CN115192584A - Nano medicine and its preparing method and use - Google Patents
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- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
The invention discloses a nano-drug and a preparation method and application thereof, and the nano-drug provided by the invention comprises a metal matrix and an organic ligand for modifying the metal matrix, wherein the organic ligand and the metal matrix generate a synergistic effect, and can effectively inhibit the proliferation of coronavirus and kill the coronavirus. The nano-drug has better prospect in preparing drugs for treating coronavirus. The invention also provides a preparation method and application of the nano-medicament.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nano-medicine and a preparation method and application thereof.
Background
Coronaviruses belong to the genus Coronaviridae (Coronaviridae) of the order Nidovirales (Nidovirales), and coronaviruses that have caused an epidemic situation include SARS-CoV (Severe acid Respiratory Syndrome), MERS-CoV (Middle East Respiratory Syndrome), and SARS-CoV-2 (used by the name 2019-nCoV, and cause neocoronarism). Coronaviruses are widely distributed, have rapid variation, strong infectivity and large genetic diversity, and may continuously vary in the process of frequent cross-species transmission or may periodically appear in human beings. In view of the epidemic situation caused by the above viruses, new and emergent virus infection caused by coronavirus may be a new biological threat.
Over the past decades, many drugs have been found to have antiviral activity and are used in the clinical treatment of coronaviruses. However, some drugs exhibit antiviral ability while causing toxic side effects to humans or animals, and in addition, resistance occurs in humans when patients take long-term medication or the spread of viruses is long.
Therefore, the development of an anti-coronavirus drug with safety, high activity and broad spectrum has important theoretical significance and clinical application value for preventing and treating the current new coronary pneumonia and the coronavirus diseases which may appear in the future.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a nano-drug which can effectively inhibit the proliferation of coronavirus and kill coronavirus.
The invention also provides a preparation method of the nano-drug.
The invention also provides the application of the nano-drug.
According to an embodiment of the first aspect of the present invention, there is provided a nano-drug comprising a metal matrix and an organic ligand modifying the metal matrix;
the organic ligand is selected from at least one of compounds shown in formulas (1) to (12):
the nano-drug provided by the embodiment of the invention has at least the following beneficial effects:
(1) Nano-drugs have become important tools for human disease resistance, and have been studied in drug carrier delivery, pathogen detection, cancer treatment, and sterilization. For coronavirus infection, such as SARS-CoV-2, etc., although nano-drugs have made some progress in infection protection, no therapeutic nano-drug can directly treat diseases caused by coronavirus infection at present.
The nano-drug provided by the invention can act on coronavirus through multiple mechanisms and multiple ways. When the nano medicament directly interacts with coronavirus, the nano medicament can physically destroy phospholipid bilayer of the virus, so that the virus can be directly killed; in the intracellular replication stage of the virus, antiviral effects can be exerted by binding to coronavirus 3CL protease and RdRp enzyme, etc. The action mechanism has broad spectrum and is not degraded by coronavirus variation. The nano-drug provided by the invention has lower damage degree to normal cells and higher safety.
Namely, the nano-drug provided by the invention has protective and therapeutic effects on coronavirus, and has broad spectrum and safety on the effect of coronavirus.
Specifically, the nano-drug provided by the invention has an EC50 (percent for 50% of maximum effect) value as low as 1.19 μ M in an in vitro model for coronavirus. In vivo animal models, viral titers can also be significantly reduced.
(2) The nano-drug provided by the invention has the advantages that the interaction between the metal matrix and the organic ligand is realized, the specific metal matrix is a carrier of the organic ligand, and the organic ligand can passivate the metal matrix to a certain extent so as to enable the metal matrix to exist stably.
According to some embodiments of the invention, when the organic ligand comprises a compound of formula (8), the configuration of the compound of formula (8) is at least one of a levorotatory configuration (L-form) and a dextrorotatory configuration (D-form).
Compared with the dextrorotatory structure, the compound shown in the formula (8) has better inhibition on coronavirus after the levorotatory compound is combined with a metal matrix.
According to some preferred embodiments of the present invention, the organic ligand is selected from at least one of compounds represented by formula (1), formula (7), formula (8), formula (9), formula (11), and formula (12).
According to some preferred embodiments of the present invention, the organic ligand is selected from the group consisting of compounds represented by formula (1) (4, 6-diamino-2-mercaptopyrimidine).
According to some preferred embodiments of the invention, the organic ligand is selected from compounds represented by formula (10).
According to some preferred embodiments of the invention, the organic ligand is selected from compounds represented by formula (5) or formula (6).
According to some further preferred embodiments of the present invention, the organic ligand is selected from the group consisting of compounds represented by formula (8) (glutathione) and formula (9) (N, N-trimethyl- (11-mercaptoundecyl) ammonium chloride).
According to some embodiments of the invention, the metal matrix comprises at least one of gold, copper and iron.
According to some embodiments of the invention, the metal substrate is gold. According to some embodiments of the invention, the nano-drug has a particle size of 0.1 to 15nm.
According to some preferred embodiments of the present invention, the nano-drug has a particle size of 1 to 6nm.
It is thus understood that the metal matrix is at least one of nanoparticles and nanoclusters.
The particle size of the nanocluster is usually less than or equal to 5nm, the nanocluster is an ultra-small metal nanoparticle formed by gathering several to hundreds of metal atoms, the particle size of the nanoparticle is more than 5nm, compared with the nanoparticle, the nanocluster is higher in activity, and the prepared nano medicine is better in performance.
According to some embodiments of the invention, the particle size of the nano-drug is 1 to 10nm.
According to some embodiments of the invention, the nanomedicine comprises a gold nanocluster matrix and an organic ligand that modifies the gold nanocluster matrix, the organic ligand comprising N, N-trimethyl- (11-mercaptoundecyl) ammonium chloride and levoglutathione.
According to a second aspect of the embodiment of the present invention, there is provided a method for preparing the above nano-drug, the method comprising purifying after reacting a dispersion liquid containing a metal source and the organic ligand.
In the preparation method of the nano-drug, the reaction mechanism is as follows: the organic ligand is simultaneously used as a ligand and a reducing agent, reduces metal in the metal source into a simple substance, and coordinates with simple substance atoms to form stable connection.
The preparation method provided by the embodiment of the invention has at least the following beneficial effects:
the preparation method provided by the invention is simple, the process is easy to control, and mass production is easy to realize.
According to some embodiments of the invention, the metal source comprises chloroauric acid.
According to some embodiments of the invention, the concentration of the metal source in the dispersion is 5 to 10mM.
According to some preferred embodiments of the invention, the concentration of the metal source in the dispersion is 7 to 8mM.
According to some embodiments of the invention, the concentration of the organic ligand in the dispersion is between 10 and 50mM.
According to some preferred embodiments of the invention, the concentration of the organic ligand in the dispersion is 20 to 40mM.
According to some preferred embodiments of the present invention, in the dispersion, the metal source is chloroauric acid, and the organic ligand is at least one of compounds represented by formulas (1) to (12).
According to some preferred embodiments of the present invention, in the dispersion, the metal source is chloroauric acid, and the organic ligands are levorotatory compound of the compound represented by formula (8) and the compound represented by formula (9).
According to some preferred embodiments of the present invention, the molar ratio of the chloroauric acid, the levorotatory compound of the compound represented by formula (8), and the compound represented by formula (9) in the dispersion is (0.5-4): 1 (0.5-4). According to some embodiments of the invention, the dispersant of the dispersion is water.
Therefore, the preparation method provided by the invention does not relate to an organic solvent, and does not relate to subsequent post-treatment stages such as removal of the organic solvent.
According to some embodiments of the invention, the method of preparing the dispersion comprises: and (3) after the organic ligand is heated and dispersed in the solvent, adding the metal source into the solvent.
According to some embodiments of the invention, the temperature-rising dispersion method is stirring, and the rotation speed of the stirring is 500-800 rpm.
According to some preferred embodiments of the present invention, the temperature-increasing dispersion method is stirring, and the rotation speed of the stirring is about 600rpm.
According to some embodiments of the invention, the end point temperature of the temperature-increasing dispersion is 50 to 95 ℃.
According to some preferred embodiments of the invention, the end point temperature of the ramping dispersion is about 70 ℃.
According to some embodiments of the invention, the temperature of the reaction is between 0 and 95 ℃.
According to some preferred embodiments of the invention, the reaction is a heating reaction.
According to some embodiments of the invention, the temperature of the heating reaction is 50 to 95 ℃.
According to some embodiments of the invention, the temperature of the heating reaction is 60 to 90 ℃.
According to some embodiments of the invention, the temperature of the heating reaction is about 70 ℃.
According to some embodiments of the invention, the reaction is carried out under stirring at a speed of 800 to 1200rpm.
According to some preferred embodiments of the invention, the reaction is carried out under stirring at a speed of about 1000rpm.
According to some embodiments of the invention, the reaction time is 18 to 24 hours.
According to some embodiments of the invention, the reaction time is about 24 hours.
According to some embodiments of the invention, the method of purifying comprises dialysis with pure water.
According to some embodiments of the invention, the dialysis membrane used for dialysis has a specification of 3500Da.
According to some embodiments of the present invention, the preparation method further comprises concentrating and preserving the obtained nano-drug dispersion after the purification.
According to some embodiments of the invention, after the concentrating, the concentration of the nano-drug in the resulting dispersion is 1000 to 2000 μ g/mL.
According to some embodiments of the invention, the temperature of the storing is about 4 ℃.
According to some embodiments of the invention, the preserving comprises diluting the concentrated dispersion to a working concentration in cell culture medium, PBS (phosphate balanced saline), dextrose injection or saline as a diluent.
According to the third aspect of the embodiment of the invention, the application of the nano-drug in preparing anti-coronavirus drugs is provided.
The nano-drug has excellent inhibiting and killing effects on in-vivo and in-vitro coronaviruses, so that the anti-coronaviruses drug prepared from the nano-drug has better preventing and treating effects on diseases caused by coronaviruses.
According to some embodiments of the invention, the coronavirus comprises at least one of SARS-CoV, SARS-CoV-2, MERS-CoV and GX _ P2V (isolated from squama Manis).
According to some embodiments of the invention, the SARS-CoV-2 comprises at least one of the SARS-CoV-2 variants B.1.617.2 and B.1.1.529.
Unless otherwise specified, "about" in the present invention means an allowable error of ± 2%, for example, about 100 means 98 to 102 in practice.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows zeta potential test results of the nano-drug obtained in example 1 of the present invention;
FIG. 2 shows the results of particle size measurement of the nano-drug obtained in example 1 of the present invention;
FIG. 3 is EDS map and HAADF image of nano-drug obtained in example 1 of the present invention;
FIG. 4 shows the results of the inhibition of coronavirus and the toxicity of normal cell by the nano-drugs tested in test examples 1-2 of the present invention;
FIG. 5 shows the results of the consistency of Ruixiwei against coronavirus and the toxicity against normal cells measured in test examples 1-2 of the present invention;
FIG. 6 shows the antiviral effects of the nano-drug and Reidesciclovir on mouse lung measured in test example 3;
FIG. 7 shows the antiviral effects of the nano-drug and Reidesciclovir on mouse trachea, measured in test example 3 of the present invention;
FIG. 8 shows the results of inhibiting coronavirus by the nano-drugs obtained in examples 1 to 21 of the present invention.
FIG. 9 is a graph showing the inhibition of coronavirus 3CL protease activity by the nano-drugs obtained in example 1 of the present invention.
Detailed Description
The idea of the invention and the resulting technical effects will be clearly and completely described below in connection with the embodiments, so that the objects, features and effects of the invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts are within the protection scope of the present invention based on the embodiments of the present invention.
In the description of the present invention, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present numbers, and the above, below, within, etc. are understood as including the present numbers. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
Unless otherwise specified, all test materials used in the examples were commercially available and were not pretreated before use.
Example 1
The preparation method of the nano-drug comprises the following specific steps:
s1, preparing an N, N, N-trimethyl- (11-mercapto-undecyl) ammonium chloride (levorotatory configuration) aqueous solution with the concentration of 25 mM;
preparing an aqueous solution of glutathione with the concentration of 100 mM;
s2, mixing the N, N, N-trimethyl- (11-mercapto-undecyl) ammonium chloride aqueous solution obtained in the step S1 and a glutathione aqueous solution according to the volume ratio of 4; the next test was carried out after the temperature reached 70 ℃.
The reaction vessel in this step can be adjusted according to the reaction amount and the test conditions, and in this embodiment, the reaction vessel can be a round-bottom flask;
the heating method in this step may be adjusted according to the conditions of the reaction vessel and the equipment, and in this embodiment, the heating method may be oil bath heating;
s3, adding 1mL of chloroauric acid solution mother liquor (with the concentration of 25 mM) into the mixed solution (2.5 mL) obtained in the step S2, increasing the stirring speed to 1000rpm to start reaction, and keeping the reaction time for 24 hours;
s4, after the reaction in the step S3 is finished, taking out the round-bottom flask from the oil bath, cooling, and dialyzing and purifying by using ultrapure water;
and S5, after the dialysis is finished, performing sterile and ultrafiltration concentration treatment on the antiviral cluster water solution to a final concentration of 1000 mug/mL, and storing at 4 ℃ for later use.
The particle size of the nano-drug obtained in this example was measured by zeta potential analyzer, the potential results are shown in fig. 1, and the corresponding particle size results are shown in fig. 2. The result shows that the peak value of zeta potential of the nano-drug is 14.7 +/-2.72 mV, namely the obtained nano-drug (colloid particles) presents certain electropositivity; the particle size is mainly concentrated around 4.79 + -0.43 nm. The nano-drug prepared by the invention is proved to have nano-size.
The element distribution of the nano-drug obtained in the present example was characterized by EDS (energy spectroscopy), and the results are shown in fig. 3, where HAADF represents a high-angle annular dark field image-scanning transmission electron image, overlap shows the gold element distribution and the sulfur element distribution, au shows the gold element distribution, and S shows the sulfur element distribution in fig. 3. The results show that:
the nano-drug obtained in this example indeed has a size of 1-5 nm; and the atlas shows that the distribution of the gold element and the distribution of the sulfur element have similar regionality, and the sulfur element is derived from the organic ligand, and the nano-drug is purified before characterization, so that the nano-drug prepared by the embodiment is proved to be combined between the organic ligand and the gold nanocluster.
In order to verify the performance of the nano-drugs obtained by combining different metal sources and organic ligands on coronavirus, the invention provides examples 2-13, which are as follows:
examples 2 to 13 differ from example 1 in that:
the compositions of the metal source and the organic ligand are different, and the specific compositions are shown in table 1:
TABLE 1 compositions of the organic ligands and the metal sources (ratio is the ratio of the amounts of the substances) in examples 1 to 13
In table 1, the information of each organic ligand is as follows:
the CAS number of the compound represented by the formula (1) is: 1004-39-3;
the CAS number of the compound of formula (2) is: 657-24-9;
the CAS number of the compound represented by the formula (3) is: 7134-41-0;
the CAS number of the compound represented by formula (4) is: 2935-90-2;
the CAS number of the compound of formula (5) is: 123-30-8;
the CAS number of the compound of formula (6) is: 645-96-5;
the CAS number of the compound of formula (7) is: 5192-03-0;
the CAS number of the compound of formula (8) is: 70-18-8;
the CAS number of the compound of formula (9) is: 225790-17-0;
the CAS number of the compound of formula (10) is: 49594-30-1;
compounds of formula (11) and formula (12) reference chem.sci.2021,12,14871-14882 methods of preparation of compounds 13 and 14.
Test example 1
In this test example, vero E6 cells (African green monkey kidney cell line) are used to simulate host cells, and the in vitro inhibitory activity of the nano-drug obtained in example 1 on coronavirus GX _ P2V (from Beijing university of chemical industry, life academy) is verified.
Because the new coronavirus (SARS-CoV-2) has strong infectivity and pathogenicity, related research works can be developed only in laboratories of level 3 biosafety (BSL-3) and above, but the number of the laboratories of level 3 biosafety is small, the operation procedure is complicated, and the progress of large-scale drug screening is greatly limited. The coronavirus GX _ P2V extracted from pangolin scales is proved to have high homology with the new coronavirus through high-throughput sequencing and subsequent analysis, wherein the homology of the whole genome is 85.94 percent, and the homology of spike protein (S) is 92.2 percent. The S protein is an important structural protein for the invasion of new coronavirus into host cells. This is the coronavirus with highest homology with new coronavirus which is successfully separated and cultured at present. Research shows that GX _ P2V uses angiotensin converting enzyme 2 (ACE 2) as an entry receptor like neocoronaviruses, and ACE2 is a receptor of cells infected by the neocoronaviruses and can recognize S protein. In addition, GX _ P2V is not found to be pathogenic to humans and can be cultured in a conventional BSL-2 laboratory. In order to verify the effectiveness of GX _ P2V in replacing a new coronavirus as a novel drug screening model, anti-GX _ P2V tests are carried out by using drugs which are widely reported to have anti-new coronavirus activity in vitro, such as Remdesivir, chloroquin, hydroxychloroquin, nefinavir, lopinavir and the like, and the results show that the anti-GX _ P2V activity of the drugs is highly consistent with the anti-new coronavirus activity. The effectiveness of GX _ P2V as a drug screening substitution model is proved, and the GX _ P2V is an ideal anti-new coronavirus drug screening substitution model. That is, the test example performed with GX _ P2V strain can verify at least the effect of the nanopharmaceutical obtained in example 1 on both GX _ P2V and SARS-CoV-2 virus.
The specific test method comprises the following steps:
D1. and (3) incubation: inoculating Vero E6 cells into a 96-well plate, culturing after inoculation until the density of monolayer adherent cells is more than 80%, adding 50 mu L of GX _ P2V (MOI = 0.01) and 50 mu L of nano-drug dispersion liquid with different concentrations into each well plate, incubating for 2h, then sucking out, and washing the obtained cells 1 time with PBS (phosphate buffer solution) mildly;
after the addition of the nano-drug dispersion, the concentrations of the nano-drug dispersion in the incubation medium were 0.047. Mu.M, 0.094. Mu.M, 0.188. Mu.M, 0.375. Mu.M, 0.750. Mu.M, 1.5. Mu.M, 3.0. Mu.M, 6.0. Mu.M and 12.0. Mu.M, respectively, with two replicates per concentration set up;
during the incubation process in the step, the nano-drug and the virus can play an interaction both outside the host cell and inside the host cell; the whole effect of the nano-drug is investigated in the process of infecting host cells by the virus.
D2. Setting up experimental groups: the washed cells incubated in step D1 were added to the medium containing the same concentration gradient of the nano-drug and placed at 37 ℃ 5% 2 A cell incubator; after culturing for 48h, collecting cell supernatant and adherent cells for RT-qPCR analysis;
wherein the specific concentration of the nano-drug in the culture medium is the same as the concentration in the step D1: two parallel tests were performed for each concentration of nano-drug;
the medium contains, in addition to the above nano-drugs, 10% FBS and 1% antibiotic-antibiotic (fungal antibiotic).
Setting a positive control group A: the difference from the test group is that the culture medium does not contain nano-drugs;
the positive control group B is set, and the difference from the experimental group is that the nano-drug in the culture medium is replaced by Rib West, and the concentration of the Rib West is as follows; 0.20. Mu.M, 0.40. Mu.M, 0.8. Mu.M, 1.6. Mu.M, 3.2. Mu.M, 6.4. Mu.M, 12.5. Mu.M, 50. Mu.M and 100. Mu.M.
In the step, RT-qPCR tests the gene copy number of the virus, and the primer sequences adopted in the test process are as follows:
GAPDH-F,5’-AGCCTCAAGATCATCAGCAATG-3’(SEQ ID NO.1)，
GAPDH-R,5’-ATGGACTGTGGTCATGAGTCCTT-3’(SEQ ID NO.2)；
GX_P2V-F,5’-GGTGATTGCCTTGGTGATATTG-3’(SEQ ID NO.3)，
GX_P2V-R,5’-GCAAGTAGTGCAGAAGTGTATTG-3’(SEQ ID NO.3)。
D3. the value of (1-experimental group virus content/positive control group A virus content) x 100% was calculated and fitted based on this to obtain the EC50 value.
The test results of the nano-drug obtained in example 1 are shown in fig. 4 and table 2. The results show that the EC50 inhibition rate of the nano-drug obtained in example 1 on GX _ P2V virus is about 1.19 μ M (using X-ray photoelectron spectroscopy and nuclear magnetic resonance techniques to prove that in the nano-drug obtained in example 1, each particle contains 42 gold atoms, 39 ligands shown in formula (8), 16 ligands shown in formula (9), and the molecular mass (particle mass) is about 25 kDa); namely, in vitro experiments, the nano-drug provided by the invention shows excellent inhibition on coronavirus.
The test results of the ridciclovir form the positive control group B are shown in fig. 5 (abscissa is logarithmic value with the concentration of the ridciclovir at base 10). The test result shows that the EC50 value of the Ribes ciclovir is 1.01 mu M in a system of Vero E6 cells and GX _ P2V, and the effect is equivalent to that of the nano-drug provided by the invention.
Test example 2
In the test example, the toxicity of the nano-drug obtained in example 1 on Vero E6 cells is tested, and half cytotoxicity (CC 50) is calculated, and the specific test method is as follows:
A1. inoculating Vero E6 cells in a 96-well plate, and starting an experiment until the density of the monolayer adherent cells is about 80%;
A2. adding the culture medium containing the nano-drugs into the 96-well plate obtained in the step A1, wherein each well is 100 mu L; then charged at 37 ℃ with 5% CO 2 The cell culture box is continuously cultured for 48 hours, and the cell viability is detected by a CellTiter-Blue method. mu.L of Resazurin cell dye was added to each well, and the absorbance of the cells at 570nm was measured every 30min using a multifunctional plate reader (OD test group). The formula for the cytotoxicity is as follows: cell lethality (%) =1- (OD experimental/ODcontrol) × 100%. And fitted to give CC50. In this test example, the cell lethality rate is also referred to as inhibition rate.
ODcontrol is the absorbance of cells of a blank control group at 570nm, and neither nano-drugs nor Ribes-cidevir are added in the blank control group.
Meanwhile, a Reidesciclovir control group is arranged, and the difference from the nano-drug group is that the added 100 mu L of culture medium does not contain nano-drugs but contains Reidesciclovir.
After the nano-drug-containing medium was added, the concentrations of the nano-drug and the ciclovir were the same in the obtained culture system as in test example 1, respectively. And 3 replicates were run simultaneously for each concentration.
The cytotoxicity results obtained in this test example are shown in FIGS. 4 to 5 and tables 2 to 3, and the results show that: the nano-drug prepared in example 1 has a CC50 value of > 12 μ M for Vero E6, and shows extremely low cytotoxicity. The trend of the results was the same as that of the cytotoxicity of the ciclovir shown in fig. 5.
The results of the test example 1 show that the nano-drug prepared by the invention shows excellent anti-coronavirus performance and extremely low cytotoxicity in Vero E6 and GX _ P2V systems.
TABLE 2 inhibition of coronavirus and toxicity of normal cells by the nano-drug obtained in example 1
SD in table 2 represents standard deviation.
TABLE 3 inhibition of coronavirus by Reidesciclovir and toxicity to normal cells
| Concentration of μ M | Inhibition ratio% | Cytotoxicity% |
| 0.20 | -0.85 | -11.94 |
| 0.39 | 17.05 | -17.59 |
| 0.78 | 47.76 | -7.96 |
| 1.56 | 39.25 | -4.6 |
| 3.13 | 74.05 | -0.85 |
| 6.25 | 97.76 | 3.48 |
| 12.5 | 99.97 | 5.21 |
| 25 | 100 | 9.62 |
| 50 | 99.98 | 13.63 |
| 100 | 99.99 | 32.99 |
Test example 3
The test example tests the anti-coronavirus effect of the nano-drug obtained in example 1 in the animal body, and the specific method comprises the following steps:
B1. animal molding: adopting Syria golden hamster as model animal and coronavirus GX _ P2V as model virus;
50 μ L GX _ P2V (final titer 2X 10) per golden hamster 5 PFU/mL) for nasal drip infection;
B2. experimental groups: c, carrying out continuous intraperitoneal administration on the golden hamster obtained in the step B1 for 4 days according to the administration concentration (the ratio of the mass of the gold element to the body weight of the model animal) of 3.2 mg/kg; the given drug is the nano-drug obtained in example 1;
control group: the nano-drug of the test group is replaced by the Reidesciclovir, and the administration concentration is 15mg/kg (when the Reidesciclovir is used on golden mice, the standard concentration of the Reidesciclovir);
B3. and D, euthanizing the site of the syrian golden hamster obtained in the step B2, dissecting and taking the lung and the trachea, grinding the lung and the trachea by a cryogrinding instrument, extracting total RNA, and then carrying out reverse transcription and RT-qPCR detection, thereby determining the in vivo effect of the nano-drug obtained in the example 1 on the coronavirus.
The results of the number of virus replications in the lungs are shown in FIG. 6, and the number of virus replications in the trachea are shown in FIG. 7.
The results of fig. 6 to 7 show that the nano-drug prepared in example 1 can effectively reduce the virus titer of coronavirus in lung and trachea in golden hamster model, wherein the lung can be reduced by about 2 to 3 orders of magnitude, and the trachea can be reduced by 0.5 to 1.5 orders of magnitude, which indicates that the nano-drug prepared by the invention has good in vivo anti-coronavirus effect.
Comprehensive test examples 1-3 show that the nano-drug provided by the invention has excellent in vivo and in vitro anti-coronavirus effects, and has a good prospect in preparation of drugs for treating coronavirus infection diseases.
Test example 4
This test example tested the inhibition of coronavirus 3CL protease activity by the nanopharmaceuticals obtained in example 1.
The test method comprises the following steps: the method for detecting the influence of the small molecules on the enzyme digestion activity of the 3CLpro extracellular protein by a Fluorescence Resonance Energy Transfer (FRET) method specifically comprises the following steps:
experimental materials and instruments:
the fluorescent substrate Dabcyl-KNSTLQSGLRKE-Edans is synthesized by Nanjing Kingsrie Biotech, inc.;
fluorescence substrate mother liquor: prepared with autoclaved buffer (20 mM Tris-HCl, pH7.4,120mM NaCl) to a final concentration of 100. Mu.M. Subpackaging into small parts, and freezing at-20 deg.C for use;
nano-drug mother liquor: 100% DMSO was added to the final concentration of 100mM, and the mixture was stored at 4 ℃ until use;
HBS-EP working buffer (10mM hepes,150mM NaCl,3mM EDTA and 0.005% (v/v) surfactant P20, pH 7.4);
microplate reader (MD corporation);
IC of compound for inhibiting 2019 novel coronavirus 3CL protease 50 And (3) determination:
the nano-drug stock solution was diluted, 1.2ul of each concentration solution was mixed with 120ul of 2 μ M3 CL protease (final DMSO concentration: 0.5%) and incubated at 4 ℃ for 2 hours. Then 100ul of each concentration sample was added to a 96-well plate, and 100ul of 20 μ M fluorogenic substrate solution was added to start the reaction, where the final concentration of 3CL protease was 1 μ M, the final concentration of fluorogenic substrate was 10 μ M, and the final concentrations of nanopharmaceuticals were 24 μ M, 12 μ M, 6,3 μ M, 1.5 μ M, 0.3 μ M, 0.15 μ M, 0.03 μ M, 0.015 μ M, 0.003 μ M, 0.0015 μ M, and 0.0003 μ M, respectively. Exciting by light with the wavelength of 340nm, detecting the change of the fluorescence value with the emission wavelength of 488nm, and continuously measuring for 1h through reaction to finish the measurement of the inhibition effect of the compound on the new coronavirus 3CL protease. Determination of Compound IC 50 Values are given as blanks, i.e. 3CL protease samples without nanopharmaceutical but with the same DMSO concentration and samples of fluorogenic substrate solution alone. Multiple wells were set for each sample and the measurements averaged. According to the reaction speed of the new coronavirus 3CL protease enzyme digestion fluorogenic substrate under the condition of the existence of compounds with different concentrations, comparing the reaction speed with the reaction speed of a blank control, and calculating to obtain the inhibition rate under each concentration(in terms of the enzyme activity in the DMSO-containing condition of 0.5%, the inhibition = 100%; relative activity of enzyme at different concentrations of nano-drug x 100%, relative activity is the ratio of the enzyme activity corresponding to different concentrations of nano-drug to the enzyme activity of the blank). Calculating the IC of a Compound for inhibition of 3CL protease Activity by Logistic equation Using origin software for non-Linear fitting 50 The value is obtained.
The test results are shown in fig. 9.
The result shows that the activity of coronavirus 3CL protease is obviously inhibited along with the increase of the addition amount of the nano-drug. Therefore, the nano-drug provided by the invention can exert antiviral effect by combining with the coronavirus 3CL protease.
Test example 5
The test example tests the inhibitory performance of the nano-drugs obtained in examples 1 to 13 on coronavirus, and the specific test method comprises the following steps:
D1. and (3) incubation: the Vero E6 cells were seeded in 96-well plates, cultured after seeding until the monolayer adherent cell density reached more than 80%, and GX _ P2V (MOI = 0.01), and examples 1-13 (final concentration 25ug/mL in terms of gold mass concentration) were added to each plate in 3 replicates;
d2.96 well plates at 37 ℃ C, 5% CO 2 A cell incubator; after 48h of culture, cell supernatants and adherent cells were collected for RT-qPCR analysis, and the relative mRNA content (ratio of experimental group value to internal standard, divided by ratio of control group to internal standard) of GX _ P2V/GAPDH at different drug treatments was calculated. The primer probes used for RT-qPCR analysis were the same as in test example 1.
The control group is the group without the nano-drug added in the step D2.
The test results are shown in FIG. 8, the abscissa is the number of the structural formula of the organic ligand, and examples 2 to 9, example 1, and examples 10 to 13 are sequentially compared from left to right; the results show that: when the variety of the organic ligand is changed, the nano-drug provided by the invention has excellent inhibition performance on coronavirus.
Therefore, the nano-drug provided by the invention has certain inhibitory performance on coronavirus, and has better prospect in preparing drugs for treating coronavirus infection diseases.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
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Claims (10)
1. A nano-drug, comprising a metal matrix and an organic ligand that modifies the metal matrix;
the organic ligand is selected from at least one of compounds shown in formulas (1) to (12):
2. the nano-drug of claim 1, wherein when the organic ligand comprises the compound of formula (8), the configuration of the compound of formula (8) is at least one of a levorotatory configuration and a dextrorotatory configuration.
3. The nano-drug according to claim 1, wherein the organic ligand is at least one compound selected from the group consisting of compounds represented by formula (1), formula (7), formula (8), formula (9), formula (11), and formula (12).
4. The nano-drug of claim 1, wherein the metal matrix comprises at least one of gold, copper and iron.
5. The nano-drug according to claim 4, wherein the metal matrix is made of gold.
6. The nano-drug according to any one of claims 1 to 5, wherein the nano-drug has a particle size of 0.1 to 15nm.
7. A process for the preparation of a nano-drug as claimed in any one of claims 1 to 6, which comprises reacting a dispersion comprising a metal source and said organic ligand and purifying the reaction mixture.
8. The method according to claim 7, wherein the reaction temperature is 0 to 95 ℃.
9. Use of a nano-drug as defined in any one of claims 1 to 6 for the preparation of an anti-coronavirus drug.
10. The use of claim 9, wherein the coronavirus comprises at least one of SARS-CoV, SARS-CoV-2, MERS-CoV, and GX _ P2V.
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