CN113995845B - Ivermectin preparation and preparation method and application thereof - Google Patents

Ivermectin preparation and preparation method and application thereof Download PDF

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CN113995845B
CN113995845B CN202111104375.9A CN202111104375A CN113995845B CN 113995845 B CN113995845 B CN 113995845B CN 202111104375 A CN202111104375 A CN 202111104375A CN 113995845 B CN113995845 B CN 113995845B
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ivermectin
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exosomes
viruses
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CN113995845A (en
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孙博
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Southeast University
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    • A—HUMAN NECESSITIES
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Abstract

The invention discloses an ivermectin preparation, a preparation method and application thereof, wherein the ivermectin preparation is prepared by taking an exosome as a drug carrier to load ivermectin. The invention uses exosomes generated by ACE2 positive human cells as a carrier, so that viruses taking ACE2 as a binding target spot can be combined with the carrier, on one hand, the combination degree of the viruses and human cells can be partially blocked, the virus virulence is reduced, and on the other hand, after the viruses are combined with ACE2 positive exosome preparations, when infection occurs, ivermectin can enter the cells together with virus genetic materials, and the replication of the viruses in target cells is prevented. The invention can effectively inhibit the infection of SARS-CoV-2 and other viruses on ACE2 positive cells.

Description

Ivermectin preparation and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to an ivermectin preparation and a preparation method and application thereof.
Background
Ivermectin is an antiparasitic agent widely used in the fields of human, livestock and poultry and plant protection. However, IN recent years many studies have demonstrated that ivermectin is capable of inhibiting recognition and binding of the input protein (IMP) a/b1 heterodimer to viral Integrase (IN), thereby inhibiting the RNA viral entry process and inhibiting viral infection. Presently, related studies have demonstrated that ivermectin can exert an effect of controlling infection by the above mechanism on a variety of viruses that rely on the realization of nuclear proliferation during infection, including HIV-1, DENV serotypes 1-4, influenza viruses, and the recently reported novel coronavirus SARS-CoV-2.
Different pharmaceutical formulations and delivery systems have been developed to optimize the efficacy of ivermectin. Research shows that the preparation technology of liposome, microemulsion, polymer micelle and the like can obviously improve the antiparasitic effect of ivermectin and the safety of ivermectin to a host. However, the antiviral properties of ivermectin have not been fully explored, while the lack of a suitable formulation can improve the intracellular internalization of ivermectin and reduce the adverse effects of the drug on the body.
However, although the traditional preparations such as ivermectin liposome can change the distribution of the medicament in vivo and improve the utilization efficiency of the medicament, no research shows that the preparations of ivermectin can target to virus-susceptible cells. The targeting of drugs is generally accomplished by modifying the relevant antibody on the carrier so that the drug carrier can bind to the target cell through receptor-ligand binding. However, this method requires the preparation of antibodies with high binding efficiency and high targeting property, and is extremely costly.
Disclosure of Invention
The invention aims to: the invention uses the exosome secreted by 16HBE as a carrier to load ivermectin, and can obtain a novel ivermectin pharmaceutical preparation which is easy to enrich in bronchial epithelial cells, thereby increasing the internalization of the ivermectin in the bronchial epithelium, accumulating to a higher level and generating the effect of limiting SARS-CoV-2 virus infection. The main technical problem solved by the invention is to provide a targeted ivermectin exosome preparation.
The invention also solves the technical problem of providing a preparation method and application of the ivermectin exosome preparation.
The technical scheme is as follows: the invention provides an ivermectin preparation, which is prepared by taking exosomes as drug carriers to load ivermectin.
Wherein the exosomes are exosomes derived from human cells or cell lines positive for ACE2 expression.
Wherein the ivermectin concentration is 1-3 mug/mL.
Wherein the diameter of the exosome is 50-200nm.
The invention also discloses a preparation method of the ivermectin preparation, which comprises the following steps:
1) Preparation of ACE2 positive exosomes: culturing human bronchial epithelial 16HBE cells in a conditioned medium, collecting the conditioned medium, and separating exosomes by centrifugation;
2) Preparation of ivermectin exosome: and (2) coating the isolated exosomes in the step (1) with ivermectin, mixing the exosomes with the ivermectin, adding a cold electroporation buffer solution for electroporation, recovering, and rotationally desalting to remove unincorporated ivermectin, thereby obtaining the ivermectin exosome preparation.
Wherein, the culture medium in the step 1) is as follows: 90% high sugar dmem+10% fetal bovine serum.
Wherein, the centrifugation step in the step 1) is as follows:
s1) centrifuging at 1000xg for 10-15 minutes to treat conditioned medium to remove cells and large debris;
s2) centrifuging at 10,000Xg for 20-30 minutes, and ultracentrifugating at 110,000Xg for 75-150 minutes;
s3) discarding the supernatant, and re-suspending the precipitate containing the exosomes to obtain the exosomes.
Wherein, in the step 2), the exosomes are obtainedThe concentration is 1-10 multiplied by 10 5 Preferably, the exosome concentration in step 2) is 5X 10 5 And each mL.
Wherein the final concentration of ivermectin in the step 2) is 1-3 mug/mL.
The invention also discloses application of the ivermectin preparation in preparing medicaments for treating SARS-CoV-2 virus infection diseases.
The exosome separation method and the exosome loading method are only used for illustrating the invention, and other exosome separation and exosome drug loading methods are also within the protection scope of the patent.
The present invention unexpectedly found that human bronchial epithelial cells 16 HBE-derived exosomes can naturally target the bronchial epithelial cells. Whereas the bronchial epithelium is a SARS-CoV-2 virus-infected cell. If the human bronchial epithelial cell 16 HBE-derived exosome is used for coating the ivermectin, the targeted delivery of the ivermectin to the airway epithelial cells can be realized in a convenient and low-cost mode, and the invasion infection of viruses is resisted.
Therefore, the invention prepares the ivermectin exosome preparation based on the exosome of the humanized ACE2 positive cells, promotes the internalization and enrichment of the ivermectin into the virus target cells, reduces the side effect of the medicine and effectively controls the virus infection.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the invention uses exosomes as the drug carrier of ivermectin, has more stable structure and is more beneficial to long-term storage and use of drugs;
2. the invention uses exosomes generated by ACE2 positive humanized cells as a carrier, so that an exosome preparation can more easily target ACE2 positive cells such as respiratory tract or vascular endothelium, thereby accelerating internalization and enrichment of ivermectin into the cells, improving drug targeting, improving drug effect, reducing drug dosage and improving safety of drug application.
3. The invention uses exosomes generated by ACE2 positive human cells as a carrier, so that viruses taking ACE2 as a binding target spot can be combined with the carrier, on one hand, the combination degree of the viruses and human cells can be partially blocked, the virus virulence is reduced, and on the other hand, after the viruses are combined with ACE2 positive exosome preparations, when infection occurs, ivermectin can enter the cells together with virus genetic materials, and the replication of the viruses in target cells is prevented. The invention can effectively inhibit the infection of viruses such as SARS-CoV-2 and the like on ACE2 positive cells by enriching the ivermectin into the cells through ACE2 positive liposome, and can improve the effect efficiency of the ivermectin by more than 2 times.
Detailed Description
The invention is further illustrated by the following examples, which are provided to illustrate the invention and are not intended to limit the scope of the invention. Those skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
The specific conditions not specified in the examples were either conventional or manufacturer-recommended. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The present invention uses all techniques known in the art, except as noted specifically.
Example 1 preparation of ACE2-positive exosomes
Human bronchial epithelial cells 16HBE cells (Wohunorace life technologies Co., ltd.) were washed twice in PBS (pH 7.2) and then cultured in 90% high-sugar DMEM+10% fetal bovine serum. After 3 days, the conditioned medium was collected and the exosomes were isolated by centrifugation. During the separation, the buffer was PBS (pH 7.2). The conditioned medium was treated by centrifugation at 1000Xg for 10 minutes to remove cells and large debris; centrifuging at 10,000Xg for 20 minutes; discarding the precipitate, and further treating the supernatant by ultracentrifugation at 110,000Xg for 75 minutes; the supernatant was discarded and the pellet resuspended to obtain the exosomes.
EXAMPLE 2 preparation of ivermectin exosome preparation
10 prepared in example 1 5 The exosomes were diluted to 200 μl in PBS (pH 7.2) and individually added with ivermectin (Sigma-Aldrich) to give final productsThe concentrations were 1. Mu.g/mL, 2. Mu.g/mL, 3. Mu.g/mL, respectively, followed by the addition of equal volumes of cold 2 Xelectroporation buffer (PBS pH7.2, 600mM sucrose), respectively. Electroporation was performed in a 4mm cuvette in Gene Pulser II Electroporator (BioRad) at 250V and 125. Mu.F. The exosomes were recovered at 37 ℃ for 20 minutes. Unincorporated ivermectin was removed by 3 consecutive rotary desalting columns (ThermoFisher Scientific), wherein in the last desalting step the sucrose concentration in the sample was gradually reduced to 150mM, 50mM and 10mM. High performance liquid chromatography was used to detect ivermectin content in exosomes. As shown in Table 1, the yield of ivermectin in the form of ivermectin exosome was 50% or more in the range of 1. Mu.g/mL to 3. Mu.g/mL, indicating that the exosome can be well loaded with ivermectin.
TABLE 1 results of recovery of ivermectin addition from exosomes
Addition value (ug/mL) Detection value (ug/mL) Recovery (%)
1 0.58±0.04 58％
2 1.06±0.10 53％
3 1.56±0.17 52％
Example 3 ivermectin exosome targeting validation
To examine the ability of an ACE 2-positive exosome ivermectin preparation to enrich ACE 2-positive cells, we applied the ACE 2-positive exosome ivermectin preparation prepared in example 2 (ivermectin content: 1.56. Mu.g/mL) to 10 7 Personal umbilical mesenchymal stem cells (Siro biosciences Co., ltd.; ACE2 Low expression) and 10 7 Personal bronchial epithelial cells 16HBE cells (Wohaze life technologies Co., ltd.; ACE2 high expression). At 37 ℃,5% CO 2 And incubated for 48 hours. After 3 washes with PBS, trypLE was used TM Express digestion for 5 minutes, cells were collected and washed centrifugally. The harvested cells were dissolved in 0.5ml of methanol, vortexed for 3 minutes, centrifuged at 2500 rpm for 15 minutes, and the supernatant was collected. The precipitate was again dissolved in 0.5ml of methanol, extracted once again, the supernatants were combined and the volume was set to 2ml. 10 μl of the sample was introduced and the ivermectin content was determined by HPLC. As shown in Table 2, the enrichment degree of ivermectin in umbilical mesenchymal stem cells is obviously lower than that of human bronchial epithelial 16HBE cells, which indicates that the ACE2 positive exosome ivermectin preparation can target ACE2 positive and inhibit infection of ACE2 positive cells by SARS-CoV-2 and other viruses.
TABLE 2 ivermectin Targeted enrichment
Cell species Detection value (ug/mL)
Umbilical cord mesenchymal stem cells 0.04±0.010
16HBE cells 0.28±0.068
Example 4 ivermectin exosome targeting validation
To examine that an ACE 2-positive exosome ivermectin preparation can promote the enrichment of ivermectin to ACE 2-positive cells, we applied the ACE 2-positive exosome ivermectin preparation (ivermectin content: 1.56. Mu.g/mL) prepared in example 2 and ivermectin (1.56. Mu.g/mL) to 10 respectively 7 Personal bronchial epithelial cells 16HBE cells (Wohaze life technologies Co., ltd.; ACE2 high expression). At 37 ℃,5% CO 2 And incubated for 48 hours. After 3 washes with PBS (pH 7.2), trypLE was used TM Express digestion for 5 minutes, cells were collected and washed centrifugally. The harvested cells were dissolved in 0.5ml of methanol, vortexed for 3 minutes, centrifuged at 2500 rpm for 15 minutes, and the supernatant was collected. The precipitate was again dissolved in 0.5ml of methanol, extracted once again, the supernatants were combined and the volume was set to 2ml. Sample 10 mu 0 was taken and the ivermectin content was determined by HPLC. The results are shown in Table 3, the ivermectin exosome preparation can obviously promote the enrichment degree of ivermectin in human bronchial epithelial cells 16HBE cells, and the intracellular concentration reaches 0.28 mug/mL; in the control group added with ivermectin only, the intracellular concentration is only 0.12 mug/mL, which shows that the use of the ACE2 positive exosome ivermectin preparation can improve the effect efficiency of the ivermectin by 2 times, is beneficial to reducing the dosage of medicaments and improves the safety of the medicaments.
TABLE 3 exosomes promote targeted intracellular enrichment of ivermectin
Species of type Detection value (ug/mL)
Ivermectin 0.12±0.027
Ivermectin exosome preparation 0.28±0.088
。

Claims (1)

1. The application of the ivermectin preparation in preparing the medicine for treating SARS-CoV-2 virus infection diseases is characterized in that the ivermectin content in the medicine is 1.56 mug/mL, and the ivermectin preparation specifically comprises the following steps:
1) Human bronchial epithelial 16HBE cells were washed twice in PBS and then cultured in conditioned medium, which is: 90% high sugar dmem+10% fetal bovine serum; after 3 days, the conditioned medium was collected and the exosomes were centrifuged, during which time the conditioned medium was treated to remove cells and large debris by centrifugation at 1000xg for 10 minutes; centrifuging at 10,000Xg for 20 minutes; discarding the precipitate, and further treating the supernatant by ultracentrifugation at 110,000Xg for 75 minutes; removing supernatant, and suspending the precipitate again to obtain exosomes;
2) 10 prepared in step 1) 5 The individual exosomes were diluted in PBS, each ivermectin was added, then each equal volume of cold 2x electroporation buffer was added, electroporation was performed at 250V and 125 μf, the exosomes were recovered at 37 ℃ for 20 min, unincorporated ivermectin was removed by 3 consecutive rotary desalting columns, wherein in the last desalting step the sucrose concentration in the sample was gradually reduced to 150mM, 50mM and 10mM, and the ivermectin content in the exosomes was detected using high performance liquid chromatography.
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