CN110101685B

CN110101685B - Bionic nano-drug, preparation method and application thereof

Info

Publication number: CN110101685B
Application number: CN201910422970.3A
Authority: CN
Inventors: 师冰洋; 王一斌; 邹艳; 郑蒙
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2021-03-30
Anticipated expiration: 2039-05-21
Also published as: CN110101685A

Abstract

The application relates to the field of medicines, in particular to a bionic nano-drug, a preparation method and application thereof. The 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 with a side chain containing a sensitive bond; the shell includes a cancer cell membrane. The carrier of the sensitive bond is in a weak acid environment, the carrier is quickly swelled, the anti-cancer drug TMZ and the second component are efficiently released, the cancer cell membrane is taken as a shell to be made into the nano drug, the cancer cell membrane has certain self recognition and high tumor selectivity to target the 'homing' homologous tumor in vivo, and the in vivo circulation time of the nano drug can be greatly prolonged. The carrier of the cancer cell membrane and the sensitive bond as the shell efficiently releases the anti-cancer drug TMZ and the second component, finally kills tumor cells, and achieves the aim of targeted cooperative treatment of human brain glioma.

Description

Bionic nano-drug, preparation method and application thereof

Technical Field

The application relates to the field of medicines, in particular to a bionic nano-drug, a preparation method and application thereof.

Background

Malignant brain glioma is a brain tumor with the strongest invasiveness. Surgery is used to remove tumor volume, but infiltrating tumor cells within the normal brain parenchyma are not completely removed, and the remaining tumor cells are protected by the Blood Brain Barrier (BBB) or the Blood Brain Tumor Barrier (BBTB), which hinders the delivery of chemotherapeutic drugs, and thus tumor recurrence is likely to occur. Although nanoparticle-based Drug Delivery Systems (DDS) show the ability to enhance tumor targeting, these DDS do not reach the full therapeutic potential of post-operative glioma treatment, presenting the problem of low drug concentration levels within the tumor.

Disclosure of Invention

The embodiment of the application aims to provide a bionic nano-drug, a preparation method and an application thereof, and aims to solve the problem that the concentration content of the existing tumor drug in tumor is low.

In a first aspect, the present application provides a biomimetic nano-drug, the nano-drug comprising 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 with a side chain containing a sensitive bond;

the first component comprises temozolomide; the second component comprises one or more of cisplatin, lomustine, vincristine and procarbazine;

the shell includes a cancer cell membrane.

The carrier containing sensitive bonds is adopted to encapsulate Temozolomide (TMZ) and a second component (one or more of cisplatin, lomustine, vincristine and procarbazine), and the TMZ and the second component are used for treating the brain glioma in a synergistic manner, so that the treatment effect is improved, and the drug resistance of tumor cells is reduced. The carrier of the sensitive bond is in a weak acid environment, the carrier is quickly swelled, the anti-cancer drug TMZ and the second component are efficiently released, the carrier of the sensitive bond is coated with temozolomide and the second component is used as an inner core, a cancer cell membrane is used as an outer shell to be made into the nano drug, the cancer cell membrane has certain self-recognition and high tumor selectivity to target the homing homologous tumor in vivo, and the in vivo circulation time of the nano drug can be greatly prolonged.

The carrier of the cancer cell membrane and the sensitive bond as the shell efficiently releases the anti-cancer drug TMZ and the second component, finally kills tumor cells, and achieves the aim of targeted cooperative treatment of human glioma.

In some embodiments of the first aspect of the present application, the sensitive bond comprises one or more of an acetal bond, a hydrazone bond, and an amide bond; preferably, the sensitive bond is an acetal bond.

Hydrazone bonds and amide bonds are hydrophobic groups and can be used as a carrier for loading drugs. The acetal bond is an acid sensitive group, the chemical reaction for increasing the acetal bond is simple, the operation is convenient, the grafting efficiency is high, and more sensitive groups can be grafted, so that the carrier is more sensitive to acid.

In some embodiments of the first aspect of the present application, the carrier having sensitive bonds in its side chains comprises dextran having acetal bonds in its side chains.

Glucans with acetal linkages in the side chains conform to the desired properties of the biomaterial, are soluble in organic solvents and are completely insoluble in water, and acid-catalyzed hydrolysis of the side chain acetals regenerates the native glucan and harmless amounts of acetone and methanol into small molecule by-products.

In a second aspect, the present application provides a method for preparing a biomimetic nano-drug, comprising:

mixing the carrier, the first component and the second component, and dialyzing to remove the free first component and the free second component to obtain an inner core;

the cancer cell membrane and the inner core are mixed and then squeezed.

In some embodiments of the second aspect of the present application, mixing the carrier, the first component, and the second component specifically comprises:

dissolving a carrier in tetrahydrofuran to obtain a first solution, dissolving the first component and the second component in anhydrous dimethyl sulfoxide to obtain a second solution, and then mixing the first solution and the second solution.

The preparation method of the bionic nano-drug provided by the embodiment of the application is convenient to operate, simple and feasible, and mild and not harsh conditions in the preparation process.

In some embodiments of the second aspect of the present application, the mixed cancer cell membrane and the core are sequentially extruded through the filters of 700-800nm and 400-500 nm.

In some embodiments of the second aspect of the present application, the cancer cell membrane is made essentially by:

and digesting glioma cell U87MG cells by EDTA, washing by phosphate buffer saline solution, and extracting cell membranes.

In some embodiments of the second aspect of the present application, the glucan having acetal linkages in the side chains is prepared mainly by:

reacting dextran with 2-ethoxypropene in a solvent under the protection of nitrogen, terminating the reaction by triethylamine after the reaction is finished, precipitating in an alkaline solution, washing, and drying the precipitate.

In some embodiments of the second aspect of the present application, the dextran is reacted with 2-ethoxypropene with pyridinium p-toluenesulfonate as a catalyst.

In some embodiments of the second aspect of the present application, the solvent is anhydrous dimethylsulfoxide.

The glucan and 2-ethoxypropene react in one step under the catalysis condition to synthesize linear and annular acetal, and the grafting ratio of the linear acetal to the annular acetal is 66.72%, wherein the linear acetal accounts for 11.28% and the annular acetal accounts for 55.44%. The cyclic acetal decomposes under acidic conditions to produce only one molecule of acetone and one molecule of ethanol. The ratio can be calculated by comparing the characteristic peak of ethanol with the characteristic peak of dextran.

The third aspect of the application provides an application, namely the application of the bionic nano-drug provided by the first aspect of the application in preparing a targeted therapeutic drug for human brain glioma.

The bionic nano-drug can cross the protection of a Blood Brain Barrier (BBB), so that the drug is delivered into cells, the tumor targeting ability is enhanced, the carrier containing sensitive bonds in side chains is quickly swelled after hydrolysis, the anti-cancer drugs TMZ and CDDP are efficiently released, the accumulation of the TMZ and CDDP at tumors is facilitated, the tumor inhibition effect is achieved, and the tumor cells are finally killed.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required 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 application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 shows the NMR spectrum of dextran with acetal linkage in the side chain;

FIG. 2 shows the nuclear magnetic resonance spectrum of the decomposition of dextran containing acetal linkages in the side chains.

FIG. 3 shows particle sizes and Zeta potentials of NP and MNP;

FIG. 4 shows the in vitro release of TMZ at different pH in a simulated physiological environment;

figure 5 shows the in vitro release of CDDP at different pH in a simulated physiological environment;

FIG. 6 shows experimental results of cytotoxicity and intracellular release experiments;

FIG. 7 shows the entry of MNPs-FITC, RNPs-FITC, NPs-FITC into U87MG cells.

FIG. 8 shows drug concentration-time curves of a drug in vivo;

FIG. 9 shows the in vivo therapeutic effect of MNPs @ TMZ/CDDP in tumor-bearing mice;

FIG. 10 shows the relative photon counts before and after treatment at the tumor sites in mice of different groups;

FIG. 11 shows the weight change of different groups of mice over the course of treatment;

figure 12 shows the life cycle of different groups of mice;

FIG. 13 shows sections of major organs with different subgroups of drugs;

FIG. 14 shows the fluorescence intensity of the DIR-loaded MNPs @ DiR at different time points.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application 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 anti-biotic nano-drug, the preparation method and the application thereof according to the embodiments of the present application are specifically described below.

The nano-drug comprises an inner core and an outer shell coated outside the inner core;

the first component comprises temozolomide; the second component comprises one or more of cisplatin, lomustine, vincristine or procarbazine.

The shell includes a cancer cell membrane.

In the embodiments of the present application, each character represents the following meaning:

m-dextran;

Ac-DEX-a glucan having an acetal linkage in a side chain;

BBB-blood brain barrier;

TMZ-temozolomide;

CDDP-cisplatin;

CM-cancer cell membrane;

DiR-infrared dyes;

NPs-nanoparticles prepared using a carrier that do not encapsulate cancer cell membranes;

MNPs @ DiR-nanoparticles loaded with infrared dyes wrapped by cancer cell membranes;

MNPs @ TMZ/CDDP-TMZ and CDDP loaded nanoparticles wrapped by cancer cell membranes, namely bionic nano-drugs;

MNPs (tumor cells) -empty nanoparticles wrapped by cancer cell membranes comprise carriers and the cancer cell membranes;

MNPs-FITC-nanoparticles labeled with FITC wrapped by cancer cell membranes;

RNPs-FITC-nanoparticles labeled with FITC wrapped by erythrocyte membrane;

free TMZ — free temozolomide;

free CDDP-free cisplatin;

RNPs-empty nanoparticles encapsulated by erythrocyte membranes;

free FITC-free FITC;

RNPs-FITC-nanoparticles labeled with FITC wrapped by erythrocyte membrane;

NPs-FITC labeled nanoparticles;

NPs @ TMZ/CDDP-nanoparticles loaded with TMZ and CDDP.

Temozolomide (TMZ), a novel imidazole tetrazine alkylating agent, can rapidly pass through the blood brain barrier, has lipid solubility and certain water solubility, is a clinical chemotherapeutic drug with better effect for treating malignant brain glioma at present, undergoes spontaneous hydrolysis in cells to form an active metabolite MITC (3-methyl- (triazine-1-yl) imidazole-4-formamide) and alkylates the O6 and N7 positions of guanine, thereby accelerating the optimal treatment goal of cell therapy to improve the curative effect of the chemotherapeutic drug by enhancing tumor specificity and avoiding ineffective treatment results. However, the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (MGMT) exists in cells, and can repair the alkylation of TMZ to DNA, so that the tumor generates drug resistance, and the treatment effect is not ideal.

Cisplatin (CDDP) CAS No.: 15663-27-1; it has been shown to reduce the activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, which mediates resistance to TMZ. Cisplatin (CDDP) is widely applied to clinic as a broad-spectrum chemotherapeutic drug at present, and the mechanism of killing and injuring tumors of the cisplatin is mainly capable of combining with DNA to form interchain and interchain cross connection, so that the functions of the DNA are damaged, the replication and the transcription of the DNA are prevented, and finally, the apoptosis of cells is caused; the high concentration can also inhibit the synthesis of RNA and protein, but can also cause toxic and side effects such as nephrotoxicity, gastrointestinal toxicity, bone marrow suppression and the like to the organism.

Lomustine, CAS No.: 13010-47-4; the molecular formula is as follows: c₉H₁₆ClN₃O₂CCNU for short.

Lomustine (CCNU) belongs to chloroethylamine-based nitros antineoplastic, after entering into body, its molecule is broken into two parts from aminomethane bond, one part is chloroethylamine, and the chlorine is dissociated to form ethylene carbonium ion (CH)₂＝CH⁺) The alkylation function is exerted, the DNA is broken, and the synthesis of nucleic acid and protein is inhibited; the other part is the conversion of the amino methyl phthalein group part into isocyanuric acid or into carbamic acid, which plays the role of amino methyl phthalein and reacts with protein, especially the terminal amino group of lysine therein, which mainly reacts with bone marrowInhibition is relevant, but the carbamoylation can also destroy some enzyme proteins to play an anti-tumor role. Lomustine (CCNU) is a cell cycle non-specific drug, can act on various stages of proliferating cells and non-proliferating cells, is most sensitive to Gl-S stage boundary or S stage cells, and has stronger inhibition effect on G2 stage than BCNU. The product has high lipid solubility, and can rapidly cross gastrointestinal mucosa and blood brain barrier.

Lomustine can damage some enzyme proteins while damaging DNA, and MGMT enzyme for repairing temozolomide can be damaged, so that the synergistic treatment effect of the two medicines is improved.

Vincristine (Vincristine, Oncovin, VCR), CAS No.: 57-22-7; the molecular formula is as follows: c₄₆H₅₆N₄O₁₀；

The anti-tumor effect target of vincristine is microtubules, and the main effect is to inhibit polymerization of tubulin to influence formation of spindle microtubules. Arrest mitosis in metaphase. It also can interfere protein metabolism and inhibit RNA polymerase activity, and inhibit cell membrane lipid synthesis and amino acid transport on cell membrane. Vincristine can also be used in combination with cell cycle nonspecific drugs to enhance therapeutic effect.

Procarbazine (Procarbazine, PCZ), CAS number: 671-16-9; the molecular formula is as follows: c₁₂H₁₉N₃O；

The procarbazine can inhibit the synthesis of DNA and protein, and can form H by autoxidation after entering human body₂O₂And OH-groups, which cause similar ionizing radiation-like effects, in particular methylation of the 3-position of guanine and the 1-position of adenine. Has multiple biological effects, such as inhibiting cell mitosis, disorganizing chromosome arrangement, teratogenicity, carcinogenesis, immunosuppression, cytotoxicity, etc. PCZ inhibits cell mitosis. Methyl positive ions are released in vivo, combined with DNA to be depolymerized, and are cell cycle nonspecific drugs which mainly act on the G1/S boundary and have a delay effect on the S phase. It has no cross resistance with alkylating agent, vincristine and corticoid, and can raise the curative effect obviously when used together with the said medicine.

Temozolomide acts synergistically with one or more of cisplatin, lomustine, vincristine, and procarbazine.

The existing temozolomide and cisplatin have a plurality of key problems of short in-vivo circulation time, difficulty in crossing BBB, low tumor cell uptake, slow drug release at a focus and the like.

In the application, a carrier containing a sensitive bond is adopted to encapsulate Temozolomide (TMZ) and a second component (one or more of cisplatin, lomustine, vincristine and procarbazine), and the TMZ and the second component are used for treating the brain glioma in a synergistic manner, so that the treatment effect is improved, and the drug resistance of tumor cells is reduced. The carrier of the sensitive bond is in a weak acid environment, the carrier is quickly swelled, the anti-cancer drug TMZ and the second component are efficiently released, the carrier of the sensitive bond is coated with Temozolomide (TMZ) and the second component is used as an inner core, a cancer cell membrane is used as an outer shell to be made into the nano-drug, the cancer cell membrane has certain self-recognition and high tumor selectivity to target 'homing' homologous tumors in vivo, and the in vivo circulation time of the nano-drug can be greatly prolonged.

Further, in some embodiments of the present application, the sensitive bond includes one or more of an acetal bond, a hydrazone bond, and an amide bond; preferably, the sensitive bond is an acetal bond.

The carrier with the side chain containing the acetal bond is degraded in an endosome acidic environment, the nanoparticles swell and release TMZ and CDDP, so that safe and efficient chemotherapy of human brain glioma is achieved.

Further, in some embodiments of the present application, the carrier having a sensitive bond in a side chain thereof includes dextran (m-dextran) having an acetal bond in a side chain thereof.

The glucan with the side chain containing the acetal bond is selected as a carrier, the glucan with the side chain containing the acetal bond accords with the characteristics required by biological materials, is dissolved in an organic solvent and is completely insoluble in water, and the acid catalyzed hydrolysis of the side chain acetal enables the natural glucan and harmless acetone and methanol to be regenerated into micromolecular byproducts.

The application also provides a preparation method of the bionic nano-medicament, which comprises the following steps:

the cancer cell membrane and the inner core are mixed and then squeezed.

Further, mixing the carrier, the first component, and the second component specifically comprises:

The carrier is dissolved in tetrahydrofuran, and the first component and the second component are dissolved in anhydrous dimethyl sulfoxide and then mixed, which is beneficial to uniformly mixing the carrier, the tetrahydrofuran, the first component and the second component.

Dissolving the carrier, the first component and the second component in tetrahydrofuran, loading the temozolomide and the second component on the carrier, removing redundant temozolomide and the second component after dialysis to obtain an inner core, mixing the inner core with a cancer cell membrane, and then co-extruding to obtain the core-shell structure.

Further, in this example, the carrier used was dextran having acetal bond in the side chain, and the second component used was CDDP, which was mainly prepared by the following steps:

Further, dextran is reacted with 2-ethoxypropene in the presence of pyridinium p-toluenesulfonate as a catalyst.

In detail, dried and dehydrated dextran (dextran,10kDa) and 2-ethoxypropene were reacted in anhydrous Dimethylsulfoxide (DMSO) solvent at room temperature for 3h under nitrogen protection, and a catalytic amount of pyridinium p-toluenesulfonate was added. After the reaction is finished, triethylamine is used for stopping the reaction, precipitation and washing are carried out for 3 times in deionized water with the pH value of 8 so as to prevent acetal bonds from being degraded, and the final product m-dextran is obtained by freeze drying of the precipitate after centrifugation. The main raw materials used in the synthesis process, such as dextran, 2-ethoxypropene, pyridinium tosylate and the like, can be directly obtained by purchase.

The synthesis of acetal grafted dextran, the reaction formula of acetal bond degradation to dextran, ethanol and acetone under acidic condition is as follows:

the method for synthesizing the acetal grafted glucan can obtain a target product through one-step reaction, the preparation method is simple, the finally obtained product is easy to separate, and the purity of the product is high.

The glucan and 2-ethoxypropene react in one step under the catalysis condition to synthesize linear and annular acetal, and the grafting ratio of the linear acetal to the annular acetal is 67.81%, wherein the linear acetal accounts for 10.39% and the annular acetal accounts for 57.42%. FIG. 1 shows the NMR spectrum of dextran with acetal linkage in the side chain; FIG. 2 shows the nuclear magnetic resonance spectrum of the decomposition of dextran containing acetal linkages in the side chains.

As shown in FIG. 1, the linear acetal decomposes under acidic conditions to produce one molecule of acetone and one molecule of ethanol, the ratio of which is calculated by comparing the characteristic peaks of acetone (2.08ppm,6H) and ethanol (0.95ppm,3H) with those of dextran (3.4-4.0ppm, 6H). The cyclic acetal produces only one molecule of acetone under acidic conditions. The ratio can be calculated by comparing the characteristic peak of ethanol with the characteristic peak of dextran.

In this example, the cancer cell membrane was mainly prepared by the following steps:

the glioma cell U87MG cell is digested by EDTA (ethylene diamine tetraacetic acid), and then washed by phosphate buffer saline solution to extract the cell membrane.

In detail, after culturing glioma cells U87MG cells in a cell culture dish, the culture medium was aspirated and washed with PBS, 5mL of EDTA (2mM) was added for digesting the cells, after collecting the cells, the cells were washed with PBS, followed by extraction of cell membranes with a membrane protein extraction kit.

Further, in the preparation process of the bionic nano-drug, the MNPs are finally obtained by repeatedly extruding for 11 times through the filter membranes with the wavelengths of 700-.

In other embodiments of the present application, the glioma cells may also be U251 cells, U1261 cells, and the like.

The application also provides an application of the bionic nano-drug in preparation of a targeted cooperative therapy drug for human brain glioma.

The bionic nano-drug is identified by self-recognition internalization and highly tumor-selective targeting homing of the in-vivo homologous tumor of the in-vitro homologous cancer cell line, and is beneficial to the aggregation of nano-particles at the tumor, thereby achieving the effect of inhibiting the tumor.

The features and properties of the present application are described in further detail below with reference to examples.

Example 1

The embodiment provides an anti-biotic nano-drug which is mainly prepared by the following method:

dried and dehydrated dextran (dextran,10kDa) and 2-ethoxypropene are reacted in anhydrous dimethyl sulfoxide (DMSO) solvent for 3h at normal temperature under the protection of nitrogen, and a catalytic amount of pyridinium p-toluenesulfonate is added. After the reaction is finished, triethylamine is used for stopping the reaction, precipitation and washing are carried out for 3 times in deionized water with the pH value of 8 so as to prevent acetal bonds from being degraded, and the final product m-dextran is obtained by freeze drying of the precipitate after centrifugation.

Extraction of cancer Cell Membranes (CM): after culturing glioma cells U87MG cells on a cell culture dish, removing the culture medium by suction, washing with PBS, adding 5mL of EDTA (2mM) for digesting the cells, collecting the cells, washing with PBS, extracting the cell membrane with a membrane protein extraction kit, freeze-drying, and storing in a refrigerator at-80 ℃ for a long time.

Formation of acid-sensitive nanoparticles and drug loading: dissolving m-dextran in tetrahydrofuran, adding Temozolomide (TMZ) and Cisplatin (CDDP) with corresponding theoretical drug-loading capacity, dialyzing to remove free drugs, and finally obtaining the acid-sensitive drug-loaded nanoparticle, namely the inner core.

And mixing the inner core and the CM according to a proportion, and repeatedly extruding under 800nm and 400nm filter membranes to finally obtain MNPs @ TMZ/CDDP.

The biological control nano-drug provided by the embodiment can be used for preparing a targeted synergistic therapeutic drug for human brain glioma.

FIG. 3 shows particle sizes and Zeta potentials of NP and MNP; further, FIG. 3a shows particle diameters of NP and MNP, and FIG. 3b shows Zeta potentials of NPs and MNPs.

m-dextran can self-assemble to form stable nanoparticles in water solution, the particle diameter of the nanoparticles is increased from 174nm to 185nm after wrapping cell membranes (figure 3a), the potential is changed from-44.3 mV to-22.7 mV (figure 3b), and the distribution of the particle diameters of the nanoparticles is uniform as shown by DLS test. When the theoretical drug loading of TMZ is 10%, the encapsulation rate is 49.5% as measured by a microplate reader and a fluorescence spectrophotometer.

The biomimetic nano-drug prepared in this example was used to conduct the study of test examples 1-3.

Test example 1

The in vitro drug release behavior acid response of the bionic nano-drug is researched: in order to quantify the Drug Loading Efficiency (DLE) and the Drug Loading Capacity (DLC) of TMZ and CDDP, the ultraviolet absorption of TMZ at 327nm is directly detected by a multifunctional microplate reader, and the nanoparticles are dissolved in HNO with the volume fraction of 5 percent₃In the solution, the CDDP content was determined by inductively coupled plasma spectrometer. Based on known concentrationAnd the DLC and DLE can be calculated by drawing standard curves of TMZ and CDDP and the following formulas:

DLC (wt.%) (total amount of drug/total amount of drug and polymer) × 100

DLE (%) - (measured dose/theoretical dose) × 100

The nano-drug is incubated (simulated) in an endosomal weak acid environment (pH 5-6.5) of the tumor cells, and the number, the particle size and the particle size distribution of the particles are tracked in real time by DLS. In vitro release experiments were performed at 37 ℃ by dialyzing 600. mu.l of MNPs @ TMZ/CDDP against 25mL of buffer solution containing acetic acid. At the set time point, 5mL of release medium was withdrawn and the same amount of fresh medium was replenished. The amounts of TMZ and CDDP in the release medium were determined by a multifunctional microplate reader and an inductively coupled plasma spectrometer, respectively. The release results are the average of three replicates.

Figure 4 shows the in vitro release of TMZ at different pH in a simulated physiological environment and figure 5 shows the in vitro release of CDDP at different pH in a simulated physiological environment.

As can be seen from FIGS. 4 and 5, the in vitro release test results showed that TMZ and CDDP release was low (less than 25% in 24 hours) at 37 ℃ under physiological conditions of pH 7.4. Under acidic conditions, the release of TMZ at 24 hours was over 65%, and the release of CDDP at 24 hours was over 75%. This is the swelling of the nanoparticles under acidic conditions leading to drug release. The MNPs simultaneously solve the two problems of leakage and slow release in cells of the traditional biodegradable nano-drug.

Test example 2

Cell experiments

(ii) cytotoxicity assay:

human brain glioma cell U87MG was selected as the cell model. U87MG was cultured in 100. mu.L of DEME medium containing 10% FBS and 1% streptomycin (100IU/mL) and plated in 96-well plates (5X 10)³Cells/well). After 24 hours, the medium was aspirated, 90. mu.L of fresh medium, 10. mu.L of empty Nanoparticles (NPs) without TMZ and CDDP and 10. mu.L of Nanoparticles (NPs) without membrane of cancer cells were added, and after incubation for 48 hours, 10. mu.L of a 5mg/mL solution of 3- (4, 5-dimethyl-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) was added and incubatedAfter 4 hours, the medium was removed, and 150. mu.L of DMSO was added to dissolve MTT-formazan produced in living cells. The microplate reader measures the absorbance at 492nm for each well, with the medium well to which MTT was added as the zero point. Five groups (n-5) were made in parallel for each experimental data.

② flow cytometry and confocal microscope characterization of endocytosis and release in cells

In the flow cytometry test, three cell types of U87MG, A549 and Hela are planted in a 6-well cell culture plate (1 × 10)⁶Cells/well) was cultured at 37 ℃ for 24 hours, 500. mu.L of MNPs-FITC, RNPs-FITC, PBS was added thereto and incubated for 2 hours, and the sample was aspirated off, and the cells were digested with 500. mu.L of trypsin. The resulting Cell suspension was centrifuged at 1000 Xg for 3 minutes, washed twice with PBS, re-dispersed in 500. mu.L PBS, and subjected to flow cytometry (BD FACS Calibur, Becton Dickinson, USA) for 1 hour, and obtained by circling 10000 cells with Cell Quest software.

The endocytosis and intracellular drug release behavior was observed by CLSM photographs. The U87MG cells were plated in 24-well cell culture plates (1X 10) containing microscope slides⁵Cells/well) for 24 hours, 50. mu.L of MNPs-FITC, RNPs-FITC, and NPs-FITC was added. After 4 hours of incubation, the medium was removed and washed twice with PBS. Nuclei were stained with DAPI for 15 minutes and washed twice. Fluorescent pictures were taken by CLSM (TCS SP 5).

Figure 6 shows the experimental results of cytotoxicity and intracellular release experiments. Wherein: FIG. 6a shows the results of toxicity of MNPs with NPs for detecting dextran having acetal bond in the side chain. Figure 6b shows the killing results of different nanoparticles on cancer cells; further shows TMZ in MNPs @ TMZ/CDDP: the CDDP drug loading mass ratio is 1: 1 and 1: 2, a cell killing effect. FIG. 6c shows the endocytosis of MNPs, RNPs and PBS by different cells (U87MG, A549, Hela) respectively to test the homologous targeting effect of nanoparticles.

As can be seen from FIG. 6, even when the concentrations of MNPs and NPs were as high as 1.0MG/mL, it was still non-toxic to U87MG cells (FIG. 6a), confirming good biocompatibility of MNPs and NPs. MNPs showed significant antitumor activity against U87MG cells (fig. 6 b). Flow cytometry experiments prove that MNPs can be well endocytosed by cells and have a good homologous targeting effect (figure 6 c).

FIG. 7 shows the entry of MNPs @ FITC, RBCm @ NPs @ FITC, NPs @ FITC into U87MG cells.

As can be seen from FIG. 7, CLSM observed strong FITC fluorescence in the nucleus of U87MG cells after 4 hours of incubation with MNPs-FITC, the entry effect was much better than that of RNPs-FITC and free FITC, indicating that MNPs-FITC rapidly swells in cells and is effectively released in FITC cytoplasm. The bionic nano-drug prepared in the embodiment 1 can enter U87MG cells more, so as to verify the homologous targeting effect of the bionic nano-drug.

Test example 3

Animal experiments

Study of pharmacokinetics

In an in vivo pharmacokinetic study, BALB/c mice were randomized into groups (3 in parallel per group) at 6-8 weeks and blood was drawn from the orbit at predetermined time points by tail vein injection of 200 μ L MNPs @ TMZ/CDDP, NPs @ TMZ/CDDP, free TMZ and free CDDP (5 mg/kg TMZ and CDDP doses, respectively). The blood sample is extracted with an organic solvent to separate the drug and passed through High Performance Liquid Chromatography (HPLC) and Inductively Coupled Plasma (ICP). Compared with the traditional nano-drug wrapped by the cancer-free cell membrane, the blood stability of the bionic nano-drug can be judged. Fig. 8 shows the drug concentration-time curve of the drug in vivo. MNPs @ DiR, NPs @ DiR and Free DiR are injected into mice, and the accumulation of the nanoparticles in the brains of the mice is observed at different time points.

As can be seen from FIG. 8, the circulation time of the targeted nano-drug MNPs @ TMZ/CDDP in vivo is longer, which is equivalent to that of NPs @ TMZ/CDDP, but longer than that of Free TMZ or Free CDDP, which indicates that the biocompatibility of the nano-particle modified by the cell membrane is better; the bionic nano-drug is beneficial to the circulation time of chemotherapeutic drugs (TMZ/CDDP) in vivo and can prolong the accumulation of brain tumors.

② antitumor effect

An in-situ model of U87MG glioma tumor was established by transplanting tumor tissue to the brain of BALB/c nude mice (18-20g, 6-8 weeks old).

An in situ model was established with luciferase-labeled human brain glioma cells (U87 MG-Luc), and tumor growth was followed qualitatively and quantitatively by IVIS III by multiple dose administration. In the treatment process layer, the toxic and side effects and the anti-tumor activity of the bionic nano-drug are evaluated through the weight change and the survival rate of the mice. After the treatment is finished, the health condition of each normal organ and the apoptosis condition of tumor tissues of the mouse after the treatment of the bionic nano-drug are analyzed by histological staining methods such as H & E, TUNEL and the like. Through treatment experiments, the system toxicity and the antitumor activity of the bionic nano-drug on a nude mouse loaded with U87 MG-Luc can be seen.

③ biodistribution

Injecting nanometer medicine into nude mouse with Henshini U87MG via tail vein, collecting main tissues of mouse heart, liver, spleen, lung, kidney, brain and tumor at different time points, and imaging with IVIS III in vitro. Then, after homogenizing each tissue, extracting with organic solvent, and centrifuging, the fluorescence spectrophotometer quantitatively analyzes the in vivo biological distribution of the drug at different time points. Through the experiment, the influence of the in vivo stability, the active targeting performance and the released TMZ and CDDP of the bionic nano-drug on the enrichment, retention and permeation of the drug at a tumor part can be deduced.

FIG. 10 shows the relative photon counts before and after treatment at the tumor sites in mice of different groups; FIG. 11 shows the weight change of different groups of mice over the course of treatment; figure 12 shows the life cycle of different groups of mice; figure 13 shows the sectioned observations of different groups of drugs on major organs, the experimental results demonstrating the low toxicity of the nanoparticles.

As can be seen from fig. 10-13: the results of treatment experiments with MNPs @ TMZ/CDDP in BALB/c nude mice loaded with U87 MG-luc show that it is effective in inhibiting tumor growth in a dose-dependent manner, and that it is significantly inhibited at TMZ doses of 5MG/kg and CDDP doses of 5 MG/kg. When the free TMZ and CDDP concentrations were 5mg/kg, the mice lost weight over 7 days. In comparison, the weight change of the mice treated by the MNPs @ TMZ/CDDP is small, which indicates that the bionic nano-drug has small toxic and side effects. In addition, mice were treated with MNPs @ TMZ/CDDP at a dose of TMZ 5mg/kg and CDDP 5mg/kg, with a 100% survival rate at 22 days of the treatment cycle. The results of H & E staining histological analysis prove that the MNPs @ TMZ/CDDP have little harm to the main organs including heart, liver, spleen, lung and kidney when the MNPs @ TMZ/CDDP and the CDDP 5mg/kgTMZ are dosed. The results again indicate that MNPs @ TMZ/CDDP have very low systemic toxicity.

Crossing effect and targeting property of BBB

The near-infrared dye DiR is adopted to replace TMZ and CDDP to self-assemble to form nano particles, nano drugs are injected into nude mice with in-situ human brain glioma U87MG through tail veins, the distribution condition of the nano drugs at different time points in the bodies is tracked by a small animal imager (IVIS III), accumulation and detention at brain tumor positions are repeatedly investigated, and qualitative and quantitative comparison is carried out with a cancer cell membrane-Free control group and a Free DiR group, so that BBB crossing efficiency and nano drug tumor targeting capacity are investigated.

The distribution of the nano-drug in vivo at different time points was followed by a small animal imager (IVIS III).

FIG. 14 shows fluorescence intensity of the MNPs @ DiR loaded with DiR at different time points; as can be seen from fig. 14, the fluorescence intensity of MNPs @ DiR is significantly higher than that of the control group, indicating that MNPs have a good ability to target tumors.

In conclusion, the bionic nano-drug provided by the embodiment of the application avoids the leakage of the drug; the release speed of the drug in the cell is high and the effective release is formed; and the bionic nano-medicament has longer circulation time in vivo, better biocompatibility and obvious synergistic treatment effect.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nano-drug, comprising an inner core and an outer shell surrounding 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 one or more of cisplatin, lomustine, vincristine or procarbazine;

the housing comprises a cancer cell membrane;

the cancer cell membrane is mainly prepared by the following steps:

culturing glioma cell U87MG cells in a cell culture dish, sucking out a culture medium, washing with PBS, adding EDTA for digesting the cells, collecting the cells, washing with PBS, and extracting cell membranes with a membrane protein extraction kit;

the structural formula of the carrier is as follows:

。

2. the method of preparing a nano-drug according to claim 1, wherein the method of preparing a nano-drug comprises:

mixing said carrier, said first component and said second component and then dialyzing to remove free said first component and said second component to obtain said inner core;

the cancer cell membrane and the inner core are mixed and then extruded.

3. The method of claim 2, wherein mixing the carrier, the first component, and the second component specifically comprises:

dissolving the carrier in tetrahydrofuran to obtain a first solution, dissolving the first component and the second component in anhydrous dimethyl sulfoxide to obtain a second solution, and then mixing the first solution and the second solution.

4. The method for preparing the nano-drug according to claim 2, wherein the cancer cell membrane and the inner core are mixed and extruded through the filters of 700-800nm and 400-500nm in sequence.

5. The method for preparing nano-drug according to claim 2, wherein the carrier is mainly prepared by the following steps:

under the protection of nitrogen, reacting dextran with 2-ethoxypropene in a solvent, after the reaction is finished, terminating the reaction by triethylamine, precipitating in an alkaline solution, washing, and drying the precipitate.

6. The method for preparing nano-drug according to claim 5, wherein the dextran is reacted with the 2-ethoxypropene in the presence of pyridinium p-toluenesulfonate as a catalyst.

7. The use of the nano-drug of claim 1 in the preparation of a targeted therapeutic drug for human brain glioma.