CN112704748B

CN112704748B - Photodynamic medicine for killing novel coronavirus COVID-19 and application thereof

Info

Publication number: CN112704748B
Application number: CN202110177951.6A
Authority: CN
Inventors: 黄明东; 余淑娟; 袁彩; 江龙光; 徐芃; 孙高辉; 隋亚群
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2022-06-14
Anticipated expiration: 2041-02-09
Also published as: CN112704748A

Abstract

The invention provides a photodynamic method for killing novel coronavirus, which kills the novel coronavirus under the irradiation of a light source by using a photosensitizer, wherein the emission wavelength of the light source covers the absorption wavelength of the photosensitizer. The method can kill extracellular and intracellular new type coronavirus, and does not damage cells. The invention provides a brand new method for treating the novel coronavirus.

Description

Photodynamic medicament for killing novel coronavirus COVID-19 and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to killing of a novel coronavirus and related application thereof.

Background

With the global pandemic of the novel coronavirus in 2019, the number of newly increased infection people in the world is tens of thousands every day at present, the number of death people is increased day by day, medical resources are seriously overloaded, and the further epidemic of the novel coronavirus is restrained and is urgently needed. In 11 months of 2020, there are 5600 ten thousands of cases of pneumonia caused by new coronavirus infection reported worldwide, and 133 thousands of death cases. The spread of new coronavirus is much higher than that of SARS virus and seasonal influenza, and the elimination of new coronavirus is very challenging. Vaccines are currently the most potential solution to prevent the further prevalence of new coronavirus. However, the new crown adenovirus vaccine developed by the cooperation of the Asricon company and the Oxford university in England has a serious side effect in the third-phase clinical test, and the clinical test is suspended in a short period. The first worldwide copy of the official approved new corona vaccine on the market, Sputnik V from russia, and the results of early clinical trials published in the "lancet" journal on day 9 and 4, were found by the same workers to be potentially problematic. Vaccine development in all countries of the world is advancing at an unprecedented rate, but before large-scale phase three clinical trials are completed, it is impossible to determine when a vaccine is available, how much protection the vaccine can provide, how much side effects are, and whether the vaccine productivity is sufficient.

In addition to the less bright prospect of vaccines, the clinical effect of the small-molecule drug, namely the Reidesvir, is also poor. Therefore, there is a great need in the clinical field to develop drugs that act effectively on new coronaviruses and are useful in the treatment of new coronaviruses infections.

Disclosure of Invention

The invention aims to provide a method for treating new coronavirus infection, which uses a photosensitizer to generate Reactive Oxygen Species (ROS) or singlet oxygen under the illumination condition to directly kill new coronavirus. The photosensitizer is a molecule which only absorbs photons in photochemical reaction and transfers energy to molecules which can not absorb photons to promote chemical reaction, but does not participate in chemical reaction per se and is recovered to the original state.

In order to achieve the above object, the present invention discloses the use of a photosensitizer for killing a novel coronavirus, wherein the photosensitizer is used for killing the novel coronavirus under the irradiation of a light source, and the emission wavelength of the light source covers the absorption wavelength of the photosensitizer.

The photosensitizer comprises one or more of phthalocyanine or a derivative thereof.

The photosensitizer comprises one or more porphyrins or derivatives thereof.

The photosensitizer kills extracellular novel coronaviruses under light conditions.

The photosensitizer kills a novel coronavirus in a host cell under light conditions.

The mechanism of killing the virus of the invention is as follows: photosensitizers distributed around the virus, upon irradiation by a suitable light source, undergo a transition from the ground state to the excited state upon absorption of energy. The excited photosensitizer transfers energy to oxygen molecules around the virus, and then a series of photochemical reactions occur to generate a large amount of active oxygen species with strong oxidizing properties, which react with the virus to cause inactivation of the virus.

The invention has the advantages that: the photosensitizer can not only aim at extracellular new coronavirus, but also act on cells infected with virus to kill intracellular virus through photosensitization of the photosensitizer, and can not damage the cells in an effective range.

The commonly used phthalocyanine photosensitizer ZnPc5K and porphyrin photosensitizer chlorin (ce 6) are used in the examples of the present invention.

Drawings

FIG. 1: ZnPc5K structural diagram.

FIG. 2: schematic structure of ce 6.

FIG. 3: cytotoxicity of ZnPc5K and ce6 against HELF cells.

FIG. 4: ZnPc5K acts on the extracellular SARS-COV-2 pseudovirus.

FIG. 5: ce6 acts on the SARS-COV-2 pseudovirus in cells.

FIG. 6: ZnPc5K acts on intracellular SARS-COV-2 pseudovirus.

FIG. 7: ce6 acts on the extracellular SARS-COV-2 pseudovirus.

FIG. 8: ZnPc5K acts on SARS-COV-2.

FIG. 9: ce6 has effect on SARS-COV-2.

Detailed Description

The method and its advantages will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the claims. The present invention may be further modified and improved without departing from the scope of the main characteristics of the present invention, and such modifications and improvements are intended to be included within the scope of the present invention.

Example 1 evaluation of the safety of ZnPc5K and ce6 on HELF cells of Normal human embryonic Lung fibroblasts

ZnPc5K (structure shown in figure 1) and ce6 (structure shown in figure 2) are added into HELF cells (human lung fibroblasts) to be mixed and incubated for 4 h, then illumination is carried out, and 24 h culture is carried out. The specific operation steps are as follows:

1. the seed cells are: HELF cells were counted, diluted to 10000/mL concentration with DMEM medium containing double antibody, and 40. mu.L of cell suspension per well was inoculated into 384-well plates for 24 h before the corresponding experiment.

2. Diluting the medicine: 100 μ M ZnPc5K and ce6 were diluted 2-fold with DMEM (with diabodies) for 12 gradients, and 10 μ L of each concentration was added to the cell culture medium in each well. The actual final concentration of the drug on the cells was 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M, 0.625. mu.M, 0.312. mu.M, 0.156. mu.M, 0.078. mu.M, 0.039. mu.M, 0.02. mu.M, 0.01. mu.M, 0. mu.M.

3. And (3) safety evaluation: after incubation for 4 h with the drug mixed with the cells, one group was not illuminated and one group was illuminated with 660nm LED light (light dose 0.48J/cm 2). Respectively marked as Control, Illumination; then, the cells were cultured in a 5% CO2 incubator at 37 ℃ for 24 hours. Nuclei were stained with Hoechst 33342, cell number was observed and counted using high content, and drug safety was calculated.

As shown in fig. 3, the cell number of ZnPc5K was calculated to show almost no photo-toxicity and dark-toxicity to HELF cells in this light dose and concentration range used, while ce6 was safe to HELF cells at concentrations below 1.25 μ M.

Example 2 photosensitizers ZnPc5K and ce6 kill the extracellular SARS-COV-2 pseudovirus

ZnPc5K and ce6 are respectively and directly mixed with SARS-COV-2 pseudovirus and then illuminated, 293T-ACE2 cells are infected, and the killing effect of the green fluorescent protein EGFP is evaluated by the fluorescence of the green fluorescent protein EGFP generated by the replication and amplification of the pseudovirus in the 293T-ACE2 cells. The specific operation steps are as follows:

1. the seed cells are: after counting the host cells (293T-ACE 2), the cells were diluted to 10000/mL in DMEM medium with 10% FBS, inoculated into 384 well plates at a cell suspension volume of 40. mu.L per well, and the corresponding experiment was performed 24 h later.

2. Diluting the medicine: ZnPc5K and ce6 were diluted to 2. mu.M, 0. mu.M with DMEM (containing diabodies), respectively.

3. Dilution of SARS-COV-2 pseudovirus: the virus solution was diluted to 40000 copies/5. mu.L.

4. Co-incubation and infection of cells: mixing 5 μ L of the drug and 5 μ L of the virus diluent, repeating for 3 times, and irradiating one group with 660 nm LED light source (light dose 0.48J/cm)²) And one group is not illuminated. The actual final concentrations of drug on the virus were 1 μ M and 0 μ M, with a final virus concentration of 10 MOI. After illumination, the materials are added into a 384-hole plate and are respectively marked as ZnPc 5K-illumination and ZnPc 5K-no-illumination; then put it in 5% CO₂And culturing in an incubator at 37 ℃ for 48-72 hours. The virus killing efficiency of the drug is observed and calculated with high content.

As shown in FIGS. 4 and 5, by calculating the fluorescence intensity of EGFP, the virus inactivation rate of both 1. mu.M ZnPc5K and ce6 under the illumination condition reached 100%.

EXAMPLE 3 photosensitizers ZnPc5K and ce6 in killing of the intracellular SARS-COV-2 pseudovirus

ZnPc5K and ce6 and viruses are respectively added into host cells (293T-ACE 2) at the same time, the host cells are incubated for 4 hours, then illumination is carried out, the host cells are cultured for 48 hours, and the killing effect of the green fluorescent protein EGFP generated by replication and amplification of the pseudoviruses in the 293T-ACE2 cells is evaluated by using fluorescence. The specific operation steps are as follows:

1. The seed cell: host cells (293T-ACE 2) were counted, diluted to 10000/mL with DMEM medium containing the diabody, plated in 384-well plates at 40. mu.L cell suspension per well, and the corresponding experiments were performed after 24 h.

2. Diluting the medicine: ZnPc5K and ce6 were diluted to 4.44. mu.M, 1.48. mu.M, 0.48. mu.M, 0. mu.M, respectively, with DMEM (containing diabodies).

3. Dilution of SARS-COV-2 pseudovirus: the virus solution was diluted to 40000 copies/5. mu.L

4. Co-incubation and infection of cells: mu.L of different concentrations of drug and 5. mu.L of virus dilution were mixed and added to 384 well plates in 3 replicates. 5% CO was placed on the plates₂After incubation at 37 ℃ for 4 h in an incubator, the cells were illuminated with 660 nm LED light source (light dose 0).48 J/cm²) And then continuously culturing for 48-72 h. The final concentration of drug was 2.22. mu.M, 0.74. mu.M, 0.24. mu.M, 0. mu.M, and the final concentration of virus was 10 MOI. High content observation and analysis are carried out to calculate the virus killing efficiency of the medicine.

The experimental results are shown in fig. 6 and fig. 7, the fluorescence intensity of EGFP is reduced and the survival rate of the virus is reduced with the increase of the drug concentration, which indicates that ZnPc5K and ce6 can both act and inactivate the intracellular virus.

EXAMPLE 4 inactivation of SARS-COV-2 Virus by photosensitizers ZnPc5K and ce6 light

ZnPc5K and ce6 are respectively mixed with viruses in a culture plate, then are illuminated, and then are added into host cells (Vero) for co-culture. The specific operation steps are as follows:

1. the seed cell: vero cells were seeded in 24-well plates and the corresponding experiments were performed 24 h later.

2. Co-incubation: respectively diluting the medicines ZnPc5K and ce6 to 500 mu L/hole SARS-COV-2 at different concentrations, the final virus concentration is 0.02 MOI, 3 repeats are set, each medicine is divided into two groups, one group is illuminated by 660 nm LED light source (light dose is 0.48J/cm)²) And one group is not illuminated. The final concentration of the drug was 5. mu.M, 1. mu.M, 0.5. mu.M, 0.1. mu.M, 0.05. mu.M, 0.01. mu.M.

3. Infecting the cells: vero cells were washed 2 times with DMEM (containing double antibody) after which the virus-drug containing incubations were added to the cells. Placing the cell culture plate in CO₂Incubator in 5% CO₂And culturing at 37 ℃ for 48 h. Collecting culture supernatant, extracting RNA from the culture supernatant, measuring the virus content through fluorescent quantitative PCR, and further calculating the virus killing rate of the medicament.

The experimental results are shown in fig. 8 and 9, and the 50% effective concentration of the photophobic drugs, namely the EC, of ZnPc5K and ce6 was calculated by using GraphPad prism₅₀And 90% effective concentration, namely EC₉₀To obtain: EC of ZnPc5K for light group₅₀= 177nM，EC₉₀= 308 nM; EC of ce6 for light group ₅₀= 156nM，EC₉₀= 352 nM. Experiments show that ZnPc5K has very good virucidal effect under the condition of illumination. Furthermore, ZnPc5K and ce6 had essentially no effect in the absence of light.

Claims

1. Use of a photosensitizer for killing a novel coronavirus covi-19, wherein the photosensitizer is used to kill the novel coronavirus covi-19 under irradiation of a light source, the light source emitting at a wavelength that covers the absorption wavelength of the photosensitizer; the photosensitizer is any one of ZnPc5K and ce 6; the photosensitizer kills extracellular novel coronavirus COVID-19 under the illumination condition; the photosensitizer kills a novel coronavirus COVID-19 in a host cell under the illumination condition;

wherein the structural formula of the photosensitizer ZnPc5K is as follows:

；

the structural formula of the photosensitizer ce6 is as follows:

。