Active oxygen scavenging drug-loaded targeted nano-delivery platform based on diselenide bond bridging as well as preparation method and application of active oxygen scavenging drug-loaded targeted nano-delivery platform

Abstract

The invention discloses an active oxygen scavenging drug-loaded targeted nano delivery platform based on diselenide bond bridging as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, tetra (4-carboxyphenyl) porphyrin (TCPP) and manganese porphyrin (Mn-TCPP) are used as raw materials to synthesize an organic metal framework MOFs, a target drug is loaded on the MOFs through electrostatic adsorption / blending, and amino and diselenide bonds are linked on the MOFs to realize combination with a specific peptide ligand and promote release of a drug oxidative stress microenvironment. The nano delivery platform with specific targeting and ROS response performance is obtained. The specific treatment effect of different diseases is achieved through different selections of the loaded drugs and the specific peptide ligands.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a drug-loaded targeted nanodelivery platform bridged by diselenide bonds for scavenging reactive oxygen species, and a preparation method and application thereof. Background Art

[0002] In recent years, the application of nanotechnology in the medical field has attracted extensive attention and research. Especially in the field of drug delivery, nanotechnology provides new opportunities and solutions for solving many limitations of traditional drug administration methods. The innovative progress of nanotechnology in the field of drug delivery brings new opportunities and challenges for improving drug efficacy and reducing side effects. However, with the development of nanodrug delivery systems, there are still some problems to be solved. For example, the therapeutic targeting of nanomaterials, the in vivo residue rate, cellular immunity, and the accompanying biosafety problems, as well as the preparation and large-scale production of nanoparticles. Therefore, future research should focus on solving these problems to promote the wide application of nanotechnology in the field of drug delivery. Therefore, we urgently need to develop a targeted, safe, and efficient nanodrug delivery and treatment strategy.

[0003] In order to target and effectively treat various increasing tumors, cancers, and various chronic diseases, preparing a polymer nanodrug delivery platform with target specificity, ROS responsiveness, oxidative stress elimination ability, extensibility for dealing with different diseases and environments, and the ability to combine key drugs to intervene in key targets can provide ideas and strategies for further enhancing the application of nanotechnology in the medical field. Summary of the Invention

[0004] The purpose of the present invention is to provide a drug-loaded targeted nanodelivery platform bridged by diselenide bonds for scavenging reactive oxygen species, and a preparation method and application thereof. Organic metal frameworks MOFs are synthesized using tetrakis(4-carboxyphenyl)porphyrin (TCPP) and manganese porphyrin (Mn-TCPP) as raw materials. The target drug is loaded on the MOFs through electrostatic adsorption / blending, and the binding with specific peptide ligands and the release of the drug in the oxidative stress microenvironment are promoted by linking amino groups and diselenide bonds on the MOFs, thereby obtaining a specifically targeted and ROS-responsive nanodelivery platform. Different therapeutic effects for different diseases are obtained by selecting different drugs and specific peptide ligands for loading.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention: Provide a drug-loaded targeted nanodelivery platform bridged by diselenide bonds for scavenging reactive oxygen species and a preparation method thereof, including the following steps:

[0007] Using tetrakis(4-carboxyphenyl)porphyrin (TCPP), manganese porphyrin (Mn-TCPP), and ZrCl4 as raw materials, an organometallic framework MOF is synthesized. The target drug is loaded onto the MOF through electrostatic adsorption / blending, and the binding to a specific peptide ligand and the promotion of the release of the drug in the oxidative stress microenvironment are achieved by linking amino groups and diselenide bonds to the MOF, resulting in a nanodelivery platform with specific targeting and ROS-responsive performance.

[0008] The specific steps of the nanodelivery platform are as follows:

[0009] Step 1: TCPP, Mn-TCPP, and ZrCl4 are dissolved in an organic solvent. Then, acetic acid is added, and the mixture is hydrothermally heated for 6 - 24 h. The purple precipitate is separated by washing with 98% ethanol and dried at room temperature to obtain the mixed ligand MOF.

[0010] Step 2: The target drug and the MOF are added to an organic solvent and stirred. Subsequently, centrifugation is performed at 10000 - 15000 rpm for 5 - 20 min to obtain the drug-loaded MOF.

[0011] Step 3: The drug-loaded MOF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are added to a cross-linking agent solution of 1-ethyl-3-(3-dimethylaminopropyl) and stirred to react, obtaining a mixture solution.

[0012] Step 4: Subsequently, a solution of selenocysteine (Sec) is added to the mixture solution. After reacting for a period of time, centrifugation is performed at 10000 - 15000 rpm for 5 - 20 min to obtain the drug-loaded MOF-Se-Se-NH2. Then, it is washed with water to remove unreacted Sec, EDC, and NHS to purify the drug-loaded MOF-Se-Se-NH2.

[0013] Step 5: The drug-loaded MOF-Se-Se-NH2 and the specific peptide ligand are dissolved in an organic solvent and reacted at room temperature to obtain the drug-loaded targeted nanodelivery platform based on diselenide bond bridging and reactive oxygen species scavenging.

[0014] In Step 1, the mass ratio of TCPP to Mn-TCPP is 1 - 5:0.2 - 1, and the mass ratio of Mn-TCPP to ZrCl4 is 1:1 - 5.

[0015] In Steps 1 and 2, the organic solvent is one or more of water-soluble organic solvents such as dimethylformamide (DMF), dihydrolevoglucosenone, ethyl acetate, and methyl ethyl ketone (MEK).

[0016] The mass ratio of the target drug to MOFs in step 2 is 0.1 - 1:1 - 5, and the stirring time is 6 - 48 h;

[0017] The mass ratio of the drug-loaded MOFs, EDC, and NHS in step 3 is 1:1 - 5:1 - 5, and the stirring time is 6 - 48 h;

[0018] The volume ratio of the mixture solution to the Sec solution in step 4 is 1:1 - 5, and the concentration of the Sec solution is 0.5 - 5 mg / mL -1 , and the reaction time is 6 - 48 h.

[0019] The beneficial technical effects of the present invention are as follows:

[0020] (1) In this study, we are committed to designing an active-targeting and ROS-responsive polymer nanodrug delivery platform to improve the bioavailability and effectiveness of the target drug for the treatment of specific diseases.

[0021] (2) Utilize the high specificity between the specific peptide ligand and the complementary target protein on the surface of the target cell to efficiently deliver the nanoplatform to the lesion site to improve the active targeting efficiency of the nanodrug delivery platform.

[0022] (3) Through linking amino groups and diselenide bonds on MOFs to achieve the binding with the specific peptide ligand and promote the release of the drug in the oxidative stress microenvironment of DKD. The selenide bond has a faster degradation rate compared to sulfur compounds and can react bidirectionally with oxidants and reductants. Compared with disulfide bonds, the prodrug nanoassemblies containing diselenide bonds have higher circulation stability, tumor accumulation ability, and drug release efficiency.

[0023] (4) The charge, morphology, and size of the nano-system can be adjusted by adjusting the raw material and drug ratios and reaction conditions in the reaction system to adjust its immune phagocytosis avoidance and passive targeting characteristics to achieve high-efficiency bioavailability.

[0024] (5) The effects of different drugs in different disease systems can be specifically investigated through this nanodelivery platform to find the action targets and related signaling pathways of various drugs during the disease development and treatment processes, and at the same time provide new ideas for the development of biomedicine in clinical medicine. Description of the Drawings

[0025] Figure 1 It is the characterization diagram of the nanoparticles in Example 1. (a, b) TEM spectra of MOFs and MOFs-Se-Se@Cel, scale bar: 200 nm; (c, d) EDS spectra; (e) Zeta potentials of MOFs and MOFs-Se-Se@Cel; (f, g) particle size diagrams of MOFs and MOFs-Se-Se@Cel;

[0026] Figure 2 FT-IR spectra before and after the nanoparticle composite in Example 1

[0027] Figure 3 Uptake and distribution of MOFs-Se-Se@Cel nanoparticles in 3T3-L1 cells in Example 1. (a) Confocal laser microscopy after co-incubation of Cel and MOFs-Se-Se@Cel nanoparticles with 3T3-L1 cells for 6 h and 12 h, red: Cy3; blue: DAPI, scale bar: 50 μm; (b) Cell viability of Cel, MOFs and MOFs-Se-Se@Cel determined by MTT assay Detailed implementation manners

[0028] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention.

[0029] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention.

[0031] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] Step 1: TCPP (4.73 mg), Mn-TCPP (2.77 mg) and ZrCl4 (5 mg) were dissolved in 15 ml of dihydrolevoglucosenone organic solvent. Then, 0.5 ml of acetic acid was added, and the mixture was hydrothermally heated for 18 h. The purple precipitate was separated by washing with 98% ethanol and dried at room temperature for 12 h to obtain the mixed ligand MOFs;

[0034] Step 2: Add 5 mg of the target drug Celastrol (Cel) and 10 mg of MOFs to 10 ml of dihydrolevoglucosenone organic solvent and stir. Then centrifuge at 14,000 rpm for 5 min to obtain drug-loaded MOFs (MOFs@Cel).

[0035] Step 3: Add MOFs@Cel (5 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (5 mg), and N-hydroxysuccinimide (NHS) (5 mg) to 10 ml of 1-ethyl-3-(3-dimethylaminopropyl) crosslinker solution and stir for 6 h to obtain a mixture solution.

[0036] Step 4: Then add 10 ml of (1 mg·mL -1 ) selenocysteine (Sec) solution to the mixture solution. After reacting for a period of time, centrifuge at 14,000 rpm for 5 min to obtain drug-loaded MOF-Se-Se-NH2 (MOF-Se-Se-NH2@Cel). Then, wash with water to remove unreacted Sec, EDC, and NHS to purify MOF-Se-Se-NH2@Cel.

[0037] Step 5: Dissolve 5 mg of MOF-Se-Se-NH2@Cel and 5 mg of the specific peptide ligand KKEEE in 10 ml of the organic solvent dihydrolevoglucosenone and react at room temperature for 12 h to obtain a nanodelivery platform (MOF-Se-Se-NH2@Cel@KKEEE) for targeted treatment of diabetic nephropathy based on diselenide-bridged reactive oxygen species scavenging and loading Celastrol.

[0038] Celastrol (Cel) can delay the development of diabetic nephropathy through multiple pathways such as improving hemodynamics, inhibiting pro-inflammatory factors, attenuating oxidative stress, and inhibiting RAS.

[0039] The kidney-targeting peptide KKEEE can be specifically bound to the proximal tubule surface receptor Megalin. Therefore, connecting this targeting peptide to the material can be used to improve the active targeting efficiency of the kidney.

[0040] The MOF-Se-Se-NH2@Cel@KKEEE nanodelivery platform in Example 1 mainly acts on the targeted treatment of diabetic nephropathy.

[0041] Example 2

[0042] Step 1: TCPP (3.15 mg), Mn-TCPP (4.15 mg) and ZrCl4 (4 mg) were dissolved in 10 ml of dimethylformamide organic solvent. Then, 0.5 ml of acetic acid was added and heated hydrothermally for 24 h. The purple precipitate was separated by washing with 98% ethanol and dried at room temperature for 10 h to obtain the mixed-ligand MOFs;

[0043] Step 2: 3 mg of the target drug doxorubicin and 5 mg of MOFs were added to 10 ml of dihydrolevoglucosenone organic solvent and stirred. Subsequently, it was centrifuged at 10000 rpm for 10 min to obtain MOFs@Doxorubicin;

[0044] Step 3: MOFs@Doxorubicin (3 mg), carbodiimide (EDC) (3 mg) and N-hydroxysuccinimide (NHS) (3 mg) were added to 10 ml of 1-ethyl-3-(3-dimethylaminopropyl) crosslinker solution and stirred for reaction for 12 h to obtain a mixture solution;

[0045] Step 4: Subsequently, 8 ml of (1 mg·mL -1 ) selenocysteine (Sec) solution was added to the mixture solution. After reacting for a period of time, it was centrifuged at 10000 rpm for 10 min to obtain the drug-loaded MOF-Se-Se-NH2 (MOF-Se-Se-NH2@Doxorubicin). Then, it was washed with water to remove the unreacted Sec, EDC and NHS to purify MOF-Se-Se-NH2@Doxorubicin.

[0046] Step 5: 3 mg of MOF-Se-Se-NH2@Doxorubicin and 3 mg of the specific peptide ligand X1DGRX5GF were dissolved in 5 ml of the organic solvent dimethylformamide and reacted at room temperature for 12 h to obtain the nano-delivery platform MOF-Se-Se-NH2@Doxorubicin@X1DGRX5GF for targeted treatment of bladder cancer based on diselenide bond-bridged reactive oxygen species scavenging and loading Doxorubicin.

[0047] Doxorubicin is a prescription drug for the treatment of bladder cancer.

[0048] X1DGRX5GF is a specific ligand peptide for bladder cancer.

[0049] The MOF-Se-Se-NH2@Doxorubicin@X1DGRX5GF nano-delivery platform in Example 2 mainly acts on the targeted treatment of bladder cancer.

[0050] Example 3

[0051] Step 1: TCPP (2.37 mg), Mn-TCPP (5.53 mg) and ZrCl4 (3 mg) were dissolved in 15 ml of dihydrolevoglucosenone organic solvent. Then, 0.5 ml of acetic acid was added and hydrothermally heated for 12 h. The purple precipitate was separated by washing with 98% ethanol and dried at room temperature for 10 h to obtain mixed-ligand MOFs;

[0052] Step 2: 3 mg of the target drug celastrol (Cel) and 5 mg of MOFs were added to 10 ml of dihydrolevoglucosenone organic solvent and stirred. Subsequently, centrifugation was carried out at 12000 rpm for 5 min to obtain drug-loaded MOFs (MOFs@Cel);

[0053] Step 3: MOFs@Cel (3 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (3 mg) and N-hydroxysuccinimide (NHS) (3 mg) were added to 10 ml of 1-ethyl-3-(3-dimethylaminopropyl) cross-linking agent solution and stirred for reaction for 6 h to obtain a mixture solution;

[0054] Step 4: Subsequently, 10 ml of (1 mg·mL -1 ) selenocysteine (Sec) solution was added to the mixture solution. After reacting for a period of time, centrifugation was carried out at 12000 rpm for 5 min to obtain drug-loaded MOF-Se-Se-NH2 (MOF-Se-Se-NH2@Cel). Then, washing with water was carried out to remove unreacted Sec, EDC and NHS to purify MOF-Se-Se-NH2@Cel.

[0055] The MOF-Se-Se-NH2@Cel nano drug delivery platform in Example 3 can be used for fat loss treatment when linked with specific peptide ligands.

[0056] Figure 1 Characterization of the nanoparticles MOFs and MOF-Se-Se-NH2@Cel in Example 3. (a, b) TEM spectra, scale bar: 200 nm; (c, d) EDS spectra; (e) Zeta potential map; (f, g) particle size map. It can be seen that MOFs with adjustable morphology were prepared (such as Figure 1 (a)), and the loading of Cel by MOFs was achieved. To improve the ROS response characteristics of the nanoparticles, a compound with Se-Se functional groups was modified on the surface of MOFs to obtain nanorods as shown in Figure 1 (b). The nanoparticles had high uniformity and dispersibility. The micro-area composition of the nanorods was determined by EDS energy spectrum, and it contained elements such as N, Mn, C, O, Zr and Se( Figure 1(c, d)), demonstrating the successful synthesis of MOFs-Se-Se@Cel. The zeta potential of the nanoparticles before and after Cel complexation was measured. Figure 1 (e). The resulting negatively charged MOFs-Se-Se@Cel is more conducive to achieving long circulation in kidney tissues. Figure 1 (f, g) further measured the particle size of the nanoparticles, which is consistent with the results of TEM.

[0057] Figure 2 FT-IR spectra of the MOFs-Se-Se@Cel nanoparticles before and after complexation in Example 3. The results show that the vibration peak near 1000 cm -1 originates from the Mn-N stretching vibration in the MOF structure, and the same results are shown for MOF and MOFs-Se-Se@Cel. The vibration in the range of 1650 - 1425 cm -1 (as indicated by the dashed line) originates from the benzene ring skeletal vibration in the MOF. The vibration peak near 591 cm -1 indicates the successful modification of the Se-Se bond. The above results further demonstrate the successful preparation of the MOFs-Se-Se@Cel nanoparticles.

[0058] Figure 3 Confocal laser microscopy and MTT test images of the nanoparticles co-incubated with 3T3-L1 cells in Example 3. (a) Confocal laser microscopy image, red: Cy3; blue: DAPI, scale bar: 50 μm; (b) Cell viability measured by the MTT method. As Figure 3 (a) shows, Cel loaded by MOF (i.e., MOFs-Se-Se@Cel) has a relatively higher uptake efficiency. From the MTT test results, it can be seen that MOF has low cytotoxicity, and the cell viability of Cel carried by MOF in the MOFs-Se-Se@Cel group is significantly reduced, indicating its better fat-reducing effect.

Claims

1. A method for preparing a drug-loaded targeted nano-delivery platform based on diselenide bond bridging active oxygen scavenging, characterized in that: The following steps are involved: Organic metal frameworks (MOFs) were synthesized using tetrakis(4-carboxyphenyl)porphyrin (TCPP), manganese porphyrin (Mn-TCPP) and ZrCl4 as raw materials. The target drugs were loaded on MOFs by electrostatic adsorption / blending, and the amino and diselenide bonds were linked on MOFs to achieve binding with specific peptide ligands and promote the release of drugs in the oxidative stress microenvironment, thus obtaining a nano-delivery platform with specific targeting and ROS responsiveness.

2. The method for preparing the nano delivery platform according to claim 1, characterized in that: The specific steps of the nano delivery platform include: Step 1: TCPP, Mn-TCPP and ZrCl4 were dissolved in an organic solvent. Then, acetic acid was added and hydrothermally heated for 6 to 24 h. The purple precipitate was separated by washing with 98% ethanol and dried at room temperature to obtain mixed ligand MOFs. Step 2: Add the target drug and MOFs to an organic solvent and stir, then centrifuge at 10,000 to 15,000 rpm for 5 to 20 min to obtain drug-loaded MOFs; Step 3: Add drug-loaded MOFs, carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to a 1-ethyl-3-(3-dimethylaminopropyl) crosslinker solution and stir to react to obtain a mixture solution; Step 4: Then, selenocysteine ​​(Sec) solution is added to the mixture solution. After a period of reaction, the mixture is centrifuged at 10,000 to 15,000 rpm for 5 to 20 min to obtain drug-loaded MOF-Se-Se-NH2. Then, the mixture is washed with water to remove unreacted Sec, EDC and NHS to purify the drug-loaded MOF-Se-Se-NH2. Step 5: The drug-loaded MOF-Se-Se-NH2 and the specific peptide ligand are dissolved in an organic solvent and reacted at room temperature to obtain a diselenide bond-bridging active oxygen scavenging drug-loaded targeted nano-delivery platform.

3. The method for preparing the nano delivery platform according to claim 2, characterized in that: In the step 1, the mass ratio of TCPP to Mn-TCPP is 1-5:0.2-1, The mass ratio of Mn-TCPP and ZrCl4 is 1:1~5.

4. The method for preparing the nano delivery platform according to claim 2, characterized in that: The organic solvent in step 1 and step 2 is one or more of dimethylformamide (DMF), dihydrolevoglucosenone, ethyl acetate, butanone (MEK) and other organic solvents that retain their original water solubility.

5. The method for preparing the nano delivery platform according to claim 2, characterized in that: The mass ratio of the target drug to MOFs in step 2 is 0.1-1:1-5, and the stirring time is 6-48 hours.

6. The method for preparing the nano delivery platform according to claim 2, characterized in that: The mass ratio of the drug-loaded MOFs, EDC and NHS in step 3 is 1:1-5:1-5, and the stirring time is 6-48 hours.

7. The method for preparing the nano delivery platform according to claim 2, characterized in that: The volume ratio of the mixture solution and the Sec solution in step 4 is 1:1-5, and the concentration of the Sec solution is 0.5-5 mg.mL -1 The reaction time is 6 to 48 hours.

8. The specific peptide ligands according to claim 1 and claim 2 include one or more ligands such as biotin and avidin, antigens and antibodies, enzymes and their inhibitors, hormone receptors, etc. that only bind to single or very few types of biological macromolecules such as proteins.

9. The target drug according to claim 1 and claim 2 comprises one or more of a small molecule drug and a monoclonal antibody drug.

10. A nano delivery system prepared by the method for preparing the nano delivery platform according to any one of claims 1 to 9.

11. A method for preparing a diselenide bond-bridged active oxygen scavenging drug-loaded targeted nano-delivery platform as claimed in claim 1 and its application in biomedicine.

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