Cell-penetrating riboflavin complex and its use in the manufacture of a photodynamic therapy drug

Abstract

The application discloses a cell-penetrating riboflavin complex and application thereof in preparation of a photodynamic therapy drug. The riboflavin is coupled with cell-penetrating peptides Arg8, (Cha-Arg)3 and a targeting small molecule triphenylphosphine (TPP) respectively, three complexes of Arg8-RF, (Cha-Arg)3-RF and TPP-RF are designed, and by comparing the cell uptake efficiency and photosensitivity toxicity of the three complexes, (Cha-Arg)3-RF can improve the uptake efficiency of riboflavin by tumor cells, and meanwhile, the photodynamic therapy effect of riboflavin is enhanced. The riboflavin complex can be used to prepare a natural derivative anti-tumor photodynamic therapy drug, and the drug has the advantages of good biocompatibility, low toxicity, and economic availability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a cell-penetrating riboflavin complex and its application in the preparation of photodynamic therapy drugs. BACKGROUND

[0002] Photodynamic therapy (PDT) is a modern, non-invasive new cancer treatment method, which has the advantages of minimally invasive, targeted, low toxicity, no drug resistance, etc. In recent years, photodynamic therapy as a non-surgical alternative therapy has been widely used in the treatment of malignant tumors. Photodynamic therapy relies on the interaction of light source and photosensitizer to produce reactive oxygen species (ROS), which acts on biological molecules such as proteins and nucleic acids and destroys their structure, ultimately causing cell death. In photodynamic therapy, photosensitizers play an important role. Some photosensitizers reported so far, such as hematin, have the disadvantages of poor water solubility, large toxic and side effects, etc. Therefore, the development of a biocompatible, economical and non-toxic natural derivative photosensitizer has attracted widespread attention from researchers.

[0003] Riboflavin (RF), also known as vitamin B2, is widely present in vegetables and food, and is an essential vitamin for the human body, which is an important component of in vivo flavin coenzyme. As an indispensable vitamin for human life activities, riboflavin plays an important role in improving the metabolism of organisms and promoting the growth and development of the human body. Riboflavin is composed of an isoalloxazine nucleus and a D-type sugar chain. The isoalloxazine structure makes riboflavin have strong absorption at 375 nm and 445 nm, and emit fluorescence at 550 nm. It has been reported that riboflavin has certain photodynamic properties and is an important photosensitizer. Riboflavin can undergo complex photochemical reactions with amino acids, proteins, DNA, etc. under the irradiation of blue light or ultraviolet light, causing the destruction of biological molecule structure and function. The triplet excited state of riboflavin can undergo photochemical reactions with oxidizable substrates or oxygen molecules to produce reactive oxygen species (ROS) such as O 2·- ·, 1 O 2 As an important natural photosensitizer, riboflavin has been widely used in the treatment of pathogen destruction, virus and bacteria inactivation. Based on its superior optical and photochemical properties, riboflavin as a photosensitizer has also been concerned in the application of anti-tumor photodynamic therapy. However, the uptake of riboflavin by various tumor cells is very low, and riboflavin is non-toxic to these cells under blue light irradiation. Therefore, there is an urgent need for a kind of riboflavin which can effectively penetrate cells and enhance the photosensitivity of riboflavin, so as to develop riboflavin into a natural source of biocompatible, low-toxicity anti-tumor photodynamic therapy drug. SUMMARY

[0004] To address the shortcomings of existing technologies, this invention proposes a cell-penetrating riboflavin complex and its application in the preparation of photodynamic therapy drugs. Riboflavin is coupled to cell-penetrating peptides Arg8, (Cha-Arg)3, and the targeting small molecule triphenylphosphine (TPP), respectively, to design three complexes: Arg8-RF, (Cha-Arg)3-RF, and TPP-RF.

[0005] The present invention provides a cell-penetrating riboflavin complex comprising riboflavin and its functional ligand, wherein the functional ligand is any one of a cell-penetrating peptide and triphenylphosphine.

[0006] Furthermore, the cell-penetrating peptide is a polypeptide rich in arginine.

[0007] Furthermore, the cell-penetrating peptide is a hexapeptide composed of alternating cyclohexylalanine and arginine.

[0008] Furthermore, the arginine-rich polypeptide is Arg8.

[0009] Furthermore, the structure of the cell-penetrating riboflavin complex is shown in formula (I):

[0010]

[0011] Furthermore, the structure of the cell-penetrating riboflavin complex is shown in formula (II):

[0012]

[0013] Furthermore, the structure of the cell-penetrating riboflavin complex is shown in formula (III):

[0014]

[0015] The present invention also provides a method for preparing the cell-penetrating riboflavin complex, wherein riboflavin is coupled with the functional ligand to synthesize a riboflavin-functional ligand complex, wherein the riboflavin-functional ligand complex is either cell-penetrating peptide-riboflavin or triphenylphosphine-riboflavin.

[0016] The present invention also provides the use of the cell-penetrating riboflavin complex in the preparation of photodynamic therapy drugs.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0018] 1. This invention improves the cellular uptake efficiency of riboflavin by coupling functional ligands, enhances the photosensitivity and toxicity of riboflavin to tumor cells, and strengthens the photodynamic therapy effect of riboflavin.

[0019] 2. The riboflavin complex of the present invention can be used to prepare naturally derived antitumor photodynamic therapy drugs, which have advantages such as good biocompatibility, low toxicity, and economic availability. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The structure (A) and synthetic route (B) of the riboflavin complex are shown.

[0022] Figure 2 Riboflavin degrades tyrosine-containing peptides and proteins. (A, B) Degradation of peptides by riboflavin under blue light irradiation. (C) Degradation of tyrosine-containing proteins by riboflavin under blue light irradiation. (D) Photoproducts of riboflavin.

[0023] Figure 3 It facilitates cellular uptake of riboflavin and riboflavin complexes.

[0024] Figure 4 It is cytotoxic to riboflavin and riboflavin complexes.

[0025] Figure 5 This is for the determination of intracellular ROS.

[0026] Figure 6 For mitochondrial membrane potential analysis.

[0027] Figure 7 The HRMS spectrum of compound RF-COOH(4) is shown.

[0028] Figure 8 The HRMS spectrum of compound RF-Ma(5) is shown.

[0029] Figure 9 The HRMS spectrum of compound Arg8-RF(1) is shown.

[0030] Figure 10 The HRMS spectrum of compound (Cha-Arg)3-RF(2) is shown.

[0031] Figure 11 The HRMS spectrum of compound TPP-NH2(8) is shown.

[0032] Figure 12 The HRMS spectrum of compound TPP-RF(3) is shown.

[0033] Figure 13 HPLC analysis of the riboflavin complex Arg8-RF(1).

[0034] Figure 14 HPLC analysis of riboflavin complex (Cha-Arg) 3-RF(2).

[0035] Figure 15 HPLC analysis of the riboflavin complex TPP-RF(3). Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0037] Riboflavin is a biocompatible, non-toxic, and naturally available photosensitizer. Although riboflavin is an essential vitamin for the human body, its intracellular concentration is very low, resulting in minimal photosensitivity. Increasing the intracellular concentration of riboflavin holds promise for developing it into a photodynamic therapy drug with significant anti-tumor activity. It has been shown that riboflavin can oxidize Aβ1-42 under visible light irradiation, leading to a decrease in Aβ1-42 aggregation ability and neurotoxicity. Studies have shown that riboflavin nanocrystals possess optical waveguide and photosensitizing properties; under light irradiation, riboflavin nanocrystals can kill tumor cells. Previous studies have found that various tumor cell types, including PC3, HeLa, MDA-MB-231, A549, and U-87, show very low riboflavin uptake, and that riboflavin is non-toxic to these cell types under blue light irradiation. Therefore, it is possible to improve the cell entry efficiency of riboflavin by using delivery tools such as cell-penetrating peptides and targeted molecules, which can enhance the photosensitivity and toxicity of riboflavin. It is hoped that riboflavin can be developed into a naturally derived, biocompatible, and low-toxicity anti-tumor photodynamic therapy drug.

[0038] Experiments have shown that riboflavin has extremely low efficiency in entering tumor cells, and correspondingly, riboflavin itself has no phototoxicity to tumor cells. Therefore, developing photodynamic therapy drugs using riboflavin as a photosensitizer and enhancing its cellular uptake efficiency is crucial. This invention discovers that arginine-rich peptides, such as Arg8, can serve as delivery tools to improve the ability of drugs and nanomaterials to enter cells. Furthermore, hexapeptides composed of alternating cyclohexylalanine (Cha) and Arg can effectively enter cells and distribute in the cytoplasm. Therefore, using the cell-penetrating peptides Arg8 and (Cha-Arg)3 as delivery ligands, this invention synthesizes Arg8-RF and (Cha-Arg)3-RF complexes. Considering the high oxygen levels around mitochondria, which are conducive to ROS generation, this invention couples the mitochondrial targeting molecule triphenylphosphine (TPP) with riboflavin to synthesize the complex TPP-RF. This invention utilizes cell-penetrating peptides Arg8 and (Cha-Arg)3, along with the mitochondrial-targeting molecule triphenylphosphine (TPP), as delivery ligands to enhance the cell entry efficiency of riboflavin. Three complexes—Arg8-RF, (Cha-Arg)3-RF, and TPP-RF—were designed and synthesized. By examining the cellular uptake efficiency and cytotoxicity of these complexes, the mechanism of cell death induction was analyzed, and antitumor photodynamic therapy drugs with good cell penetration and strong photosensitivity were screened out, providing a new strategy for the development of drugs based on naturally derived riboflavin.

[0039] To achieve the above objectives, this invention provides a cell-penetrating riboflavin complex and evaluates its cellular uptake efficiency and photodynamic therapy efficacy. The method includes the following steps: (1) degradation of peptides and proteins by riboflavin; (2) synthesis of riboflavin complexes Arg8-RF, (Cha-Arg)3-RF, and TPP-RF; (3) investigation of cellular uptake efficiency; (4) study of antitumor photodynamic therapy efficacy; (5) assessment of cytotoxicity; (6) generation of intracellular ROS; and (7) mitochondrial membrane potential analysis.

[0040] Example 1: Degradation of peptides and proteins by riboflavin

[0041] 3.1 μL of 50 mM riboflavin solution was added to 300 μL of 1.3 mM PBS solution containing peptides. This mixture was irradiated with an LED lamp (13W, 450-475 nm) for 20 min, and then 10 μL of the solution was analyzed by HPLC. 300 μL of PBS solution containing insulin (30 μM) and riboflavin (12 μM) was irradiated with an LED lamp for 20 min, and then 10 μL of the solution was analyzed by HPLC. PBS solution containing green fluorescent protein (GFP, 1 μM) and riboflavin (10 μM) was irradiated with an LED lamp for 20 min, and then the reaction solution was analyzed by electrophoresis. The results are as follows: Figure 2As shown, the degradation of peptides and proteins by riboflavin indicates that riboflavin can degrade peptides rich in histidine, tryptophan, and tyrosine, and can also degrade proteins rich in tyrosine such as insulin and green fluorescent protein. This suggests that riboflavin can disrupt the structure and function of peptides and proteins, and may become a naturally derived photodynamic therapy drug.

[0042] Example 2 Synthesis of Riboflavin Complex

[0043] The structure and synthetic route of the riboflavin complex are as follows: Figure 1 As shown:

[0044] Step 1. Synthesis of compound RF-COOH(4). Riboflavin (200 mg, 0.53 mmol) and triethylamine (440 μL, 3.18 mmol) were dissolved in anhydrous dimethyl sulfoxide (2 mL). The reaction mixture was stirred at 80 °C for 3 h, then adipic anhydride (67.9 mg, 0.53 mmol) was added, and stirring was continued at 80 °C for 5 h. The reaction solution was purified by HPLC and lyophilized to give carboxylated riboflavin 4 (96 mg, 36.2%). HRMS(ESI) m / z:calcd.for C 23 H 29 N4O9[M+H] + 505.1929, found 505.19345 ( Figure 7 ).

[0045] Step 2. Synthesis of compound RF-Ma(5). Carboxylated riboflavin 4 (19.3 mg, 0.038 mmol) and HATU (14.4 mg, 0.038 mmol) were dissolved in anhydrous DMF (2 mL), and then DIEA (66 μL, 0.38 mmol) was added. After activation for 1 min, N-(2-aminoethyl)maleimide hydrochloric acid (6.7 mg, 0.038 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction solution was purified by HPLC and lyophilized to obtain a yellow solid RF-Ma(5) (22 mg, 92.4%). HRMS(ESI) m / z:calcd.for C 29 H 35 N6O 10 [M+H] + 627.2409, found 627.24170 ( Figure 8 ).

[0046] Step 3. Synthesis of Arg8-RF(1) and (Cha-Arg)3-RF(2). Thiol-containing transmembrane peptides HS-Arg8(6) (13.5 mg, 0.01 mmol) and HS-(Cha-Arg)3(7) (10 mg, 0.01 mmol) were added to anhydrous DMF (500 μL) solutions of RF-Ma (3.1 mg, 0.005 mmol) and TEA (0.05 mmol, 7 μL), respectively. The mixture was stirred in an ice bath for 2 h, and the reaction solution was purified by RP-HPLC. After lyophilization, Arg8-RF(1) (9 mg) and (Cha-Arg)3-RF(2) (8 mg) were obtained, with yields of 90.8% and 96.4%, respectively. The HPLC results of Arg8-RF(1) are shown below. Figure 13 As shown in Table 1, the HPLC results of (Cha-Arg)3-RF(2) are as follows: Figure 14 As shown in Table 2.

[0047] Table 1. HPLC results of Arg8-RF(1)

[0048]

[0049] Table 2. HPLC results of (Cha-Arg)3-RF(2)

[0050]

[0051] Arg8-RF HRMS(ESI)m / z:calcd.for C 80 H 139 N 38 O 20 S[M+3H] 3+ 661.3577, found661.35724( Figure 9 ).(Cha-Arg)3-RF HRMS(ESI)m / z:calcd.for C 77 H 125 N 22 O 17 S[M+3H] 3+ 553.9765, found 553.97723 ( Figure 10 ).

[0052] Step 4. Synthesis of compound TPP-NH2(8). (3-Propylcarboxy)triphenylphosphine bromide (22.17 mg, 0.052 mmol) and HATU (19.8 mmol, 0.052 mmol) were dissolved in anhydrous DMF, and then activated with DIEA (27.2 μL, 0.156 mmol). N,N-bis(3-aminopropyl)methylamine (42 μL, 0.26 mmol) was added to the activation solution, and the mixture was stirred at room temperature for 1 h. The reaction solution was purified by RP-HPLC and lyophilized to obtain TPP-NH2 (19 mg, 76.7%). HRMS(ESI) m / z:calcd.for C 29 H 40 N3OP[M+H] 2+ 238.6449, found 238.64470 ( Figure 11 ).

[0053] Step 5. Synthesis of TPP-RF(3). RF-COOH(4) (4.2 mg, 0.0083 mmol) and HATU (3.56 mg, 0.0083 mmol) were dissolved in anhydrous DMF (500 μL), and then DIEA (30 μL, 0.166 mmol) was added for activation for 1 min. TPP-NH2(8) (4 mg, 0.0083 mmol) was then added to the activation solution. The reaction mixture was stirred at room temperature for 1 h and purified by RP-HPLC. The collected product was lyophilized to obtain TPP-RF(3) (7 mg, 87.5%). The HPLC results of TPP-RF(3) are shown below. Figure 15 As shown in Table 3.

[0054] Table 3 HPLC results of TPP-RF(3)

[0055]

[0056] HRMS(ESI)m / z:calcd.for C 52 H 66 N7O9P[M+H] 2+ 481.7324, found 481.73309 ( Figure 12 ).

[0057] The preparation methods of riboflavin complexes Arg8-RF, (Cha-Arg)3-RF and TPP-RF in this invention have been experimentally proven to be feasible, and the experimental conditions are reproducible with stable yields.

[0058] Example 3: Study on Cell Uptake

[0059] HeLa cells were seeded in 12-well plates and cultured for 24 h. Riboflavin and its complex Arg8-RF, (Cha-Arg)3-RF, and TPP-RF were prepared at 25 μM using culture medium and added to the cells, then incubated at 37 °C for 2 h. Cell nuclei were stained with Hoechst 33258 (10 μg / mL) for 30 min. After staining, excess dye was washed away with PBS, and cells were fixed with 4% paraformaldehyde for 15 min. Finally, fluorescence imaging was performed using an Olympus Qlmaging Retiga R6. Riboflavin and its complex were excited with 488 nm blue light, and their fluorescence was monitored in the green channel. The results are shown below. Figure 3 As shown in the figure. Cellular uptake experiments showed that riboflavin has extremely low cellular uptake efficiency and no cytotoxicity under light conditions; after riboflavin is coupled with functional molecules to form a complex, (Cha-Arg)3-RF can enter cells in large quantities, Arg8-RF accumulates very little in cells, while TPP-RF cannot enter cells.

[0060] Example 4: Study on the effect of anti-tumor photodynamic therapy

[0061] 3000 to 4000 HeLa cells were seeded into 96-well plates. After 24 hours, the culture medium was removed. Riboflavin and its complex Arg8-RF, (Cha-Arg)3-RF, and TPP-RF were prepared at 5, 10, 25, and 50 μM concentrations using culture medium and added to the cells, with a blank solvent as a control. Cells were incubated at 37°C in a 5% CO2 incubator. After 4 hours, the drugs were washed away with PBS, and 100 μL of PBS was added, followed by LED blue light irradiation. After 20 minutes of irradiation, the PBS was removed, and fresh culture medium was added. Cells were incubated at 37°C in a 5% CO2 incubator. After 48 hours, CCK8 reagent was added, and incubation continued for 1 hour. The absorbance at 450 nm was then measured using a Thermol Multiskan GO. Each experiment was repeated three times independently.

[0062] Example 5: Dead Cell Staining Analysis

[0063] HeLa cells were seeded in 12-well plates and cultured for 24 h. Riboflavin and its complex (Cha-Arg) 3-RF were prepared at 25 μM using culture medium and added to the cells, followed by 4 h of incubation. The drugs were then removed, PBS was added, and the cells were irradiated with blue light for 20 min. After irradiation, the cells were stained with PI (50 μg / mL) and Hoechst 33258. After 30 min of incubation, the staining solution was washed off, fresh culture medium was added, and imaging was performed using an Olympus BX63 fluorescence microscope. PI was excited with 560 nm green light, and its fluorescence was monitored in the red light channel. The results are shown below. Figure 4As shown in the figure. Cytotoxicity experiments showed that (Cha-Arg)3-RF exhibited strong photosensitivity to tumor cells at the same concentration, while Arg8-RF and TPP-RF had almost no effect on cell viability under light conditions. This is consistent with the cellular uptake results, meaning that improving the cellular uptake efficiency of riboflavin can enhance its photosensitivity. Further experiments demonstrated that the phototoxicity of (Cha-Arg)3-RF was directly proportional to the drug concentration and the duration of light exposure. PI staining experiments showed that a large number of cells treated with (Cha-Arg)3-RF died under light conditions, while no dead cells were observed in the control group treated with RF.

[0064] Example 6: Intracellular ROS Generation

[0065] HeLa cells were seeded in 12-well plates and cultured for 24 h. Riboflavin and its complex (Cha-Arg) 3-RF were prepared at 10 μM using culture medium and added to the cells, followed by 4 h of incubation. The drug was then removed, and the ROS probe H2DCFDA (10 μg / mL) was added. After 1 h of incubation, the cells were washed three times with PBS and then irradiated with PBS. After 10 min of irradiation, the cells were stained with a Hoechst 33258 microscope. After 30 min of incubation, the staining solution was washed off, and fresh culture medium was added. Imaging was performed using an Olympus BX63 fluorescence microscope. H2DCFDA was excited with 488 nm blue light, and its fluorescence was monitored in the green channel. The results are shown below. Figure 5 As shown in the figure, intracellular ROS measurement and analysis showed that (Cha-Arg)3-RF generates ROS under blue light irradiation. ROS can non-selectively destroy protein structure and function, cause mitochondrial damage, and ultimately induce cell death.

[0066] Example 7 Mitochondrial membrane potential analysis

[0067] HeLa cells were seeded in 12-well plates and cultured for 24 h. Riboflavin and its complex (Cha-Arg) 3-RF were prepared at 10 μM using culture medium and added to the cells, followed by 4 h of incubation. The drugs were then removed, PBS was added, and the cells were irradiated with blue light for 10 min. After irradiation, the cells were stained with mitochondrial membrane potential analysis dye TMRE (10 μM) and Hoechst 33258. After 30 min of incubation, the staining solution was washed off, fresh culture medium was added, and imaging was performed using an Olympus BX63 fluorescence microscope. TMRE was excited with 560 nm green light, and its fluorescence was monitored in the red light channel. The results are shown below. Figure 6 As shown in the figure. Intracellular mitochondrial membrane potential analysis showed that (Cha-Arg)3-RF generates ROS under blue light irradiation. ROS can non-selectively destroy protein structure and function, cause mitochondrial damage, and ultimately induce cell death.

[0068] The functional ligands used in this invention can be replaced by other functional groups such as tumor-targeting molecules and tumor-targeting peptides, including folic acid, targeting peptides, nucleic acid aptamers, and targeting fluorescent dyes; riboflavin can be replaced by riboflavin derivatives such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), lumichrome, and riboflavin tetrabutyrate. Both the above functional ligands and riboflavin derivatives can achieve the technical effects of this invention.

[0069] In summary, this invention discloses a cell-penetrating riboflavin complex and its application in the preparation of photodynamic therapy drugs. Riboflavin was conjugated with cell-penetrating peptides Arg8, (Cha-Arg)3, and the targeting small molecule triphenylphosphine (TPP) to design three complexes: Arg8-RF, (Cha-Arg)3-RF, and TPP-RF. By comparing their cellular uptake efficiency and photosensitivity toxicity, (Cha-Arg)3-RF was found to improve the uptake efficiency of riboflavin by tumor cells and enhance the photodynamic therapy effect of riboflavin. The riboflavin complex of this invention can be used to prepare naturally derived antitumor photodynamic therapy drugs, which have advantages such as good biocompatibility, low toxicity, and economic availability.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell-penetrating riboflavin complex, characterized in that, comprising riboflavin and a functional ligand which is a cell-penetrating peptide; the structure of the cell-penetrating riboflavin complex is shown as formula (II):

2. The method of preparing a cell-penetrating riboflavin complex of claim 1, characterized in that, the riboflavin is coupled with the functional ligand to synthesize a riboflavin-functional ligand complex which is a cell-penetrating peptide-riboflavin.

3. Use of the cell-penetrating riboflavin complex of claim 1 in the preparation of a photodynamic therapy drug.

Images