Synthesis method and application of mitochondrial targeting and responding porphyrinyl supramolecular self-assembled nano system

Abstract

The invention discloses a synthesis method and application of a mitochondrial targeting and responding porphyrinyl supramolecular self-assembly nano system. The specific synthesis process comprises the following steps: connecting monocarboxyl porphyrin and a triphenylphosphine group through bis (2-hydroxyethyl) disulfide through esterification reaction to obtain a compound TPP-S-S-PPh3; then self-assembling in a mixed solvent of tetrahydrofuran and water by utilizing the difference of hydrophilic and hydrophobic properties of different groups in a compound TPP-S-PPP3 to obtain a nano-structure compound TPP-S-PPP3 NPs, and adding catalase in the self-assembling process to carry out loading to obtain the target product porphyrin-based organic supramolecular self-assembled nano system TPP-S-PPP3 (at) CAT NPs. The synthesized nano system can accurately locate and enrich mitochondria in tumor cells, the level of active oxygen in the cells is remarkably improved, and the treatment effect of photodynamic therapy is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of synthesis of functional organic supramolecular systems and organic molecular self-assembly, and specifically relates to a synthesis method and application of a mitochondrial targeting and responsive porphyrin-based supramolecular self-assembly nanosystem. Background Art

[0002] In today's medical field, tumor treatment has always been a focus of attention. Targeted and highly biocompatible drugs show great potential in tumor treatment (a) XDMa, PFWang, QWWu, JNZhou, DQWang, D.Yadav, HBZhang, YZZhang, Advanced Healthcare Materials 2023, 12, 13. b) B.Ji, MJWei, B.Yang, Theranostics 2022, 12, 434-458.). As a disease that seriously threatens human health, traditional treatments such as surgery, radiotherapy and chemotherapy can control the development of tumors to a certain extent, but they are often accompanied by serious side effects and limitations. Surgery may not be able to completely remove the tumor, and radiotherapy and chemotherapy will cause great damage to normal tissues, affecting the quality of life of patients. Therefore, the development of targeted drugs has become an important direction for tumor treatment. This type of drug can accurately identify tumor cells, act specifically on the tumor site, and reduce damage to normal tissues. At the same time, high biocompatibility ensures that the drug can work safely and effectively in the body, reducing the risk of adverse reactions such as immune rejection. With the continuous development of biotechnology, significant progress has been made in exploring targeted and highly biocompatible drugs. For example, antibody-drug conjugates, nanocarriers and other technologies are used to achieve precise targeted delivery of tumor cells. These drugs can not only improve the therapeutic effect, but also reduce the drug dosage and reduce side effects, bringing new hope to cancer patients (a) WGQiao, T.Ma, G.Xie, JWXu, ZR Yang, C.Zhong, H.Jiang, JLXia, LBZhang, JTZhu, ZALi, AcsNano2024, 18, 25671-25684b) YJYang, FM Chen, N.Xu, QC Cao, R.Wang, XC Xie, F.Zhang, Y.He, D.Shao, WF Dong, JL Fan, W.Sun, XJPeng, Biomaterials2022, 281, 10.). In short, drugs with targeted properties and high biocompatibility have broad application prospects in tumor treatment and are expected to become an important means of tumor treatment in the future.

[0003] Photodynamic therapy (PDT) is an emerging cancer strategy that uses light of a specific wavelength to irradiate photosensitizers accumulated in diseased tissues to produce reactive oxygen species, thereby destroying diseased cells and achieving a therapeutic effect. This treatment method is highly selective and can act precisely on the lesion site, reducing damage to surrounding normal tissues. At the same time, photodynamic therapy is relatively non-invasive, and patients recover quickly, providing a new option for the treatment of tumor diseases. In recent years, with the continuous development of photosensitizers and the advancement of light transmission technology, the effect and safety of photodynamic therapy have been further improved (a) LX Sun, J. Wang, BCYang, XX Wang, GX Yang, XQ Wang, YY Jiang, TY Wang, JZ Jiang, RSC Adv. 2021, 11, 10061-10074.). As an effective organic photosensitizer, porphyrin compounds have a unique chemical structure, which lays the foundation for their designability. In the treatment of diseases such as tumors, porphyrins can be chemically modified to adjust their absorption spectra, hydrophilicity, hydrophobicity, subcellular organelle targeting and other properties to better meet different treatment needs.

[0004] Supramolecular self-assembly is a self-organizing process based on weak intermolecular interactions, which can form supramolecular systems with specific structures and functions (a) C. Zhou, YH Zhao, M. Yang, W. Yin, YS Li, ​​Y. Xiao, YB Liu, M Lang, Advanced Healthcare Materials 2024, 13, 11. b) LX Sun, J. Wang, B C Yang, XX Wang, GX Yang, XQ Wang, YY Jiang, TY Wang, JZ Jiang, RSC Adv. 2021, 11, 10061-10074.). In recent years, supramolecular self-assembled nanomaterials with amphiphilic organic molecules as building blocks have shown obvious design advantages. Functional organic molecules can induce changes in the nanostructure and physical and chemical properties of the assembly, realizing artificial regulation of the system's functions and properties. Therefore, porphyrin photosensitizers can be combined with other functional molecules through supramolecular self-assembly to form supramolecular systems with higher stability, better targeting and stronger photodynamic activity (a) K. Wang, J. Chen, L. Lin, N. Yan, WHYang, KY Cai, HY Tian, ​​XSChen, Nano Today 2022, 46, 9b) DY Hou, DB Cheng, NY Zhang, ZJ Wang, XJ Hu, X. Li, MY Lv, XP Li, LR Jian, JP Ma, TLS Sun, ZY Qiao, WH Xu, H. Wang, Nature Communications 2024, 15, 16.). In addition, supramolecular self-assembly structures have the ability to deliver drugs, so they are often used as effective carriers of drugs. The modified porphyrin-based supramolecular assemblies will be able to respond to changes in external pH, light, and the microenvironment of tumor cells, such as changes in aggregation state, drug release, changes in absorption spectrum, and enhanced ability to produce reactive oxygen species. Therefore, the artificial design and synthesis of porphyrin self-assembled supramolecular systems with unique properties has important practical significance and research value. Summary of the invention

[0005] The purpose of the present invention is to provide a synthesis method and application of a porphyrin-based supramolecular self-assembly nanosystem that targets and responds to mitochondria. The method constructs a photodynamic therapy nanosystem that integrates multiple functions such as precise targeting of mitochondria, dynamic response of glutathione, and synergistic enhancement of the level of reactive oxygen in the tumor microenvironment. The triphenylphosphine (PPh3) unit with mitochondrial targeting function and the disulfide bond (SS) unit that responds to overexpressed glutathione (GSH) in the tumor microenvironment are introduced into the molecular system by modifying the substituents on the aromatic ring of the porphyrin meso-position, and the TPP-SS-PPh3 self-assembly building unit (TSP) is designed and synthesized, and catalase (CAT) is loaded during the self-assembly process to finally obtain the target product porphyrin supramolecular assembly TPP-SS-PPh3@CAT NPs (TSPC). Studies have shown that the assembly system can accurately locate and enrich mitochondria in tumor cells, significantly improve the level of reactive oxygen in cells, and effectively improve the therapeutic effect of photodynamic therapy (PDT). It has shown good potential application value in the fields of drug delivery, intracellular tracing imaging and anti-tumor therapy.

[0006] The present invention adopts the following technical scheme to achieve the above-mentioned purpose, a method for synthesizing a porphyrin-based organic supramolecular self-assembled nanosystem, and the specific synthesis process is: a monocarboxyl porphyrin and a triphenylphosphine group are connected by bis(2-hydroxyethyl) disulfide through an esterification reaction to obtain a compound TPP-SS-PPh3, and then the difference in hydrophilicity of different groups in the compound TPP-SS-PPh3 is used to self-assemble in a mixed solvent of tetrahydrofuran and water to obtain a nanostructured compound TPP-SS-PPh3NPs (TSP), and catalase (CAT) is added during the self-assembly process for loading to obtain a target product having good water solubility and stability. The porphyrin-based organic supramolecular self-assembled nanosystem TPP-SS-PPh3@CATNPs (TSPC), the specific synthesis route is as follows:

[0007]

[0008] The method for synthesizing the porphyrin-based organic supramolecular self-assembly nanosystem of the present invention comprises the following specific steps:

[0009] Step S1: Triphenylphosphine (PPh3) and 6-bromohexanoic acid are heated to reflux under inert gas protection conditions with acetonitrile as solvent until the raw materials react completely, and the reaction solution is filtered, washed and crystallized to obtain a compound PPh3-COOH;

[0010] Step S2: monocarboxylporphyrin (TPP), excess bis(2-hydroxyethyl) disulfide and a catalyst are reacted under inert gas protection with N,N-dimethylformamide as a solvent at room temperature with stirring until the raw materials react completely, and the reaction solution is separated by column chromatography and purified by recrystallization to obtain a compound TPP-SS-OH, wherein the catalyst is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP);

[0011] Step S3: The TPP-SS-OH obtained in step S2, an excess of PPh3-COOH obtained in step S1 and a catalyst are reacted under the protection of an inert gas with dichloromethane as a solvent at room temperature with stirring until the raw materials react completely, and the reaction solution is separated by column chromatography and purified by recrystallization to obtain a compound TPP-SS-PPh3, wherein the catalyst is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine;

[0012] Step S4: dissolving the compound TPP-SS-PPh3 obtained in step S3 in tetrahydrofuran, adding an aqueous solution of catalase, and then removing the tetrahydrofuran to obtain the target product porphyrin-based organic supramolecular self-assembled nanosystem TPP-SS-PPh3@CATNPs (TSPC) dispersed in the aqueous solution.

[0013] It is further defined that the molar ratio of the monocarboxyl porphyrin to bis(2-hydroxyethyl) disulfide in step S2 is 1:6-10.

[0014] It is further defined that the molar ratio of the compound TPP-SS-OH to the compound PPh3-COOH in step S3 is 1:1.5 to 2.5.

[0015] The invention discloses an application of the porphyrin-based organic supramolecular self-assembly nanosystem in the preparation of mitochondria-targeted photodynamic therapy drugs for tumors.

[0016] The invention discloses an application of the porphyrin-based organic supramolecular self-assembly nanosystem in the preparation of a drug delivery system.

[0017] The invention discloses an application of the porphyrin-based organic supramolecular self-assembly nanosystem in the preparation of intracellular tracing imaging preparations.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention has relatively few synthesis steps, high yield, mild preparation conditions, and the porphyrin molecule itself as a building unit has unique photochemical properties. Porphyrin can be used as a photosensitizer in photodynamic therapy and as a hydrophobic group of a self-assembly building unit, with multiple uses in one body and a simplified model; at the same time, bis(2-hydroxyethyl) disulfide is used as a bridge linking the mitochondrial targeting group (PPh3) and porphyrin, and also as a response part of the tumor microenvironment. After entering the tumor cell, the assembly is cleaved to release the porphyrin molecule, which greatly enhances the killing power of the tumor cell. This makes the present invention as a multifunctional organic self-assembly nanoplatform for enhancing tumor photodynamic therapy show excellent performance and good potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scanning electron microscope image of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1.

[0020] Figure 2 This is a transmission electron microscopy image of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1.

[0021] Figure 3 This is the UV-visible absorption spectrum of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1.

[0022] Figure 4 This is the singlet oxygen performance diagram of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1.

[0023] Figure 5 This is a comparative analysis diagram of the co-localization of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1 targeting mitochondria.

[0024] Figure 6 This is a graph showing the in vitro biocompatibility and cell light / dark toxicity test of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1.

[0025] Figure 7 This is a diagram showing the in vivo anti-tumor effect of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs prepared in Example 1. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0027] Example 1

[0028] Synthesis of Compound PPh3-COOH

[0029]

[0030] In a 100mL round-bottom flask, 6-bromohexanoic acid (2g, 10.25mmol) and triphenylphosphine (2.825g, 10.75mmol) were added in sequence, and 15mL of dry acetonitrile was added under N2 protection. The reaction mixture was stirred and refluxed at 95°C for 16h, then cooled to room temperature, and the solvent was removed under reduced pressure to precipitate a white solid. The solid was washed with ether three times and then dried to obtain a white solid product (3.146g, yield 85.43%). 1 H NMR (400MHz, CDCl3) δ7.92–7.60 (m, 14H), 3.66 (s, 2H), 2.44 (t, J = 6.4Hz, 2H), 1.71 (s, 6H).

[0031] Example 2

[0032] Synthesis of Compound TPP-SS-OH

[0033]

[0034] Compound monocarboxylporphyrin (100 mg, 0.152 mmol), bis(2-hydroxyethyl) disulfide (0.3 mL, 1.25 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (48.3 mg, 0.253 mmol) and 4-dimethylaminopyridine (DMAP) (28 mg, 0.23 mmol) were added to a 50 mL round-bottom flask, and 15 mL of dry N,N-dimethylformamide (DMF) was added under N2 protection. After stirring at room temperature for 48 h under N2 protection, the solvent was removed under reduced pressure, extracted with dichloromethane, washed three times with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and separated by a chromatographic column. The developing solvent was dichloromethane: methanol (volume ratio 100:1), and a purple solid product (50 mg, yield 41.3%) was obtained. 1 HNMR (400MHz, DMSO) δ8.84 (s, 8H), 8.37 (d, J = 8.5Hz, 4H), 8.23 ​​(d, J = 9.2Hz, 7H), 7.85 (s, 10H), -2.92 (s, 2H).

[0035] Example 3

[0036] Synthesis of Compound TPP-SS-PPh3

[0037]

[0038] Compound TPP-SS-OH (134 mg, 0.168 mmol), compound PPh3-COOH (127 mg, 0.337 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (128.8 mg, 0.674 mmol) and 4-dimethylaminopyridine (DMAP) (41.1 mg, 0.337 mmol) were added to a 50 mL round-bottom flask, and 20 mL of dry dichloromethane was added under N2 protection. The reaction was stirred at room temperature under N2 protection for 36 h, then extracted with dichloromethane, washed three times with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and separated by column chromatography. The developing solvent was dichloromethane: methanol (volume ratio of 20:1) to obtain a purple solid product (80 mg, yield of 48.7%). 1 H NMR (400MHz, CDCl3) δ8.85(s,8H),8.43(d,J=8.1Hz,2H),8.30(d,J=8.1Hz,2H),8.24-8.17(m,6H),7.88-7.60(m,26H ), 4.76 (s, 2H), 4.37 (s, 2H), 3.83 (s, 2H), 3.20 (s, 2H), 3.01 (s, 2H), 2.35 (s, 2H), 1.65 (d, J = 7.2Hz, 6H), -2.78 (s, 2H).

[0039] Example 4

[0040] The assembly process of the porphyrin-based organic supramolecular self-assembled nanosystem TPP-SS-PPh3@CATNPs (TSPC) was as follows: first, 0.8 mg of catalase (CAT) was weighed and dissolved in 2 mL of water to prepare a catalase (CAT) solution (0.4 mg / mL); then, TPP-SS-PPh3 (1.2 mg) was weighed into a 10 mL centrifuge tube, 2 mL of tetrahydrofuran (THF) was added to dissolve it, and then the above-mentioned CAT solution was added to prepare a mixed solution of TPP-SS-PPh3 and CAT. The organic solvent in the solution was then removed by bubbling, and the precipitate was collected by high-speed centrifugation and washed three times with ultrapure water to obtain the target product, the self-assembled nanomaterial TPP-SS-PPh3@CATNPs (TSPC).

[0041] Figure 1 and 2They are scanning and transmission electron micrographs of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs. It can be seen from the figure that the average particle size of the synthesized TSPC nanomaterial is about 82.73 nm, indicating that the TSPC nanomaterial has good dispersion and stability.

[0042] Figure 3 The UV-visible absorption spectrum of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs shows that the UV absorption of the self-assembled nanomaterials (TSP, TSPC) decreases with its maximum peak after assembly, and the B band peak red-shifts from 418nm to 423nm and 432nm, proving that the compound TPP-SS-PPh3 is assembled into nanoaggregates.

[0043] Figure 4 This is the performance diagram of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs in generating singlet oxygen. The change in the absorbance of the singlet oxygen scavenger 1,3-diphenylisobenzofuran (DPBF) at 412nm was detected by UV-visible absorption spectroscopy to characterize the ability of the sample to generate singlet oxygen. The same concentration of TP, TSP+GSH, TSPC+GSH, and TSPC+GSH+H2O2 were used to detect the ability to generate singlet oxygen. After irradiation with a 650nm laser for 100s, the absorbance of DPBF at 412nm in the TSPC+GSH+H2O2 group was significantly lower than that in other groups. This shows that the sample TSPC releases porphyrin molecules under the action of glutathione, and has the ability to generate singlet oxygen and catalyze hydrogen peroxide to produce oxygen under 650nm laser irradiation.

[0044] Figure 5 This is a co-localization analysis diagram of different self-assembled materials in cell mitochondria. The CLSM test results show that the supramolecular systems formed by molecular units modified with triphenylphosphine groups (TP, TSP and TSPC) have good mitochondrial targeting effects, while TPP has no obvious targeting enrichment effect on mitochondria without being modified with mitochondrial targeting groups.

[0045] Figure 6 The figure shows the in vitro biocompatibility and cell light / dark toxicity test of the self-assembled nanomaterial TPP-SS-PPh3@CATNPs. In order to prove that the self-assembled nanomaterials (TP, TSP, TSPC) have good biocompatibility and phototoxicity, the three nanoparticles were co-incubated with normal rat liver cells (3A cells) and mouse breast cancer cells (4T1 cells) to test the biocompatibility of the three nanoparticles and their killing ability to tumor cells. Figure 6As shown, the three nanomaterials showed good biocompatibility when incubated with normal cells and without laser irradiation, and the killing effect of the 650nm laser irradiation group on tumor cells was significantly better than that in the dark group, and the toxicity of the three nanoparticles to 4T1 cells increased successively.

[0046] Twenty-five female BALB / c mice inoculated with 4T1 cells were randomly divided into five groups (PBS, TSPC, TP+L, TSP+L, TSPC+L). When the average tumor volume in each group reached 100 mm 3 Each mouse was injected with the drug through the tail vein. 24 hours after injection, the mice were treated with 650 nm laser irradiation for 10 min, while the PBS and TSPC groups were not irradiated with laser. Figure 7 As shown in the figure, there was no significant change in the weight of mice in each group during the 14 days of treatment, which once again proved that the synthesized self-assembled nanoparticles had good biocompatibility and no obvious toxic side effects. At the same time, the tumor volume of each group was measured every other day during the 14 days, and the changes in the tumor volume of different treatment groups over time were recorded. The tumor volume of the PBS and TSPC groups grew rapidly to 600mm on the 14th day. 3 With 350mm 3 This indicates that TSPC did not exert its anti-tumor properties without laser irradiation. For the laser irradiation group, the tumor volume of the TP+L group was about 150mm on day 14. 3 Around, TSP+L group at 90mm 3 The tumors in the TSPC+L group were almost gone on day 14, and were significantly smaller than those in the control group. This proves that TSPC has a good photodynamic therapy effect under 650nm laser irradiation and has the best inhibitory effect on tumors. In addition, this result can also be verified by observing the tumor volume and tumor weight of each group.

[0047] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for synthesizing a mitochondrial targeting and responsive porphyrin-based supramolecular self-assembled nanosystem, characterized in that The specific synthesis process is: the monocarboxyl porphyrin is connected with the triphenylphosphine group through bis(2-hydroxyethyl) disulfide through an esterification reaction to obtain the compound TPP-SS-PPh3, and then the difference in hydrophilicity of different groups in the compound TPP-SS-PPh3 is used to self-assemble in a mixed solvent of tetrahydrofuran and water to obtain the nanostructured compound TPP-SS-PPh3 NPs. During the self-assembly process, catalase is added for loading to obtain the target product porphyrin-based organic supramolecular self-assembled nanosystem TPP-SS-PPh3@CATNPs. The specific synthesis route is as follows:

2. The method for synthesizing the porphyrin-based organic supramolecular self-assembly nanosystem according to claim 1, characterized in that The specific synthesis steps are: Step S1: heating triphenylphosphine and 6-bromohexanoic acid under inert gas protection with acetonitrile as solvent for reflux reaction until the raw materials react completely, filtering, washing and crystallizing the reaction solution to obtain compound PPh3-COOH; Step S2: monocarboxyl porphyrin, excess bis(2-hydroxyethyl) disulfide and a catalyst are reacted under inert gas protection with N,N-dimethylformamide as a solvent at room temperature with stirring until the raw materials react completely, and the reaction solution is separated by column chromatography and purified by recrystallization to obtain a compound TPP-SS-OH, wherein the catalyst is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; Step S3: The TPP-SS-OH obtained in step S2, an excess of PPh3-COOH obtained in step S1 and a catalyst are reacted under the protection of an inert gas with dichloromethane as a solvent at room temperature with stirring until the raw materials react completely, and the reaction solution is separated by column chromatography and purified by recrystallization to obtain a compound TPP-SS-PPh3, wherein the catalyst is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine; Step S4: dissolving the compound TPP-SS-PPh3 obtained in step S3 in tetrahydrofuran, adding an aqueous solution of catalase, and then removing the tetrahydrofuran to obtain the target product porphyrin-based organic supramolecular self-assembled nanosystem TPP-SS-PPh3@CATNPs dispersed in the aqueous solution.

3. The method for synthesizing the porphyrin-based organic supramolecular self-assembly nanosystem according to claim 1, characterized in that: The molar ratio of the monocarboxyl porphyrin to bis(2-hydroxyethyl) disulfide in step S2 is 1:6-10.

4. The method for synthesizing the porphyrin-based organic supramolecular self-assembly nanosystem according to claim 1, characterized in that: In step S3, the molar ratio of the compound TPP-SS-OH to the compound PPh3-COOH is 1:1.5-2.

5.

5. Use of the porphyrin-based organic supramolecular self-assembled nanosystem prepared by the method according to any one of claims 1 to 4 in the preparation of mitochondria-targeted photodynamic therapy drugs for tumors.

6. Use of the porphyrin-based organic supramolecular self-assembled nanosystem prepared by the method according to any one of claims 1 to 4 in the preparation of a drug delivery system.

7. Use of the porphyrin-based organic supramolecular self-assembled nanosystem prepared by the method according to any one of claims 1 to 4 in the preparation of intracellular tracing imaging preparations.

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