CN111298139B - Targeted nanomedicine for overcoming drug resistance caused by tumor hypoxia based on MRI guidance and its preparation method and application - Google Patents

Abstract

The invention discloses a targeted nanomedicine for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, a preparation method and application thereof, and belongs to the technical field of biomedicine. The preparation method of the targeted nanomedicine includes the following steps: S1. Dispersing PLGA, a nuclear magnetic imaging contrast agent and an antitumor drug in an organic solvent respectively, then adding a surfactant, and stirring and dialysis to obtain a solution A; S2. Use NHS and EDC to activate the carboxyl group of solution A to obtain an activated solution A; S3. Add targeting molecules to the above activated solution A, and after stirring and dialysis, MRI-guided anti-tumor hypoxia can be obtained Targeted nanomedicines causing drug resistance. The targeted nanomedicine PLZ4@SeD nanoparticle of the present invention has better therapeutic effect than clinical medicines such as doxorubicin, mitomycin and pirarubicin under the condition of hypoxia.

Description

Targeted nanomedicine for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance and its preparation method and application

technical field

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

Background technique

Bladder cancer (BCa) is the urinary system tumor with the highest incidence in my country. According to statistics, there were nearly 550,000 new cases worldwide in 2018. In the conventional treatment regimen for bladder cancer, in addition to transurethral resection of bladder tumor (TURBT) for non-muscle-invasive BCa (NMIBC) and radical total cystectomy for muscle-invasive BCa (MIBC), Drug treatment is an important part of intravesical infusion therapy, systemic chemotherapy and local interventional chemotherapy after TUR. However, in the process of using anti-BCa drugs, infusion drugs have poor sensitivity, unstable function, high cost, and large toxic and side effects. The problem still exists. In the past three decades, the effect of anti-advanced BCa drug treatment has not been significantly improved, especially for patients who progressed after first-line chemotherapy, there is still no effective drug treatment; whether immunotherapy represented by expensive PD-1/L1 inhibitors is effective? The survival benefit of conventional chemotherapy, in particular, remains to be confirmed by future data. Therefore, it is of great clinical value and practical significance to find anti-BCa drugs with high efficiency, low toxicity, stability and low price.

In organisms, selenium is an important active center of glutathione peroxidase (GSH-Px), iodothyronine and mammalian thioredoxin reductase (TrxR). Since the negative correlation between selenium and tumor prevalence was first discovered more than 40 years ago, the anti-tumor value of selenium compounds has been verified by a large number of studies. Oxidative, DNA damage and apoptosis pathways. Compared with inorganic selenium compounds, organic selenium compounds have the characteristics of high absorption rate, strong biological activity and low toxicity. At present, researchers have synthesized a large number of organic selenium compounds with biological activity, including aromatic selenium compounds, diselenide, selenocyanide, etc. Among them, the antioxidant drug ebselen and the antitumor drug selenazofuran have entered the third phase and Phase I clinical study.

In the previous research, our group synthesized a selenium heterocyclic compound: organoselenium 1b (SeD-1b), the molecular formula is C ₂₀ H ₁₄ ON ₄ Se, which is a class of selenadiazole derivatives and is a dark yellow solid. SeD-1b is easily soluble in organic solvents such as DMSO, and almost insoluble in water. Previous research results showed that SeD-1b has significant anti-bladder cancer selectivity and activity, and it has high stability in the urine environment, which induces BCa cell apoptosis through ROS-mediated p53-AKT/MAPK signaling pathway. Compared with traditional anti-BCa drugs, it has low toxicity to normal bladder epithelial cells. However, although SeD-1b is obviously superior to traditional anti-BCa drugs, it also has the following shortcomings: 1. SeD-1b is a chemical small molecule drug, which is usually dispersed and adsorbed to the bladder cancer site in a free diffusion manner. However, due to the small molecule drug Short metabolic lifespan leads to poor targeting of SeD-1b; 2. Due to the special structure of the bladder, drugs that reach the tumor site are easily diluted and cleared by urine; 3. Although SeD-1b has a certain stability in the urine environment However, the solubility still needs to be improved; 4. Although SeD-1b itself has fluorescence, its luminescence ability is weak, and it cannot be used as a localization agent for diagnosis, nor does it have the ability to apply it to enhanced imaging examinations, so it cannot Serve the efficacy monitoring of BCa and reduce the pain of patients undergoing cystoscopy. The above shortcomings limit the application of SeD-1b in anti-tumor.

Tumor microenvironment (TME) refers to the structure, function, metabolism of the tumor tissue and other related physicochemical indicators of the internal environment of the tumor during the occurrence, growth and metastasis of tumors, such as low pH (<7), partial pressure of oxygen Low (<1.3%), high H ₂ O ₂ expression, etc. The hypoxic microenvironment inside the tumor makes cancer cells resistant to both radiotherapy and chemotherapy, significantly reducing the therapeutic effect of radiotherapy and chemotherapy. The current clinical research on the unique hypoxic microenvironment of tumors shows that 80% of bladder cancer patients are in a hypoxic state, and the hypoxic microenvironment in bladder tumors is one of the main factors affecting the effect of chemotherapy and the prognosis of patients. In order to improve the hypoxic environment inside the tumor, the existing technology often adopts the method of delivering oxygen to the tumor tissue, such as hyperbaric oxygen chamber, to increase the hypoxic environment inside the tumor.

However, there is no targeted nanomedicine that can not only overcome the drug resistance problem caused by tumor hypoxia, but also monitor the tumor treatment process in real time.

SUMMARY OF THE INVENTION

The primary purpose of this application is to provide a method for preparing a targeted nanomedicine based on the guidance of nuclear magnetic resonance imaging to overcome the drug resistance caused by tumor hypoxia.

Another object of the present application is to provide a targeted nanomedicine that can overcome the drug resistance caused by tumor hypoxia based on the guidance of nuclear magnetic resonance imaging.

Another object of the present application is to provide the application of the above-mentioned targeted nanomedicine based on nuclear magnetic imaging guidance for overcoming the drug resistance caused by tumor hypoxia in anti-tumor.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

A preparation method of a targeted nanomedicine for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, comprising the following steps:

S1. Disperse PLGA, MRI contrast agent and antitumor drug in an organic solvent respectively, then add a surfactant, and obtain solution A after stirring and dialysis;

S2. Use NHS and EDC to activate the carboxyl group of solution A to obtain activated solution A;

S3. Add targeting molecules to the activated solution A, and after stirring and dialysis, a targeted nanomedicine based on the guidance of nuclear magnetic resonance imaging to overcome the drug resistance caused by tumor hypoxia is obtained.

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

In step S1, the nuclear magnetic imaging contrast agent is at least one of Fe ₃ O ₄ nanoparticles and gadopentetate meglumine injection; preferably Fe ₃ O ₄ nanoparticles.

The particle size of the Fe ₃ O ₄ nanoparticles is 5-200 nm; preferably 5-50 nm; more preferably 10 nm.

In step S1, the antitumor drugs are daunorubicin, doxorubicin, daunorubicin, epirubicin, vinblastine, vincristine, tamoxifen, formestane, and anastrozole , flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, tegafur and selenodiazole derivatives (SeD-1b) At least one of ; preferably a selenodiazole derivative (SeD-1b).

The SeD-1b is an anti-tumor drug synthesized by our research group based on the previous research results, and the preparation method is shown in the application number 201610127128.3. On the one hand, due to the hydrophobic nature of SeD-1b, it can enter the hydrophobic cavity of polymer nanoparticles and realize the loading of antitumor drugs on polymer carriers. On the other hand, SeD-1b can induce BCa cell apoptosis through ROS-mediated p53-AKT/MAPK signaling pathway due to its remarkable anti-bladder cancer selectivity and activity, and its high stability in the urine environment. , and low toxicity to normal bladder epithelial cells. Therefore, combining SeD-1b with PLGA polymer nanoparticles can ensure its good antitumor activity and efficiently inhibit tumor growth.

In step S1, the PLGA, the MRI contrast agent and the antitumor drug in the solution A are in a molar ratio of (1-40):(1-10):(1-10); preferably a molar ratio of (20- 40):(1～2):(1～2) ratio; more preferably, the molar ratio is 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 an aqueous solution of Tween; in the aqueous solution of Tween, the concentration of Tween is 1-10 mM; preferably 2-8 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 dropped into the surfactant by dropwise addition.

The speed of the dropwise addition is that the interval between each drop is 1-20 seconds; preferably, it is 10-15 seconds.

In 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 equivalent weight of the dialysis membrane is 300-10000kDa; preferably 3000-10000kDa; more preferably 10000kDa.

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 consumption of described N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and PLGA is according to NHS :EDC:PLGA=molar ratio of 1:1:1～40; preferably by NHS:EDC:PLGA=molar ratio of 1:1:20～40; more preferably by NHS:EDC:PLGA=molar ratio of 1 :1:20 ratio.

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

In step S3, the targeting molecules are cyclic RGD polypeptide (cRGD), folic acid (FA), transferrin, activatable cell penetrating peptide (ACPP), MUC-1 annexin, galactosamine, neovascularization At least one of targeting peptide, granulocyte macrophage stimulating factor and PLZ4; preferably PLZ4.

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

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

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

In step S3, the dialysis is dialysis membrane dialysis.

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

When the nuclear magnetic imaging contrast agent of the present invention is Fe ₃ O ₄ nanoparticles, the anti-tumor drug is SeD-1b, and the targeting molecule is PLZ4, the solution A is PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution, The finally obtained targeted nanomedicine based on MRI-guided overcoming the drug resistance caused by tumor hypoxia is the targeted nanomedicine PLZ4@SeD nanoparticles.

In steps S1 to S3, the operations described above can be performed at normal temperature. The normal temperature is 15-35°C; more preferably 20-30°C.

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

The targeted nanomedicine needs to be stored in a sol form at 1-25°C.

The application of the above-mentioned targeted nanomedicine based on the guidance of nuclear magnetic resonance imaging to overcome the drug resistance caused by tumor hypoxia in the preparation of antitumor drugs.

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

The mechanism of the present invention is:

1. PLGA has good biocompatibility and is a clinical pharmaceutical excipient verified by the US FDA; it can not only connect different target molecules through the polymer chain, and play a steric stabilization role; The charge plays a role in electrostatic stabilization, which can effectively improve the stability and dispersibility of various nanoparticles.

2. Targeting polypeptide PLZ4 is a cyclic peptide specific to bladder cancer cells and has the ability to target bladder cancer cells; PLZ4 has an amino group, which can undergo dehydration condensation reaction with the carboxyl group of activated PLGA to form an amide bond.

3. SeD-1b can effectively up-regulate the production of intracellular reactive oxygen species (ROS) after entering cells through endocytosis to activate the phosphorylation of p53 protein in the apoptosis pathway, thereby further inducing bladder tumor cell apoptosis and inhibiting tumors. proliferation of cells.

4. Ferric oxide nanoparticles (Fe ₃ O ₄ nanoparticles) have superparamagnetic properties and can reduce T2 relaxation, making them an ideal contrast agent for nuclear magnetic resonance imaging (MRI); in addition, Fe ₃ O ₄ nanoparticles can The Fenton reaction and the Haber-Weiss reaction catalyze H ₂ O ₂ to generate O ₂ , thereby improving the hypoxic microenvironment inside the tumor; in addition, Fe ₃ O ₄ nanoparticles have good biocompatibility and are almost suitable for normal cells and tissues. Nontoxic. It can be seen that Fe ₃ O ₄ nanoparticles are not only a good contrast agent, but also a good catalyst. Fe ₃ O ₄ nanoparticles enter into the lipophilic core-shell of PLGA polymer nanoparticles through hydrophobic interaction to realize the loading of NMR imaging drugs by polymer carriers. _At the same time, _Fe3O4 nanoparticles, also known as T2 negative contrast agents, have good superparamagnetic properties and are widely considered to be ideal contrast agents for magnetic resonance imaging (MRI) and one of the most sensitive MR contrast agents at present. It can significantly shorten the T2 relaxation time and darken the T2-weighted image; in addition, Fe ₃ O ₄ nanoparticles have the properties of Fenton reaction, which can catalyze the degradation of H ₂ O ₂ .

5. Using the oil-in-water method, with acetone as the inner oil phase and water as the continuous outer phase, the hydrophobic SeD-1b and Fe ₃ O ₄ nanoparticles were dispersed in acetone and then wrapped in PLGA, and finally dispersed in water , the structure of PLGA-wrapped _SeD -1b and _Fe3O4 nanoparticles was obtained.

6. NMR-guided bladder cancer therapy targeting nanodrugs is to use the targeting ability of PLZ4 to enable PLGA-encapsulated SeD-1b to reach the tumor area, and enhance Fe ₃ O ₄ on the basis of the confinement effect of PLGA micelles The ability of nanoparticles to catalyze H ₂ O ₂ can provide a large amount of O ₂ , improve the hypoxic environment in the tumor area, reduce the drug resistance caused by tumor hypoxia, and further improve the ability of SeD-1b to induce tumor cell apoptosis, thereby inhibiting the proliferation of tumor cells.

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

(1) The therapeutic effect of the targeted nano-drug PLZ4@SeD nanoparticles of the present invention is better than that of clinical drugs such as doxorubicin, mitomycin and pirarubicin under the condition of hypoxia.

(2) In the present invention, Fe ₃ O ₄ nanoparticles have the ability to catalyze H ₂ O ₂ to generate O _2. After being encapsulated by PLGA, the overall catalytic ability is significantly enhanced due to the confinement effect formed in the PLGA micelles. The ability to improve the hypoxic microenvironment of the tumor site is improved, the drug resistance caused by tumor hypoxia is reduced, and the effect of tumor treatment is finally improved.

(3) The selectivity of PLZ4 for bladder cancer is better than that of other peptides. The introduction of PLZ4 makes the nanoparticles have better targeting and lower toxic and side effects. The biocompatibility of PLGA is better, the synthesis method is simple, and it is cheaper and easier to obtain. Compared with the clinical drug mitomycin for bladder cancer infusion, SeD-1b has higher safety and less toxic and side effects.

(4) The targeted nano-drug PLZ4@SeD nanoparticles prepared by the method of the present invention not only overcomes the drug resistance problem caused by tumor hypoxia, but also realizes real-time monitoring of the tumor treatment process, and finally achieves high efficiency and low Toxic, stable and inexpensive treatment of cancer.

Description of drawings

Fig. 1 is a graph showing the characterization results of the targeted nanomedicine PLZ4@SeD nanoparticles prepared in Example 1; wherein, Fig. A is the TEM photo and particle size measurement results of PLZ4@SeD nanoparticles; Fig. B is the PLGA nanoparticle Potential measurement results of particles, Fe ₃ O ₄ nanoparticles, PLGA@SeD nanoparticles, activated PLGA@SeD/Fe ₃ O ₄ nanoparticles, and PLZ4@SeD nanoparticles; Figure C shows PLGA nanoparticles, SeD- 1b, UV-Vis absorption spectra of PLZ4@SeD nanoparticles; D is the fluorescence spectra of Fe ₃ O ₄ nanoparticles, PLGA nanoparticles, SeD-1b, PLZ4@SeD nanoparticles; E is PLZ4@ The in vitro MRI images of SeD nanoparticles and their T ₂ relaxation rate maps; Figure F shows the infrared spectra of PLZ4@SeD nanoparticles, PLZ4@SeD/Fe ₃ O ₄ nanoparticles, SeD-1b, and PLGA, respectively.

Figure 2 is a graph showing the stability of the targeted nano-drug PLZ4@SeD nanoparticles described in Example 2 under different conditions; wherein, Figure A shows the targeted nano-drug PLZ4@SeD nanoparticles at pH 5.6, respectively, in phosphoric acid Stability curves in salt buffered saline (PBS) and DMEM medium; Figure B shows the stability of targeted nanomedicine PLZ4@SeD nanoparticles in phosphate buffered saline (PBS) and DMEM medium with pH 7.4, respectively Sex graph.

Figure 3 is a graph showing the results of cell viability after adding Fe ₃ O ₄ nanoparticles to different cancer cells.

Figure 4 shows the results of the half-inhibitory concentration of each drug on each cell after adding SeD-1b, PLGA@SeD nanoparticles, PLGA@SeD/Fe ₃ O ₄ nanoparticles and PLZ4@SeD nanoparticles to different cancer cells, respectively.

Figure 5 is a graph showing the results of the half-inhibitory concentration of different drugs on bladder cancer EJ cells under hypoxia.

Figure 6 is a graph showing the immunofluorescence effect of the receptor integrin αvβ3 corresponding to the polypeptide PLZ4 expressed in the isolated human normal bladder tissue and bladder cancer tissue.

Figure 7 shows the results of selective absorption of PLZ4@SeD nanoparticles by bladder cancer EJ cells and normal bladder cells SV-HUC-1; among them, Figure A shows the nanomedicine PLZ4@SeD in bladder cancer EJ cells under different time conditions Fluorescence intensity results of nanoparticles; Figure B shows the fluorescence intensity results of nanomedicine PLZ4@SeD nanoparticles in normal bladder epithelial cells SV-HUC-1 under different time conditions.

Figure 8 is a graph showing the performance of the targeted nanomedicine PLZ4@SeD nanoparticles in catalyzing H ₂ O ₂ in vitro; in which, Figure A shows the PLGA nanoparticles, SeD-1b, Fe ₃ O ₄ nanoparticles, and PLZ4@SeD nanoparticles with H 2 O 2 nanoparticles, respectively. The effect diagram of the effect of ₂ O ₂ ; Picture B is the result of oxygen generation after PLZ4@SeD nanoparticles, Fe ₃ O ₄ nanoparticles, and PLGA nanoparticles reacted with H ₂ O ₂ within 15 minutes; Picture C is 15 Within minutes, different concentrations of H ₂ O ₂ solutions were mixed with PLZ4@SeD nanoparticles and the results of oxygen production; D is the cyclic voltammetry results of PLZ4@SeD nanoparticles under argon protection.

Figure 9 is a graph showing the effect of Fe ₃ O ₄ nanoparticles, SeD-1b, and PLZ4@SeD nanoparticles on the viability of bladder cancer EJ cells under different concentrations under hypoxic conditions; among them, picture A is in a hypoxic environment The results of the effect of Fe ₃ O ₄ nanoparticles, SeD-1b, and PLZ4@SeD nanoparticles on the viability of bladder cancer EJ cells under different concentrations of Fe 3 O 4 nanoparticles, respectively; Figure B shows the results under hypoxia and hydrogen peroxide conditions. Figures showing the effect of Fe ₃ O ₄ nanoparticles, SeD-1b, and PLZ4@SeD nanoparticles on the viability of bladder cancer EJ cells under different concentrations.

Figure 10 shows the results of the content of targeted nano-drug PLZ4@SeD nanoparticles in bladder cancer EJ cells; among them, Figure A shows the absorption results of bladder cancer EJ cells for targeted nano-drug PLZ4@SeD nanoparticles under different time conditions Figure; Figure B shows the results of the absorption of the targeted nanodrug PLZ4@SeD nanoparticles at different final concentrations by bladder cancer EJ cells at 8h.

Figure 11 shows the intracellular localization of the targeted nanodrug PLZ4@SeD nanoparticles in bladder cancer EJ cells under different time conditions.

Figure 12 is a graph showing the change of cellular reactive oxygen species level with time after the targeted nanodrug PLZ4@SeD nanoparticles acted on bladder cancer EJ cells; in which, Figure A is the DHE probe to detect different concentrations of the targeted nanodrug PLZ4@SeD Figure B shows the change trend of reactive oxygen species generated after incubation of bladder cancer EJ cells with nanoparticles; Figure B shows the trend of reactive oxygen species generated after incubation of bladder cancer EJ cells with different concentrations of targeted nanodrug PLZ4@SeD nanoparticles.

Figure 13 is the cell cycle diagram of bladder cancer EJ cells after different concentrations of targeted nano-drug PLZ4@SeD nanoparticles act on bladder cancer EJ cells; among them, Figure A shows the effect of different concentrations of PLZ4@SeD nanoparticles on bladder cancer EJ cells The results of the effect of cycle; Figure B is the effect of different concentrations of nanoparticles PLZ4@SeD nanoparticles on the distribution of bladder cancer EJ cell cycle.

Fig. 14 is a graph showing the confocal laser results of bladder cancer EJ cell tumor spheres in the sealed group and the unsealed group.

Figure 15 is a diagram showing the effect of the targeted nanodrug PLZ4@SeD nanoparticles perfusion of human in vitro bladder tissue containing bladder cancer tumor; wherein, Figure A is the human in vitro bladder tissue containing bladder cancer tumor before drug perfusion Figure; B is the anatomical view of the isolated human bladder tissue containing bladder cancer tumor after drug infusion; C is the partial view of the bladder after 4 times magnification of B; D is the nano-drug PLZ4@SeD nanoparticle after infusion , the tissue axial MRI results of human isolated bladder containing bladder cancer tumor; E is the result of R ₂ ^* values corresponding to different bladder regions after the targeted nano-drug PLZ4@SeD nanoparticles acted on the isolated bladder ; Figure F shows the results of T ₂ values corresponding to different bladder regions after the targeted nano-drug PLZ4@SeD nanoparticles acted on the isolated bladder.

Figure 16 is a graph showing the particle size measurement results of the targeted nanomedicines prepared when different organic solvents are used as dispersants.

Detailed ways

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

The reagents used in the examples can be routinely purchased from the market unless otherwise specified.

The bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells, liver cancer cells HepG2, and normal bladder epithelial cells SV-HUC-1 in the examples of the present invention are all collected from American model culture collections. Inventory ATCC purchases.

Acetone, acetonitrile and dichloromethane used in this example were purchased from Tianjin Jindong Tianzheng Fine Chemical Reagent Factory; Fe ₃ O ₄ nanoparticles were purchased from Sigma Company; PLGA was purchased from Sigma Company of the United States; DMEM medium was purchased from Gibco Company of the United States Company; Malvern laser particle size analyzer was purchased from Malvern Company, UK; MTT was purchased from Sigma Company; Multifunctional fluorescence microplate reader model ELX800 was purchased from Bio-Tek Company of the United States; BSA was purchased from Guangzhou Jabes Biotechnology Co., Ltd. (GBCBIO Company ); integrin αvβ3 antibody was purchased from Cell Signaling Technology; integrin goat anti-mouse immunoglobulin was purchased from Cell Signaling Technology; Hoechst was purchased from Merck, Germany; cell lysate was purchased from Biyuntian; 608, purchased from China Leica Company; flow cytometer model CytoFLEX S, purchased from Beckman Coulter Company of the United States; propidium iodide purchased from Sigma Company; laser confocal microscope purchased from Leica Company; PLZ4 purchased from Gill Biochemical (Shanghai) Co., Ltd. company.

Example 1: Preparation of targeted nanomedicine

(1) Preparation of raw materials:

1) Preparation of 3.5 mM Tween-80: Weigh 5 g of Tween-80, and use 1 L of secondary water to prepare an aqueous solution of Tween-80 with a concentration of 3.5 mM.

2) Preparation of 1.05mM Fe ₃ O ₄ nanoparticles-acetone dispersion: Weigh 0.2436g Fe ₃ O ₄ nanoparticles and disperse them in 1L of acetone.

3) Preparation of 20 mM PLGA-acetone dispersion: Weigh 20 g of PLGA (lactic acid LA: glycolic acid GA=50:50, Mn=13000) and disperse it in 1 L of acetone.

4) Preparation of 0.65 mM SeD-1b solution: Weigh 24.375 mg of SeD-1b (SeD-1b is prepared according to Example 1 in the patent "201610127128.3") and dissolve it in 10 mL of dimethyl sulfoxide.

5) Preparation of 20 mg/mL PLZ4 solution: Weigh 20 g of polypeptide PLZ4 powder and dissolve it in 1 L of phosphate buffered saline (PBS) with a pH of 7.4 and a concentration of 0.01 M.

(2) Preparation of targeted nanomedicine PLZ4@SeD nanoparticles

1) Preparation of PLGA nanoparticle aqueous solution

Take 1 mL of the above-mentioned 20 mM PLGA-acetone dispersion to 3 mL with acetone, drop it into 10 mL of the above-mentioned 3.5 mM Tween-80 aqueous solution dropwise at a rate of 10 seconds/drop, stir at 800 rpm for 12 hours, and the dialysis membrane equivalent is 10000 kDa After dialysis in the dialysis bag for 12 hours, the PLGA polymer nanoparticle aqueous solution was obtained after dialysis.

₂ ) Preparation of PLGA@ _Fe3O4 nanoparticles

Take 1mL of the above 20mM PLGA-acetone dispersion and mix it with 1mL of the above 1.05mM Fe ₃ O ₄ nanoparticle-acetone dispersion, then dilute to 3mL with acetone, and then add the mixed solution to the volume at a rate of 10 seconds/drop. Dropwise into 10 mL of the above-mentioned 3.5 mM Tween-80 aqueous solution, stirred at 800 rpm overnight, and dialyzed in a dialysis bag with a dialysis membrane equivalent of 10000 kDa for 12 hours to obtain a PLGA@Fe ₃ O ₄ nanoparticle aqueous solution; wherein, the obtained PLGA@ In the Fe ₃ O ₄ nanoparticle aqueous solution, the concentration of Fe ₃ O ₄ was 0.08 mM.

3) Preparation of PLGA@SeD nanoparticles

Take 1 mL of the above-mentioned 20 mM PLGA-acetone dispersion and 200 μL of the above-mentioned 0.65 mM SeD-1b solution to 3 mL with acetone, and then drop the mixture after the constant volume dropwise into 10 mL of the above-mentioned 3.5 mM Tween at a rate of 10 seconds/drop. -80 aqueous solution, stirred at 800 rpm for 12 hours, and dialyzed in a dialysis bag with a dialysis membrane equivalent of 8000 kDa 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 to 10 μM.

₄ ) Preparation of PLGA@SeD/ _Fe3O4 nanoparticles

Take 1 mL of the above-mentioned 20 mM PLGA-acetone dispersion, 1 mL of 1.05 mM Fe ₃ O ₄ nanoparticle-acetone dispersion and 200 μL of 0.65 mM SeD-1b solution, respectively, and adjust the volume to 3 mL with acetone, and then adjust the volume of the mixture to 3 mL for 10 seconds. PLGA@SeD/Fe ₃ O ₄ nanoparticles were obtained after 12 hours of dialysis in a dialysis bag with a dialysis membrane equivalent of 8000 kDa after 10 mL of the above-mentioned 3.5 mM Tween-80 aqueous solution was added dropwise at a rate of 1 drop/drop. aqueous solution; wherein, the concentrations of Fe ₃ O ₄ and SeD-1b in the obtained PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution were 0.08 mM and 10 μM, respectively.

5) Activation of PLGA@ _SeD / _Fe3O4 nanoparticles

To the PLGA@SeD/Fe ₃ O ₄ nanoparticles prepared in step 4), 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 12 mg of N -Hydroxysuccinimide (NHS) powder, stirred at 25 °C, 800 rpm for 3 hours to obtain an activated PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution.

6) Preparation of targeted nanomedicine PLZ4@SeD nanoparticles

Take 1 mL of the above-mentioned 20 mg/mL PLZ4 solution, add it to 19 mL of the above-mentioned activated PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution, stir at 800 rpm for 12 hours, and dialyze for 12 hours in a dialysis membrane with a dialysis membrane equivalent of 10000 kDa, The targeted nano-drug PLZ4@SeD nanoparticles were obtained and stored in a refrigerator at 4°C.

(3) Relevant tests and results

The prepared targeted nanomedicine PLZ4@SeD nanoparticles were tested as follows, and the results are shown in Figure 1:

1) The morphology of PLZ4@SeD nanoparticles was characterized by Hitachi H-7650 transmission electron microscope, and the results are shown in Figure A in Figure 1.

From Figure A in Figure 1, it can be seen that the PLZ4@SeD nanoparticles have good dispersibility and the particle size is about 120 nm, which is consistent with the electron microscope results, indicating that the PLZ4@SeD nanoparticles have good dispersibility and the particle size is about 120 nm. spherical nanoparticles.

2) Using Nano-ZS (Malvern Instruments Limited) to characterize PLGA nanoparticles, Fe ₃ O ₄ nanoparticles, PLGA@SeD nanoparticles, activated PLGA@SeD/Fe ₃ O ₄ nanoparticles, and PLZ4@SeD nanoparticles aqueous solutions, respectively The change of the potential is shown in Figure B in Figure 1.

From Figure B in Figure 1, it can be seen that Fe ₃ O ₄ nanoparticles, activated PLGA@SeD/Fe ₃ O ₄ nanoparticles and PLZ4@SeD nanoparticles have positive potentials, while PLGA nanoparticles and PLGA@SeD nanoparticles have negative potentials potential. Compared with PLGA nanoparticles and PLGA@SeD nanoparticles, the surface potential of PLZ4@SeD nanoparticles is higher; the surface potential of Fe ₃ O ₄ nanoparticles is higher than that of activated PLGA@SeD/Fe ₃ O ₄ nanoparticles and The PLZ4@SeD nanoparticles are higher, while the surface positive potentials of the activated PLGA@SeD/Fe ₃ O ₄ nanoparticles and PLZ4@SeD nanoparticles are not much different, indicating that the Fe ₃ O ₄ nanoparticles have strong surface positive potentials. It can reverse the negative surface potential of PLGA nanoparticles; while the negative surface potential of PLGA@SeD nanoparticles is stronger than that of PLGA, indicating that the SeD-1b encapsulated by PLGA has a negative potential; the negative potential of PLZ4@SeD nanoparticles The positive surface potential of the activated PLGA@SeD/Fe ₃ O ₄ nanoparticles is lower than that of the activated PLGA@SeD/Fe 3 O 4 nanoparticles, indicating that the polypeptide PLZ4 has a certain negative potential. Due to the presence of a large number of proteins on the surface of the cell membrane, the surface of the cell membrane has a negative potential, while PLZ4 has a negative potential. The positive surface potential of @SeD nanoparticles facilitates its binding to the cell membrane with negative potential, which is more conducive to the absorption of targeted nanodrugs by cancer cells.

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

It can be seen from Figure C in Figure 1 that the excitation wavelength of PLGA@SeD nanoparticles is the same as that of SeD-1b alone, which is 623 nm, indicating that SeD-1b is successfully loaded into the PLGA nanosystem and retains its own optical properties. .

4) The optical properties of the prepared Fe ₃ O ₄ nanoparticles, PLGA polymer nanoparticles, SeD-1b, and PLZ4@SeD nanoparticles were analyzed by UV-Vis absorption spectrometer (UH4150), respectively, and the results are shown in Figure D in Figure 1. shown.

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

5) Panel E in Figure 1 is the result of 1/T ₂ signal characterization of PLZ4@SeD nanoparticles using the SGNA HOR120N magnetic resonance imaging instrument.

From E in Figure 1, it can be seen that the 1/T ₂ signal of PLZ4@SeD nanoparticles increases with the increase of iron concentration in a dose-dependent manner, indicating that PLZ4@SeD nanoparticles have the effect of reducing the T ₂ relaxation rate , that is, it has the ability to enhance the effect of _T2 contrast.

6) The chemical structures of PLZ4@SeD nanoparticles, SeD-1b, and PLGA polymer nanoparticles were characterized by Fourier transform infrared spectrometer. The results are shown in Figure F in Figure 1.

It can be seen from Figure F in Figure 1 that peaks appear at 3258cm ^-1 and 1590cm ^-1 , both of which are amide bonds, that is, PLZ4 and PLGA form amide bonds through dehydration condensation, indicating that the targeting polypeptide PLZ4 has been successfully connected to the activated peptide. PLGA@SeD/Fe ₃ O ₄ nanoparticles.

Example 2: In vitro stability study of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

The mixed solutions for detecting the particle size of nanomedicine PLZ4@SeD nanoparticles were prepared according to the following methods:

Group A1: 1 mL of dialyzed PLZ4@SeD nanoparticle aqueous solution was mixed with pH=5.6, 0.01M phosphate buffer at a volume ratio of 1:2;

Group A2: 1 mL of dialyzed PLZ4@SeD nanoparticle aqueous solution was mixed with DMEM medium with pH=5.6 adjusted with hydrochloric acid in a volume ratio of 2:1;

Group B1: 1 mL of dialyzed PLZ4@SeD nanoparticle aqueous solution was mixed with pH=7.4, 0.01M phosphate buffer at a volume ratio of 2:1;

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

Using Malvern laser particle size analyzer (Zetasizer Nano-ZS), the mixed solutions of A1, A2, B1, B2 groups were analyzed at 0h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, At the time point of 144 h, the particle size of the nano-drugs was detected, and the stability of the PLZ4@SeD nanoparticles was analyzed. The results are shown in Figure 2.

It can be seen from Figure 2 that the stability curve of PLZ4@SeD nanoparticles under acidic conditions is similar to the stability curve under neutral conditions, indicating that the prepared PLZ4@SeD nanoparticles are not easily degraded in acidic solution. The drug-loaded effect lasts longer.

Example 3: Comparison of in vitro antitumor activity of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Take bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells, and liver cancer cells HepG2 with a growth density of 2×10 ⁴ cells/mL in logarithmic phase, and inoculate the above cells respectively. In a 96-well plate, 100 μL per well; after the cells adhered, the cells in the first group were added to the wells containing bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells, and liver cancer cells HepG2 respectively. Add 100 μL of Fe ₃ O ₄ nanoparticles diluted in DMEM medium to a final concentration of 0.08 mmol/L. Taking bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer cells HepG2 without Fe ₃ O ₄ nanoparticles as controls, 48 hours later, 30 μL of 5 mg/well was added to each well. mL of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) was incubated at 37°C for 3 hours in the dark, and the MTT-containing supernatant was discarded , add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake at constant temperature at 37°C for 15 minutes, and use a multifunctional fluorescence microplate reader to test the viability of Fe ₃ O ₄ nanoparticles on the above five kinds of cells under the condition of wavelength of 570 nm. The results are shown in Figure 3.

It can be seen from Figure 3 that Fe ₃ O ₄ nanoparticles have almost no toxicity to the above five types of tumor cells, and the survival rates of the five types of cells are all close to 100%.

In addition, the second group contained 100 μL of adherently grown bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer cells HepG2 cells at a density of ² x 104 cells/mL, respectively. With 20 μM as the highest concentration in each well of 20 μM, 100 μL of SeD-1b diluted in DMEM was added in gradient concentrations of 10 μM, 5 μM, 2.5 μM, and 1.25 μM in dimethyl sulfoxide; ×10 ⁴ cells/mL of adherent-growing bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells, and hepatoma cells HepG2 were added to each well at the highest concentration of 20 μM in a gradient concentration 100 μL of PLGA@SeD nanoparticles diluted in DMEM, the concentration gradient is the same as that of the second group; the fourth group contains adherent-growing bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer cells at a density of 2×10 ⁴ cells/mL, respectively. To each well of cancer T921 cells, breast cancer MCF-7 cells, and liver cancer cells HepG2, 100 μL of PLGA@SeD/Fe ₃ O ₄ nanoparticles diluted in DMEM were added at the highest concentration of 20 μM in a gradient concentration, the concentration gradient was the same as that of the second group; To the fifth group of wells containing adherent-growing bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells, and liver cancer cells HepG2 at a density of 2×10 ⁴ cells/mL, respectively. 100 μL of PLZ4@SeD nanoparticles diluted in DMEM were added at the highest concentration of 20 μM, and the concentration gradient was the same as that of the second group; without SeD-1b, PLGA@SeD nanoparticles, and PLGA@SeD/Fe ₃ O ₄ nanoparticles And PLZ4@SeD nanoparticles containing adherent growth bladder cancer EJ cells, bladder cancer J82 cells, bladder cancer T921 cells, breast cancer MCF-7 cells and liver cancer cells HepG2 as a control, 48 hours later, 30 μL was added to the above wells 5 mg/mL of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) was incubated at 37°C in the dark for 3 hours, and the MTT-containing supernatant was discarded. Add 150 μL of dimethyl sulfoxide (DMSO) to each well of the supernatant, and shake at 37°C for 15 minutes. Under the condition of a wavelength of 570 nm, use a multifunctional fluorescence microplate reader to test the above concentrations of SeD-1b and PLGA. The absorption values of cells treated with @SeD nanoparticles, PLGA@SeD/Fe ₃ O ₄ nanoparticles and PLZ4@SeD nanoparticles were obtained, and the regression curves were obtained. The half-inhibitory concentrations of PLGA@SeD/Fe ₃ O ₄ nanoparticles and PLZ4@SeD nanoparticles on different cells are shown in Figure 4.

It can be seen from Figure 4 that compared with SeD-1b, the half-inhibitory concentrations of PLZ4@SeD nanoparticles on different cells were significantly decreased, and PLZ4@SeD nanoparticles were more toxic to bladder cancer EJ cells.

In addition, to each well of the fourth group containing 100 μL of adherent-growing bladder cancer EJ cells at a density of 2×10 ⁴ cells/mL, 20 μM was the highest concentration in a gradient concentration of 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively. Clinical drugs diluted in DMEM medium were added in a volume of 100 μL: doxorubicin hydrochloride, mitomycin, pirarubicin and targeted nano-drug PLZ4@SeD nanoparticles; without doxorubicin hydrochloride, mitomycin Adherent-grown bladder cancer EJ cells containing 100 μL, 2×10 ⁴ cells/mL, and PLZ4@SeD nanoparticles were used as controls in a gas composition of 5% carbon dioxide, 1% oxygen, 94 30 μL of 5 mg/mL 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) was added to the above wells after 48 hours under hypoxic conditions of % nitrogen. After incubating in the dark at 37°C for 3 hours, discard the supernatant containing MTT, add 150 μL DMSO to each well, and shake at 37°C for 15 minutes. Under the above hypoxic conditions, the absorption values of bladder cancer EJ cells treated with the above different concentrations of doxorubicin hydrochloride, mitomycin, pirarubicin and PLZ4@SeD nanoparticles were obtained, and the regression curve was obtained and substituted into the curve The effect of each drug on the half-inhibitory concentration of bladder cancer EJ cells was obtained, and the results are shown in FIG. 5 .

It can be seen from Figure 5 that under hypoxic conditions, the IC ₅₀ of the targeted nanomedicine PLZ4@SeD nanoparticles is lower than the half-inhibitory concentrations of doxorubicin hydrochloride, mitomycin, and pirarubicin, indicating the present invention The prepared targeted nano-drug PLZ4@SeD nanoparticles have stronger efficacy.

Example 4: Evaluation of immunofluorescence effect of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Dehydration of the isolated human normal bladder tissue containing normal bladder epithelial cells SV-HUC-1 and muscle-invasive bladder cancer tissue containing bladder cancer EJ cells: frozen at -80℃ with OCT, 8μm serial sections , the obtained frozen sections were stored at -20 °C; the sections were placed in a dark and humid box to rewarm and rehydrate, and the excess water was dried, and then sealed with bovine serum albumin solution (5% BSA) at room temperature for 30 minutes, and then dried, directly 50 μL of integrin αvβ3 primary antibody at a dilution of 1:1000 was added dropwise, wherein integrin αvβ3 is the target of PLZ4 targeting cells. overnight at 4°C, then placed at room temperature for half an hour to warm up, and washed three times with PBS to remove excess integrin αvβ3 primary antibody that is not bound to the tissue on the slice, and then drop 50 μL of the tissue on each slice according to 1:1500 diluted integrin goat anti-mouse immunoglobulin was incubated at room temperature for 2 hours. After the incubation time, 50 μL of 1 μg/mL Hochest was added dropwise to the tissue on each slice, and incubated at room temperature for 30 minutes. After that, the fluorescence microscope was used to take pictures and analyze the protein expression in normal tissue and tumor tissue. The results are shown in Figure 6.

It can be seen from Figure 6 that the expression level of integrin αvβ3 in bladder cancer tissue area is significantly higher than that in normal bladder tissue. It is inferred that the high expression of integrin αvβ3 in muscle-invasive bladder cancer tissue is conducive to the active targeting of PLZ4 in the targeted nanomedicine PLZ4@SeD nanoparticles to bladder cancer cells.

Example 5: Comparison of in vitro selective absorption effects of targeted nanomedicines

The preparation method of the activated PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution in this example is the same as the preparation method of the activated PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution described in Example 1.

Weigh 2.5 mg of PLZ4 powder, and prepare 0.3125 mg/mL, 0.625 mg/mL, 1.250 mg/mL, 2.500 mg/mL PLZ4 solution with PBS buffer (pH 7.4, 0.01 M), respectively, for standby use; take 1 mL each for activation The resulting PLGA@SeD/Fe ₃ O ₄ nanoparticle aqueous solution was mixed with the above PLZ4 solutions of different concentrations in a volume ratio of 4:1, stirred at room temperature for 12 h, dialyzed with an equivalent 8000 kDa dialysis membrane for 12 h, and cultured in DMEM. After base dilution, the targeted nanomedicine PLZ4@SeD nanoparticles with targeted concentrations of 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, and 0.5 mg/mL were obtained, respectively.

The logarithmic growth of bladder cancer EJ cells and normal bladder epithelial cells SV-HUC-1 were seeded in 96-well plates at a density of 8×10 ⁴ cells/mL, 100 μL per well; Time points: 2h, 4h, and 8h, adding 100 μL of the above-mentioned targeted nanodrug PLZ4@SeD nanoparticles with different targeted concentrations, and at the same time, the bladder cancer EJ cells and normal bladder epithelium without the targeted nanodrug PLZ4@SeD nanoparticles were added respectively. Cell SV-HUC-1 was used as a control group; washed with PBS at 4°C for 2 to 3 times to remove the excess targeted nano-drug PLZ4@SeD nanoparticles on the surface of cancer cells, added 100 μL/well of cell lysate, and shaken at 37°C After 15 minutes, the cells were fully disrupted, and the fluorescence value with an excitation wavelength of 400 nm and an emission wavelength of 623 nm was measured by a multifunctional fluorescent microplate reader. The results are shown in Figure 7.

It can be seen from panel A in Figure 7 that as the concentration of the targeting polypeptide PLZ4 in the nanomedicine PLZ4@SeD nanoparticles increases, the detected fluorescence intensity also increases, that is to say, the absorption of PLZ4@SeD nanoparticles by bladder cancer EJ cells With the increase of the concentration of the targeting polypeptide PLZ4 in the nanomedicine PLZ4@SeD nanoparticles; and the fluorescence intensity also became stronger with the passage of time. It can be seen that the effect of bladder cancer EJ cells on the nanomedicine PLZ4@SeD nanoparticles Absorption is also time-dependent.

It can be seen from panel B in Figure 7 that the fluorescence intensity of the nanodrug PLZ4@SeD nanoparticles in the detected normal bladder epithelial cells SV-HUC-1 did not increase with the concentration of the targeting polypeptide PLZ4 in the nanodrug PLZ4@SeD nanoparticles. There was a significant improvement and no enhancement over time, i.e., the uptake of PLZ4@SeD nanoparticles by normal bladder cells SV-HUC-1 was less pronounced than that of bladder cancer EJ cells. It shows that the targeted nanomedicine PLZ4@SeD has selectivity.

Example 6: In vitro catalytic H ₂ O ₂ performance of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application; the results of its in vitro catalytic H ₂ O ₂ performance are shown in FIG. 8 .

H2O2 at a concentration of 1.7M was prepared in PBS buffer (pH 7.4, _{0.01M )} . Take 5 quartz dishes containing 3.8 mL of H ₂ O ₂ with a concentration of 2 mol/L, respectively, and add 200 μL of PLGA nanoparticles (10.7 mM), SeD-1b (10 μM), and Fe ₃ O ₄ nanoparticles to them. (0.08mM), PLZ4@SeD nanoparticles (16μM), and 4mL of PBS buffer (pH7.4, 0.01M) and 1.7M H ₂ O ₂ solution were used as controls, respectively. After 10 minutes of reaction, pictures were taken. The results are as follows As shown in Figure A in Figure 8.

From Figure A in Figure 8, it can be seen that the ability of PLZ4@SeD nanoparticles to catalyze H ₂ O ₂ to generate O ₂ is significantly stronger than that of Fe ₃ O ₄ nanoparticles, while PLGA nanoparticles and SeD-1b hardly generate O ₂ , It can be seen that PLZ4@SeD nanoparticles can efficiently catalyze H ₂ O ₂ to generate O ₂ , which in turn can improve the hypoxic environment of bladder cancer cells.

Prepare 1.7mol/L H ₂ O ₂ with PBS buffer (pH 7.4, 0.01M), and deoxidize it; take 200 μL of PLGA nanoparticles (1.5 mg/mL), Fe ₃ O ₄ nanoparticles (0.08mM), PLZ4@SeD nanoparticles (16μM), and 3.8mL of 1.7MH ₂ O ₂ solution were thoroughly mixed, and the O ₂ value in the solution was measured continuously for 15 minutes using a dissolved oxygen meter. The results are shown in Figure 8 shown in Figure B.

From panel B in Fig. ₈ , it can be seen that the rate of _PLZ4 @ _SeD nanoparticles catalyzing H2O2 to produce _O2 is significantly higher _than that of PLGA nanoparticles and _Fe3O4 nanoparticles _catalyzing H2O2 in the same time period _The rate of O generation, that is, the confinement effect of PLZ4@ _SeD due to the PLGA _- wrapped _Fe3O4 makes its ability to catalyze the generation of _O2 from H2O2 significantly stronger _than that of _Fe3O4 nanoparticles alone _.

Prepare 3.8 mL of H ₂ O ₂ solutions with concentrations of 1.0 mM, 0.8 mM, 0.6 mM, and 0.4 mM, respectively, add 200 μL of 16 μM PLZ4@SeD nanoparticles to them, and immediately use a dissolved oxygen meter to measure the O ₂ value in the solution. The measurement was performed for 15 minutes with water as a control, and the results are shown in panel C in FIG. 8 .

From Figure C in Figure 8, it can be seen that the amount of O ₂ produced by PLZ4@SeD nanoparticles catalyzing H ₂ O ₂ is related to the concentration of H ₂ O _2. Taking 0.4-0.8 mM H ₂ O ₂ as an example, H ₂ The higher the concentration of O ₂ , the more oxygen generated, indicating that the PLZ4@SeD nanoparticles have a strong ability to catalyze H ₂ O ₂ to generate O ₂ .

The PBS buffer solution with pH of 7.0 and 0.01M was prepared, and the prepared PBS buffer solution was divided into three groups; the blank group was: PBS buffer solution with a volume of 2 mL; the control group I was: PBS buffer solution with a volume of 2 mL was used to prepare the concentration 1.0 mM H ₂ O ₂ solution; variable II group: 2 mL of 16 μM PLZ4@SeD nanoparticles were added to 2 mL of 1.0 mM H ₂ O ₂ solution prepared in PBS buffer. The redox potentials of the three groups of solutions were measured with an electrochemical workstation respectively, and argon gas was introduced for protection during the measurement. The results are shown in Figure D in Figure 8 .

It can be seen from the D figure in Figure 8 that the PBS buffer has almost no redox potential; H ₂ O ₂ has a certain redox potential due to its own disproportionation reaction; while the variable II group is due to the addition of the targeted nanomedicine PLZ4 The redox potential of the @SeD nanoparticles was significantly higher than that of the blank group and the control group I, and the redox potential curve had no significant bulge. It can be seen that the PLZ4@SeD nanoparticles have the ability to catalyze H ₂ O ₂ to generate O ₂ .

Example 7: In vitro hypoxia experiment of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Bladder cancer EJ cells were cultured under hypoxic environmental conditions consisting of 5% CO ₂ , 1% O ₂ , 94% N ₂ , and bladder cancer EJs in logarithmic growth phase at a density of 2×10 ⁴ cells/mL were cultured The cells were seeded in 96-well plates, 100 μL per well; after the cells adhered, the supernatant medium was discarded, and 100 μL of DMEM medium containing 400 μM H ₂ O ₂ was added as the treatment group, and DMEM without H ₂ O ₂ was added as the treatment group. The medium was used as the control group; then 100 μL of Fe ₃ O ₄ nanoparticles, SeD-1b, and PLZ4@SeD nanoparticles were added to the wells of the control group and the treatment group, respectively, at different concentrations, so that Fe ₃ O ₄ nanoparticles, SeD-1b The final concentrations of , PLZ4@SeD nanoparticles in the groups were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively. 48 hours after the addition, 30 μL of MTT with a concentration of 5 mg/mL was added to each well and incubated in the dark for 3 hours. After the supernatant containing MTT was discarded, 150 μL of dimethyl sulfoxide was added to each well, and it was shaken at room temperature for 15 minutes. The fluorescence microplate reader tested the absorbance at the wavelength of 570 nm and calculated the survival rate. The results of the in vitro hypoxia experiment are shown in Figure 9.

It can be seen from the in vitro hypoxia experiments of A and B in Figure 9 that under hypoxic conditions, the cell survival rate of bladder cancer EJ cells in the treatment group is lower than that in the control group, indicating that the lack of Bladder cancer EJ cells in oxygen environment are resistant to drugs, and the drug efficacy is significantly improved after the local hypoxic environment is improved by decomposing H ₂ O ₂ to generate O ₂ .

Example 8: In vitro absorption experiments of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Bladder cancer EJ cells in logarithmic phase growth were taken and seeded in a 6 cm diameter petri dish at a density of 8×10 ⁴ cells/mL, 6 mL per well. After the cells had adhered and grown for 24 hours, PLZ4@ SeD nanoparticles at a concentration of 2 μM, respectively, incubated for 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h, and then washed with PBS buffer at 4°C for 2 to 3 times to collect bladder cancer EJ cells. After centrifugation at 1500 rpm for 5 minutes , detected by flow cytometer, and finally analyzed the content of intracellular drug by software Flow Jo, and the result is shown in Figure A in Figure 10 .

According to the results of Panel A in Figure 10, when the drug concentration remained the same, the uptake of the targeted nanodrug PLZ4@SeD nanoparticles by bladder cancer EJ cells increased with time.

In addition, the above-mentioned bladder cancer EJ cells growing in logarithmic phase were inoculated according to the same method and conditions as above. After the cells had adhered to the wall for 24 hours, different concentrations of PLZ4@SeD nanoparticles were added to make the final concentration of 0.25 μM respectively. , 0.5μM, 1μM, 2μM, 4μM, and bladder cancer EJ cells without PLZ4@SeD nanoparticles were used as control; after incubation for 8 hours, washed with 4 ℃ PBS buffer for 2 to 3 times, and collected bladder cancer EJ cells The cells were centrifuged at 1500 rpm for 5 minutes, and then detected by flow cytometry. Finally, the software Flow Jo was used to analyze the content of the targeted nano-drug PLZ4@SeD nanoparticles in bladder cancer EJ cells. The results are shown in Figure B in Figure 10.

It can be seen from panel B in Figure 10 that, with the increase of the concentration of the targeted nanodrug PLZ4@SeD nanoparticles, the amount of the targeted nanodrug PLZ4@SeD nanoparticles absorbed by the cells increases in a dose-dependent manner at the same time. effect.

Example 9: Intracellular localization experiments of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Take bladder cancer EJ cells growing in logarithmic phase and inoculate them in a 2 cm diameter petri dish at a density of 8 × 10 ⁴ cells/mL, with 2 mL per well. The LysoTracker Red probe was added to the petri dish and incubated for 2 h, the Hoechst probe was added to the other petri dish and incubated for 1 h. After incubation, PLZ4@SeD nanoparticles were added to the two petri dishes to make the final concentration 2.00 μM, after incubation for 0 hours, 1 hour, 2 hours, 4 hours, 8 hours, and 12 hours, remove the supernatant medium, wash the cells with PBS at 4°C for 2 to 3 times, and observe the cells under a fluorescence microscope. Fluorescence signal of PLZ4@SeD nanoparticles. In this example, since the PLZ4@SeD nanoparticles are loaded with SeD-1b and emit green fluorescence in cells, the green fluorescence can be further analyzed by overlapping the fluorescence of lysosome (red fluorescence) and nucleus (blue fluorescence). The localization of the drug in the cells; the results are shown in Figure 11.

From the intracellular localization experiment in Figure 11, it was found that the intracellular lysosomes of bladder cancer EJ cells emitted green fluorescence, indicating that the nano-targeted drug PLZ4@SeD nanoparticles were localized in the lysosomes of bladder cancer EJ cells.

Example 10: Cellular reactive oxygen species experiment after targeting nanomedicine to cells

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Bladder cancer EJ cells in log phase growth were taken and seeded in different 96-well plates at a density of 2×10 ⁵ cells/mL, 100 μL per well. After the cells adhered, 1 μL of 1 mmol/L DHE was added to each well. The probes were incubated in the dark for 30 minutes; then, 100 μL of the targeted nanomedicine PLZ4@SeD nanoparticles were added to each well as the treatment group, so that the final concentrations of the targeted nanomedicine PLZ4@SeD nanoparticles were 16 μM, 8 μM, and 4 μM, respectively. Without targeting nano-drug PLZ4@SeD nanoparticles as blank group, the fluorescence absorption value was measured every 5 minutes at the excitation wavelength of 360 nm and the emission wavelength of 610 nm using a multifunctional fluorescence microplate reader for 2 hours continuously. The measurement results are as follows: Figure 12.

The results of panel A in Figure 12 show that the fluorescence intensity of bladder cancer EJ cells increases with the increase in the amount of targeted nanodrug PLZ4@SeD nanoparticles; With the increase of the amount of SeD nanoparticles, the level of intracellular reactive oxygen species in bladder cancer EJ cells increased, and the intracellular reactive oxygen species content increased with the increase of drug concentration, indicating that the antitumor activity of the targeted nanodrug PLZ4@SeD nanoparticles is It does this by increasing the intracellular reactive oxygen species levels and inducing apoptosis.

Example 11: Evaluation of in vitro antitumor effect of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Cycle arrest and apoptosis are two important ways that antitumor drugs induce cell death. Therefore, the present invention uses flow cytometry to analyze the way in which the targeted nanomedicine causes the death of bladder cancer EJ cells.

Bladder cancer EJ cells growing in logarithmic phase were taken and seeded in different culture dishes with a diameter of 6 cm at a density of 2 × 10 ⁴ cells/mL, and 6 mL of cells were added to each culture dish. PLZ4@SeD nanoparticles were added to 4 of them as treatment groups, so that the final concentrations of PLZ4@SeD nanoparticles in the medium were 1.0 μM, 0.5 μM, 0.25 μM, and 0.125 μM, respectively; The above medium of PLZ4@SeD nanoparticles was used as the control group; bladder cancer EJ cells of the treatment group and the control group were incubated in a 37°C, 5% _CO2 incubator for 72 hours, and then the cells were collected, and 1mL-20°C of The volume fraction was 70% ethanol, and placed at -20 °C for 24 hours, centrifuged at 1500 rpm for 10 minutes, and then stained with 300 μL propidium iodide in the dark for 30 minutes. (40-50μm pore size) Nylon mesh filter, the filtered samples are detected by flow cytometer, and the content of DNA in cells is analyzed by Modfit software, and the G0/G1 phase, S phase, G2/M phase and apoptosis peak are obtained. The ratio of Sub G1 and the detection results are shown in Figure 13, respectively.

It can be seen from the results of Panel A in Figure 13 that the ratio of the apoptotic peak Sub-G1 in the control group was 0.57%, while the ratio of the apoptotic peak in the treatment group gradually increased from 5.76% when the drug concentration was 0.25 μmol/L The drug concentration was 18.23% of 1.0 μmol/L, indicating that the targeted nano-drug PLZ4@SeD nanoparticles exerted significant anti-tumor activity by inducing apoptosis.

It can also be seen from the results of Panel B in Figure 13 that the proportion of cells in S phase has also increased, and the proportions of G2/M and G0/G1 phases have decreased. This is due to the cleavage of DNA during apoptosis. The S phase, which is the DNA synthesis phase, cannot enter the next phase, so the proportion of S phase increases. This phenomenon indicates that the targeted nanodrug PLZ4@SeD nanoparticles exert their antitumor activity by inducing apoptosis. .

Example 12: In vitro tumor sphere experiments targeting nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

Bladder cancer EJ cells in log phase growth were taken and seeded in ultra-low adsorption six-well plates at a density of 2×10 ⁶ cells/mL, with 2 mL per well. Shake every 0.5 hours in the ultra-clean workbench, shake in the same direction for 10 minutes each time, shake every 2 hours after 3 hours, shake in the same direction for 10 minutes each time, and shake every 12 hours after 12 hours, every time Shake in the same direction for 10 minutes each time, and observe whether tumor spheres with a diameter of 100 μm are formed after one week. The PLZ4 solution prepared with 0.01M PBS, the final concentration of PLZ4 was 0.5 mg/mL, the above group was taken as the blocking group, the tumor spheres without PLZ4 were taken as the unblocked group, and the blocked and unblocked groups were placed in 37 After 3 hours in a 5% _CO2 incubator at ℃, PLZ4@SeD nanoparticles were added to the above two groups at a final concentration of 2 μM, and incubated for 4 hours. After the incubation, the results were observed by confocal microscopy. , as shown in Figure 14.

According to the laser confocal results in Figure 14, after the tumor spheres in the blocked group were blocked with the targeting polypeptide PLZ4, their fluorescence was significantly lower than that in the unblocked group, and the amount of the targeted nanodrug PLZ4@SeD nanoparticles entering the tumor spheres in the blocked group less than that in the unblocked group, indicating the selectivity of PLZ4@SeD nanoparticles.

Example 13: Evaluation of in vitro bladder perfusion effect of targeted nanomedicines

The preparation method of the targeted nanomedicine PLZ4@SeD nanoparticles used in this example is the same as the preparation method described in Example 1 of this application.

The nano-drug PLZ4@SeD nanoparticles were diluted to 1 mM with normal saline; the human-derived isolated bladder tissue containing normal bladder epithelial cells SV-HUC-1 and bladder cancer EJ cells was taken, and the above-mentioned human-derived isolated bladder cancer-containing tumor was collected. After wiping the periphery of the body bladder tissue, squeeze out the residual urine and blood in the bladder tissue, and wash with normal saline for 2 to 3 times until there is no blood. 100 mL of the above-mentioned targeted nano-drug PLZ4@SeD nanoparticles with a concentration of 1 mM was perfused through the urethra to allow the drug to fully diffuse in the bladder. The distribution of drugs in the bladder before and after perfusion was observed, and the results are shown in Figure 15.

It can be seen from the pictures A to C in FIG. 15 that the tumor size in the isolated bladder is about 5cm×4cm;

From the magnetic resonance imaging (MRI) results of the D image in Figure 15, it can be seen that the drug absorption area of the tumor part is significantly larger than that of the normal tissue part, that is, the amount of the targeted nanodrug PLZ4@SeD nanoparticles entering the tumor part is significantly larger According to the amount of entering normal tissues, it can be inferred that the R ₂ * value in MRI should theoretically increase with the increase of drug concentration, and the T ₂ value should decrease with the increase of drug concentration.

From Figure E and F in Figure 15, it can be seen that the trends formed by the drug trend, the tumor margin, the normal tissue margin, the tumor inner region, and the R ₂ * value and T ₂ value corresponding to the normal tissue inner region are consistent with the theoretical trend. _The T2 values of the tumor interior area and the normal tissue interior area were equal, and the R2* values _were also almost equal. The R ₂ * value of the tumor edge was greater than the R ₂ * value of the normal tissue edge, and the T ₂ value was smaller than the T ₂ value of the normal tissue edge, indicating that the targeted nanomedicine PLZ4@SeD nanoparticles selectively entered the bladder tumor area. .

Example 14: Size evaluation of targeted nanomedicines prepared with different organic solvents as dispersants

The preparation method of the targeted nanomedicine prepared in this example is the same as the preparation method described in Example 1 of this application except that the types of organic solvents used to disperse PLGA and Fe ₃ O ₄ are different; The organic solvents for dispersing PLGA and Fe ₃ O ₄ are acetone, acetonitrile, and dichloromethane, respectively. The sizes of the obtained targeted nanomedicines were characterized by Nano-ZS (Malvern Instruments Limited), and the results are shown in Figure 16 .

It can be seen from Figure 16 that the size of the targeted nanomedicine obtained by using acetone as a dispersant is about 120 nm, while the size of the targeted nanomedicine obtained by using acetonitrile and dichloromethane as a dispersing agent is 220 nm and 210 nm, respectively, indicating that under the same conditions The size of the targeted nanomedicine obtained by using acetone as a dispersant is smaller and the effect is better.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> Jinan University

<120> Targeted nano-drugs based on MRI-guided overcoming drug resistance caused by hypoxia in tumors and their preparation methods and applications

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence

<223> Targeting polypeptide PLZ4

<400> 1

Cys Gln Asp Gly Arg Met Gly Phe Cys

1 5

Claims (7)

1. a preparation method of a targeted nanomedicine for overcoming tumor hypoxia to cause drug resistance based on nuclear magnetic imaging guidance, is characterized in that, comprises the following steps: S1. Disperse PLGA, MRI contrast agent and anti-tumor drug in organic solvent respectively, then add surfactant, and obtain solution A after stirring and dialysis; S2. Utilize NHS and EDC to activate the carboxyl group of solution A to obtain activated solution A; S3. Add targeting molecules to the above activated solution A, and after stirring and dialysis, a targeted nano-drug that overcomes the drug resistance caused by tumor hypoxia based on the guidance of nuclear magnetic resonance imaging is obtained; The nuclear magnetic imaging contrast agent described in step S1 is ferric oxide nanoparticles; The antitumor drug described in step S1 is a selenodiazole derivative; The targeting molecule described in step S3 is PLZ4. 2. preparation method according to claim 1 is characterized in that, in the solution A described in step S1, PLGA, MRI contrast agent and antitumor drug are prepared in a molar ratio of 1～40: 1～10: 1～10 Compare. 3. The preparation method according to claim 1, wherein the organic solvent described in step S1 is at least one of acetone, acetonitrile and dichloromethane. 4. The preparation method according to claim 1, wherein the surfactant described in step S1 is an aqueous Tween solution. 5. preparation method according to claim 1 is characterized in that, the consumption of NHS described in step S2 and EDC and PLGA is proportioned by NHS:EDC:PLGA=mol ratio 1:1:1～40. 6 . A targeted nanomedicine for overcoming drug resistance caused by tumor hypoxia based on nuclear magnetic imaging guidance, characterized in that it is prepared by the preparation method according to any one of claims 1 to 5 . 7 . The application of the MRI-guided targeting nanomedicine for overcoming the drug resistance caused by tumor hypoxia according to claim 6 in the preparation of antitumor drugs. 8 .

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