CN111298139B

CN111298139B - Nuclear magnetic imaging guidance-based targeted nano-drug for overcoming drug resistance caused by tumor hypoxia and preparation method and application thereof

Info

Publication number: CN111298139B
Application number: CN201911227246.1A
Authority: CN
Inventors: 陈填烽; 林伟强; 曾钦松; 刘宏星
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2022-07-12
Anticipated expiration: 2039-12-04
Also published as: CN111298139A

Abstract

The invention discloses a targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, and a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method of the targeted nano-drug comprises the following steps: s1, respectively dispersing PLGA, a nuclear magnetic imaging contrast agent and an anti-tumor drug in an organic solvent, then adding a surfactant, and stirring and dialyzing to obtain a solution A; s2, activating the carboxyl of the solution A by using NHS and EDC to obtain an activated solution A; and S3, adding a targeting molecule into the activated solution A, and stirring and dialyzing to obtain the targeted nano-medicament for overcoming the medicament resistance caused by tumor hypoxia based on the guidance of nuclear magnetic imaging. The treatment effect of the targeted nano-drug PLZ4@ SeD nano-particle is superior to that of clinical drugs such as doxorubicin, mitomycin, pirarubicin and the like under the condition of cell hypoxia.

Description

Nuclear magnetic imaging guidance-based targeted nano-drug for overcoming drug resistance caused by tumor hypoxia and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a targeted nano-medicament for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, and a preparation method and application thereof.

Background

Bladder cancer (BCa) is the most common urinary system tumor in China, and according to statistics, about 55 ten thousand new cases occur in 2018 worldwide. In the conventional treatment scheme of bladder cancer, after transurethral cystectomy (TURBT) of non-muscle-layer invasive BCa (NMIBC) and radical total cystectomy of muscle-layer invasive BCa (MIBC), the drug treatment is an important component part of bladder perfusion treatment, systemic chemotherapy, local intervention chemotherapy and the like after TUR operation, however, in the using process of the anti-BCa drug, the problems of poor perfusion drug sensitivity, unstable function, high cost, large toxic and side effects and the like still exist. In recent thirty years, the curative effect of the medicine for resisting the advanced BCa is not obviously improved, and particularly, effective medicine treatment is not available for patients who progress after first-line chemotherapy; whether immunotherapy, represented by expensive PD-1/L1 inhibitors, is superior to conventional chemotherapy, particularly in terms of survival benefit, remains to be demonstrated with future data. Therefore, the search for the anti-BCa medicament with high efficiency, low toxicity, stability and low price has very important clinical value and practical significance.

In the organism, selenium is an important active center of glutathione peroxidase (GSH-Px), iodothyronine, and mammalian thioredoxin reductase (TrxR). Since the first discovery of negative correlation between selenium and tumor prevalence over 40 years ago, selenium compounds have been verified by a great number of research institutes for their antitumor value, and their potential antitumor mechanisms include regulation of growth inhibitory effects of cancer suppressor genes, oxidation resistance, DNA damage, apoptosis pathways, and the like. Compared with inorganic selenium compound, the organic selenium compound has the characteristics of high absorption rate, strong biological activity, low toxicity and the like. At present, researchers synthesize a large number of organic selenium compounds with biological activity, including aromatic selenium compounds, diselenide, selenocyanide and the like, wherein the antioxidant drug ebselen and the antitumor drug selenazolofuran have been respectively subjected to three-phase and one-phase clinical research.

This group synthesized a selenium heterocyclic compound in earlier studies: organic selenium 1b (SeD-1b), molecular formula is C₂₀H₁₄ON₄Se is a selenadiazole derivative and is a dark yellow solid. SeD-1b is easily soluble in organic solvents such as DMSO and is hardly soluble in water. The early research results show that SeD-1b has high selectivity and activity for resisting bladder cancer, has high stability in urine environment, induces BCa cell apoptosis through ROS-mediated p53-AKT/MAPK signal pathway and is applied to normal bladderThe skin cell toxicity is low, and the medicine shows remarkable superiority compared with the traditional anti-BCa medicine. However, although SeD-1b is clearly superior to the conventional anti-BCa drugs, it also has the following disadvantages: 1. SeD-1b is a chemical small molecule drug, and is generally dispersed and adsorbed to the bladder cancer part in a free diffusion mode, however, the small molecule drug has short metabolic life, so that the SeD-1b has poor targeting property; 2. due to the special structure of the bladder, the medicine reaching the tumor part is easy to be diluted and removed by urine; 3. although SeD-1b has a certain stability in urine environment, the solubility still needs to be improved; 4. although SeD-1b has fluorescence, the light-emitting capability is weak, and the SeD-1b cannot be used as a diagnostic positioning agent and cannot be used for enhancing the identification capability of the diagnostic positioning agent in imaging examination, so that BCa cannot be served for monitoring the curative effect, and the pain of a patient suffering from cystoscopy is reduced. The above disadvantages limit the application of SeD-1b in anti-tumor.

Tumor Microenvironment (TME) refers to the structure, function, metabolism of the tissues and other relevant physicochemical indices of the internal environment of the tumor, such as low pH, during the development, growth and metastasis of the Tumor (TME)<7) Low oxygen partial pressure: (<1.3％)、H₂O₂High expression, etc. The hypoxic microenvironment in the tumor enables cancer cells to have tolerance to radiotherapy and chemical drug treatment, and the treatment effect of radiotherapy and chemotherapy is obviously reduced. The current clinical research aiming at the unique hypoxic microenvironment of the tumor shows that 80 percent of patients with bladder cancer have hypoxic states at the tumor parts, and the hypoxic microenvironment in the bladder cancer is one of the main factors influencing the chemotherapy effect and the prognosis of the patients. In order to improve the hypoxic environment inside the tumor, the prior art often adopts a method of delivering oxygen to the tumor tissue, such as a hyperbaric oxygen chamber, and the like, to increase the hypoxic environment inside the tumor.

However, no targeted nano-drug which can overcome the drug resistance problem caused by tumor hypoxia and can monitor the tumor treatment process in real time is available at present.

Disclosure of Invention

The application aims at providing a preparation method of a targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance.

Another objective of the present application is to provide a targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on mri guidance.

Still another objective of the present application is to provide the application of the targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on mri guidance in anti-tumor.

The above object of the present invention is achieved by the following technical solutions:

a preparation method of targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance comprises the following steps:

s1, respectively dispersing PLGA, a nuclear magnetic imaging contrast agent and an anti-tumor drug in an organic solvent, then adding a surfactant, and stirring and dialyzing to obtain a solution A;

s2, activating the carboxyl of the solution A by using NHS and EDC to obtain an activated solution A;

and S3, adding targeted molecules into the activated solution A, and stirring and dialyzing to obtain the targeted nano-medicament for overcoming the drug resistance caused by tumor hypoxia based on the guidance of nuclear magnetic imaging.

In step S1, the size of the PLGA is 100-140 nm; preferably 120 nm.

In step S1, the mri contrast agent is Fe₃O₄At least one of nanoparticles and gadopentetate meglumine injection; preferably Fe₃O₄Nanoparticles.

Said Fe₃O₄The particle size of the nano particles is 5-200 nm; preferably 5-50 nm; more preferably 10 nm.

In step S1, the anti-tumor drug is at least one of daunorubicin, doxorubicin, demethoxydaunorubicin, epirubicin, vinblastine, vincristine, tamoxifen, formestane, anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, tegafur, and selenadiazole derivative (SeD-1 b); the selenadiazole derivative (SeD-1b) is preferred.

SeD-1b is an antitumor drug synthesized by the subject group according to earlier research results, and the preparation method is disclosed in application No. 201610127128.3. On one hand, SeD-1b has hydrophobic property, so that SeD-1b can enter a hydrophobic cavity of the polymer nanoparticle, and the loading of the polymer carrier on the antitumor drug is realized. On the other hand, since SeD-1b has high selectivity and activity against bladder cancer and high stability in urine environment, BCa apoptosis can be induced by ROS-mediated p53-AKT/MAPK signaling pathway, and toxicity to normal bladder epithelial cells is low. Therefore, the SeD-1b and the PLGA polymer nano-particles are combined to ensure good anti-tumor activity and effectively inhibit tumor growth.

In step S1, the PLGA, the nuclear magnetic imaging contrast agent and the antitumor drug in the solution A are mixed according to the molar ratio of (1-40) to (1-10); preferably, the molar ratio of (20-40) to (1-2) is selected; more preferably in a molar ratio of 20:1.6: 1.

In step S1, the organic solvent is at least one of acetone, acetonitrile, and dichloromethane.

In step S1, the surfactant is a tween aqueous solution; in the Tween aqueous solution, the concentration of Tween is 1-10 mM; preferably 2 to 8 mM; more preferably 3.5 mM.

The Tween is at least one of Tween-80, Tween-81 and Tween-20; preferably tween-80.

In step S1, the organic solvent is added dropwise to the surfactant.

The dropping speed is 1-20 seconds per drop; preferably 10 to 15 seconds.

In the step S1, the stirring speed is 200-800 r/min, and the stirring time is 8-24 hours; preferably 12 hours.

In step S1, the dialysis is dialysis membrane dialysis.

The dialysis membrane equivalent is 300-10000 kDa; preferably 3000-10000 kDa; more preferably 10000 kDa.

In step S1, the mass ratio of the organic solvent to the surfactant is 1: 4.5-9; preferably 1: 9.

In step S2, the amounts of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and PLGA are as follows: EDC: the PLGA is mixed according to a molar ratio of 1:1: 1-40; preferably as NHS: EDC: the PLGA is mixed according to a molar ratio of 1:1: 20-40; more preferably as NHS: EDC: the molar ratio of PLGA to PLGA is 1:1: 20.

In step S2, stirring operation is required when the solution a is activated; the stirring operation refers to stirring at normal temperature; the stirring conditions are as follows: 200-800 r/min for 2-8 hours; preferably 3 hours.

In step S3, the targeting molecule is at least one of cyclic RGD polypeptide (cRGD), Folic Acid (FA), transferrin, Activatable Cell Penetrating Peptide (ACPP), MUC-1 annexin, galactosamine, neovascular targeting peptide, granulocyte-macrophage stimulating factor, and PLZ 4; preferably PLZ 4.

In the step S3, the addition amount of the targeting molecule is 0.0625-40 mg/mL; preferably 20 mg/mL.

In the step S3, the mass ratio of the activated solution A to the target molecules is 1-3: 1; preferably 1.5: 1.

In step S3, the stirring conditions are: 200-800 rpm, and 8-24 hours of stirring time; preferably 12 hours.

In step S3, the dialysis is dialysis membrane dialysis.

When the nuclear magnetic imaging contrast agent is Fe₃O₄When the nano particles, the anti-tumor drug are SeD-1b and the targeting molecule is PLZ4, the solution A is PLGA @ SeD/Fe₃O₄The nano particle water solution, and the finally obtained targeted nano medicament for overcoming the medicament resistance caused by tumor hypoxia based on the guidance of nuclear magnetic imaging is the targeted nano medicament PLZ4@ SeD nano particle.

In steps S1 to S3, the operation can be performed at normal temperature. The normal temperature is 15-35 ℃; more preferably 20 to 30 ℃.

A targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance is prepared by the preparation method.

The targeted nano-drug is stored in a sol form at 1-25 ℃.

The targeted nano-drug for overcoming the drug resistance caused by tumor hypoxia based on the guidance of nuclear magnetic imaging is applied to the preparation of the anti-tumor drug.

The tumor is at least one of bladder cancer, breast cancer and liver cancer.

The mechanism of the invention is as follows:

PLGA has good biocompatibility and is a clinical pharmaceutic adjuvant which passes the verification of the US FDA; the polymer chain can be connected with different targeting molecules to play a role in space stabilization; and the electrostatic stabilization effect can be achieved through the charges carried by the nano-particles, and the stability and the dispersity of various nano-particles can be effectively improved.

2. The targeting polypeptide PLZ4 is a cyclic peptide with bladder cancer cell specificity, and has the capability of targeting bladder cancer cells; PLZ4 has an amino group and can undergo a dehydration condensation reaction with the carboxyl group of activated PLGA to form an amide bond.

After entering cells through endocytosis, SeD-1b can effectively up-regulate the generation amount of Reactive Oxygen Species (ROS) in the cells so as to activate the phosphorylation of a cell apoptosis pathway p53 protein, thereby further inducing the apoptosis of bladder tumor cells and inhibiting the proliferation of the tumor cells.

4. Ferroferric oxide nanoparticles (Fe)₃O₄Nanoparticles) have superparamagnetism, can reduce T2 relaxation, and is an ideal Magnetic Resonance Imaging (MRI) contrast agent; in addition, Fe₃O₄The nanoparticles can catalyze H through Fenton reaction and Haber-Weiss reaction₂O₂Production of O₂Thereby improving hypoxic microenvironment in the tumor; further, Fe₃O₄The nano particles have good biocompatibility and almost no toxicity to normal cells and tissues. Thus, it can be seen that Fe₃O₄The nano-particle is a good contrast agent and a good catalystAn oxidizing agent. Fe₃O₄The nano particles enter a lipophilic core shell of the PLGA polymer nano particles through a hydrophobic effect, so that the loading of the polymer carrier on the nuclear magnetic imaging medicine is realized. At the same time, Fe₃O₄Nanoparticles, also known as T2 negative contrast agents, have excellent superparamagnetism, are widely considered as ideal contrast agents for Magnetic Resonance Imaging (MRI), and are one of the most sensitive MR contrast agents at present, and can significantly shorten T2 relaxation time, so that T2 weighted images become dark; in addition, Fe₃O₄The nano particles have the property of Fenton reaction and can catalyze and degrade H₂O₂。

5. By using the oil-in-water method, taking acetone as an internal oil phase and water as a continuous external phase, and mixing hydrophobic SeD-1b and Fe₃O₄Dispersing the nano particles in acetone, wrapping the nano particles in PLGA, and finally dispersing the nano particles in water to obtain the PLGA wrapped SeD-1b and Fe₃O₄The structure of the nanoparticles.

6. The nuclear magnetism-guided bladder cancer treatment of the targeted nano-drug is to make the SeD-1b wrapped by the PLGA reach the tumor region by utilizing the targeting capability of the PLZ4, and enhance Fe on the basis of the confinement effect of PLGA micelle₃O₄Catalysis of nanoparticles H₂O₂Ability to provide a large amount of O₂The tumor cell apoptosis inhibitor can improve hypoxic environment of tumor region, reduce drug resistance caused by tumor hypoxia, and improve apoptosis inducing ability of SeD-1b, thereby inhibiting proliferation of tumor cells.

Compared with the prior art, the invention has the following beneficial effects and advantages:

(1) the treatment effect of the targeted nano-drug PLZ4@ SeD nano-particle is superior to that of clinical drugs such as doxorubicin, mitomycin, pirarubicin and the like under the condition of cell hypoxia.

(2) In the present invention, Fe₃O₄The nanoparticles have catalytic H₂O₂Production of O₂After PLGA encapsulation, the integral catalytic capability is obviously enhanced due to the limited domain effect formed in the PLGA micelle, the capability of improving the hypoxic microenvironment of the tumor part is improved, and the tumor hypoxia caused by tumor hypoxia is reducedThe drug resistance of the compound can improve the effect of tumor treatment finally.

(3) The selectivity of PLZ4 on bladder cancer is better than that of other polypeptides, and the introduction of PLZ4 enables the targeting of the nanoparticles to be better and the toxic and side effects to be lower. The PLGA has better biocompatibility, simple synthesis method, and lower price and easy obtaining. Compared with the clinical mitomycin used for bladder cancer perfusion, the SeD-1b has higher safety and smaller toxic and side effects.

(4) The target nano-drug PLZ4@ SeD nano-particle prepared by the method not only overcomes the drug resistance problem caused by tumor hypoxia, but also can realize real-time monitoring on the tumor treatment process, and finally achieves the aim of treating the cancer with high efficiency, low toxicity, stability and low price.

Drawings

FIG. 1 is a graph showing the characterization results of the targeted nano-drug PLZ4@ SeD nanoparticles prepared in example 1; wherein, A is a transmission electron micrograph and a particle size measurement result chart of PLZ4@ SeD nano particles; b shows PLGA nanoparticles and Fe₃O₄Nanoparticle, PLGA @ SeD nanoparticle, and activated PLGA @ SeD/Fe₃O₄Potential measurement result graphs of the nanoparticle and PLZ4@ SeD nanoparticle; FIG. C is the ultraviolet absorption-visible absorption spectra of PLGA nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles, respectively; d is respectively Fe₃O₄Fluorescence spectra of the nanoparticles, PLGA nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles; e picture is in vitro MRI imaging picture of PLZ4@ SeD nano particles and T picture thereof₂A relaxation rate map; f is PLZ4@ SeD nano-particle, PLZ4@ SeD/Fe₃O₄Infrared spectrogram of the nano-particles, SeD-1b and PLGA.

FIG. 2 is a graph of the stability of the targeted nano-drug PLZ4@ SeD nanoparticles described in example 2 under different conditions; wherein, A is a stability curve chart of the targeted nano-drug PLZ4@ SeD nano-particle in Phosphate Buffered Saline (PBS) and DMEM medium with pH of 5.6 respectively; panel B is a graph of the stability of the targeted nanomedicine PLZ4@ SeD nanoparticles in Phosphate Buffered Saline (PBS) and DMEM media, both pH7.4, respectively.

FIG. 3 shows different cancersAdding Fe to cells₃O₄And (4) a cell survival rate result graph after nanoparticles are obtained.

FIG. 4 shows SeD-1b, PLGA @ SeD nanoparticles, PLGA @ SeD/Fe were added to different cancer cells₃O₄Results of the half inhibitory concentration of each drug on each cell after nanoparticles and PLZ4@ SeD nanoparticles.

FIG. 5 is a graph showing the results of the half inhibitory concentration of different drugs on EJ cells of bladder cancer under hypoxic conditions.

FIG. 6 is a graph showing the immunofluorescence effect of the receptor integrin α v β 3 corresponding to the polypeptide PLZ4 expressed in isolated human normal bladder tissue and bladder cancer tissue.

FIG. 7 is a graph showing the results of selective uptake of PLZ4@ SeD nanoparticles by EJ cells of bladder cancer, SV-HUC-1 of normal bladder cells; wherein, the A picture is a fluorescence intensity result picture of nano-drug PLZ4@ SeD nano-particles in EJ cells of bladder cancer under different time conditions; b is a graph showing the fluorescence intensity results of nano-drug PLZ4@ SeD nanoparticles in normal bladder epithelial cells SV-HUC-1 under different time conditions.

FIG. 8 shows that targeting nano-drug PLZ4@ SeD nano-particle catalyzes H in vitro₂O₂A performance map of (a); wherein, A picture is PLGA nano particle, SeD-1b, Fe₃O₄Nanoparticles, PLZ4@ SeD nanoparticles with H, respectively₂O₂Effect graph of action; panel B shows PLZ4@ SeD nanoparticles, Fe in 15 min₃O₄Nanoparticles, PLGA nanoparticles and H, respectively₂O₂A graph of the resulting amount of oxygen produced after the action; graph C shows H at different concentrations for 15 min₂O₂The oxygen generation amount result graph is obtained after the solution is mixed with PLZ4@ SeD nano particles respectively; and the D picture is a graph of cyclic voltammetry results of PLZ4@ SeD nanoparticles under the protection of argon.

FIG. 9 shows Fe at different concentrations under hypoxic conditions₃O₄Graph showing the effect of nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles, on the viability of EJ cells of bladder cancer; wherein, A picture is Fe under different concentration conditions in an oxygen-deficient environment₃O₄Presence of nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles, respectively, on EJ cells of bladder cancerA graph of the activity rate influence results; b is Fe under different concentrations in the oxygen-deficient environment and the hydrogen peroxide condition₃O₄Graph showing the effect of nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles on the viability of EJ cells of bladder cancer.

FIG. 10 is a graph showing the results of the content of targeted nano-drug PLZ4@ SeD nanoparticles in EJ cells of bladder cancer; wherein, the A picture is a graph of the absorption capacity result of the EJ cells of the bladder cancer to the PLZ4@ SeD nano-particles of the targeted nano-drug under different time conditions; and B is a graph of the absorption capacity results of bladder cancer EJ cells on different final concentrations of targeted nano-drug PLZ4@ SeD nanoparticles at 8 h.

Fig. 11 is a graph of the intracellular localization of targeted nanomedicine PLZ4@ SeD nanoparticles in EJ cells of bladder cancer under different time conditions.

FIG. 12 is a graph showing the results of the change in the level of cellular reactive oxygen species over time following the action of targeted NanoDRUG PLZ4@ SeD nanoparticles on EJ cells of bladder cancer; wherein, the graph A is a graph of the change result of active oxygen generated after the bladder cancer EJ cells are incubated by using a DHE probe to detect targeted nano-drug PLZ4@ SeD nano-particles with different concentrations; and the B picture is a trend graph of active oxygen generated after bladder cancer EJ cells are incubated by the targeted nano-drug PLZ4@ SeD nano-particles with different concentrations.

FIG. 13 is a cell cycle chart of bladder cancer EJ cells after various concentrations of targeted nano-drug PLZ4@ SeD nanoparticles act on the bladder cancer EJ cells; wherein, A is a graph of the effect of PLZ4@ SeD nanoparticles with different concentrations on EJ cell cycle of bladder cancer; panel B is a graph showing the effect of different concentrations of nanoparticles PLZ4@ SeD nanoparticles on the cell cycle distribution of EJ cells from bladder cancer.

FIG. 14 is a graph of laser confocal results of EJ cell tumor spheres from bladder cancer in both the occluded and non-occluded groups.

FIG. 15 is a graph showing the perfusion effect of human isolated bladder tissue containing bladder cancer tumor body targeting nano drug PLZ4@ SeD nanoparticles; wherein, A is a figure of human isolated bladder tissues containing bladder cancer tumor bodies before drug infusion; b is a human isolated bladder tissue anatomical map containing bladder cancer tumor after drug infusion; section C is a partial view of the bladder at 4 times magnification; d is as a graph of nanometerAfter the drug PLZ4@ SeD nano-particles are infused, an MRI result graph of the tissue axial plane of the human in-vitro bladder containing the bladder cancer tumor body is obtained; the E picture shows the corresponding R of different bladder areas after the target nano-drug PLZ4@ SeD nano-particles act on the bladder in vitro₂ ^*A value result graph; f is T corresponding to different bladder areas after target nano-drug PLZ4@ SeD nano-particles act on the bladder in vitro₂Value result graph.

Fig. 16 is a graph showing the results of measuring the particle size of targeted nano-drugs prepared using different organic solvents as dispersants.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The reagents used in the examples are commercially available without specific reference.

The cells of the bladder cancer EJ cell, bladder cancer J82 cell, bladder cancer T921 cell, breast cancer MCF-7 cell, liver cancer HepG2 cell and normal bladder epithelial cell SV-HUC-1 in the examples of the present invention were purchased from American type culture Collection ATCC.

Acetone, acetonitrile and dichloromethane used in this example were purchased from Tianjindongzheng fine chemical reagent factory; fe₃O₄Nanoparticles were purchased from Sigma; PLGA was purchased from Sigma, USA; DMEM medium was purchased from Gibco, USA; malvern laser granulometer, available from Malvern, uk; MTT was purchased from Sigma; a multifunctional fluorescent microplate reader model ELX800, available from Bio-Tek, usa; BSA was purchased from Kyoho Biotechnology Inc. (GBCBIO Co., Guangzhou); integrin α v β 3 antibody was purchased from Cell Signaling Technology; integrin sheep anti-mouse immunoglobulin was purchased from Cell Signaling Technology; hoechst is available from Merck, germany; cell lysates were purchased from Byuntian; the model JPBJ-608 of the dissolved oxygen instrument is purchased from Remaye, China; flow cytometer model CytoFLEX S, available from Beckman Coulter, usa; propidium iodide was purchased from Sigma company; confocal laser microscopy was purchased from Leica corporation; PLZ4 was purchased from Gill Biochemical (Shanghai) Inc.

Example 1: preparation of targeted nano-drug

(1) Preparation of raw materials:

1) preparation of 3.5mM Tween-80: 5g of Tween-80 is weighed and 1L of secondary water is used for preparing a Tween-80 aqueous solution with the concentration of 3.5 mM.

2) Preparation of 1.05mM Fe₃O₄Nanoparticle-acetone dispersion: 0.2436g Fe were weighed out₃O₄Dispersing the nano particles in 1L of acetone to obtain the nano particles.

3) Preparation of 20mM PLGA-acetone dispersion: 20g of PLGA (lactic acid LA: glycolic acid GA: 50, Mn: 13000) was weighed and dispersed in 1L of acetone.

4) Preparation of 0.65mM SeD-1b solution: SeD-1b (prepared according to example 1 of patent "201610127128.3") 24.375mg was weighed and dissolved in 10mL of dimethyl sulfoxide.

5) Prepare 20mg/mL PLZ4 solution: weighing 20g of polypeptide PLZ4 powder, and dissolving in 1L Phosphate Buffer Solution (PBS) with pH of 7.4 and concentration of 0.01M.

(2) Preparation of targeted nano-drug PLZ4@ SeD nano-particle

1) Preparation of aqueous PLGA nanoparticle solutions

And (3) taking 1mL of the 20mM PLGA-acetone dispersion, diluting to 3mL with acetone, dropwise adding into 10mL of the 3.5mM Tween-80 aqueous solution at the speed of 10 seconds per drop, stirring at 800rpm for 12 hours, dialyzing in a dialysis bag with the dialysis membrane equivalent of 10000kDa for 12 hours, and dialyzing to obtain the PLGA polymer nanoparticle aqueous solution.

2)PLGA@Fe₃O₄Preparation of nanoparticles

1mL of the 20mM PLGA-acetone dispersion and 1mL of the 1.05mM Fe₃O₄Mixing the nano particles and the acetone dispersion liquid, then using acetone to fix the volume to 3mL, dropwise adding the mixed solution after fixing the volume into 10mL of the 3.5mM Tween-80 aqueous solution at the speed of 10 seconds/drop, stirring at 800rpm overnight, dialyzing in a dialysis bag with the dialysis membrane equivalent of 10000kDa for 12 hours to obtain PLGA @ Fe₃O₄An aqueous solution of nanoparticles; wherein the resulting PLGA @ Fe₃O₄In an aqueous solution of nanoparticles, Fe₃O₄In a concentration of0.08mM。

3) Preparation of PLGA @ SeD nanoparticles

Taking 1mL of the 20mM PLGA-acetone dispersion solution and 200 mu L of the 0.65mM SeD-1b solution, using acetone to fix the volume to 3mL, then dropwise adding the mixed solution after fixing the volume into 10mL of the 3.5mM Tween-80 aqueous solution at the speed of 10 seconds/drop, stirring at 800rpm for 12 hours, and dialyzing in a dialysis bag with the dialysis membrane equivalent of 8000kDa for 12 hours to obtain a PLGA @ SeD nanoparticle aqueous solution; wherein, in the obtained PLGA @ SeD nanoparticle aqueous solution, the concentration of SeD-1b is 10 mu M.

4)PLGA@SeD/Fe₃O₄Preparation of nanoparticles

1mL of the 20mM PLGA-acetone dispersion and 1.05mM Fe were each taken₃O₄1mL of nano particle-acetone dispersion liquid and 200 mu L of 0.65mM SeD-1b solution are subjected to volume fixing to 3mL by acetone, then the mixed solution after volume fixing is dripped into 10mL of the 3.5mM Tween-80 aqueous solution at the speed of 10 seconds/droplet, stirred at 800rpm for 12 hours, dialyzed in a dialysis bag with the dialysis membrane equivalent of 8000kDa for 12 hours to obtain PLGA @ SeD/Fe₃O₄An aqueous solution of nanoparticles; wherein the resulting PLGA @ SeD/Fe₃O₄Fe in nanoparticle aqueous solution₃O₄SeD-1b was added at a concentration of 0.08mM and 10. mu.M, respectively.

5)PLGA@SeD/Fe₃O₄Activation of nanoparticles

The PLGA @ SeD/Fe prepared in the step 4) is added₃O₄Adding 20mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 12mg of N-hydroxysuccinimide (NHS) powder into the nanoparticles respectively, and stirring at 25 ℃ and 800rpm for 3 hours to obtain activated PLGA @ SeD/Fe₃O₄An aqueous solution of nanoparticles.

6) Preparation of targeted nano-drug PLZ4@ SeD nano-particle

1mL of the 20mg/mL PLZ4 solution was added to 19mL of the activated PLGA @ SeD/Fe solution₃O₄Stirring the nano particle water solution at 800rpm for 12 hours, dialyzing the nano particle water solution in a dialysis membrane with the dialysis membrane equivalent of 10000kDa for 12 hours to obtain the target nano drug PLZ4@ SeD nano particle, and storing the target nano drug PLZ4@ SeD nano particle in a refrigerator at 4 ℃.

(3) Correlation test and results

The prepared target nano-drug PLZ4@ SeD nano-particle is detected as follows, and the result is shown in figure 1:

1) the morphology of PLZ4@ SeD nanoparticles was characterized using a Hitachi transmission electron microscope model H-7650, and the results are shown in Panel A of FIG. 1.

As can be seen from the graph A in FIG. 1, the PLZ4@ SeD nanoparticles have good dispersibility and a particle size of about 120nm, which is consistent with the results of electron microscopy, and the PLZ4@ SeD nanoparticles are spherical nanoparticles with good dispersibility and a particle size of about 120 nm.

2) Respectively characterizing PLGA Nano particles and Fe by using Nano-ZS (Malvern instruments Limited)₃O₄Nanoparticle, PLGA @ SeD nanoparticle, and activated PLGA @ SeD/Fe₃O₄The potential change of the nanoparticle, PLZ4@ SeD nanoparticle aqueous solution is shown in Panel B of FIG. 1.

As can be seen from B in FIG. 1, Fe₃O₄Nanoparticle, activated PLGA @ SeD/Fe₃O₄The nanoparticles and PLZ4@ SeD nanoparticles are at positive potential, and the PLGA nanoparticles and PLGA @ SeD nanoparticles are at negative potential. The positive surface potential of PLZ4@ SeD nanoparticles is higher compared to PLGA nanoparticles and PLGA @ SeD nanoparticles; fe₃O₄The positive surface potential of the nano particle is higher than that of the activated PLGA @ SeD/Fe₃O₄Nanoparticles and PLZ4@ SeD nanoparticles were higher, while PLGA @ SeD/Fe after activation₃O₄The surface positive potentials of the nanoparticles and PLZ4@ SeD nanoparticles are not very different, which is illustrated by Fe₃O₄The nano particles have stronger positive surface potential and can reverse the negative surface potential of the PLGA nano particles; the surface negative potential of the PLGA @ SeD nano particles is stronger than that of PLGA, which shows that the SeD-1b encapsulated by the PLGA has negative potential; surface positive potential of PLZ4@ SeD nanoparticles is higher than that of activated PLGA @ SeD/Fe₃O₄The positive potential on the surface of the nanoparticle is low, which indicates that the polypeptide PLZ4 has a certain negative potential, and the surface of the cell membrane is negative due to the existence of a large amount of protein on the surface of the cell membrane, while PLZ4@ SeThe positive potential on the surface of the D nano particles is beneficial to the combination of the D nano particles and the cell membrane with negative potential, and is more beneficial to the absorption of cancer cells to the targeted nano-drug.

3) The optical properties of the obtained PLGA nanoparticles, SeD-1b and PLGA @ SeD nanoparticles were analyzed by fluorescence spectroscopy (Thermo Scientific Lumina), respectively, and the results are shown in FIG. 1C.

As can be seen from the graph C in FIG. 1, the excitation wavelengths of the PLGA @ SeD nanoparticles and SeD-1b alone are 623nm, which indicates that SeD-1b is successfully loaded into the PLGA nano-system and retains its own optical properties.

4) The prepared Fe was analyzed by UV-visible absorption spectrometer (UH4150) respectively₃O₄The optical properties of the nanoparticles, PLGA polymer nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles, are shown in graph D of FIG. 1.

As can be seen from plot D in FIG. 1, the absorption wavelengths of PLZ4@ SeD nanoparticles and SeD-1b alone are the same, both 400nm, indicating that SeD-1b was successfully loaded into the PLZ4@ SeD nanoparticle nanosystem and retained its own optical properties.

5) FIG. 1, Panel E, is a 1/T plot of PLZ4@ SeD nanoparticles characterized by SGNA HOR120N magnetic resonance imaging instrumentation₂Signal result graph.

As can be seen from Panel E of FIG. 1, the 1/T of PLZ4@ SeD nanoparticles₂The signal increased with increasing iron concentration, and was dose-dependent, indicating that PLZ4@ SeD nanoparticles have a decreasing T₂The effect of relaxation rate, i.e. having an enhanced T₂The ability to visualize the effects.

6) The chemical structures of PLZ4@ SeD nanoparticles, SeD-1b and PLGA polymer nanoparticles respectively characterized by Fourier transform infrared spectroscopy are shown in graph F in FIG. 1.

As can be seen from Panel F in FIG. 1, 3258cm^-1And 1590cm^-1The peak is shown, namely the PLZ4 and PLGA form amide bond through dehydration condensation, which indicates that the targeting polypeptide PLZ4 has been successfully connected to the activated PLGA @ SeD/Fe₃O₄On the nanoparticles.

Example 2: in vitro stability study of targeting nano-drugs

The preparation method of the targeted nano-drug PLZ4@ SeD nanoparticle used in this example is the same as the preparation method described in example 1 of the present application.

The mixed solution for detecting the particle size of the nano-drug PLZ4@ SeD nano-particle is prepared according to the following methods respectively:

group A1: 1mL of dialyzed PLZ4@ SeD nanoparticle aqueous solution and 0.01M phosphate buffer solution with the pH value of 5.6 are mixed according to the volume ratio of 1: 2;

group A2: 1mL of the PLZ4@ SeD nanoparticle aqueous solution after dialysis and a DMEM medium which is adjusted to be acidic by hydrochloric acid and has the pH value of 5.6 are mixed according to the volume ratio of 2: 1;

group B1: 1mL of dialyzed PLZ4@ SeD nanoparticle aqueous solution and 0.01M phosphate buffer solution with the pH value of 7.4 are mixed according to the volume ratio of 2: 1;

group B2: 1mL of the dialyzed PLZ4@ SeD nanoparticle aqueous solution was mixed with a DMEM medium having a pH of 7.4 at a volume ratio of 2: 1.

The mixed solution of groups A1, A2, B1 and B2 was subjected to Nano-drug particle size detection at time points of 0h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 96h and 144h by using a Markov laser particle sizer (Zetasizer Nano-ZS), and the stability of PLZ4@ SeD nanoparticles was analyzed, and the results are shown in FIG. 2.

As can be seen from FIG. 2, the stability curve of PLZ4@ SeD nanoparticles under acidic condition is similar to the stability curve trend under neutral condition, which indicates that the prepared PLZ4@ SeD nanoparticles are not easily degraded in acidic solution and the drug effect of the loaded drug lasts longer.

Example 3: comparison of in vitro antitumor Activity of Targeted Nanotempounds

Taking the density of the growth in logarithmic phase as 2X 10⁴Each cell/mL of the EJ cell, the J82 cell, the T921 cell, the MCF-7 cell and the HepG2 cell of the bladder cancer, which are respectively inoculated100 μ L per well in 96-well plates; after the cells are attached to the wall, 100 mu L of Fe diluted by DMEM medium is added into the first group of wells respectively containing EJ cells of bladder cancer, J82 cells of bladder cancer, T921 cells of bladder cancer, MCF-7 cells of breast cancer and HepG2 cells of liver cancer₃O₄The final concentration of the nanoparticles was 0.08 mmol/L. So as not to add Fe₃O₄Bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer cells HepG2 of nano particles are used as a control, 30 mu L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT) with the concentration of 5mg/mL is added into each hole after 48 hours, after 3 hours of light-shielding incubation at 37 ℃, the supernatant containing the MTT is discarded, 150 mu L of dimethyl sulfoxide (DMSO) is added into each hole, constant temperature oscillation is carried out for 15 minutes at 37 ℃, and Fe is respectively tested by a multifunctional fluorescence microplate reader under the condition that the wavelength is 570nm₃O₄The effect of nanoparticles on the viability of the above 5 cells is shown in FIG. 3.

As can be seen from FIG. 3, Fe₃O₄The nano-particles have almost no toxicity to the 5 tumor cells, and the survival rate of the 5 cells is close to 100%.

In addition, the second group contained 100. mu.L of the mixture and had a density of 2X 10⁴Each cell/mL of the adherently grown bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer HepG2, 20 muM was used as the highest concentration, and 100 muL of DMEM-diluted SeD-1b dissolved in dimethyl sulfoxide was added at gradient concentrations of 10 muM, 5 muM, 2.5 muM and 1.25 muM; respectively containing to the third group at a density of 2 × 10⁴100 mu L of PLGA @ SeD nano particles diluted by DMEM are added into each hole of the bladder cancer EJ cells, the bladder cancer J82 cells, the bladder cancer T921 cells, the breast cancer MCF-7 cells and the liver cancer HepG2 with each cell/mL, wherein the highest concentration is 20 mu M, and the concentration gradient is the same as that of the second group; respectively containing to the fourth group at a density of 2 × 10⁴Each cell/mL of adherently grown bladder cancer EJ cell, bladder cancer J82 cell, bladder cancer T921 cell, breast cancer MCF-7 cell and liver cancer cell HepG2, wherein 20 muM is used as the highest concentration, and 100 muL of PLGA @ SeD/Fe diluted by DMEM is added into each hole of the cell/mL of adherently grown bladder cancer EJ cell, bladder cancer J82 cell, bladder cancer T921 cell, breast cancer MCF-7 cell and liver cancer cell HepG2 according to gradient concentration₃O₄Nanoparticles having a concentration gradient of the second group; respectively contain to the fifth group the density of 2X 10⁴Each cell/mL of adherently grown bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer HepG2 is added with 100 mu L of PLZ4@ SeD nano particles diluted by DMEM according to gradient concentration with 20 mu M as the highest concentration, and the concentration gradient is the same as the second group; without adding SeD-1b, PLGA @ SeD nanoparticles, PLGA @ SeD/Fe₃O₄The nano particles and PLZ4@ SeD nano particles are used as a reference for bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer HepG2 which contain adherent growth, 30 mu L of 5mg/mL 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT) is added into each hole after 48 hours, the mixture is incubated for 3 hours in a dark place at 37 ℃, supernatant containing the MTT is discarded, 150 mu L of dimethyl sulfoxide (DMSO) is added into each hole, the mixture is oscillated for 15 minutes at the constant temperature of 37 ℃, and under the condition that the wavelength is 570nm, a multifunctional fluorescence microplate reader is used for respectively testing SeD-1b, PLGA @ SeD nano particles, PLGA @ SeD/Fe with each concentration₃O₄The absorption values of the cells treated by the nano particles and the PLZ4@ SeD nano particles are obtained, regression curves are obtained, and the curves are substituted to respectively obtain SeD-1b, PLGA @ SeD nano particles, PLGA @ SeD/Fe₃O₄The half inhibitory concentrations of nanoparticles and PLZ4@ SeD nanoparticles on different cells are shown in figure 4.

As can be seen from FIG. 4, compared with SeD-1b, the half-inhibitory concentration of PLZ4@ SeD nanoparticles on different cells was significantly reduced, and PLZ4@ SeD nanoparticles were more toxic to EJ cells of bladder cancer.

In addition, the fourth group contained 100. mu.L of a 2X 10 density⁴Each cell/mL of adherently growing bladder cancer EJ cells was added to each well with 20 μ M as the highest concentration and with gradient concentrations of 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M, to a volume of 100 μ L of clinical drug diluted in DMEM medium: doxorubicin hydrochloride, mitomycin, pirarubicin and the targeted nano-drug PLZ4@ SeD nanoparticles; the nano-particles without doxorubicin hydrochloride, mitomycin, pirarubicin and PLZ4@ SeD, which contain 100 mu L and have the density of 2 multiplied by 10⁴Individual cell/mL patchUsing bladder cancer EJ cells growing on the wall as a reference, adding 30 mu L of 5mg/mL of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) into each hole after 48 hours under the hypoxic condition that the gas composition is 5% of carbon dioxide, 1% of oxygen and 94% of nitrogen, incubating for 3 hours in the dark at 37 ℃, discarding the supernatant containing the MTT, adding 150 mu L of DMSO into each hole, shaking for 15 minutes at the constant temperature of 37 ℃, testing the absorption values of the bladder cancer EJ cells treated by the doxorubicin hydrochloride, the mitomycin, the pirarubicin and the PLZ4@ SeD nano particles under the hypoxic condition by using a multifunctional fluorescence microplate reader under the condition that the wavelength is 570nm, calculating a regression curve, substituting the curve to respectively calculate the influence of each drug on the half-inhibited concentration of the bladder cancer EJ cells, the results are shown in FIG. 5.

As can be seen from FIG. 5, the IC of the targeted nano-drug PLZ4@ SeD nanoparticles under hypoxic conditions₅₀Compared with doxorubicin hydrochloride, mitomycin and pirarubicin, the semi-inhibitory concentration of the target nano-drug PLZ4@ SeD nano-particle is lower, which shows that the drug effect of the target nano-drug PLZ4@ SeD nano-particle prepared by the invention is stronger.

Example 4: evaluation of immunofluorescence Effect of Targeted Nanoparticulates

The preparation method of the targeted nano-drug PLZ4@ SeD nanoparticles used in this example is the same as the preparation method described in example 1 of the present application.

Dehydrating normal bladder tissues of isolated human origin containing normal bladder epithelial cells SV-HUC-1 and muscle layer invasive bladder cancer tissues containing bladder cancer EJ cells: embedding the frozen sections at-80 ℃ by OCT, continuously slicing the frozen sections by 8 mu m, and storing the frozen sections at-20 ℃; and (3) putting the section into a light-proof wet box, carrying out rewarming and rehydration, drying excessive water, then sealing for 30 minutes at room temperature by using bovine serum albumin (5% BSA), drying again, and directly dripping 50 mu L of the first antibody of the integrin alpha v beta 3 with the dilution multiple of 1:1000, wherein the integrin alpha v beta 3 is the target point of PLZ4 targeted to cells. After overnight at 4 ℃, then the sections were left at room temperature for half an hour for rewarming, and washed 3 times with PBS to wash off excess integrin α ν β 3 primary antibody not bound to the tissues on the sections, 50 μ L of integrin goat anti-mouse immunoglobulin diluted at 1:1500 was added dropwise to the tissues on each section, and incubated at room temperature for 2 hours, after the incubation time was over, 50 μ L of 1 μ g/mL Hochest was added dropwise to the tissues on each section, incubated at room temperature for 30 minutes, and after the incubation time was over, the expression of protein in normal and tumor tissues was photographed and analyzed using a fluorescence microscope, and the results are shown in fig. 6.

As can be seen from fig. 6, the expression level of integrin α v β 3 in the bladder cancer tissue region was significantly higher than that in the normal bladder tissue. It is therefore assumed that high expression of integrin α v β 3 in muscle-invasive bladder cancer tissues is beneficial for active targeting of bladder cancer cells by PLZ4 in targeted nanomedicine PLZ4@ SeD nanoparticles.

Example 5: comparison of in vitro Selective absorption Effect of Targeted Nanotemporugs

Activated PLGA @ SeD/Fe in this example₃O₄Preparation of aqueous nanoparticle solutions and activated PLGA @ SeD/Fe as described in example 1₃O₄The preparation method of the nanoparticle aqueous solution is the same.

Weighing 2.5mg of PLZ4 powder, and preparing into 0.3125mg/mL, 0.625mg/mL, 1.250mg/mL and 2.500mg/mL PLZ4 solutions with PBS buffer (pH7.4 and 0.01M) respectively for later use; 1mL of activated PLGA @ SeD/Fe was taken₃O₄And respectively mixing the nanoparticle aqueous solution with the PLZ4 solutions with different concentrations according to the volume ratio of 4:1, stirring for 12h at room temperature, respectively dialyzing for 12h by using a dialysis membrane with the equivalent weight of 8000kDa, and diluting by a DMEM (DMEM) culture medium to obtain the target nano-drug PLZ4@ SeD nanoparticles with the target concentrations of 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL and 0.5mg/mL respectively.

Taking the logarithm growth of the EJ cells of the bladder cancer and the SV-HUC-1 cells of the normal bladder epithelial cells at the ratio of 8 multiplied by 10 respectively⁴The cells were seeded in 96-well plates at a density of 100. mu.L per well; after the cells adhered to the wall, at different time points: adding 100 mu L of the targeted nano-drug PLZ4@ SeD nanoparticles with different target concentrations for 2h, 4h and 8h, and simultaneously taking bladder cancer EJ cells and normal bladder epithelial cells SV-HUC-1 without adding the targeted nano-drug PLZ4@ SeD nanoparticles as control groups; washing the cells for 2-3 times by PBS (phosphate buffer solution) at 4 ℃ until redundant targeting nano-drugs PLZ4@ SeD sodium on the surfaces of the cancer cells are washed awayAnd (3) adding 100 mu L/hole of cell lysate into the rice grains, oscillating at the constant temperature of 37 ℃ for 15 minutes to fully break the cells, and testing the fluorescence values of the rice grains with the excitation wavelength of 400nm and the emission wavelength of 623nm by using a multifunctional fluorescence microplate reader, wherein the results are shown in FIG. 7.

As can be seen from panel a in fig. 7, as the concentration of targeting polypeptide PLZ4 in the nanomedicine PLZ4@ SeD nanoparticles increased, the detected fluorescence intensity also increased, that is to say the absorption of PLZ4@ SeD nanoparticles by bladder cancer EJ cells increased with the increase in the concentration of targeting polypeptide PLZ4 in the nanomedicine PLZ4@ SeD nanoparticles; and the fluorescence intensity also becomes stronger with the passage of time, so that the absorption of the nano-drug PLZ4@ SeD nano-particles by the bladder cancer EJ cells also has the characteristic of time dependence.

As can be seen from the B plot in FIG. 7, the fluorescence intensity of the nano-drug PLZ4@ SeD nanoparticles in normal bladder epithelial cells SV-HUC-1 was neither significantly increased with the increase in the concentration of the targeting polypeptide PLZ4 in the nano-drug PLZ4@ SeD nanoparticles, nor increased with time, i.e., the absorption of PLZ4@ SeD nanoparticles by normal bladder cells SV-HUC-1 was not as significant as bladder cancer EJ cells. Indicating that the targeted nano-drug PLZ4@ SeD has selectivity.

Example 6: in vitro catalysis of targeted nanomedicines₂O₂Performance exploration

The preparation method of the targeted nano-drug PLZ4@ SeD nanoparticle used in the embodiment is the same as the preparation method described in the embodiment 1 of the application; its in vitro catalysis H₂O₂The performance results are shown in fig. 8.

1.7M H was prepared in PBS buffer (pH7.4, 0.01M)₂O₂. Taking 5 containers of 3.8mL of H with the concentration of 2mol/L₂O₂200. mu.L of PLGA nanoparticles (10.7mM), SeD-1b (10. mu.M), Fe were added to the quartz dish₃O₄Nanoparticles (0.08mM), PLZ4@ SeD nanoparticles (16. mu.M), in 4mL of PBS buffer (pH7.4, 0.01M) and 1.7M of H, respectively₂O₂The solution was used as a control, and the photograph was taken after 10 minutes of reaction, and the result is shown in graph A in FIG. 8.

From FIG. 8As can be seen in Panel A, PLZ4@ SeD nanoparticles catalyze H₂O₂Production of O₂The capability of the alloy is remarkably stronger than that of Fe₃O₄Nanoparticles, and PLGA nanoparticles, SeD-1b, produced almost no O₂Thus, it can be seen that PLZ4@ SeD nanoparticles can efficiently catalyze H₂O₂Production of O₂Further improving the hypoxic environment of the bladder cancer cells.

1.7mol/L H was prepared in PBS buffer (pH7.4, 0.01M)₂O₂And carrying out deoxidization treatment on the mixture; 200 microliter of PLGA nano-particles (1.5mg/mL) and Fe₃O₄Nanoparticles (0.08mM), PLZ4@ SeD nanoparticles (16. mu.M), 3.8mL 1.7M H₂O₂After the solution is fully mixed, immediately using an oxygen dissolving instrument to treat O in the solution₂The values were measured continuously for 15 minutes, and the results are shown in the B graph in FIG. 8.

As can be seen from panel B in FIG. 8, PLZ4@ SeD nanoparticles catalyze H at the same time₂O₂Production of O₂The rate of (A) is significantly higher than that of PLGA nano-particles and Fe₃O₄Nanoparticle catalysis H₂O₂Production of O₂That is, PLZ4@ SeD is due to PLGA encapsulating Fe₃O₄The generated confinement effect causes the catalyst to catalyze H₂O₂Production of O₂Is significantly stronger than Fe alone₃O₄Nanoparticles.

3.8mL of H was prepared at a concentration of 1.0mM, 0.8mM, 0.6mM, 0.4mM, respectively₂O₂The solution to which 200. mu.L of 16. mu.M PLZ4@ SeD nanoparticles were added, was immediately treated with O in solution using an oxygen dissolver₂The values were measured continuously for 15 minutes while using water as a control, and the results are shown in graph C of fig. 8.

As can be seen from Panel C in FIG. 8, PLZ4@ SeD nanoparticles catalyze H₂O₂Production of O₂Amount of (A) and H₂O₂In a concentration of 0.4 to 0.8mM of H₂O₂For example, H₂O₂The higher the concentration of oxygen, the more the oxygen generation amount is, the PLZ4@ SeD nano-particles have stronger catalytic H₂O₂Production of O₂Capability.

Preparing PBS buffer solution with pH of 7.0 and 0.01M, and dividing the prepared PBS buffer solution into three groups; blank groups were: PBS buffer with the volume of 2 mL; control i group was: volume 2mL of H prepared at a concentration of 1.0mM in PBS buffer₂O₂A solution; the variable II group is: to 2mL of 1.0mM H in PBS buffer₂O₂To the solution was added 2mL of PLZ4@ SeD nanoparticles at a concentration of 16. mu.M. The redox potentials of the three solutions were measured by electrochemical stations, respectively, and argon gas was introduced for protection during the measurement, and the results are shown in graph D in fig. 8.

As can be seen from graph D in FIG. 8, the PBS buffer had almost no redox potential; h₂O₂Because the disproportionation reaction can occur, the catalyst has certain oxidation-reduction potential; and the variable II group is added with the targeted nano-drug PLZ4@ SeD nano-particle, so that the redox potential of the variable II group is obviously higher than that of the blank group and the control group I, and the redox potential curve has no obvious bulge, therefore, the PLZ4@ SeD nano-particle has catalytic H₂O₂Production of O₂The ability of the cell to perform.

Example 7: in vitro hypoxia experiment of targeted nano-drug

In the presence of 5% CO₂、1％O₂、94％N₂Culturing EJ cells of bladder cancer under hypoxic environment, wherein the EJ cells of bladder cancer are cultured in logarithmic growth phase at a density of 2 × 10⁴Each cell/mL of bladder cancer EJ cells were seeded in 96-well plates at 100 μ Ι _ per well; after the cells were attached to the wall, the supernatant medium was discarded to add 100. mu.L of 400. mu. M H₂O₂DMEM medium as treatment group without addition of H₂O₂The DMEM medium of (1) was used as a control group; then 100. mu.L of Fe at different concentrations was added to the wells of the control and treated groups, respectively₃O₄Nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles, to Fe₃O₄Nanoparticles, SeD-1b, PLZ4@ SeD nanoparticles in the groupThe final concentrations of (A) and (B) are respectively 40 μm, 20 μm, 10 μm, 5 μm, 2.5 μm and 1.25 μm. Adding 30 mu L of MTT with the concentration of 5mg/mL into each hole after 48 hours, incubating for 3 hours in a dark place, discarding the supernatant containing MTT, adding 150 mu L of dimethyl sulfoxide into each hole, oscillating for 15 minutes at the constant temperature at room temperature, testing the light absorption value at the position with the wavelength of 570nm by using a multifunctional fluorescence microplate reader, calculating the survival rate, and obtaining the in vitro hypoxia experiment result as shown in FIG. 9.

As can be seen from the in vitro hypoxia experiments of panels A and B in FIG. 9, the cell survival rate of EJ cells of bladder cancer in the treated group is lower than that of EJ cells of bladder cancer in the control group under hypoxic environment, which indicates that EJ cells of bladder cancer in hypoxic environment are drug-resistant, while H is utilized₂O₂Decomposition to O₂The drug effect of the drug is obviously improved after the local hypoxic environment is improved.

Example 8: in vitro absorption experiment of targeting nano-drug

Bladder cancer EJ cells grown in log phase were taken at 8X 10⁴The cells/mL are inoculated in a culture dish with the diameter of 6cm, 6mL is added in each hole, after the cells grow for 24 hours in an adherent manner, PLZ4@ SeD nanoparticles are added to make the concentration of each particle be 2 mu M, the cells are incubated for 0h, 1h, 2h, 4h, 8h and 12h respectively, then the cells are washed for 2-3 times by PBS buffer solution with the temperature of 4 ℃, bladder cancer EJ cells are collected, the cells are centrifuged for 5 minutes at 1500rpm and then detected by a Flow cytometer, and finally the content of the drugs in the cells is analyzed by using software Flow Jo, and the result is shown in A picture in figure 10.

From the results of graph a in fig. 10, it can be seen that the uptake of targeted nano-drug PLZ4@ SeD nanoparticles by bladder cancer EJ cells increases with time while the drug concentration remains the same.

Inoculating the bladder cancer EJ cells growing in the logarithmic phase according to the same method and conditions, adding PLZ4@ SeD nanoparticles with different concentrations after the cells grow in an adherent manner for 24 hours to enable the final concentrations to be 0.25 mu M, 0.5 mu M, 1 mu M, 2 mu M and 4 mu M respectively, and taking the bladder cancer EJ cells without the PLZ4@ SeD nanoparticles as a control; after 8 hours of incubation, washing the cells with 4 ℃ PBS buffer solution for 2-3 times, collecting the bladder cancer EJ cells, centrifuging the cells at 1500rpm for 5 minutes, detecting the cells by using a Flow cytometer, and finally analyzing the content of the targeted nano-drug PLZ4@ SeD nanoparticles in the bladder cancer EJ cells by using software Flow Jo, wherein the result is shown in a B diagram in FIG. 10.

As can be seen from the B plot in fig. 10, the amount of targeted nanopharmaceutical PLZ4@ SeD nanoparticles taken up by cells increased with increasing concentration of targeted nanopharmaceutical PLZ4@ SeD nanoparticles at the same time, with a dose-dependent effect.

Example 9: intracellular localization experiments of targeted nano-drugs

The EJ cells of bladder cancer growing in logarithmic phase were taken at 8X 10⁴Inoculating the cells in a culture dish with the density of 2mL per hole and the diameter of 2 cm, after the cells grow adherent to the wall for 24 hours, adding a Lyso Tracker Red probe into one culture dish to incubate for 2 hours, adding a Hoechst probe into the other culture dish to incubate for 1 hour, after the incubation is finished, respectively adding PLZ4@ SeD nanoparticles into the two culture dishes to enable the final concentration to be 2.00 mu M, respectively after incubation for 0 hour, 1 hour, 2 hours, 4 hours, 8 hours and 12 hours, removing a supernatant culture medium, washing the cells for 2-3 times by PBS at 4 ℃, and observing the fluorescence signals of the PLZ4@ SeD nanoparticles in the cells under a fluorescence microscope. In this example, since PLZ4@ SeD nanoparticles load SeD-1b to emit green fluorescence in cells, the localization of drugs in cells can be further analyzed by overlapping the green fluorescence with the fluorescence of lysosomes (red fluorescence) and nuclei (blue fluorescence); the results are shown in FIG. 11.

From the intracellular localization experiment of fig. 11, it was found that lysosomes within the EJ cells of bladder cancer fluoresced green, indicating that the nano-targeted drug PLZ4@ SeD nanoparticles localized to lysosomes of the EJ cells of bladder cancer.

Example 10: cellular active oxygen experiment after targeting nano-drug acting on cells

Bladder cancer EJ cells grown in log phase at 2X 10⁵Inoculating each cell/mL in different 96-well plates at a density of 100 mu L per well, and adding 1 mu L of 1mmol/L DHE probe into each well after the cells adhere to the wall and culturing for 30 minutes in the dark; then, 100 μ L of targeted nano-drug PLZ4@ SeD nanoparticles are respectively added into each hole to serve as a treatment group, the final concentrations of the targeted nano-drug PLZ4@ SeD nanoparticles are respectively 16 μ M, 8 μ M and 4 μ M, meanwhile, the targeted nano-drug PLZ4@ SeD nanoparticles are not added to serve as a blank group, a multifunctional fluorescence microplate reader is used for measuring fluorescence absorption values at the excitation wavelength of 360nm and the emission wavelength of 610nm every 5 minutes, and the measurement results are continuously measured for 2 hours and are shown in FIG. 12.

The results in panel a of fig. 12 show that the fluorescence intensity of bladder cancer EJ cells increases with increasing addition of targeted nanopharmaceutical PLZ4@ SeD nanoparticles; from the results in panel B of fig. 12, it can be seen that the level of reactive oxygen species in the bladder cancer EJ cells is increased with the increase of the amount of the nano-drug PLZ4@ SeD nanoparticles, and the content of reactive oxygen species in the cells is increased with the increase of the drug concentration, indicating that the anti-tumor activity of the targeted nano-drug PLZ4@ SeD nanoparticles is achieved by inducing apoptosis by increasing the level of reactive oxygen species in the cells.

Example 11: evaluation of in vitro antitumor Effect of Targeted NanoTaharmaceutical

Cycle arrest and apoptosis are two important ways in which antineoplastic drugs induce cell death. Therefore, the invention analyzes the mode of the targeted nano-drug causing the death of the EJ cells of the bladder cancer by using a flow cytometer.

Bladder cancer EJ cells grown in log phase at 2X 10⁴The individual cells/mL were seeded at different 6cm diameter culturesAdding 6mL of cells into each culture dish, and after the cells are attached to the wall, adding PLZ4@ SeD nanoparticles into 4 culture media respectively to serve as treatment groups, so that the final concentrations of PLZ4@ SeD nanoparticles in the culture media are 1.0 mu M, 0.5 mu M, 0.25 mu M and 0.125 mu M respectively; the culture medium without target nano-drug PLZ4@ SeD nano-particles is used as a control group; the EJ cells of the bladder cancer of the treated group and the control group were treated at 37 ℃ with 5% CO₂The culture box is incubated for 72 hours, then the cells are collected, 1mL of ethanol with the volume fraction of 70% at the temperature of 20 ℃ below zero is added, the mixture is placed in an environment with the temperature of 20 ℃ below zero for 24 hours for fixation, the mixture is centrifuged at 1500rpm for 10 minutes, then 300 mu L of propidium iodide is used for shading and dyeing for 30 minutes, after the dyeing is finished, a 300-mesh (with the aperture of 40-50 mu M) nylon net is used for filtration, the filtered sample is detected by a flow cytometer, the content of DNA in the cells is analyzed by Modfit software, the proportions of a G0/G1 phase, an S phase, a G2/M phase and an apoptosis peak Sub G1 are obtained, and the detection results are respectively shown in figure 13.

As can be seen from the results of graph a in fig. 13, the proportion of the apoptotic peak Sub-G1 of the control group was 0.57%, while the proportion of the apoptotic peak of the treatment group gradually increased from 5.76% at a drug concentration of 0.25 μmol/L to 18.23% at a drug concentration of 1.0 μmol/L, indicating that the targeting nanopharmaceutical PLZ4@ SeD nanoparticles exert significant antitumor activity by inducing apoptosis.

From the results of the B diagram in FIG. 13, it can also be seen that the proportion of S phase of the cells is also increased, and the proportion of G2/M and G0/G1 phases is decreased, because the DNA is sheared during apoptosis, and the S phase as the DNA synthesis phase cannot enter the next phase, so that the proportion of S phase is increased, which indicates that the targeted nano-drug PLZ4@ SeD nanoparticles exert their anti-tumor activity by inducing apoptosis.

Example 12: in vitro tumor ball experiment of targeting nano-drug

Bladder cancer EJ cells grown in log phase at 2X 10⁶The density of each cell/mL is inoculated2mL per well in a six-well ultra-low adsorption plate. Shaking every 0.5 hr for 10 min, shaking every 2 hr for 3 hr for 10 min, shaking every 12 hr for 12 hr, shaking every 10 min, and observing whether tumor spheres with diameter of 100 μ M are formed after one week, adding 50 μ L of 20mg/mL PLZ4 solution prepared with PBS (pH7.4 and 0.01M) to the formed tumor spheres to make the final concentration of PLZ4 0.5mg/mL, using the above-mentioned group as sealed group, using the tumor spheres without PLZ4 as unsealed group, and placing the sealed group and unsealed group at 37 deg.C and 5% CO at the same time₂After 3 hours in the incubator, the nanoparticles at PLZ4@ SeD were added to the two groups to a final concentration of 2. mu.M, and the mixture was incubated for 4 hours, and the results were observed with a confocal laser microscope after the incubation was completed, as shown in FIG. 14.

From the confocal laser results shown in fig. 14, the fluorescence of the tumor spheres in the closed group is significantly smaller than that in the non-closed group after the tumor spheres are closed by the targeting polypeptide PLZ4, and the quantity of the targeted nano-drug PLZ4@ SeD nanoparticles entering the tumor spheres in the closed group is smaller than that in the non-closed group, indicating that PLZ4@ SeD nanoparticles have selectivity.

Example 13: evaluation of in vitro bladder perfusion effect of targeting nano-drug

Diluting the nano-drug PLZ4@ SeD nanoparticles to 1mM with physiological saline; the method comprises the steps of taking a human isolated bladder tissue containing normal bladder epithelial cells SV-HUC-1 and bladder cancer EJ cells, wiping the periphery of the human isolated bladder tissue containing a bladder cancer tumor body, squeezing out residual urine and blood in the bladder tissue, and washing for 2-3 times by using normal saline until no blood exists. 100mL of the targeted nano-drug PLZ4@ SeD nanoparticles with the concentration of 1mM are perfused through the urethra to fully diffuse the drug in the bladder, and after the drug is placed in a DMEM medium at 37 ℃ and is kept still for 8 hours, the distribution of the drug in the bladder before and after perfusion is observed by using a nuclear magnetic resonance imager, and the result is shown in FIG. 15.

As can be seen from panels A-C in FIG. 15, the size of the tumor in the isolated bladder was about 5cm by 4 cm;

from the Magnetic Resonance Imaging (MRI) results of the graph D in FIG. 15, it can be concluded that the drug absorption area of the tumor part is significantly larger than that of the normal tissue part, i.e., the targeted nano-drug PLZ4@ SeD nanoparticles enter the tumor part in a significantly larger amount than the normal tissue, and thus, it can be inferred that R in MRI is significantly larger than that of the normal tissue₂The value should theoretically increase with increasing drug concentration, T₂The value should then decrease with increasing drug concentration.

As can be seen from the E and F plots in FIG. 15, R corresponds to the drug trend, the tumor margin, the normal tissue margin, the tumor interior region, and the normal tissue interior region₂Value sum T₂The trend of value formation is consistent with the theoretical trend, and the T of the internal area of the tumor and the internal area of the normal tissue₂Equal value, R₂Values are also nearly equal. And R at the tumor margin₂R with value greater than normal tissue margin₂Value, T₂T with a value less than the normal tissue margin₂Values indicate that targeted nanopharmaceutical PLZ4@ SeD nanoparticles selectively entered the bladder tumor region.

Example 14: size evaluation of targeting nano-drugs prepared by using different organic solvents as dispersants

The preparation method of the targeted nano-drug prepared in this example and the preparation method described in example 1 of this application are used for dispersing PLGA and Fe₃O₄The organic solvents are different in types, and other conditions are consistent; wherein, the dispersing agent is used for dispersing PLGA and Fe₃O₄The organic solvents of (a) are acetone, acetonitrile and dichloromethane respectively. The sizes of the obtained target Nano-drugs were respectively characterized by Nano-ZS (Malvern instruments Limited), and the results are shown in FIG. 16.

As can be seen from fig. 16, the size of the targeted nano-drug obtained by using acetone as the dispersant is about 120nm, while the sizes of the targeted nano-drug obtained by using acetonitrile and dichloromethane as the dispersants are 220nm and 210nm, respectively, which indicates that under the same conditions, the size of the targeted nano-drug obtained by using acetone as the dispersant is smaller and the effect is better.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<223> targeting polypeptide PLZ4

<400> 1

Cys Gln Asp Gly Arg Met Gly Phe Cys

1 5

Claims

1. A preparation method of a targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance is characterized by comprising the following steps:

s1, respectively dispersing PLGA, a nuclear magnetic imaging contrast agent and an antitumor drug in an organic solvent, then adding a surfactant, stirring and dialyzing to obtain a solution A;

s3, adding targeting molecules into the activated solution A, and stirring and dialyzing to obtain the targeted nano-medicament for overcoming the drug resistance caused by tumor hypoxia based on the guidance of nuclear magnetic imaging;

the nuclear magnetic imaging contrast agent in the step S1 is ferroferric oxide nano particles;

the antitumor drug in the step S1 is a selenadiazole derivative;

the targeting molecule described in step S3 was PLZ 4.

2. The method according to claim 1, wherein the PLGA, the MRI contrast agent and the anti-tumor drug in the solution A in step S1 are mixed in a molar ratio of 1-40: 1-10.

3. The method according to claim 1, wherein the organic solvent in step S1 is at least one of acetone, acetonitrile, and dichloromethane.

4. The method according to claim 1, wherein the surfactant in step S1 is an aqueous Tween solution.

5. The method of claim 1, wherein the amounts of the NHS, EDC and PLGA used in step S2 are as follows: EDC: PLGA = molar ratio 1:1:1 ~ 40.

6. A targeted nano-drug for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance is characterized by being prepared by the preparation method of any one of claims 1 to 5.

7. The use of the targeted nanomedicine for overcoming the drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance as claimed in claim 6 in the preparation of an anti-tumor drug.