A pH-responsive phase transition sulfonate liposome for destroying virus membrane structure, and a preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses a pH response phase transition sulfonic acid liposome for destroying a virus envelope structure as well as a preparation method and application thereof. The liposome is composed of dioleoyl phosphatidyl ethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, oleic acid, sodium hexadecyl sulfonate and cholesterol. The liposome constructed by the application can neutralize viruses in the form of adhesion and wrapping of viruses in a high-pH environment in the early stage of virus infection, is endocytosed by cells, and is phase transitioned in lysosomes to realize intracellular inactivation of the viruses. In a low-pH microenvironment of inflammatory tissues caused by the late stage of virus infection, the liposome can directly destroy the virus envelope to realize virus inactivation through phase transition outside cells after adhesion of the viruses. Therefore, the liposome can realize effective killing of viruses in the early stage and the late stage of virus infection, has a spectrum antiviral effect, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pH-responsive phase-transition sulfonic acid liposome for disrupting viral envelope structure, its preparation method, and its application. Background Technology

[0002] In recent years, diseases and outbreaks caused by various viruses have become a major challenge to public health. In response to this problem, several antiviral drugs have been reported. For example, patent application CN115894587A discloses the preparation of a nucleoside derivative and its use in the field of antiviral drugs. The resulting compound exhibits good antiviral activity and is suitable for development into a novel antiviral drug.

[0003] Patent application CN114605555A discloses a bispecific neutralizing antibody against the novel coronavirus SARS-CoV-2 and its application. It is a bispecific antibody A4-A7 or a bispecific antibody A7-A4, both of which include nanobody A4 and nanobody A7. Both nanobody A4 and nanobody A7 include a variable region, and the variable region has three complementarity-determining regions CDR1, CDR2 and CDR3.

[0004] Although vaccines, antiviral drugs, and virus-neutralizing antibodies have controlled the epidemic to some extent, the emergence of various viral variants due to the virus's self-evolution has greatly limited the effectiveness of these treatments.

[0005] Antiviral nanoparticles have attracted much attention due to their simple preparation, mild storage conditions, and excellent broad-spectrum antiviral effects. Most antiviral nanoparticles inhibit viral diseases through virus neutralization mechanisms. These nanoparticles can prevent contact between viruses and cells by binding to viruses. For example, heparan sulfate proteoglycan (HSPG) is a highly sulfated proteoglycan located on the cell surface and is the first binding site for many viruses to invade cells. By modifying nanoparticles with sulfate or sulfonic acid groups, HSPG-inspired and broad-spectrum virus neutralization can be achieved. However, since most nanoparticles do not destroy the viral structure, viruses may still regain their ability to infect cells after the nanoparticles detach from the viral surface.

[0006] Viruses are tiny particles without a complete cellular structure. Based on the presence or absence of an envelope on their surface, viruses can be broadly classified into enveloped viruses and non-enveloped viruses. Compared to non-enveloped viruses, enveloped viruses that cause human diseases are more diverse, such as coronaviruses, influenza viruses, HIV, and hepatitis B viruses. These enveloped viruses use membrane proteins to adhere to susceptible cells. With the help of these proteins, the viral envelope fuses with the cell membrane or phagosome membrane, releasing its genome into the cell. Because the envelope plays a crucial role in viral invasion, disrupting the viral envelope may be a method to inactivate the virus. Summary of the Invention

[0007] This invention addresses the problem of insufficient virus inactivation by providing a pH-responsive phase-transition sulfonic acid liposome for disrupting the viral envelope structure. It can not only achieve preliminary neutralization of the virus by using a biomimetic strategy of heparan sulfate proteoglycans and prevent the virus from contacting susceptible cells by competitively binding with viral particles, but also respond to the low pH microenvironment in lysosomes or inflammatory tissues caused by viral infection, thereby disrupting the viral envelope structure and achieving virus inactivation.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, a pH-responsive phase-inversion sulfonic acid liposome for disrupting viral envelope structure is composed of double-chain phospholipids, single-chain lipids and excipients.

[0010] The double-chain phospholipids are dioleoylphosphatidylethanolamine (DOPE) and (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP); the single-chain lipids are oleic acid (OA) and sodium hexadecyl sulfonate (HSA); and the excipient is cholesterol (Cho).

[0011] The DOPE structure is as follows, having a conical molecular shape.

[0012]

[0013] The DOTAP is used to regulate the pH response of liposomes, and its structure is as follows:

[0014]

[0015] The OA, as a weak electrolyte lipid with tunable molecular shape, has the following structure:

[0016]

[0017] The HSA, a sulfonic acid lipid that enables liposomes to have virus adhesion activity, has the following structure:

[0018]

[0019] The mechanism by which this invention disrupts the viral envelope structure is as follows: In the high pH environment of normal physiological tissues, the hydrophilic head of the OA group maintains its ionic form, resulting in an inverted conical molecular shape with a large hydrophilic head and a small hydrophobic tail. This shape matches the conical DOPE molecule, which also has a small hydrophilic head and a large hydrophobic tail, forming a lipid bilayer structure. However, when the liposome enters a low pH environment, the OA carboxyl group is protonated, causing its hydrophilic head to shrink and its molecular shape to transform into a conical shape. At this point, the liposome struggles to maintain its original bilayer structure and begins to transition to the crystalline hexagonal phase. Furthermore, the sulfonic acid groups provided by HSA in the liposome allow it to come into close contact with the viral envelope. Therefore, during the phase transition, the liposome further promotes membrane fusion with the virus, causing structural damage to the viral envelope and ultimately leading to viral inactivation.

[0020] Therefore, the pH-responsive phase transition sulfonic acid liposomes of the present invention can maintain a stable and dispersed lipid bilayer structure in a higher pH environment (above pH 7.0, such as 7.4), while in a lower pH environment (below 7.0, such as pH 6.8), they will first aggregate and further transition to the hexagonal phase.

[0021] Preferably, the molar amount of (2,3-dioleoyl-propyl)-trimethylammonium chloride is 20-30% of the total molar amount of dioleoylphosphatidylethanolamine, oleic acid, and sodium hexadecyl sulfonate; wherein the incorporation of the cationic lipid DOTAP can change the pH response of the liposomes by altering their net charge; the higher the DOTAP content, the weaker the negative charge of the liposomes, and the higher the pH response. Preferably, DOTAP is 25% of the total molar amount of DOPE, OA, and HSA.

[0022] Preferably, the molar ratio of dioleoylphosphatidylethanolamine, oleic acid, and sodium hexadecyl sulfonate is 2:1-1.5:1-1.5. Variations in the oleic acid content can regulate the phase transition ability of liposomes in response to pH, while variations in the sulfonic acid ratio are related to their ability to neutralize viruses.

[0023] Preferably, the molar ratio of dioleoylphosphatidylethanolamine, oleic acid, and sodium hexadecyl sulfonate is 2:1:1.

[0024] The cholesterol incorporated comprises 5-25% of the total molar amount of double-chain phospholipids and single-chain lipids. Cholesterol provides excellent protection for the oxidative and physical stability of liposomes. Insufficient cholesterol incorporation is detrimental to the stability of liposomes, while excessive cholesterol incorporation carries the risk of causing hyperlipidemia, tumors, and other diseases.

[0025] Secondly, the present invention also provides a method for preparing pH-responsive phase-transition sulfonic acid liposomes for disrupting viral envelope structure, comprising the steps of:

[0026] Step 1: Dioleoylphosphatidylethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, oleic acid, sodium hexadecyl sulfonate and cholesterol are dissolved and mixed, and the solvent is removed by rotary evaporation to obtain a lipid film;

[0027] Step 2: The lipid film is dispersed by soaking in an aqueous solution and then ultrasonically broken to obtain the pH-responsive phase transition sulfonic acid liposomes.

[0028] The solvent used in step 1 includes one or more of dichloromethane, chloroform, and methanol; preferably, the solvent is a mixture of chloroform and methanol, such as a trichloromethane / methanol mixture with a volume ratio of 8:2-3, wherein the highly polar methanol promotes the dissolution of charged lipids.

[0029] The aqueous solution in step 2 includes one or more of the following: phosphate buffer, deionized water, and ultrapure water.

[0030] Preferably, in step 2, the ultrasonic power is 40-60W and the ultrasonic time is 1-15min. Low ultrasonic power results in low injected energy, which cannot fully break down the liposomes; excessive ultrasonic power will release heat, which is not conducive to the stability of phospholipids or lipids.

[0031] The pH-responsive phase transition sulfonic acid liposomes have a particle size of 30-60 nm. Larger liposomes require stronger binding forces to ensure they adhere to the virus surface and do not detach, thus their antiviral ability may be affected to some extent.

[0032] More preferably, the method for preparing pH-responsive phase-transition sulfonate liposomes for disrupting viral envelope structure includes the following steps:

[0033] Step 1: Dissolve the raw material in a chloroform / methanol mixed solvent with a volume ratio of 8:2, remove the solvent by rotary evaporation in a 55°C water bath, and dry the solvent on the surface of the product with nitrogen to obtain a lipid film.

[0034] Step 2: The lipid film is hydrated with phosphate buffer and the lipid film adhering to the container wall is peeled off into the aqueous solution using an ultrasonic cleaner, initially forming a crude product with a relatively wide size distribution.

[0035] Step 3: The prepared lipid membrane solution is thoroughly ultrasonically disrupted using an ultrasonic cell disruptor to prepare liposomes. At this point, the liposomes have a uniform size distribution and regular morphology.

[0036] Thirdly, the present invention also provides the use of the pH-responsive phase-inversion sulfonate liposomes in the preparation of drugs for the prevention and / or treatment of viral diseases.

[0037] This invention utilizes a biomimetic strategy based on heparan sulfate proteoglycans to regulate the phase transition of liposomes by altering the shape of lipid molecules, constructing a pH-responsive phase-transition sulfonic acid liposome for disrupting the viral envelope structure. In the high-pH environment of the early stages of viral infection, close to the normal physiological environment, this liposome can adhere to the virus, blocking viral contact with cells by encapsulating the virus and neutralizing it. At this time, the large aggregates formed by the virus and liposomes can be endocytosed by the cell, and the liposomes can undergo a phase transition in the acidic environment of the intracellular lysosomes to inactivate the virus. In the low-pH environment of inflammation in the later stages of viral infection, after the liposomes adhere to the virus, a phase transition occurs directly extracellularly, disrupting the viral envelope and causing leakage of the viral genome, thus achieving viral inactivation.

[0038] In addition, this liposome also has broad-spectrum antiviral capabilities because it can bind to a wide variety of viruses that invade cells by binding to heparan sulfate proteoglycans.

[0039] Preferably, the virus includes one or more of the following: Severe Acute Respiratory Syndrome Virus (SARS-CoV), Middle East Respiratory Syndrome Virus (MERS-CoV), porcine deltacoronavirus (PDCoV), human coronavirus OC43, human coronavirus NL63, novel coronavirus, porcine epidemic diarrhea virus (PEDV), Lassa fever virus (LADV), herpes simplex virus (HSV), HIV, and human papillomavirus (HPV).

[0040] The pH-responsive phase-transition sulfonic acid liposomes of this invention are mixed with viruses and incubated to reduce the virus's ability to infect cells. Especially in cases of severe viral infection in vivo, where the pH of the in vivo microenvironment decreases, the liposomes of this invention can aggregate and undergo further phase transitions, thereby disrupting the viral envelope structure and achieving viral inactivation, resulting in a stronger effect.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The pH-responsive phase transition sulfonic acid liposomes constructed by this invention for disrupting the viral envelope structure have clear components and formulations, simple preparation methods, suitable size, and uniform distribution. They improve the drug-likeness of heparan sulfate proteoglycan biomimetic nanoparticles and may also enable mass production.

[0043] (2) The liposomes constructed in this invention can neutralize viruses in the high pH environment of the early stage of viral infection by adhering to and encapsulating the viruses. Utilizing the similarity in composition and morphology to the viral envelope, they fuse with the viral envelope and undergo phase transition within lysosomes via endocytosis to achieve intracellular inactivation of the viruses. In the low pH microenvironment of inflammatory tissues caused by the later stage of viral infection, the liposomes, after adhering to the viruses, can directly destroy the viral envelope extracellularly through phase transition to achieve viral inactivation. Therefore, these liposomes can effectively kill viruses in both the early and late stages of viral infection.

[0044] (3) Since heparan sulfate proteoglycan is a common binding site for various viruses to invade cells, the sulfonate liposome with the heparan sulfate proteoglycan structure can exhibit an inhibitory effect on various viruses and has a broad-spectrum antiviral ability.

[0045] (4) Compared with the weak virus inactivation ability of traditional heparan sulfate proteoglycan biomimetic nanoparticles, this liposome can destroy the viral envelope through a phase transition strategy, causing the leakage of the viral genome and achieving efficient virus inactivation. Attached Figure Description

[0046] Figure 1 The images shown are cryo-transmission electron microscopy (Cyro-EM) images of the pH-responsive phase transition sulfonic acid liposomes prepared in Example 1 at pH 7.4, pH 6.8, and pH 6.0.

[0047] Figure 2 The hydrodynamic diameters (a) of pH-responsive phase transition sulfonate liposomes with different DOTAP ratios prepared in Example 1 in pH 7.4 and pH 6.0 environments, and the size distribution (b) of pH-responsive phase transition sulfonate liposomes with a DOTAP ratio of 25% in pH 7.4 and pH 6.0 environments.

[0048] Figure 3 The images are cryo-transmission electron microscopy images of wild-type SARS-CoV-2 co-incubated with pH-responsive phase transition sulfonate liposomes in Application Example 1 at pH 7.4 and pH 6.0.

[0049] Figure 4 This document presents the electrophoretic bands and band intensities of RNA released from the SARS-CoV-2 pseudovirus in pH 7.4 and pH 6.0 environments after incubation with pH-responsive phase-transition sulfonic acid liposomes in Application Example 2.

[0050] Figure 5 To demonstrate the toxicity of pH-responsive phase-transition sulfonate liposomes to HEK-293T cells in Example 3.

[0051] Figure 6 The inhibition curves and EC50 of the pH-responsive phase transition sulfonate liposomes in Example 4 against Omicron variant novel coronavirus pseudovirus, lentiviral vector LV, and type I herpes simplex virus HSV-I in a pH 7.4 environment are shown.

[0052] Figure 7 The inhibition curves and EC50 of the pH-responsive phase transition sulfonic acid liposomes in Example 4 against Omicron variant novel coronavirus pseudovirus, lentiviral vector LV, and type I herpes simplex virus HSV-I in a pH 6.0 environment are shown.

[0053] Figure 8 To demonstrate the application of pH-responsive phase-transition sulfonate liposomes in Example 5, co-localization with lysosomes (top) and empty lentivirus LV (bottom) in cells during endocytosis in a pH 7.4 environment.

[0054] Figure 9 Laser confocal images showing HEK-293T cells co-incubated with empty lentivirus LV, LV and pH-responsive phase-transition sulfonate liposomes at pH 7.4, and LV and pH-responsive phase-transition sulfonate liposomes at pH 6.0, respectively.

[0055] Figure 10 This is for the virus inactivation test of wild-type novel coronavirus pseudovirus, lentiviral vector LV, and type I herpes simplex virus HSV-I used in Example 7. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0057] All raw materials used in the following specific embodiments were purchased from the market. Among them, wild-type SARS-CoV-2 pseudovirus refers to a virus assembled by integrating the green fluorescent protein (GFP) gene into the corresponding viral envelope. It can infect susceptible cells and express green fluorescent protein, but does not have the ability to replicate.

[0058] Example 1: Preparation of pH-responsive phase transition sulfonate liposomes

[0059] 1) The preparation process of pH-responsive phase transition sulfonate liposomes is as follows:

[0060] Step 1: Weigh DOPE, DOTAP, OA, HSA and Cho separately according to the molar ratio of 2:1:1:1:0.8, and dissolve each component separately in a chloroform / methanol mixed solvent with a volume ratio of 8:2 to prepare a 10mM solution for later use.

[0061] Step 2: Add solutions of DOPE, DOTAP, OA, HSA, and Cho to a pear-shaped flask, and then add approximately 20 mL of chloroform / methanol mixed solvent to ensure complete lipid dissolution. Remove the solvent by rotary evaporation in a 55°C water bath, and then dry the remaining solvent gas in the flask with high-purity nitrogen gas. A lipid-based thin film will then be obtained on the inner wall of the flask.

[0062] Step 3: Add a small amount of PBS to the flask and use a water bath ultrasonic cleaner to peel off the lipid film on the inner wall of the flask into the PBS.

[0063] Step 4: Transfer the PBS solution containing lipids to a 5mL centrifuge tube and sonicate the liposomes at 60W for 2 minutes using a probe-type ultrasonic cell disruptor until the solution changes from milky white to opalescent. This will prepare pH-responsive phase transition sulfonic acid liposomes.

[0064] 2) Experimental Results and Analysis

[0065] Cryo-transmission electron microscopy characterized the morphology of pH-responsive phase transition sulfonic acid liposomes in pH 7.4, pH 6.8, and pH 6.0 environments, as shown in the figure. Figure 1 As shown, the prepared liposomes are stably dispersed in a high pH environment, exhibiting a spherical shape with a diameter mainly ranging from 30 to 60 nm.

[0066] from Figure 1 It is evident that as the environmental pH decreases, the hydrophilic head of the weakly acidic anionic lipid OA in the liposome composition is protonated, leading to a decrease in the negative charge of the liposomes and a weakening of the electrostatic repulsion between particles, thus causing liposome aggregation. With a further decrease in environmental pH, OA is further protonated, and the molecular shape changes from a conical to an inverted conical shape. The liposomes struggle to maintain their lipid bilayer structure, resulting in large-scale aggregation and phase transitions. Figure 1 It can be seen that the liposomes transform from the original lipid bilayer structure to the hexagonal phase, and the lattice stripes rearranged by the liposomes can be observed, indicating that the prepared liposomes have excellent pH-responsive phase transition ability.

[0067] Example 2

[0068] Following the preparation process of Example 1, in order to adjust the pH responsiveness of liposomes so that they can remain stably dispersed in the physiological environment and aggregate and undergo phase transition in the low pH environment of lysosomes or inflammatory tissues, we investigated the effect of the proportion of cationic phospholipid DOTAP on pH responsiveness. Therefore, liposomes with DOTAP accounting for 0% and 12.5% ​​of the total molar ratio of DOPE, OA and HSA were also prepared, while the proportions of other components remained unchanged, i.e., DOPE, DOTAP, OA and HSA were in molar ratios of 2:0:1:1 and 2:0.5:1:1, and other steps were the same.

[0069] The hydrodynamic diameters of pH-responsive phase transition sulfonic acid liposomes with different DOTAP contents were analyzed using a nanoparticle size potentiometry assay at pH 7.4 and pH 6.0. The results are as follows: Figure 2 As shown in (a), in an environment of pH 7.4, although the hydrodynamic diameter of the three types of liposomes increased to some extent with the increase of the proportion of cationic phospholipid DOTAP, they all remained within the range of 30-60 nm. However, in an environment of pH 6.0, the hydrodynamic diameter of all liposomes showed a significant increase. Among them, the hydrodynamic diameter of the liposomes doped with 25% DOTAP showed the most significant change, as shown in (a). Figure 2 As shown in (b), when the ambient pH decreased from 7.4 to 6.0, its hydrodynamic diameter increased from 55.74±5.01 nm to 976.33±5.94 nm, further demonstrating that the liposomes underwent large-scale aggregation and phase transition.

[0070] Application Example 1: Characterizing the interaction mechanism of liposomes with viruses at different pH levels using cryo-electron microscopy

[0071] 1) Experimental Procedure

[0072] First, the formaldehyde-inactivated wild-type SARS-CoV-2 was concentrated to 10^10 PFU / mL using ultracentrifugation. A small amount of the SARS-CoV-2 concentrate was mixed with an equal volume of liposomes and incubated at 37°C for 1 hour. After incubation, the pH of the solution was adjusted to the desired range by adding 100 mM citric acid solution dropwise to the sample, and the sample was incubated for another 10 minutes. Then, a small amount of the sample was transferred to a glow discharge-treated porous carbon membrane, and the virus solution was rapidly cooled to a glassy state using liquid ethane via a cryo-sample preparation machine. Finally, the interaction between the virus and liposomes was observed using cryo-electron microscopy at -185°C.

[0073] 2) Experimental Results and Analysis

[0074] like Figure 3As shown, the morphology of the wild-type novel coronavirus did not differ significantly under different pH conditions, indicating that the virus still has the ability to infect cells in low pH environments. After co-incubating the virus with liposomes, it was observed that even in high pH environments, the liposomes could interact strongly with the virus and even fuse with its membrane. However, in low pH environments, the liposomes underwent a significant phase transition and simultaneously disrupted the viral envelope structure.

[0075] Application Example 2: RNA gel electrophoresis test to detect viral genome leakage caused by liposomes disrupting viral structure in a low pH environment.

[0076] 1) Experimental Procedure

[0077] 30 μg / mL liposomes were mixed with 10^8 TU / mL lentivirus and incubated at 37°C for 1 h. The pH was then adjusted to a suitable range by adding 100 mM citric acid solution dropwise. The treated solution was then added to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, centrifuged at 5000 rpm for 15 min, and the lower filtrate was collected.

[0078] 0.8 g of agarose was added to 100 mL of 1×TEA, and the solution was heated in a microwave oven to melt the agarose. Before solidification, 5 μL of Goldview nucleic acid dye was added, mixed thoroughly, and poured into a mold to solidify into a gel. Then, 5 μL of filtrate containing viral RNA was taken, mixed thoroughly with 1 μL of 6×loading buffer, and added to the wells of the agarose gel. Electrophoresis was performed at 120 V for 15 min, followed by imaging using a fluorescence and chemiluminescence imaging system, and the gray values ​​of each band were quantitatively analyzed.

[0079] 2) Experimental Results and Analysis

[0080] See Figure 4 The fluorescence intensity of viral RNA filtrate incubated with liposomes at low pH was significantly higher than that at high pH. Quantitative analysis of the grayscale values ​​revealed that the amount of viral RNA leakage caused by liposomes at low pH was approximately twice that at high pH. This pH-responsive phase-inversion sulfonic acid liposome can disrupt the viral envelope structure and induce leakage of the viral genome at low pH.

[0081] Application Example 3: CCK-8 assay for detecting the cytotoxicity of pH-responsive phase transition sulfonate liposomes

[0082] 1) Experimental Procedure

[0083] NIH-3T3 cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured overnight to allow cell adhesion. Liposomes were diluted to concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 300 μg / mL in fresh DMEM high-glucose medium containing 10% FBS. Each well in the 96-well plate was divided into groups of five replicates, with 200 μL of the diluted liposomes added to each well. The plates were incubated at 37°C for 24 h. Afterward, the culture medium was aspirated, and 100 μL of CCK-8 solution was added to each well. The plates were incubated at 37°C for 2 h, and the absorbance at 450 nm was measured using a microplate reader.

[0084] 2) Experimental Results and Analysis

[0085] See Figure 5 The pH-responsive phase transition sulfonate liposomes designed in this invention did not exhibit significant cytotoxicity to NIH-3T3 cells.

[0086] Application Example 4: pH-responsive phase transition sulfonate liposomes' broad-spectrum antiviral activity

[0087] 1) Experimental Procedure

[0088] HEK-293T cells overexpressing the SARS-CoV-2 receptor ACE2 were seeded at a density of 20,000 cells per well in 48-well plates and incubated for 24 hours beforehand. pH-responsive phase-inversion sulfonate liposomes were serially diluted with Omicron variant SARS-CoV-2 pseudoviruses and incubated at 37°C for 1 hour before being directly added to the seeded cells, or the solution pH was adjusted to 6.0 with 1M HCl before being added to the seeded cells. After 2 days of incubation at 37°C, the expression of green fluorescent protein (GFP) in the cells was detected by flow cytometry to characterize the cell infection status. The GFP expression was compared with that of cells treated with only the virus to obtain the inhibitory efficiency and half-maximum effective concentration (EC50) of different concentrations of pH-responsive phase-inversion sulfonate liposomes against SARS-CoV-2.

[0089] HEK-293T cells were seeded at a density of 20,000 cells per well in 48-well plates and incubated for 24 hours beforehand. pH-responsive phase-inversion sulfonate liposomes were serially diluted with lentiviral vector LV, incubated for 1 hour, and then directly added to the seeded cells, or the solution pH was adjusted to 6.0 with 1M HCl before addition. After culturing at 37°C for 2 days, the expression of green fluorescent protein (GFP) in the cells was detected by flow cytometry to characterize the viral infection status. The GFP expression was compared with that of cells treated with only the virus to obtain the inhibitory efficiency and half-maximum effective concentration (EC50) of different concentrations of pH-responsive phase-inversion sulfonate liposomes against lentiviral vector LV.

[0090] Vero cells were seeded in 48-well plates and incubated for 24 hours beforehand. pH-responsive phase-inversion sulfonate liposomes were serially diluted with type I herpes simplex virus and incubated at 37°C for 1 hour before being added directly to the seeded cells, or the pH of the solution was adjusted to 6.0 with 1M HCl before being added to the seeded cells. After 6 days of incubation at 37°C, 100 μL of the viral cell culture was aspirated, and viral nucleic acid was extracted using a nucleic acid extraction kit (magnetic bead method) and an automated nucleic acid extractor. Nucleic acid levels were detected using qPCR. The inhibitory efficiency and half-maximum effective concentration (EC50) of different concentrations of pH-responsive phase-inversion sulfonate liposomes against type I herpes simplex virus were compared with those of the virus-only group.

[0091] 2) Experimental Conclusions

[0092] See Figure 6 , Figure 7 The pH-responsive phase-inversion sulfonate liposomes exhibited EC50 values ​​of 54.31 μg / mL and 11.77 μg / mL against Omicron variant novel coronavirus pseudovirus at pH 7.4 and pH 6.0, respectively; 27.15 μg / mL and 5.162 μg / mL against lentiviral vector LV at pH 7.4 and pH 6.0, respectively; and 6.08 μg / mL and 1.81 μg / mL against herpes simplex virus type I (HSV-I) at pH 7.4 and pH 6.0, respectively. This pH-responsive phase-inversion sulfonate liposome effectively inhibited infection by various viruses at safe concentrations in both high and low pH environments, demonstrating broad-spectrum antiviral activity. Application Example 5: Co-localization of pH-responsive phase-inversion sulfonate liposomes and viruses in lysosomes.

[0093] 1) Experimental Procedure

[0094] HEK-293T cells were seeded at a density of 200,000 cells per dish in laser confocal culture dishes and incubated at 37°C for 24 h to allow for cell adhesion. Rhodamine B-labeled PE phospholipids were incorporated into liposomes at a concentration of 1% to prepare Rhodamine B-loaded liposomes. The Rhodamine B-loaded liposomes were diluted to 200 μg / mL in fresh DMEM high-glucose medium free of serum and phenol red, and the liposome solution was added to the laser confocal culture dishes and incubated at 37°C for 2 h to allow for sufficient cell endocytosis. The solution was then aspirated, and the cells were washed three times with PBS. Cells were then incubated with 50 nM Lysotraker lysosomal dye for 1 h, and the incubation solution was discarded followed by washing three times with PBS. Subsequently, 4% paraformaldehyde solution was added to the culture dishes, and the cells were incubated at room temperature for 10 min to fix the cells. The paraformaldehyde solution was discarded, and the cells were washed three times with PBS. After washing, 1 μg / mL DAPI solution was added to the culture dish, and the cells were incubated at room temperature for 15 min. Then, the cells were washed three times with PBS. Finally, 1 mL of PBS was added to the culture dish to maintain the cell state. The co-localization of liposomes (red fluorescence) and lysosomes (green fluorescence) was observed using a laser confocal microscope.

[0095] Empty lentivirus was mixed with 10 μM of the lipophilic dye DiO and incubated on a shaker at 37 °C for 15 min. Then, it was centrifuged at 4 °C and 5000 rpm for 15 min using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times with PBS to remove excess fluorescent dye, thus preparing DiO-LV with a green fluorescent viral envelope.

[0096] HEK-293T cells were seeded at a density of 200,000 cells per dish in laser confocal microscopy culture dishes and incubated at 37°C for 24 h to allow for cell adhesion. Liposomes loaded with 200 μg / mL Rhodamine B and 10^6 TU / mL DiO-LV were incubated in fresh DMEM high-glucose medium (serum-free and phenol red-free) for 1 h, then added to the culture dishes and incubated at 37°C for 2 h to allow for full cell endocytosis. The solution was then aspirated, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde, washed three times, stained with DAPI, washed three more times, and then 1 mL of PBS was added to maintain cell morphology. Finally, the colocalization of liposomes (red fluorescence) and viruses (green fluorescence) was observed using laser confocal microscopy.

[0097] 2) Experimental Results and Analysis

[0098] See Figure 8The top image shows that liposomes can be taken up into the cell and exhibit significant fluorescent co-localization with intracellular lysosomes. The bottom image shows the co-localization of liposomes and viruses, indicating that liposomes can be internalized into the cell along with the virus. Both figures demonstrate that the pH-responsive phase-inversion sulfonic acid liposomes prepared in this invention can bind to viruses in a high pH environment and then inactivate the viruses through phase transition in the acidic environment of the lysosome via endocytosis.

[0099] Application Example 7: Extracellular inactivation of viruses by pH-responsive phase transition sulfonate liposomes under low pH conditions

[0100] 1) Experimental Procedure

[0101] HEK-293T cells were seeded at a density of 200,000 cells per dish in laser confocal microscopy culture dishes and incubated at 37°C for 24 h to allow them to adhere. Liposomes loaded with 200 μg / mL Rhodamine B and 10^6 TU / mL DiO-LV were incubated in fresh DMEM high-glucose medium (serum-free and phenol red-free) for 1 h. The pH of the solution was adjusted to 6.0 with 1M HCl, and the mixture was then added to the culture dishes and incubated at 37°C for 2 h. The solution was then aspirated, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde, stained with DAPI, washed three more times, and then 1 mL of PBS was added to maintain cell morphology. Finally, the interaction between liposomes (red fluorescence) and viruses (green fluorescence) and cells (blue fluorescence) was observed using laser confocal microscopy.

[0102] 2) Experimental Results and Analysis

[0103] See Figure 9 At pH 6.0, the green fluorescence of the virus was not visible inside the cells, indicating that in a low pH environment, liposomes can destroy the viral structure extracellularly, thereby inactivating the virus and preventing it from infecting cells.

[0104] Application Example 8: pH-responsive phase transition sulfonate liposome virus inactivation ability

[0105] 1) Experimental Procedure

[0106] HEK-293T cells overexpressing ACE2 were seeded in 96-well plates and incubated for 24 h. pH-responsive phase-inversion sulfonate liposomes were incubated with wild-type novel coronavirus pseudovirus at 37°C for 1 h. The virus solution was diluted 10-fold with PBS at pH 7.4 and pH 6.0, and then serially diluted 10^5-fold with culture medium. The diluted virus was added to the cells seeded in the wells, and after 48 h of culture, the expression of green fluorescent protein in the cells was analyzed using fluorescence microscopy. The virus titer was calculated based on the fluorescence images and the dilution factors.

[0107] HEK-293T cells were seeded in 96-well plates and incubated for 24 h. pH-responsive phase-inversion sulfonate liposomes were incubated with lentiviral vector LV for 1 h. The virus solution was diluted 10-fold with PBS at pH 7.4 and pH 6.0, and then serially diluted 10^5-fold with culture medium. The diluted virus was added to the cells seeded in the wells, and after 48 h of culture, the expression of green fluorescent protein in the cells was analyzed using fluorescence microscopy. The viral titer was calculated based on the fluorescence images and the dilution factors.

[0108] Vero cells were seeded in 96-well plates and cultured at 37°C for 24 h. pH-responsive phase-inversion sulfonate liposomes were incubated with type I herpes simplex virus for 1 h. The virus solution was diluted 10-fold with PBS at pH 7.4 and pH 6.0, and then serially diluted 10^5-fold with culture medium. The diluted virus was added to the cells seeded in the well plates, and after 6 days of culture, 100 μL of the virus-cell culture was aspirated. Viral nucleic acid was extracted using a nucleic acid extraction kit (magnetic bead method) and an automated nucleic acid extractor, and the nucleic acid level was detected using qPCR.

[0109] 2) Experimental Results and Analysis

[0110] See Figure 10 At pH 7.4, viruses reacting with liposomes regained their ability to infect cells after significant dilution with PBS, and the infection titer was not significantly different from that of the pure virus group. This indicates that at pH 7.4, liposomes mainly neutralize viruses through adhesion. However, if viruses co-incubated with liposomes are placed in a low pH environment, even with high dilution, the viral titer still decreases significantly, and the viruses still do not regain their ability to infect cells. This demonstrates that liposomes can inactivate viruses in a low pH environment, and this inactivation has a broad-spectrum effect.

Claims

1. A pH-responsive phase-transition sulfonate liposome for disrupting a viral membrane structure, characterized by, Composed of double-chain phospholipid, single-chain lipid and adjuvant; The double-chain phospholipid is dioleoylphosphatidylethanolamine and (2,3-dioleoyl-propyl)-trimethylammonium chloride; the single-chain lipid is oleic acid and sodium hexadecylsulfate; and the adjuvant is cholesterol; The molar amount of the (2,3-dioleoyl-propyl)-trimethylammonium chloride is 20-30% of the total molar amount of the dioleoylphosphatidylethanolamine, the oleic acid and the sodium hexadecylsulfate; The molar ratio of the dioleoylphosphatidylethanolamine, the oleic acid and the sodium hexadecylsulfate is 2:1-1.5:1-1.5; The cholesterol is 5-25% of the total molar amount of the double-chain phospholipid and the single-chain lipid.

2. The method for preparing a pH-responsive phase transition sulfonate liposome for destroying a viral membrane structure according to claim 1, characterized by, The method comprises the steps of: Step 1, dissolving and mixing the dioleoylphosphatidylethanolamine, the (2,3-dioleoyl-propyl)-trimethylammonium chloride, the oleic acid, the sodium hexadecylsulfate and the cholesterol, and removing the solvent by rotary evaporation to obtain a lipid film; Step 2, dispersing the lipid film by soaking in an aqueous solution, and obtaining the pH-responsive phase transition sulfonic acid liposome by ultrasonic crushing.

3. The method for preparing a pH-responsive phase transition sulfonate liposome for destroying a viral membrane structure according to claim 2, characterized by, The solvent used in step 1 comprises one or more of dichloromethane, chloroform and methanol; The aqueous solution in step 2 comprises one or more of phosphate buffer, deionized water and ultrapure water.

4. The method for preparing a pH-responsive phase transition sulfonate liposome for destroying a viral membrane structure according to claim 2, characterized by, The ultrasonic power in step 2 is 40-60 W, and the ultrasonic time is 1-15 min.

5. The method for preparing a pH-responsive phase transition sulfonate liposome for destroying a viral membrane structure according to claim 2, characterized by, The pH-responsive phase transition sulfonic acid liposome has a particle size of 30-60 nm.

6. Use of the pH-responsive phase-transition sulfonate liposome according to claim 1 in the manufacture of a medicament for preventing or / and treating a disease caused by a viral infection, characterized in that, The virus comprises one or more of severe acute respiratory syndrome virus, novel coronavirus and herpes simplex virus.

Images