WO2023202090A1

WO2023202090A1 - Carbonyl iron carrier for responsively releasing carbon monoxide, method for preparing same, and use thereof

Info

Publication number: WO2023202090A1
Application number: PCT/CN2022/137364
Authority: WO
Inventors: 龚萍; 马功成; 张鹏飞; 蔡林涛; 刘中轲
Original assignee: 深圳先进技术研究院
Priority date: 2022-04-22
Filing date: 2022-12-07
Publication date: 2023-10-26
Also published as: CN115010939B; CN115010939A

Abstract

Provided are a carbonyl iron carrier for responsively releasing carbon monoxide, a method for preparing same, and use thereof. The carbonyl iron carrier comprises a carbonyl iron compound and a polymer with thiol end groups. The carbonyl iron compound and the polymer with thiol end groups are connected by means of a coordination reaction. A responsive carrier capable of supplying CO is prepared by means of using the carbonyl iron compound and the polymer with thiol end groups, which greatly simplifies the delivery process and improves the delivery efficiency. The carrier is capable of carrying other therapeutic or contrast agents, thus achieving synergism in treating diseases.

Description

A carbonyl siderophore that responds to the release of carbon monoxide and its preparation method and application

Technical field

The present invention relates to the field of nanomedicine, and in particular to a carbonyl siderophore that responds to the release of carbon monoxide and its preparation method and application.

Background technique

The World Health Organization estimates that 84 million people will die from cancer in 2021 without intervention. Uncontrolled proliferation and spread of cancer cells can affect almost any part of the human body. The complexity, diversity, and heterogeneity of tumors severely compromises the potential for treatment. Gas therapy as a new and promising treatment method utilizes therapeutic/therapeutic auxiliary gases (NO, CO, H ₂ S, H ₂ , O ₂ , SO ₂ and CO ₂ ) and their prodrugs to inhibit cancer cells The proliferation and metastasis are receiving more and more attention in treatment. As a signaling molecule, CO is produced during hemoglobin degradation and triggers a series of cell protection mechanisms during stress and inflammation. The normal body produces CO under conditions of increased pressure through the expression of the heme oxygenase-1 (HO-1) gene, however, this gene is inactive in cancer cells. Within a suitable concentration range, CO can reverse the Warburg effect, selectively induce cell apoptosis, and inhibit the metastasis of cancer cells, while normal cells are induced to enter a dormant state and are protected from cytotoxicity. The significant anti-cancer selectivity of CO is superior to traditional chemotherapy/radiotherapy drugs. Therefore, combination therapy is emerging as a promising therapeutic strategy.

CO is small in size, has high transmembrane diffusivity and has no purpose. High blood drug concentrations and low intratumoral accumulation lead to the risk of CO poisoning and limited combined therapeutic effects, respectively. Previous CO treatment research mainly focused on how to effectively deliver CO to tumor tissues. The existing means of achieving CO gas therapy mainly involve the use of other complex drug carriers to deliver CO from the blood to the tumor site. The current supply of CO gas mainly relies on the delivery of carriers such as mesoporous silicon and metal nanocages. Traditional delivery methods require additional drug carriers and encapsulation methods, resulting in extremely complex design of nanotherapeutic agents, difficult preparation, and cumbersome processes, which are not conducive to industrial production or clinical experiments. And too many materials add additional uncontrollable factors and physiological toxicity. The biosafety of carrier materials such as metallic nanocages and mesoporous silicon is questionable. The light-controlled CO release also means that the material is unstable under light conditions. CO concentration in tumor tissue is extremely difficult to control, and the preparation of complex nanosystems will also increase the uncertainty of pharmacokinetics and operational difficulty, reducing the reproducibility of clinical translation. Therefore, there is an urgent need for a drug carrier that can intelligently respond to the tumor microenvironment and accurately release CO.

technical problem

In view of the defects in the prior art, the present invention proposes a carbonyl siderophore that responds to the release of carbon monoxide and its preparation method and application. The present invention uses carbonyl iron compounds and thiol end-group polymers to prepare an intelligent responsive carrier capable of supplying CO.

Technical solutions

The present invention provides a carbonyl siderophore that responds to the release of carbon monoxide, including a carbonyl iron compound and a thiol terminal polymer, and the carbonyl iron compound and the thiol terminal polymer are connected through a coordination reaction.

Further, the carbonyl iron compound is selected from any one of tricarbonyl iron, pentacarbonyl iron, nonacarbonyl iron, and dodecacarbonyl iron.

Further, the thiol-terminated polymer is selected from any one of polyethylene glycol polymers, polypropylene polymers, polystyrene polymers, and polypropylene ester polymers.

Further, the molecular weight of the thiol-terminated polymer is 1000~8000. As the molecular weight of the thiol-terminated polymer increases, the loading of CO in the carrier decreases, and the reverse is true as the molecular weight of the thiol-terminated polymer decreases. The relative ratio between thiol-terminated polymer and CO loading is set to X. As the molecular weight and CO loading of the thiol-terminated polymer change, the amphiphilicity of the carrier changes. When X increases, the carrier will become more hydrophilic, otherwise it will become more hydrophobic. Both states are not conducive to the use of the carrier. When the molecular weight of the thiol-terminated polymer is less than 1,000, the carrier has poor water solubility and is difficult to dissolve. When the molecular weight of the thiol-terminated polymer is greater than 8,000, the proportion of hydrophobic ends of the carrier is too small and the drug cannot be effectively loaded. Therefore, the molecular weight of the thiol-terminated polymer needs to be between 1000-8000.

The invention also provides a method for preparing the carbonyl iron carrier, which includes the following steps: dissolve the carbonyl iron compound and the thiol-terminated polymer in tetrahydrofuran, and stir under a nitrogen flow; at the end of the reaction, the solution changes from dark blue to Brownish yellow; cool to room temperature, add liquid alkane to obtain brown precipitate, wash with organic solvent and dry to obtain the carbonyl siderophore.

Further, the preparation step also includes a purification step, specifically as follows: redissolve the carbonyl siderophore prepared in tetrahydrofuran, freeze it in a -20°C environment for 5-24 h, and filter the precipitated crystals to obtain Purified carbonyl siderophores.

Further, the mass ratio of the carbonyl iron compound and the thiol-terminated polymer is 1:(4~8).

Further, the nitrogen flow temperature is 50-120°C, and the stirring time is 1-12 h.

Further, the liquid alkane is n-hexane or n-pentane.

Further, the organic solvent is diethyl ether or n-butyl ether.

The present invention also provides the use of the carbonyl siderophore in the preparation of anti-tumor drugs.

beneficial effects

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

1. Use carbonyl iron compounds and thiol-terminated polymers to directly prepare intelligent responsive carriers that can supply CO, which greatly simplifies the delivery process and improves delivery efficiency. The carrier can also load other drugs or contrast agents. Achieve coordinated treatment of diseases. At the same time, polyethylene glycol polymers have passed the Food Additive Supplementary Regulations of the U.S. Federal Food, Drug, and Cosmetic Regulations, approving the use of food chemical compendial grade polyethylene glycol directly or indirectly as food additives. The safety of the materials is good. protection.

2. The current supply technology for CO relies on the design of small molecule CO prodrugs, but the present invention directly designs the CO prodrug as a polymer carrier, which can not only be released as a responsive prodrug, but also encapsulate other drugs, contrast agents or Nanosystems are more conducive to imaging monitoring and collaborative treatment of CO gas therapy.

3. The carbonyl siderophores prepared by the present invention have high yield and short preparation time.

4. The nanoparticles prepared by the present invention can exist stably, and no sedimentation or flocculation occurs after 15 days.

5. The preparation method is simple and easy to operate and promote.

6. This carbonyl siderophore responds to ROS in the tumor microenvironment, releases CO, and can be successfully enriched in the tumor site to achieve long-term treatment.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings required to be 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 therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the process of using PG-CO to encapsulate dyes in the present invention;

Figure 2 shows the results of the change in absorption of the nanoparticles with concentration after using PG-CO to encapsulate the fluorescent dye IR-813 in Example 3;

Figure 3 is a verification experiment of the response to active oxygen after the PG-CO carrier of Example 4 is loaded with Bodipy dye;

Figure 4 shows the gradual enrichment and precipitation of PG-CO in Example 5 after responding to hydrogen peroxide;

Figure 5 is a picture of the aggregation of PG-CO nanoparticles in Example 6 in response to ROS under a high-resolution transmission electron microscope;

Figure 6 shows the HE stained sections of Example 7, showing that PG-CO has no obvious effect on mouse organs;

Figure 7 is a picture of the PG-CO nanoparticles of Comparative Example 1 under a high-resolution transmission electron microscope.

Embodiments of the invention

In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

The present invention constructs a polymer carrier based on metal carbonyl through coordination reaction. The carrier can simultaneously store and release CO in response to reactive oxygen species. Through the coordination reaction between thiol and carbonyl metal complex, the carbonyl iron compound at the end of the polymer was modified, and a new amphiphilic polymer PG-CO was obtained. Due to the strong hydrophobicity of the metal complex, PG-CO disperses into a molecular state in chloroform and aggregates into nanoparticles in water. In the presence of reactive oxygen species (ROS), PG-CO will release enough CO gas through a Fenton-like reaction, and the carbonyl iron side will be oxidized to iron oxide, leading to particle deposition. Due to the lack of ROS, PG-CO does not release CO in normal cell tissues, but in the tumor microenvironment with overexpression of ROS, PG-CO will gradually release CO and deposit it in the tumor site for long-term treatment. As a multifunctional smart nanocarrier, PG-CO simultaneously meets the following requirements: 1. As a drug carrier; 2. Reactive oxygen species respond to CO gas release system; 3. Accumulate in tumor tissue. 4. Simple structure and convenient synthesis; 5. Stable and safe.

The preparation method of the carbonyl siderophore of the present invention has the following steps:

Dissolve carbonyl iron compounds and thiol-terminated polymers in tetrahydrofuran and stir under nitrogen flow; at the end of the reaction, the solution changes from dark blue to brown; cool to room temperature, add liquid alkane to obtain a brown precipitate, and wash with an organic solvent After drying, the carbonyl siderophore is obtained.

The preparation step also includes a purification step, specifically as follows: redissolve the prepared carbonyl siderophore in tetrahydrofuran, freeze it in a -20°C environment for 5-24 h, and filter the precipitated crystals to obtain the purified carbonyl siderophore. Siderophores.

Among them, the carbonyl iron compound is selected from any one of tricarbonyl iron, pentacarbonyl iron, nonacarbonyl iron, and dodecacarbonyl triiron. The following examples take iron dodecacarbonyl as an example. Iron tricarbonyl, iron pentacarbonyl, and iron nonacarbonyl all have similar physical and chemical properties. Therefore, those skilled in the art can know that iron tricarbonyl, iron pentacarbonyl, and iron nonacarbonyl can be used. The technical solution of the present invention can be realized.

The thiol-terminated polymer is selected from any one of mercapto polyethylene glycol, mercapto polypropylene, mercapto polystyrene, and mercapto polypropylene ethyl ester. The following examples take mercapto polyethylene glycol as an example. Mercapto polypropylene, mercapto polystyrene, and mercapto polypropylene ethyl ester all have similar physical and chemical properties. Therefore, those skilled in the art can know that mercapto polypropylene, mercapto polystyrene, Both mercapto polypropylene ethyl esters can realize the technical solution of the present invention.

Example 1 Preparation and purification of carbonyl siderophores of the present invention

Iron dodecacarbonyl, due to its strong hydrophobicity, is generally coated on other carriers for transportation. This example uses the substitution reaction of ferric dodecacarbonyl and thiol to modify one end of the methyl polyethylene glycol polymer methoxypolyethylene glycol thiol (mPEG-SH) as reactive oxygen species (ROS). Responsive amphiphilic polymer carrier PG-CO. The specific preparation process is as follows. Iron dodecacarbonyl (5-100 mg) and mPEG-SH (M.W.≈2000) (100-600 mg) are dissolved in tetrahydrofuran (THF) and stirred at 50-120°C under nitrogen flow. 1-12 hours. At the end of the reaction, the solution changed from dark blue to brown. Cool to room temperature, add n-hexane to obtain brown precipitate, wash with diethyl ether and dry to obtain PG-CO. Redissolve PG-CO in THF, freeze it in a -20°C environment for 5-24 hours, and filter the precipitated crystals to obtain a new smart carbonyl siderophore PG-CO. PG-CO is a brown solid that is soluble in water and organic solutions.

Example 2 Synthesis of intelligent nanopreparations using the carbonyl siderophore of the present invention

PG-CO can be used to coat various small molecule drugs, polymer drugs, fluorescent probes, nanoparticles, etc. For example, FPG-CO is used to coat polymer fluorescent probes TPB, MCH-PPV, Bodipy, etc. First combine 1-50 mg PG-CO and 5-10 mg MCH-PPV dye is dissolved in THF. After ultrasonic for 5-10 minutes, add 10 ml of deionized water, blow out the THF with nitrogen, and co-precipitate. Centrifuge the ultrafiltration tube for 5 minutes and repeat washing three times. Co-precipitation is used to form stable and uniform nanoparticles with a particle size of 50-200 nm.

Example 3 Results of changes in the absorption of nanoparticles with concentration after using PG-CO to encapsulate the fluorescent dye IR-813

The experimental steps of using PG-CO to encapsulate the fluorescent dye IR-813 are as in Example 2. The concentration of the obtained nanoparticles is 100-120mM. Dilute the nanoparticle solution and take 5mM, 10mM, 20mM, 40mM, 50mM and 60mM concentrations for UV measurement. Absorption, use a UV spectrophotometer to measure the UV absorption of the above-mentioned concentration nanoparticles in the wavelength range of 500-1000nm. The results are shown in Figure 2. The concentrations from bottom to top are 1uM, 2uM, 3uM, 4uM, 5uM and 6uM. It shows that its UV absorption increases with the increase of nanoparticle concentration.

Example 4 Verification experiment of response to active oxygen after PG-CO carrier is loaded with Bodipy dye

The experimental steps for using PG-CO to encapsulate Bodipy dye are as in Example 2. Dilute the concentration of the obtained nanoparticles to 20-30mM, take 1mL and put it into a cuvette, add 1-2 drops of 0.3‰ hydrogen peroxide solution, and then Record the color change of the solution every hour from 0 to 4 hours. The Fenton reaction between carbonyl iron and hydrogen peroxide gradually produces ROS. The specific phenomenon is that the nanoparticle solution changes from light green to light pink over time, as shown in Figure 3 (the solution gradually becomes brighter). It shows that after adding hydrogen peroxide solution, the nanoparticles gradually release ROS.

Example 5 PG-CO gradually enriches and precipitates in response to hydrogen peroxide

Dissolve the prepared PG-CO in water to make a 40-50mM solution. Take 0.5-1mL of each into two centrifuge tubes, add 1-2 drops of 0.3‰ hydrogen peroxide solution to one of them, and leave the other untreated. After waiting for 1-2 hours, observe the solution in the centrifuge tube. You can see that there is no precipitation in the untreated PG-CO solution, while the PG-CO solution after adding hydrogen peroxide solution has precipitation on the wall of the centrifuge tube, as shown in Figure 4. . It shows that PG-CO gradually enriches and precipitates after responding to hydrogen peroxide.

Example 6 Pictures of PG-CO nanoparticles under high-resolution transmission electron microscopy in response to aggregation of ROS

Take the PG-CO solution after adding hydrogen peroxide solution in Example 4, prepare an electron microscope sample, and send it to a professional institution to take high-resolution transmission electron microscope pictures, as shown in Figure 5. It can be seen that the PG-CO nanoparticles aggregated after responding to ROS.

Example 7 HE stained sections show that PG-CO has no obvious effect on mouse organs

Dissolve the prepared PG-CO in water to make a 40-50mM solution. Take 0.5-1mL and inject it into the mice through tail vein injection. After normal feeding for 1-2 days, dissect the mice, remove the organs, and send them to a professional. The institution makes HE-stained sections, as shown in Figure 6. It can be seen that the mouse organs are normal, indicating that PG-CO has no obvious adverse effects on the mouse organs.

Comparative Example 1 Preparation of carbonyl siderophores using thiol-terminated polymers with molecular weights greater than 8000

Iron dodecacarbonyl (5-100 mg) and mPEG-SH (M.W.≈1w-1.2w) (100-600 mg) were dissolved in tetrahydrofuran (THF) and stirred at 50-120°C under nitrogen flow 1-12 h. At the end of the reaction, the solution changed from dark blue to brown. Cool to room temperature, add n-hexane to obtain brown precipitate, wash with diethyl ether and dry to obtain PG-CO. Redissolve PG-CO in THF, freeze it in a -20°C environment for 5-24 hours, and filter the precipitated crystals to obtain a new smart carbonyl siderophore PG-CO.

Dissolve 1-50 mg PG-CO and 5-10 mg Bodipy dye in THF. After ultrasonic for 5-10 minutes, add 10 ml of deionized water, blow out the THF with nitrogen, and co-precipitate. Centrifuge the ultrafiltration tube for 5 minutes and repeat washing three times, and co-precipitation is used to form stable and uniform nanoparticles. Dilute the concentration of the obtained nanoparticles to 20-30mM, put 1mL into a cuvette, add 1-2 drops of 0.3‰ hydrogen peroxide solution, prepare an electron microscope sample, send it out and ask a professional institution to take high-resolution transmission electron microscope pictures. As shown in Figure 7. It can be seen that compared with Figure 5, PG-CO nanoparticles did not aggregate after responding to ROS, indicating that the use of thiol-terminated polymers with molecular weights greater than 8000 to prepare carbonyl siderophores cannot effectively produce uniform and stable nanoparticles.

Based on the above examples and comparative examples, the characterization and detection results of the carbonyl siderophore synthesized by the present invention prove its ability as a carrier and the ability to release CO in response to ROS, and the carbonyl siderophore is used to successfully encapsulate berberine drugs, such as TPE, TPA, MCH-PPV, Bodipy fluorescent molecules and PBPTV fluorescent polymers, etc.; proved the biosafety of the vector, and had no effect on mouse organs after tail vein injection; proved the enrichment of the vector in response to ROS ability. The present invention uses carbonyl iron compounds and thiol-terminated polymers to prepare an intelligent responsive carrier capable of supplying CO, which greatly simplifies the delivery process and improves delivery efficiency. Moreover, the carrier can load other drugs or contrast agents to achieve Collaborative treatment of disease.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims

A carbonyl siderophore that releases carbon monoxide in response is characterized in that it includes a carbonyl iron compound and a thiol terminal polymer, and the carbonyl iron compound and the thiol terminal polymer are connected through a coordination reaction.
The carbonyl iron carrier according to claim 1, characterized in that the carbonyl iron compound is selected from any one of tricarbonyl iron, pentacarbonyl iron, nonacarbonyl iron, and dodecacarbonyl triiron.
The carbonyl siderophore according to claim 1, wherein the thiol-terminated polymer is selected from the group consisting of polyethylene glycol polymers, polypropylene polymers, polystyrene polymers, and polypropylene ethyl esters. Any type of polymer.
The carbonyl siderophore according to claim 1, wherein the molecular weight of the thiol-terminated polymer is 1000~8000.
The preparation method of the carbonyl siderophore according to any one of claims 1 to 4, characterized in that it includes the following steps:

Dissolve carbonyl iron compounds and thiol-terminated polymers in tetrahydrofuran and stir under nitrogen flow; at the end of the reaction, the solution changes from dark blue to brown; cool to room temperature, add liquid alkane to obtain a brown precipitate, and wash with an organic solvent After drying, the carbonyl siderophore is obtained.
The preparation method according to claim 5, characterized in that the preparation step further includes a purification step, specifically as follows:

Re-dissolve the prepared carbonyl siderophore in tetrahydrofuran, freeze it in a -20°C environment for 5-24 hours, and filter the precipitated crystals to obtain the purified carbonyl siderophore.
The preparation method according to claim 5, characterized in that the mass ratio of the carbonyl iron compound and the thiol terminal polymer is 1: (4~8).
The preparation method according to claim 5, characterized in that the nitrogen flow temperature is 50-120°C, and the stirring time is 1-12 h.
The preparation method according to claim 5, characterized in that the liquid alkane is n-hexane or n-pentane; the organic solvent is diethyl ether or n-butyl ether.
Use of the carbonyl siderophore according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.