Single sulfide bond bridged chrysin prodrug, preparation method and application thereof

Abstract

The present invention relates to a monosulfide-bridged chrysin prodrug, a preparation method and application thereof. The method comprises the following steps: connecting a long-chain fatty alcohol and the 7-hydroxyl group of chrysin by thioglycolic anhydride to obtain a chrysin prodrug having a monosulfide bond as a connecting bond and a long-chain fatty alcohol as a side chain. The long-chain fatty alcohol is tetradecanol C 14 , hexadecanol C 16 、Octadecanol C 18 Or oleyl alcohol (OA). The resulting monosulfide-bridged chrysin prodrug can be prepared into self-assembled nanoparticles via a one-step nanoprecipitation method. The resulting chrysin prodrug organic solution is added dropwise to water with stirring, spontaneously forming uniform nanoparticles. The solvent is then removed by rotary evaporation to obtain a nanoparticle solution. This simple preparation process allows for the release of the parent drug under the influence of high glutathione (GSH) concentrations in the tumor microenvironment, ensuring anti-tumor efficacy while minimizing toxic side effects on normal tissues. Furthermore, the nanoparticles can be used to prepare injectable formulations. Conjugating the nanoparticles with the modifier PEG enhances their stability.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a preparation method and application of a monosulfide-bridged chrysin prodrug and self-assembled nanoparticles thereof, and specifically relates to the synthesis of a chrysin prodrug with a monosulfide bond as an intermediate and different lipid side chains located at position 7 of chrysin, the preparation of self-assembled nanoparticles of a chrysin prodrug with different lipid side chain monosulfide bonds located at position 7 of chrysin, and the application of the same in drug delivery. Background Art

[0002] Cancer is one of the most serious diseases that threaten human life today, with high morbidity and mortality. Many natural medicines have a good effect in preventing cancer. For example, natural polyphenols have been widely used in the prevention and treatment of cancer and are potential candidates for anticancer drugs. [1] It also has a wide range of pharmacological effects such as anti-oxidation, anti-neurodegenerative diseases, anti-cardiovascular and cerebrovascular diseases, and anti-virus. [2,3] .

[0003] Studies have shown that polyphenolic flavonoids have strong anti-cancer and anti-tumor activities. [4] Chrysin (CHR) is a polyphenolic flavonoid compound with the following structural formula:

[0004]

[0005] Chrysin has a good killing effect on melanoma [5-7] , and also has anti-inflammatory properties [8] , antioxidant [8] , anti-tumor [9 ,10 ,11] and neuroprotection

[12] However, due to its own structure, chrysin has the disadvantages of poor solubility, rapid metabolism in the body, and low bioavailability, which limits its clinical application.

[13] Studies have confirmed that glucuronidation metabolism is the main metabolic pathway and clearance method of chrysin in the body.

[14] The 7-hydroxyl group of chrysin is the main site of glucuronidation metabolism, and the product is chrysin-7-O-glucuronide.

[15] .

[0006] In order to solve the problems of low solubility and rapid metabolism of chrysin, the applicable strategies mainly include the following two: 1) formulation strategy, such as the use of solid lipid nanoparticles

[16] , nanoemulsion [17,18] , liposomes

[19] , self-microemulsifying drug delivery system

[20] , micelles

[21] , eutectic [22,23] and nanoparticles

[24] Although the above-mentioned formulation strategy has solved the problem of poor solubility of chrysin to a certain extent and improved the bioavailability of chrysin (2.16 to 4.3 times), there are still some problems with traditional formulations. For example, liposomes and emulsions often use non-covalent physical encapsulation to encapsulate drugs, so they often have disadvantages such as low drug loading, poor stability, easy drug leakage, and toxic side effects of the carrier. [25,26] 2) Structural modification of chrysin has been studied.

[27] The synthesis of 5,7-dimethoxychrysin to block its metabolic sites improves the bioavailability of chrysin, but this reaction results in the binding of the active groups of chrysin, resulting in reduced efficacy. Therefore, a new strategy is urgently needed to deliver chrysin that can maintain its activity and enhance its efficacy while improving its bioavailability. Therefore, it is necessary to design chrysin (hydroxyl group at position 7) into a prodrug and use a prodrug nanodelivery system to reduce chrysin metabolism, increase drug loading, and improve the bioavailability and anti-tumor efficacy of chrysin.

[0007] Prodrug nanodrug delivery system based on self-assembly of prodrugs, which mainly includes polymer-anticancer drug macromolecular prodrug nanodrug delivery system, small molecule prodrug self-assembly nanodrug delivery system and nanodrug delivery system encapsulating small molecule prodrugs

[28] Among them, the small molecule prodrug self-assembly nano-delivery system has unique advantages, such as high drug loading, good stability, long blood circulation time, high safety, and intelligent response of prodrug activation at the tumor target site. [29,30] Small molecule prodrugs include small molecule prodrug drugs, linkers, and non-reactive parts. In order to improve the response to the redox microenvironment of tumor cells, the prodrug is designed to use redox-responsive linkers such as sulfur bonds. [31,41] , Selenium bond

[31] and disulfide bonds [41-44] etc., which enhances the advantages of drug tumor targeting [31,41-47] Small molecule prodrugs are divided into amphiphilic prodrugs, hydrophobic prodrugs and heterodimer prodrugs. For poorly soluble drugs, the non-reactive part of the prodrug

[54] , modified with common lipid side chains and designed as hydrophobic prodrugs. It has been used in many natural medicines and other chemotherapy drugs, such as paclitaxel [31-33] , docetaxel [34,35] , dihydroartemisinin [36,37] , curcumin

[38] , cytarabine [39,40]Lipid prodrugs enhance physical stability, improve pharmacokinetic characteristics, prolong circulation half-life, selective distribution (such as brain penetration), reduce liver first-pass metabolism, and increase absorption across biological barriers (such as the gastrointestinal wall and blood-brain barrier). For example, some prodrug preparations based on fatty acids and fatty alcohols can prolong systemic circulation time for several months or a year. For example, paliperidone palmitate is a currently available lipid-modified prodrug

[48] , and the effectiveness of a single injection can last up to half a year. Therefore, this lipid prodrug self-assembly nanodelivery system with oxidation and reduction properties has become the preferred strategy for many drug modification and delivery

[39] .

[0008] Commonly used lipids are saturated fatty acids and unsaturated fatty acids such as fatty acids

[49] , folic acid

[50] , vitamin E

[51] It is safe and compatible with the human body. It is mainly involved in the production and storage of energy, the synthesis of phospholipids required for membrane formation, lipid transport, and the covalent modification of many regulatory proteins. Fatty acids are effective activity regulators of drug metabolizing enzymes. [52,53] , such as sodium oleate, which, in addition to having certain emulsifying and stabilizing effects, is also a uridine diphosphate glucuronyl transferase that can inhibit the glucuronic acid-dependent metabolism of related drugs. Wu Baojian et al.

[17] In this study, sodium oleate was used to prepare chrysin nanoemulsions, which greatly improved the oral bioavailability of chrysin. However, the present application did not disclose the introduction of long-chain fatty alcohols into chrysin to obtain redox-responsive prodrugs for delaying the in vivo metabolism of chrysin and enhancing the anti-tumor activity of chrysin. Summary of the Invention

[0009] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a single-sulfide bridged chrysin prodrug, which introduces a long-chain fatty alcohol at the 7-hydroxyl group of chrysin to obtain a redox-responsive prodrug with self-assembly ability, thereby solving the problems of low solubility and rapid metabolism of chrysin in the body.

[0010] Another object of the present invention is to provide a method for preparing a monosulfide-bridged chrysin prodrug, which uses an oxidation reaction bond (monosulfide bond) as a bond bridge and a long-chain saturated fatty alcohol (tetradecanol C 14 , hexadecanol C 16 、Octadecanol C 18 ) and long-chain unsaturated fatty alcohol (oleyl alcohol, OA) as side chains of chrysin prodrug, which has a low melting point, high lipid solubility, amorphous crystal form and self-assembly ability.

[0011] Another object of the present invention is to provide a method for preparing chrysin prodrugs with a monosulfide bridged chrysin prodrug. Using the linker and fatty alcohol moieties (linkers include C—C, -S—, and -SS—) of the chrysin prodrug, self-assembled chrysin prodrug nanoparticles are prepared using a nanoprecipitation method. This method can help delay chrysin metabolism in the body, prolong its in vivo residence time, increase its bioavailability, enhance its anti-tumor activity, and improve the stability of the system after PEGylation. Chrysin itself does not have these groups, making it difficult to prepare nanoparticles via self-assembly, which complicates the preparation process and makes stability difficult to control.

[0012] In order to achieve the above object, the present invention provides the following technical solutions:

[0013] A single-sulfide-bridged chrysin prodrug (RS-CHR) uses a single-sulfide bond as a connecting bridge and different lipids as side chains. The general structural formula is shown below.

[0014]

[0015] Specifically, long-chain saturated fatty alcohols (tetradecanol C 14 , hexadecanol C 16 、Octadecanol C 18 ) and long-chain unsaturated fatty alcohol (oleyl alcohol, OA) as side chains of chrysin prodrugs, the structural formulas are as follows:

[0016] The prodrug structure of chrysin with a single sulfur bond as the connecting bridge and tetradecanol as the side chain is:

[0017]

[0018] Single sulfur bond is the connecting bridge, hexadecanol C 16 The prodrug structure of chrysin with side chain is:

[0019]

[0020] Single sulfur bond is the connecting bridge, octadecanol C 18 The prodrug structure of chrysin with side chain is:

[0021]

[0022] The prodrug structure of chrysin with a single sulfur bond as the connecting bridge and oleyl alcohol OA as the side chain is:

[0023]

[0024] The preparation process of the above-mentioned single sulfide bond bridged chrysin prodrug is as follows:

[0025]

[0026] The present invention provides a preparation method of a chrysin prodrug containing a single sulfide bond bridge. Thiohydroxyacetic anhydride and a saturated long-chain fatty alcohol are reacted to form an ester to obtain an intermediate product; then the intermediate product is reacted with chrysin to form an ester to obtain a chrysin-fatty alcohol prodrug. The yield is about 50% and the purity reaches more than 95%.

[0027] Specifically, the following steps are included:

[0028] (1) Thiohydroxyacetic anhydride, long-chain fatty alcohol and DMAP were weighed, and 10 mL of dichloromethane was added as the reaction solvent. The mixture was reacted at room temperature for 48 h to obtain a crude product. The crude product was purified and separated by column chromatography using an eluent (ethyl acetate:petroleum ether 1:15 v / v). The product was recrystallized from dichloromethane and stored in a vacuum drying oven (37°C, 24 h) or at -4°C.

[0029] (2) Weigh chrysin, long-chain fatty alcohol, DAMP, and EDCI (reaction solvent: dichloromethane, nitrogen protection, and react at room temperature for 36 hours). Use eluent (ethyl acetate: petroleum ether 1:15 v / v) for column chromatography purification and separation. After recrystallization from dichloromethane, the obtained product, chrysin prodrug, is dried in a vacuum drying oven (37°C, 24 hours) and stored at 4°C for subsequent studies.

[0030] Self-assembling nanoparticles comprising the aforementioned monosulfide-bridged chrysin prodrug and pharmaceutically acceptable excipients. The self-assembling nanoparticles are PEG-modified or unmodified prodrug self-assembling nanoparticles. The preparation method is a nanoprecipitation method.

[0031] Specifically, the monosulfide-bridged chrysin prodrug and the PEG modifier are dissolved in an organic solvent, and the resulting solution is slowly added dropwise to water to spontaneously form uniform nanoparticles. The solvent is removed by reduced pressure rotary evaporation to obtain nanoparticles free of organic solvent.

[0032] The PEG modifier is DSPE-PEG 2000. The solvent is selected from acetone.

[0033] (Non-PEGylated prodrug self-assembled nanoparticles do not contain PEG modifiers)

[0034] An injection comprises the self-assembled nanoparticles and pharmaceutically acceptable excipients.

[0035] The application of the above-mentioned single-sulfur-bond linked chrysin prodrug and self-assembled nanoparticles in the preparation of anti-tumor drugs, wherein the tumor is melanoma.

[0036] Beneficial effects: The advantages of the present invention are:

[0037] (1) The present invention connects fatty alcohols of different lengths to the 7-hydroxyl group of chrysin via thioglycolic anhydride. The synthesis method is simple and easy, with a yield of about 50% and a purity greater than 95%.

[0038] (2) The obtained prodrug has good self-assembly ability. With the help of the linker bond + long-chain fatty alcohol part of the chrysin prodrug, the nanoprecipitation method was used to prepare chrysin prodrug self-assembled nanoparticles with uniform particle size and strong stability, achieving efficient drug encapsulation with a drug loading of more than 26%. In vitro and in vivo experiments found that the octadecanol prodrug has higher bioavailability and anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The structure of the chrysin prodrug (C14-S-CHR) with a single sulfide bond as the connecting bond and tetradecanol as the side chain in Example 1 of the present invention was confirmed;

[0040] A:C 14 -Mass spectrum of S-CHR.

[0041] B:C 14 1H-NMR spectrum of -S-CHR.

[0042] Figure 2 This is the structural confirmation of the chrysin prodrug (C16-S-CHR) in Example 2 of the present invention, which has a single sulfide bond as a connecting bond and hexadecanol as a side chain.

[0043] A:C 16 -Mass spectrum of S-CHR.

[0044] B:C 16 1H-NMR spectrum of -S-CHR.

[0045] Figure 3 This is the structural confirmation of the chrysin prodrug (C18-S-CHR) in Example 3 of the present invention, which has a single sulfide bond as a connecting bond and octadecanol as a side chain.

[0046] A:C 18 -Mass spectrum of S-CHR.

[0047] B:C 18 1H-NMR spectrum of -S-CHR.

[0048] Figure 4 This is the structural confirmation of the chrysin prodrug (OA-S-CHR) in Example 4 of the present invention, in which the single sulfide bond is the connecting bond and oleyl alcohol is the side chain.

[0049] A: Mass spectrum of OA-S-CHR.

[0050] B: 1H-NMR spectrum of OA-S-CHR.

[0051] Figure 5 Differential scanning calorimetry curves of chrysin and chrysin prodrug.

[0052] Figure 6 XRD spectra of chrysin and chrysin drug.

[0053] Figure 7 This is a transmission electron micrograph of the PEG-modified chrysin prodrug self-assembled nanoparticles in Example 5 of the present invention. It can be seen from the figure that the average particle size of the obtained nanoemulsion is less than 200 nanometers;

[0054] Figure 8 This is the stability result of the PEGylated chrysin prodrug self-assembled nanoparticles in Example 5 of the present invention.

[0055] A: Stability of PEGylated chrysin prodrug self-assembled nanoparticles in water at room temperature for 28 days.

[0056] B: Stability of PEGylated chrysin prodrug nanoparticles in PBS for 28 days.

[0057] C: Stability of PEGylated chrysin prodrug nanoparticles in 10% FBS for 24 h.

[0058] Figure 9 This is an in vitro release test of the PEG-modified monosulfide-bridged chrysin prodrug self-assembled nanoparticles in Example 6 of the present invention under reducing and oxidizing conditions.

[0059] A: Blank medium (without H2O2 and DTT).

[0060] B: 1 mM H2O2 was added to the medium.

[0061] C: 10 mM H2O2 was added to the medium.

[0062] D: Blank medium (without H2O2 and DTT).

[0063] E: 1 mM DTT was added to the medium.

[0064] F: 10 mM DTT was added to the medium.

[0065] Figure 10 This is an in vivo pharmacokinetic experiment of the PEG-modified single-sulfide-bridged chrysin prodrug self-assembled nanoparticles in Example 7 of the present invention. It can be seen from the figure that the in vivo bioavailability of the modified prodrug nanoparticles is improved compared with the chrysin raw material, and the average residence time in the body is prolonged.

[0066] Figure 11 This is the in vivo tissue distribution result of the chrysin prodrug self-assembled nanoparticles in Example 8 of the present invention.

[0067] A: In vivo fluorescence imaging of free DiR and prodrug nanoparticles by chrysin prodrug self-assembled nanoparticles at different time points.

[0068] B: Fluorescence quantitative analysis of organs and tumors 24 h after administration of chrysin prodrug self-assembled nanoparticles.

[0069] Figure 12 These are the in vivo anti-tumor experimental results of the chrysin prodrug self-assembled nanoparticles in Example 9 of the present invention.

[0070] A: Growth curve of tumor-bearing mice after treatment with chrysin prodrug self-assembled nanoparticles.

[0071] B: Tumor weight of tumor-bearing mice after treatment with chrysin prodrug self-assembled nanoparticles.

[0072] C: Photograph of tumor in tumor-bearing mice after treatment with chrysin prodrug self-assembled nanoparticles.

[0073] D: Body weight changes of tumor-bearing mice after treatment with chrysin prodrug self-assembled nanoparticles.

[0074] Figure 13 This is a diagram showing the effect of the weight ratio of chrysin prodrug and modifier PEG on self-assembled nanoparticles (the figure shows weight ratios of 5:1, 5:2, and 5:3), A shows the effect on particle size; B shows the effect on zeta potential. DETAILED DESCRIPTION

[0075] The present invention is further described below by way of examples, but the invention is not limited to the scope of the examples.

[0076] Example 1: C 14 Synthesis of -S-CHR

[0077] Weigh 1.5 g (12.3 mmol) of thioglycolic anhydride and saturated fatty alcohol C 14 Alcohol (4.1 mmol), DAMP 122 mg (1 mmol), in a 100 mL round-bottom flask, added reaction solvent dichloromethane 10 mL, reacted at room temperature for 48 h to obtain a crude product, which was purified by column chromatography (eluent: ethyl acetate: petroleum ether = 1:15 (v / v)) and separated. The product obtained after recrystallization from dichloromethane was dried in a vacuum drying oven (37 ° C, 24 h) and stored at 4 ° C.

[0078] Weigh chrysin (1 mmoL), C 14Alcohol (1mmol), DAMP 12mg (0.1mmol), EDCI (1.3mmol) were placed in a 100mL round-bottom flask, and the reaction solvent dichloromethane was added. The mixture was protected by nitrogen and reacted at room temperature for 36h. Ethyl acetate: petroleum ether (1:15, v / v) was used as the eluent. After purification by column chromatography, the product was recrystallized from dichloromethane and dried in a vacuum oven (37°C, 24h) and stored at 4°C for subsequent research.

[0079] Mass spectrometry was used to determine the molecular weight of the compound obtained in Example 1. Figure 1 As shown. [M+H] was detected + Peak 583.3, proved to be successfully synthesized, with a melting point of about 79 ° C ( Figure 5 ), the crystal form is amorphous ( Figure 6 ).

[0080] The results of NMR spectroscopy analysis are as follows:

[0081] C 14 -S-CHR 1 H NMR (400 MHz, Chloroform-d) results are as follows δ 12.75 (s, 1H), 7.90 (d, J = 6.5 Hz, 2H), 7.61-7.50 (m, 3H), 6.91 (d, J = 2.0 Hz, 1H), 6.75 (s, 1H), 6.62 (d, J = 2.1 Hz, 1H), 4.15 (t, J = 6.8 Hz, 2H), 3.65 (s, 2H), 3.47 (s, 2H), 1.67 (q, J = 7.0 Hz, 2H), 1.24 (s, 22H), 0.87 (t, J = 6.7 Hz, 3H).

[0082] Example 2: C 16 Synthesis of -S-CHR

[0083] The preparation method of Example 1 was adopted, except that tetradecanol was replaced by hexadecanol.

[0084] Mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy were used to determine the C 16 -S-CHR structure, the results are as follows Figure 2 shown.

[0085] The results of NMR spectroscopy analysis are as follows:

[0086] C 16 -S-CHR 1H NMR (600 MHz, Chloroform-d) results are as follows δ 12.74 (s, 1H), 7.91-7.88 (m, 2H), 7.60-7.52 (m, 3H), 6.91 (d, J = 2.0 Hz, 1H), 6.74 (s, 1H), 6.62 (d, J = 2.1 Hz, 1H), 4.16 (t, J = 6.8 Hz, 2H), 3.64 (s, 2H), 3.47 (s, 2H), 1.69-1.63 (m, 2H), 1.38-1.22 (m, 26H), 0.88 (t, J = 7.0 Hz, 3H).

[0087] Mass spectrometry results are MS (ESI): m / z [M+H] + =611.2.

[0088] Example 3: C 18 Synthesis of -S-CHR

[0089] The preparation method of Example 1 was adopted, except that tetradecanol was replaced by octadecanol.

[0090] Mass spectrometry and H NMR spectroscopy were used to confirm the structure of the product obtained in Example 3. Figure 3 shown.

[0091] The results of NMR spectroscopy analysis are as follows:

[0092] C 18 -S-CHR 1 H NMR (600 MHz, Chloroform-d) results are as follows δ 12.74 (s, 1H), 7.91-7.88 (m, 2H), 7.60-7.51 (m, 3H), 6.91 (d, J = 2.0 Hz, 1H), 6.74 (s, 1H), 6.62 (d, J = 2.1 Hz, 1H), 4.16 (t, J = 6.8 Hz, 2H), 3.64 (s, 2H), 3.47 (s, 2H), 1.70-1.62 (m, 2H), 1.31-1.22 (m, 30H), 0.88 (t, J = 7.0 Hz, 3H).

[0093] Mass spectrometry results are MS (ESI): m / z [M+H] + =639.5.

[0094] Example 4: Synthesis of OA-S-CHR

[0095] The preparation method of Example 1 was adopted, except that tetradecanol was replaced with oleyl alcohol.

[0096] Mass spectrometry and H NMR spectroscopy were used to confirm the structure of the product obtained in Example 4. Figure 4shown.

[0097] The results of NMR spectroscopy analysis are as follows:

[0098] OA-S-CHR 1 H NMR (600 MHz, Chloroform-d) results are as follows δ 12.76 (s, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.55 (dt, J = 14.9, 7.2 Hz, 3H), 6.91 (s, 1H), 6.75 (s, 1H), 6.62 (s, 1H), 5.40-5.29 (m, 2H), 4.15 (t, J = 6.8 Hz, 2H), 3.65 (s, 2H), 3.47 (s, 2H), 2.00 (q, J = 6.6 Hz, 4H), 1.66 (p, J = 7.0 Hz, 2H), 1.39-1.21 (m, 22H), 0.87 (t, J = 6.9 Hz, 3H).

[0099] Mass spectrometry results are MS (ESI) m / z [M+H] + =637.3.

[0100] Example 5: Preparation and Stability Investigation of PEG-Modified Monosulfide-Bridged Chrysin Prodrug Self-Assembled Nanoparticles

[0101] Preparation of PEGylated Self-Assembling Nanoparticles: 5 mg of the prodrugs prepared in Examples 1-4 and 1 mg of DSPE-PEG2000 were precisely weighed and dissolved in 1000 μL of acetone. Chrysin prodrug self-assembled nanoparticles were prepared using a one-step nanoprecipitation method. The solution was added dropwise to 5 mL of deionized water under stirring at 1000 rpm, causing the prodrug to self-assemble into PEGylated nanoparticles. The solvent was removed by rotary evaporation to obtain organic solvent-free nanoparticles. The solution was then filtered through a 0.8 μm filter to remove any unformed drug. The resulting nanoparticle solution was stored at 4°C.

[0102] Preparation of non-PEGylated self-assembled nanoparticles: only the prodrug was dissolved in 1000 μL of acetone, and the other steps were the same.

[0103] The PEG modifiers are TPGS, DSPE-PEG, PLGA-PEG and PE-PEG, and the molecular weights of the PEG modifiers are 1000, 2000 and 5000. The PEG modifier is preferably DSPE-PEG, with a molecular weight of 2000.

[0104] The molecular weight of PEG in DSPE-PEG is 2000;

[0105] The organic solvent is acetone;

[0106] The effect of the weight ratio of the single sulfide bridged chrysin prodrug to the modifier PEG on the self-assembled nanoparticles was investigated. The weight ratios of 5:1, 5:2, and 5:3 were investigated. Figure 13 As shown, the preferred weight ratio is 5:1 because the self-assembled nanoparticles have smaller particle size and lower zeta potential at this ratio.

[0107] The performance of non-PEGylated and PEGylated chrysin prodrug self-assembled nanoparticles is compared as shown in Table 1. The particle size of DSPE-PEG2000 modified prodrug nanoparticles is about 150 nm, which is smaller than that of non-PEGylated chrysin prodrug self-assembled nanoparticles. Figure 5 As shown in Figure 2, the surface charge is around -30 mV, which is beneficial to prevent the aggregation of nanoparticles. The stability of PEGylated chrysin prodrug nanoparticles at room temperature was investigated. Figure 6 As shown, both are stable.

[0108] Table 1 Particle size, PDI, and Zeta potential of PEGylated poplar prodrug self-assembled nanoparticles

[0109]

[0110]

[0111] Example 6: In vitro release test of PEG-modified monosulfide-bridged chrysin prodrug self-assembled nanoparticles

[0112] The in vitro release behavior of four chrysin prodrug self-assembled nanoparticles was investigated using the small cup method. Figure 7 In the presence of 1mM H2O2, the four chrysin prodrug nanoparticles slowly released chrysin, and the release rates of the four chrysin prodrug nanoparticles were C 14 -S-CHR NPs>C 16 -S-CHR NPs≈C 18 -S-CHR NPs>OA-S-CHR NPs. In the presence of 10mM H2O2, the release rates of the four prodrug nanoparticles were C 18 -S-CHR NPs>C 16 -S-CHR NPs>C 14 -S-CHR NPs>OA-S-CHR NPs, at 48h, C 18 The release amounts of the -S-CHR NPs group and the OA-S-CHR NPs group were slightly greater than those of the other two groups.

[0113] Under reducing conditions with 1 mM DTT, the C 14-S-CHR NPs released a higher amount of NPs, while the other three nanoparticles had no significant difference in release. Under 10mM DTT, the release rates of the nanoparticles were similar, C 14 -S-CHR NPs, C 16 -S-CHR NPs and C 18 The release of CHR from the OA-S-CHR NPs group was slightly greater than that from the OA-S-CHR NPs group, and the release of the four prodrug nanocombinations was close to 60%. In the blank release medium without H2O2 and DTT, the nanoparticles released less than 6% of CHR.

[0114] The results showed that under redox conditions, the chrysin content in the four nanoparticles increased over time. The chrysin prodrug self-assembled nanoparticles all had redox capabilities, and the oxidation sensitivity was higher than the reduction sensitivity. They were able to release the parent drug under the action of high concentrations of GSH in the tumor microenvironment, which not only ensured the anti-tumor effect but also reduced the toxic side effects on normal tissues. At the same time, under oxidative conditions, the C 14 -S-CHR NPs, C 16 -S-CHR NPs and C 18 -S-CHR NPs showed a higher release rate than that of the unsaturated fatty alcohol-linked OA-S-CHR NPs.

[0115] Example 7: Pharmacokinetic Study of PEG-Modified Monosulfide-Bridged Chrysin Prodrug Self-Assembled Nanoparticles

[0116] The results of tail vein injection of free chrysin and C n The temporal changes in rat plasma chrysin levels after administration of OA-S-CHR (n=14, 16, 18) and OA-S-CHR NPs were investigated. SD rats weighing 180-220 g were randomly divided into a free chrysin group and a self-assembled nanoparticle group (5 groups, 3 rats per group). All rats were fasted before the experiment and had free access to water. Chrysin solution and chrysin prodrug nanoparticles were injected into the tail vein at a dose of 10 mg / kg (calculated as chrysin). Blood was collected from the rats' orbits at pre-determined time points (0.083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h) after administration, and plasma was frozen and stored at -20°C. Plasma drug concentrations were determined by liquid chromatography-mass spectrometry.

[0117] Due to the large molecular weight of the prodrug, it is difficult to detect a stable ion peak under the existing mass spectrometry conditions and it is impossible to quantify the prodrug in plasma. Therefore, only the blood concentration of chrysin was detected. The drug-time curve is shown in Figure 2. Figure 10 The results are shown in Table 3. The area under the curve and peak concentration of chrysin released from the four prodrug nanoparticles are higher than those of the chrysin solution. 18The area under the curve and peak concentration of -S-CHR NPs were significantly higher than those of the others, indicating that C 18 -S-CHR NPs have better stability and can more effectively improve the pharmacokinetic behavior of chrysin.

[0118] Table 3 Pharmacokinetic parameters of PEG-modified chrysin prodrug self-assembled nanoparticles

[0119]

[0120] (Note: *: P<0.05VS CHR, #: P<0.05VS OA-S-CHR NPs, a: P<0.05VS C 14 -S-CHR NPs, b: P < 0.05 VS C 16 -S-CHR NPs)

[0121] Example 8: Tissue distribution experiment of PEG-modified monosulfide-bridged chrysin prodrug self-assembled nanoparticles

[0122] B16F10 cells in good growth condition were digested with trypsin and collected with RPMI 1640 medium. After centrifugation at 1000 rpm, the cells were redispersed, counted, and collected and dispersed in serum-free RPMI 1640 medium. Mice were subcutaneously inoculated with 0.1 mL of 1×10 6 B16F10 cell suspension of cells. When the tumor volume of the tumor-bearing mice grows to 800mm 3 In vivo distribution studies were initiated. Tumor-bearing mice were randomly divided into three groups. DiR solution and DiR-labeled nanoparticles were injected via the tail vein. The DiR dose was 1.5 mg / kg. Images were taken 2, 4, and 8 hours after administration. 24 hours later, the animals were sacrificed, and the heart, liver, spleen, lungs, kidneys, and tumors were removed. Fluorescence intensity in the ex vivo tissues was observed and analyzed using a small animal in vivo imaging device, and fluorescence images and fluorescence intensity data were obtained.

[0123] The results are as follows Figure 11 As shown in the figure, no fluorescence distribution was observed in the tumor site after DiR entered the body. OA-S-CHRNPs and C 18 -S-CHR NPs nanoparticles group showed weak fluorescence, while the fluorescence intensity at the tumor site continued to increase with time, reaching the highest fluorescence intensity at 8h. This should be due to the prolonged blood circulation time of the nanoassembly and the passive targeted drug delivery based on the EPR effect. At the same time, compared with the mice treated with OA-S-CHR NPs prodrug nanoparticles, C 18-S-CHR NPs showed higher fluorescence intensity in tumor tissue, which is related to its good pharmacokinetics and long circulation. At the same time, mice were killed 24 hours after administration, and the heart, liver, spleen, lung, and kidney tumors were removed for fluorescence quantification. The results showed that the fluorescence of the nanoparticle group was mainly distributed in the liver, spleen, and tumor, while C 18 -S-CHR NPs had a higher fluorescence intensity than OA-S-CHR NPs (P<0.05). In summary, chrysin prodrug self-assembled nanoparticles can be targeted and accumulated in tumor sites, and C 18 -S-CHR NPs have significantly longer retention time and more significant tumor targeting than OA-S-CHR NPs, and the tumor cells are melanoma cells.

[0124] The preparation steps of the fluorescently labeled nanoparticles are as follows: accurately weigh 3 mg of DiR, dissolve it in 1.5 mL of anhydrous ethanol and dilute it to 2 mg / mL, accurately weigh 7.5 mg of prodrug and dissolve it in 1 mL of acetone solution, 1.5 mg of DSPE-PEG2000 and dissolve it in 0.3 mL of the above DiR ethanol solution, and slowly add the mixed solution of DiR, prodrug and DSPE-PEG2000 to 3 mL of deionized water under stirring (800 rpm). In the water and ethanol system, the prodrug can self-assemble uniformly into DIR-labeled prodrug self-assembled nanoparticles. At room temperature, the organic solvent in the solvent is removed by vacuum rotary evaporation, and after passing through a 0.8 μm filter membrane, DiR-labeled prodrug self-assembled nanoparticles DiR-C are obtained. 18 -S-CHR NPs, DiR-OA-S-CHR NPs, wherein the concentration of DiR was 0.2 mg / mL and the prodrug concentration was 2 mg / mL.

[0125] Example 9: In vivo antitumor experiment of PEG-modified monosulfide-bridged prodrug self-assembled nanoparticles

[0126] The anti-tumor effect of chrysin and self-assembled nanoparticles was evaluated using a C57BL / 6 mouse model. Thirty C57BL / 6 mice were subcutaneously inoculated with 0.1 mL of 1×10 6 The B16-F10 cell suspension of cells was used to establish a melanoma B16F10 tumor-bearing mouse model. About 8 days after inoculation, the tumor volume reached about 150 mm 3 The mice were randomly divided into 5 groups (n=6): control group (normal saline), chrysin group, cyclophosphamide group and self-assembled nanoparticle group (C 18 -S-CHR NPs, OA-S-CHR NPs. Chrysin and chrysin prodrug self-assembled nanoparticles were both administered at a dose of 118 μmol / kg (calculated as chrysin) daily. Cyclophosphamide was administered every other day at a dose of 30 mg / kg.

[0127] like Figure 12 As shown in the figure, compared with the saline group, the other groups all inhibited the tumor volume to some extent, among which, compared with chrysin, C 18 -S-CHR NPs group significantly improved the anti-tumor effect of chrysin, and the anti-tumor activity was more significant. In vivo anti-tumor studies showed that C 18 -S-CHR NPs group has higher anti-tumor activity. This should be attributed to its good pharmacokinetic behavior, long retention time in the body, and higher accumulation in tumor sites. The OA-S-CHR NPs group, on the other hand, has slower release, faster clearance in the body, shorter retention time in the body, weak tumor targeting, and lower bioavailability, which can also be reflected in tissue distribution. Prodrugs modified with long lipid chains, especially saturated fatty alcohols (octadecyl alcohol), can make the prodrug self-assembled nanoparticles more stable, greatly improve the pharmacokinetic behavior of the nanoparticles, and make the nanoparticles more targeted to accumulate in tumor sites, thereby enhancing the anti-cancer effect of chrysin itself.

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Claims

1. A monosulfide-bridged chrysin prodrug, characterized in that: The structure is shown in formula (I): Formula (I) Specifically including the following compounds: 。 2. The method for preparing the monosulfide-bridged chrysin prodrug according to claim 1, characterized in that: The method comprises connecting a long-chain fatty alcohol and chrysin through thiohydroxyacetic anhydride to obtain a chrysin prodrug with a single sulfur bond as a connecting bond and a long-chain fatty alcohol as a side chain.

3. The method for preparing the monosulfide-bridged chrysin prodrug according to claim 2, characterized in that: The reaction process is as follows: 。 4. A self-assembled nanoparticle, characterized in that: include: The monosulfide-bridged chrysin prodrug according to claim 1 and pharmaceutically acceptable excipients.

5. The self-assembled nanoparticles according to claim 4, characterized in that The preparation method comprises dissolving the chrysin prodrug and the modifier PEG in an organic solvent, slowly adding the solution dropwise into water under stirring to spontaneously form uniform nanoparticles, and removing the solvent by rotary evaporation to obtain a nanoparticle solution free of organic solvent. Among them, non-PEGylated prodrug nanoparticles do not contain PEG modifiers.

6. The self-assembled nanoparticles according to claim 5, characterized in that The molecular weight of the PEG is 2000; The organic solvent is acetone; The weight ratio of the monosulfide-bridged chrysin prodrug to the modifying agent PEG was 5:

1.

7. Use of the single-sulfur-bond linked chrysin prodrug according to claim 1 in the preparation of an anti-tumor drug, wherein the tumor is melanoma.

8. Use of the self-assembled nanoparticles according to claim 5 in the preparation of anti-tumor drugs, wherein the tumor is melanoma.

9. An injection, characterized in that: The self-assembled nanoparticles according to claim 5 and pharmaceutically acceptable excipients.

Images