A preparation method of copper gadolinium nano-diagnostic agent and the prepared diagnostic agent

Abstract

The present invention discloses a preparation method of a copper-gadolinium nanotheranostic agent, which relates to the technical field of biomedical materials. The present invention comprises the following steps: (1) adding cupric acetylacetonate, gadolinium acetylacetonate, and a polymer surfactant into an organic solvent, heating and stirring, and obtaining a reaction solution after dissolution; (2) transferring the reaction solution to a microwave reaction kettle, heating and reacting and dispersing in deionized water to obtain CGO; (3) dissolving PDGFB, EDC, and NHS in DMSO, stirring in the dark and then adding PEG-NH2, and concentrating after reaction; (4) adding CGO, carrying out a constant-temperature reaction and then centrifuging. The beneficial effects of the present invention are as follows: The copper-gadolinium nanotheranostic agent prepared by the present invention realizes active targeting at the tumor site with the aid of PDGFB, and is responsive to release in a weakly acidic tumor microenvironment, and finally realizes chemodynamic and photothermal synergistic theranostics guided by magnetic resonance imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a method for preparing a copper gadolinium nano-diagnostic agent and the prepared diagnostic agent. Background Art

[0002] It is well known that the microenvironment of the tumor lesion area has its own specificity, generally showing characteristics such as hypoxia, weak acidity (pH 6.5-6.9), excess reduced glutathione (GSH), and excess hydrogen peroxide (H2O2), which plays an extremely important role in tumor occurrence, development, and metastasis. In recent years, many emerging tumor treatment strategies have been developed using the characteristics of the tumor microenvironment (TME). Chemodynamic therapy (CDT) is an emerging and promising treatment model that uses the endogenous excess H2O2 in the tumor to produce highly toxic hydroxyl radicals (·OH) through metal ion-catalyzed Fenton / Fenton-like reactions to kill tumor cells. Compared with other reactive oxygen species (ROS) therapies, CDT has a stronger ability to catalyze ROS generation, is less dependent on external stimuli, and has deep tissue treatment capabilities. In addition, the unique ROS generation mode in CDT is different from the death induced by chemotherapeutic drugs, which can escape biological barriers and overcome tumor resistance. However, differences between the H2O2 concentration and pH value in the TME and the optimal pH required for Fenton / Fenton-like reactions have hampered CDT from realizing its full potential.

[0003] In the past decade, iron-based reagents have been widely used as classic CDT reagents in tumor treatment research. However, iron-based CDT reagents have disadvantages such as weak reactivity, insufficient Fe(II) release, and low catalytic activity, and have been gradually replaced by copper-based CDT reagents. Cu(I) can catalyze the occurrence of Fenton-like reactions under both weakly acidic and neutral conditions, and in theory, the Fenton-like catalytic rate of Cu(I) is 160 times that of traditional iron-based CDT reagents. More importantly, the excess antioxidant GSH in tumor cells drives the conversion of Cu(II) to Cu(I). This process not only realizes the in situ supply of Cu(I) in the tumor, but also consumes GSH in the cells, further enhancing the effect of CDT.

[0004] In addition, photothermal therapy (PTT) has been widely studied as a non-invasive in situ tumor treatment strategy. Photothermal agents convert external light energy into heat energy under the irradiation of near-infrared light, causing local high temperature in the tumor to ablate tumor cells. PTT can achieve externally controllable irradiation of tumor tissue sites, minimizing damage to normal tissues. While increasing the temperature of tumor tissue sites, PTT can also promote the occurrence of Fenton-like effects and enhance the therapeutic effect of CDT. For example, the patent application with application number 202110672005.9 discloses a targeted drug for a chemokinetically enhanced photothermal therapy system for the treatment of malignant tumors, its preparation method and application. Moreover, CDT is triggered by endogenous chemicals in the TME, rather than relying on external energy input, which effectively avoids the defect of energy attenuation of PTT when penetrating tumor tissue. Therefore, the integration of CDT and PTT into a nanoplatform can give play to their respective advantages, construct a tumor-targeted diagnostic and therapeutic agent based on CGO, and realize the synergistic and efficient diagnosis and treatment of CDT and PTT for tumors.

[0005] For example, the patent application with application number 202111583795.X discloses a copper gadolinium core-shell nanoparticle with both Fenton catalysis and photothermal conversion properties, and its preparation method and application. It can achieve dual-modal tumor treatment with the help of its Fenton catalysis and photothermal conversion properties, but its preparation time is relatively long and it only has tumor treatment function. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a simple preparation method of a copper gadolinium nano-diagnostic and therapeutic agent and the prepared copper gadolinium nano-diagnostic and therapeutic agent, which can be responsively released in a weakly acidic tumor microenvironment, and ultimately achieve the synergistic therapeutic effect of magnetic resonance imaging-guided chemodynamics and photothermal therapy.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] A method for preparing a copper gadolinium nano-diagnostic agent comprises the following steps:

[0009] (1) adding copper acetylacetonate, gadolinium acetylacetonate, and a polymer surfactant to an organic solvent, heating and stirring, and obtaining a reaction solution after dissolution;

[0010] (2) transferring the reaction solution to a microwave reactor, heating the reaction to obtain copper gadolinium nanoclusters, and dispersing the purified solution in deionized water to obtain copper gadolinium nanoclusters (CGO);

[0011] (3) Dissolve platelet growth factor receptor β recognition cyclic peptide (PDGFB), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in dimethyl sulfoxide (DMSO), add polyethylene glycol amino (PEG-NH2) after stirring in the dark, and concentrate after reaction;

[0012] (4) CGO is added to the concentrated solution of step (3), reacted at a constant temperature, and centrifuged to obtain a product, which is a copper gadolinium nano-diagnostic agent.

[0013] Beneficial effects: The present invention uses a cyclic peptide to modify copper gadolinium nanoclusters to obtain a targeted copper gadolinium nanodiagnostic agent PDGFB-CGO. PDGFB gives it active tumor targeting ability. The material has pH-responsive release ability, responsive release in a weakly acidic tumor microenvironment, and has high Fenton-like catalytic performance and photothermal performance.

[0014] The present invention has good T1 magnetic resonance imaging diagnostic capabilities, helps to achieve the coordinated diagnosis and treatment of CDT and PTT guided by magnetic resonance imaging, and lays a material foundation for tumor patients to achieve a visualized, efficient and safe integrated diagnosis and treatment strategy.

[0015] The present invention uses copper acetylacetonate and gadolinium acetylacetonate as precursors and prepares copper gadolinium nanoclusters by a microwave one-step synthesis method. Subsequently, platelet growth factor receptor β (PDGFR-β) recognition cyclic peptide (PDGFB) is modified on the surface of the copper gadolinium nanoclusters to prepare a copper gadolinium nanodiagnostic agent targeting the tumor microenvironment. The preparation process of the present invention is simple and convenient, the preparation method is suitable for mass production, and has good biosafety.

[0016] Compared with the prior art, in terms of synthesis method, the present invention adopts a microwave one-step synthesis method, which has the advantages of rapid and uniform heating and no need for heat conduction process. It can be completed quickly within tens of minutes, which is helpful to achieve the green synthesis of nanomaterials. The patent application with application number 202111583795.X synthesizes copper gadolinium nanomaterials through the traditional solvent thermal method, which requires maintaining a high temperature and high pressure environment for a long time, and the reaction time is more than 24 hours.

[0017] In terms of application, the present invention not only has the efficient synergistic treatment of tumor CDT and PTT, but also has good magnetic resonance imaging performance, which can realize the integration of tumor diagnosis and treatment. The copper gadolinium nanomaterial in the patent application with application number 202111583795.X only has the function of tumor treatment.

[0018] Preferably, the mass ratio of copper acetylacetonate, gadolinium acetylacetonate and polymer surfactant is 1-2:1:4-6.

[0019] Preferably, the polymer surfactant includes polyvinyl pyrrolidone, polyethylene imine, and sodium polyacrylate.

[0020] Preferably, the organic solvent in step (1) can be ethylene glycol, diethylene glycol, or triethylene glycol, 25 to 40 mL.

[0021] Preferably, the temperature after heating in step (1) is 110-135°C.

[0022] Preferably, in step (2), the heating reaction temperature is 200-260° C., and the heating time is 5-20 min.

[0023] Preferably, the platelet-derived growth factor receptor β recognition cyclic peptide is a cyclic small molecule polypeptide formed by a single disulfide bond or a derivative having the small molecule polypeptide sequence, and the peptide sequence of the platelet-derived growth factor receptor β recognition cyclic peptide is C*SRNLIDC*.

[0024] Preferably, the mass ratio of EDC to NHS is 1:1.

[0025] Preferably, the molar ratio of PDGFB to PEG-NH2 is 1:1.

[0026] Preferably, in the step (3), after stirring in the dark, polyethylene glycol amino is added and then shaken in a constant temperature shaker at 37° C. overnight, and the solution is dialyzed with a dialysis bag to obtain a concentrated solution.

[0027] Preferably, an inorganic base solution is added to the concentrate obtained in step (3) to adjust the pH to 7.0-7.4.

[0028] A copper gadolinium nano-diagnostic agent prepared by the above preparation method.

[0029] Beneficial effects: The diagnostic and therapeutic agent prepared by the present invention has pH-responsive release capability, responsive release in a weakly acidic tumor microenvironment, high Fenton-like catalytic performance and photothermal performance, and good T1 magnetic resonance imaging diagnostic capability, which helps to achieve magnetic resonance imaging-guided CDT and PTT coordinated diagnosis and treatment, and lays a material foundation for tumor patients to achieve a visualized, efficient, and safe integrated diagnosis and treatment strategy.

[0030] The advantages of the present invention are as follows: the present invention uses a cyclic peptide to modify copper gadolinium nanoclusters to obtain a targeted copper gadolinium nano-diagnostic agent PDGFB-CGO, PDGFB gives it active tumor targeting ability, the material has pH-responsive release ability, responsive release in a weakly acidic tumor microenvironment, and has high Fenton-like catalytic performance and photothermal performance.

[0031] The present invention has good T1 magnetic resonance imaging diagnostic capabilities, helps to achieve the coordinated diagnosis and treatment of CDT and PTT guided by magnetic resonance imaging, and lays a material foundation for tumor patients to achieve a visualized, efficient and safe integrated diagnosis and treatment strategy.

[0032] The present invention uses copper acetylacetonate and gadolinium acetylacetonate as precursors and prepares copper gadolinium nanoclusters by a microwave one-step synthesis method. Subsequently, platelet growth factor receptor β (PDGFR-β) recognition cyclic peptide (PDGFB) is modified on the surface of the copper gadolinium nanoclusters to prepare a copper gadolinium nanodiagnostic agent targeting the tumor microenvironment. The preparation process of the present invention is simple and convenient, the preparation method is suitable for mass production, and has good biosafety.

[0033] Compared with the prior art, in terms of synthesis method, the present invention adopts a microwave one-step synthesis method, which has the advantages of rapid and uniform heating and no need for heat conduction process. It can be completed quickly within tens of minutes, which is helpful to achieve the green synthesis of nanomaterials. The patent application with application number 202111583795.X synthesizes copper gadolinium nanomaterials through the traditional solvent thermal method, which requires maintaining a high temperature and high pressure environment for a long time, and the reaction time is more than 24 hours.

[0034] In terms of application, the present invention not only has the efficient synergistic treatment of tumor CDT and PTT, but also has good magnetic resonance imaging performance, which can realize the integration of tumor diagnosis and treatment. The copper gadolinium nanomaterial in the patent application with application number 202111583795.X only has the function of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 TEM image of CGO prepared in Example 1 of the present invention;

[0036] Figure 2 The UV-visible spectrum curves of the TMB solution of CGO under different pH conditions of the present invention;

[0037] Figure 3 is the release amount of Cu ions of CGO of the present invention in buffer solutions of different pH values;

[0038] Figure 4 It is the Fourier transform infrared spectra of CGO, PEG-PDGFB and PDGFB-CGO prepared in Example 1 and Example 2 of the present invention;

[0039] Figure 5 The uptake of CGO-FITC and PDGFB-CGO-FITC of the present invention by tumor cells;

[0040] Figure 6 The effect of PDGFB-CGO of the present invention on the viability of tumor cells;

[0041] Figure 7 is the longitudinal relaxation rate of CGO of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0044] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0045] Example 1

[0046] This embodiment provides a method for preparing copper gadolinium nanoclusters, which specifically includes the following steps:

[0047] (1) Add 0.13 g of copper acetylacetonate, 0.1 g of gadolinium acetylacetonate and 0.6 g of polyvinyl pyrrolidone to 25 mL of triethylene glycol and stir magnetically at 110 °C for 10 min;

[0048] (2) The reaction solution in step (1) was transferred to a microwave reactor, and the microwave reactor was set to heat to 200° C. over a heating time of 20 min, and maintained at 200° C. for 10 min.

[0049] (3) After the reaction was completed, the reaction system was cooled to 60° C. under a nitrogen atmosphere, and the prepared CGO was collected and washed three times with ethanol and deionized water, respectively, and then dispersed in deionized water.

[0050] The morphology of the CGO prepared in Example 1 was characterized by transmission electron microscopy (TEM). Figure 1 As shown. TEM results show that the nanomaterials synthesized under this condition have uniform morphology and good dispersion. Figure 2 As shown in Figure 2, through the 3,3',5,5'-tetramethylbenzidine (TMB) experiment, it was found that the catalytic ability of CGO is concentration-dependent, and the catalytic ability increases with the decrease of pH value. Figure 3 As shown in the figure, through the Cu ion release experiment, we found that the release of Cu in CGO is pH responsive, and the release amount can reach more than 90% after 12 hours in the buffer solution of pH 5.5.

[0051] Example 2

[0052] This embodiment provides a method for preparing a copper gadolinium nanodiagnostic and therapeutic agent targeting the tumor microenvironment, which specifically comprises the following steps:

[0053] (1) Dissolve 5 mg PDGFB, 20 mg EDC, and 20 mg NHS in 5 mL DMSO, stir magnetically for 2 h in the dark, then add 10 mg PEG-NH2 and shake in a 37°C constant temperature shaker overnight;

[0054] (2) Using a dialysis bag, dialyze the solution in step (1) against deionized water to obtain a PEG-PDGFB polymer concentrate, and adjust the pH to 7.0 using an inorganic base solution.

[0055] (3) Add 10 mg of CGO to the concentrated solution obtained in step (2) and shake in a constant temperature shaker at 37°C for 2-4 h.

[0056] (4) Finally, the reactants were collected and centrifuged at 12000 rpm / min for 10 min to obtain PDGFB-CGO, which was washed three times with deionized water and then dispersed in water and stored in a refrigerator at 4°C for later use.

[0057] The structure of PDGFB-CGO prepared in Example 2 was characterized by infrared spectrometer. Figure 4 As shown, after structural and compound modification, the corresponding characteristic absorption peak changes were finally reflected in the absorption spectrum of PDGFB-CGO, proving the successful preparation of PDGFB-CGO.

[0058] Test Example 1

[0059] The tumor targeting ability of PDGFB-CGO was evaluated, including the following steps:

[0060] (1) Culture of tumor cells

[0061] PC3 cells, MCF7 cells, and MDA-MB-231 cells were cultured at 1.5×10 5 The cells were inoculated at a density of 1:1 / disk in a confocal dish and cultured for 12 to 18 hours.

[0062] (2) Observation of tumor cell uptake of copper gadolinium nanodiagnostic agents using laser confocal microscopy

[0063] CGO and PDGFB-CGO were labeled with fluorescein isothiocyanate (FITC) to obtain CGO-FITC and PDGFB-CGO-FITC, respectively. Tumor cells were incubated with different concentrations of CGO-FITC and PDGFB-CGO-FITC for 4 h, then the cells were fixed, the cell nuclei were stained with DAPI, and the cell uptake was observed using a laser confocal microscope.

[0064] like Figure 5As shown, compared with CGO-FITC at the same concentration, there is a stronger fluorescence signal in the tumor cells incubated with PDGFB-CGO-FITC, which indicates that the nanoclusters modified with PDGFB have tumor cell targeting.

[0065] Test Example 2

[0066] The anti-tumor effect of PDGFB-CGO was evaluated, including the following steps:

[0067] (1) Culture of tumor cells

[0068] PC3 cells were cultured at 10 4 The cells were inoculated at a density of 100 cells / well in a 96-well plate and cultured for 12 hours. After 12 hours, the cells were incubated with materials of different concentrations and cultured for another 24 hours.

[0069] (2) CCK8 assay to detect the effect of PDGFB-CGO on PC3 cell viability

[0070] Prepare CCK8 detection solution with fresh culture medium, add 110 μL CCK8 detection solution to each well and continue culturing for 1 hour. After gently shaking, detect the absorbance value at 490 nm.

[0071] like Figure 6 As shown in the figure, the results of CCK8 experiments showed that ultrasmall iron oxide (USIO) had no obvious toxicity to PC3 cells. Free Cu(Ⅱ) was toxic to PC3 cells at high concentrations. CGO and PDGFB-CGO showed strong inhibitory effects on cell viability after treatment. Compared with CGO, PDGFB-CGO had a stronger inhibitory effect, which may be due to the excellent tumor targeting ability given by PDGFB.

[0072] Test Example 3

[0073] Evaluation of the potential of CGO as a T1 MRI contrast agent involves the following steps:

[0074] (1) Sample preparation

[0075] CGO solutions of different concentrations were prepared using ultrapure water and fixed in 2 mL centrifuge tubes using 0.5% agarose gel;

[0076] (2) Magnetic resonance imaging scan

[0077] Scanning was performed using a 3.0T and 7.0T MRI scanner, first performing a localization scan, then acquiring a series of inversion fast echo images, and performing longitudinal relaxation time (T1) measurements.

[0078] (3) Calculation of relaxation rate

[0079] The relaxation rate of CGO was calculated according to the formula r1 = (R1-b) / C, where R1 is the reciprocal of T1, C is the concentration of CGO, and b is a constant.

[0080] like Figure 7 As shown in the results, the relaxation rates of CGO prepared in Example 1 under the field strength of 3.0T and 7.0T were 2.00mM -1 s -1 and 0.29mM -1 s -1 , indicating that the nanoclusters have good T1 contrast performance.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a copper gadolinium nano-diagnostic agent, characterized in that: The following steps are involved: (1) adding copper acetylacetonate, gadolinium acetylacetonate, and a polymer surfactant to an organic solvent, heating and stirring, and dissolving to obtain a reaction solution; the polymer surfactant includes polyvinyl pyrrolidone, polyethyleneimine, and sodium polyacrylate; (2) transferring the reaction solution to a microwave reactor, heating the reaction to obtain copper gadolinium nanoclusters, and dispersing the purified solution in deionized water to obtain copper gadolinium nanoclusters CGO; (3) dissolving platelet growth factor receptor-β recognition cyclic peptide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide in dimethyl sulfoxide, adding polyethylene glycol amino group after stirring in the dark, and concentrating after reaction; (4) Add copper gadolinium nanoclusters CGO to the concentrated solution of step (3), react at a constant temperature, and centrifuge to obtain a product that is a copper gadolinium nanodiagnostic agent.

2. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The mass ratio of the copper acetylacetonate, gadolinium acetylacetonate and polymer surfactant is 1-2:0.3-0.5:0.

1.

3. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The organic solvent in step (1) includes ethylene glycol, diethylene glycol or triethylene glycol.

4. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The temperature after heating in the step (1) is 110-135°C.

5. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: In the step (2), the heating reaction temperature is 200-260° C., and the heating time is 5-20 min.

6. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The platelet-derived growth factor receptor-β recognition cyclic peptide is a cyclic small molecule polypeptide formed by a single disulfide bond, and the peptide sequence of the platelet-derived growth factor receptor-β recognition cyclic peptide is C*SRNLIDC*.

7. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:

1.

8. The method for preparing the copper gadolinium nano-diagnostic agent according to claim 1, characterized in that: The molar ratio of the platelet growth factor receptor-β recognition cyclic peptide to the polyethylene glycol amino group is 1:

1.

9. A copper gadolinium nanodiagnostic agent prepared by the preparation method according to any one of claims 1 to 8.

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