CN109620972B - T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis and preparation method thereof - Google Patents
T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a T1-T2 bimodal targeting imaging contrast agent for lung cancer diagnosis and a preparation method thereof, firstly, in the self-assembly process of a nano carrier, hydrophobic magnetic nano particles are wrapped in a hydrophobic inner core of a polymer carrier, the hydrophobic nano particles comprise superparamagnetic nano particles and manganese dioxide nano particles, then, targeting peptide cNGQGEQc is coupled on a maleimide group at the tail end of a hydrophilic chain, the targeting peptide is positioned on an outer shell layer of the micelle after the micelle is formed, and the T1-T2 bimodal targeting contrast agent SPIO-PEG-MnO is synthesized2-cNGQGEQc. The imaging contrast agent has the effects of lung cancer cell active target recognition and development combined with T1 weighted images and T2 weighted images.
Description
Technical Field
The invention belongs to the fields of high molecular materials and medical engineering, and particularly relates to a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis and a preparation method thereof.
Background
The incidence and mortality of lung cancer are the first of malignant tumors in the world, and nearly 60 million people die of lung cancer in China every year. In industrially developed countries, the number of patients who die from lung cancer accounts for 29% of the total number of deaths from various cancers; lung cancer survival rate is only 13% in 5 years, with 80% of patients dying within 1 year after diagnosis; the early diagnosis rate of lung cancer is only 15%, but the 5-year survival rate of the patients can reach 60-90%.
China is a cigarette selling nation, the incidence rate of lung cancer is on the rise year by year, the incidence rate of lung cancer is increased by 11.9 percent year by year in 17 years from 1973 to 1990, and the lung cancer is the most important of various cancers. As can be seen, the control of lung cancer is an urgent and difficult task in clinical medicine, and the key to improving the prognosis of lung cancer lies in early detection and early diagnosis.
With the development of physical examination means, people's health problems can be found more quickly in medicine, but the early lung cancer diseases are very weak, so that the examination is not accurate and rapid enough, and the patients suffering from the early lung cancer miss the optimal treatment time, so that a means for accurately examining the early lung cancer cells is urgently needed. Therefore, there is a need to develop accurate targeted detection methods.
Magnetic Resonance Imaging (MRI) is a popular and mature imaging technique currently used in clinic, and a noninvasive molecular imaging technique is used for identifying and tracing a living body under the enhancement effect of a contrast agent, so that the possibility of effectively detecting early cancer cells is provided. Generally, contrast agents for MRI are divided into two categories: one is a paramagnetic compound such as gadolinium and manganese, and the contrast agent can cause the relaxation change of surrounding protons and mainly change the relaxation time of T1, and is applied to T1 weighted imaging to cause the brightness of T1 weighted imaging; another contrast agent is a superparamagnetic substance, mostly iron oxide based nanoparticles, applied to T2-weighted imaging, causing T2-weighted imaging to darken. Both contrast agents have their own advantages and disadvantages, and thus combining the two will have the potential to improve MRI detection efficiency.
GUO et al (Advances in biochemistry and Biophysics, 2007, 34(10): 1080-.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis and a preparation method thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis comprises the following steps:
s1) dissolving superparamagnetic nano-particles or manganese dioxide nano-particles into a dissolving solution to obtain a first nano-particle solution; dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide in the dissolving solution to obtain a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution; adding the first nanoparticle solution into a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution, and uniformly mixing to obtain a second nanoparticle solution; adding the second nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain first freeze-dried powder;
s2) corresponding to the step S1, dissolving manganese dioxide nanoparticles or superparamagnetic nanoparticles in a dissolving solution to obtain a third nanoparticle solution; dissolving the first freeze-dried powder in the dissolving solution to obtain a first freeze-dried powder solution; mixing the third nano-particle solution with the first freeze-dried powder solution to obtain a fourth nano-particle solution; adding the fourth nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain second freeze-dried powder;
s3) dissolving the targeting peptide shown as SEQ ID NO.1 in water to obtain a targeting peptide aqueous solution; dissolving the second freeze-dried powder in water to obtain a second freeze-dried powder aqueous solution; adding the targeting peptide aqueous solution into a second freeze-dried powder aqueous solution, and stirring under the protection of nitrogen; after the reaction is finished, sequentially dialyzing and freeze-drying to obtain the T1-T2 bimodal targeted imaging contrast agent; the T1-T2 bimodal targeted imaging contrast agent is of a core-shell structure, an inner core layer is formed by a hydrophobic distearoyl phosphatidyl ethanolamine segment, an outer shell layer is formed by a hydrophilic polyethylene glycol-maleimide segment, hydrophobic superparamagnetic nanoparticles and manganese dioxide nanoparticles are wrapped in the inner core layer, and the targeted peptide is coupled with the maleimide segment through a sulfydryl of cysteine;
the dissolving solution is chloroform, dimethylformamide or dimethyl sulfoxide.
As an improvement of the above technical solution, in step S1, dissolving superparamagnetic nanoparticles in a dissolving solution to obtain a first nanoparticle solution; in step S2, the manganese dioxide nanoparticles are dissolved in the solution to obtain a third nanoparticle solution.
As a further improvement of the above technical solution, the superparamagnetic nanoparticles are prepared by the following method: the preparation method is characterized in that ferric triacetylacetonate and manganese acetylacetonate are used as raw materials, 1, 2-dodecahexaalkane and dibenzyl ether are used as solvents, oleic acid and oleylamine are used as surfactants, and the preparation method is carried out by adopting a high-temperature thermal decomposition method.
As a further improvement of the above technical solution, the superparamagnetic nanoparticles are prepared by: putting ferric triacetylacetonate, manganese acetylacetonate and 1, 2-hexadecanediol into a reaction vessel for mixing, then respectively adding oleic acid, oleylamine and dibenzyl ether, magnetically stirring under the protection of argon, heating to 200 ℃, preserving heat for 1h, and then heating to 300 ℃ for reflux reaction for 1 h; cooling to room temperature, adding ethanol, and centrifuging to remove supernatant; adding n-hexane into the brownish black precipitate for dissolving, adding ethanol after dissolving, and centrifuging to remove supernatant; after multiple times of washing, dissolving the brownish black precipitate by n-hexane, and drying to obtain the superparamagnetic nano-particles; the mass ratio of ferric triacetylacetonate to manganese acetylacetonate was 2.
As a further improvement of the above technical solution, the manganese dioxide nanoparticles are prepared by the following method: preparing a potassium permanganate aqueous solution with the concentration of 0.1-5 mg/mL and a polyallyl ammonium chloride aqueous solution with the concentration of 10-100 mg/mL; mixing the potassium permanganate aqueous solution and the polyallyl ammonium chloride aqueous solution, stirring at normal temperature, filtering, and freeze-drying to obtain manganese dioxide nanoparticles; the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 1-9.
As a further improvement of the above technical solution, in step S1, the concentration of the superparamagnetic nanoparticles in the first nanoparticle solution is 0.01-10 mg/mL, and the concentration of the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution is 0.1-100
mg/mL, wherein the mass ratio of the superparamagnetic nanoparticle to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 0.1-2; the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide is polyethylene glycol 2000, and the molecular weight of the dialysis bag during dialysis is 3500.
As a further improvement of the above technical solution, in step S2, the concentration of the manganese dioxide nanoparticles in the third nanoparticle solution is 0.01-10 mg/mL, and the concentration of the first lyophilized powder in the first lyophilized powder solution is 0.1-100 mg/mL; the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide is polyethylene glycol 2000, and the molecular weight of the dialysis bag during dialysis is 1000.
As a further improvement of the above technical scheme, in step S3, the concentration of the targeting peptide in the targeting peptide aqueous solution is 0.1-5 mg/mL, and the concentration of the second lyophilized powder in the second lyophilized powder aqueous solution is 0.1-10 mg/mL; the molecular weight of the dialysis bag during dialysis was 2000.
In addition, the invention also provides a T1-T2 bimodal targeted imaging contrast agent for early diagnosis of lung cancer, which is prepared by the preparation method.
As an improvement of the technical scheme, the T1-T2 bimodal targeted imaging contrast agent is dissolved in PBS (phosphate buffer solution) with the concentration of 0.01mol/L and the pH value of 7.4, and the concentration of the T1-T2 bimodal targeted imaging contrast agent is 0.1-10 mg/mL.
Preferably, the concentration of the T1-T2 bimodal targeted imaging contrast agent is 1 mg/mL.
The invention has the beneficial effects that: the invention provides a T1-T2 bimodal targeting imaging contrast agent for lung cancer diagnosis and a preparation method thereof, the invention provides a method for preparing the bimodal targeting imaging contrast agent for lung cancer diagnosis, in the self-assembly process of a nano carrier, hydrophobic magnetic nano particles are wrapped in a hydrophobic inner core of a polymer carrier, the hydrophobic nano particles comprise superparamagnetic bead nano particles and manganese dioxide nano particles, then targeting peptide cNGQGEQc is coupled on a maleimide group at the tail end of a hydrophilic chain, the targeting peptide is positioned on an outer shell layer of a micelle after the micelle is formed, and the T1-T2 bimodal targeting contrast agent SPIO-PEG-MnO of the invention is synthesized2cNGQGEQc, which makes lung cancer cells actively target and identify and develop T1-weighted image and T2-weighted image.
Drawings
Fig. 1 is a TEM image of superparamagnetic nanoparticles of example 1;
FIG. 2 is a TEM image of manganese dioxide nanoparticles of examples 2-4;
FIG. 3 is a TEM image of SPIO-PEG nanoparticles in example 5;
FIG. 4 shows SPIO-PEG-MnO in example 52TEM magnification of nanoparticles;
FIG. 5 shows an MRI image of a rat after injection of an imaging contrast agent; wherein, the non-targeting nano material represents the injection imaging contrast agent B, and the targeting nano material represents the injection imaging contrast agent A.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.
It should be understood that the terms "first", "second", "third", and "fourth", etc. are used herein to describe various information, but the information should not be limited to these terms, and these terms are only used to distinguish one type of information from another. For example, "first" information may also be referred to as "second" information, and similarly, "second" information may also be referred to as "first" information, without departing from the scope of the present invention.
The invention provides a preparation method of a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis, which comprises the following steps:
s1) dissolving superparamagnetic nano-particles or manganese dioxide nano-particles into a dissolving solution to obtain a first nano-particle solution; dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide in the dissolving solution to obtain a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution; adding the first nanoparticle solution into a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution, and uniformly mixing to obtain a second nanoparticle solution; adding the second nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain first freeze-dried powder;
s2) corresponding to the step S1, dissolving manganese dioxide nanoparticles or superparamagnetic nanoparticles in a dissolving solution to obtain a third nanoparticle solution; dissolving the first freeze-dried powder in the dissolving solution to obtain a first freeze-dried powder solution; mixing the third nano-particle solution with the first freeze-dried powder solution to obtain a fourth nano-particle solution; adding the fourth nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain second freeze-dried powder;
s3) dissolving the targeting peptide shown as SEQ ID NO.1 in water to obtain a targeting peptide aqueous solution; dissolving the second freeze-dried powder in water to obtain a second freeze-dried powder aqueous solution; adding the targeting peptide aqueous solution into a second freeze-dried powder aqueous solution, and stirring under the protection of nitrogen; after the reaction is finished, sequentially dialyzing and freeze-drying to obtain the T1-T2 bimodal targeted imaging contrast agent; the T1-T2 bimodal targeted imaging contrast agent is of a core-shell structure, an inner core layer is formed by a hydrophobic distearoyl phosphatidyl ethanolamine segment, an outer shell layer is formed by a hydrophilic polyethylene glycol-maleimide segment, hydrophobic superparamagnetic nanoparticles and manganese dioxide nanoparticles are wrapped in the inner core layer, and the targeted peptide is coupled with the maleimide segment through a sulfydryl of cysteine;
the dissolving solution is chloroform, dimethylformamide or dimethyl sulfoxide.
Preferably, in step S1, dissolving the superparamagnetic nanoparticles in the dissolving solution to obtain a first nanoparticle solution; in step S2, the manganese dioxide nanoparticles are dissolved in the solution to obtain a third nanoparticle solution.
Further, the superparamagnetic nanoparticle is prepared by: the preparation method is characterized in that ferric triacetylacetonate and manganese acetylacetonate are used as raw materials, 1, 2-dodecahexaalkane and dibenzyl ether are used as solvents, oleic acid and oleylamine are used as surfactants, and the preparation method is carried out by adopting a high-temperature thermal decomposition method.
More preferably, the superparamagnetic nanoparticle is prepared by: putting ferric triacetylacetonate, manganese acetylacetonate and 1, 2-hexadecanediol into a reaction vessel for mixing, then respectively adding oleic acid, oleylamine and dibenzyl ether, magnetically stirring under the protection of argon, heating to 200 ℃, preserving heat for 1h, and then heating to 300 ℃ for reflux reaction for 1 h; cooling to room temperature, adding ethanol, and centrifuging to remove supernatant; adding n-hexane into the brownish black precipitate for dissolving, adding ethanol after dissolving, centrifuging, and removing supernatant; after multiple times of washing, dissolving the brownish black precipitate by n-hexane, and drying to obtain the superparamagnetic nano-particles; the mass ratio of ferric triacetylacetonate to manganese acetylacetonate was 2.
Further preferably, the manganese dioxide nanoparticles are prepared by the following method: preparing a potassium permanganate aqueous solution with the concentration of 0.1-5 mg/mL and a polyallyl ammonium chloride aqueous solution with the concentration of 10-100 mg/mL; mixing the potassium permanganate aqueous solution and the polyallyl ammonium chloride aqueous solution, stirring at normal temperature, filtering, and freeze-drying to obtain manganese dioxide nanoparticles; the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 1-9.
Further preferably, in step S1, the concentration of the superparamagnetic nanoparticle in the first nanoparticle solution is 0.01-10 mg/mL, the concentration of the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution is 0.1-100 mg/mL, and the mass ratio of the superparamagnetic nanoparticle to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 0.1-2; the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide is polyethylene glycol 2000, and the molecular weight of the dialysis bag during dialysis is 3500.
Further preferably, in step S2, the concentration of the manganese dioxide nanoparticles in the third nanoparticle solution is 0.01-10 mg/mL, and the concentration of the first lyophilized powder in the first lyophilized powder solution is 0.01-100 mg/mL; the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide is polyethylene glycol 2000, and the molecular weight of the dialysis bag during dialysis is 1000.
Further preferably, in step S3, the concentration of the targeting peptide in the targeting peptide aqueous solution is 0.1-5 mg/mL, and the concentration of the second lyophilized powder in the second lyophilized powder aqueous solution is 0.1-10 mg/mL; the molecular weight of the dialysis bag during dialysis was 2000.
Example 1
The embodiment provides a preparation method of superparamagnetic nanoparticles, which includes the following steps: mixing Fe (acac)3And Mn (acac)2And 1, 2-hexadecyl diol are put into a reaction vessel to be mixed, then oleic acid, oleylamine and dibenzyl ether are respectively added, the mixture is heated to 200 ℃ under the protection of nitrogen and is kept warm for 1 hour, then the mixture is heated to 300 ℃ to carry out reflux reaction for 1 hour; after the reaction is stopped, cooling the product solution to room temperature, sucking the solution and dropping the solution into ethanol for washing, centrifuging to remove the supernatant, dissolving brownish black solid by using a small amount of n-hexane, washing by using ethanol, centrifuging again to remove the supernatant, and repeatedly washing for 2 to 3 times; dissolving with n-hexane, transferring to a culture dish, drying to obtain superparamagnetic nanoparticles, and storing at low temperature; fe (acac)3And Mn (acac)2The amount ratio of the substances of (1) is 2.
The particle size distribution of the superparamagnetic nanoparticles prepared in example 1 was measured using a dynamic laser light scattering apparatus, and the results were: the polydispersity was 0.231, and TEM images showed that the average particle size of the nanoparticles was 10.1nm, the particles were spherical, and the dispersion was relatively uniform (as shown in figure 1).
Example 2
This example provides a method for preparing manganese dioxide nanoparticles, which includes the following steps: weighing a certain amount of potassium permanganate and dissolving in distilled water, wherein the concentration of the potassium permanganate aqueous solution is 0.1 mg/mL; weighing a certain amount of polyallyl ammonium chloride (PAH) and dissolving in distilled water to prepare a polyallyl ammonium chloride aqueous solution, wherein the concentration of the polyallyl ammonium chloride aqueous solution is 10 mg/mL; mixing the potassium permanganate aqueous solution with the polyallyl ammonium chloride aqueous solution, stirring at normal temperature for 30min, filtering the obtained mixed solution with a 0.22 mu m filter membrane, and freeze-drying to obtain manganese dioxide nanoparticles; the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 1.
Example 3
This example provides a method of preparing manganese dioxide nanoparticles, similar to example 2, with the following differences: the mass concentration of the potassium permanganate aqueous solution is 3mg/mL, the mass concentration of the polyallyl ammonium chloride aqueous solution is 37.4mg/mL, and the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 9.
Example 4
This example provides a method of preparing manganese dioxide nanoparticles, similar to example 2, with the following differences: the mass concentration of the potassium permanganate aqueous solution is 5mg/mL, the mass concentration of the polyallyl ammonium chloride aqueous solution is 100mg/mL, and the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 4.
The particle size distribution of the manganese dioxide nanoparticles prepared in examples 2 to 4 was measured by a dynamic laser light scattering apparatus, and the results were as follows: the polydispersity was 0.258, and TEM images showed that the average particle size of the nanoparticles was 23nm, the particles were spherical, and the dispersion was relatively uniform (as shown in figure 2).
Example 5
The embodiment provides a preparation method of a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis, which comprises the following steps:
s1) dissolving superparamagnetic nanoparticles (modified by oleic acid and oleylamine on the surface of SPIO) in chloroform to obtain a first nanoparticle solution, wherein the concentration of the superparamagnetic nanoparticles in the first nanoparticle solution is 0.01 mg/mL; dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide in chloroform to obtain a distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide solution, wherein the concentration of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide in the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide solution is 0.1mg/mL, and the mass ratio of the superparamagnetic nanoparticles to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 0.1; adding the first nano-particle solution into a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution, performing ultrasonic treatment in the adding process to uniformly mix and disperse, and uniformly mixing to obtain a second nano-particle solution; adding the second nanoparticle solution into water, and carrying out ultrasonic probe treatment for 30min in the adding process; after the ultrasound is finished, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 3500, dialyzing with pure water at normal temperature for 24h, changing water for 5 times, after the dialysis is finished, forming micelles in the mixed solution, collecting the solution in the dialysis bag, and freeze-drying with a freeze dryer to obtain first freeze-dried powder (SPIO-distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, SPIO-PEG);
s2) dissolving manganese dioxide nanoparticles in chloroform to obtain a third nanoparticle solution, wherein the concentration of the manganese dioxide nanoparticles in the third nanoparticle solution is 0.01 mg/mL; dissolving the first freeze-dried powder in chloroform to obtain a first freeze-dried powder solution, wherein the concentration of SPIO-PEG in the first freeze-dried powder solution is 0.1 mg/mL; mixing the third nano-particle solution with the first freeze-dried powder solution to obtain a fourth nano-particle solution; adding the fourth nano-particle solution into water, and carrying out ultrasonic probe treatment for 30min in the adding process; transferring the mixed solution to dialysis bag with cut-off molecular weight of 1000, dialyzing with pure water for 24 hr, changing water for 5 times, freeze drying the solution after dialysis to obtain second lyophilized powder (SPIO-distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide-manganese dioxide, SPIO-PEG-MnO)2)；
S3) dissolving the targeting peptide shown as SEQ ID NO.1 in water to obtain a targeting peptide aqueous solution, wherein the concentration of the targeting peptide in the targeting peptide aqueous solution is 0.1 mg/mL; mixing the second lyophilized powder (SPIO-PEG-MnO)2) Dissolving in water to obtain a second lyophilized powder water solution, wherein the concentration of the second lyophilized powder in the second lyophilized powder water solution is 0.1 mg/mL; adding the targeting peptide aqueous solution into the second freeze-dried powder aqueous solution, and stirring under the protection of nitrogen; after 4h of reaction, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 2000, dialyzing for 24h with ice bath and pure water, changing water for 5 times, collecting the solution in the dialysis bag, and freeze-drying to obtain the T1-T2 bimodal targeted imaging contrast agent.
The first lyophilized powder (SPIO-PEG) prepared in example 5 was measured for particle size distribution using a dynamic laser light scattering apparatus, and the results were: the polydispersity was 0.206, and TEM images showed that the average particle size of the nanoparticles was 160nm, the particles were spherical, and the dispersion was relatively uniform (as shown in figure 3).
For the second lyophilized powder (SPIO-PEG-MnO) prepared in example 52) The particle size distribution of the particles was measured using a dynamic laser light scattering apparatus, and the results were: the polydispersity was 0.261, and the TEM photograph showed that the average particle size of the nanoparticles was 187nm, the particles were spherical, and the dispersion was relatively uniform (as shown in fig. 4).
Example 6
This example provides a method for preparing a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis, which is similar to example 5 with the difference that:
1) in step S1, the concentration of superparamagnetic nanoparticles in the first nanoparticle solution is 6 mg/mL; the concentration of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide in the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide solution is 18mg/mL, and the mass ratio of the superparamagnetic nanoparticle to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 1;
2) in step S2, the concentration of manganese dioxide nanoparticles in the third nanoparticle solution is 5mg/mL, and the concentration of SPIO-PEG in the first lyophilized powder solution is 10 mg/mL;
3) in step S3, the concentration of the targeting peptide in the aqueous solution of the targeting peptide is 1.6mg/mL, and the second lyophilized powder (SPIO-PEG-MnO) in the aqueous solution of the second lyophilized powder2) The concentration of (2) is 1 mg/mL.
Example 7
This example provides a method for preparing a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis, which is similar to example 5 with the difference that:
1) in step S1, the concentration of superparamagnetic nanoparticles in the first nanoparticle solution is 10 mg/mL; the concentration of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide in the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide solution is 100mg/mL, and the mass ratio of the superparamagnetic nanoparticle to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide is 2;
2) in step S2, the concentration of manganese dioxide nanoparticles in the third nanoparticle solution is 10mg/mL, and the concentration of SPIO-PEG in the first lyophilized powder solution is 100 mg/mL;
3) in the step ofS3, the concentration of the target peptide in the target peptide aqueous solution is 5mg/mL, and the second freeze-dried powder (SPIO-PEG-MnO) in the second freeze-dried powder aqueous solution2) The concentration of (2) is 10 mg/mL.
Performance assays for targeted imaging agents
Imaging contrast agent a: taking SPIO-PEG-MnO of the invention2-cNGQGEQc dissolved in 0.01M PBS at pH 7.4 to give a dispersion with a concentration of 0.1-10 mg/mL;
imaging contrast agent B: taking SPIO-PEG-MnO2(not linked to the targeting peptide) and dissolved in 0.01M PBS (pH 7.4) to give a dispersion having a concentration of 0.1 to 10 mg/mL.
1) Taking a rat which is cultured with a three-month lung cancer model, injecting a contrast agent into the rat through a tail vein, wherein the injection dosage is that 1mL of the contrast agent A is injected into each kilogram of the rat, and carrying out T1 and T2 weighted imaging on a rat tumor part by using a small animal imaging means; the obtained magnetic resonance imaging result shows that relaxation time of T1 and T2 of a detected tumor part is obviously changed after 3 hours of injection, and T1 relaxation and T2 relaxation are shortened; therefore, the nanoparticle composite can be used for preparing imaging contrast agents and can be used for T1 and T2 bimodal imaging of tumor sites.
2) 2Targeting of SPIO-PEG-MnO-cNGQGEQc
Taking 6 rats which are cultured with lung cancer models for three months, dividing the rats into two groups, and dividing each group into 3 rats; a first group: injecting imaging contrast agent A through tail vein, injecting 1mL imaging contrast agent A for each kilogram of rat weight, and carrying out T1 and T2 weighted imaging on the whole body of the rat by using small animal imaging means; second group: imaging contrast B was injected via the tail vein at a dose of 1mL per kg of rat weight, and the whole body of the rat was subjected to T1, T2 weighted imaging using small animal imaging means. The obtained magnetic resonance imaging result shows that T1 and T2 relaxation time of the whole body of a rat is obviously changed, T1 relaxation is shortened, T2 relaxation is shortened after the first group of contrast agent A is injected for 24 hours, wherein T1 and T2 weighted imaging at a tumor part has more obvious imaging effect compared with other parts; and a second group of 24-hour posttests with contrast agent BThe relaxation time of T1 and T2 of the whole body of a rat is obviously changed, the relaxation of T1 is shortened, the relaxation of T2 is shortened, but the imaging effect of T1 and T2 weighted imaging at the tumor site inoculated in advance is not greatly different from that of other sites. At 0, the imaging effect of the first group injected with the contrast agent A and the imaging effect of the second group injected with the contrast agent B, namely T1 and T2, are poor, after 24 hours, the imaging effect of T1 and T2 in the targeted group injected with the contrast agent A is obviously improved, and the imaging effect of T1 and T2 in the non-targeted group injected with the contrast agent B is not obviously enhanced, which is caused by the fact that the targeted contrast agent can be specifically aggregated in a lesion area. As can be seen, the SPIO-PEG-MnO2The contrast agent prepared by cNGQGEQc has certain targeting property (as shown in figure 5).
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Guangzhou Chuangsai biomedical materials Co., Ltd
<120> T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis and preparation method thereof
<130> 2019.01.04
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213> artificially synthesized sequence
<400> 1
Cys Asn Gly Gln Gly Glu Gln Cys
1 5
Claims (7)
1. A preparation method of a T1-T2 bimodal targeted imaging contrast agent for lung cancer diagnosis is characterized by comprising the following steps:
s1) dissolving the superparamagnetic nano-particles into a dissolving solution to obtain a first nano-particle solution; dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide in the dissolving solution to obtain a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution; adding the first nanoparticle solution into a distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide solution, and uniformly mixing to obtain a second nanoparticle solution; adding the second nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain first freeze-dried powder;
s2) corresponding to the step S1, dissolving manganese dioxide nanoparticles in the solution to obtain a third nanoparticle solution; dissolving the first freeze-dried powder in the dissolving solution to obtain a first freeze-dried powder solution; mixing the third nano-particle solution with the first freeze-dried powder solution to obtain a fourth nano-particle solution; adding the fourth nanoparticle solution into water, and performing ultrasonic treatment in the adding process; after the ultrasonic treatment is finished, sequentially carrying out dialysis and freeze drying to obtain second freeze-dried powder;
s3) dissolving the targeting peptide shown as SEQ ID NO.1 in water to obtain a targeting peptide aqueous solution; dissolving the second freeze-dried powder in water to obtain a second freeze-dried powder aqueous solution; adding the targeting peptide aqueous solution into a second freeze-dried powder aqueous solution, and stirring under the protection of nitrogen; after the reaction is finished, sequentially dialyzing and freeze-drying to obtain the T1-T2 bimodal targeted imaging contrast agent; the T1-T2 bimodal targeted imaging contrast agent is of a core-shell structure, an inner core layer is formed by a hydrophobic distearoyl phosphatidyl ethanolamine segment, an outer shell layer is formed by a hydrophilic polyethylene glycol-maleimide segment, hydrophobic superparamagnetic nanoparticles and manganese dioxide nanoparticles are wrapped in the inner core layer, and the targeted peptide is coupled with the maleimide segment through a sulfydryl of cysteine;
the dissolving solution is chloroform, dimethylformamide or dimethyl sulfoxide;
the manganese dioxide nanoparticles are prepared by the following method: preparing a potassium permanganate aqueous solution with the concentration of 0.1-5 mg/mL and a polyallyl ammonium chloride aqueous solution with the concentration of 10-100 mg/mL; mixing the potassium permanganate aqueous solution and the polyallyl ammonium chloride aqueous solution, stirring at normal temperature, filtering, and freeze-drying to obtain manganese dioxide nanoparticles; the volume ratio of the potassium permanganate aqueous solution to the polyallyl ammonium chloride aqueous solution is 1-9.
2. The method of claim 1, wherein the superparamagnetic nanoparticles are prepared by: the preparation method is characterized in that ferric triacetylacetonate and manganese acetylacetonate are used as raw materials, 1, 2-dodecahexanediol and dibenzyl ether are used as solvents, oleic acid and oleylamine are used as surfactants, and the preparation method is carried out by adopting a high-temperature thermal decomposition method.
3. The method of claim 2, wherein the superparamagnetic nanoparticles are prepared by: putting ferric triacetylacetonate, manganese acetylacetonate and 1, 2-hexadecanediol into a reaction vessel for mixing, then respectively adding oleic acid, oleylamine and dibenzyl ether, magnetically stirring under the protection of argon, heating to 200 ℃, preserving heat for 1h, and then heating to 300 ℃ for reflux reaction for 1 h; cooling to room temperature, adding ethanol, and centrifuging to remove supernatant; adding n-hexane into the brownish black precipitate for dissolving, adding ethanol after dissolving, centrifuging, and removing supernatant; after multiple times of washing, dissolving the brownish black precipitate by n-hexane, and drying to obtain the superparamagnetic nano-particles; the mass ratio of ferric triacetylacetonate to manganese acetylacetonate was 2.
4. The method of claim 1, wherein in step S1, the concentration of superparamagnetic nanoparticles in the first nanoparticle solution is 0.01 to 10mg/mL, the concentration of distearoylphosphatidylethanolamine-polyethylene glycol-maleimide in the distearoylphosphatidylethanolamine-polyethylene glycol-maleimide solution is 0.1 to 100mg/mL, and the mass ratio of the superparamagnetic nanoparticles to the distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide is 0.1 to 2; the polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide is polyethylene glycol 2000, and the molecular weight of the dialysis bag during dialysis is 3500.
5. The method of claim 1, wherein in step S3, the concentration of the targeting peptide in the targeting peptide aqueous solution is 0.1-5 mg/mL, and the concentration of the second lyophilized powder in the second lyophilized powder aqueous solution is 0.1-10 mg/mL; the molecular weight of the dialysis bag during dialysis was 2000.
6. A T1-T2 bimodal targeted imaging contrast agent for early diagnosis of lung cancer, prepared by the preparation method of any one of claims 1 to 5.
7. The T1-T2 bimodal targeted imaging contrast agent of claim 6, wherein the T1-T2 bimodal targeted imaging contrast agent is dissolved in PBS with 0.01mol/L and pH of 7.4, and the concentration of the T1-T2 bimodal targeted imaging contrast agent is 0.1-10 mg/mL.
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