CN117045817A - Nucleic acid-encapsulated metal organic framework nanoparticle and preparation method and application thereof - Google Patents
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- CN117045817A CN117045817A CN202310870126.3A CN202310870126A CN117045817A CN 117045817 A CN117045817 A CN 117045817A CN 202310870126 A CN202310870126 A CN 202310870126A CN 117045817 A CN117045817 A CN 117045817A
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Abstract
The invention provides a nucleic acid-entrapped metal organic framework nanoparticle, a preparation method and application thereof, wherein the metal organic framework is EGCG/Zn entrapped with a cell-promoting functional nucleic acid CpGODN1826 2+ MOF nanoparticles. EGCG/Zn 2+ MOF is EGCG and Zn 2+ Stirring and self-assembling in buffer solution with pH of 7.4, and preparing EGCG/Zn with CpGODN1826 2+ The MOF is entrapped during the process. The nano metal organic framework is formed by stirring, coordination and self-assembly in a neutral environment, the preparation method is simple and controllable, and the particle size of the prepared nano particles is about 100nm. The book is provided withThe nano-metal organic framework has various activities, including promotion of macrophage cytokinesis, lipid degradation and cholesterol excretion, free radical removal and promotion of macrophage repolarization. When the nano metal organic framework is used for in vivo treatment of atherosclerosis through intravenous injection, the nano metal organic framework has good anti-atherosclerosis effect, can reduce plaque burden and increase plaque stability, and provides a new treatment strategy for atherosclerosis.
Description
Technical Field
The invention relates to the field of nano materials and nano biological medicines, in particular to a metal organic framework nanoparticle for encapsulating nucleic acid, and a preparation method and application thereof.
Background
Atherosclerosis (AS) is a major cause of a variety of cardiovascular diseases, and plaque formation is the pathological basis of AS. The existing AS therapeutic drug mainly adopts lipid-lowering treatment, can only reduce lipid in blood plasma, delay plaque formation, and has weak plaque clearance capability. Plaque removal is currently mainly dependent on surgery, but treatment cost is high, postoperative complications and postoperative recurrence risks are often caused, and the removal of specific plaques has the problem of high surgical difficulty, so that clinical application effects are poor. Therefore, there is an urgent clinical need for a means of drug treatment that is effective in clearing AS plaque.
The cytocidal effect of restoring macrophages is key to eliminating AS plaques, and a great deal of research focuses on the implementation by blocking the "don't eat me" signal of CD47, but inevitably causes side effects such AS anemia. The oligonucleotide CpG can assist macrophages to avoid a CD 47-mediated anticytosis pathway and restore phagocytic function of apoptotic cells in plaques. However, restoration of cyto-burying is only the first step in plaque clearance, and if phagocytosed lipid plaque fails to digest, degrade and excrete in time, it may lead to more foam cell formation, increasing plaque burden.
Furthermore, cpG ODN1826 as a nucleic acid molecule has problems in application that its half-life is short due to degradation by ribozymes, and because it is more difficult to enter cells negatively charged, it is often required for a vector to deliver it effectively to function. Traditional nucleic acid drug delivery vectors mainly comprise cationic polymers, liposomes and the like, but the vectors are easy to interact with negatively charged blood components after systemic administration, so that the structures of the vectors are recombined, aggregated or dissociated, the nucleic acid drugs are released in advance and degraded, and acute toxicity is generated. Therefore, the development of the high-efficiency low-toxicity drug carrier with the self-activity can promote the degradation and the discharge of lipid and finally remove the plaque to realize AS treatment while delivering CpG ODN1826 to promote cytoburied, and has important clinical significance.
Disclosure of Invention
In order to solve the technical problems, the invention uses Zn 2+ Coordination with epigallocatechin gallate (EGCG) to construct a nano-metal organic framework (EZC) for delivering the cytoburied functional nucleic acid CpG ODN 1826. In this structure, cpG ODN1826 promotes uptake of plaque apoptotic cells by macrophages through cytokinesis; zn (zinc) 2+ Activating autophagy, and degrading intracellular lipid burden into free cholesterol; EGCG upregulates ABCA1 transporter, allowing free cholesterol to be exported from the cell; the three are cooperated to activate the path of 'cyto-degradation-discharge', so AS to realize plaque lipid removal, and provide a high-efficiency low-toxicity non-operative strategy for AS plaque removal.
In order to achieve the above objective, the present invention firstly provides a nucleic acid-entrapped metal-organic framework nanoparticle, comprising a nucleic acid CpG ODN1826 and a metal-organic framework EGCG/Zn2+MOF, wherein the EGCG/Zn2+MOF is formed by self-assembly of EGCG and Zn2+ in buffer solution through stirring, and the nucleic acid CpG ODN18 is entrapped in the EGCG/Zn2+MOF to form a nucleic acid-entrapped metal-organic framework EGCG/Zn 2+ CpG nanoparticles. .
Preferably, the sequence of the nucleic acid CpG ODN18 is shown as SEQ ID NO. 1.
Preferably, the EGCG/Zn 2+ The CpG nanoparticle is spherical.
Preferably, the EGCG and Zn 2+ The molar ratio of CpG ODN18 is 500:500:1.
based on a general inventive concept, the invention provides a preparation method of metal organic frame nanoparticles for encapsulating nucleic acid, which comprises the following steps:
s1, dissolving EGCG in acetone to obtain EGCG mother liquor, and dissolving PLGA in acetone to obtain PLGA solution; mixing EGCG mother liquor with PLGA solution to obtain organic phase solution;
s2, sequentially mixing Hepes buffer solution with Zn 2+ Mixing with CpG ODN1826 to obtain mixed solution, and dripping while water-bath ultrasonic treatmentThe organic phase solution prepared in the step S1 is added and then subjected to water bath ultrasonic treatment;
s3, stirring the system prepared in the step S2 in a water bath in a dark place to volatilize acetone, and centrifuging to obtain the metal organic framework EGCG/Zn of the CpG ODN1826 of the invention 2+ CpG nanoparticles.
Preferably, the PLGA solution has a concentration of 20mg/mL.
Preferably, in the step S1, the volume ratio of EGCG mother liquor to PLGA solution is 1:1.
preferably, in step S2, hepes buffer ph=7.4.
Preferably, the water bath temperature in the step S2 is 30 ℃, and the ultrasonic time is 5min.
Preferably, the Hepes buffer concentration in step S2 is 10mM.
Preferably, the water bath temperature in the step S3 is 30 ℃, and the stirring time is 3 hours; the centrifugation speed was 16000rpm and the centrifugation time was 10min.
Based on a general inventive concept, the invention also provides application of the nucleic acid-entrapped metal organic framework nanoparticle in preparation of medicines for treating atherosclerosis.
EGCG/Zn according to the invention 2+ The mechanism by which CpG nanoparticles can be used for preparing medicines for treating atherosclerosis is as follows:
by Zn 2+ Coordination with epigallocatechin gallate (EGCG) to construct a nano-metal organic framework (EZC) for delivering nucleic acid CpG with a cytogenic function, and activating a 'cytogenic-degradation-excretion' path in cooperation with the nano-metal organic framework: 1) CpG ODN18 regulates and controls macrophages, promotes the cytocidal effect of the macrophages on apoptotic cells and normal apoptotic cells with high expression of CD47, and simultaneously increases the phagocytosis of foam cells; 2) Zn (zinc) 2+ Activating autophagy function of macrophage, promoting lipid degradation into free cholesterol; 3) Under the action of EGCG, the ABCA1 protein of macrophages is obviously up-regulated, mediates the excretion and transportation of free cholesterol to the outside of cells, enters a circulatory system through the transportation of high-density lipoprotein and finally is excreted along with feces, thereby realizing the effective elimination of AS plaque to realize the removal of plaque lipid, and being the elimination of AS plaqueBesides providing a non-operative strategy with high efficiency and low toxicity.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a preparation method and application of a nano metal organic framework for delivering nucleic acid with a cytokinesis function, and polyphenols EGCG and Zn 2+ During the coordination process, 100% entrapment of the oligonucleotide CpG ODN18 CpG can be achieved. The entrapped nucleic acid is formed by coordination and crosslinking of metal ion-organic ligand bonds, synthesis and complex preparation process and purification of materials are not needed, the preparation process is simple and controllable, green and safe, has low cytotoxicity and good biological safety, and solves the problems that the traditional nucleic acid medicaments are released and degraded in advance to generate acute toxicity.
2. EGCG/Zn provided by the invention 2+ The carrier has good protection effect on the CpG ODN18, and can prevent the CpG ODN18 from being degraded by in vivo enzymes; EZC CpG ODN18 can be effectively delivered into RAW264.7 macrophages, and can stay in lysosomes for a long time after entering cells for release; because the receptor TLR9 of the CpG ODN18 is positioned in the lysosome, the long-time stay of the CpG ODN18 in the lysosome is favorable for the combination of the CpG ODN18 and the TLR9, and the better pharmaceutical activity of the CpG ODN is favorable.
3. EGCG/Zn provided by the invention 2+ The CpG nanometer preparation can also remove free radical and promote macrophage to transform from pro-inflammatory M1 type to anti-inflammatory M2 type, thereby cooperating with the function of promoting 'cytocidal-degradation-excretion', and better realizing the function of resisting AS.
4. The invention provides an application of metal organic frame nanoparticles for delivering nucleic acid with a cytocidal function in preparing medicines for treating AS, wherein the metal organic frame nanoparticles can reduce circulating lipid level, reduce vascular plaque burden and increase plaque stability; the anti-AS effect can be exerted by promoting macrophage 'cytoburied-degradation-excretion' serial process, scavenging free radical and promoting macrophage M1 to M2 repolarization, thus providing a new potential non-operative therapy for AS treatment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows EGCG/Zn obtained by the test of test example 1 2+ Nanoparticles (EZ) and EGCG/Zn 2+ Characterization of CpG nanoparticles (EZC), fig. 1A shows the entrapment results of the carrier for CpG at different concentrations; FIG. 1B, C shows the particle size and potential results for EZ and EZC; FIG. 1D is an ultraviolet spectrum of EZ and EZC; FIG. 1E, F shows the transmission electron microscope images of EZ and EZC and the elemental analysis results; FIG. 1G shows the result of the CpG protection effect of the vector;
FIG. 2 is a graph showing the results of the test of experimental example 2 of the present invention, wherein EZ and EZC have different cell viability effects, and FIG. 2A is a graph showing the results of EZ's viability effects on RAW264.7, MOVAS, HUVEC cells; FIG. 2B is a graph showing the effect of EZC on the viability of RAW264.7, MOVAS, HUVEC cells;
FIG. 3 shows the cellular uptake results of the free CpG ODN18 CpG and EZC nanoparticles obtained by the test of Experimental example 3, and FIG. 3A shows the microscopic photographing results; FIG. 3B is a flow chart of the results of the flow test; FIG. 3C is the quantification result of FIG. 3B;
FIG. 4 is a laser confocal image of EZC and lysosome co-localization obtained by the detection of experimental example 4 of the present invention;
FIG. 5 is a graph showing the results of the test of Experimental example 5 in the present invention, wherein EZC promotes the cytoburied effect, and the target phagocytosed cells of FIG. 5A are apoptotic MOVAS cells highly expressing CD 47; FIG. 5B is a normal apoptotic MOVAS cell as phagocytosed target cells; FIG. 5C phagocytosed target cells are foam MOVAS cells; FIGS. 5D-F are the quantitative results of FIGS. 5A-C, respectively;
FIG. 6 shows the autophagy-promoting results of EZC obtained by the test of Experimental example 6, and FIG. 6A shows autophagy after treatment of each cell detected by the autophagy detection kit; FIG. 6B shows the expression of autophagy representative proteins LC3 alpha, LC3 beta, p62 after each cell treatment as detected by Western Blot; FIG. 6C is a plot of LC3 beta/alpha ratio results; FIG. 6D is a plot of the p 62/beta-actin ratio result;
FIG. 7 shows the results of the excretion promotion of EZC obtained by the test of Experimental example 7, and FIG. 7A shows the relative excretion rate of free cholesterol detected; FIG. 7B shows the expression of the efflux protein ABCA1 detected by Western Blot; FIG. 7C is a plot of ratio statistics for ABCA 1/beta-actin;
FIG. 8 shows the results of the repolarization of EZC scavenging free radicals and M1-M2 promotion obtained by the test of Experimental example 8, and FIGS. 8A-B show the results of DPPH free radical scavenging; FIGS. 8C-D are NO radical scavenging results; FIGS. 8E-F are OH radical scavenging results; FIGS. 8G-H are ABTS radical scavenging results; FIG. 8I is a photomicrograph of intracellular ROS; FIG. 8J, K is a flow chart of the results of detecting intracellular ROS scavenging, FIG. 8J is a quantitative analysis of FIG. 8K; FIG. 8L is a representative result of flow-through detection of macrophage polarization-associated molecules CD80 and CD206, FIG. 8M is a quantitative analysis of CD80 expression; FIG. 8N is a quantitative analysis of CD206 expression;
FIG. 9 is a flowchart of an animal experiment in which the detection of experimental example 9 of the present invention obtains EZC in vivo anti-AS results, and FIG. 9A is a flowchart of an animal experiment; figure 9B, C shows the change in animal body weight during dosing; FIG. 9D is a quantitative analysis of mouse abdominal aortic oil red O staining; fig. 9E is a photograph of the result of the red O staining of the abdominal aortic oil of the mice; FIGS. 9F-I are graphs showing the results of four tests on blood lipid levels of AS mice, including TG (F), CHO (G), HDL (H), and LDL (I), respectively; FIG. 9J-L shows the results of detection of inflammatory factors in AS mice, including IL6 (J), IL1 beta (K), and TNF alpha (L);
FIG. 10 is a graph showing the results of the detection of the aorta Dou Qiepian of the mice in experimental example 9 of the present invention, and FIG. 10A shows the results of the oil red O staining, masson staining, mac-3 immunohistochemistry, MMP9 immunohistochemistry and HE staining represented by aorta Dou Qiepian; FIGS. 10B-F are quantitative analyses of the above results, respectively;
FIG. 11 shows the immunohistochemical staining of aortic sinus sections of mice in Experimental example 9 of the present invention, and FIG. 11A shows the representative immunohistochemical staining of aorta Dou Qiepian, including ABCA1, LC3 beta, p62; FIGS. 11B-D are quantitative analyses of the above results, respectively;
FIG. 12 shows immunofluorescence double staining of aortic sinus sections of mice according to experimental example 9 of the present invention, and FIG. 12A shows immunofluorescence double staining results of aorta Dou Qiepian Mac-3 and caspase-3; FIG. 12B shows immunofluorescence double staining results of aortic arch Dou Qiepian CD80 and CD 206;
FIG. 13 shows the in vivo safety results of EZC obtained by the test of Experimental example 10 of the present invention, and FIGS. 13A-D show the results of the biochemical markers CRE, BUN, AST, ALT, respectively; FIG. 13E shows HE staining of heart, liver, spleen, lung, kidney of each group of mice;
figure 14 shows the mechanism of the invention EZC for treating atherosclerosis.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
Example 1
Preparation of EGCG/Zn 2+ Nanoparticles (EZ)
To the EP tube was added sequentially 1mL of 10mM Hepes (pH=7.4), 20. Mu.L of ZnCl 2 (100 mM), adding 40 mu L of an organic phase (the organic phase is obtained by mixing 20mg/mL PLAG and 100mM EGCG in a volume ratio of 1:1) while carrying out water bath ultrasound, carrying out ultrasound for 5min, carrying out light-shielding stirring for 3h in a 30 DEG water bath to volatilize acetone, and centrifuging at 16000rpm for 10min to collect precipitate, thereby obtaining the nanoparticle. Then re-dissolving with ultrapure water, and obtaining EGCG/Zn by ultrasonic probe for 3 cycles (20W, 3s on,3s off) 2+ (EZ) nanoparticle solution.
Example 2
Preparation of EGCG/Zn 2+ CpG nano-particle (EZC)
To the EP tube was added sequentially 1mL of 10mM Hepes (pH=7.4), 20. Mu.L ZnCl 2 (100 mM), 40. Mu.L of CpG ODN18 (100. Mu.M), 40. Mu.L of organic phase (obtained by mixing 20mg/mL PLAG with 100mM EGCG in volume ratio of 1:1) was added while water-bath ultrasound, after 5min ultrasound, stirring in a 30℃water bath in a dark place for 3h to volatilize acetone, and then centrifugation at 16000rpm for 10min to collect the precipitate, thus obtaining nanoparticles.Then re-dissolving with ultrapure water, and obtaining EGCG/Zn by ultrasonic probe for 3 cycles (20W, 3s on,3s off) 2+ CpG (EZC) nanoparticle solution.
Experimental example 1
1. Investigation of optimal dosing concentration of CpG ODN18
The measuring method comprises the following steps: under the condition of example 2, cpG-FAM is added to make its final concentration be 1, 2 and 4 mu M, after the reaction is completed, the supernatant is centrifugally taken, and EGCG/Zn is analyzed by gel electrophoresis 2+ The inclusion amount of CpG ODN18 in MOF.
As shown in FIG. 1A, when the concentration of CpG ODN18 is increased from 1. Mu.M to 4. Mu.M, no CpG expression is detected in the supernatant, which indicates that the inclusion of the CpG ODN18 by the vector is 100%, and in order to maximize the inclusion of the therapeutic drug, the concentration of CpG ODN18 is finally selected as 4. Mu.M.
2. Examination of particle size and potential of EZ and EZC
The EZ and EZC nanoparticle solutions prepared in examples 1-2 were taken and tested as follows: the sample solution was placed in a Marlven Nano ZS instrument and the particle size and potential were measured by dynamic light scattering with the cell temperature set at 25 ℃.
As shown in FIG. 1B and FIG. 1C, the EZ nanoparticles have a particle size of about 170nm and a potential of about-28 mV; EZC nanoparticles have a particle size of about 160nm and a potential of about-27 mV.
3. Investigation of the ultraviolet spectra of EZ and EZC
The ultraviolet spectrum scanning is respectively carried out on EZ and EZC nano particles, and the measuring method comprises the following steps: and scanning an ultraviolet absorption spectrum of 200-500nm by taking a blank solvent as a reference.
As shown in the measurement result in FIG. 1D, EZ and EZC nanoparticles have characteristic absorption peaks at-210 nm and-320 nm, which indicates that the nanoparticles EZC and the carrier EZ after being coated with CpG have no obvious property difference.
4. Detection of EZ and EZC morphology and elemental analysis
Observing morphology and analysis elements of EZ and EZC nano particles, wherein the detection method comprises the following steps: EZ and EZC samples are dripped on a copper mesh covered with a carbon film, dried in a dryer for 2-3 times, and then placed under a transmission electron microscope Titan G260-300, and the morphology is observed and elemental analysis and detection are carried out.
As shown in FIG. 1E, F, EZ and EZC have particle diameters of about 100nm under an electron microscope, and each contains C, N, O, zn elements; compared with EZ nanoparticles, the EZC nanoparticles contain P element, which indicates successful entrapment of CpG ODN 18.
5. Investigating the protection of vectors against CpG
The measurement method is as follows: according to the method of example 2, EZC nanoparticles prepared by using FAM-CpG were additionally prepared, free FAM-CpG with the same final concentration was co-incubated at 37℃for 3 hours under 5% FBS and 10% FBS, followed by incubation at 95℃for 10 minutes, EDTA was added to the system (to make the final concentration 100 mM), and the mixture was sonicated in a water bath for 30 minutes to allow the nanoparticles to be fully cleaved. Mixing the cracked supernatant with DNAloading buffer, loading urea gum with free FAM-CpG as control, and performing fluorescence development to observe degradation degree.
As shown in FIG. 1G, no degradation band was observed in group EZC, indicating that the vector has good protection to CpG ODN18, and thus the CpG ODN18 can be prevented from being degraded by in vivo enzymes.
Experimental example 2
Investigation of toxic effects of EZ and EZC nanoparticles on cells
EZ and EZC nanoparticles were prepared according to the conditions of example 1-2, RAW264.7 cells, MOVAS cells and HUVEC cells were seeded in 96-well plates one night in advance, respectively, and the cells were treated with different concentrations (0, 2.5, 5, 10, 20, 40. Mu.g/mL) of EZ and EZC nanoparticles for 24 hours, and then the preparation solution was discarded; incubating each cell with 0.5mg/mL MTT solution for 4 hours, and discarding the MTT solution; 100. Mu.L/well DMSO was added to dissolve the crystals generated and absorbance was measured at 490nm using a multifunctional microplate reader to assess the toxic effect of the nanoparticles on the cells.
The detection results are shown in fig. 2, and fig. 2A and 2B show that the cell survival rate of RAW264.7 cells, MOVAS cells and HUVEC cells is higher than 80% even under the condition of the highest administration concentration of 40 μg/mL, respectively, and the EZ and EZC nanoparticles are proved to have small cytotoxicity and good biosafety.
Experimental example 3
Investigation of EZC cellular uptake
FAM-EZC nanoparticles were prepared using FAM-CpG according to the conditions of example 2. RAW264.7 cells were plated in 24 well plates one night in advance, treated with free FAM-CpG and FAM-EZC nanoparticles for 4h, and treated wells without any treatment were used as control groups, after which the cells of each treatment group were examined for CpG uptake by flow and microscopic photographing, respectively. (1) microscopic photograph to detect cellular uptake: after the treatment of the cells with each preparation, removing the culture medium containing the preparation, and washing with PBS for 3 times; 4% paraformaldehyde fix cells for 15min (dark, room temperature); washing with PBS for 2 times, adding DAPI staining solution, 0.5 mL/hole, and incubating at room temperature in dark place for 5min; PBS was washed 3 times; finally, 0.5ml fbs/well was added and photographed under a microscope. (2) flow-through detection of cellular uptake: after the treatment of the cells with each preparation, the preparation-containing medium was removed, washed 3 times with PBS, 0.5mLPBS was added to each well, and the adherent RAW264.7 cells were blown down, collected into EP tubes, and run on a machine for flow detection.
The microscope photograph results are shown in fig. 3A, the flow-on results are shown in fig. 3B, and fig. 3C is a quantitative statistic for fig. 3B. From the results of these two assays, fluorescence was stronger in the FAM-EZC group than in the free CpG ODN18 group, indicating EZC that CpG ODN18 could be efficiently delivered into RAW264.7 macrophages.
Experimental example 4
Investigation of the lysosome co-localization of EZC
FAM-EZC nanoparticles were prepared using FAM-CpG according to the conditions of example 2. RAW264.7 cells were plated one night in advance into laser confocal dishes, treated with EZC nanoparticles for 2h, 4h, 12h, respectively, and washed 3 times with PBS to remove excess drug. Adding lysosome probes into each small dish in a light-shielding environment, and dyeing for 30min; washing with PBS 3 times, and fixing the cells with 4% paraformaldehyde for 15min (light-shielding, room temperature); washing with PBS for 2 times, adding DAPI staining solution, 0.5 mL/hole, and incubating at room temperature in dark place for 5min; PBS was washed 3 times, and finally 0.5 mLPBS/well was added and photographed by a laser confocal microscope.
As shown in FIG. 4, the fluorescence of lysosomes and CpG fluorescence almost completely overlap, indicating that EZC nanoparticle-entrapped FAM-CpG can stay in lysosomes for a long time after entering cells for release. Because the receptor TLR9 of the CpG ODN18 is positioned in the lysosome, the long-time stay of the CpG ODN18 in the lysosome is favorable for the combination of the CpG ODN18 and the TLR9, and the better pharmaceutical activity of the CpG ODN is favorable.
Experimental example 5
Investigation of the Cytometric action of EZC
Phagocytized 3 targeted cells were first constructed. (1) MOVAS cells are inoculated into a cell pore plate overnight, then treated by TNF-alpha to induce high expression of CD47, and then added with staurosporine to induce apoptosis, so AS to obtain the apoptotic MOVAS cells with high expression of CD47, so AS to simulate the characteristics of AS plaque part lesion cells. (2) MOVAS cells were induced to apoptosis using staurosporine to obtain normal apoptotic MOVAS cells. (3) MOVAS cells were treated with oXLDL for 48h to obtain foamed smooth muscle cells. These three phagocytosed target cells were then individually fluorescently labeled with cell tracker deep red dye. In addition, after RAW264.7 macrophages were plated overnight, EZC nanoparticles were added, and treated for 24 hours with the vector EZ nanoparticles as a control, and then cell tracker CMFDA cell dye was used to fluorescently label macrophages as phagocytes. And incubating the fluorescent-marked phagocytes and the fluorescent-marked phagocytes, collecting the cells, and detecting the proportion of the double positive cells by using a flow cytometry to obtain the cytochromes.
The results are shown in FIG. 5. FIGS. 5A-C are representative results of phagocytosis assays of apoptotic MOVAS cells, normal apoptotic MOVAS cells, and foam MOVAS cells, respectively, by RAW264.7 cells that highly expressed CD 47; FIGS. 5D-F are the quantitative results of FIGS. 5A-C, respectively. Therefore, compared with EZ nanoparticles, EZC nanoparticles can promote cytoburied effect of macrophages, and increase phagocytosis of apoptotic cells and foam cells, which proves phagocytosis of CpG ODN18 after encapsulation by EZ.
Experimental example 6
Investigation of the degradation-promoting (autophagy-promoting) effect of EZC
RAW264.7 cells were inoculated into an orifice plate in advance, the RAW264.7 cells were treated with EZ and EZ nanoparticles for 24 hours, respectively, no treatment was added as a control, and after the cell treatment was completed, the autophagy condition of each group was detected by staining with an autophagy kit (operating according to instructions) and performing fluorescence detection (ex=335 nm, em=512 nm) using a multifunctional microplate reader. In addition, western Blot was used to detect the expression of autophagy classical marker proteins (LC 3a, LC3 β and p 62), respectively. RAW264.7 cells were treated with EZ and EZC nanoparticles for 24 hours, and the total protein of the cells was extracted and the protein concentration was measured after the cell treatment was completed without any treatment. And spotting the proteins with equal mass on SDS-PAGE gel sample holes according to the protein concentration, carrying out electrophoresis and membrane transfer, adding a primary antibody after sealing by a sealing solution, rinsing at 4 ℃ overnight, adding a corresponding secondary antibody by TBST, and detecting by using ECL (electron cyclotron resonance imaging) color development liquid. And (3) measuring the optical density value of the target protein band, and analyzing the LC3 beta/alpha ratio and the expression change of the p62 protein by taking beta-actin as an internal reference.
The results are shown in FIG. 6. Fig. 6A shows the relative fluorescence intensities detected, with a significant increase in fluorescence after EZ and EZC nanoparticle treatment compared to the no treatment group, demonstrating that EZ and EZC nanoparticle treatment can enhance autophagy. FIG. 6B shows representative expression patterns of LC 3. Alpha., LC 3. Beta. And p62 detected by Western Blot. FIG. 6C is a plot of LC3 beta/alpha ratio statistics and FIG. 6D is a plot of p62/actin ratio statistics. It follows that EZ and EZC can enhance autophagy by up-regulating LC3 beta/alpha and down-regulating p62 expression, thereby increasing degradation of intracellular lipids.
Experimental example 7
Investigation of the efflux-promoting action of EZC
RAW264.7 cells were seeded overnight in well plates and then incubated with NBD cholesterol in 1640 medium without serum and phenol red for 24h. Cells were then treated with EZ and EZC nanoparticles, respectively, in serum-free medium. Thereafter, the medium containing the formulation was replaced with 10% fbs to initiate cholesterol efflux. Finally, fluorescence of the supernatant and the lysed cells was measured by a multifunctional microplate reader (ex=472nm, em=540 nm). Cholesterol efflux ratio is defined as the ratio of supernatant fluorescence to total fluorescence in the supernatant and cells. Furthermore, western Blot was used to detect expression of the efflux protein ABCA 1. RAW264.7 cells were treated with EZ and EZC nanoparticles for 24 hours, and the total protein of the cells was extracted and the protein concentration was measured after the cell treatment was completed without any treatment. And spotting the proteins with equal mass on SDS-PAGE gel sample holes according to the protein concentration, carrying out electrophoresis and membrane transfer, adding a primary antibody after sealing by a sealing solution, rinsing at 4 ℃ overnight, adding a corresponding secondary antibody by TBST, and detecting by using ECL (electron cyclotron resonance imaging) color development liquid. And (3) measuring the optical density value of the target protein band, and analyzing the expression change of the ABCA1 protein by taking beta-actin as an internal reference.
The results are shown in FIG. 7. Fig. 7A shows the relative cholesterol excretion rate detected, and the significant increase in cholesterol excretion rate after EZ and EZC nanoparticle treatment compared to the no treatment group, demonstrating that EZ and EZC nanoparticles can enhance cholesterol excretion. FIG. 7B shows representative ABCA1 expression in Western Blot detection. FIG. 7C is a plot of ABCA1/actin ratio statistics. It follows that EZ and EZC can promote cholesterol efflux by up-regulating ABCA1 expression.
Experimental example 8
Investigation of EZC scavenging free radicals and macrophage-promoting M1-M2 repolarization
EZC nanoparticles were prepared according to the conditions of example 2, and the free radical scavenging effect of EZC at the tube level was examined using commercially available DPPH, NO, OH, ABTS free radical detection kits (operating according to the instructions).
In addition, the ROS scavenging capacity of EZ and EZC at the cellular level was investigated using the DCFH-DA probe. The specific operation is as follows: RAW264.7 cells were seeded overnight in 6-well plates and then treated with 10 μg/mL Lipopolysaccharide (LPS) for 48 hours, resulting in excessive ROS production. LPS was removed and cells were treated with EZ and EZC nanoparticles (40. Mu.g/mL). Subsequently, cells were incubated with DCFH-DA probe for 30min, after which the probe was removed. The ROS scavenging image was directly taken with a fluorescence microscope for qualitative analysis, and the ROS scavenging capacity was quantitatively analyzed using a flow cytometer.
In addition, the capacity of EZC nanoparticles for promoting the transformation of macrophages M1-M2 is examined, and the specific operation flow is as follows: RAW264.7 cells were plated overnight in 24-well plates in advance and co-cultured with 10. Mu.g/mL LPS for 48 hours to induce macrophage M1 polarization. Cells were then incubated with EZ and EZC nanoparticles, respectively, for 24h. The nanofabrics were then removed and the cells were washed three times with PBS. Subsequently, the washed cells were collected and incubated with CD80 antibodies (typical M1 molecules) and CD206 antibodies (typical M2 molecules) respectively for 30min in the dark at 4 ℃. Finally, the repolarization of macrophages M1-M2 was assessed by flow cytometry to detect the average fluorescence intensity of CD80 and CD206 in each group of cells.
The results are shown in FIG. 8, and FIGS. 8A-B show DPPH radical scavenging results; FIGS. 8C-D are NO radical scavenging results; FIGS. 8E-F are OH radical scavenging results; FIGS. 8G-H are ABTS radical scavenging results. FIG. 8I is a photomicrograph of intracellular ROS; FIG. 8J, K shows the results of flow-through detection of intracellular ROS scavenging, and FIG. 8J shows the quantitative analysis of FIG. 8K. FIG. 8L is a representative result of flow-through detection of macrophage polarization-associated molecules CD80 and CD206, FIG. 8M is a quantitative analysis of CD80 expression; FIG. 8N is a quantitative analysis of CD206 expression. It follows that EZC scavenges DPPH, NO, OH, ABTS free radicals in a dose dependent manner at the tube level; at the same time EZC can remove ROS at the cellular level, which indicates EZC has good free radical removal effect. Furthermore, following EZC treatment, the expression of CD80, a representative molecule of the pro-inflammatory M1 type, was down-regulated and CD206, a representative molecule of the anti-inflammatory M2 type, was up-regulated, suggesting EZC may promote macrophage transformation from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.
Experimental example 9
Investigation of the anti-atherosclerosis Effect in EZC vivo
Feeding ApoE of 6 weeks of age on a high fat diet -/- Mice were modeled for 4 weeks to develop atherosclerosis, which were then randomized into 3 groups, physiological saline, EZ, and EZC, respectively. The atherosclerosis mice were injected via tail vein with EZ and EZC nanoparticles, 10 times per 5 days, and maintained on a high fat diet during the dosing period. After the start of dosing, mice were weighed and recorded weekly. After the end of dosing, mice were fasted overnight and then euthanized to collect blood, abdominal aorta and major viscera. Mouse plasma lipids including Triglycerides (TG), cholesterol (CHO), low Density Lipoproteins (LDL), high Density Lipoproteins (HDL) were assayed. The ELISA kit is used for detecting inflammatory cytokines in blood plasma, including IL1 beta, IL6 and TNF alpha. To assess vascular plaque burden, the abdominal aorta was stained using oil red O staining. In addition, plaque thickness and necrotic core were assessed by staining frozen sections of aortic sinuses with oil red O and HE.Also, to assess plaque stability, immunohistochemical staining (including Mac-3 and MMP-9) and masson staining were performed. To investigate their therapeutic mechanisms, immunohistochemical staining of ABCA1, LC3 beta and p62 was performed. To examine the repolarization of macrophages in vivo, immunofluorescent counterstaining of CD80 and CD206 was performed. In addition, mac-3 and caspase-3 immunofluorescence double staining was performed to examine the in vivo cytocidal condition, free apoptotic cells were indicated by asterisks, and macrophage-related apoptotic cells were indicated by arrows.
The results are shown in FIGS. 9-12. Figures 9-10 show the results of anti-atherosclerosis treatment in EZC. Fig. 9A is a flow chart of an animal experiment. Figure 9B, C shows weight changes in animals during dosing, and weight loss of EZ and EZC compared to the model group, demonstrating that EZ and EZC nanoparticles can maintain healthy weight in AS mice. Fig. 9D is a quantitative analysis of the staining of the abdominal aortic oil red O in mice, and fig. 9E is a picture of the results of the staining of the abdominal aortic oil red O in mice, demonstrating that EZ and EZC can significantly reduce the plaque burden in AS mice. FIGS. 9F-I show the results of four tests of AS mice blood lipid, EZ and EZC can obviously reduce plasma CHO and plasma LDL, and have good blood lipid reducing effect. FIGS. 9J-L show the results of detection of inflammatory factors in AS mice, and thus, it can be seen that EZ and EZC significantly reduce plasma inflammatory factor IL6.
FIG. 10A shows results of oil red O staining, masson staining, mac-3 immunohistochemistry, MMP9 immunohistochemistry and HE staining representative of aorta Dou Qiepian, and graphs B-F are quantitative analyses of the above results, respectively. It follows that EZ and EZC can reduce plaque thickness and necrotic core area; and simultaneously, the expression of collagen can be increased, and the expression of Mac-3 and MMP9 can be reduced, so that the plaque stability is improved.
FIGS. 11-12 show the therapeutic mechanism of atherosclerosis in EZC. FIG. 11A is a representative immunohistochemical staining of aorta Dou Qiepian, including ABCA1, LC 3. Beta., p62, and graphs B-D are quantitative analyses of the above results, respectively. EZ and EZC can increase expression of efflux protein ABCA1, promoting efflux; at the same time it increases the expression of LC3 beta and decreases the expression of p62, thereby promoting autophagy.
FIG. 12A shows the results of immunofluorescence double staining of aorta Dou Qiepian Mac-3 and caspase-3, showing that EZC promotes cytocidal action in EZC groups with more apoptotic cells phagocytosed by macrophages than EZ groups. FIG. 12B shows immunofluorescence double staining of aortic Dou Qiepian CD80 and CD206, and EZ and EZC treatment down-regulates CD80 expression and up-regulates CD206 expression compared to model group, demonstrating that EZ and EZC promote macrophage transformation from M1 to M2.
Fig. 14 is an overall mechanism diagram of EZC treatment of atherosclerosis.
Experimental example 10
Investigation of EZC in vivo safety
Mice were treated as in experimental example 9, and after the end of administration, serum from mice was collected for biochemical index analysis; the main organs of mice, namely heart, liver, spleen, lung and kidney, are fixed by 4% paraformaldehyde, and then paraffin embedded, sliced and H & E stained, and pathological changes are observed by using an optical microscope.
The results are shown in FIG. 13. FIGS. 13A-D show the results of biochemical measurements CRE, BUN, AST, ALT, respectively, without significant changes in blood biochemistry from EZ and EZC groups compared to the model group. Fig. 13E shows HE staining results of heart, liver, spleen, lung, kidney of mice of each group, and no significant pathological changes were found compared to the model group. The biological safety of EZ and EZC nanometer preparation in vivo is good.
The above embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention.
Claims (10)
1. The metal organic framework nanoparticle is characterized by comprising nucleic acid CpGODN1826 and metal organic framework EGCG/Zn 2+ MOF of the EGCG/Zn 2+ MOF is composed of EGCG and Zn 2+ Self-assembling in buffer solution under stirring, wherein the nucleic acid CpGODN18 is entrapped in EGCG/Zn 2+ Metal organic frameworks EGCG/Zn for nucleic acid entrapment in MOF 2+ CpG nanoparticles.
2. The nucleic acid-entrapped metal organic framework nanoparticle of claim 1, wherein the nucleic acid CpGODN18 sequence is shown in SEQ ID No. 1.
3. The nucleic acid entrapped metal organic framework nanoparticle of claim 1, wherein the EGCG/Zn is 2+ The CpG nanoparticle is spherical.
4. The nucleic acid entrapped metal organic framework nanoparticle of claim 1, wherein the EGCG, zn 2+ The molar ratio of CpGODN18 is 500:500:1.
5. a method for preparing the nucleic acid-entrapped metal organic framework nanoparticle according to any one of claims 1 to 4, comprising the steps of:
s1, dissolving EGCG in acetone to obtain EGCG mother liquor, and dissolving PLGA in acetone to obtain PLGA solution; mixing EGCG mother liquor with PLGA solution to obtain organic phase solution;
s2, sequentially mixing Hepes buffer solution with Zn 2+ Mixing CpGODN1826 to obtain a mixed solution, dropwise adding the organic phase solution prepared in the step S1 into the mixed solution system while carrying out water bath ultrasonic treatment, and continuing water bath ultrasonic treatment after adding;
s3, stirring the system prepared in the step S2 in a water bath in a dark place to volatilize acetone, and centrifuging to obtain the metal organic framework EGCG/Zn loaded with CpGODN1826 2+ CpG nanoparticles.
6. The method according to claim 5, wherein the volume ratio of EGCG mother liquor to PLGA solution in step S1 is 1:1.
7. the method of claim 5, wherein the Hepes buffer ph=7.4 in step S2.
8. The method according to claim 5, wherein the water bath temperature in the step S2 is 30 ℃ and the ultrasonic time is 5min.
9. The method according to claim 5, wherein the water bath temperature in the step S3 is 30 ℃ and the stirring time is 3 hours; the centrifugation speed was 16000rpm and the centrifugation time was 10min.
10. Use of the nucleic acid-entrapped metal-organic framework nanoparticles of any one of claims 1-4 or the nucleic acid-entrapped metal-organic framework nanoparticles prepared by the preparation method of any one of claims 5-9 in the preparation of a medicament for treating atherosclerosis.
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| CN116942842B (en) * | 2023-07-17 | 2024-05-07 | 无锡市第二人民医院 | Nano silver bonded with cytoblast promoting functional nucleic acid, and preparation method and application thereof |
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