD-L1 to generate a powerful anti-tumor immune response. ABNM@TMZ/OTX induces high levels of cytokines in serum, promotes maturation of DCs, and increases expression of CD4+CD8+T cells in tumors and blood, to further enhance the effect of brain tumor immunotherapy. ABNM@TMZ/OTX proves that the chemotherapy and immunity combined treatment can provide more effective glioma treatment effect than single treatment in a primary and recurrent in-situ glioma mouse model. It is worth noting that the bionic nano-drugs have little damage to normal tissues and high safety in vivo. The intelligent bionic nano-drug provides a multifunctional platform for controlling the immunosuppressive microenvironment and treating tumors which are easy to generate drug resistance.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. The application of the bionic nano-drug in preparing a human brain glioma multi-drug resistance reversal agent, preparing an anti-human brain glioma drug sensitizer or preparing a recurrent human brain glioma therapeutic agent is characterized in that the bionic nano-drug comprises an inner core and an outer shell coated outside the inner core, the inner core comprises a first component, a second component and a carrier, the first component comprises temozolomide, the second component comprises OTX-015, and the carrier is pH sensitive nano-particles;

the shell comprises a biological membrane, wherein the biological membrane is selected from erythrocyte membranes, and a targeting agent is modified on the biological membrane so that the bionic nano-drug can target cells; the target cells are glioblastoma cells; the targeting agent is DSPE-PEG-ApoE, the targeting agent is covalently connected with the biological membrane through DSPE, and the DSPE-PEG-ApoE is prepared by reacting DSPE-PEG-Mal with apolipoprotein E polypeptide; the pH sensitive nanoparticle is a-glucan.

2. The use according to claim 1, wherein the theoretical drug loading of the first component in the biomimetic nano-drug is 1-40wt.%, and the theoretical drug loading of the second component in the biomimetic nano-drug is 1-40wt.%.

3. A method of preparing a biomimetic nano-drug according to any one of claims 1-2, comprising coating a biological membrane outside the inner core to prepare the biomimetic nano-drug.

4. The method of preparing a biomimetic nano-drug according to claim 3, wherein the method of preparing comprises preparing an inner core and coating a biological membrane around the inner core to form an outer shell;

the preparation of the inner core comprises the steps of mixing a first component, a second component and a carrier to prepare the inner core; the method also comprises the following steps of modifying the biological film before coating the inner core with the biological film: and mixing the biological film with the targeting agent to prepare the biological film modified with the targeting agent.

5. The method for preparing the bionic nano-drug according to claim 4, wherein the first component, the second component and the carrier are mixed, and then the solvent of the carrier is evaporated, and the first component and the second component which are not entrapped are removed by dialysis;

The modification of the biological membrane is to react DSPE-PEG-Mal with apolipoprotein E polypeptide to prepare targeting agent DSPE-PEG-ApoE, and then mix and incubate the targeting agent DSPE-PEG-ApoE with the biological membrane.

6. The method for preparing the bionic nano-drug according to claim 5, wherein the inner core is mixed with a biological membrane with a modifier and extruded by a filter membrane.

7. The method for preparing the bionic nano-drug according to claim 6, wherein the membrane is extruded with a 100-200nm filter.

8. Use of a biomimetic nano-drug as defined in any one of claims 1-2 as a tumor multi-drug resistance reversal agent or an anti-tumor drug sensitizer in the preparation of a medicament for the combined treatment of drug resistant tumors;

the tumor is human brain glioma.

9. A chemoimmune combination therapeutic formulation, characterized in that it comprises a biomimetic nano-drug according to any one of claims 1-2.

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