CN112263679A - Targeted oxygen-carrying nano acoustic sensitizer and preparation method thereof - Google Patents
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- A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
- A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
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Abstract
The invention discloses a targeted oxygen-carrying nano acoustic sensitizer and a preparation method thereof. The method has simple preparation process, and the prepared targeted oxygen-carrying nano acoustic sensitizer has stable property, high bioavailability and good targeting property, and can deliver exogenous oxygen molecules and MnPP into the tumor in a highly efficient targeted manner, thereby improving the tumor hypoxia microenvironment and improving the acoustic dynamic curative effect.
Description
Technical Field
The invention relates to the field of nano medicine, in particular to a targeted oxygen-carrying nano sound-sensitive agent and a preparation method thereof.
Background
In the prior art, tumor optical treatment is gradually becoming a research hotspot for cancer treatment due to the advantages of non-invasion and space-time control. The tumor optical treatment mainly comprises photodynamic therapy (PDT) and photothermal therapy (PTT). However, the penetration depth of near infrared tissue is limited, and the therapeutic effect of deep tumors is not good. In contrast, sonodynamic therapy (SDT) has attracted much attention, and utilizes the stronger tissue penetration of ultrasound to generate Reactive Oxygen Species (ROS) or cavitation effect from sonosensitizers accumulated in deep tumors to treat tumors. Currently, sonodynamic therapy has been used clinically, combining endocrine therapy and immunotherapy to successfully cure 3 patients with advanced breast cancer. Commonly used sonosensitizer molecules include: porphyrin, porphyrin derivatives, anticancer drugs DOX, acid yellow, methylene blue, polyhydroxy fullerene and other organic molecules. However, the traditional organic sonosensitizer molecules have the problems of low bioavailability, oxygen dependence, lack of tumor targeting and the like, so that the sonodynamic therapy efficiency is low, and the clinical popularization of the method is seriously hindered.
In recent years, the rapid development of global nanotechnology brings a new revolution to cancer treatment. The novel drug carrier of the nano material can realize specific targeting positioning, and efficiently enrich the drug to the tumor focus; meanwhile, by regulating the size, morphology, surface charge, ligand modification and other physical and chemical properties of the nanoparticles, high-density interstitial matrix barriers formed by collagen, mucopolysaccharide, proteoglycan and the like in the tumor are broken through, and loaded drugs are delivered to the interior of the tumor, so that the anti-tumor curative effect is enhanced. Aiming at the bottleneck problems of low bioavailability, poor targeting, oxygen dependence and the like of the traditional sonosensitizer, the nanotechnology provides a new strategy and a new direction, and brings a new opportunity for clinical popularization of a novel nano sonodynamic medicine preparation. The development of novel nano-acoustic sensitivity agents with high bioavailability, good targeting and tumor hypoxia microenvironment becomes an important research direction in the field of acoustic dynamic therapy.
However, the existing nano-acoustic sensitivity agent has complex preparation process, cannot prepare large volume, has unstable property, is easy to precipitate and flocculate, still has a plurality of problems of acoustic sensitivity agent safety and the like, and hinders the clinical popularization of acoustic dynamic therapy.
In the prior art, a hybrid system of human serum albumin and oxygen-carrying hemoglobin is reported, but a high-efficiency and low-toxicity sound-sensitive agent is not added, so that the high-efficiency treatment of deep tumors cannot be realized. Three major factors of acoustic dynamics are: ultrasound, oxygen, and sonosensitizers. The depth of ultrasound can be as high as 10cm, so when the nanoparticles penetrate deep into the tumor, other means such as light, even two-zone, cannot achieve such deep penetration. The sound sensitive agent can effectively play a role in the condition of ultrasonic deep penetration, and realizes the high-efficiency treatment of deep tumors. The prior art also reports that the manganese porphyrin sonosensitizer nanoparticles are coated by human serum albumin, but the manganese porphyrin sonosensitizer nanoparticles cannot carry oxygen efficiently, so that the manganese porphyrin sonosensitizer nanoparticles cannot achieve efficient sonodynamic therapy.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the targeted oxygen-carrying nano acoustic sensitizer which has the advantages of simple preparation process, stable property, high safety, high bioavailability and good targeting property, and can realize efficient acoustic dynamic treatment. Adopts a disulfide bond 'disconnection-reconstruction' technology, hybridizes Human Serum Albumin (HSA) and oxygen-carrying hemoglobin (Hb) by a one-step ultrasonic method, wraps hydrophobic manganese porphyrin (MnPP) serving as a sound-sensitive agent, prepares a targeted oxygen-carrying nano sound-sensitive agent, and efficiently delivers exogenous oxygen molecules and MnPP to the interior of a tumor in a targeted manner, thereby improving the hypoxic microenvironment of the tumor and improving the sound-dynamic curative effect.
The invention provides a targeted oxygen-carrying nano sound-sensitive agent, wherein the outside of the targeted oxygen-carrying nano sound-sensitive agent is a hybrid protein body, and the interior of the targeted oxygen-carrying nano sound-sensitive agent is wrapped by manganese porphyrin, and the hybrid protein body consists of human serum albumin and oxygen-carrying hemoglobin.
The targeting oxygen-carrying nano sound-sensitive agent is in a spherical structure, and forms stronger intermolecular force with the manganoporphyrin sound-sensitive agent with a macrocyclic conjugated structure, so that the manganoporphyrin sound-sensitive agent is efficiently wrapped in a hybrid protein body, the water solubility of the manganoporphyrin sound-sensitive agent is improved, and the bioavailability of the manganoporphyrin sound-sensitive agent is improved.
The oxygen-carrying hemoglobin can carry oxygen with high efficiency, and can enhance the effect of the acoustic dynamic therapy.
The human serum albumin has a tumor specific targeting function, and can efficiently target tumors through a tumor endothelial cell penetration effect mediated by a gp60 glycoprotein receptor and an enhanced uptake mechanism mediated by a tumor cell secretory protein SPARC.
Further, the manganese porphyrin is formed by complexing metal manganese ions with a macrocyclic compound with a porphyrin structure. After the metal manganese ions are complexed with the macrocyclic compound with the porphyrin structure, a fluorescence resonance energy transfer effect is generated, so that the fluorescence of the porphyrin sonosensitizer is quenched, and the phototoxicity of the porphyrin sonosensitizer is reduced.
The oxygen-carrying nano sound-sensitive agent can deliver exogenous oxygen molecules and manganoporphyrin to the interior of a tumor in a targeted manner.
The invention also provides a preparation method of the targeted oxygen-carrying nano sound-sensitive agent, which comprises the following steps:
(1) preparation of manganese porphyrin: mixing the macrocyclic compound with porphyrin structure and metal manganese salt, filtering, precipitating and drying;
(2) preparing the targeted oxygen-carrying nano acoustic sensitizer by using human serum albumin, oxygen-carrying hemoglobin and the manganoporphyrin obtained in the step (1).
Further, the macrocyclic compound with a porphyrin structure and the metal manganese salt in the step (1) are in an equimolar ratio.
Further, the macrocyclic compound with the porphyrin structure is 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin.
Further, the metal manganese salt in the step (1) comprises at least one of manganese chloride, manganese sulfate or manganese acetate.
Further, the step (1) includes a step of performing reaction protection by using an inert gas, wherein the inert gas is at least one of nitrogen and argon.
Further, the separation step in the step (1) uses column chromatography with silica gel as a stationary phase and dichloromethane and methanol as mobile phases.
Further, the mass ratio of the human serum albumin and the oxygen-carrying hemoglobin in the step (2) is 1-5: 1. this ratio will affect the particle size of the targeted oxygen-carrying nanoscopic acoustic agent.
Further, the step (2) includes a step of subjecting the mixed solution of the human serum albumin, the oxygen-carrying hemoglobin, and the manganoporphyrin to ultrasound using a cell disruptor.
Further, the ultrasonic time is 5-20 min. The ultrasonic time can affect the particle size of the oxygen-carrying nano sound-sensitive agent.
Further, the pH value of the mixed solution is 7-8. The pH value directly influences the particle size of the oxygen-carrying nano acoustic sensing agent, and the particle size directly determines the enrichment amount of the oxygen-carrying nano acoustic sensing agent at a tumor part.
The invention also provides application of the oxygen-carrying nano sound-sensitive agent in preparing anti-tumor drugs.
In summary, compared with the prior art, the invention achieves the following technical effects:
1. the preparation method of the invention can prepare the targeting oxygen-carrying nano sound-sensitive agent with large volume, and can prepare more than several liters.
2. The targeted oxygen-carrying nano sound-sensitive agent prepared by the invention can be stably stored, and the phenomena of sedimentation and flocculation do not occur in one year.
3. The preparation method is simple and easy to implement, the raw materials are added in two steps under stirring, the targeted oxygen-carrying nano acoustic sensitizer with uniform particle size can be prepared, no high-temperature and complex equipment or harsh reaction conditions are needed, and the operation and popularization are convenient.
4. After the metal manganese ions are complexed with the macrocyclic compound with the porphyrin structure, the phototoxicity of the porphyrin sound-sensitive agent is reduced, and the safety is improved.
5. The invention improves the water solubility of the manganoporphyrin sound-sensitive agent, thereby improving the bioavailability of the manganoporphyrin sound-sensitive agent.
6. The human serum albumin has the tumor specific targeting function, so the oxygen-carrying nano sound-sensitive agent has good targeting property.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram showing the UV-Vis absorption spectrum of the manganese porphyrin sound-sensitive molecule in example 4.
FIG. 2 is a transmission electron micrograph of the targeted oxygen-carrying nanoacoustic sensitizer in example 4.
FIG. 3 is a graph showing the results of example 5 in which the targeted oxygen-carrying nano acoustic sensor of the present invention effectively carries oxygen molecules into tumors.
FIG. 4 is a graph showing the results of the targeting oxygen-carrying nano-sized sonosensitizer of the present invention effectively inhibiting tumor growth in example 6.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The targeting oxygen-carrying nano sound-sensitive agent can realize safe and efficient targeting inside a tumor, thereby improving the tumor hypoxia microenvironment and improving the sound power curative effect according to the following principle:
1. after the metal manganese ions are complexed with the macrocyclic compound with the porphyrin structure, a fluorescence resonance energy transfer effect is generated, so that the fluorescence of the porphyrin sound-sensitive agent is quenched, and the phototoxicity of the porphyrin sound-sensitive agent is reduced;
2. after porphyrin molecules are subjected to metal coordination, metalloporphyrin triplet (3 MTTP) generated by ultrasonic action is effectively combined with oxygen molecules, so that the active oxygen yield of the metalloporphyrin is remarkably improved, and the metalloporphyrin is an ideal sound sensitive agent molecule;
3. the targeting oxygen-carrying nano sound-sensitive agent is of a spherical structure, and forms stronger intermolecular force with the manganoporphyrin sound-sensitive agent with a macrocyclic conjugated structure, so that the manganoporphyrin sound-sensitive agent is efficiently wrapped in a hybrid protein body, the water solubility of the manganoporphyrin sound-sensitive agent is improved, and the bioavailability of the manganoporphyrin sound-sensitive agent is improved;
4. the invention adopts the following components in human body: the human serum albumin and the oxygen-carrying hemoglobin are used as hybrid carriers, so that the safety is high;
5. the human serum albumin has the tumor specific targeting function: the tumor is efficiently targeted through the tumor endothelial cell penetration effect mediated by gp60 glycoprotein receptor and the enhanced uptake mechanism mediated by tumor cell secretory protein SPARC;
6. manganese ions released by degradation of the targeted oxygen-carrying nano sonosensitizer can be used for magnetic resonance imaging.
The preparation method of the targeted oxygen-carrying nano sound-sensitive agent is characterized in that the targeted oxygen-carrying nano sound-sensitive agent is prepared by adopting a one-step ultrasonic method, and hydrophobic manganoporphyrin is wrapped in a hybrid protein body along with the volatilization of a chloroform solvent to form the spherical targeted oxygen-carrying nano sound-sensitive agent. The method comprises the following specific steps:
(1) preparation of manganoporphyrins
Mixing macrocyclic compounds (such as 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin and PP) with equal molar ratio and metal manganese salt (at least one of manganese chloride, manganese sulfate or manganese acetate), stirring under the protection of inert gas, heating and refluxing, stopping reaction, and cooling to room temperature;
filtering the precipitate, washing with deionized water, and vacuum drying. And separating by using column chromatography, carrying out low-temperature rotary evaporation, drying, and collecting a sample to obtain manganoporphyrin (MnPP).
(2) Preparation of targeted oxygen-carrying nano acoustic sensitizer
Weighing manganoporphyrin, and dissolving the manganoporphyrin in chloroform;
weighing oxygen-carrying hemoglobin powder, dissolving in ultrapure water, and placing in a shaking table for reaction;
thirdly, mixing human serum albumin and oxygen-carrying hemoglobin according to the mass ratio of 1-5: 1, mixing, and transferring the manganoporphyrin solution in the step one to the bottom of the mixed solution;
putting the mixed solution in an ice water bath, carrying out ultrasonic treatment by using a cell crushing instrument to form turbid liquid, adjusting the pH value to 7-8, and putting the turbid liquid in an ultrasonic cleaner for ultrasonic treatment until the turbid liquid becomes clear, thereby obtaining the targeted oxygen-carrying nano sound-sensitive agent.
Example 1 preparation method of Targeted oxygen-carrying NanoSonensitizer
The method specifically comprises the following steps:
(1) preparation of manganoporphyrins
Mixing 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin (PP) with equal molar ratio, which is purchased from carbofuran technology limited, and manganese chloride in an anhydrous DMF solution, stirring under the protection of nitrogen, heating and refluxing for 2 hours, monitoring the reaction progress by a UV-Vis absorption spectrometer, stopping the reaction after the reaction solution is changed from black red to turquoise and Q band peaks are changed from 4 to 2 along with a certain degree of red shift, and cooling to room temperature;
filtering the precipitate, washing with deionized water, and vacuum drying. And then separating by using column chromatography, taking silica gel as a stationary phase, and performing separation by using the volume ratio of 3: 1, taking dichloromethane and methanol as mobile phases for separation and purification. Performing low-temperature rotary evaporation, drying and collecting a sample to obtain manganoporphyrin (MnPP).
(2) Preparation of targeted oxygen-carrying nano acoustic sensitizer
Weighing 1mg of manganoporphyrin, and dissolving the manganoporphyrin in 100 mu L of chloroform;
weighing 5mg of Hb powder, dissolving the Hb powder in 1mL of ultrapure water, placing the Hb powder in a shaking table for reacting for 0.5h, wherein the reaction conditions are as follows: 37 ℃ at 100 rpm;
③ mixing HSA and Hb according to the mass ratio of 1: 1, uniformly mixing, and transferring 100 mu L of manganoporphyrin solution in the step one to the bottom of the mixed solution;
putting the mixed solution in ice water bath, performing ultrasonic treatment for 5min by using a cell crushing instrument to form turbid liquid, and adding Na2CO3And adjusting the pH value of the solution to 7, and then placing the solution in an ultrasonic cleaner for ultrasonic treatment until the suspension becomes clear, thereby obtaining the targeted oxygen-carrying nano acoustic sensitizer.
The zeta potential of the targeted oxygen-carrying nano sound-sensitive agent detected by a particle size analyzer is more than-30 mV, the particle size range is 50-200nm, and the results of the potential and the particle size can indicate that the targeted oxygen-carrying nano sound-sensitive agent is successfully prepared. However, the targeted oxygen-carrying nano acoustic sensitizer has a slightly larger particle size range and a low uniform degree of particle size. This is related to the ratio of human serum albumin to oxygen-carrying hemoglobin, the pH of the solution, and the time of sonication.
Example 2 preparation method of Targeted oxygen-carrying NanoSonensitizer
In this example, the ratio of human serum albumin to 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 acoustic sensor, and the particle size directly determines the enrichment amount of the oxygen-carrying nano acoustic sensor at the tumor site. The ultrasonic time directly influences the particle size of the oxygen-carrying nano acoustic sensitizer, and the proportion of the human serum albumin and the oxygen-carrying hemoglobin also influences the particle size.
The method specifically comprises the following steps:
(1) preparation of manganoporphyrins
Mixing 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin (PP) and manganese acetate in an equal molar ratio in an anhydrous DMF solution, stirring under the protection of argon, heating and refluxing for 6 hours, monitoring the reaction progress by an UV-Vis absorption spectrometer, stopping the reaction after the reaction solution is changed from black red to turquoise and Q band peaks are changed from 4 to 2 along with a certain degree of red shift, and cooling to room temperature;
filtering the precipitate, washing with deionized water, and vacuum drying. And then separating by using column chromatography, taking silica gel as a stationary phase, and performing separation by using the volume ratio of 3: 1, taking dichloromethane and methanol as mobile phases for separation and purification. Performing low-temperature rotary evaporation, drying and collecting a sample to obtain manganoporphyrin (MnPP).
(2) Preparation of targeted oxygen-carrying nano acoustic sensitizer
Weighing 5mg of manganoporphyrin, and dissolving the manganoporphyrin in 500 mu L of chloroform;
weighing 10mg of Hb powder, dissolving the Hb powder in 5mL of ultrapure water, placing the Hb powder in a shaking table for reacting for 2 hours, wherein the reaction conditions are as follows: at 37 ℃ and 200 rpm;
③ mixing the HSA and the Hb according to the mass ratio of 5: 1, uniformly mixing, and transferring 500 mu L of manganoporphyrin solution in the step one to the bottom of the mixed solution;
putting the mixed solution in ice water bath, performing ultrasonic treatment for 20min by using a cell crushing instrument to form turbid liquid, and adding Na2CO3And adjusting the pH value of the solution to 8, and then placing the solution in an ultrasonic cleaner for ultrasonic treatment until the suspension becomes clear, thereby obtaining the targeted oxygen-carrying nano acoustic sensitizer.
The zeta potential of the targeted oxygen-carrying nano sound-sensitive agent is detected by a particle size analyzer to be more than-30 mV, and the particle size range is 80-200 nm. The results of potential and particle size can show that the targeted oxygen-carrying nano sound-sensitive agent is successfully prepared. The particle size range of the targeted oxygen-carrying nano sonosensitizer is slightly reduced compared with that of example 1, but the particle size range is still slightly large, and the particle size uniformity is not high, which is related to the proportion of human serum albumin and oxygen-carrying hemoglobin, the pH value of the solution and the ultrasonic time. The targeting oxygen-carrying nano acoustic sensitizer with more uniform particle size can be prepared by continuously optimizing the conditions.
Example 3 preparation method of Targeted oxygen-carrying NanoSonensitizer
In this example, the ratio of human serum albumin to 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 acoustic sensor, and the particle size directly determines the enrichment amount of the oxygen-carrying nano acoustic sensor at the tumor site. The ultrasonic time directly influences the particle size of the oxygen-carrying nano acoustic sensitizer, and the proportion of the human serum albumin and the oxygen-carrying hemoglobin also influences the particle size.
The method specifically comprises the following steps:
(1) preparation of manganoporphyrins
Mixing 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin (PP) and manganese acetate in an equal molar ratio in an anhydrous DMF solution, stirring under the protection of argon, heating and refluxing for 6 hours, monitoring the reaction progress by an UV-Vis absorption spectrometer, stopping the reaction after the reaction solution is changed from black red to turquoise and Q band peaks are changed from 4 to 2 along with a certain degree of red shift, and cooling to room temperature;
filtering the precipitate, washing with deionized water, and vacuum drying. And then separating by using column chromatography, taking silica gel as a stationary phase, and performing separation by using the volume ratio of 3: 1, taking dichloromethane and methanol as mobile phases for separation and purification. Performing low-temperature rotary evaporation, drying and collecting a sample to obtain manganoporphyrin (MnPP).
(2) Preparation of targeted oxygen-carrying nano acoustic sensitizer
Weighing 1mg of manganoporphyrin, and dissolving the manganoporphyrin in 200 mu L of chloroform;
weighing 5mg of Hb powder, dissolving the Hb powder in 1mL of ultrapure water, placing the Hb powder in a shaking table for reacting for 0.5h, wherein the reaction conditions are as follows: 37 ℃ at 100 rpm;
③ mixing the HSA and the Hb according to the mass ratio of 3: 1, uniformly mixing, and transferring 200 mu L of manganoporphyrin solution in the step one to the bottom of the mixed solution;
putting the mixed solution in ice water bath, performing ultrasonic treatment for 5min by using a cell crushing instrument to form turbid liquid, and adding Na2CO3And adjusting the pH value of the solution to 8, and then placing the solution in an ultrasonic cleaner for ultrasonic treatment until the suspension becomes clear, thereby obtaining the targeted oxygen-carrying nano acoustic sensitizer.
The zeta potential of the targeted oxygen-carrying nano sound-sensitive agent is detected to be more than-30 mV by a particle size analyzer, the particle size range is 100-200nm, and the results of the potential and the particle size can indicate that the targeted oxygen-carrying nano sound-sensitive agent is successfully prepared. The particle size range of the targeted oxygen-carrying nano sonosensitizer is slightly reduced compared with that of example 2, but the particle size range is still slightly large, and the particle size uniformity is not high, which is related to the proportion of the human serum albumin and the oxygen-carrying hemoglobin, the pH value of the solution and the ultrasonic time. The targeting oxygen-carrying nano acoustic sensitizer with more uniform particle size can be prepared by continuously optimizing the conditions.
Example 4 preparation method of Targeted oxygen-carrying NanoSonensitizer
In this example, the ratio of human serum albumin to 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 acoustic sensor, and the particle size directly determines the enrichment amount of the oxygen-carrying nano acoustic sensor at the tumor site. The ultrasonic time directly influences the particle size of the oxygen-carrying nano acoustic sensitizer, and the proportion of the human serum albumin and the oxygen-carrying hemoglobin also influences the particle size.
The method specifically comprises the following steps:
(1) preparation of manganoporphyrins
Mixing 5,10,15, 20-tetra (4-carboxyphenyl) porphyrin (PP) and manganese sulfate in an equal molar ratio in an anhydrous DMF solution, stirring under the protection of argon, heating and refluxing for 4 hours, monitoring the reaction progress by a UV-Vis absorption spectrometer, stopping the reaction after the reaction solution is changed from black red to turquoise and Q band peaks are changed from 4 to 2 along with a certain degree of red shift, and cooling to room temperature;
filtering the precipitate, washing with deionized water, and vacuum drying. And then separating by using column chromatography, taking silica gel as a stationary phase, and performing separation by using the volume ratio of 3: 1, taking dichloromethane and methanol as mobile phases for separation and purification. Performing low-temperature rotary evaporation, drying and collecting a sample to obtain manganoporphyrin (MnPP).
(2) Preparation of targeted oxygen-carrying nano acoustic sensitizer
Weighing 3mg of manganoporphyrin, and dissolving the manganoporphyrin in 300 mu L of chloroform;
weighing 7.5mg of Hb powder, dissolving the Hb powder in 3mL of ultrapure water, placing the Hb powder in a shaking table for reacting for 1.5h, wherein the reaction conditions are as follows: 37 ℃ and 150 rpm;
③ mixing the HSA and the Hb according to the mass ratio of 3: 1, uniformly mixing, and transferring 300 mu L of manganoporphyrin solution in the step one to the bottom of the mixed solution;
fourthly, placing the mixed solution in ice water bath, carrying out ultrasonic treatment for 12.5min by using a cell crushing instrument to form turbid liquid, and adding Na2CO3And adjusting the pH value of the solution to 7.5, and then placing the solution in an ultrasonic cleaner for ultrasonic treatment until the suspension becomes clear, thereby obtaining the targeted oxygen-carrying nano sound-sensitive agent.
The zeta potential of the targeted oxygen-carrying nano sound-sensitive agent is detected to be more than-30 mV by a particle size analyzer, the particle size range is 100-150nm, and the results of the potential and the particle size can indicate that the targeted oxygen-carrying nano sound-sensitive agent is successfully prepared. The oxygen-carrying nano sound-sensitive agent can be finally enriched to a tumor part in vivo more efficiently.
The particle size means that the amount of the loaded acoustic sensitivity agent is different, so that the oxygen carrying amount is different, but the particle size directly determines the enrichment amount of the nano acoustic sensitivity agent at the tumor site, and generally, when the particle size is 100-150nm, the targeted oxygen-carrying nano acoustic sensitivity agent is easier to enrich to the tumor site.
In examples 1 to 4, the prepared oxygen-carrying nano-sized acoustic sensing agents have different particle sizes, and factors influencing the particle sizes include: the proportion of human serum albumin and oxygen-carrying hemoglobin, the pH value of the system and the ultrasonic time. Various preparation conditions are explored, fig. 1 is a UV-Vis absorption spectrogram of the manganoporphyrin sonosensitive molecule in the embodiment, and the diagram can show that the Q-band peak of the manganoporphyrin prepared by the preparation method is converted into 2 from 4 and accompanied with a certain degree of red shift (see a dashed line frame of fig. 1), so that the manganoporphyrin sonosensitive molecule can be effectively prepared by the preparation method disclosed by the invention. FIG. 2 is a transmission electron microscope image of the targeted oxygen-carrying nano acoustic sensitizer in this embodiment, in which the shape of the prepared targeted oxygen-carrying nano acoustic sensitizer is regular and the particle size is 100-150nm, which illustrates that the preparation method of the present invention can effectively prepare the targeted oxygen-carrying nano acoustic sensitizer.
The targeted oxygen-carrying nano sound-sensitive agent prepared by the embodiment has the most uniform particles and the highest oxygen carrying amount, and can be better accumulated to a tumor site in a targeted manner. The preparation conditions and ratios of this example are most preferred. Animal experimental results can also be shown, and are specifically shown in example 5 and example 6.
Example 5 the targeted oxygen-carrying nano-sized sonosensitizer of the present invention effectively carries oxygen molecules into tumors
The targeted oxygen-carrying nano acoustic sensor prepared in the embodiment 4 is injected into a tumor-bearing mouse (directly purchased) through tail vein, the results are shown in fig. 3 after injection and determination by a small animal photoacoustic apparatus, the control group is a control group only added with phosphate buffer, and the results show that the targeted oxygen-carrying nano acoustic sensor of the invention has obvious photoacoustic signals (such as areas indicated by white arrows) compared with the control group, so that the targeted oxygen-carrying nano acoustic sensor of the invention can effectively improve the tumor hypoxia microenvironment.
Example 6 the targeted oxygen-carrying nano-acoustic sensitivity agent of the invention can effectively inhibit the growth of tumor
The targeted oxygen-carrying nano sound-sensitive agent prepared in the embodiment 4 is injected into a tumor-bearing mouse (which can be purchased directly) via tail vein, ultrasonic treatment is started for 5 minutes after 3 hours, the mouse is killed 20 days after treatment, the tumor is taken out to measure the volume of the tumor, the result is shown in figure 4, PBS is a control group only added with phosphate buffer, US represents ultrasonic, and the result shows that the tumor volume of the targeted oxygen-carrying nano sound-sensitive agent is obviously reduced compared with the control group under the action of ultrasonic, so that the targeted oxygen-carrying nano sound-sensitive agent can effectively inhibit the growth of the tumor.
In conclusion, the invention adopts a one-step ultrasonic method to hybridize Human Serum Albumin (HSA) and oxygen-carrying hemoglobin (Hb) and wrap hydrophobic manganese porphyrin (MnPP) serving as a sound-sensitive agent to prepare the targeted oxygen-carrying nano sound-sensitive agent. The preparation process is simple, and the prepared targeted oxygen-carrying nano sound-sensitive agent has stable property, high bioavailability and good targeting property, can deliver exogenous oxygen molecules and MnPP into the tumor in a highly efficient targeted manner, improves the tumor hypoxia microenvironment and improves the sound power curative effect.
1. The preparation method of the invention can prepare the targeting oxygen-carrying nano sound-sensitive agent with large volume, and can prepare more than several liters.
2. The targeted oxygen-carrying nano sound-sensitive agent prepared by the invention can be stably stored, and the phenomena of sedimentation and flocculation do not occur in one year.
3. The preparation method is simple and easy to implement, the raw materials are added in two steps under stirring, the targeted oxygen-carrying nano acoustic sensitizer with uniform particle size can be prepared, no high-temperature and complex equipment or harsh reaction conditions are needed, and the operation and popularization are convenient.
4. After the metal manganese ions are complexed with the macrocyclic compound with the porphyrin structure, the phototoxicity of the porphyrin sound-sensitive agent is reduced, and the safety is improved.
5. The invention improves the water solubility of the manganoporphyrin sound-sensitive agent, thereby improving the bioavailability of the manganoporphyrin sound-sensitive agent.
6. The human serum albumin has the tumor specific targeting function, so the oxygen-carrying nano sound-sensitive agent has good targeting property.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (13)
1. The targeted oxygen-carrying nano sound-sensitive agent is characterized in that a hybrid protein body is arranged outside the targeted oxygen-carrying nano sound-sensitive agent, manganoporphyrin serving as the sound-sensitive agent is wrapped inside the targeted oxygen-carrying nano sound-sensitive agent, and the hybrid protein body consists of human serum albumin and oxygen-carrying hemoglobin.
2. The oxygen-carrying nanoscopic acoustic agent of claim 1, wherein the manganoporphyrin is formed by complexation of metal manganese ions with a macrocyclic compound of porphyrin structure.
3. A preparation method of a targeted oxygen-carrying nano sound-sensitive agent is characterized by comprising the following steps:
(1) preparation of manganese porphyrin: mixing the macrocyclic compound with porphyrin structure and metal manganese salt, filtering, precipitating, drying and separating;
(2) preparing the targeted oxygen-carrying nano acoustic sensitizer by using human serum albumin, oxygen-carrying hemoglobin and the manganoporphyrin obtained in the step (1).
4. The method according to claim 3, wherein the macrocyclic compound having a porphyrin structure and the metal manganese salt in step (1) are present in an equimolar ratio.
5. The process according to any one of claims 3 to 4, wherein the macrocyclic compound having a porphyrin structure is 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin.
6. The method according to claim 3, wherein the metal manganese salt in the step (1) comprises at least one of manganese chloride, manganese sulfate or manganese acetate.
7. The method according to claim 3, wherein the step (1) comprises a step of performing reaction protection using an inert gas.
8. The production method according to claim 3, wherein the separation step in the step (1) uses column chromatography.
9. The method according to claim 3, wherein the ratio of the human serum albumin to the oxygen-carrying hemoglobin in step (2) is 1-5: 1.
10. the method according to claim 3, wherein the step (2) comprises a step of sonicating the mixed solution of human serum albumin, the oxygen-carrying hemoglobin, and the manganoporphyrin using a cell disruptor.
11. The method of claim 10, wherein the sonication time is 5-20 min.
12. The method according to claim 10, wherein the pH of the mixed solution is 7 to 8.
13. Use of the oxygen-carrying nano-sized sonosensitizer according to any one of claims 1-2 in the preparation of an anti-tumor medicament.
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