CN113786496A - Target drug nano system and preparation method and application thereof - Google Patents

Target drug nano system and preparation method and application thereof Download PDF

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CN113786496A
CN113786496A CN202111014052.0A CN202111014052A CN113786496A CN 113786496 A CN113786496 A CN 113786496A CN 202111014052 A CN202111014052 A CN 202111014052A CN 113786496 A CN113786496 A CN 113786496A
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cells
fpd
targeted drug
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tumor
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于德新
赵洁
李明
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Qilu Hospital of Shandong University
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Abstract

The invention relates to the technical field of carrier drugs, in particular to a targeted drug nano system and a preparation method and application thereof. In order to solve the problems of high toxicity, poor specificity, low accuracy and poor targeting property of the existing MRI contrast agent, the invention provides a targeted drug nano system and a preparation method and application thereof, and ferroferric oxide (Fe)3O4) As a carrier, with the aid of DMSA @ Fe3O4The magnetic nanoparticles have the characteristic of superparamagnetism, and the immunotherapy drugs and the chemotherapeutic drugs are modified on the surfaces of the nanoparticles in a surface charge attraction mode. The targeted drug nano system can promote CD4+ cells to gather at a tumor part through combined treatment of immunity and chemotherapy, enhances curative effect in a mutual promotion mode, and can realize real-time tracking of the drug and real-time monitoring of the curative effect by means of MRI, thereby realizing the effect of integrated diagnosis and treatment.

Description

Target drug nano system and preparation method and application thereof
Technical Field
The invention relates to the technical field of carrier drugs, in particular to a targeted drug nano system and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The breast cancer is the most common malignant tumor in women and the second leading cause of cancer-related death in women, wherein triple-negative breast cancer (TNBC) has high tumor grade and late disease stage, accounts for 10% -20% of all invasive breast cancers, and is particularly troublesome to treat.
The current diagnosis of breast cancer mainly depends on pathological biopsy and imaging diagnosis. Although the former is the gold standard for diagnosis, in view of the invasiveness of the pathological biopsy and the limited availability of materials, imaging still remains the main means for diagnosing and judging the treatment effect of breast cancer. Imaging examination techniques for breast cancer include infrared scanning, X-ray molybdenum target photography, X-ray Computed Tomography (CT), Magnetic Resonance Imaging (MRI), ultrasound, and Positron Emission Tomography (PET), among others. Among them, MRI is one of the most important examination techniques for breast diseases due to its remarkable soft tissue resolution, and its detection sensitivity can reach 96%, so that the number of false positives in X-ray breast examination can be reduced, thereby avoiding unnecessary biopsy. In actual clinical work, contrast-enhanced MRI is also needed to provide more hemodynamic information to improve the diagnostic accuracy of breast disease. MRI contrast agents can shorten the relaxation time of, increase tissue contrast, and improve signal-to-noise ratio in or around tissues.
However, the inventor researches and discovers that the currently clinically commonly used contrast agents are gadolinium-pentetic acid meglumine (gadolinium-DTPA, Gd-DTPA) and gadolinium-gadolinium (iii)1,4,7, 10-tetraazacyclodecane-1, 4,7,10-tetraacetate, Gd-DOTA) which are representative gadolinium contrast agents, but the problems of renal fibrosis toxicity and potential neurotoxicity caused by free Gd ions exist to a certain extent. In addition, with the advent of the molecular targeted therapy era of malignant tumors, the small molecular contrast agents have the defects of short cycle time, lack of targeting property, poor specificity and the like, so that the requirements of accurate diagnosis and real-time efficacy monitoring of malignant tumors cannot be met.
In addition, during the TNBC treatment, doxorubicin (doxorubicin, DOX) is a broad-spectrum anti-tumor chemotherapeutic, and the mechanism of action is mainly that the drug intercalates DNA to inhibit nucleic acid synthesis, and has strong cytotoxic effects. The compound has a wide antitumor spectrum, belongs to a periodic nonspecific medicine, and is clinically used for treating breast cancer. But it has serious side effects such as damage of white blood cells and platelets, cardiotoxicity, etc.
Meanwhile, the TNBC tumor cell microenvironment has high tumor infiltration lymphocyte infiltration degree, so that the TNBC tumor cell microenvironment is used as a new treatment means, and immunotherapy has great clinical development potential. The T cell autoimmunity in a human body can be activated to play an anti-cancer role by inhibiting the immune checkpoint programmed cell death protein-1 (PD-1) and the ligand PD-L1 thereof. However, the inventors found that single-drug treatment with immune checkpoint inhibitors was of limited help for TNBC patients, with Objective Remission Rates (ORR) ranging from 5% in the PD-1/PD-L1 positive population to 23% in the first-line PD-L1 positive population.
Disclosure of Invention
To solve the existing MRI contrastThe invention provides a targeted drug nano system and a preparation method and application thereof, and ferroferric oxide (Fe) is used for solving the problems of high toxicity, poor specificity, low accuracy and poor targeting property of the existing drugs3O4) As a carrier, with the aid of DMSA @ Fe3O4The magnetic nanoparticles have the characteristic of superparamagnetism, and the immunotherapy drugs and the chemotherapeutic drugs are modified on the surfaces of the nanoparticles in a surface charge attraction mode. On one hand, the targeted drug nano system can target breast cancer cells by virtue of an antibody modified on the surface of the targeted drug nano system, and belongs to active targeting; on the other hand, the targeted drug nano system can promote the CD4+ cells to gather at the tumor part through the combined treatment of immunity and chemotherapy, and enhance the curative effect in a mutual promotion mode. In addition, the real-time tracking of the medicine and the real-time monitoring of the curative effect can be realized by means of MRI, and the diagnosis and treatment integrated effect is realized.
Specifically, the invention is realized by the following technical scheme:
in the first aspect of the invention, a targeted drug nano system is provided, and takes ferroferric oxide as a carrier, and the surface of the targeted drug nano system is modified with an immunotherapy drug and a chemotherapy drug.
In a second aspect of the present invention, a method for preparing a targeted drug nanosystem is provided, which comprises: mixing ferroferric oxide modified by 2, 3-dimercaptosuccinic acid with immunotherapy drugs and chemotherapy drugs.
In a third aspect of the invention, an application of a targeted drug nano system in preparation of a breast cancer drug is provided.
In a fourth aspect of the invention, a T2WI negative contrast agent is provided, comprising a targeted drug nanosystem.
In a fifth aspect of the invention, an application of a targeted drug nano system in preparation of a T2WI negative contrast agent is provided.
In a sixth aspect of the invention, an application of a targeted drug nano system in MRI real-time monitoring is provided.
One or more of the technical schemes have the following beneficial effects:
1) with ferroferric oxide (Fe)3O4) As a carrier byDMSA@Fe3O4The magnetic nanoparticles have the characteristic of superparamagnetism, and the immunotherapy drugs and the chemotherapeutic drugs are modified on the surfaces of the nanoparticles in a surface charge attraction mode. On one hand, the targeted drug nano system can target breast cancer cells by virtue of an antibody modified on the surface of the targeted drug nano system, and belongs to active targeting; on the other hand, the targeted drug nano system can promote the CD4+ cells to gather at the tumor part through the combined treatment of immunity and chemotherapy, and enhance the curative effect in a mutual promotion mode.
2) Experiments show that if the targeted drug nano system is used as a T2 contrast agent, real-time tracking of the drug and real-time monitoring of the curative effect can be realized by means of MRI, and the diagnosis and treatment integrated effect is realized.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows superparamagnetic Fe according to example 1 of the present invention3O4Characterization of nanospheres: (a) fe3O4TEM images of nanospheres; (b) fe3O4HRTEM (high resolution transmission electron microscope) images of nanospheres; (c) fe3O4-TEM images of DMSA nanospheres; (d) the resulting Fe3O4XRD (X-ray diffractometer) patterns of nanospheres; (e) fe3O4Saturation magnetization curve of nanospheres; (f) fe3O4And Fe3O4-FTIR (fourier infrared transform spectrometer) spectra of DMSA nanospheres;
FIG. 2 is a diagram illustrating the preparation of Fe in example 1 of the present invention3O4Performing cytotoxicity test on the material; (a) MCF-7 and (b)4T1 cells with varying concentrations of Fe3O4Cell activity quantification plots of nanospheres after 6, 12, 24 and 48 hours of culture (5 sets of data were obtained for each time parameter, 0, 25, 50, 100, 200 μ g/mL from left to right);
FIG. 3 is (a) a T2 MR image and (b) a pseudo color image of an FPD at different Fe concentrations according to example 1 of the present invention,(c) inverse relaxation time T2-1Fitting a curve with the Fe concentration, wherein the slope is the relaxation rate (r) of the FPD2)；
FIG. 4 is an experimental image of hemolysis of red blood cells of each group of nanospheres of the inventive and comparative example products and control group (saline) at different concentrations (200, 100, 50, 25, 12.5, 6.25 μ g/mL from left to right, respectively), (a) saline; (b) fe3O4；(c)FP；(d)FD；(e)FPD；
FIG. 5 is an immunofluorescence image of mouse breast cancer 4T1 cells PD-L1 of the present invention;
FIG. 6 is TEM images of different groups of nanospheres of the present invention taken up by 4T1 cells at 1h (a), 4h (b), and 24h (c);
FIG. 7 shows a Prussian blue staining image (a) and an ICP-MS quantitative image (b) of 4T1 cells according to the present invention;
FIG. 8 is a T2 image (a) of 4T1 cells treated with different groups of nanoparticles and a quantitative map of the signal-to-noise ratio of magnetic resonance (b) according to the present invention;
FIG. 9 is (a) a Hoechst33342/PI fluorescence image of different nanosphere-treated 4T1 cells; (b) the activity quantitative graph of Hoechst33342/PI fluorescence double-staining cells is Fe from left to right2O3FP, FD, FPD; (c) ROS reactive oxygen species staining fluorescence images of different nanosphere treated 4T1 cells; (d) ROS reactive oxygen species staining positive cell quantitative diagram, from left to right in sequence, is Fe2O3，FP，FD，FPD；
FIG. 10 is a schematic representation of a mouse treatment regimen of the present invention;
FIG. 11 is a graph of the tumor and 4T1 breast cancer of each treatment group of the present invention (a), a trend graph of the tumor volume of the mice (b), a survival curve of the mice (c) and a weight change graph of the mice (d), wherein in FIG. 11(c), the curves are PBS and Fe from left to right3O4FP, FD and FPD;
FIG. 12 shows that (a)4T1 tumor-bearing mice of the present invention are injected with Fe in tail vein3O4And T2 MR imaging at different time points after FPD, white boxes marking tumor tissue; (b)4T1 tumor-bearing mouse tail vein injection Fe3O4And relative T at different time points after FPD2Signal strength plots.
FIG. 13 is a graph showing HE staining of the heart, liver, spleen and kidney of mice before and after treatment for 1, 5, 10 and 15d in FPD group of the present invention.
FIG. 14 is a tumor TUNEL staining image (a) and its quantification image (b) of 4T1 tumor-bearing mice of different treatment groups of the present invention.
FIG. 15 shows Fe of the present invention3O4FD and FPD treatment group 4T1 TUNEL staining images (a) and quantification images (b) of heart, liver, spleen and kidney of tumor-bearing mice, where the drug type for each site is Fe from left to right in fig. 15(b)3O4FD and FPD.
FIG. 16 shows Fe of the present invention3O4FD and FPD treatment group 4T1 tumor-bearing mice and real-time fluorescence quantitative PCR of three apoptosis-related genes (Bax, Bcl-2 and Caspase-3) of heart, liver, spleen and kidney.
FIG. 17 shows a PD-L1 immunofluorescent staining image (a) (left: negative control) and a Prussian blue staining image (b) (left: Fe) of tumor tissue of FPD group mice of the present invention3O4Group, right: FPD set).
FIG. 18 is a flow cytometric analysis of Fe in accordance with the present invention3O4CD3 in tumor tissue after three groups of treatment, FD and FPD+CD8+Results for T cells (a) and their quantification plots (b).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The inventor researches and discovers that the contrast agents commonly used in clinic at present are gadolinium contrast agents represented by gadolinium-DTPA (gadolinium-DTPA, Gd-DTPA) and gadolinium-gadolinium (iii)1,4,7, 10-tetraazacyclodecane-1, 4,7,10-tetraacetate, Gd-DOTA, but the problems of renal fibrosis toxicity and potential neurotoxicity caused by free Gd ions exist to a certain extent. In addition, with the advent of the molecular targeted therapy era of malignant tumors, the small molecular contrast agents have the defects of short cycle time, lack of targeting property, poor specificity and the like, so that the requirements of accurate diagnosis and real-time efficacy monitoring of malignant tumors cannot be met.
In addition, during the TNBC treatment, doxorubicin (doxorubicin, DOX) is a broad-spectrum anti-tumor chemotherapeutic, and the mechanism of action is mainly that the drug intercalates DNA to inhibit nucleic acid synthesis, and has strong cytotoxic effects. The compound has a wide antitumor spectrum, belongs to a periodic nonspecific medicine, and is clinically used for treating breast cancer. But it has serious side effects such as damage of white blood cells and platelets, cardiotoxicity, etc.
Meanwhile, the TNBC tumor cell microenvironment has high tumor infiltration lymphocyte infiltration degree, so that the TNBC tumor cell microenvironment is used as a new treatment means, and immunotherapy has great clinical development potential. The T cell autoimmunity in a human body can be activated to play an anti-cancer role by inhibiting the immune checkpoint programmed cell death protein-1 (PD-1) and the ligand PD-L1 thereof. However, the inventors found that single-drug treatment with immune checkpoint inhibitors was of limited help for TNBC patients, with Objective Remission Rates (ORR) ranging from 5% in the PD-1/PD-L1 positive population to 23% in the first-line PD-L1 positive population.
Therefore, the invention provides a targeted drug nano system and a preparation method and application thereof, and ferroferric oxide (Fe)3O4) As a carrier, with the aid of DMSA @ Fe3O4The magnetic nanoparticles have the characteristic of superparamagnetism, and the immunotherapy drugs and the chemotherapeutic drugs are modified on the surfaces of the nanoparticles in a surface charge attraction mode.
Specifically, the invention is realized by the following technical scheme:
in the first aspect of the invention, a targeted drug nano system is provided, and takes ferroferric oxide as a carrier, and the surface of the targeted drug nano system is modified with an immunotherapy drug and a chemotherapy drug.
Some embodiments of the invention utilize Fe3O4The preparation of a novel targeted drug nano system is characterized in that: the immunotherapy medicament is used for realizing the targeting of diagnosis and treatment, synchronously realizing the combined treatment of immunotherapy and chemotherapy medicament, and realizing the real-time visual monitoring of the curative effect based on MRI, thereby realizing the accurate diagnosis and treatment of breast cancer, obviously improving the diagnosis and treatment level, and having important value and significance.
In one or more embodiments of the invention, the chemotherapeutic is selected from one or more of daunorubicin, doxorubicin, mitoxantrone, paclitaxel.
In one or more embodiments of the invention, the immunotherapeutic agent is selected from one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody.
Wherein the iron oxide is ferroferric oxide (Fe)3O4) As a carrier, surface modification of anti-PD-L1 antibody and chemotherapeutic drug DOX is carried out to synthesize a diagnosis and treatment integrated nano system (PD-L1@ Fe) of targeted PD-L13O4-DOX，FPD)。
In some embodiments, ferroferric oxide (Fe)3O4) Is a nanoparticle or nanosphere.
In a second aspect of the present invention, a method for preparing a targeted drug nanosystem is provided, which comprises: mixing ferroferric oxide modified by 2, 3-dimercaptosuccinic acid (DMSA) with immunotherapy drugs and chemotherapy drugs.
The prior art discloses passive targeting vector drugs, which are taken into tumor cells by virtue of liposome through caveolin-mediated endocytosis, and the liposome finally achieves the aim of tumor treatment by inducing the tumor cells to generate apoptosis. But cannot monitor tumors and drugs in real time.
According to some embodiments of the invention, ferroferric oxide is used as a carrier, and an immunotherapy drug and a chemotherapy drug are surface-modified, so that breast cancer cells can be targeted, and the aggregation of CD4+ cells at a tumor part is promoted through combined therapy of immunization and chemotherapy, and the curative effect is enhanced in a mutual promotion manner.
Experiments show that if 2, 3-dimercaptosuccinic acid (DMSA) modification is not carried out on ferroferric oxide, immunotherapy drugs and chemotherapy drugs cannot be loaded under the condition that components such as liposome are not contained.
In one or more embodiments of the invention, 2, 3-dimercaptosuccinic acid is dissolved in deionized water, ferroferric oxide is added, tetrahydrofuran is added, and the mixture is subjected to ultrasonic treatment and reaction to obtain the ferroferric oxide modified by the 2, 3-dimercaptosuccinic acid;
dispersing ferroferric oxide modified by 2, 3-dimercaptosuccinic acid in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and sulfo-N-hydroxysuccinic acid, adding an immunotherapy medicament and a chemotherapeutic medicament, and reacting at low temperature overnight to obtain the product.
The 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the sulfo-N-hydroxysuccinic acid have the effects of avoiding excessive aggregation of nanoparticles, enabling the exposed ferroferric oxide modified by the 2, 3-dimercaptosuccinic acid to have more surfaces and being beneficial to modification of immunotherapy drugs and chemotherapy drugs.
In one or more embodiments of the invention, the mass ratio of 2, 3-dimercaptosuccinic acid to ferroferric oxide is 1.5-4:1, preferably 2: 1.
If the dosage of the 2, 3-dimercaptosuccinic acid is too high, the DMSA cannot be well combined with the ferroferric oxide, and if the dosage of the 2, 3-dimercaptosuccinic acid is too low, the DMSA cannot be fully combined with the ferroferric oxide, so that the modification of the immunotherapy medicament and the chemotherapy medicament is influenced.
The proportions of the ferroferric oxide, the immunotherapy medicament and the chemotherapy medicament are as follows: 400g, 1g and 2L.
In a third aspect of the invention, an application of a targeted drug nano system in preparation of a breast cancer drug is provided.
In a fourth aspect of the invention, a T2WI negative contrast agent is provided, comprising a targeted drug nanosystem.
In a fifth aspect of the invention, an application of a targeted drug nano system in preparation of a T2WI negative contrast agent is provided.
In a sixth aspect of the invention, an application of a targeted drug nano system in MRI real-time monitoring is provided.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Experiment raw materials:
FeCl3·6H2o: national pharmaceutical group chemical reagents limited, shanghai; sodium oleate: mclin biochemistry technologies, ltd, shanghai; octadecene: alatin holdings group ltd, beijing; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, sulfo-N-hydroxysuccinic acid: source leaf biotechnology limited, shanghai; CCK-8 kit: bebo corporation, shanghai; 4% rabbit red blood cell kit: solebao technologies, Inc., Beijing; 4T1 cells, MCF-10A cells: zhongqiao new boat biotechnology limited, shanghai; 1640 medium, fetal bovine serum, 0.25% trypsin (containing 0.02% EDTA): gibio corporation, USA; 100X penicillin-streptomycin mixture: solarbio corporation, beijing; t25 flask, 6-well plate, 96-well plate, 15mL centrifuge tube, 50mL centrifuge tube, cryopreservation tube: corning corporation, usa.
An experimental instrument:
JEM-2100 Transmission Electron microscope: JEOL Ltd, Japan; analyzing by a Malvern nanometer sphericity and potential analyzer: malvern instruments, uk; inductively coupled plasma mass spectrometer (ICP-MS): becton Dickinson, usa; beijing; 3.0T NMR scanner: GE corporation, usa; an ultrasonic cleaner. The scanning coil adopts a small animal coil.
Example 1: FPD (PD-L1@ Fe)3O4-DOX) Synthesis
Fe3O4Synthesis of nanospheres
10.8g FeCl3·6H2O and 36.5g sodium oleate were added to 60mL deionized water, 80mL EtherAnd (3) adding alcohol and 140mL of n-hexane into the mixed solution, heating and stirring the mixed solution at 70 ℃ for 4h, and purifying the target product, namely the iron oleate by liquid separation and distillation. Adding the obtained iron oleate into 5.7g of oleic acid and 200g of octadecene, uniformly mixing, heating to 320 ℃, preserving heat for half an hour, cooling to room temperature after the reaction is finished, adding excessive ethanol to precipitate Fe3O4The nanoparticles were washed three times with ethanol.
Washed Fe3O4The nanospheres react with 2, 3-dimercaptosuccinic acid (DMSA) to generate the water-dispersed nano material. 40mg of DMSA was dissolved in Na2CO3To the deionized water solution of (1), 20mg of Fe was added3O4Adding 1mL of tetrahydrofuran into the nano particles, reacting for half an hour under the ultrasonic condition, and then reacting for 1 hour on a shaking table to obtain the DMSA modified Fe3O4Nanosphere (Fe)3O4-DMSA)。
Mixing Fe3O4Dispersing the-DMSA into 2mL of water with the concentration of 1mg/mL, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and sulfo-N-hydroxysuccinic acid, then adding 100 mu g of DOX +50 mu L of PD-L1 antibody, reacting overnight at 4 ℃, after reaction, centrifuging and purifying the obtained product to obtain the FPD, and storing the FPD in a refrigerator at 4 ℃.
Comparative example 1: FP (PD-L1@ Fe)3O4) Synthesis of (2)
The difference from example 1 is that: mixing Fe3O4Dispersing the-DMSA into 2mL of water with the concentration of 1mg/mL, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and sulfo-N-hydroxysuccinic acid, then adding 50 mu L of PD-L1 antibody, reacting overnight at 4 ℃, and after reaction, centrifugally purifying the obtained product to obtain FP (PD-L1@ Fe)3O4，Fe3O4PD-L1 antibody surface-modified nanospheres) and stored in a refrigerator at 4 ℃.
The rest is the same as in example 1.
Comparative example 2: FD (Fe)3O4-DOX) Synthesis
The difference from example 1 is that: mixing Fe3O4Dispersing the-DMSA into 2mL of water with the concentration of 1mg/mL, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and sulfo-N-hydroxysuccinic acid, then adding 100 mu g of DOX, reacting overnight at 4 ℃, and after reaction, centrifuging and purifying the obtained product to obtain FD (Fe)3O4-DOX,Fe3O4Nanosphere loaded DOX) and stored in a 4 ℃ refrigerator.
The rest is the same as in example 1.
The first method for detecting the product comprises the following steps:
1. characterization of
The morphology and crystal structure of the nanospheres were characterized using transmission electron microscopy and high resolution transmission electron microscopy (TEM, HRTEM, JEM-2100, JEOL, japan). On a German Bruker D8 advanced powder diffractometer (D8 Advance, Bruker, Germany) with Cu Ka as the X-ray source for Fe3O4The structural information of the nanospheres was characterized by X-ray diffraction (XRD). The surface functional groups of the nanomaterials were analyzed by Fourier Transform Infrared (FTIR) spectroscopy using an infrared spectrophotometer (Nicolet Nexus 670, Thermo Fisher Scientific, Inc). The prepared nanospheres were saturated magnetized by vibrating a sample magnetometer (micromagt model 2900AGM system). The ultraviolet-visible (UV-vis) light absorption spectrum of the synthesized water was analyzed with an ultraviolet-visible spectrophotometer (UV-6100, meprobat, sienna). The surface charge characteristics and particle size distribution of the nanoparticle suspension were characterized using a Zeta potential analyzer (ZetaPALS, Brookhaven Instrument Crop, Holtsville NY, USA).
2. In vitro T2 relaxation Rate detection
(1) Preparing FPD solution: FPD solutions having Fe ion concentrations of 0.025325, 0.05065, 0.1013, 0.2026, 0.4052mM were prepared by adding different volumes of ultrapure water to 2mg/mL of the FPD solution, wherein the Fe ion concentration was measured by ICP-MS. Before MR scanning, the FPD was fully dissolved by shaking for 15min with an ultrasonic oscillator.
(2) T2 relaxation rate: scanning was performed using a 3.0T nuclear magnetic resonance scanner (GE Signa HDx). T2 weighted imaging and T2 relaxation times were acquired using a T1 Fast Spin Echo (FSE) sequence with the following parameters: repetition ofTime (TR) 4240 ms; echo Time (TE) is 108 ms. After the scanning is finished, T of the workstation is utilized2The mapping function measures T2 values for different solution concentrations. The transverse relaxation rate (r) is obtained by linear fitting of the inverse relaxation time of T2 and the Fe ion concentration2)。
3. Cytotoxicity assays
(1) The 4T1 cells in logarithmic growth phase were seeded in 96-well plates to a cell density of approximately 6X 103Cells/well, 100uL of DMEM medium containing 10% FBS and 1% penicillin-streptomycin was added to each well, and 96-well plates were placed at 37 ℃ in 5% CO2After culturing for 24h in a cell culture box, observing the adherent condition and the growth state of the cells by an inverted phase contrast microscope.
(2) The medium was aspirated from each well of the 96-well plate by pipette and Fe was added to each well to final concentrations of 0, 25, 50, 100, 200. mu.g/mL3O4Solution, negative control group was DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and each group was set with 5 duplicate wells. Place 96-well plates at 37 ℃ 5% CO2Culturing for a certain time (6, 12, 24 and 48 hours) in a cell culture box.
(3) Completely sucking up the solution in each hole of a 96-hole plate by using a pipettor, washing for 2 times by using sterile PBS solution, adding 100uL of fresh culture medium into each hole, sequentially adding 10uL of CCK-8 solution into each hole, and placing the 96-hole plate at 37 ℃ under the condition of keeping out of the sun and 5% CO2Culturing in a cell culture box for 2 h.
(4) And (3) placing the 96 plate in a microplate reader, measuring the light absorption value of each hole with the wavelength of 450nm, calculating the cell activity, subtracting the OD value of the blank hole from the OD value of each test hole, and averaging the OD values of the repeated holes. Cell viability = (plus Mn @ CNS cell OD-blank OD)/(control cell OD-blank OD) × 100.
4. Hemolysis test
(1) 4% rabbit red blood cell is prepared into 2% red blood cell suspension for standby.
(2) Mixing Fe3O4FP, FD, FPD and FPD are respectively prepared into nano materials with different concentration gradients (200, 100, 50, 25, 12.5 and 6.25 mu g/mL).
(3) The prepared nano material is placed in a water bath kettle at 37 ℃ for 30min, and 1mL of distilled water and physiological saline are set as controls.
(4) 1mL of erythrocyte suspension is added into each tube, the mixture is centrifuged at 3000r/min for 5min after 1h at 37 ℃, and the centrifugation is carried out for photographing and observation.
Secondly, analyzing a characterization result:
1. construction and characterization of nanospheres
Fe produced by oil heating method3O4Nanospheres. The morphology and crystal structure of the synthesized nanospheres were characterized by Transmission Electron Microscopy (TEM) and hrtem (hrtem). As shown in FIG. 1a, the prepared nanoparticles have uniform shapes and particle sizes of about 20 nm. HRTEM images of the resulting nanospheres (fig. 1b) show that nanosphere crystallinity is good, with interplanar spacing d 0.29nm corresponding to the (422) crystal plane. Modified by DMSA molecules, Fe3O4The DMSA had good dispersibility (FIG. 1 c). The XRD pattern (fig. 1d) shows that the synthesized nanospheres correspond to standard PDF cards (19-0629), indicating that the nanospheres have good crystallinity. This result is consistent with the HRTEM results. Measurement of the synthesized Fe by saturation magnetization Curve on a vibrating sample magnetometer3O4The superparamagnetic property of the nanospheres confirms that the saturation magnetization can reach 53emu/g at room temperature (FIG. 1 e). To confirm the molecular pair of DMSA to Fe3O4The FTIR spectra were analyzed for the modification of the nanoparticle surface, as shown in fig. 1 f. For Fe3O4Samples, at 2916 and 2847cm-1The strong peak corresponds to the C-H stretching vibration of methylene, which is caused by nano Fe3O4The surface of the film is remained by oleic acid molecules. 2037cm-1The left and right broad peaks correspond to C-O stretching vibrations. For Fe3O4DMSA samples, 1569 and 1360cm-1The peaks at (a) correspond to a large number of symmetrical and asymmetrical carboxylate (COO-) stretches. At 3243cm-1The broad peak at (a) is caused by the O-H stretching vibration. The peak at 1046cm-1 corresponds to the C-O stretching vibration. The position of these peaks indicates the abundant presence of DMSA molecules. The infrared spectrum result proves that the DMSA molecule is successfully modified in Fe3O4The surface of the nanoparticles.
2、Fe3O4Cell toxicity test of
The biological safety has been evaluatedOne of the most critical indexes of the application of the rice material in vivo, the CCK-8 experiment can be used for detecting the cytotoxicity of the material by utilizing the characteristic principle that the quantity of formazan generated is proportional to the quantity of living cells. Mammary Normal cells MCF-10A (FIG. 2a) and Breast 4T1 (FIG. 2b) with Fe3O4After 6, 12, 24 and 48h of co-incubation, as shown in FIG. 2, in Fe3O4At concentrations of 25, 50, 100, 200. mu.g/mL, as compared to the control (no addition of Fe)3O4) The cell viability of MCF-10A and 4T1 cells was close to 100%, indicating that Fe3O4No significant cytotoxicity was observed against 4T1 and MCF-10A cells at concentrations ranging from 0-200. mu.g/mL.
3. T2 relaxation rate detection for FPD
The performance of the FPD as a contrast agent for magnetic resonance imaging is examined by determining the relaxation rate of the FPD. The fit curves (fig. 3c) made from T2WI images and pseudocolor maps of FPD at 0.025325, 0.05065, 0.1013, 0.2026, 0.4052mM, as shown in fig. 3a and 3b, show that the relaxation rate of FPD is 146.43mM-1s-1Indicating that the FPD can be used as a T2WI negative contrast agent.
4. Hemolysis test of FPD
Hemolysis experiments were necessary before animal experiments were performed using FPDs and other groups of nanomaterials. Experimental results show that nanospheres of varying concentrations (6.25-200. mu.g/mL) include Fe3O4The results of no obvious hemolysis and aggregation of FP, FD, FPD and normal saline treated rabbit erythrocytes (FIG. 4) show that the nanomaterials prepared in the examples and the comparative examples have no toxicity to erythrocytes, have good biological safety and can be used for further experiments.
Fe3O4NPs also have several recognized drawbacks, including: the size is difficult to control. ② the magnetic susceptibility is low. Thirdly, the synthetic route is complex. (iv) further modification is required to increase stability. The FPD synthesized in the embodiment 1 is relatively stable, and can be used as a good nano synthetic material for diagnosis and treatment integrated research of breast cancer. TEM shows that FPD is spherical particle with particle size of about 20nm, and the spherical shape is favorable to the materialTransport in vivo, and 20nm at Fe3O4The T2 contrast agent has the optimal particle size range for imaging, and can have enough accumulation time in vivo under the premise of not being phagocytized by a phagocytosis system, so that the T2 contrast agent is better taken up by tumor cells.
In addition, because the material is used as a T2 imaging contrast agent, the imaging effect is probably not as direct and clearer as that of T1 imaging. Therefore, it is necessary to explore the optimal imaging time and dose in further experiments with the help of animal tumor burden models.
III, PD-L1@ Fe3O4In vitro imaging and curative effect method of-DOX nanospheres
1. Experimental reagent and consumable
Mouse 4T1 cells, zhong qiao new boat biotechnology limited, shanghai; 1640 medium, fetal bovine serum, 0.25% trypsin (containing 0.02% EDTA): gibio corporation, USA; prussian blue kit, 100X penicillin-streptomycin mixed solution: solebao, Beijing; 33342/PI double staining kit: solebao, Beijing; DCFH-DA active oxygen ROS fluorescent probe: baiolai Boke technologies, Inc., Beijing; dimethyl sulfoxide, RIPA lysate, Trizol, tween: solebao, Beijing; concentrated nitric acid, concentrated hydrochloric acid, pyridine, ethanol, methanol: national drug group, beijing; glass bottom culture dish: corning corporation, usa.
2. Laboratory animal
The animal experiments were approved by the animal ethics committee and the animal protection committee of the qilu hospital, Shandong university.
Female Balb/c mice 5-6 weeks old were purchased from Beijing Huafukang laboratory animals, Inc., Beijing.
SPF level irradiation feed: beijing Huafukang laboratory animals Co., Ltd, Beijing.
Cell culture and general handling (see above for methods of cell culture in "cytotoxicity assay procedure")
Immunofluorescence assay for cellular PD-L1
To assess the feasibility of the PD-L1 target, 4T1 cells were immunofluorescent stained. Immunofluorescence experiments are described below. The antibodies used in this example or comparative example were as follows: anti-PD-L1 antibody (1: 2000; MCE) and Alexa Fluor 488 antibody. 4T1 cells were then incubated with DAPI at 1: 500 dilution; thermo Fisher Scientific), labeling nuclei for 20 minutes. The photographs were taken with a fluorescence microscope and a confocal microscope.
The first day:
(1) the 4T1 cells in the logarithmic growth phase were seeded in 6-well plates previously plated with a slide at about 5X 10 cells per well5Individual cells, wait for them to adhere overnight.
(2) PBS wash 3 times for 3min each.
(3) 4% fixation with paraformaldehyde.
(4) PBS wash 3 times for 3min each.
(5) 0.5% Triton X-100 (in PBS) was allowed to permeate for 20min at room temperature.
(6) The slides were washed 3 times with PBS for 3min each time.
(7) Sealing normal goat serum at room temperature for 30 min.
(8) Primary antibody was added dropwise and placed in a wet box and incubated overnight at 4 ℃.
The next day:
(9) PBST immersion-washed slide 3 times, each time for 3 min.
(10) Adding a fluorescent secondary antibody.
(11) DAPI was added and incubated for 5min in the dark.
(12) And sealing the sheet by using sealing liquid, and collecting images under a fluorescence microscope.
3. ICP-MS experiment of Fe uptake by cells
(1) 4T1 cells in logarithmic growth phase were seeded in 2 cell plates (6 wells) to ensure that the cells were exactly confluent (approximately 6X 10) after overnight incubation at cell density5One cell/well), 2mL of a prepared 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) was added per well, and the cell plate was placed at 37 ℃ in 5% CO2The cells are cultured in an incubator overnight, and the adherent growth condition of the cells is observed by an inverted microscope.
(2) The original culture medium in the 6-well plate was aspirated by a pipette, and after washing 2 times with sterile PBS, FP was added to the experimental group and Fe was added to the negative control group3O4Adding material into each hole according to the concentration of 10 mu g/mL, and arranging a P groupFP was added at a concentration of 20. mu.g/mL. Each well was made up to 1mL with 1640 complete medium, with 3 duplicate wells per set. 26 well plates were placed at 37 ℃ 5% CO2The cells were cultured in a cell incubator for 4 hours.
(3) Sucking the original solution in a 6-well plate by using a pipettor, then washing for 2 times by using a sterile PBS solution, adding 0.5mL of trypsin containing 0.02% EDTA into each well for digestion for 3min, adding 1640 complete culture medium accounting for 1-1.5 times of the volume of cells when the cells are observed to be small, round and sandy under a microscope to stop digestion, counting the cells in each well of each group by using a blood small ball counting plate, centrifuging for 5min at 800rpm, and then removing the supernatant.
(4) The aqua regia is prepared in advance, and the preparation method comprises the following steps: slowly add 1 volume of concentrated nitric acid to 3 volumes of concentrated hydrochloric acid with constant stirring on a glass rod to form a yellow mixture. 1mL of aqua regia was added to each well of cells for complete digestion. After 3 days, the cell residue which had not been digested was filtered through a 220 μm bacterial filter, and then 4mL of ultrapure water was added thereto to thereby adjust the volume to 5mL, and the Fe concentration was measured by ICP-MS. The formula: mass per intracellular Fe/pg ═ 5mL per cell number (ICP-MS assay concentration of Fe). Cell counting and ICP-MS measurement were performed for 3 duplicate wells to calculate the Fe mass per cell, and the mean and standard deviation were calculated.
4. Prussian blue experiment for cellular uptake of Fe
(1) 4T1 cells in logarithmic growth phase were seeded in 3 cell plates (6 wells) to ensure that the cells were exactly confluent (approximately 6X 10) after overnight incubation at cell density5One cell/well), 2mL of a prepared 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) was added per well, and the cell plate was placed at 37 ℃ in 5% CO2The cells are cultured in an incubator overnight, and the adherent growth condition of the cells is observed by an inverted microscope.
(2) Aspirating the original medium from each well with a pipette, washing twice with sterile PBS, adding FPD to the experimental group, and adding equal amount of Fe to the negative control group3O4Setting 3 concentration gradients of 10 mug/mL, 20 mug/mL and 40 mug/mL respectively, fixing the volume to 1mL in each hole, placing the cell plate at 37 ℃ with 5% CO2Culturing in a cell culture box for 4 h.
(3) The original solution in the 6-well plate was pipetted away, then washed 2 times with sterile PBS solution and fixed with 4% paraformaldehyde for 20 min. Equal amounts of Perls A and Perls B were mixed and formulated as Perls statin. The fixed six-hole plate was washed with tap water 2 times for 3min each time. Washing with distilled water for 3min for 2 times. Perls stain 1mL first in each well of the six-well plate for 30min, and then rinsed well with distilled water for 10 min. Then, the cell nuclei were lightly stained with eosin staining solution for 30 seconds, and washed with tap water for 5 seconds. The staining of the cells was observed under an inverted phase contrast microscope.
5. Magnetic resonance imaging of cellular uptake
To evaluate the targeting effect of FPD in vitro, Fe was set3O4FP and FPD groups. Different groups of 200. mu.L of nano-particles (2mg/mL) were added and incubated for 2 h. After incubation, cells were harvested using a cell scraper. PBS was washed, centrifuged (800rpm/500min), resuspended in PBS and MR imaged.
(1) Taking a bottle of 4T1 cells in logarithmic phase, sucking the original culture medium with a straw, washing twice with sterile PBS, adding 1mL of EDTA-pancreatin digestive juice, and shaking the culture bottle lightly to make the pancreatin digestive juice fully infiltrate the cells in the bottle. Meanwhile, when the cells were retracted and rounded to a quicksand shape, 2mL of serum-containing medium was added and gently blown with a pipette to completely detach the cells. The cell suspension was transferred to a 15mL centrifuge tube at 800rpm/5 min. Removing supernatant, adding 1mL of culture medium, blowing into cell suspension, uniformly subpackaging in three culture bottles, observing cell growth condition under an inverted microscope, normally changing liquid, and carrying out next experiment when the cell growth reaches about 80%.
(2) The old medium in the three flasks was aspirated off with a pipette, washed 2 times with sterile PBS, and 200. mu.L (2mg/mL) of Fe was added3O4FP and FPD and 4mL 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin), flasks were placed at 37 ℃ in 5% CO2Culturing the cells in the incubator for 4 hours, and observing the adherent growth condition of the cells by using an inverted microscope.
(3) The old medium was aspirated off with a pipette, washed twice with sterile PBS, cells were collected with a cell scraper into an EP tube, resuspended in cell suspension by adding 1mL PBS, and prepared for magnetic resonance imaging.
(4) At a 3.0T magnetic resonance imaging apparatus (TR 4240 ms; TE 108 ms; slice thickness 1 mm; slice spacing 1 mm; matrix, 256 × 256; FOV, 8cm × 8 cm).
6. Electron microscopy detection of Fe uptake by cells
To observe cellular uptake and retention of different groups of nanomaterials, 4T1 cells were placed at 37 ℃ in 5% CO2In a constant temperature incubator and containing Fe3O41640 complete medium from FP, FPD for 1,4 and 24 hours. Cells were collected with a cell scraper and observed by TEM for uptake of nanomaterials by cells. Observed under a JEM-1400 transmission electron microscope (JEOL, Japen). The cell uptake maps at different times were taken by an 830.10U3 specific imaging system (CCD camera) (Caltan USA).
7. In vitro curative effect detection experiment of FPD
(1) Hoechst33342/PI double staining experiment
4T1 cells were seeded in 24-well plates of cell-slide.
② the cells were divided into 4 groups: fe3O4Group (d); FP group; FD group; FPD group. Adding 200 μ L of nano material (2mg/mL) with the same concentration according to different groups, adding 4mL of 1640 complete culture medium (containing 10% FBS) prepared according to the proportion, and culturing for 4 h.
Taking out the co-incubated cells of different groups, sucking the old culture medium by using a suction pipe, and washing the cells for 3 times by using sterile cold PBS. According to the kit instruction steps, Hoechst33342 and PI dye are added in sequence, then the mixture is placed in a refrigerator at 4 ℃ for reaction for 15min, and the reaction is stopped by cold PBS.
And fourthly, observing the apoptosis and death condition of the cells by using red and ultraviolet channels of an inverted fluorescence microscope.
(2) Dyeing experiment with active oxygen
This study used DCFH-DA fluorescent probe for intracellular reactive oxygen species staining.
Firstly, 1-10 mM DMSO stock solution is prepared. Unused DMSO stock solutions should be dispensed into disposable EP vials and stored at 20 ℃ (protected from light).
② the working concentration of the dye is 1-10 uM in physiological buffer solution (such as PBS, HBSS, HEPES).
③ 4T1 cells in logarithmic growth phase were seeded in 6-well plates (5X 10) in which cell slides had been previously placed5Each well) and cultured overnight for their adherence.
Fourthly, 4T1 cells which are adhered to the wall and have climbed the plate are mixed with nano materials (Fe) of different groups3O4Group (d); FP group; FD group; FPD group), then adding a dye working solution to the cells, and incubating the cells at room temperature or 37 ℃ for 50-60 min.
Fifthly, removing the dye working solution; wash 3 times with pre-warmed PBS.
Sixthly, photographing and observing under a fluorescence confocal microscope.
IV, PD-L1@ Fe3O4-in vitro imaging of DOX nanospheres and results of efficacy studies
1. Immunofluorescence assay for cellular PD-L1
In the immunofluorescence experiment, the specific combination of a fluorescence labeled antibody and a target antigen is utilized, and the fluorescence of the antigen is photographed through a fluorescence microscope, so that the positioning and expression conditions of the target antigen in cells can be known, and the feasibility of a target spot is verified. Considering targeting of FPD, expression of PD-L1 in breast cancer 4T1 cells was analyzed using immunofluorescence staining. As shown in FIG. 5, the results of immunofluorescence experiments show that PD-L1 is expressed in the cell membrane and cytoplasm of breast cancer 4T1 (qualitative data), the length of the scale in the figure is 5 μm, and the fact that PD-L1 is feasible as a target of nano active targeting of tumor cells is verified.
2. Cell uptake assay
And (3) observing the way and action time of the 4T1 cells for taking up the nano material through TEM detection, and detecting the Fe taking-up amount of the cells through ICP-MS so as to further evaluate the in-vivo measurement and taking-up process of the nano material of different groups.
(1) Cell uptake electron microscope detection experiment
As shown in fig. 6, the TEM images show that each set of nanomaterials showed a range of properties, with increasing time,the amount of nanospheres taken up by the cells in each group is obviously more than 1h at 4h, as shown in fig. 6a and b, so that certain time dependence is achieved. After the nano materials and cells of different groups are cultured for about 1h, the nano materials can enter the cells and are mainly phagocytized by lysosomes through a TEM (transmission electron microscope) observation. Further, Fe3O4The degrees of uptake of FP, FD and FPD by cells are also obviously different, as shown in the figure, the uptake of the FPD group to the nano material is obviously greater than that of other groups, which shows that the active targeting effect of PD-L1 enables the nano material to reach the position of the target more accurately and efficiently, and the efficiency is greatly improved compared with that of other groups of EPR passive targeting. Furthermore, at 24h, little accumulation of nanomaterial was found in 4T1 cells (fig. 6c), indicating that the nanomaterial had been substantially expelled from the cells at this point.
(2) Prussian blue assay for cellular uptake
To observe the in vitro absorption properties of these nanoparticles, prussian blue staining experiments were performed on 4T1 cells. As shown in FIG. 7a, cellular uptake of Fe was shown as blue staining, with the highest uptake (shown as darkened gray) in the FP group (40. mu.g/mL), followed by FP (20. mu.g/mL), FP (10. mu.g/mL), Fe3O4(40μg/mL)、Fe3O4(20. mu.g/mL) and Fe3O4(10. mu.g/mL). For quantitative analysis of the uptake of material by the cells, the uptake of Fe by the cells was subsequently detected using ICP-MS.
(3) ICP-MS detection experiment for cell uptake
The uptake of the nanomaterials of the different groups by the cells was determined by ICP-MS. Fe in 4T1 cells after addition of nanosphere concentrations of 10, 20 and 40 μ g/mL3O4The Fe contents of group and FPD group were 7.12 + -1.13 and 13.95 + -2.88, 8.15 + -2.14 and 27.95 + -3.00, 8.55 + -2.05 and 40.55 + -8.97, respectively (FIG. 7 b). The results show that the FPD group takes up Fe in an amount significantly higher than Fe3O4Group (P)<0.001)。
(4) Magnetic resonance imaging of cellular uptake
The magnetic resonance imaging result of 4T1 cells treated by different groups of nanospheres is shown in FIG. 8a, and FP group is thinThe signal intensity of T2 of the cell is obviously lower than that of Fe3O4And (4) grouping. As the FP concentration increased, the signal intensity decreased. In addition, the signal-to-noise ratio of the signal intensity of T2 was quantified (FIG. 8b), and Fe was found at a concentration of 100. mu.g/mL3O4The difference between the group (-10.12 + -1.21) and FP group (-17.35 + -2.85) is statistically significant (P)<0.05); at a concentration of 200. mu.g/mL, the FP group had a further decrease in signal intensity (-27.12. + -. 3.35).
3. In vitro therapeutic efficacy detection of FPD
(1) Hoechst33342/PI double staining experiment
In order to explore the curative effect of FPD on the in vitro treatment of tumor cells, different groups of nano materials (Fe)3O4FP, FD and FPD) and breast cancer 4T1 cells were co-incubated for 6h, and then Hoechst33342/PI fluorescence double staining was performed according to the kit instructions, thereby obtaining fluorescence images of 4T1 cells. After treatment with Hoechst33342/PI, normal cells stained a uniform weak blue, whereas apoptotic cells appeared as bright blue fluorescence with condensation of the nucleus. At the same time, the necrotic cells appear as bright red fluorescence. In FIG. 9a, Fe3O4The cells of the group (survival rate 95-97%) and the FP group (survival rate 95-97%) showed weak blue fluorescence (gray scale image shows low brightness, small number of detected particles), and the cell survival rate between the two groups was not significantly different (P)>0.05) (FIG. 9b), which also confirms Fe3O4The biological safety is better without obvious influence on the cell activity. While the FD group and the FPD group showed bright red, bright blue fluorescence (the gray scale pattern showed high brightness and the number of particles detected was large), and the proportion of viable cells (survival rate 30% to 50%) of the FPD group was significantly less than that of the FD group (survival rate 40% to 55%), with a significant statistical difference between the two groups (fig. 9 b). The results of Hoechst33342/PI fluorescence double-staining show that FD and FPD have obvious killing effect on tumor cells, and the killing effect of the target group FPD is obviously higher than that of the non-target group FD, so that the tumor cells can be killed to a greater extent.
(2) Dyeing experiment with active oxygen
To further quantify the therapeutic efficacy of DOX in vitro, ROS staining was performed using DCFH-DA probes as per the instructions, followed by fluorescent visualizationCells were observed with a microscope. The intensity of the fluorescence signal with an emission wavelength of 488 was quantified over the same range of fields. The results show that Fe3O4No significant ROS-positive cells were seen under the group and FP group mirror (fig. 9c), indicating that neither group of materials caused significant damage to 4T1 cells. The ROS fluorescence signal intensity observed under the nano material mirror of the FD group and the FPD group has obvious difference (P) between the groups<0.05), FPD ROS signal intensity (higher than FD group (fig. 9d, fold change of 3.200 ± 0.5657 compared to control), further demonstrates that the targeting effect of PD-L1 improves the efficiency of the material to target 4T1 tumor cells.
V, PD-L1@ Fe3O4Method for researching immune combination chemotherapy of DOX nanospheres in breast cancer mouse model
1. Experimental reagent and consumable
10% pentobarbital, 10% paraformaldehyde: qilu hospital, shandong university, denna; eosin, hematoxylin staining solution: solarbio corporation, beijing; 4T1 cells: zhongqiao new boat biotechnology limited, shanghai; 1640 medium, fetal bovine serum, 0.25% trypsin (containing 0.02% EDTA): gibio corporation, USA; 75% medical alcohol: beijing, Zhenyu, Ministry of health and drug industry, Beijing; PD-L1 antibody (clone 10 f.9g2)): bioxcell corporation, USA; anti-tissue embedding cassette, slide, coverslip: jiangsu Shitai, Inc., Nantong; pathological section knife: featurer corporation, japan; mouse tail vein fixer: jade research scientific instruments ltd, shanghai; TUNEL kit, DAPI: bi yun tian biotechnology limited, shanghai; IV collagenase: force sensitive industries, ltd, shanghai; prussian blue staining kit: solebao technologies, Inc., Beijing; PCR reverse transcription and amplification kit: total gold Biotechnology, Inc., Beijing.
2. Cell culture and general handling (see above for methods of cell culture in "cytotoxicity assay procedure")
3. Construction and grouping treatment of 4T1 colorectal cancer mouse model
(1) Feeding of mice
25 male Balb/c mice of SPF grade 4-5 weeks old, the weight is about 20g, purchased from Beijing Huafu laboratory animal technology Limited company, bred in animal laboratory rat room in Qilu hospital, and bred under the following conditions: temperature 22 deg.C, relative humidity 50%, and light and dark alternating each day for 12 hr, supplying SPF feed and drinking water, changing bedding material 2 times per week, and weighing once every 3 days.
(2) Tumor-bearing mouse model construction
And (3) cell planting: balb/c male mice, 4-5 weeks old, were raised for about 1 week (about 20 g) and inoculated with cells after acclimatization to the feeding environment. The right back hair of the mice was first shaved with a shaver. Digesting and centrifuging the cells in advance, blowing the cells into cell suspension by PBS, collecting the cell suspension in a 15mL centrifuge tube, uniformly blowing the cell suspension of the centrifuge tube by a 1mL syringe, sucking all the cell suspension, and performing cell suspension treatment according to the proportion of about 1 × 107The cell suspension of 200uL was subcutaneously inoculated in the right back of mice (care was taken not to insert too deep to inject into muscle tissue), and a local apparent skin dome appeared at the injection site after injection.
Tumor formation in model mice: observing the growth condition of the tumor of the model mouse, weighing by an electronic balance every 3 days and recording the weight of the white mouse; the length and length of the tumor were measured and recorded with a vernier caliper, and the tumor volume was calculated (volume ═ long diameter × short diameter)2) 2), when the tumor volume reaches 80mm3The time indicates that the model is successfully constructed (about 5-7 days), and subsequent animal experiments can be carried out.
(3) Grouping, numbering and treatment scheme of model mice
Each mouse was divided into 5 groups of 5 mice each on a random basis. The components are respectively (1) PBS component and (2) Fe3O4Group, (3) FP group, (4) FD group, (5) FPD group. The injection dose of each group of the nano materials and PBS is 5 ml/kg. Model mice were numbered. 5 groups are respectively marked by paint marker pens (red, yellow, blue, black and green) with 5 colors, and 5 mice in each group are respectively marked on a left key, a right shoulder, a left back leg, a right back leg and a head.
The treatment scheme comprises the following steps:
(1) PBS group: tail vein injection of the same dose of PBS only;
(2)Fe3O4group (2): tail vein injection of same dose of Fe3O4A nanomaterial;
(3) FP group: injecting the FP nanometer material with the same dosage into the tail vein;
(4) group FD: injecting FD nanometer materials with the same dosage into tail vein;
(5) FPD group: the same dose of FPD nanomaterial was injected into the tail vein.
The body weight and tumor volume of the mice were measured every three days, the mice were injected with different groups of nanomaterials or equivalent amounts of PBS, magnetic resonance imaging was performed on each group of mice, and the survival status of the mice was recorded. When the tumor volume reaches 2500mm3Or when suffering symptoms such as food refusal, obvious weight loss, sinking elimination and the like are presented, the mouse is killed by dislocation of the cervical vertebra due to humanism, data of death of the cervical vertebra on a day and natural death of the mouse are recorded, and growth curves of all groups are drawn.
4. Targeted imaging studies of FPD in mice
In vivo magnetic resonance imaging to study tumor targeting effects of FPD
(1) FPD group and Fe3O4The group nano materials are respectively injected into mice through tail vein, each group is 100 mul, and each group is provided with 5 nano materials.
(2) Anesthesia: before scanning, 100uL of 1% sodium pentobarbital is injected into the abdominal cavity of a model mouse, after the mouse is successfully anesthetized, the prone position of the mouse is placed in a magnetic resonance imaging animal coil, and isoflurane is used for maintaining anesthesia.
(3) Positioning: the tumor with the back of the mouse is positioned in the center of the coil, a positioning line is drawn, and after the positioning is successful, the mouse enters the bed to the scanning position of 3.0T MRI of GE company to start scanning.
5. Biological safety of FPD
To investigate make internal disorder or usurp that the above treatment has no toxicity to the vital tissue organs of the organism, 3 mice were sacrificed on days 0, 5, 10, and 15 of the treatment, and the four major organs of the heart, liver, spleen, and kidney were subjected to HE staining to observe whether the tissues were damaged or not.
Conventional HE staining:
(1) baking the slices overnight, hydrating, and sequentially dewaxing in xylene for 2 times and 10min each;
(2) then washing with anhydrous ethanol for 5min, 95% ethanol for 5min, 90% ethanol for 5min, 80% ethanol for 2min, 75% ethanol for 2min, and distilled water for 3 min;
(3) immersing in hematoxylin staining solution for 10-15 min;
(4) differentiating with 1% hydrochloric acid ethanol for several seconds, and washing with tap water for 5 min;
(5) staining with eosin for 3-5min, and washing with tap water for 5 min;
(6) 80% ethanol for 5min, 95% ethanol for 5min respectively for 2 times, and anhydrous ethanol for 5min respectively for 2 times;
(7) the xylene is transparent for 3 times and each time for 2 min;
(8) the neutral gum was encapsulated and observed under an optical microscope.
6. In vivo toxicity test of FPD
(1) TUNEL staining of tissue
To further investigate make internal disorder or usurp the in vivo efficacy of the above treatments, mice treated with different treatment modalities were decapped, tumor tissue sections and major organs were incubated with proteinase K solution for 15min, followed by co-incubation with TUNEL reaction mixture. After three PBS washes, the slides were mounted with blocking solution containing the nuclear dye DAPI and observed with confocal laser fluorescence microscopy.
The method comprises the following specific steps:
1. soaking and washing with xylene for 5min for 2 times;
2. soaking and washing with gradient ethanol (100%, 95%, 90%, 80.70%) for 3min for 1 time;
3. treating the tissue with a protease K working solution for 15-30 min at 21-37 ℃ (temperature, time and concentration need to be searched) or adding cell permeation solution for 8 min;
4, rinsing with PBS for 2 times;
5. preparing TUNEL reaction mixed solution, and uniformly mixing the treatment group with 50uL of TdT +450uL of fluorescein labeled dUTP solution; and only 50uL of fluorescein-labeled dUTP solution is added into the negative control group, 100uLDNase 1 is added into the positive control group, the reaction is carried out at 15-25 ℃ for x10 min, and the subsequent steps are the same as those of the treatment group.
6. After the slide is dry, 50uL TUNEL reaction mixed solution (only 50uL fluorescein labeled dUTP solution is added in the negative control group) is added on the specimen, and the specimen is covered with a cover glass or a sealing film and reacted in a dark and wet box at 37 ℃ for 1 h;
rinsing with PBS 3 times;
8.1 drop of PBS can be added to count the apoptotic cells under a fluorescence microscope (the wavelength of the excitation light is 450-500 nm, and the detection wavelength is 515-565 nm);
9. adding 50uL coverer-POD on the specimen after the slide is dry, covering the slide or a sealing film, and reacting for 30min at 37 ℃ in a dark and wet box;
rinsing with PBS 3 times;
11. adding 50-100 uL DAB substrate to the tissue, and reacting for 10min at 15-25 ℃;
rinsing with PBS 3 times;
after taking the picture, counterstaining is carried out again by hematoxylin or methyl green, and the picture is rinsed by tap water immediately after a few seconds. Gradient alcohol dehydration, xylene transparency, neutral gum sealing. And (4) observing by using an optical microscope.
7. PCR detection of tissue apoptosis
Real-time fluorescent quantitative PCR is a method of measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle in DNA amplification reaction using fluorescent chemicals. A method for quantitatively analyzing a specific DNA sequence in a sample to be detected by an internal reference method or an external reference method. The research adopts a PCR method to detect the apoptosis conditions of main organs such as mouse tumor, heart, liver, spleen, kidney and the like which are treated differently by detecting three apoptosis-related genes, Caspase3, Bax and Bcl-2.
(1) Cell/tissue lysis:
the mouse tissues and heart, liver, spleen, kidney were ground by shearing and then lysed. Rapidly adding each group of different tissues into a 1.5mL centrifuge tube, adding 1mL lysate (Trizol), crushing by using a tissue crusher, repeatedly blowing by using a pipette gun until no obvious precipitate is formed in the lysate, and standing for 2min at room temperature.
(2) Centrifuge at 12000rpm for 5min, and discard the precipitate.
(3) Chloroform was added to 200uL of chloroform/mL of Trizo1, and the mixture was shaken and mixed well, followed by standing at room temperature for 15 min.
(4) Centrifuge at 12,000g for 15min at 4 ℃.
(5) The upper aqueous phase was aspirated into another centrifuge tube.
(6) Adding 0.5mL of isopropanol/mL of Trizol into isopropanol (precipitated RNA), mixing uniformly, and standing at room temperature for 5-10 min.
(7) Centrifugation at 12,000g for 10min at 4 ℃ removed the supernatant and RNA deposited at the bottom of the tube.
(8) Add 75% ethanol (precipitated RNA) to 1mL of 75% ethanol/mL Trizol, gently shake the centrifuge tube and suspend the precipitate.
(9) Centrifuge at 8,000g for 5min at 4 ℃ and discard the supernatant as much as possible.
(10) And (5) drying at room temperature or in vacuum for 5-10 min.
(11) Dissolving the RNA sample by 50uL H2O, and carrying out reaction at 55-60 ℃ for 5-10 min.
(12) The OD was measured to quantify the RNA concentration.
(13) Experiments for DNA removal and reverse transcription were performed according to the procedure of the Takara reverse transcription kit.
(14) The Real-Time fluorescent quantitative PCR was performed according to the procedure of Takara PCR kit, using an Applied Biosystems 7300/7500Real Time PCR System, with the primer sequences shown in Table 1.
(15) And (3) after the reaction is finished, confirming an amplification curve and a melting curve of Real Time PCR, and carrying out PCR quantitative application on the 2-delta Ct method.
8. PD-L1 immunofluorescence of tissue
To further verify the feasibility of PD-L1 as a target, immunofluorescence experiments were also performed with PD-L1 in tumor tissues of tumor-bearing 4T1 mice.
The method comprises the following specific steps:
baking slices: 1h at 37 ℃ and taking the anti-falling tablets. PBS was washed 3 times for 5min each. The glue on the upper surface is wiped clean by toilet paper.
The following procedure refers to the PD-L1 immunofluorescence assay of the third fraction of cells.
9. Prussian blue staining of tissue absorbing nanomaterials
In order to further verify the difference of in vivo absorption of different groups of nanomaterials, mouse tumor tissues were also subjected to prussian blue staining.
The specific experimental procedure refers to the prussian blue staining experiment of the third part of cells.
10. In vivo immunotherapy efficacy of FPD
To examine the efficacy of FPD treatment in vivo, flow experiments were performed on tumor tissues of mice by detecting CD3+CD8+The number of T cells before and after different treatment modes is different, so that the difference of the curative effect of different treatment modes is determined.
(1) Single cell suspensions were prepared as per flow sample requirements: the tumor tissue of a mouse is cut into pieces which are as small as possible, the pieces are respectively placed into six-hole plates according to different groups, IV-type collagenase enough to cover the pieces is added, and the six-hole plates are placed into a 37-degree cell incubator to be fully digested (the tissue can be observed to be digested into single cell suspension under a microscope).
(2) The binding buffer was diluted 1:4 with deionized water (4mL binding buffer +12mL deionized water).
(3) Cells were washed twice with 4 ℃ pre-chilled PBS, resuspended in 250uL binding buffer, and adjusted to a concentration of 1X 106/mL。
(4) mu.L of cell suspension was put in a 5mL flow tube, and 5. mu.L of anti-CD3 antibody diluent and anti-CD8 antibody diluent were added.
(5) After mixing, incubation is carried out for 15min at room temperature in the dark.
(6) And (4) resuspending a proper amount of flow type staining solution, and performing on-machine detection. The peer-to-peer comparison was performed according to the procedure described above. And (5) analyzing the result by software.
VI, PD-L1@ Fe3O4Results of studies of DOX nanosphere immunization in combination with chemotherapy in a mouse model of breast cancer
1. Mouse weight, survival curve and tumor volume
The therapeutic protocol roadmap for mice is shown in FIG. 10, which is a 1X 10 map7A BALB/c mouse right shoulder is inoculated with 4T1 cells, and a mouse breast cancer model is established. Tumor-bearing mice were divided into 5 groups and treated with different nanomaterials and PBS for 15 days, respectively.
The images of the treated mice and tumors are shown in FIG. 11 a. The length and the short diameter of the tumor were measured with a vernier caliper every 3 days after the administration of each group of mice, and the volume (mm) was calculated according to the formula3) Tumor volume was calculated as 0.5 × long diameter × short diameter. The results are shown in FIG. 11b, in PBS group, Fe3O4The tumor volumes of the mice in the group, FP group and FPD group are increased along with the change of observation time. FPD group tumors were minimal in volume (186.4 ± 38.0 mm) on day 153) Statistical differences (P) compared to the other groups<0.05)。FD(1708.6±17.6mm3) Group, FP (1935.2 + -80.7 mm)3) In the following order, with Fe3O4Group (1962.2 + -186.6 mm)3) And PBS group (2141.7. + -. 108.3 mm)3) The tumor volume was maximal.
To observe the subsequent efficacy of the different treatment groups, survival curves were plotted (fig. 11 c). Therefore, the survival rate of the FPD group mouse is higher than that of other treatment groups, the tumor growth can be obviously inhibited, and the survival days of the mouse can be prolonged. At day 40, the survival rates of mice in the FPD group and the FD group are respectively 60-65% and 55-65%, and no statistical significance exists between the two groups; by day 60, the survival rate of FPD group mice was 35%, and the survival rate of other groups of mice was 0. In addition, the body weights of the mice in each group were also recorded, and no significant difference was seen between the groups as shown in fig. 11 d.
2. Targeted imaging of FPD in mice
In order to further research the in-vivo tumor magnetic resonance targeted imaging capability of the FPD nano material, the subcutaneous breast cancer model of the 4T1 mouse is injected with FPD and Fe intravenously3O4At various time points (1, 4, 8, 24h) within the last 24 hours, T2WI scan images were obtained. As shown in FIGS. 12a and b, MRI showed Fe at the tumor site3O4And FPD both at 4h after probe injection (Fe)3O4Group (2): -42.1 ± 2.8; FPD group: 65.5. + -. 3.3) reached the minimum, but a more significant decrease in the MRI signal (P) was detectable in the tumor area 4h after FPD injection<0.001), both groups of tumors gradually returned to the initial state (Fe) 24h after probe injection3O4Group (2): -26.3 ± 2.0; FPD group: -33.5 ± 1.8). The results are consistent with the results of early cell imaging, and support that FPD can specifically target tumors, and the accumulation ratio of FPD to Fe at tumor sites3O4More, can be used as a good probe for the magnetic resonance imaging of the living body, and improves the accuracy of the biomedical imaging.
3. Biological safety of FPD
In general, nanoparticles having high therapeutic effects and low toxicity are more suitable for tumor treatment. In this example and comparative examples, mouse body weight was monitored as a systemic toxicity index and dynamically evaluated over a 15 day treatment period. Mice in the different treatment groups did not show significant weight loss or abnormal behavior. In order to further research whether the treatment has no toxicity on important tissues and organs, four time points are taken in the treatment process to carry out tissue HE staining on heart, liver, spleen, kidney and other main organs of the FPD group mice to detect whether the FPD group mice are damaged or not, and the mice before treatment are used as a control. As shown in fig. 13. The organ tissue sections of the mice among the treatment groups have no obvious difference with the organ tissue sections of the normal mice, and no obvious tissue damage or toxic effect such as tissue necrosis, fibrosis and the like is found.
4. FPD exerts therapeutic effects by inducing apoptosis
Experimental TUNEL staining was used to label apoptotic cells. TUNEL staining of tumor tissue in mice treated with different treatment regimens is shown in FIG. 14a, with PBS (13.7. + -. 5.8) and Fe in each field of view3O4No positive cells were evident in any of the groups (27. + -. 12.9), only a small number of positive cells were seen in the FP group (57.6. + -. 18.9), the number of TUNEL staining positive cells was the largest in the FPD group (234.1. + -. 32.8), and the number of positive cells was found in the FD group (176.6. + -. 32.2), as shown in FIG. 14 b.
To further examine the damage of each group of nanomaterials to the major organs of mice, TUNEL staining was performed. As shown in FIG. 15a, Fe3O4No obvious damage was observed to the major organs, indicating Fe3O4The nano material has no obvious toxicity to biological tissues; the damage of the main organs of the FD group is obviously larger than that of the FPD group, and the difference has statistical significance (P)<0.05) (fig. 15b), the results show that under the active targeting effect of PD-L1, the efficiency of the material reaching tumor cells is greatly improved, so that the tumor cells can reach the target site more accurately, and the damage of DOX to normal tissues and organs is significantly reduced, which is also very important in the research.
In addition, three apoptosis related genes (Bax, Bcl-2 and Caspase-3) of the tumor and main organs are detected by a real-time fluorescence quantitative PCR method, and the result is obtainedConsistent with the previous TUNEL experiment, for tumor tissues, Fe3O4The expression level of apoptosis-related genes was lower, and the expression level of FD group apoptosis genes was lower than that of FPD group (fig. 16). For the major organs, as shown in fig. 15a, the apoptosis of FD group heart is especially severe, and it can be seen that DOX has significant cardiotoxicity, but the apoptosis of FPD group heart and other organs is significantly reduced, again demonstrating that the active targeting of FPD greatly reduces the killing of DOX to major organ tissues, thereby protecting normal tissues outside the target.
5. FPD exerts therapeutic effects by immunotherapy
(1) Immunofluorescence of tumor tissue PD-L1
In order to detect the expression of PD-L1 in tumor tissues, immunofluorescence staining of tissue sections is carried out, and the result shows that PD-L1 in 4T1 tumor tissues is also widely expressed (FIG. 17a), which is consistent with the expression of the PD-L1 of the previous cells, and the feasibility of taking PD-L1 as a target point is verified again.
(2) Prussian blue staining of tumor tissue
To further examine the uptake of material by tumor tissue, prussian blue staining (fig. 17b) was performed on tumor tissue sections, Fe3O4The group (left in FIG. 17b) is significantly different from the FPD group (right in FIG. 17b), Fe3O4No obvious blue stain was observed in the tumor tissues of the FPD group, and a large amount of material was absorbed in the tumor tissues of the FPD group. The result shows that the active targeting material can reach the tumor more accurately and has good killing effect on the tumor.
(3) CD3 of tumor tissue+CD8+T cell assay
To examine the efficacy of in vivo immunotherapy with FPD, tumor tissue was labeled CD3+CD8+Flow assay of T cells. The results are shown in FIG. 18a, CD3 in tumor tissue of FD group (10% -15%) mice after treatment+CD8+T cells, as compared with Fe3O4The groups (9-12%) had significant increase, indicating that chemotherapy had a certain effect on promoting immune system activation, and the quantitative figure indicates that the differences between groups had statistical significance (P)<0.05) (fig. 18 b). Is treated by FPDCD3 in post-treatment mouse tumor tissue+CD8+The increase of T cells (19-28%) is more obvious, and the result shows that on one hand, the active targeting effect of PD-L1 improves the utilization rate of materials, and on the other hand, the immunotherapy and the chemotherapy of PD-L1 further improve the curative effect under the combined and synergistic effect, thereby achieving the effect of twice the result with half the effort.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A targeted drug nano system is characterized in that ferroferric oxide is used as a carrier, and immunotherapy drugs and chemotherapy drugs are surface-modified.
2. The targeted drug nanosystem of claim 1, wherein the chemotherapeutic is selected from one or more of daunorubicin, doxorubicin, mitoxantrone, paclitaxel.
3. The targeted drug nanosystem of claim 1, wherein the immunotherapeutic drug is selected from one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody.
4. The method for preparing the targeted drug nanosystem of any one of claims 1 to 3, comprising: mixing ferroferric oxide modified by 2, 3-dimercaptosuccinic acid with immunotherapy drugs and chemotherapy drugs.
5. The preparation method of the targeted drug nano system according to claim 4, characterized by dissolving 2, 3-dimercaptosuccinic acid in deionized water, adding ferroferric oxide, adding tetrahydrofuran, performing ultrasonic treatment, and reacting to obtain ferroferric oxide modified by 2, 3-dimercaptosuccinic acid;
dispersing ferroferric oxide modified by 2, 3-dimercaptosuccinic acid in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and sulfo-N-hydroxysuccinic acid, adding an immunotherapy medicament and a chemotherapeutic medicament, and reacting at low temperature overnight to obtain the product.
6. The preparation method of the targeted drug nano system according to claim 4, wherein the mass ratio of the 2, 3-dimercaptosuccinic acid to the ferroferric oxide is 1.5-4: 1.
7. Use of the targeted drug nanosystem of any one of claims 1 to 3 for the preparation of a medicament for breast cancer.
8. A T2WI negative contrast agent, comprising a targeted drug nanosystem of any one of claims 1 to 3.
9. Use of the targeted drug nanosystems of any one of claims 1 to 3 for the preparation of a T2WI negative contrast agent.
10. Use of the targeted drug nanosystems of any of claims 1 to 3 for MRI real-time monitoring.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115068630A (en) * 2022-08-24 2022-09-20 山东大学齐鲁医院 Preparation method of temperature-sensitive controlled-release ferroferric oxide/PD-L1 monoclonal antibody modified nanoparticles

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* Cited by examiner, † Cited by third party
Title
赵洁: "PD--L1靶向Fe3O4@DOX多功能纳米体系在三阴性乳腺癌联合治疗及MRI实时监控中的价值研究", 万方数据 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115068630A (en) * 2022-08-24 2022-09-20 山东大学齐鲁医院 Preparation method of temperature-sensitive controlled-release ferroferric oxide/PD-L1 monoclonal antibody modified nanoparticles

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