An ultrasound-responsive CO-releasing nanoformulation and its preparation method

Abstract

The present invention discloses an ultrasound-responsive CO-releasing nanoformulation and a preparation method thereof. First, a vanadium tetrasulfide nanoformulation with sonodynamic and chemodynamic effects is prepared. Then, a CO-releasing molecule is loaded to construct an ultrasound-responsive CO-releasing nanoformulation, realizing the controllable release of CO. The preparation process of the ultrasound-responsive CO-releasing nanoformulation is simple, and it can achieve the combination of CO therapy / sonodynamic therapy / chemodynamic therapy for tumors. Using it for tumor treatment has safety and effectiveness, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an ultrasound-responsive CO-releasing nanoformulation and a preparation method thereof. Background Art

[0002] The application of nanomedicine has opened up a new era of cancer diagnosis and treatment. Tumor treatment nanoformulations targeting subcellular organelles have advantages such as precise drug delivery, maximized therapeutic index, and reduced off-target side effects, and thus have received increasing attention. Mitochondria are the source of cell energy and are crucial for cell survival. Strategies based on mitochondrial apoptosis have brought good prospects for cancer treatment. Current research has proposed several strategies for mitochondrial apoptosis, including the generation of carbon monoxide (CO) and reactive oxygen species (ROS).

[0003] CO is a very stable inert gas molecule that can freely diffuse into all cells. Its endogenous source is mainly heme. Due to the regulatory effect of CO on cell function and the tumor microenvironment, it has received increasing attention in cancer treatment. Mitochondria are the target for CO to exert its physiological functions. CO inhibits mitochondrial respiration by competitively binding to cytochrome c oxidase with oxygen, thereby disrupting the mitochondrial energy of tumor cells and leading to apoptosis of tumor cells. CO can reversibly bind to transition metals with a specific redox state, such as manganese, iron, vanadium, molybdenum, cobalt, nickel, and copper, etc., to generate CO-releasing molecules. CO-releasing molecules are a relatively safe and potential strategy in CO therapy. However, high blood drug concentration and low tumor accumulation will respectively lead to the risk of CO poisoning and limited CO treatment effect. Therefore, the targeted delivery and controlled release of CO are crucial for improving the effectiveness and biosafety of CO therapy.

[0004] Current research has proposed a variety of non-invasive cancer treatments, including photodynamic therapy, sonodynamic therapy, and chemodynamic therapy. Due to the high tissue penetration of ultrasound, the efficacy of sonodynamic therapy is usually better than that of photodynamic therapy. However, sonodynamic therapy usually lacks ideal sonosensitizers, which hinders its application in clinical practice. Titanium dioxide is a commonly used inorganic sonosensitizer. Due to its wide bandgap and rapid recombination of excited electrons and holes, the ROS quantum yield is low. In contrast, narrow bandgap sonosensitizers require less energy to achieve electron excitation. The principle of chemodynamic therapy is that Fenton's reagent reacts with a higher concentration of hydrogen peroxide in the tumor microenvironment to generate highly toxic hydroxyl radicals. In addition to iron-related chemodynamic strategies, Fenton-like reactions based on various metal elements such as copper, manganese, cobalt, and titanium have also been proposed. Here, we studied the ROS generated during sonodynamic therapy and chemodynamic therapy and the CO generated by CO therapy for mitochondrial apoptosis of tumor cells. Summary of the Invention

[0005] The present invention first prepared a vanadium tetrasulfide nano - preparation (VS4), and then used VS4 to load a CO - releasing molecule to prepare an ultrasound - responsive CO - releasing nano - preparation (V - Mn).

[0006] To achieve the above - mentioned purpose, the technical solution adopted by the present invention is as follows:

[0007] Step 1: Dissolve Pluronic F127 and ammonium metavanadate in ultrapure water, heat and stir, add an ethylene glycol solution of thioacetamide, continue stirring, then add the reaction solution into a hydrothermal reaction kettle, react under heating conditions, cool and then centrifuge. Wash the obtained precipitate with ethanol and water to obtain the vanadium tetrasulfide nano - preparation;

[0008] Step 2: Disperse the vanadium tetrasulfide nano - preparation obtained in Step 1 in ethylene glycol, add an ethanol solution of the CO - releasing molecule, heat and stir for a certain period of time, cool and then centrifuge to obtain the ultrasound - responsive CO - releasing nano - preparation.

[0009] As a further improvement of the present invention, Step 1 is specifically: Dissolve 50 - 100 mg of Pluronic F127 and 35 - 140 mg of ammonium metavanadate in 5 - 15 mL of ultrapure water, heat and stir at 25 - 75 °C for 15 - 60 min, add an ethylene glycol solution of thioacetamide (0.2 - 0.6 M, 5 - 15 mL), continue stirring for 15 - 60 min, then add the reaction solution into a hydrothermal reaction kettle, heat and react at 120 - 180 °C for 2 - 12 h, cool and then centrifuge. Wash the obtained precipitate with ethanol and water to obtain the vanadium tetrasulfide nano - preparation;

[0010] As a further improvement of the present invention, Step 2 is specifically: Disperse the vanadium tetrasulfide nano - preparation (10 - 40 mg) obtained in Step 1 in 10 - 20 mL of ethylene glycol, add an ethanol solution of the CO - releasing molecule (0.5 - 4 mg / mL, 10 - 20 mL), heat and stir at 25 - 75 °C for 2 - 5 h, cool and then centrifuge to obtain the ultrasound - responsive CO - releasing nano - preparation.

[0011] The innovations of the present invention are as follows:

[0012] 1. For the first time, an ultrasound - responsive CO - releasing nano - preparation is constructed. This nano - preparation can release CO molecules under ultrasonic excitation to achieve spatiotemporally controllable CO therapy.

[0013] 2. VS4 is a new type of narrow - bandgap sonosensitizer with a high ROS yield. Moreover, we first discovered that it has a Fenton - like effect, so it can achieve dual therapeutic effects of sonodynamic therapy and chemodynamic therapy.

[0014] 3. V-Mn can release CO, generate ROS under the action of timed and positioned ultrasound, and can generate hydroxyl radicals by using the high-concentration H2O2 in the tumor microenvironment, thereby causing apoptosis of tumor cell mitochondria. Brief Description of the Drawings

[0015] Figure 1 Appearance of V-Mn

[0016] Figure 2 UV-Vis absorption spectra of products obtained by reacting VS4 with manganese pentacarbonyl bromide at different times

[0017] Figure 3 Investigation diagram of sonodynamic effect of VS4

[0018] Figure 4 Investigation diagram of sonodynamic effect of V-Mn

[0019] Figure 5 Investigation diagram of ultrasonic response CO release of V-Mn

[0020] Figure 6 Investigation diagram of chemodynamic effect of VS4

[0021] Figure 7 MTT method was used to investigate the cell compatibility and cytotoxicity of VS4 and V-Mn Detailed Description of the Invention

[0022] The present invention will be further described in detail by the following examples:

[0023] Example 1

[0024] This example is a preparation method of VS4, including the following steps:

[0025] Dissolve 50 mg of Pluronic F127 and 35 mg of ammonium metavanadate in 15 mL of ultrapure water, heat and stir at 25 °C for 15 min, add an ethylene glycol solution of thioacetamide (0.2 M, 15 mL), continue to stir for 15 min, then add the reaction solution into a hydrothermal reaction kettle, heat and react at 180 °C for 2 h, cool and then centrifuge, and wash the obtained precipitate with ethanol and water to obtain VS4.

[0026] Example 2

[0027] This example is a preparation method of VS4, including the following steps:

[0028] Dissolve 100 mg of Pluronic F127 and 140 mg of ammonium metavanadate in 5 mL of ultrapure water. Heat and stir at 75 °C for 30 min, add an ethylene glycol solution of thioacetamide (0.6 M, 5 mL), continue stirring for 30 min, then add the reaction solution to a hydrothermal reaction kettle, heat and react at 120 °C for 12 h. After cooling, centrifuge, and wash the obtained precipitate with ethanol and water to obtain VS4.

[0029] Example 3

[0030] This example is a preparation method of VS4, including the following steps:

[0031] Add 100 mg of Pluronic F127 and 70 mg of ammonium metavanadate to 15 mL of ultrapure water. Heat and stir at 60 °C for 60 min, add an ethylene glycol solution of thioacetamide (0.2 M, 15 mL), continue stirring for 60 min, then add the reaction solution to a hydrothermal reaction kettle, heat and react at 160 °C for 6 h. After cooling, centrifuge, and wash the obtained precipitate with ethanol and water to obtain VS4.

[0032] Example 4

[0033] This example is a preparation method of V-Mn, including the following steps:

[0034] Disperse 10 mg of VS4 in 20 mL of ethylene glycol, add an ethanol solution of manganese pentacarbonyl bromide (0.5 mg / mL, 20 mL), heat and stir at 75 °C for 2 h, and centrifuge after cooling to obtain V-Mn.

[0035] Example 5

[0036] This example is a preparation method of V-Mn, including the following steps:

[0037] Disperse 40 mg of VS4 in 10 mL of ethylene glycol, add an ethanol solution of manganese pentacarbonyl bromide (4 mg / mL, 10 mL), heat and stir at 25 °C for 5 h, and centrifuge after cooling to obtain V-Mn.

[0038] Example 6

[0039] This example is a preparation method of V-Mn, including the following steps:

[0040] Disperse 20 mg of VS4 in 15 mL of ethylene glycol, add an ethanol solution of manganese pentacarbonyl bromide (1.33 mg / mL, 15 mL), heat and stir at 75 °C for 3 h. Centrifuge after cooling to obtain V-Mn.

[0041] Example 7

[0042] This example is the appearance inspection of V-Mn:

[0043] Disperse V-Mn in water and take pictures to record its appearance. As Figure 1 shown, the nanoformulation suspension is brownish black and has good dispersibility.

[0044] Example 8

[0045] This example is the inspection of the ultraviolet-visible absorption spectra of VS4 and V-Mn obtained by reacting VS4 with manganese pentacarbonyl bromide for different times:

[0046] Take the reaction solutions of VS4 reacting with manganese pentacarbonyl bromide for different times, centrifuge them, disperse the precipitates in water, and use an ultraviolet-visible spectrophotometer to measure the ultraviolet-visible absorption spectra of each sample. As Figure 2 shown, after VS4 reacts with manganese pentacarbonyl bromide for 2 - 5 h, characteristic absorption peaks of manganese pentacarbonyl bromide appear at 200 nm, indicating the successful preparation of V-Mn.

[0047] Example 9

[0048] This example is the inspection of the sonodynamic effects of VS4 and V-Mn:

[0049] Use 1,3-diphenylisobenzofuran as a ROS indicator and adopt ultraviolet-visible spectrophotometry to investigate the extracellular sonodynamic effects of VS4 and V-Mn. Mix the nano suspension and the 1,3-diphenylisobenzofuran solution in equal volumes and sonicate (1.0 W / cm 2 ) for different times, and take samples to measure the ultraviolet-visible absorption spectra of each sample. Figure 3 、 Figure 4 are the sonodynamic effect diagrams of VS4 and V-Mn respectively. The research results show that the longer the sonication time, the more obvious the decrease in the absorption peak, which proves the sonodynamic effect and shows power dependence.

[0050] Example 10

[0051] This example is the inspection of CO release of V-Mn under ultrasonic excitation:

[0052] Mix the suspension of the ultrasonic-responsive CO nano-release system with deoxyhemoglobin in equal volumes and sonicate (1.0 W / cm 2 ) for different times, and take samples to measure the ultraviolet-visible absorption spectra of each sample. Figure 5 is the CO release diagram of V-Mn under ultrasonic excitation. The experimental results show that as the sonication time increases, the absorption peak gradually moves to 420 nm, indicating that V-Mn can release CO under ultrasonic excitation. The above experimental results further prove the successful preparation of V-Mn.

[0053] Example 11

[0054] This example is for investigating the chemical kinetic effect of VS4:

[0055] Mix 3,3',5,5'-tetramethylbenzidine with the test solution, add phosphate buffer solution at pH 5.5 and H2O2, and measure the generation of hydroxyl radicals using an ultraviolet-visible spectrophotometer at different time points. Figure 6 It is a detection graph of the chemical kinetic effect of VS4. The experimental results show that VS4 has Fenton-like activity, showing time-dependent generation of hydroxyl radicals, which proves the chemical kinetic effect of VS4.

[0056] Example 12

[0057] This example is for investigating the cell compatibility and cytotoxicity of VS4 and V-Mn:

[0058] Taking the relative cell survival rate as the investigation index, the MTT method was used to investigate the killing effect of VS4 and V-Mn on 4T1 cells with or without ultrasonic excitation. The experimental results are as Figure 7 shown. When there is no ultrasonic excitation, the cell viability of VS4 and V-Mn is close to 100%. However, after ultrasonic excitation, the cell viability decreases significantly, showing concentration dependence, and the cell viability of the V-Mn + ultrasonic group is lower than that of the VS4 + ultrasonic group. It can be seen that each nanon preparation has good biocompatibility. After ultrasonic treatment, the sonodynamic effect of VS4 has a killing effect on cells, while the sonodynamic effect of V-Mn combined with CO treatment enhances the killing effect on cells.

Claims

1. An ultrasound-responsive CO-releasing nanoplatform, and its preparation method comprises the following steps: (1) Dissolve 50 - 100 mg of Pluronic F127 and 35 - 140 mg of ammonium metavanadate in 5 - 15 mL of ultrapure water, heat and stir at 25 - 75 °C for 15 - 60 min, add 5 - 15 mL of thioacetamide ethylene glycol solution with a concentration of 0.2 - 0.6 M, continue to stir for 15 - 60 min, then add the reaction solution into a hydrothermal reaction kettle, heat and react at 120 - 180 °C for 2 - 12 h, after cooling, centrifuge, and wash the obtained precipitate with ethanol and water to obtain vanadium tetrasulfide nanoplatform; (2) Disperse the vanadium tetrasulfide nanoplatform obtained in step (1) in ethylene glycol, add an ethanol solution of a CO-releasing molecule, wherein the CO-releasing molecule is selected from one or a mixture of two or more of manganese pentacarbonyl bromide, manganese carbonyl, dimanganese decacarbonyl, and iron pentacarbonyl, heat and stir for a certain time, and after cooling, centrifuge to obtain an ultrasound-responsive CO-releasing nanoplatform.

2. The ultrasound-responsive CO-releasing nanoplatform according to claim 1, wherein the specific step (2) is: disperse 10 - 40 mg of the vanadium tetrasulfide nanoplatform obtained in step (1) in 10 - 20 mL of ethylene glycol, add 10 - 20 mL of an ethanol solution of a CO-releasing molecule with a concentration of 0.5 - 4 mg / mL, wherein the CO-releasing molecule is selected from one or a mixture of two or more of manganese pentacarbonyl bromide, manganese carbonyl, dimanganese decacarbonyl, and iron pentacarbonyl, then heat and stir at 25 - 75 °C for 2 - 5 h, and after cooling, centrifuge to obtain an ultrasound-responsive CO-releasing nanoplatform.

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