CN112263679B - A targeted oxygen-carrying nano-sound sensitizer and its preparation method - Google Patents

Abstract

The invention discloses a targeted oxygen-carrying nano sound sensitizer and a preparation method thereof. The preparation method is simple in preparation process, and the prepared targeting oxygen-carrying nano sound sensitizer is stable in property, high in bioavailability and good in targeting property, and can be used for efficiently and targeted delivery of exogenous oxygen molecules and MnPP into tumors, so that the tumor hypoxia microenvironment is improved, and the sound power curative effect is improved.

Description

A targeted oxygen-carrying nano-sound sensitizer and its preparation method

Technical field

The invention relates to the field of nanomedicine, and in particular to a targeted oxygen-carrying nanometer sonosensitizer and a preparation method thereof.

Background technique

Among existing technologies, tumor optical therapy has gradually become a research hotspot in cancer treatment due to its advantages of non-invasiveness and spatiotemporal controllability. Optical tumor treatment mainly includes photodynamic therapy (PDT) and photothermal therapy (PTT). However, the tissue penetration depth of near-infrared light is limited, and the treatment effect of deep-seated tumors is poor. In contrast, sonodynamic therapy (SDT) has attracted widespread attention. It uses the stronger tissue penetration ability of ultrasound to cause the sonosensitizer accumulated in deep tumors to produce reactive oxygen species (ROS) or cavitation effect. Treat tumors. Currently, sonodynamic therapy has been used in clinical practice, and three patients with advanced breast cancer were successfully cured by combining endocrine therapy and immunotherapy. Commonly used sonosensitizer molecules include: porphyrin, porphyrin derivatives, anti-cancer drug DOX, acid yellow, methylene blue, polyhydroxyfullerene and other organic molecules. However, these traditional organic sonosensitizer molecules have problems such as low bioavailability, oxygen dependence, and lack of tumor targeting, resulting in low sonodynamic therapy efficiency and seriously hindering the clinical promotion of this method.

In recent years, the rapid development of global nanotechnology has brought a new revolution to cancer treatment. New drug carriers based on nanomaterials can achieve specific targeting and efficiently concentrate drugs into tumor lesions; at the same time, by regulating the size, morphology, surface charge, ligand modification and other physical and chemical properties of nanoparticles, they can break through the internal collagen, The high-density interstitial matrix barrier composed of mucopolysaccharides, proteoglycans, etc., delivers the loaded drugs into the tumor, thus enhancing the anti-tumor efficacy. In view of the bottleneck problems of traditional sonosensitizers such as low bioavailability, poor targeting, and oxygen dependence, nanotechnology provides new strategies and directions, and brings new opportunities for the clinical promotion of new nanosonodynamic pharmaceutical preparations. The development of new nano-sound sensitizers with high bioavailability, good targeting and improving the hypoxic microenvironment of tumors has become an important research direction in the field of sonodynamic therapy today.

However, the current nano-sound sensitizers have complicated preparation processes, cannot be prepared in large volumes, are unstable in nature, and prone to sedimentation and flocculation. There are still a series of problems such as the safety of sono-sensitizers, which hinder the clinical promotion of sonodynamic therapy.

Hybrid systems of human serum albumin and oxygen-carrying hemoglobin have been reported in the prior art, but without the addition of highly efficient and low-toxic sonosensitizers, efficient treatment of deep-seated tumors cannot be achieved. The three main factors of sonodynamics are: ultrasound, oxygen and sonosensitizer. The depth of ultrasound can be as high as 10cm, so when nanoparticles penetrate deep into the tumor, other methods such as light, even in the second zone, cannot achieve such deep penetration. Sonosensitizers can effectively function under the condition of deep ultrasound penetration to achieve efficient treatment of deep-seated tumors. Nanoparticles containing manganese porphyrin sonosensitizer coated with human serum albumin have also been reported in the prior art, but they cannot carry oxygen efficiently and therefore cannot provide efficient sonodynamic therapy.

Contents of the invention

In view of the deficiencies in the existing technology, the present invention proposes a targeted oxygen-carrying nano-sound sensitizer with simple preparation process, stable properties, high safety, high bioavailability and good targeting property, which can achieve efficient sonodynamic therapy. . Using disulfide bond "disconnection-reconstruction" technology, one-step ultrasonic method hybridizes human serum albumin (HSA) and oxygen-carrying hemoglobin (Hb), and wraps the hydrophobic sonosensitizer manganese porphyrin (MnPP) to prepare the targeted carrier. Oxygen nano-sonosensitizer can efficiently deliver exogenous oxygen molecules and MnPP into tumors, thereby improving the tumor hypoxic microenvironment and improving sonodynamic efficacy.

The invention provides a targeted oxygen-carrying nano-sound sensitizer. The outer part is a hybrid protein body and the inner part is wrapped with the sound sensitizer manganese porphyrin. The hybrid protein body is composed of human serum albumin and oxygen-carrying hemoglobin.

The targeted oxygen-carrying nano-sound sensitizer has a spherical structure and forms a strong intermolecular force with the manganese porphyrin sonosensitizer with a macrocyclic conjugated structure, thereby effectively wrapping the manganese porphyrin sonosensitizer in the hybrid protein In the body, the water solubility of the manganese porphyrin sonosensitizer is improved, thereby improving its bioavailability.

Oxygen-carrying hemoglobin can carry oxygen efficiently and can enhance the effect of sonodynamic therapy.

Human serum albumin has a tumor-specific targeting function and efficiently targets tumors through the tumor endothelial cell penetration effect mediated by the gp60 glycoprotein receptor and the enhanced uptake mechanism mediated by the tumor cell secreted protein SPARC.

Further, the manganese porphyrin is formed by complexing a metal manganese ion with a macrocyclic compound of a porphyrin structure. After the metal manganese ion is complexed with the macrocyclic compound of the porphyrin structure, a fluorescence resonance energy transfer effect is generated, thereby quenching the fluorescence of the porphyrin sound sensitizer and reducing the phototoxicity of the porphyrin sound sensitizer.

The oxygen-carrying nano-sound sensitizer can deliver exogenous oxygen molecules and manganese porphyrins into tumors in a targeted manner.

The invention also provides a method for preparing a targeted oxygen-carrying nano-acoustic sensitizer, which includes the following steps:

(1) Preparation of manganese porphyrin: Mix the macrocyclic compound of the porphyrin structure and the metal manganese salt, filter and precipitate, and dry;

(2) Use human serum albumin, oxygen-carrying hemoglobin and the manganese porphyrin obtained in step (1) to prepare a targeted oxygen-carrying nano-sound sensitizer.

Further, the macrocyclic compound with the porphyrin structure and the metal manganese salt in the step (1) are in an equimolar ratio.

Further, the macrocyclic compound of the porphyrin structure is 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin.

Further, the metal manganese salt in step (1) contains at least one of manganese chloride, manganese sulfate or manganese acetate.

Further, the step (1) includes the step of using an inert gas for reaction protection, and the inert gas is at least one of nitrogen and argon.

Further, the separation step in step (1) uses column chromatography, using silica gel as the stationary phase and dichloromethane and methanol as the mobile phase.

Further, the mass ratio of the human serum albumin and the oxygen-carrying hemoglobin in step (2) is 1-5:1. This ratio will affect the particle size of the targeted oxygen-carrying nanosonosensitizer.

Further, the step (2) includes the step of ultrasonicating the mixed solution of human serum albumin, oxygen-carrying hemoglobin and manganese porphyrin using a cell disruptor.

Further, the ultrasonic time is 5-20 minutes. Ultrasound time will affect the particle size of oxygen-carrying nano-sound sensitizer.

Further, the pH value of the mixed solution is 7-8. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines the enrichment amount of the oxygen-carrying nano-sound sensitizer in the tumor site.

The invention also provides the application of the oxygen-carrying nano-sound sensitizer in the preparation of anti-tumor drugs.

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

1. The preparation method of the present invention can prepare a large volume of targeted oxygen-carrying nano-acoustic sensitizers, and can prepare more than several liters. The volume of nanoparticles obtained by the preparation method of the prior art is up to several hundred milliliters, so the present invention is a nanometer-sized sonosensitizer. Provides a basis for scale-up production and clinical research of particles.

2. The targeted oxygen-carrying nano-acoustic sensitizer prepared by the present invention can be stored stably without settling or flocculation for one year.

3. The preparation method of the present invention is simple and easy to implement. By adding raw materials in two steps under stirring, a targeted oxygen-carrying nano-acoustic sensitizer with uniform particle size can be prepared. It does not require any high temperature, complicated equipment or harsh reactions. conditions for easy operation and promotion.

4. After the metal manganese ions of the present invention are complexed with the macrocyclic compound of the porphyrin structure, the phototoxicity of the porphyrin sound sensitizer is reduced and the safety is improved.

5. The present invention improves the water solubility of the manganese porphyrin sound sensitizer, thereby improving its bioavailability.

6. Human serum albumin has tumor-specific targeting function, so the oxygen-carrying nano-sound sensitizer of the present invention has good targeting properties.

Description of the drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, 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 also be obtained based on these drawings without exerting creative efforts.

Figure 1 is the UV-Vis absorption spectrum of the manganese porphyrin sound-sensitive molecule in Example 4.

Figure 2 is a transmission electron microscope image of the targeted oxygen-carrying nano-sound sensitizer in Example 4.

Figure 3 is a diagram showing the results of the targeted oxygen-carrying nanosonosensitizer of the present invention effectively carrying oxygen molecules into tumors in Example 5.

Figure 4 is a diagram showing the results of effective inhibition of tumor growth by the targeted oxygen-carrying nano-sound sensitizer of the present invention in Example 6.

Detailed ways

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 principle that the targeted oxygen-carrying nano-sonophonic agent of the present invention can achieve safe and efficient targeting inside the tumor, thereby improving the hypoxic microenvironment of the tumor and improving the sonodynamic efficacy is:

1. After the metal manganese ion is complexed with the macrocyclic compound of the porphyrin structure, a fluorescence resonance energy transfer effect is generated, thereby quenching the fluorescence of the porphyrin sound sensitizer and reducing the phototoxicity of the porphyrin sound sensitizer;

2. After metal coordination of porphyrin molecules, the metalloporphyrin triplet state (3MTTP*) generated by ultrasound is effectively combined with oxygen molecules, which significantly increases the active oxygen production rate of metalloporphyrin, making it an ideal sonosensitizer. molecular;

3. The targeted oxygen-carrying nano-sound sensitizer has a spherical structure and forms a strong intermolecular force with the manganese porphyrin sonosensitizer having a macrocyclic conjugated structure, thereby effectively wrapping the manganese porphyrin sonosensitizer in the complex. In the protein complex, the water solubility of the manganese porphyrin sonosensitizer is improved, thereby improving its bioavailability;

4. The present invention uses components in the human body: human serum albumin and oxygen-carrying hemoglobin as hybrid carriers, which are highly safe;

5. Human serum albumin has a tumor-specific targeting function: it can efficiently target tumors through the tumor endothelial cell penetration effect mediated by the gp60 glycoprotein receptor and the enhanced uptake mechanism mediated by the tumor cell secreted protein SPARC;

6. The manganese ions released by the degradation of targeted oxygen-carrying nanosonosensitizers can be used for magnetic resonance imaging.

The preparation method of the targeted oxygen-carrying nanometer sonosensitizer of the present invention is prepared by a one-step ultrasonic method. As the chloroform solvent evaporates, the hydrophobic manganese porphyrin is wrapped in the hybrid protein body to form a spherical targeted oxygen-carrying nanometer sonosensitizer. Sound sensitizer. Specific steps are as follows:

(1) Preparation of manganese porphyrin

① Combine an equal molar ratio of a macrocyclic compound with a porphyrin structure (such as 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin, PP) and a metal manganese salt (manganese chloride, manganese sulfate or acetic acid). At least one type of manganese) is mixed, stirred under the protection of inert gas, heated to reflux, the reaction is stopped, and cooled to room temperature;

② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate, low-temperature rotary evaporation, drying, and collect samples to obtain manganese porphyrin (MnPP).

(2) Preparation of targeted oxygen-carrying nano-sound sensitizers

①Weigh the manganese porphyrin and dissolve it in chloroform;

②Weigh the oxygen-carrying hemoglobin powder, dissolve it in ultrapure water, and place it in a shaker for reaction;

③ Mix human serum albumin and oxygen-carrying hemoglobin at a mass ratio of 1-5:1, and pipet the manganese porphyrin solution in ① at the bottom of the mixed solution;

④ Place the above mixture in an ice water bath, use a cell crusher to sonicate to form a suspension, adjust the pH value to 7-8, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear and the target is obtained. Oxygen-carrying nano-sound sensitizer.

Example 1 Preparation method of targeted oxygen-carrying nano-sound sensitizer

Specifically, it includes the following steps:

(1) Preparation of manganese porphyrin

① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP), purchased from Bailingwei Technology Co., Ltd., and manganese chloride in an anhydrous DMF solution, and place under nitrogen protection Next, stir and heat to reflux for 2 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction Stop and cool to room temperature;

② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).

(2) Preparation of targeted oxygen-carrying nano-sound sensitizers

①Weigh 1 mg of manganese porphyrin and dissolve it in 100 μL chloroform;

②Weigh 5 mg of Hb powder, dissolve it in 1 mL of ultrapure water, and place it in a shaker for 0.5 h. Reaction conditions: 37°C, 100 rpm;

③Mix HSA and Hb evenly at a mass ratio of 1:1, and pipet 100 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;

④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 5 minutes to form a suspension, use Na ₂ CO ₃ solution to adjust the pH value to 7, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.

The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 50-200 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. However, the particle size range of targeted oxygen-carrying nano-sound sensitizers is slightly larger, and the particle size uniformity is not high. This is related to the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the solution, and the time of ultrasound.

Example 2 Preparation method of targeted oxygen-carrying nano-sound sensitizer

In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.

Specifically, it includes the following steps:

(1) Preparation of manganese porphyrin

① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese acetate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 6 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;

② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).

(2) Preparation of targeted oxygen-carrying nano-sound sensitizers

①Weigh 5 mg of manganese porphyrin and dissolve it in 500 μL chloroform;

②Weigh 10 mg of Hb powder, dissolve it in 5 mL of ultrapure water, and place it in a shaker to react for 2 hours. Reaction conditions: 37°C, 200 rpm;

③Mix HSA and Hb evenly at a mass ratio of 5:1, and pipet 500 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;

④ Place the above mixture in an ice water bath, use a cell crusher to sonicate for 20 minutes to form a suspension, adjust the pH value to 8 with Na ₂ CO ₃ solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.

The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 80-200nm. The results of potential and particle size can indicate the successful preparation of targeted oxygen-carrying nano-sound sensitizers. The particle size range of the targeted oxygen-carrying nano-sound sensitizer is slightly narrower than in Example 1, but the particle size range is still somewhat large, and the particle size uniformity is not high. This is inconsistent with the ratio of human serum albumin and oxygen-carrying hemoglobin, the solution It is related to the pH value and ultrasound time. Targeted oxygen-carrying nano-sound sensitizers with more uniform particle sizes can be prepared by continuing to optimize the above conditions.

Example 3 Preparation method of targeted oxygen-carrying nano-sound sensitizer

In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.

Specifically, it includes the following steps:

(1) Preparation of manganese porphyrin

① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese acetate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 6 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;

② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).

(2) Preparation of targeted oxygen-carrying nano-sound sensitizers

①Weigh 1 mg of manganese porphyrin and dissolve it in 200 μL chloroform;

②Weigh 5 mg of Hb powder, dissolve it in 1 mL of ultrapure water, and place it in a shaker for 0.5 h. Reaction conditions: 37°C, 100 rpm;

③Mix HSA and Hb evenly at a mass ratio of 3:1, and pipet 200 μL of the manganese porphyrin solution in ① at the bottom of the mixed solution;

④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 5 minutes to form a suspension, adjust the pH value to 8 with Na ₂ CO ₃ solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes clear. , to obtain targeted oxygen-carrying nano-sound sensitizers.

The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 100-200 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. The particle size range of the targeted oxygen-carrying nano-sound sensitizer is slightly smaller than in Example 2, but the particle size range is still somewhat large, and the particle size uniformity is not high. This is inconsistent with the ratio of human serum albumin and oxygen-carrying hemoglobin, the solution It is related to the pH value and ultrasound time. Targeted oxygen-carrying nano-sound sensitizers with more uniform particle sizes can be prepared by continuing to optimize the above conditions.

Example 4 Preparation method of targeted oxygen-carrying nano-sound sensitizer

In this example, the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, and the ultrasound time are different from those in Example 1. The pH value directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the particle size directly determines The enrichment amount of the oxygen-carrying nanosonosensitizer in the tumor site was determined. The ultrasonic time directly affects the particle size of the oxygen-carrying nano-sound sensitizer, and the ratio of human serum albumin and oxygen-carrying hemoglobin also affects the particle size.

Specifically, it includes the following steps:

(1) Preparation of manganese porphyrin

① Mix equal molar ratios of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (PP) and manganese sulfate in anhydrous DMF solution, stir under argon protection, and heat to reflux for 4 hours. A UV-Vis absorption spectrometer monitors the progress of the reaction. After the reaction liquid changes from black-red to grass green, and the Q-band peaks change from 4 to 2 with a certain degree of red shift, the reaction is stopped and cooled to room temperature;

② Filter the precipitate, wash with deionized water, and dry in vacuum. Then use column chromatography to separate and purify, using silica gel as the stationary phase and dichloromethane and methanol with a volume ratio of 3:1 as the mobile phase. Rotate evaporate at low temperature, dry, and collect the sample to obtain manganese porphyrin (MnPP).

(2) Preparation of targeted oxygen-carrying nano-sound sensitizers

①Weigh 3 mg of manganese porphyrin and dissolve it in 300 μL chloroform;

②Weigh 7.5 mg of Hb powder, dissolve it in 3 mL of ultrapure water, and place it in a shaker to react for 1.5 hours. Reaction conditions: 37°C, 150 rpm;

③Mix HSA and Hb evenly at a mass ratio of 3:1, and pipet 300 μL of the manganese porphyrin solution in ① to the bottom of the mixed solution;

④ Place the above mixed solution in an ice water bath, use a cell crusher to sonicate for 12.5 minutes to form a suspension, adjust the pH value to 7.5 with Na ₂ CO ₃ solution, and then place it in an ultrasonic cleaner to sonicate until the suspension becomes After clarification, the targeted oxygen-carrying nano-sound sensitizer was obtained.

The zeta potential of the targeted oxygen-carrying nano-sound sensitizer detected by a particle size analyzer is greater than -30mV, and the particle size range is 100-150 nm. The results of the potential and particle size can indicate that the targeted oxygen-carrying nano-sound sensitizer is successfully prepared. Such oxygen-carrying nano-sound sensitizers can be more efficiently concentrated into tumor sites in the body.

The particle size means that the amount of loaded sonosensitizer is different, resulting in differences in oxygen carrying capacity. However, the size of the particle size directly determines the enrichment of the nano sonosensitizer in the tumor site. Generally speaking, the particle size is 100-150nm. , the targeted oxygen-carrying nano-sound sensitizer is more likely to be enriched in the tumor site.

In Examples 1-4, the prepared oxygen-carrying nano-sound sensitizers have different particle sizes. The factors that affect the particle size include: the ratio of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system, the ultrasonic time. The present invention explored various preparation conditions. Figure 1 is the UV-Vis absorption spectrum of the manganese porphyrin sound-sensitive molecule in this embodiment. It can be seen from the figure that the Q-band peaks of the prepared manganese porphyrin changed from 4 to 2 and accompanied by a certain degree of red shift (see the dotted box in Figure 1), indicating that the preparation method of the present invention can effectively prepare manganese porphyrin sound-sensitive molecules. Figure 2 is a transmission electron microscope image of the targeted oxygen-carrying nano-sound sensitizer in this embodiment. It can be seen from the figure that the prepared targeted oxygen-carrying nano-sound sensitizer has a regular shape and a particle size of 100-150 nm, illustrating that the present invention The preparation method can effectively prepare targeted oxygen-carrying nano-sound sensitizers.

The targeted oxygen-carrying nano-sound sensitizer particles prepared in this embodiment are the most uniform, have the highest oxygen-carrying capacity, and can be better targeted and accumulated at tumor sites. Therefore, the preparation conditions and proportions of this embodiment are the most preferred. The results of animal experiments can also be explained, see Example 5 and Example 6 for details.

Example 5 The targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively carry oxygen molecules into tumors

The targeted oxygen-carrying nano-sound sensitizer prepared in Example 4 was injected into the tail vein of tumor-bearing mice (which can be purchased directly). Before the injection and 3 hours after the injection, the small animal photoacoustic instrument was used to measure the results. The results are shown in Figure 3 As shown, the control group is a control group that only adds phosphate buffer. The results show that the targeted oxygen-carrying nano-sound sensitizer of the present invention has a significant photoacoustic signal (such as the area pointed by the white arrow) compared with the control group. Therefore, the Targeted oxygen-carrying nanosonosensitizers can effectively improve the tumor hypoxic microenvironment.

Example 6 The targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively inhibit tumor growth

The targeted oxygen-carrying nano-sound sensitizer prepared in Example 4 was injected into the tail vein of tumor-bearing mice (which can be purchased directly). After 3 hours, ultrasonic treatment was started for 5 minutes. 20 days after treatment, the mice were killed and taken out. The volume of the tumor was measured. The results are shown in Figure 4. PBS is the control group with only phosphate buffer added, and US represents ultrasound. The results show that the targeted oxygen-carrying nano-sound sensitizer of the present invention has a better tumor volume than the control under the action of ultrasound. The group is significantly reduced, so the targeted oxygen-carrying nano-sound sensitizer of the present invention can effectively inhibit tumor growth.

In summary, the present invention uses a one-step ultrasonic method to hybridize human serum albumin (HSA) and oxygen-carrying hemoglobin (Hb), and wrap the hydrophobic sonosensitizer manganese porphyrin (MnPP) to prepare a targeted oxygen-carrying nano-sound sensitizer. The preparation process is simple, and the prepared targeted oxygen-carrying nano-sonophore has stable properties, high bioavailability, and good targeting properties. It can efficiently deliver exogenous oxygen molecules and MnPP to the inside of the tumor and improve the tumor hypoxic microenvironment. , improve the sonodynamic efficacy.

1. The preparation method of the present invention can prepare a large volume of targeted oxygen-carrying nano-acoustic sensitizers, and can prepare more than several liters. The volume of nanoparticles obtained by the preparation method of the prior art is up to several hundred milliliters, so the present invention is a nanometer-sized sonosensitizer. Provides a basis for scale-up production and clinical research of particles.

2. The targeted oxygen-carrying nano-acoustic sensitizer prepared by the present invention can be stored stably without settling or flocculation for one year.

3. The preparation method of the present invention is simple and easy to implement. By adding raw materials in two steps under stirring, a targeted oxygen-carrying nano-acoustic sensitizer with uniform particle size can be prepared. It does not require any high temperature, complicated equipment or harsh reactions. conditions for easy operation and promotion.

4. After the metal manganese ions of the present invention are complexed with the macrocyclic compound of the porphyrin structure, the phototoxicity of the porphyrin sound sensitizer is reduced and the safety is improved.

5. The present invention improves the water solubility of the manganese porphyrin sound sensitizer, thereby improving its bioavailability.

6. Human serum albumin has tumor-specific targeting function, so the oxygen-carrying nano-sound sensitizer of the present invention has good targeting properties.

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 (5)

1. The preparation method of the targeted oxygen-carrying nano sound sensitizer is characterized by comprising the following steps of:

(1) Preparation of manganese porphyrin: mixing 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin and metal manganese salt, filtering to precipitate, drying and separating; the 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin and the manganese metal salt are in an equimolar ratio; the metal manganese salt comprises at least one of manganese chloride, manganese sulfate or manganese acetate;

(2) Preparing a targeted oxygen-carrying nano sound sensitizer by using human serum albumin, oxygen-carrying hemoglobin and manganese porphyrin obtained in the step (1);

weighing 3mg of manganese porphyrin, and weighing 7.5mg of oxyhemoglobin-loaded powder, wherein the mass ratio of human serum albumin to the oxyhemoglobin-loaded powder is 3:1, a step of; the step (2) comprises the step of performing ultrasonic treatment on the mixed solution of the human serum albumin, the oxygen-carrying hemoglobin and the manganese porphyrin by using a cytobreaker; the ultrasonic time is 12.5 min; the pH value of the mixed solution is 7.5;

the particle size range of the targeted oxygen-carrying nano sound-sensitive agent obtained by the steps (1) and (2) is 100-150nm.

2. The method according to claim 1, wherein the step (1) comprises a step of performing reaction protection using an inert gas.

3. The method according to claim 1, wherein the separation step in the step (1) uses column chromatography.

4. A targeted oxygen-carrying nano-sonosensitizer, characterized in that it is prepared by the preparation method of any one of claims 1-3; the targeted oxygen-carrying nano sound sensitizer is externally provided with a heterozygous protein body, and the interior of the targeted oxygen-carrying nano sound sensitizer is wrapped with manganese porphyrin; the hybrid protein body consists of human serum albumin and oxyhemoglobin; the manganese porphyrin is formed by complexing metal manganese ions with 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin.

5. The use of the targeted oxygen-carrying nano sound-sensitive agent in the preparation of antitumor drugs.