CN111317817B

CN111317817B - Targeted photoacoustic imaging nanomolecular probes and their applications

Info

Publication number: CN111317817B
Application number: CN202010134484.4A
Authority: CN
Inventors: 曹丰; 林乐健; 许梦琪
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2021-04-27
Anticipated expiration: 2040-03-02
Also published as: CN111317817A

Abstract

The invention discloses a nano-probe that can simultaneously realize targeted recognition and can be used for photoacoustic imaging, and correspondingly provides a convenient, efficient, economical and environmentally friendly nano-probe preparation method for atherosclerotic vulnerable plaques Detection of internal neovascularization in order to provide more timely treatment. In this application, ICG is used as a contrast agent for photoacoustic detection, which provides a new detection method for the detection of atherosclerotic vulnerable plaques.

Description

Targeting photoacoustic imaging nano molecular probe and application thereof

Technical Field

The application relates to a targeted photoacoustic imaging nano molecular probe technology.

Background

Acute cardiovascular events such as myocardial infarction and sudden cardiac death caused by atherosclerosis become the first killers endangering human health, and plaque rupture and secondary thrombosis caused by atherosclerotic vulnerable plaques are main factors causing the acute cardiovascular events. Pathological features of vulnerable plaque include: thin fibrous caps, a large necrotic lipid core, massive infiltration of inflammatory cells within the plaque, and pathologic neovascularization. The mononuclear macrophage phagocytes phagocytize and oxidize low-density lipoprotein to form foam cells, participate in formation and development of lipid cores, release a large amount of inflammatory factors to induce inflammatory reaction of plaques, and reduce the stability of the plaques, so that the plaques are easy to break. The rapid growth of new blood vessels is often associated with the metabolism and progression of the plaque, and adversely affects the stability of the plaque because new blood vessels provide nutrients required for plaque metabolism. Therefore, pre-identifying vulnerable plaques prior to disease onset, assessing the risk of cardiovascular events in patients and performing effective intervention is critical.

Current imaging modalities clinically used to diagnose vulnerable plaque include Optical Coherence Tomography (OCT) and intravascular ultrasound (IVUS). Among other things, IVUS can show the structure and properties of the interior of plaque. OCT can evaluate the property and stability of plaque, distinguish fibrous plaque, calcified plaque, plaque rich in lipid, etc., and has the characteristics of high resolution and strong penetrating power. However, these examination methods are all to observe vulnerable plaque on the whole structure, are difficult to monitor the process of occurrence and development of vulnerable plaque at the molecular level, and are not suitable for early warning of vulnerable plaque.

The emerging and developed photoacoustic imaging technology organically combines the characteristics of optical imaging and acoustic imaging, can provide a high-resolution and high-contrast tissue tomographic image of deep tissues, obtains the high-resolution and high-contrast tissue image through photoacoustic imaging, and shows great potential for detecting and diagnosing atherosclerotic plaques. However, due to the fact that the background noise of the photoacoustic imaging tissue is large, the excitation source attenuation and the penetration depth are poor, and only a limited range of biological activity processes can be tracked, the application of the photoacoustic imaging in the coronary arteriosclerosis plaque is mostly limited to the detection of lipid components and inflammatory cells at present, however, the rapid growth of the neointimal vessels is usually related to the metabolism and the development of the plaque, the stability of the plaque is adversely affected because the neointimal vessels provide nutrients required by the metabolism of the plaque, and the photoacoustic imaging technology cannot directly detect the neointimal vessels inside the arteriosclerosis plaque because of the defects. Due to the lack of effective photoacoustic contrast agents, photoacoustic imaging has been reported to achieve atherosclerosis on a living body level. Therefore, effective targeting nano photoacoustic probes are developed and the early detection of the neovascularization inside vulnerable plaque by photoacoustic imaging can be realized.

The molecular imaging technology can monitor the dynamic change of the disease-related markers from the molecular level in the state of a living organism, reflect the pathological evolution process of the disease, and can effectively intervene aiming at specific molecular targets to treat vulnerable plaques. In recent years, molecular imaging techniques are increasingly used in the study of cardiovascular diseases. However, the nano materials currently used for research mainly have the following defects: firstly, many heavy metal materials and fluorescent agents have high toxicity and low clinical application possibility; secondly, the material has poor structural stability, short detention time after entering the organism and difficult observation; thirdly, the nanometer material has too large particle size, is not easy to enter the plaque, and cannot sensitively identify and effectively treat the plaque. Therefore, the preparation of the nano material with high target specificity, good carrier biocompatibility and small particle size for early diagnosis and treatment of vulnerable plaques is urgently needed. Human Serum Albumin (HSA) is a main component of human serum protein, has good biocompatibility, and is a good platform for fusion of multi-mode imaging technologies such as magnetic resonance and optics.

Integrin has high expression in neovascular endothelial cells and plays an important role in angiogenesis, wherein the role of alpha v beta 3 is particularly important. The polypeptide (Arg-Gly-Asp, RGD) containing the arginine-glycine-aspartic sequence can be identified by an integrin alpha v beta 3 receptor, the research of the polypeptide marked by the radionuclide and containing the RGD sequence as an imaging agent and a therapeutic probe for angiogenesis becomes one of the research hotspots of nuclear medicine, the technology is mainly used for the research of the neoangiogenesis of tumor tissues at present, but the function of the angiogenesis in the generation and development process of vulnerable plaques is still needed to be researched for the high-resolution vessel imaging in vivo.

The disclosure of nano-drugs for vulnerable plaque by CN 109453375 a discloses that ICG can be used as a fluorescent agent and a photosensitizer in nano-drugs for vulnerable plaque.

Disclosure of Invention

In view of the above problems, the present application aims to propose a targeted photoacoustic imaging nano-molecular probe so that the detection of a target can be accomplished by photoacoustic imaging. The application also aims to provide a preparation method of the targeted photoacoustic imaging nano molecular probe. The present application also aims to propose a photoacoustic imaging contrast agent.

The targeted photoacoustic imaging nanomolecular probe of the present application comprises ICG as a photoacoustic contrast agent.

Preferably, HSA is further included as a vector for encapsulating the ICG.

Preferably, the RGD active short peptide is further included for targeting the probe to a target neovasculature.

Preferably, the RGD active short peptide is coupled to HSA by Sulfo-SMCC.

Preferably, ICG is used for both fluorescent and photosensitizers.

The targeted photoacoustic imaging nano molecular probe is used for detecting atherosclerotic vulnerable plaques.

The preparation method of the targeted photoacoustic imaging nano molecular probe comprises the steps of coupling HSA with RGD active short peptide through Sulfo-SMCC, and then co-encapsulating ICG through electrostatic acting force to finally form the nano molecular probe.

Use of the ICG of the present application as a contrast agent for photoacoustic imaging.

A photoacoustic imaging contrast agent of the present application, comprising ICG.

The inventor of the invention finds the photoacoustic imaging enhancement effect of ICG, applies the ICG to photoacoustic imaging detection of atherosclerotic vulnerable plaque, and provides a new detection means for detecting the atherosclerotic vulnerable plaque.

Drawings

Fig. 1 is a scanning electron micrograph of the targeted photoacoustic nanoprobe prepared in example 1 of the present invention.

Fig. 2 is a hydrated particle size distribution diagram of a nano system formed by the targeted photoacoustic nanoprobe prepared in example 1 of the present invention.

FIG. 3 is a graph of the UV absorption spectrum of the targeted photoacoustic nanoprobe prepared in example 1 of the present invention between 250nm and 850 nm.

Fig. 4 is a graph of fluorescence intensity generated by the targeted photoacoustic nanoprobe prepared in example 1 of the present invention under irradiation of laser with different wavelengths.

FIG. 5 is a graph comparing the uptake of non-targeting nanoprobes (nNPs), active targeting nanoprobes (fNPs) and competitive inhibition groups (fNPs + RGD peptide) by macrophages and foam cells formed after phagocytosis of oxidized low density lipoprotein (ox-LDL).

FIG. 6 is a CCK-8 experiment to verify the killing ability of non-targeted nanoprobes (nNPs) and active targeted nanoprobes (fNPs) to endothelial cells at different ICG concentrations.

Fig. 7 is a photo-acoustic imaging diagram of non-targeted nanoprobes (nps) and active targeted nanoprobes (fNPs) on arteriosclerotic rabbit abdominal aorta vulnerable plaque in vivo.

FIG. 8 is a flow chart of the preparation of the targeted fluorescence/photoacoustic bimodal diagnosis and treatment integrated nanoprobe.

Detailed Description

In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The materials, methods, and examples set forth in this application are illustrative only and not intended to be limiting.

Various materials, reagents, instruments, equipment and the like used in the present invention are commercially available.

Example 1:

the targeted photoacoustic imaging nano probe is formed by coupling HSA (human serum albumin) and RGD active short peptide (the amino acid sequence is Arg-Gly-Asp) capable of being specifically combined, and co-encapsulating ICG (indocyanine green) through electrostatic acting force. HSA is a framework material support; RGD active short peptide is used for targeting vulnerable plaque; ICG can be used for fluorescent imaging of plaque and nano-probe of photoacoustic imaging. Photoacoustic imaging has become a novel imaging technology for arterial plaque, and RGD targeting nanoprobes have been used in partial research work of tumor at present, but the photoacoustic imaging probe tool formed by combining with ICG has not been used for detecting clinical atherosclerotic vulnerable plaque neovascularization.

The steps comprise:

the coupling method of HSA and RGD active short peptide is as follows:

(1) sulfhydrylation of RGD active short peptides:

preferably, the molar ratio of RGD active short peptide to Traut's Reagent is 1:2, and 0.02mmol RGD active short peptide (50mg) and 0.04mmol Traut's Reagent (5.6mg) are weighed respectively. 50mg of RGD-active short peptide was dissolved in 5mL of SB Buffer (0.2M, pH 8), and 5.6mg of Traut's Reagent was dissolved in 1mL of SB Buffer (0.2M, pH 8). Mixing the two, oscillating and reacting for 1h at room temperature, and moving to 4 ℃ for storage for later use;

(2) HSA is linked to Sulfo-SMCC (maleimidomethyl):

preferably, the molar ratio of Sulfo-SMCC to HSA is 7.5:1, 40mg of HSA is weighed out and dissolved in PBS (0.1M, pH7.4), 2mg of Sulfo-SMCC is dissolved in 0.2mL of DMSO, and after mixing, the reaction is stirred at room temperature for 30 min. Then, the mixture is centrifugally purified three times by using an ultrafiltration tube with the molecular weight of 30KD to remove free Sulfo-SMCC;

(3) coupling maleimide on HSA-SMCC with sulfhydryl on RGD active short peptide:

adding 1.8mL of the RGD active short peptide solution subjected to sulfhydrylation in the step (1) into 4mL of HSA-SMCC solution, and stirring at room temperature for reaction for 2 h. Then, the mixture was purified by centrifugation three times using an ultrafiltration tube having a molecular weight of 30kD, and finally dissolved in 4mL of ultrapure water. The final concentration of HSA-RGD peptide is about 10 mg/mL;

preparing an ICG-HSA-RGD peptide nanoprobe (fNPs):

(1) 1mL of 10% HSA solution is prepared;

(2) preferably, the mass ratio of HSA to RGD peptide in two solutions of HSA 10% is set as 5: 2, the total mass of HSA is 30 mg. Calculating corresponding volumes according to the respective concentrations;

(3) the glass vial after high-temperature sterilization and evaporation to dryness is put into a magneton, two solutions with the volume calculated in the previous step are sequentially added, and then a PBS solution (1X, pH7.4) is added to make the total volume of the solution be 3 mL. Placing the vial on a magnetic stirrer for stirring;

(4) weighing 2mg ICG, and dissolving in 20 mu L DMSO by ultrasonic oscillation;

(5) slowly adding 10 mu L of ICG solution into the HSA solution obtained in the step (3) dropwise under the stirring state;

(6) mu.L of glutaraldehyde was added to 2mL of PBS (1X, pH7.4), and after dissolving by shaking sufficiently, 200. mu.L of glutaraldehyde was added slowly to the above solution, and stirred for 30 hours in the dark.

(7) The product was purified by centrifugation three times using an ultrafiltration tube with a molecular weight of 100kD and finally dissolved in 1mL of PBS (1X, pH 7.4).

FIG. 1 is a scanning electron micrograph (Ht-7700, Hitachi) of the above photoacoustic nanoprobe, which shows that the nanoprobe has a uniform particle size of about 50 nm.

FIG. 2 is a distribution diagram of the hydrated particle size of the photoacoustic nanoprobe (Nano ZS, Malvern), which shows that the particle size distribution is mainly between 100 and 120nm, the system is stable and the aggregation phenomenon is not easy to occur.

FIG. 3 shows the ultraviolet absorption spectrum (UV-1800, Mapada) of the photoacoustic nanoprobe between 250-850nm, and the characteristic absorption peaks of HSA and ICG at 278nm and 780nm are respectively measured, which shows that the preparation of the nanoprobe is successful, and the concentration of each component can be measured by the ultraviolet absorption value.

FIG. 4 shows fluorescence intensities of the photoacoustic nanoprobes under irradiation of laser beams with different wavelengths. The graph indicates that the visible fluorescence intensity of both probes reaches the peak value between 810 and 830nm, and the fluorescence spectrum characteristics of ICG are met, so that the successful ICG entrapment in the probes is prompted.

Example 2:

preparation of ICG-HSA non-targeting nanoprobes (nNPs).

(1) 1mL of 10% HSA solution is prepared;

(2) and measuring the ultraviolet absorbance value to obtain the HSA concentration. Preferably, the total mass of HSA is set to 30 mg.

(3) Weighing 2mg ICG, and dissolving in 20 mu L DMSO by ultrasonic oscillation;

(4) adding 10 mu L of ICG solution into the HSA solution dropwise and slowly under the stirring state;

(5) mu.L of glutaraldehyde was added to 2mL of PBS (1X, pH7.4), and after dissolving by shaking sufficiently, 200. mu.L of glutaraldehyde was added slowly to the above solution, and stirred for 30 hours in the dark.

(6) The product was purified by centrifugation three times using an ultrafiltration tube with a molecular weight of 100kD and finally dissolved in 1mL of PBS (1X, pH 7.4). The UV absorbance was measured and the amounts of HSA and ICG were determined separately. The respective concentrations were calculated.

Example 3:

the endothelial cell uptake assay for the nanoprobes prepared in the three examples was as follows:

(1) endothelial cells were selected from the Huvec cell line at 1X 10⁵The density is inoculated in a plurality of confocal dishes. After the cells are attached to the wall, the Huvec cell line in one half of the confocal dish is co-cultured with low-density lipoprotein ox-LDL (80 mu g/ml)And (5) incubating for 24h to construct a foam cell model.

(2) Dividing each dish cell into six groups for comparison, wherein the pure culture Medium does not contain a nano probe plus an endothelial cell group (Medium), a non-targeting nano probe (nNPs) + the endothelial cell group, an active targeting nano probe (fNPs) + the endothelial cell group, an RGD active short peptide plus a non-targeting nano probe (nNPs) + the endothelial cell group, a non-targeting nano probe (nNPs) + a foam cell group, an active targeting nano probe (fNPs) + a foam cell group, an OPN active short peptide plus an active targeting nano probe (fNPs) + a foam cell group, the concentration of the nano probe is 30 mu g/mL, and the nano probe and the cells are incubated for 6 hours.

(3) After washing away the nanoprobes with PBS (1X, pH7.4), the cells were fixed with 4% paraformaldehyde for 10 min.

(4) After staining with DAPI for 10min, washing twice with PBS (1X, pH7.4), the cells were observed in different groups under a confocal microscope.

The experimental result is shown in fig. 5, the uptake of the active targeting nanoprobe (ox-LDL + fNPs) by the foam cells is obviously higher than that of other groups, which shows that the RGD active short peptide has good capability of identifying the foam cells, and further verifies that the targeting RGD nanoprobe of the patent can accurately identify the foam cells in the vulnerable plaque of the artery.

Example 4:

the CCK-8 experiment detects the killing capacity of different groups of nano probes on macrophages:

(1) three treatment groups are set, namely a pure culture medium group (Control), non-targeting nanoprobes (nNPs) and active targeting nanoprobes (fNPs) under different concentrations.

(2) Adding 100 mu L of Huvec cell suspension into each hole of a 96-hole plate, adding 100 mu L of non-targeting nanoprobes (nNPs) and active targeting nanoprobes (fNPs) with different concentrations into each hole after the cells adhere to the wall, arranging 6 multiple holes in each group, and placing in a cell culture box for 24 hours.

(3) Using 808nm laser (1W/cm) for each hole of the illumination group²) Irradiating for 5min.

(4) After washing with PBS solution (1X, pH7.4), fresh medium was added.

(5) And (3) rapidly adding 10ul of CCK-8 solution into each hole, and measuring the absorbance of each hole solution at 450nm by using an enzyme-labeling instrument after 2 hours to obtain the cell survival rate.

As shown in FIG. 6, the cell survival rates of pure nanoprobes are all above 90% with increasing concentration, and after a certain concentration, although the cell survival rates basically tend to be stable with increasing probe concentration, which indicates that the nanoprobes described in this patent have low cytotoxicity and high safety.

Example 5:

in vivo photoacoustic imaging of vulnerable plaque of abdominal aorta of arteriosclerosis rabbit:

(1) a rabbit abdominal aorta vulnerable plaque model is constructed by feeding 5-7 weeks old male New Zealand rabbits (n is 10) for more than 20 weeks through percutaneous abdominal aorta balloon injury and high-fat high-cholesterol diet. Male new zealand rabbits were fed normal diet as a control.

(2) The arteriosclerosis rabbits were randomly divided into two groups, namely an active targeting nanoprobe group (AS + fNPs) and a non-targeting nanoprobe group (AS + nNPs). Active targeting nanoprobes (Control + fNPs) were injected in rabbits in the Control group. Injecting each group of nanoprobes into the mouse body through ear edge intravenous injection, and performing photoacoustic/ultrasonic bimodal imaging in the living body through the abdominal and cutaneous aorta after 24 hours.

As shown in fig. 7, in the non-targeting nanoprobe set, the intravascular ultrasound can clearly display the arteriosclerotic plaque, and the synchronous photoacoustic imaging does not find an obvious photoacoustic signal, while in the targeting nanoprobe set, the intravascular ultrasound can clearly display the arteriosclerotic plaque, and the synchronous photoacoustic imaging can also display an obvious photoacoustic signal, which indicates the existence of the vulnerable plaque.

Compared with the prior art, the invention has the following advantages and effects:

(1) on the diagnostic plane, the invention selects ICG which can generate photoacoustic signals as a photoacoustic signal enhancer (or contrast agent) based on a molecular imaging technology platform, RGD active short peptide which can identify the new vessels in atherosclerotic vulnerable plaques as a targeting molecule, HSA with high biocompatibility is adopted as a carrier, and a molecular nano probe which can be used for early warning vulnerable plaques is designed and constructed. Enters the body by intravenous injection, combines ultrasonic/photoacoustic imaging to realize the complementation of the identification of two layers of anatomy and function, macro and micro, and improves the sensitivity and specificity of diagnosis. Overcomes the defect that the imaging means for diagnosing vulnerable plaque is difficult to detect the generation and development process of the vulnerable plaque neovascularization at early stage clinically.

(2) The targeted photoacoustic imaging nano probe is stable in structure and high in safety; the entrapped ICG is a few dyes approved by the FDA in the United states for clinical application, can be used for near infrared fluorescence imaging and also can be used as a photosensitizer for photodynamic therapy, but is not applied to intracavity photoacoustic imaging for detecting new blood vessels in arterial plaques. The RDG can discover new vessels inside the arterial plaque in an early stage, so that the nanoprobe has a high early warning effect on the new vessels inside the vulnerable plaque.

Although the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the teachings of this application and yet remain within the scope of this application.

Claims

1. a preparation method of targeted photoacoustic imaging nano-molecular probe, is characterized in that comprising the steps:

HSA is coupled to RGD active short peptide through Sulfo-SMCC, wherein the coupling method of HSA and RGD active short peptide is as follows:

(1) Thiolylation of RGD active short peptides:

The molar ratio of RGD active short peptide to Traut's Reagent was 1:2. Weigh 0.02mmol RGD active short peptide 50mg and 0.04mmol Traut's Reagent 5.6mg respectively, dissolve 50mg RGD active short peptide in 5mL SB Buffer, and dissolve 5.6mg Traut's Reagent in 5mL SB Buffer. 1mL SB Buffer, mix the two, shake at room temperature for 1h, and move to 4°C to store for later use;

(2) HSA is connected to Sulfo-SMCC:

The molar ratio of Sulfo-SMCC to HSA was 7.5:1, 40 mg of HSA was weighed and dissolved in PBS solution, and 2 mg of Sulfo-SMCC was dissolved in 0.2 mL of DMSO. After mixing, the reaction was stirred at room temperature for 30 min, and then centrifuged using an ultrafiltration tube with a molecular weight of 30 KD. Purified three times to remove free Sulfo-SMCC;

(3) The maleimide on HSA-SMCC is coupled to the sulfhydryl group on the RGD active peptide:

Take 1.8 mL of the thiolated RGD active short peptide solution in step (1) and add it to 4 mL of HSA-SMCC solution, stir at room temperature for 2 h, and then use an ultrafiltration tube with a molecular weight of 30KD to purify three times by centrifugation, and finally dissolve it in 4 mL of ultrafiltration. In pure water, the final concentration of HSA-RGD peptide is 10mg/mL;

The ICG-HSA-RGD peptide nanoprobe fNPs were prepared as follows:

(4) Prepare 1 mL of 10% HSA solution;

(5) Set the HSA-RGD peptide, the mass ratio of HSA in the two solutions of 10% HSA is 5:2, the total mass of HSA is 30 mg, and the corresponding volume is calculated according to their respective concentrations;

(6) put magnetron into the glass vial after high-temperature sterilization and evaporate to dryness, add two kinds of solutions of the volume calculated in the previous step successively, then add PBS solution, make the total volume of the solution be 3mL, and place the vial on a magnetic stirrer and stir;

(7) Weigh 2 mg of ICG and dissolve it in 20 μL of DMSO by ultrasonic vibration;

(8) 10μLICG solution was slowly added dropwise to the HSA solution in step (6) under stirring;

(9) Add 20 μL of glutaraldehyde into 2 mL of PBS solution, and after sufficient shaking to dissolve, take 200 μL of glutaraldehyde and slowly add it to the above solution, and stir for 30 h in the dark.

(10) centrifugally purify three times using an ultrafiltration tube with a molecular weight of 100KD, and finally dissolve in PBS solution;

Finally, nano-molecular probes are formed;

Wherein, the SB Buffer is 0.2M SB Buffer with pH 8, and the PBS solution is 0.1M PBS solution with pH 7.4.