Double-targeting bimodal developing nanoparticles and preparation method thereof
阅读说明：本技术 一种双靶向双模态显影纳米粒及其制备方法 (Double-targeting bimodal developing nanoparticles and preparation method thereof ) 是由 高彬洋 于 2020-06-16 设计创作，主要内容包括：本发明涉及一种双靶向双模态显影纳米粒及其制备方法。具体提供了一种靶向双模态显影纳米粒,它是以聚乳酸-羟基乙酸共聚物、磁共振造影剂、超声造影剂和靶向多肽为原料制得的,所述靶向多肽选自cRGD和PP1中的一种或两种。该靶向双模态显影纳米粒形态规则、大小均匀、分散性好,具有超声、磁共振双模态显影功能,能靶向至易损斑块内的巨噬细胞,并在超声介导下诱发巨噬细胞凋亡,还能在超声介导下溶解易损斑块表面血栓,在制备诊断易损斑块(特别是易破裂斑块和蚀损斑块)的显像试剂,稳定易损斑块的药物,预防和/或治疗与易损斑块相关的心血管疾病的药物中具有广阔的应用前景。(The invention relates to a double-targeting bimodal developing nanoparticle and a preparation method thereof. The targeted bimodal developing nanoparticle is prepared from a polylactic acid-glycolic acid copolymer, a magnetic resonance contrast agent, an ultrasonic contrast agent and targeted polypeptide, wherein the targeted polypeptide is selected from one or two of cRGD and PP 1. The targeted bimodal developing nanoparticle has regular shape, uniform size and good dispersibility, has ultrasonic and magnetic resonance bimodal developing functions, can target macrophages in vulnerable plaques, induces apoptosis of the macrophages under ultrasonic mediation, can dissolve thrombus on the surfaces of the vulnerable plaques under ultrasonic mediation, and has wide application prospect in preparing developing reagents for diagnosing the vulnerable plaques (particularly vulnerable plaques and eroded plaques), stabilizing the vulnerable plaques and preventing and/or treating cardiovascular diseases related to the vulnerable plaques.)
1. A targeted bimodal developing nanoparticle is characterized in that: the magnetic resonance imaging contrast agent is prepared by taking polylactic acid-glycolic acid copolymer, a magnetic resonance contrast agent, an ultrasonic contrast agent and targeting polypeptide as raw materials, wherein the targeting polypeptide is selected from one or two of cRGD and PP 1.
2. The targeted bimodal imaging nanoparticle of claim 1, wherein: the magnetic resonance contrast agent is a Fe-based magnetic resonance contrast agent, and is preferably ferroferric oxide.
3. The targeted bimodal imaging nanoparticle of claim 1, wherein: the ultrasonic contrast agent is selected from perfluoropentane, perfluorohexane, perfluoroheptane or perfluorooctabromoalkane, and is preferably perfluoropentane.
4. The targeted bimodal imaging nanoparticle of claim 1, wherein: the molecular weight of the polylactic acid-glycolic acid copolymer is 6000 Da-10000 Da, preferably 8000 Da.
5. The targeted bimodal imaging nanoparticle of claim 1, wherein: the targeted polypeptide is cRGD and PP1, wherein the mass ratio of the cRGD to the PP1 is 1: (0.8 to 1.2), preferably 1: 1.
6. the targeted bimodal developing nanoparticle according to any one of claims 1 to 5, wherein: the mass-volume ratio of the polylactic acid-glycolic acid copolymer to the magnetic resonance contrast agent to the ultrasonic contrast agent to the targeting polypeptide is 50 mg: (0.8-1.2) mg: (180-220) μ L: (2-6) mg, preferably 50 mg: 1 mg: 200 μ L: 4 mg.
7. The preparation method of the targeted bimodal developing nanoparticle as claimed in any one of claims 1 to 6, wherein the preparation method comprises the following steps: the method comprises the following steps:
(1) mixing polylactic acid-glycolic acid copolymer, magnetic resonance contrast agent and ultrasonic contrast agent in organic solvent, and performing ultrasonic oscillation;
(2) adding an emulsifier into the system obtained in the step (1), and performing ultrasonic oscillation to obtain non-targeted nanoparticles;
(3) and (3) reacting the non-targeted nanoparticles obtained in the step (2) with targeted polypeptide in a buffer solution with the pH value of 7-9 to obtain the target polypeptide.
8. The method of claim 7, further comprising: in the step (1), the organic solvent is dichloromethane; the mass-volume ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 20-30 mg/mL, preferably 25 mg/mL; the power of the ultrasonic oscillation is 50-70W, preferably 60W, the time is 3-5 min, preferably 3min, and the pulse type is 5 seconds on/5 seconds off; the temperature of the ultrasonic oscillation is 0-4 ℃;
and/or, in the step (2), the emulsifier is polyvinyl alcohol, preferably a 4% polyvinyl alcohol aqueous solution in mass fraction; the ultrasonic oscillation power is 50-70W, preferably 60W, the time is 3-5 min, preferably 5min, and the pulse type is 5 seconds on/5 seconds off; the temperature of the ultrasonic oscillation is 0-4 ℃;
and/or in the step (2), after the ultrasonic oscillation, adding an alcohol solvent into the system, stirring, volatilizing the organic solvent, centrifuging, and removing the supernatant; wherein, the alcohol solvent is preferably isopropanol, and is more preferably isopropanol water solution with volume fraction of 2%; the centrifugation condition is preferably 1000rpm centrifugation for 10 min;
and/or, in the step (3), before the reaction, mixing the non-targeted nanoparticles with an amidation reaction activator, incubating in a shaking table, centrifuging, and removing the supernatant; preferably, the amidation reaction activators are EDC and NHS, and the shaker incubation conditions are as follows: incubating in MES buffer solution with pH 5 at 0-4 deg.C for 1 hr, and centrifuging at 1000rpm for 10 min;
and/or in the step (3), the buffer solution with the pH value of 7-9 is MES buffer solution with the pH value of 8, the reaction temperature is 0-4 ℃, and the reaction time is overnight.
9. Use of the targeted bimodal imaging nanoparticle according to any one of claims 1 to 6 in the preparation of an imaging agent for diagnosing vulnerable plaques and/or a medicament for stabilizing vulnerable plaques; preferably, the vulnerable plaque is a vulnerable plaque and/or an erosion plaque; more preferably, the imaging agent and/or drug is used under the following ultrasound irradiation conditions: the power is 3W, the frequency is 3HZ, the time is 3-4 min, and the duty ratio is 30%.
10. Use of the targeted bimodal imaging nanoparticle according to any one of claims 1 to 6 for the preparation of a medicament for the prevention and/or treatment of cardiovascular diseases associated with vulnerable plaques and/or an imaging agent for the diagnosis of cardiovascular diseases associated with vulnerable plaques; preferably, the cardiovascular disease is atherosclerosis; more preferably, the drug is used under the following ultrasound irradiation conditions: the power is 3W, the frequency is 3HZ, the time is 3-4 min, and the duty ratio is 30%.
Technical Field
The invention belongs to the field of targeted drugs, and particularly relates to a dual-targeted bimodal developing nanoparticle and a preparation method thereof.
Background
According to the statistics data of 'Chinese cardiovascular disease report' in 2018, serious cardiovascular diseases become the most main death cause in China, the number of the existing patients is at least 2.9 hundred million, wherein 1300 million stroke and 1100 million ischemic cardiomyopathy (myocardial infarction) exist; the total hospitalization cost per year respectively reaches 601.05 million yuan and 190.85 million yuan, and the average hospitalization cost for the times is 2.6 ten thousand yuan and 0.9 ten thousand yuan. Greatly reduces the life quality and the life span of the patients, and simultaneously brings great economic burden to families and society of the patients due to expensive treatment cost. More than 75% of fatal and disabling cardiovascular and cerebrovascular diseases are atherosclerotic diseases. Atherosclerosis (AS) is a typical pathological feature of intravascular plaque formation, with high and low fatality, a disabling risk, and is closely related to the vulnerability of the plaque (i.e., the risk of rapid plaque progression and rupture or thrombosis leading to serious cardiovascular events). Therefore, screening Vulnerable Plaque (ASVP) and delaying or reversing Plaque vulnerability are key links for improving cardiovascular disease prevention and treatment, namely first-level prevention of cardiovascular disease, in early pathological stage before clinical symptoms appear. How to effectively screen, intervene and improve the stability of plaques, and establish a cardiovascular disease prevention and treatment mode with clinical value, and has great clinical and socioeconomic significance.
Vulnerable plaques refer to those that are unstable and thrombogenic, and are classified into vulnerable plaques (Plaque Rupture), Plaque lesions (Plaque Erosion) and partially calcified nodular lesions depending on the pathology of the culprit Plaque. A large number of patients with acute coronary myocardial infarction have autopsy results, and about 60 percent of plaque rupture, 30 to 35 percent of plaque erosion and 2 to 7 percent of calcified nodules in plaque causing are shown. Wherein, the easy-to-rupture plaque is mainly characterized by a lipid core, a thin fiber cap and inflammatory cell infiltration. Among the large number of infiltrating inflammatory cells, macrophages (macrophages,) Accounts for more than 80 percent and plays an important role in the formation and vulnerability of plaques. Erosion plaque is also called erosion plaque, and Van Der Wal and the like discover the plaque with an unbroken fiber cap for the first time in 1994 and can also be accompanied with thrombosis; and enriching such surface with platelets and fibrin, andlipid core, a mildly inflammatory plaque, is defined as an erosion plaque. The key link of the disease is that the plaque on the surface of the erosion plaque is aggregated to form thrombus, and the thrombus blocks the lumen or falls off to form a distal vessel embolism.
At present, the key of vulnerable plaque prevention and treatment is thrombosis prevention, and for patients with obvious symptoms, medicines (such as antiplatelet therapy, anticoagulation, thrombolysis therapy and the like) and interventional operation therapy can be adopted. However, whether aspirin and other medicines are used for anti-platelet treatment is controversial all the time, and a plurality of large-scale research results show that aspirin can reduce the main cardiovascular disease events by 15 percent, and the curative effect is definite; but the targeting property is lacked, the local blood concentration is lower, and the relative risk of systemic complications such as gastrointestinal hemorrhage, extracranial hemorrhage and the like is increased by 69%; similarly, oral drugs for anti-inflammatory therapy cannot be used for conventional therapy due to the defects of lack of targeting property, slight therapeutic effect, large systemic side effect and the like.
In addition, because of the large differences in the pathological bases of different types of vulnerable plaques, targeted symptomatic treatment is also required at the time of treatment. At present, no report for distinguishing vulnerable plaques by adopting a non-invasive screening means is seen, and no accurate treatment method aiming at different plaque pathological types is available.
Therefore, there is a need to develop a drug that can be used for targeted and accurate diagnosis and treatment of vulnerable plaque.
Disclosure of Invention
The invention aims to provide a medicament capable of carrying out targeted and accurate diagnosis and treatment on vulnerable plaques.
The invention provides a targeted bimodal developing nanoparticle which is prepared from polylactic acid-glycolic acid copolymer, a magnetic resonance contrast agent, an ultrasonic contrast agent and targeted polypeptide, wherein the targeted polypeptide is selected from one or two of cRGD and PP 1.
Further, the magnetic resonance contrast agent is a Fe-based magnetic resonance contrast agent, and is preferably ferroferric oxide.
Further, the ultrasonic contrast agent is selected from perfluoropentane, perfluorohexane, perfluoroheptane or perfluorooctabromoalkane, and is preferably perfluoropentane.
Further, the molecular weight of the polylactic acid-glycolic acid copolymer is 6000 Da-10000 Da, preferably 8000 Da.
Further, the targeting polypeptide is cRGD and PP1, wherein the mass ratio of cRGD to PP1 is 1: (0.8 to 1.2), preferably 1: 1.
further, the mass-to-volume ratio of the polylactic acid-glycolic acid copolymer to the magnetic resonance contrast agent to the ultrasound contrast agent to the targeting polypeptide is 50 mg: (0.8-1.2) mg: (180-220) μ L: (2-6) mg, preferably 50 mg: 1 mg: 200 μ L: 4 mg.
The invention also provides a preparation method of the targeted bimodal developing nanoparticle, which comprises the following steps:
(1) mixing polylactic acid-glycolic acid copolymer, magnetic resonance contrast agent and ultrasonic contrast agent in organic solvent, and performing ultrasonic oscillation;
(2) adding an emulsifier into the system obtained in the step (1), and performing ultrasonic oscillation to obtain non-targeted nanoparticles;
(3) and (3) reacting the non-targeted nanoparticles obtained in the step (2) with targeted polypeptide in a buffer solution with the pH value of 7-9 to obtain the target polypeptide.
Further, in the step (1), the organic solvent is dichloromethane; the mass-volume ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 20-30 mg/mL, preferably 25 mg/mL; the power of the ultrasonic oscillation is 50-70W, preferably 60W, the time is 3-5 min, preferably 3min, and the pulse type is 5 seconds on/5 seconds off; the temperature of the ultrasonic oscillation is 0-4 ℃;
and/or, in the step (2), the emulsifier is polyvinyl alcohol, preferably a 4% polyvinyl alcohol aqueous solution in mass fraction; the ultrasonic oscillation power is 50-70W, preferably 60W, the time is 3-5 min, preferably 5min, and the pulse type is 5 seconds on/5 seconds off; the temperature of the ultrasonic oscillation is 0-4 ℃;
and/or in the step (2), after the ultrasonic oscillation, adding an alcohol solvent into the system, stirring, volatilizing the organic solvent, centrifuging, and removing the supernatant; wherein, the alcohol solvent is preferably isopropanol, and is more preferably isopropanol water solution with volume fraction of 2%; the centrifugation condition is preferably 1000rpm centrifugation for 10 min;
and/or, in the step (3), before the reaction, mixing the non-targeted nanoparticles with an amidation reaction activator, incubating in a shaking table, centrifuging, and removing the supernatant; preferably, the amidation reaction activators are EDC and NHS, and the shaker incubation conditions are as follows: incubating in MES buffer solution with pH 5 at 0-4 deg.C for 1 hr, and centrifuging at 1000rpm for 10 min;
and/or in the step (3), the buffer solution with the pH value of 7-9 is MES buffer solution with the pH value of 8, the reaction temperature is 0-4 ℃, and the reaction time is overnight.
The invention also provides the application of the targeted bimodal developing nanoparticles in preparing developing reagents for diagnosing vulnerable plaques and/or medicines for stabilizing vulnerable plaques; preferably, the vulnerable plaque is a vulnerable plaque and/or an erosion plaque; more preferably, the imaging agent and/or drug is used under the following ultrasound irradiation conditions: the power is 3W, the frequency is 3HZ, the time is 3-4 min, and the duty ratio is 30%.
The invention also provides application of the targeted bimodal developing nanoparticle in preparing a medicament for preventing and/or treating cardiovascular diseases related to vulnerable plaques and/or an imaging reagent for diagnosing the cardiovascular diseases related to vulnerable plaques; preferably, the cardiovascular disease is atherosclerosis; more preferably, the drug is used under the following ultrasound irradiation conditions: the power is 3W, the frequency is 3HZ, the time is 3-4 min, and the duty ratio is 30%.
cRGD, a cyclic polypeptide, with the sequence cyclic (Arg-Gly-Asp-D-Phe-Lys).
PP1, a 16 amino polypeptide, sequence LSLERFLRCWSDAPAK.
Compared with the prior art, the invention has the following beneficial effects:
(1) the specific targeting of macrophage SR-A receptor and platelet GPIIb/IIIA receptor can realize site-specific delivery and has double targeting specificity to easy-to-break plaque and erosion plaque.
(2) By triggering phase change through ultrasound, US and MR bimodal imaging is realized, and the method can be used for preventive screening of vulnerable plaques, identification of plaque types and real-time monitoring of treatment effects.
(3) The biological effect generated by ultrasonic mediation is utilized to realize the prevention and treatment integration of vulnerable plaques:
a. the permeability of vascular endothelium is increased, and the double-targeting bimodal developing nanoparticles can reach the plaque at high flux;
b. the apoptosis of macrophages is induced, the inflammatory reaction is reduced, the easy-to-crack plaque is stabilized, and the first-level prevention effect is realized;
c. is beneficial to dissolving thrombus on the surface of the plaque, and realizes the accurate treatment of the plaque, thereby avoiding the systemic toxic and side effects of anti-inflammatory or antiplatelet drugs.
In a word, the invention takes polylactic acid-glycolic acid copolymer, magnetic resonance contrast agent, ultrasonic contrast agent and targeting polypeptide as raw materials to prepare a dual-target bimodal developing nanoparticle with regular shape, uniform size and good dispersibility, the dual-target bimodal developing nanoparticle has the functions of ultrasonic development and magnetic resonance bimodal developing, can target macrophages in vulnerable plaques, induce macrophage apoptosis under ultrasonic mediation, dissolve surface thrombus of vulnerable plaques under ultrasonic mediation, and has wide application prospect in preparing developing reagents for diagnosing vulnerable plaques (especially vulnerable plaques and eroded plaques), developing agents for stabilizing vulnerable plaques and medicines for preventing and/or treating cardiovascular diseases related to vulnerable plaques.
The preparation method is simple, mild in condition and suitable for industrial production.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a transmission electron microscope picture (a), an average particle size (b), a particle size and distribution (c) and a transmission electron microscope element analysis pseudo-color picture (d) of double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP 1.
Fig. 2 shows a transmission electron microscope picture (a), a surface charge (b), a confocal laser fluorescence microscope picture (c), and an infrared spectrum (d) of each nanoparticle.
Figure 3 is the in vitro US imaging results for each sample: (a) contrast pictures in B-mode and CEUS mode, (B) echo intensities in B-mode and CEUS mode, (c) contrast pictures of Fe-PFP-PLGA @ cRGD-PP1 under different ultrasound conditions, and (d) echo intensities of Fe-PFP-PLGA @ cRGD-PP1 under different ultrasound conditions.
FIG. 4 shows the in vitro MR imaging results of the double-targeting bimodal visualization nanoparticles Fe-PFP-PLGA @ cRGD-PP 1: (a) t2 signal at different concentrations; (b) concentration versus R2.
FIG. 5 is a confocal picture of laser light after co-culturing each fluorescently labeled sample with activated macrophages or inactivated macrophages.
FIG. 6 is a graph showing the effect on macrophage apoptosis under different treatment conditions, wherein green indicates live cells and red indicates dead cells; "control" is blank control group, "US" is ultrasonic irradiation group only, "non-PFP + US" is non-PFP + ultrasonic irradiation group, "PFP + US" is PFP + ultrasonic irradiation group.
FIG. 7 shows the effect of different treatment conditions on thrombus, wherein "US" is an ultrasonic irradiation-only group, "Fe-PLGA @ cRGD-PP 1" is an Fe-PLGA @ cRGD-PP1+ ultrasonic irradiation group, "Fe-PFP-PLGA @ PP 1" is an Fe-PFP-PLGA @ PP @ 1+ ultrasonic irradiation group, and "Fe-PFP-PLGA @ cRGD-PP 1" is an Fe-PFP-PLGA @ cRGD-PP1+ ultrasonic irradiation group.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Of these, poly (lactic-co-glycolic acid, PLGA) is available from Bio-technology Ltd, molecular weight 8000Da, of the big Dipper, Jinan, Dai, Perfluoropentane (Perfluoropentane,PFP) purchase fromRelative density: 1.63g/cm3(ii) a The polypeptide cRGD was purchased from gill biochemical (shanghai) ltd; the polypeptide PP1 was purchased from Gill Biochemical (Shanghai) Co.Ltd; polyvinyl alcohol (PVA) purchased fromThe average molar mass is 30000-70000.
2- (N-morpholine) ethanesulfonic acid (MES), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) are all commercially available products.
Example 1 preparation of Dual-Targeted bimodal visualization nanoparticles of the invention
1. Preparation of non-targeted nanoparticles
50mg PLGA and 1mg ferroferric oxide (Fe)3O4) Dissolve well in 2mL of dichloromethane (CH)2Cl2) In (1), 200. mu.L of PFP was added; and performing ultrasonic oscillation by using an ultrasonic oscillator, wherein the oscillation power is 60W, the oscillation time is 3min (pulse type 5s on/5s off), and the temperature of the ultrasonic oscillation is 0 ℃.
Then, 6mL of a 4% polyvinyl alcohol aqueous solution was again subjected to ultrasonic oscillation at 0 ℃ for 5min (oscillation power 60W, pulse-like 5s on/5s off). Finally, 10mL of isopropanol water solution with the volume fraction of 2% is added, and the mixture is stirred for 3 hours in ice bath until the organic solvent is completely volatilized; centrifuging at 1000rpm for 10min, removing the supernatant, and washing the solid with double distilled water for three times to obtain the non-targeted nanoparticle Fe-PFP-PLGA.
2. Preparation of double-target bimodal developing nano particle
Dispersing all the Fe-PFP-PLGA prepared in the step 1 in 10mL of 0.1M MES buffer solution (pH is 5), adding 8mg of activating agents EDC and 24mg of activating agents NHS, and incubating for 1 hour at 0 ℃ in a shaking table; centrifuging at 1000rpm for 10min, and discarding the supernatant; the remaining system was dispersed in 10mL of 0.1M MES buffer (pH 8), 2mg of PP1 and 2mg of cRGD were added, and the mixture was subjected to shake reaction at 0 ℃ overnight; centrifuging at 1000rpm for 10min, discarding the supernatant, and obtaining the solid, namely the double-target bimodal developing nano-particle Fe-PFP-PLGA @ cRGD-PP 1.
And adding double distilled water into the double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 for resuspension to obtain the Fe-PFP-PLGA @ cRGD-PP1 nanoemulsion.
The resulting Fe-PFP-PLGA @ cRGD-PP1 nanoparticles and nanoemulsion were collected and stored in a 4 ℃ refrigerator.
The following are the preparation of control nanoparticles:
comparative example 1 preparation of Fe-PFP-PLGA nanoparticles
Fe-PFP-PLGA nanoparticles were prepared in the same manner as in step 1 of example 1. Comparative example 2 preparation of Fe-PLGA nanoparticles
Fe-PLGA nanoparticles were prepared by replacing 200. mu.L PFP in the starting material with 200. mu.L double distilled water as in step 1 of example 1.
Comparative example 3 preparation of Fe-PFP-PLGA @ PP1 nanoparticles
Fe-PFP-PLGA @ PP1 nanoparticles were prepared according to the method of example 1, replacing 2mg PP1 and 2mg cRGD in step 2 with 2mg PP 1.
Comparative example 4 preparation of Fe-PFP-PLGA @ cRGD nanoparticles
Fe-PFP-PLGA @ cRGD nanoparticles were prepared according to the method of example 1, replacing 2mg of PP1 and 2mg of cRGD in step 2 with 2mg of cRGD.
Comparative example 5 preparation of Fe-PLGA @ cRGD-PP1 nanoparticles
Fe-PLGA @ cRGD nanoparticles were prepared as in example 1, replacing 200. mu.L of PFP in the starting material of step 1 with 200. mu.L of double distilled water.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1: physical and chemical property test of double-target bimodal developing nanoparticles
1. Test objects:
Fe-PFP-PLGA @ cRGD-PP1 obtained in example 1, Fe-PFP-PLGA, Fe-PFP-PLGA @ PP1, Fe-PFP-PLGA @ cRGD, and Fe-PLGA @ cRGD-PP1 obtained in comparative examples 1 to 5.
2. The test method comprises the following steps:
dynamic light scattering: detecting the particle size and potential of each nanoparticle by using a dynamic light scattering instrument (Malvern), repeating for 3 times, and taking an average value;
nanoparticle morphology: detecting the form of each nanoparticle by using a transmission electron microscope and a scanning electron microscope; and carrying out pseudo-color processing on the transmission electron microscope image, and carrying out element analysis.
Infrared spectrum detection: detecting the structure of each nanoparticle by using infrared spectroscopy;
laser confocal fluorescence microscope: the fluorescence-labeled material is prepared by adopting fluorescence-labeled polypeptide (cRGD is connected with fluorescence rhodamine B and red, PP1 is connected with fluorescence FITC and green), the PE and FITC channels are respectively used for observing the form of the nano emulsion under a laser confocal microscope, images of the two channels are fused, and the position relation of the two fluorescence polypeptides and the nano particles is analyzed.
3. Results of the experiment
Table 1 dynamic light scattering test results for each nanoparticle
The results are shown in table 1 and fig. 1 and 2. It can be seen that the double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention are of a shell-core structure, and have the advantages of regular shape, uniform size, good dispersibility, negative surface charge (-34.90mV), average particle size of 289.54nm and uniform particle size distribution.
As can be seen from the laser confocal fluorescence microscope picture in FIG. 2c, the polypeptide cRGD (red) and the polypeptide PP1 (green) are successfully connected to the Fe-PFP-PLGA @ cRGD-PP1 nanoparticles. Further, as can be seen from the pseudo-color image of the transmission electron microscope in FIG. 1d, the invention successfully converts the metal Fe3O4The (Fe in the figure), PFP (F in the figure), polypeptide cRGD and PP1 (S in the figure) are encapsulated in PLGA material to form liquid PFP and a large amount of compact metallic Fe with PLGA as a shell3O4The particle is a core-shell structure nanoparticle with an inner core, polypeptide cRGD and PP1 connected to a PLGA shell.
Experimental example 2: in vitro US imaging
1. Test method
(1) Verifying the imaging effect of the nano material under the ultrasonic condition after the Ultrasonic (US) irradiation triggers the phase change
a. Test samples: Fe-PFP-PLGA @ cRGD-PP1, normal saline, Fe-PLGA @ cRGD-PP1 and Fe-PFP-PLGA;
b. ultrasonic phase change conditions: (ultrasonic therapeutic apparatus) 3W, 3HZ, 3min, duty ratio 30%;
c. the contrast pictures in B-mode and CEUS mode + echo intensities were recorded.
(2) Verifying the relationship between the development intensity and the ultrasonic irradiation intensity or time during the induced phase change
a. Test samples: Fe-PFP-PLGA @ PP1-cRGD
b. Ultrasonic phase change conditions: an ultrasonic therapeutic apparatus with frequency of 3HZ, duty ratio of 30%, and power and time combination of 3W 2min, 3W 3min, 3W 4min, 3W 5min and 3W 6 min;
c. the contrast pictures in B-mode and CEUS mode + echo intensities were recorded.
2. Results of the experiment
The results are shown in FIG. 3. As can be seen from FIGS. 3a and 3b, under the same phase transition condition, compared with Fe-PLGA @ cRGD-PP1 and Fe-PFP-PLGA, the double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention have stronger ultrasonic signals in a CEUS mode, can clearly display local information, and have the highest echo intensity in the CEUS mode. The Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention has excellent ultrasonic development effect after the phase change is triggered by ultrasonic irradiation.
As can be seen from FIGS. 3c and 3d, the dual-targeting dual-mode developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 of the present invention have clear and fuzzy local information and reduced echo intensity as the ultrasonic time increases. The developing effect is best under the following ultrasonic phase change conditions: 3W, 3HZ, 3-4 min and duty ratio of 30%.
Experimental example 3: in vitro Magnetic Resonance (MR) imaging
1. Test method
a. Test samples: Fe-PFP-PLGA @ cRGD-PP1, which is prepared into gradient concentrations of 0,0.2,0.4,0.6,0.8 and 1.0mg/mL by using double distilled water, and the theoretical value of the corresponding Fe content (unit: mM) is calculated according to the actual loading rate of Fe;
b. magnetic resonance imaging: 1.5T MRI scanner, head coil;
c. setting parameters:
t2 weighting parameters of imaging: using a gradient echo sequence; echo Time (TE), 10.7 milliseconds; repetition Time (TR), 520 milliseconds; imaging field of view (FOV), 180 mm; flip Angle (FA), 45 °.
Parameters of transverse relaxation rate (R2): echo Time (TE) is 1.5-18.8 milliseconds; repetition Time (TR): 35 milliseconds; number of pulse repetition excitations (NEX) 1; matrix, 320 × 192; slice thickness, 1.5 mm.
d. Images were recorded, R2 values were calculated for different concentrations of nanomaterials and a linear fit was performed.
2. Results of the experiment
The results are shown in FIG. 4. It can be seen that the T2 signal is gradually reduced (fig. 4a) with the reduction of the concentration of the Fe-PFP-PLGA @ cRGD-PP1 solution, which indicates that the double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention have good nuclear magnetic resonance imaging function. Furthermore, the transverse relaxation rate (R2) of Fe-PFP-PLGA @ cRGD-PP1 has a good linear relationship with the concentration thereof, which indicates that the material has good paramagnetism (FIG. 4b), and can be used as a magnetic resonance contrast agent.
Experimental example 4: targeting of vulnerable plaques
1. Test samples: FITC fluorescein-labeled Fe-PFP-PLGA @ cRGD-PP1, Fe-PFP-PLGA @ cRGD.
2. The experimental method comprises the following steps:
(1) culture of macrophages
Macrophage: RAW 264.7 cell line; DMEM high glucose cell culture medium, 10% fetal bovine serum, (1% diabody, penicillin-streptomycin may be added if necessary); t25 cm2Cell culture flasks (6mL of medium);cell culture dish (2mL medium).
Non-activated macrophages: macrophages in logarithmic growth phase, amounting to 2.0X 105Is inoculated tomm confocal dishes were incubated for 48 hours.
Activation of macrophages: macrophages in logarithmic growth phase, amounting to 2.0X 105Is inoculated toCulturing in mm confocal dish for 24 hr; lipopolysaccharide (LPS; 100ng/mL) was added to the medium (200ng with 2mL of fresh medium) and incubated for 24 hours.
Lipopolysaccharide can promote bone marrow-derived macrophages to differentiate into a phenotype closer to inflammatory macrophages in plaques, and better simulate macrophages in vulnerable plaques.
(2) Grouping co-culture:
(a) without addition of PP1 solution: respectively adding the fluorescence labeled samples (1mg/mL and 1mL) into culture dishes of inactivated macrophages and activated macrophages, co-culturing for 2h, washing for 3 times by using PBS (phosphate buffer solution), and washing away redundant fluorescent nano materials; after staining cell nuclei by Hoechst 33258, observing by a laser confocal microscope, and quantitatively calculating the fluorescence intensity value of the intracellular sample (repeating the measurement for 5 times, and taking an average value).
(b) Adding a PP1 solution: adding PP1(50 mu g/mL, 1mL) into a culture dish for activating macrophages before adding the fluorescence labeled sample, then adding the fluorescence labeled sample (1mg/mL, 1mL), co-culturing for 2h, washing for 3 times by PBS, and washing away redundant fluorescence nano-materials and PP 1; after staining the cell nucleus, observing by using a laser confocal microscope, and quantitatively calculating the fluorescence intensity value of the intracellular sample.
3. Results of the experiment
TABLE 2 fluorescence intensity values of the groups of intracellular samples
As shown in FIG. 5 and Table 2, it can be seen that the fluorescence intensity of the Fe-PFP-PLGA @ cRGD-PP1 prepared by the present invention was higher in both non-activated and activated macrophages compared to the Fe-PFP-PLGA @ cRGD in the absence of PP1 solution. Particularly, the fluorescence intensity of the Fe-PFP-PLGA @ cRGD-PP1 is obviously improved compared with that of the Fe-PFP-PLGA @ cRGD in activated macrophages. In addition, the fluorescence intensity of Fe-PFP-PLGA @ cRGD-PP1 in activated macrophages was higher without PP1 solution compared to the PP1 solution.
The Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention has obviously improved targeting effect on macrophages, particularly activated macrophages, and is beneficial to improving the targeting property on the easily ruptured plaques.
Experimental example 5: induction of macrophage apoptosis under ultrasound trigger
1. Test samples: PE fluorescence-labeled Fe-PFP-PLGA @ cRGD-PP1 and PE fluorescence-labeled Fe-PLGA @ cRGD-PP 1.
2. The experimental method comprises the following steps:
(1) culturing activated macrophages: the culture method was as described in Experimental example 4.
(2) Grouping processing:
blank control group: culturing for 48 hours by using a complete culture medium;
ultrasonic irradiation group only: after 24 hours of culture, ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%) and further 12 hours of culture;
non-PFP + ultrasonic irradiation group: after culturing in a culture medium for 24 hours, adding PE fluorescence labeled Fe-PLGA @ PP1-cRGD (1mg/mL, 1mL), incubating for 4 hours together, washing with PBS for three times, replacing a fresh culture medium, performing ultrasonic irradiation (3W, 3HZ, 3min, duty ratio of 30%), and culturing for 12 hours;
PFP + ultrasonic irradiation group: after 24 hours of culture, adding PE fluorescence labeled Fe-PFP-PLGA @ cRGD-PP1, after co-incubation for 4 hours, washing with PBS for three times, replacing a fresh culture medium, performing ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%), and culturing for 12 hours after irradiation.
(3) Preparing a sample: PBS wash three times, stain (Calcein-AM, 10mM in PBS; PI, 2mM in PBS) for 15min (37 ℃, 5% CO)2)；
(4) And (4) observation: the excitation wavelengths of Calcein-AM and PI are 490 + -10 nm and 545nm respectively observed by CLSM.
3. The experimental results are as follows:
the results are shown in fig. 6, and it can be seen that the macrophage cells of the blank control group, the ultrasonic irradiation only group, and the non-PFP + ultrasonic irradiation group were substantially viable cells after co-culture, and were unable to induce apoptosis; whereas the macrophage cells of the PFP + ultrasound irradiation group were essentially apoptotic after co-culture. The preparation method is proved that only by combining the Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention with ultrasonic irradiation, the apoptosis of macrophages can be induced and activated, and the inflammatory reaction can be reduced. Therefore, the Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention can realize the purposes of targeting and stabilizing vulnerable plaques and treating plaques by inducing apoptosis of macrophages in the plaques.
Experimental example 6: inducing thrombolysis under ultrasound trigger
1. Test samples: Fe-PFP-PLGA @ cRGD-PP1, Fe-PFP-PLGA @ PP1 and Fe-PLGA @ cRGD-PP 1.
2. The experimental method comprises the following steps:
(1) preparing a thrombus section:
collecting 10mL of arterial blood from the auricular artery of a New Zealand big-ear white rabbit (female 2.5Kg), and carrying out water bath at 37 ℃ for 4 hours to obtain a thrombus model;
cutting the thrombus model into pieces of 1 × 0.3 × 0.3cm and about 200mg in weight, washing the pieces with PBS three times, and preparing frozen sections; to ensure the thrombus structure, ice acetone was used for fixation for 10min and double distilled water was used for another two washes.
(2) Grouping processing:
blank control group: adding 25mL of PBS solution into the thrombus slices, and incubating for 2 hours together by a shaking table;
the following groups were each ultrasonically irradiated for different times (the ultrasonic irradiation times are shown in table 3):
ultrasonic irradiation group only: adding 25ml of LPBS solution into the thrombus slice, performing ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%), and incubating together by a shaking table;
Fe-PFP-PLGA @ cRGD-PP1+ ultrasonic irradiation group: adding 25mL of Fe-PFP-PLGA @ cRGD-PP1 solution (2mg/mL) into the thrombus slices, performing ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%), and performing shaking table incubation;
Fe-PFP-PLGA @ PP1+ ultrasonic irradiation group: adding 25mLFe-PFP-PLGA @ PP1 solution (2mg/mL) into the thrombus slice, carrying out ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%), and carrying out shaking table incubation;
Fe-PLGA @ cRGD-PP1+ ultrasonic irradiation group: adding 25mLFe-PLGA @ cRGD-PP1 solution (2mg/mL) into the thrombus slices, performing ultrasonic irradiation (3W, 3HZ, 3min, duty ratio 30%), and performing shaking table incubation.
(3) And (4) observation: after the end of the co-incubation, the supernatant was removed, washed 3 times with PBS, the surface water was removed with filter paper, and the gross image was taken and weighed.
The above experiments were performed in parallel, n is 6, and the weight was averaged.
3. The experimental results are as follows:
TABLE 3 mean value of weights of thrombus slices of each group after different time of ultrasonic irradiation
The results are shown in FIG. 7 and Table 3. It can be seen that after 3 hours of ultrasonic irradiation, the thrombus weight reduction degrees of the ultrasonic irradiation group, the Fe-PLGA @ cRGD-PP1+ ultrasonic irradiation group and the Fe-PFP-PLGA @ PP1+ ultrasonic irradiation group are equivalent and are only reduced by 9.77-10.66%; however, the thrombus weight of the group irradiated by Fe-PFP-PLGA @ cRGD-PP1+ ultrasonic waves is reduced by 31.41%, and the reduction degree is obviously higher than that of the 3 control groups. After 30 hours of ultrasonic irradiation, the thrombus weight of the ultrasonic irradiation group, the Fe-PLGA @ cRGD-PP1+ ultrasonic irradiation group and the Fe-PFP-PLGA @ PP1+ ultrasonic irradiation group is only reduced by 28.36% -30.36%, while the thrombus weight of the Fe-PFP-PLGA @ cRGD-PP1+ ultrasonic irradiation group is reduced by 73.98%, and the reduction degree is obviously higher than that of 3 control groups.
Therefore, the thrombus weight can be obviously reduced and the thrombus dissolution can be induced only by co-culturing the double-targeting bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention and thrombus slices under ultrasonic induction; moreover, compared with the Fe-PLGA @ cRGD-PP1+ ultrasonic irradiation group and the Fe-PFP-PLGA @ PP1+ ultrasonic irradiation group, the prepared double-target bimodal developing nanoparticles Fe-PFP-PLGA @ cRGD-PP1 have the synergistic effect on induced dissolution of thrombus by ultrasonic induction, and unexpected technical effects are obtained. The Fe-PFP-PLGA @ cRGD-PP1 prepared by the invention can effectively induce thrombolysis under ultrasonic triggering, is beneficial to target dissolution of thrombus on the surface of an erosion plaque, and realizes local antiplatelet treatment of the erosion plaque.
In conclusion, the invention prepares the double-target bimodal developing nanoparticles with regular shape, uniform size and good dispersibility by taking the polylactic acid-glycolic acid copolymer, the magnetic resonance contrast agent, the ultrasonic contrast agent and the target polypeptide as raw materials, the double-target bimodal developing nanoparticles have the functions of ultrasonic development and magnetic resonance bimodal development, can target macrophages in vulnerable plaques, induce macrophage apoptosis under ultrasonic mediation, dissolve surface thrombus of the vulnerable plaques under ultrasonic mediation, prepare developing reagents for diagnosing the vulnerable plaques (particularly vulnerable plaques and eroded plaques), stabilize the vulnerable plaques and have wide application prospects in medicines for preventing and/or treating cardiovascular diseases related to the vulnerable plaques.