Dual-targeting bionic hydrotalcite inorganic nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to dual-targeting bionic hydrotalcite inorganic nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: obtaining a fresh erythrocyte membrane; obtaining an aged erythrocyte membrane; adding a fresh erythrocyte membrane and an aged erythrocyte membrane into the hydrotalcite inorganic nano-particles according to a certain proportion, mixing and stirring, and centrifuging to obtain the bionic hydrotalcite inorganic nano-particles. The dual-targeting bionic hydrotalcite inorganic nano-particles have the beneficial effects that the dual-targeting bionic hydrotalcite inorganic nano-particles have dual-targeting capability of being delivered to parenchymal hepatic cells and hepatic macrophages in a balanced manner, and oxidative stress and inflammatory response can be relieved at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug preparation, and more specifically, to dual-target bionic hydrotalcite inorganic nanoparticles and a preparation method and application thereof. Background Art

[0002] Acute liver failure (ALF) is a severe acute liver disease syndrome caused by extensive liver cell damage. It can cause extensive liver cell necrosis, liver dysfunction, a sharp decrease in liver weight, and a high mortality rate in a short period of time. The main clinical manifestations are oxidative stress in the liver, while inflammatory cells infiltrate and produce an inflammatory response, leading to liver damage and systemic inflammation. Since patients do not have pre-existing liver disease and the onset is rare and rapid, it is largely impossible to effectively prevent and avoid the occurrence of the disease, which poses a huge challenge to public health in my country and even the world. However, existing drugs are difficult to simultaneously treat the liver oxidative stress and inflammatory response caused by ALF. Therefore, how to simultaneously relieve liver oxidative stress and reduce inflammatory response is the key to the treatment of ALF. Summary of the Invention

[0003] The purpose of the present invention is to address the deficiencies of the prior art and to provide a dual-target bionic hydrotalcite inorganic nanoparticle and a preparation method and application thereof.

[0004] In a first aspect, a method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles is provided, comprising:

[0005] S1. Obtain fresh red blood cells, add ethylenediaminetetraacetic acid solution to the fresh red blood cells, perform hypotonic lysis for a certain period of time, and then centrifuge to collect the precipitate to obtain fresh red blood cell membranes;

[0006] S2. Storing fresh red blood cells for a certain period of time to obtain senescent red blood cells, adding ethylenediaminetetraacetic acid solution to the senescent red blood cells, performing hypotonic lysis for a certain period of time, and then centrifuging to collect the precipitate to obtain senescent red blood cell membranes;

[0007] S3. Adding the fresh erythrocyte membrane and the aged erythrocyte membrane into the hydrotalcite inorganic nanoparticles in a certain ratio, mixing and stirring, and centrifuging to obtain biomimetic hydrotalcite inorganic nanoparticles.

[0008] Preferably, in S1 and S2, the concentration of the EDTA solution is 0.1-0.2 mM, the hypotonic lysis time is 10-30 min, the centrifugal force of the centrifugation is 15000-20000 g, and the centrifugation time is 10-20 min.

[0009] Preferably, in S3, bovine serum albumin is further added, and the mass concentration of the bovine serum albumin is 5-10 times the mass concentration of the hydrotalcite inorganic nanoparticles.

[0010] Preferably, in S3, the mass ratio of the fresh erythrocyte membrane to the aged erythrocyte membrane is 1-5:1.

[0011] Preferably, in S3, the total concentration of the fresh erythrocyte membranes and the aged erythrocyte membranes is 0.02-0.05 times the mass concentration of the hydrotalcite inorganic nanoparticles, and the mixing and stirring time is 8-14 hours.

[0012] In a second aspect, dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared by any method described in the first aspect are provided.

[0013] In a third aspect, a use of the dual-targeted biomimetic hydrotalcite inorganic nanoparticles as described in the second aspect in the preparation of anti-ALF drugs is provided.

[0014] The beneficial effects of the present invention are:

[0015] 1. The dual-targeted biomimetic hydrotalcite inorganic nanoparticles of the present invention have the dual-targeting ability of balanced delivery to hepatic parenchymal cells and liver macrophages, which can simultaneously alleviate oxidative stress and inflammatory response.

[0016] 2. The dual-targeted bionic hydrotalcite inorganic nanoparticles of the present invention have clear composition, uniform particle size and a particle size distribution of about 100 nm.

[0017] 3. The preparation method of the dual-targeted bionic hydrotalcite inorganic nanoparticles in the present invention is simple, the process is controllable, and the materials used have high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Transmission electron microscopy image of dual-targeted biomimetic hydrotalcite inorganic nanoparticles;

[0019] Figure 2 is the particle size distribution diagram of dual-targeted biomimetic hydrotalcite inorganic nanoparticles;

[0020] Figure 3 The figure shows the co-localization results of dual-targeted biomimetic hydrotalcite inorganic nanoparticles in hepatic parenchymal cells and macrophages;

[0021] Figure 4 The test results of dual-targeted biomimetic hydrotalcite inorganic nanoparticles in reducing intracellular malondialdehyde levels

[0022] Figure 5 This is a test result diagram of dual-targeted biomimetic hydrotalcite inorganic nanoparticles reducing intracellular interleukin-6 levels. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the following examples. The following examples are provided only to facilitate understanding of the present invention. It should be noted that, without departing from the principles of the present invention, it is possible for a person skilled in the art to make various modifications to the present invention, and such improvements and modifications fall within the scope of the claims of the present invention.

[0024] LDHs are an emerging class of inorganic layered nanomaterials with a two-dimensional structure. They possess a large specific surface area and are capable of accommodating a wide variety of compounds. LDHs possess numerous favorable biological properties, including high biocompatibility, high chemical stability, high loading capacity, and low toxicity. They possess anti-inflammatory and antioxidant properties, and loading drugs can significantly reduce their dosage. In the biomedical field, LDHs are a promising drug delivery vehicle. Small molecules, genes, peptides, and other drugs can be loaded onto LDHs through surface electrostatic adsorption or interlayer anion exchange. LDHs have been used in the study of various diseases, including in the treatment of liver disease.

[0025] Erythrocyte membranes (RMs) have been used in the construction of various drug delivery systems. The nanomedicines coated with them have good biocompatibility and can achieve long-term circulation in the body, making them an ideal targeted delivery tool. It is worth noting that fresh erythrocyte membranes highly express CD47 protein, which can be recognized by signal regulatory protein α (SIRPα) on macrophages and release a "don't eat me" signal to escape phagocytosis by macrophages. However, aged erythrocytes can be directly identified and cleared due to the lack of CD47 expression. Based on this characteristic of macrophages, different liver cell targeting effects can be achieved by utilizing erythrocyte membranes in different physiological states. Starting from two different cell systems in the liver, normal hepatocytes and macrophages, oxidative stress and inflammatory responses can be treated simultaneously, maximizing the therapeutic ability of the drug.

[0026] In summary, based on the problems existing in existing treatment methods, we are committed to providing a preparation method of hydrotalcite inorganic nanoparticles with simple synthesis method and dual targeting, and applying it to the balanced delivery of hepatocytes and macrophages in the treatment of ALF, so as to simultaneously alleviate oxidative stress and inflammatory response.

[0027] The present invention is further described in detail below with reference to specific embodiments.

[0028] Example 1:

[0029] As an embodiment, the present application provides a method for preparing dual-targeted biomimetic hydrotalcite inorganic nanoparticles, comprising:

[0030] S1. Add 0.2 mM ethylenediaminetetraacetic acid solution to fresh red blood cells, perform hypotonic lysis for 30 minutes, and then centrifuge (centrifugal force of 20,000 g for 20 minutes) to collect the precipitate to obtain fresh red blood cell membranes.

[0031] S2. Fresh red blood cells were placed at 4°C for 14 days to obtain senescent red blood cells. A 0.2 mM ethylenediaminetetraacetic acid solution was added to the senescent red blood cells, and the cells were hypotonicly lysed for 30 minutes and then centrifuged (20,000 g for 20 minutes). The precipitate was collected to obtain the senescent red blood cell membranes.

[0032] S3. Add 5 mg / mL bovine serum albumin with a concentration of 5 times the mass concentration of hydrotalcite inorganic nanoparticles to the hydrotalcite inorganic nanoparticles, and then add the fresh and aged red blood cell membranes obtained in steps S1 and S2 to the hydrotalcite inorganic nanoparticles at a mass ratio of 1:1. The total concentration of fresh and aged red blood cell membranes is 0.02 times the mass concentration of hydrotalcite inorganic nanoparticles. Mix and stir for 8 hours, and centrifuge to obtain biomimetic hydrotalcite inorganic nanoparticles (RBLN).

[0033] The dual-targeted bionic hydrotalcite inorganic nanoparticles prepared in the present application have clear composition, uniform particle size, simple preparation method, and dual-targeting ability of balanced delivery to liver parenchymal cells and liver macrophages.

[0034] Example 2:

[0035] As another embodiment, the present application provides a method for preparing dual-targeted biomimetic hydrotalcite inorganic nanoparticles, comprising:

[0036] S1. Add 0.1 mM ethylenediaminetetraacetic acid solution to fresh red blood cells, perform hypotonic lysis for 10 minutes, and then centrifuge (centrifugal force 15000g, centrifugation time 10 minutes) to collect the precipitate to obtain fresh red blood cell membranes;

[0037] S2. Fresh red blood cells were placed at 4°C for 7 days to obtain senescent red blood cells. A 0.1 mM ethylenediaminetetraacetic acid solution was added to the senescent red blood cells, and the cells were hypotonicly lysed for 10 minutes and then centrifuged (at a centrifugal force of 15,000 g for 10 minutes). The precipitate was collected to obtain the senescent red blood cell membranes.

[0038] S3. Add 10 mg / mL bovine serum albumin with a concentration 10 times the mass concentration of hydrotalcite inorganic nanoparticles to the hydrotalcite inorganic nanoparticles, and then add the fresh and aged red blood cell membranes obtained in steps S1 and S2 to the hydrotalcite inorganic nanoparticles at a mass ratio of 5:1. The total concentration of fresh and aged red blood cell membranes is 0.05 times the mass concentration of the hydrotalcite inorganic nanoparticles. Mix and stir for 14 hours, and centrifuge to obtain biomimetic hydrotalcite inorganic nanoparticles (RBLN).

[0039] Comparative Example 1:

[0040] The only difference from Example 1 is that in step S3, the mass ratio of fresh and aged red blood cell membranes is 10:1.

[0041] Comparative Example 2:

[0042] The only difference from Example 1 is that in step S3, the total concentration of fresh and aged erythrocyte membranes is 0.1 times the mass concentration of the hydrotalcite inorganic nanoparticles.

[0043] Effect experiment

[0044] Experiment 1

[0045] Experimental methods:

[0046] Transmission electron microscopy was used to determine the red blood cell membrane coating of the samples, and a dynamic light scattering analyzer, Malvern Panalytical Zetasizer Pro, was used to determine the particle size of the samples. The equilibrium time was 40 s, and each sample was measured three times and the average value was taken.

[0047] Experimental results analysis:

[0048] The results of transmission electron microscopy are shown in Figure 1 , Figure 1 Wherein A is Example 1, Figure 1 B is comparative example 2, Figure 1 C is comparative example 2. Figure 1 It can be seen that the dual-targeted biomimetic hydrotalcite inorganic nanoparticles obtained in the present invention and comparative examples 1-2 are coated with erythrocyte membranes, and are uniform in size and evenly distributed. The particle size of the samples was measured by dynamic light scattering analyzer. The particle size of the dual-targeted biomimetic hydrotalcite inorganic nanoparticles obtained in the present invention is uniformly distributed at about 100 nm, the particle size of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in comparative example 1 is uniformly distributed at about 110 nm, and the particle size of the naringin-loaded hydrotalcite inorganic nanoparticles obtained in comparative example 2 is uniformly distributed at about 130 nm. Figure 2 .

[0049] Experiment 2

[0050] Experimental methods:

[0051] The dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared by the present invention were incubated with mouse liver parenchymal cells and macrophages in a co-culture system at 37°C for 8 hours to evaluate the uptake of the dual-targeted biomimetic hydrotalcite inorganic nanoparticles by different liver cells, thereby preliminarily determining the dual-targeted delivery effect of the nanoparticles. The results are shown in FIG. Figure 3 .

[0052] Experimental results analysis:

[0053] like Figure 3 As shown, after 100 μg / mL of dual-targeted bionic hydrotalcite inorganic nanoparticles were co-incubated with the mouse hepatocytes and macrophages co-culture system for 8 hours, the dual-targeted bionic hydrotalcite inorganic nanoparticles obtained by the present invention were evenly distributed in the two cells, with the uptake by mouse hepatocytes being 55% and the uptake by mouse macrophages being 45%. The uptake by mouse hepatocytes of the dual-targeted bionic hydrotalcite inorganic nanoparticles obtained in Comparative Example 1 was 75%, and the uptake by mouse macrophages was 25%. The uptake by mouse hepatocytes of the dual-targeted bionic hydrotalcite inorganic nanoparticles obtained in Comparative Example 2 was 35%, and the uptake by mouse macrophages was 65%, indicating that the dual-targeted bionic hydrotalcite inorganic nanoparticles obtained by the present invention can be delivered to hepatocytes and macrophages in a balanced manner, with a dual-targeting effect, laying the foundation for the subsequent simultaneous treatment of oxidative stress and inflammatory response of ALF.

[0054] Experiment 3

[0055] Experimental methods:

[0056] The dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared by the present invention were co-incubated with 1mM acetaminophen (APAP)-induced damaged mouse liver parenchymal cells at 37°C for 24h to evaluate the restoration effect of the dual-targeted biomimetic hydrotalcite inorganic nanoparticles on the malondialdehyde (MDA) level of damaged cells, thereby preliminarily determining the in vitro antioxidant stress effect of the material. The results are shown in FIG. Figure 4 .

[0057] Experimental results analysis:

[0058] like Figure 4 As shown, the MDA content in each group was 0.16nmol / mgprot, 0.53nmol / mgprot, 0.19nmol / mgprot, 0.27nmol / mgprot and 0.34nmol / mgprot, respectively. Compared with Comparative Examples 1-2, the 100μg / mL dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared by the present invention showed a stronger effect of reducing MDA, and the intracellular MDA level could be reduced to 0.19nmol / mgprot, which is close to the level of the normal group, indicating that the dual-targeted biomimetic hydrotalcite inorganic nanoparticles obtained by the present invention have a stronger therapeutic advantage in alleviating cellular oxidative stress damage.

[0059] Experiment 4

[0060] Experimental methods:

[0061] The dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared by the present invention were co-incubated with 1mM APAP-induced damaged mouse liver parenchymal cells at 37°C for 24h to evaluate the recovery effect of the loaded dual-targeted biomimetic hydrotalcite inorganic nanoparticles on interleukin-6 (IL-6) in the damaged cells, thereby preliminarily determining the in vitro anti-inflammatory effect of the material. The results are shown in FIG. Figure 5 .

[0062] Experimental results analysis:

[0063] like Figure 5 As shown, the IL-6 content in each group was 44.5 pg / mL, 249.8 pg / mL, 54.8 pg / mL, 105.2 pg / mL, and 122.3 pg / mL, respectively. Compared with Comparative Examples 1-2, the 100 μg / mL dual-targeted biomimetic hydrotalcite inorganic nanoparticles prepared in the present invention showed a stronger effect in reducing IL-6, reducing the IL-6 content in the cell culture medium to about 54.8 pg / mL, restoring it to a near-normal level of 44.5 pg / mL, indicating that the dual-targeted biomimetic hydrotalcite inorganic nanoparticles obtained in the present invention have a stronger therapeutic advantage in alleviating cellular inflammatory responses.

[0064] In summary, this application provides a preparation and application of dual-targeted biomimetic hydrotalcite inorganic nanoparticles. The biomimetic hydrotalcite inorganic nanoparticles prepared by this method have stable structure, uniform particle size, high biosafety, and can effectively achieve balanced delivery to hepatocytes and macrophages.

Claims

1. A method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles, characterized in that: include: S1. Obtain fresh red blood cells, add ethylenediaminetetraacetic acid solution to the fresh red blood cells, perform hypotonic lysis for a certain period of time, and then centrifuge to collect the precipitate to obtain fresh red blood cell membranes; S2. Storing fresh red blood cells for a certain period of time to obtain senescent red blood cells, adding ethylenediaminetetraacetic acid solution to the senescent red blood cells, performing hypotonic lysis for a certain period of time, and then centrifuging to collect the precipitate to obtain senescent red blood cell membranes; S3. Adding the fresh erythrocyte membrane and the aged erythrocyte membrane into the hydrotalcite inorganic nanoparticles in a certain ratio, mixing and stirring, and centrifuging to obtain biomimetic hydrotalcite inorganic nanoparticles.

2. The method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles according to claim 1, characterized in that: In S1 and S2, the concentration of the EDTA solution is 0.1-0.2 mM, the hypotonic lysis time is 10-30 min, the centrifugal force of the centrifugation is 15000-20000 g, and the centrifugation time is 10-20 min.

3. The method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles according to claim 2, characterized in that: In S3, bovine serum albumin needs to be added, and the mass concentration of the bovine serum albumin is 5-10 times the mass concentration of the hydrotalcite inorganic nanoparticles.

4. The method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles according to claim 3, characterized in that: In S3, the mass ratio of the fresh erythrocyte membrane to the aged erythrocyte membrane is 1-5:

1.

5. The method for preparing dual-target biomimetic hydrotalcite inorganic nanoparticles according to claim 4, characterized in that: In S3, the total concentration of the fresh erythrocyte membranes and the aged erythrocyte membranes is 0.02-0.05 times the mass concentration of the hydrotalcite inorganic nanoparticles, and the mixing and stirring time is 8-14 hours.

6. Dual-targeting biomimetic hydrotalcite inorganic nanoparticles prepared by the method according to any one of claims 1 to 5.

7. Use of the dual-targeting biomimetic hydrotalcite inorganic nanoparticles according to claim 6 in the preparation of anti-ALF drugs.

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