CN109364272B - Application of nano enzyme corpuscle in catalysis photoacoustic imaging - Google Patents
Application of nano enzyme corpuscle in catalysis photoacoustic imaging Download PDFInfo
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Abstract
The invention relates to a nano enzyme bionic corpuscle loaded with nano enzyme and a azine diammonium salt contrast agent and application thereof, wherein the preparation method of the nano enzyme bionic corpuscle comprises the following steps: (1) preparing a nano enzyme and a azine diammonium salt contrast agent; (2) preparing a bionic corpuscle; (3) and (3) filling the nano enzyme and the azine diammonium salt complex contrast agent into the bionic corpuscle to prepare the nano enzyme bionic corpuscle. The nano enzyme bionic corpuscle can be used for preparing a tumor light imaging contrast agent or a tumor targeting drug delivery preparation.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a nanoenzyme bionic corpuscle loaded with carbon dot nanoenzyme and a azine diammonium salt contrast agent and application thereof.
Background
The photoacoustic imaging combines the advantages of high contrast of pure optical imaging and high penetration depth of pure ultrasonic imaging, can provide high-resolution and high-contrast tissue imaging, and is an imaging mode with great application prospect at present. The photoacoustic imaging contrast agent is the key for determining the photoacoustic imaging performance, improves the imaging contrast and resolution by changing the optical and acoustic characteristics of lesion tissues, and becomes a research hotspot in the field of current biological imaging. The relatively common photoacoustic imaging contrast agents include gold nanomaterials, carbon nanotubes, and dye nanomaterials. With the rapid development of nano materials, the discovery of nano enzyme as a nano material with enzyme activity promotes the further development of a nano material family. The nano-material has the characteristics of both the basic properties of the nano-material and the enzyme catalytic capability, so that the nano-material has wide application prospects in the biomedical fields of immunodetection, tumor diagnosis, biosensing and the like. At present, most of nanoparticles are only applied to laboratory research, and various factors limit the transformation of the nano preparation from basic research to clinic, such as transport efficiency, biocompatibility, long-term use safety and the like. In recent two years, international top-level periodicals such as Nature and the like continuously and deeply analyze the current situations of research and clinical transformation of nano-drugs, and the biological safety of the nano-drugs is considered to be an important reason for failure of clinical transformation. Therefore, the development of a nano-enzyme photoacoustic imaging contrast agent with higher biological safety is a necessary condition for realizing nano-enzyme tumor photoacoustic imaging.
An ideal photoacoustic imaging contrast agent should have the following characteristics: the conversion capability of photothermal signals and acoustic signals of the excellent ground is achieved; sensitively reflect the performance of the physiological structure of the organism; good in vivo distribution and metabolic characteristics and excellent biocompatibility. Nanoenzymes have the potential to integrate these features into one nanomaterial.
Catalytic properties of nanoenzyme peroxidase: in the presence of H2O2Can efficiently catalyze the conversion of azinam diammonium salt (ABTS) into ABTS with near infrared light absorption capacity+The local temperature of the tumor tissue is increased due to the released heat energy after the light energy is absorbed, and the tumor tissue is thermally expanded to generate pressure waves after the temperature is increased, so that a photoacoustic signal is formed, and a light absorption distribution image in the tissue can be reconstructed by detecting the photoacoustic signal, so that photoacoustic imaging diagnosis of the tumor is realized.
In addition, researches show that the photoacoustic signal can be further enhanced by the strong light absorption effect of the nanoenzyme in the near infrared region. However, due to the extreme complexity of living organisms, the behavior of the carefully designed nano-drug preparation in the final clinical test is often greatly different from the results of the early in vitro experiments, so that the clinical transformation is difficult to perform.
Disclosure of Invention
In order to solve the problem of the nano enzyme in practical application, the inventor of the patent prepares a bionic corpuscle by using a natural erythrocyte membrane, and carries out biobionic coating on the nano enzyme photoacoustic contrast agent so as to improve the stability of the contrast agent in a blood system and the biocompatibility of an organism.
The invention firstly relates to a method for preparing a nano enzyme bionic corpuscle loaded with nano enzyme and a azine diammonium salt contrast agent, which comprises the following steps:
(1) preparing a nano enzyme and a azine diammonium salt contrast agent;
(2) preparing a bionic corpuscle;
(3) and (3) filling the nano enzyme and ABTS complex contrast agent into the bionic corpuscle to prepare the nano enzyme bionic corpuscle.
The preparation method of the nanoenzyme in the step (1) is preferably a carbon dot nanoenzyme (GQDzyme), the contrast agent is preferably a carbon dot nanoenzyme and ABTS complex, and the preparation method of the carbon dot nanoenzyme and ABTS complex comprises the following steps:
1) adding polyacrylonitrile carbon fiber into a mixed solution of concentrated sulfuric acid and nitric acid; ultrasonic mixing and dissolving, heating to 100 ℃ and stirring fully;
2) intercepting the ultrafiltration solution with molecular weight of 3KD and freeze-drying to obtain GQDzyme;
3) mixing and dissolving GQDzyme and ABTS in an aqueous solution, stirring overnight, and then carrying out ultrafiltration and freeze-drying to obtain GQDzyme/ABTS;
the method for preparing the bionic corpuscles in the step (2) comprises the following steps:
1) adding the red blood cells into a precooled hypotonic buffer solution, uniformly mixing, and then placing at 4 ℃ for 1-2 h to ensure that the red blood cells are completely hemolyzed;
2) centrifuging at 4 deg.C to remove hemoglobin supernatant, and collecting precipitate as erythrocyte membrane;
3) repeatedly centrifuging and washing for 3-5 times to prepare a bionic corpuscle with a larger particle size;
4) and (3) carrying out ultrasonic treatment on the bionic corpuscle to prepare the bionic corpuscle with the particle size of about 400 nm.
The method for filling the GQDzyme/ABTS contrast agent into the bionic corpuscles in the step (3) comprises the following steps:
and loading the GQDzyme/ABTS contrast agent into the bionic corpuscles in a physical extrusion mode to obtain the nano enzyme bionic corpuscles.
The invention also relates to the GQDzyme/ABTS contrast agent prepared by the method or a nano enzyme bionic corpuscle containing the GQDzyme/ABTS contrast agent.
The GQDzyme/ABTS contrast agent has the particle size of about 10nm, the particle size of the bionic corpuscle is about 100nm, and the particle size of the nano enzyme bionic corpuscle is about 50 nm.
The invention also relates to an application of the GQDzyme/ABTS contrast agent or the nano enzyme bionic corpuscle containing the GQDzyme/ABTS contrast agent in preparing a tumor targeting preparation, wherein the tumor targeting preparation comprises the following components in parts by weight: and loading a tumor treatment preparation into the nano enzyme bionic corpuscle, wherein the tumor treatment preparation is an antibody, a polypeptide, an aptamer or a functional protein. Preferably, the functional protein is folic acid.
The invention also relates to the application of the GQDzyme/ABTS contrast agent or the nano enzyme bionic corpuscle containing the GQDzyme/ABTS contrast agent in preparing a tumor photoimaging contrast agent, wherein the tumor is a solid tumor.
The tumor light imaging contrast agent is a contrast agent for marking the lesion part of a solid tumor in an intravenous administration mode.
Drawings
FIG. 1, preparation and characterization of carbon dot nanoenzyme photoacoustic contrast agent wrapped by exosome biomimetic corpuscles:
FIG. 1a is a transmission electron microscope image of a carbon dot nanoenzyme and ABTS photoacoustic contrast agent complex (GQDzyme/ABTS);
FIG. 1b is a schematic diagram showing that erythrocyte membranes are processed by hypotonic treatment to prepare exosome biomimetics with smaller particle size;
FIG. 1c loading exosome biomimetic corpuscles of GQDzyme/ABTS complex by physical extrusion;
FIG. 1d shows ABTS converted by carbon dot nanoenzyme catalysis at different concentrations+A light absorption spectrum at near infrared;
FIG. 1e GQDzyme/ABTS complex light absorption at 808nm with H pair2O2A dependency relationship;
FIG. 1f photo acoustic signal intensity in solution is positively correlated with concentration.
FIG. 2, stability investigation of nano-enzyme photoacoustic imaging contrast agent in different dosage forms:
FIG. 2a is a photo of photoacoustic imaging of various sets of contrast agents;
figure 2b graph of photoacoustic signal intensity versus time.
Fig. 3 shows the recognition ability of the specially labeled exosome-biomimetic corpuscle-modified nanoenzyme photoacoustic imaging contrast agent on nasopharyngeal carcinoma cells and the endocytosis condition of the nasopharyngeal carcinoma cells:
FIG. 3a confocal laser imaging analysis of CNE-2 cell membrane and NIH3T3 cell membrane folate receptor expression;
FIG. 3b laser confocal imaging of endocytosis of Nanolase particles RM: GQDzyme/ABTS and FA-RM: GQDzyme/ABTS (RM, erythrocyte membrane; FA-RM, folate-modified erythrocyte membrane) by CNE-2 nasopharyngeal carcinoma cells;
FIG. 3c is a transmission electron microscope image of endocytosis of targeted molecule-modified photoacoustic imaging contrast agent by CNE-2 nasopharyngeal carcinoma cells;
FIG. 3d lysosomal co-localization laser confocal imaging characterization of the endocytosis of targeted molecule-modified photoacoustic imaging contrast agent into cells by CNE-2 nasopharyngeal carcinoma cells.
FIG. 4H2O2Photoacoustic imaging of the responsive exosome-biomimetic corpuscle-modified nanoenzyme photoacoustic imaging contrast agent in nasopharyngeal carcinoma tumor-bearing mice:
FIG. 4a shows photoacoustic imaging analysis of tumor sites before or after injection of contrast agent into tumor-bearing mice in tail vein at 2h, 4h, and 8 h;
fig. 4b quantitative analysis of photoacoustic signals at different time points of tumor sites.
Figure 5, investigation of the in vivo distribution and corresponding tissue distribution of different nanoenzyme photoacoustic imaging contrast agents by fluorescence imaging:
in figure 5a 8h, the results of real-time distribution of different drugs in mice:
FIG. 5b fluorescence quantification of tumor tissue regions;
FIG. 5c fluorescence imaging results of tumor-bearing mice ex vivo tissues;
FIG. 5d shows the distribution of fluorescence values of the contrast agent for each tissue.
Detailed Description
Example 1 preparation and characterization of exosome-biomimetic corpuscle-encapsulated nanoenzyme photoacoustic contrast agent
1. Preparation of GQDzyme by chemical oxidation stripping method
In order to prepare the nano enzyme with catalase activity, polyacrylonitrile carbon fiber is used as a raw material, and a chemical oxidation stripping method is used for preparing GQDzyme, wherein the specific process is as follows:
(1) adding 0.2g of polyacrylonitrile carbon fiber into a mixed solution of concentrated sulfuric acid (40mL) and nitric acid (12 mL); ultrasonic mixing and dissolving for 2h, heating to 100 ℃, and stirring for 24 h;
(2) and (4) freeze-drying by ultrafiltration (molecular cut-off of 3000Da) to obtain the GQDzyme.
As shown in FIG. 1a, the lyophilized GQDzyme can be uniformly dispersed in water again to form a transparent pale yellow solution, which indicates that the GQDzyme has good water solubility. The GQDzyme prepared by transmission electron microscopy analysis has the size of about 10nm and is uniform in size.
2. Completing adsorption loading of ABTS by using electrostatic interaction and pi-pi accumulation acting force to prepare GQDzyme/ABTS developer
(1) Mixing 10mg of GQDzyme and 2mg of ABTS in an aqueous solution, ultrasonically mixing, dissolving for 2 hours, and stirring overnight;
(2) ultrafiltering (molecular interception amount 3000Da) and freeze-drying to obtain GQDzyme/ABTS.
ABTS in appropriate H2O2ABTS (ABTS) with light absorption in near infrared region by GQDzyme under action+. To investigate the procedure with H2O2In relation to (3), we are in different H2O2Detecting ABTS in mixed aqueous solution of ABTS and GQDzyme+Light absorption and H2O2The relationship of concentration. Further by free of H2O2And catalase(CAT) pretreated H2O2Group, verification of Nanolase photoacoustic contrast agent Pair H2O2The dependence of (c). And using different concentrations of H2O2The influence of the dependence on the strength of the photoacoustic signal is examined. The results are shown in FIGS. 1d-f at H2O2ABTS can be catalytically converted into ABTS by GQDzyme when the catalyst exists+And ABTS ·+Light absorption in the near infrared region with H2O2The concentration of the photo-acoustic signal is increased, the light absorption value is gradually enhanced, and the photo-acoustic signal is also related to H2O2The concentration of (A) is in a positive correlation.
3. Preparing bionic corpuscle
(1) Taking 2mL of BALB/c mouse whole blood by using a mouse eyeball-picking and blood-taking method;
(2) placed in a 5mL centrifuge tube containing 0.5mL anticoagulant (3.8 wt.% sodium citrate solution), and mixed well;
(3) centrifuging at 4 deg.C and 3000rpm for 20min, and removing upper blood chorion by suction;
(4) placing the bottom layer of red blood cells in a 3-fold volume of isotonic phosphate buffer (pH 7.4) pre-cooled at 4 deg.C, resuspending, and centrifuging at 4 deg.C for 3 times (15min × 5000 rpm);
(5) adding the red blood cells after centrifugal cleaning into precooled 10mmol/L hypotonic Tris-HCl buffer solution (pH 7.4) according to the proportion of 1:40, slowly stirring in the adding process, and then placing in a refrigerator at 4 ℃ for 1-2 h to ensure that the red blood cells are completely hemolyzed;
(6) centrifuging at 9000rpm at 4 deg.C for 15min to remove hemoglobin supernatant, and collecting precipitate as erythrocyte membrane;
(7) carrying out repeated centrifugal washing for 3-5 times to prepare an exosome bionic corpuscle with a larger particle size;
(8) carrying out water bath ultrasound on the small erythrocyte body with large particle size for 20min under the rated power of 60W to prepare the exosome bionic body with the particle size of about 400 nm.
The hemoglobin is removed through hypotonic treatment to form a hollow vesicle structure, so that erythrocyte vesicles with the grain size of about 3 microns are formed, and experiments show that after water bath ultrasonic treatment is carried out for 20min, the result is shown in figure 1b, and the erythrocyte vesicles with the micron size can be changed into exosome bionic bodies with the grain size of 400 nm.
4. Loading GQDzyme/ABTS developer into bionic corpuscle
The bionic corpuscle can effectively reduce or shield the immunogenicity of the bionic corpuscle while completely retaining the bioactivity, improve the biocompatibility, avoid the recognition and removal of an endothelial reticulum system, improve the water solubility and the stability of the nano particles and obviously prolong the circulation time in vivo. And then loading a GQDzyme/ABTS developer with the prepared bionic corpuscle, blending the prepared 100nm exosome bionic corpuscle and the GQDzyme/ABTS according to the concentration ratio of 1:1, and continuously extruding for 7-10 times through a MINI extruder with the pore diameter of 50nm to form GQDzyme/ABTS (RM: GQDzyme/ABTS) coated with an erythrocyte membrane. By the same method, RM, GQDzyme/ABTS and FA are mixed according to the concentration ratio of 1:10 and then extruded for 10-15 times by a MINI extruder to prepare RM marked with FA, GQDzyme/ABTS.
Transmission electron microscope data show that the surface of the extruded nanoparticles is obviously covered with a layer of complete membrane structure, and the structure of the nanoenzyme is not affected in the extrusion process (as shown in figure 1 c).
Example 2 stability examination of Nanolase photoacoustic imaging contrast Agents of different formulations
Suspending the nano-enzyme photoacoustic imaging contrast agent in PBS (phosphate buffered saline) with pH 7.2 and cell culture medium (DMEM) containing 10% FBS, filling the nano-enzyme photoacoustic imaging contrast agent into a hollow agar gel rod, collecting photoacoustic signals at 0h, 12h, 24h and 48h by a photoacoustic imager, and drawing a graph of photoacoustic signal intensity along with time. The result is shown in fig. 2, as the time is prolonged (within 48 h), the photoacoustic imaging images and the photoacoustic signal intensity of the three contrast agents have no obvious trend of decreasing, which shows that the prepared nano enzyme photoacoustic contrast agent wrapped by the exosome bionic corpuscle has good photoacoustic signal stability, and the photoacoustic signal cannot be rapidly leaked out of the exosome bionic corpuscle along with the time, so that the feasibility of subsequent application can be determined to a great extent, and a foundation is laid for clinical application.
Example 3 evaluation of targeting and endocytosis abilities of exosome-biomimetic corpuscle-encapsulated nanoenzyme photoacoustic contrast agent at in vitro cell level
Although the modification of the exosome biomimetic corpuscle can prolong the circulation time of the nano-enzyme photoacoustic contrast agent in blood, the nano-enzyme photoacoustic contrast agent also has the capability of improving the uptake of tumor cells to realize the targeted delivery of nano-particles. The main target of the folic acid molecule is the folic acid receptor, and the expression level of the folic acid receptor on the surface of the CNE-2 cell directly influences the targeting capability and the treatment effect of the contrast agent. Therefore, we examined CNE-2 cell surface integrin expression using confocal laser technique and used NIH3T3 as a non-tumor cell model (negative control). As shown in FIGS. 3a and 3b, the expression level of the folate receptor on the surface of the tumor cell CNE-2 is higher than that of NIH3T3, which is helpful for improving the affinity of FA-RM GQDzyme/ABTS with the tumor cell CNE-2, thereby achieving the purpose of promoting the uptake and improving the drug effect. The metabolism and transportation path of the nano-particles after entering the cells through the endocytosis plays a key role in the drug effect. From the data analysis in fig. 3c and 3d, it can be seen that most of the exosome-biomimetic corpuscle-coated nanoenzymes are not co-localized with lysosomes. The reason is that the erythrocyte membrane of the constructed exosome bionic corpuscle has fluidity and is easy to fuse with the cell membrane of the tumor, in addition, the contact area of the nano enzyme contrast agent and the cell is increased due to the flowing of the erythrocyte membrane, so that more folic acid is combined with the receptor on the surface of the tumor, and finally, the nano enzyme contrast agent can escape from the lysosome and directly enter the cytoplasm of the tumor to release the medicine.
Example 4 examination of tumor diagnosis ability of Nanolase contrast Agents by photoacoustic imaging in vivo
In order to research the effect of FA-RM, namely GQDzyme/ABTS in-vivo tumor photoacoustic imaging, after a nasopharyngeal carcinoma tumor-bearing mouse model is successfully constructed, the mouse is subjected to tail vein administration by using RM, namely ABTS, RM, GQDzyme, RM, namely GQDzyme/ABTS, and FA-RM, namely GQDzyme/ABTS, respectively, anesthetized by using 2.5% isoflurane, and mouse images are collected by using a photoacoustic imager before injection and after injection for 2h, 4h and 8 h. As shown in FIG. 4, the FA-RM, GQDzyme/ABTS, was distributed most in tumor tissues, and exhibited excellent tumor targeting. Further verifies that a complex composed of GQDzyme and ABTS can be used as a nano enzyme photoacoustic contrast agent to generate photoacoustic signals, and the coating of exosome bionic bodies constructed by erythrocyte membranes prolongs the blood circulation time of the drug-loaded nano enzyme contrast agent, effectively avoids the removal of an endothelial reticulum system, and has more time windows to enter tumor tissues by utilizing the EPR effect. Meanwhile, the photo-thermal absorption property of GQDzyme in a near infrared region is found to generate a photoacoustic signal, so that the photoacoustic signal of the nano enzyme photoacoustic contrast agent can be further enhanced.
Example 5 in vivo near-infrared fluorescence imaging verification of imaging Effect of Nanolase photoacoustic contrast Agents
CNE-2 tumor-bearing mice were injected with contrast agent (10. mu.g/100. mu.L) via the tail vein, and at various time points, the mice were anesthetized and scanned using a small animal in vivo imaging system. And (4) killing the mice after the last in vivo imaging data acquisition is finished, picking the tumor and tissue of each mouse and carrying out corresponding fluorescence imaging. As shown in FIG. 5, the exosome-biomimetic corpuscle chain endows the nanoenzyme contrast agent with good stealth capacity, and the RM: GQDzyme/ABTS and the FA-RM: GQDzyme/ABTS do not show a large accumulation in the liver and spleen of a mouse. Due to the introduction of the folic acid targeting molecule, the tumor targeting capability of the nano enzyme contrast agent is improved, so that the accumulation amount of the nano enzyme contrast agent at a tumor part is increased.
Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.
Claims (6)
1. A method for preparing nanoenzyme biomimetic vesicles loaded with nanoenzyme and azine diammonium salt contrast agent, comprising the steps of:
(1) preparing a carbon dot nano enzyme and nitrogen-linked diammonium salt complex contrast agent;
(2) preparing a bionic corpuscle with the particle size of 400 nm;
(3) loading the nano enzyme and azine diammonium salt complex contrast agent into a bionic corpuscle to prepare a nano enzyme bionic corpuscle;
the method for preparing the carbon dot nanoenzyme and azine diammonium salt complex contrast agent in the step (1) comprises the following steps:
1) adding polyacrylonitrile carbon fiber into a mixed solution of concentrated sulfuric acid and nitric acid; ultrasonic mixing and dissolving, heating to 100 ℃ and stirring fully;
2) ultrafiltration solution with molecular weight cut-off of 3KD and freeze-drying to obtain carbon nano enzyme;
3) mixing carbon dot nano enzyme and azino diammonium salt in an aqueous solution, stirring overnight, ultrafiltering, and freeze-drying to obtain a nano enzyme and azino diammonium salt complex contrast agent;
the method for preparing the bionic corpuscle with the particle size of 400nm in the step (2) comprises the following steps:
1) adding the red blood cells into a precooled hypotonic buffer solution, uniformly mixing, and then placing at 4 ℃ for 1-2 h to ensure that the red blood cells are completely hemolyzed;
2) centrifuging at 4 deg.C to remove hemoglobin supernatant, and collecting precipitate as erythrocyte membrane;
3) repeating centrifugal washing for 3-5 times;
4) and carrying out ultrasonic treatment to obtain the bionic corpuscle.
2. The method of claim 1,
the method for filling the nano enzyme and the azinam diammonium salt complex contrast agent into the bionic corpuscles in the step (3) comprises the following steps:
and loading the nano enzyme and the azino diammonium salt complex contrast agent into the bionic corpuscles in a physical extrusion mode to obtain the nano enzyme bionic corpuscles.
3. Nanoenzyme biomimetic bodies prepared using the method of claim 1 or 2.
4. The use of the nanoenzyme biomimetic corpuscles as claimed in claim 3 for preparing tumor targeting preparations,
the tumor targeting preparation comprises: loading the tumor therapeutic agent into the nano enzyme bionic corpuscle,
the tumor therapeutic agent is an antibody, a polypeptide, a nucleic acid aptamer or folic acid.
5. Use of the nanoenzyme biomimetic bodies of claim 3 in the preparation of a tumor photoimaging contrast agent, wherein the tumor is a solid tumor.
6. The use of claim 5, wherein the tumor photoimaging contrast agent is a contrast agent administered intravenously to mark the focal site of a solid tumor.
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