Patent Application
20230256003
The present disclosure relates to an anti-inflammatory use and a prophylactic or therapeutic use for sepsis and septic shock of a functionalized transition metal dichalcogenide (TMD).
Sepsis is caused by the abnormal defensive action of the body against microbial infection. It is associated with the activation of macrophages and the excessive production of inflammation-related factors, reactive oxygen species (ROS) and reactive nitrogen species (RNS) resulting therefrom, which result in severe inflammatory responses throughout the body. Systemic inflammatory response syndrome (SIRS) is diagnosed when two or more symptoms of body temperature above 38° C., body temperature below 36° C., respiratory rate exceeding 24 times per minute (tachypnea), heart rate exceeding 90 beats per minute (tachycardia) and increased or significantly decreased number of white blood cells occur, and sepsis is diagnosed when the systemic inflammatory response syndrome is one caused by microbial infection. Sepsis may result in septic shock. Severe sepsis causes poor function of organs (heart, kidneys, liver, brain, lungs, etc.) and even shock when aggravated. Sepsis may be caused by various pathogens. Although it is usually caused by bacteria, it may also be caused by viruses or fungi. Also, sepsis is often caused by postoperative Infection. The risk of death from sepsis caused by infection or postoperative infection is 40-90%.
It is understood that sepsis results from the complicated interactions between the invading pathogens, the immune system of the host, inflammations and the coagulation system. Both the degree of response of the host and the characteristics of the pathogens has important effects on the prognosis of sepsis. Organ failure observed in sepsis is caused by the inappropriate response of the host to the pathogens. Excessive response of the host to the pathogens may cause damage to the organs of the host. Based on this concept, antagonists against the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, etc., which play a major role in the inflammatory response of the host, have been attempted as therapeutic agents for sepsis, but most of them were unsuccessful. In addition, development of effective antioxidant therapy for sepsis has been attempted to scavenge ROS and RNS, but no effective therapeutic agent has been developed yet. Therefore, there is a need of a new therapeutic agent for treating sepsis and septic shock.
Meanwhile, transition metal dichalcogenides (TMDs) are naturally abundant compounds with the formula MX2 (M: transition metal, X: chalcogen element) wherein unit layers are stacked. TMDs have a layered structure liked the well-known graphite, and can be obtained as 2D single layers like graphene because the bonding between the layers is weak.
The TMD compounds having a 2D layered structure are also highly applicable as semiconductor logic devices and electrochemical catalysts, including the applicability shown in researches about graphene. Recently, the applicability of 2D TMDs functionalized with amphiphilic block polymer compounds for antioxidant use has been proposed.
The present disclosure is directed to providing an anti-inflammatory pharmaceutical composition of a 2D TMD functionalized with an amphiphilic polymer compound, and a pharmaceutical composition for preventing or treating sepsis and septic shock, which contains the TMD.
However, the problem to be solved by the present disclosure is not limited to that mentioned above. Other problems not mentioned above will be clearly understood by those having ordinary knowledge in the art from the following description.
The present disclosure provides a pharmaceutical composition for preventing or treating sepsis or septic shock, which contains a 2D transition metal dichalcogenide (TMD) functionalized with an amphiphilic block polymer compound including a hydrophilic block and a hydrophobic block as an active ingredient.
The present disclosure also provides an anti-inflammatory pharmaceutical composition, which contains the 2D transition metal dichalcogenide (TMD) functionalized with the amphiphilic block polymer as an active ingredient.
In an exemplary embodiment of the present disclosure, the hydrophilic block refers to a moiety of a specific polymer compound which has strong affinity for water and thus is dissolved in water. It may be polyethylene oxide (PEO) or polyethylene glycol (PEG).
In another exemplary embodiment of the present disclosure, the hydrophobic block refers to a moiety of a specific polymer compound which lacks affinity for water and thus is not dissolved in water. It may be poly(ε-caprolactone) (PCL).
In another exemplary embodiment of the present disclosure, the amphiphilic block polymer compound refers to a polymer compound or a copolymer including both a hydrophilic moiety (block) and a hydrophobic moiety (block) in the polymer compound or copolymer. It may be a block polymer compound or a block copolymer including PEG as a hydrophilic block and PCL as a hydrophobic block (“PCL-b-PEG”), and may have a structure of Chemical Formula 1:
In another exemplary embodiment of the present disclosure, n in Chemical Formula 1 may be an integer from 400 to 3000, specifically an integer from 460 to 2000.
In another exemplary embodiment of the present disclosure, the TMD may be one or more selected from a group consisting of molybdenum disulfide (MoS2), tungsten disulfide (WS2), molybdenum diselenide (MoSe2) and tungsten diselenide (WSe2), specifically tungsten disulfide, molybdenum diselenide or tungsten diselenide, more specifically tungsten disulfide.
In another exemplary embodiment of the present disclosure, the functionalized 2D TMD may have a thickness of 1-50 nm, specifically 1-10 nm, more specifically 3-5 nm.
In another exemplary embodiment of the present disclosure, the functionalized 2D TMD may have a lateral length of 1-500 nm, specifically 1-100 nm, more specifically 30-43 nm.
In another exemplary embodiment of the present disclosure, the functionalized 2D TMD may have the activity of scavenging intracellular and/or mitochondrial ROS and/or RNS.
In another exemplary embodiment of the present disclosure, the functionalized 2D TMD may inhibit the secretion of an inflammatory cytokine. The inflammatory cytokine may be TNF-α, IL-1β, IL-6, IL-18 or IL-12p40, and the functionalized 2D TMD may not affect the secretion of an anti-inflammatory cytokine.
The present disclosure also provides a method for preventing or treating sepsis or septic shock, which includes a step of administering the functionalized 2D TMD to a subject, wherein the subject may be any mammal exhibiting acute inflammatory response to microbial infection, without limitation, specifically human.
In an exemplary embodiment of the present disclosure, the administration may be intravenous administration or intraperitoneal administration.
A 2D TMD nanosheet of the present disclosure functionalized with an amphiphilic block polymer compound has superior activity of scavenging intracellular ROS and RNS and mitochondrial ROS, and the scavenging activity is maintained consistently at low pH. In addition, it can increase survivability in an animal model of sepsis and prevent the aggravation of symptoms by inhibiting the excessively increased secretion of inflammatory cytokines due to microbial infection and reducing the number of pathogens. Therefore, it can be provided as an anti-inflammatory/antioxidant agent for preventing and treating sepsis and septic shock.
WS2/IR783.
The present disclosure was completed based on the finding that a 2D transition metal dichalcogenide (2D TMD) functionalized with an amphiphilic block polymer compound has scavenging activity for reactive oxygen species and reactive nitrogen species and has therapeutic effect for sepsis caused by acute microbial infection.
Specifically, the inventors of the present disclosure have prepared exfoliated 2D TMD (WS2, MoSe2 or WSe2) nanosheets functionalized with PCL-b-PEG in aqueous solutions and found out that they are biocompatible through structural analysis (see Example 1).
Then, the inventors of the present disclosure have confirmed the sustainability of scavenging activity for various radicals by comparing the three TMD nanosheets with Trolox through ABTS radical scavenging activity assay. As a result, the three TMD nanosheets showed consistent ROS and RNS scavenging activity at significantly lower concentrations as compared to Trolox. In addition, the three TMD nanosheets showed superior activity for scavenging of hydrogen peroxide, hydroxyl radical, superoxide and nitric oxide. In particular, the WS2 nanosheet showed better scavenging activity than other nanosheets and the W-based TMD nanosheets showed better superoxide scavenging activity than the Mo-based TMD nanosheets (see Example 2).
In severe sepsis, abnormal inflammatory response decreases the pH of cells and tissues than normal levels. Accordingly, it is expected from the sustainability of the radical scavenging activity of the TMD nanosheets even under low pH conditions that they can be effective in treating sepsis in vivo. In addition, the effect of the 2D TMD nanosheets was investigated in inflammation-induced cells.
For this, the cytotoxic of the three TMD nanosheets was investigated experimentally and the level of mitochondrial ROS and intracellular hydrogen peroxide, hydroxyl radical, superoxide and nitric oxide was investigated after treating LPS-induced inflammatory BMDMs with the TMD nanosheets. As a result, it was confirmed that the TMD nanosheets have superior scavenging activity for intracellular ROS and RNS, and the WS2 nanosheet has the best activity. The three TMD nanosheets scavenged ROS and RNS, but were not involved in the ROS generation mechanism in the mitochondria (see Example 3).
In addition to the ROS and RNS scavenging activity in inflammation-induced cells, it was confirmed that the TMD nanosheets can decrease the secretion of acute and chronic inflammatory cytokines in a concentration-dependent manner and have no effect on the secretion of anti-inflammatory cytokines. In addition, they also scavenged intracellular and mitochondrial ROS and inhibited the expression of pro-inflammatory cytokines in human monocytes infected with E. coli, P. aeruginosa or S. aureus (see Example 4).
Also, the inventors of the present disclosure have found out that the treatment of human monocytes infected with E. coli, P. aeruginosa or S. aureus with the WS2 nanosheet reduces the number of bacteria in the monocytes. That is to say, it was confirmed that the TMD nanosheets can be used as effective therapeutic agents for sepsis because they can consistently scavenge ROS and RNS, decrease the secretion of inflammatory cytokines and reduce the number of bacteria in infected cells (see Example 4).
The therapeutic effect of the TMD nanosheets for sepsis was investigated again in a CLP-induced bacteremic mouse model. Specifically, it was confirmed that the number of bacteria is decreased remarkably and survival rate is increased when the TMD nanosheets are injected intravenously or intraperitoneally to CLP-induced bacteremic mice. In addition, it was confirmed from the histopathological conditions of kidney, lungs, liver and spleen that the TMD nanosheets can prevent the aggravation of sepsis in the bacteremic mice (see Example 5).
Meanwhile, the TMD nanosheets showed different behaviors depending on administration routes. They were discharged from the body in 3 days when injected intravenously, but could remain in the body for 10 days when they were injected intraperitoneally. Accordingly, the administration route of the TMD nanosheets can be determined in consideration of the conditions and/or pathologies of the subject (see Example 5).
Therefore, the inventors of the present disclosure provide a prophylactic or therapeutic use for an inflammatory disease and a prophylactic or therapeutic use for sepsis and/or septic shock of an exfoliated 2D TMD nanosheet functionalized with an amphiphilic block polymer compound.
In the present specification, the “prevention or treatment of sepsis or septic shock” refers to the reduction, improvement or removal of clinical symptoms associated with sepsis and all or some symptoms of a condition associated with multiple organ dysfunction syndrome (e.g., fever of various grades, hypoxemia, tachycardia, endothelitis, myocardial infarction, delirium, vascular collapse, organ damage, acute respiratory distress syndrome, coagulopathy, heart failure, renal failure, shock and/or lethargy).
In the present specification, the “functionalization” includes changing, improving or modifying the physical, chemical or biological properties (e.g., interfacial characteristics) by introducing, treating, coating or binding a physical means (heat, pressure, vibration, light, etc.) or a chemical means (specific compound, polymer, functional group, etc.). For example, introduction of a specific polymer compound for hydrophobic interaction may be a kind of functionalization.
In the present disclosure, prevention refers to any action of delaying infection by sepsis-inducing bacteria or delaying the onset of inflammatory response and disease caused by the infection by administering a pharmaceutical composition according to the present disclosure, and treatment refers to any action of improving or favorably changing the inflammatory response against the infection or the symptoms of infection or sepsis or septic shock by administering the pharmaceutical composition according to the present disclosure.
In the present disclosure, the pharmaceutical composition may further contain, in addition to the functionalized 2D TMD nanosheet, one or more known antibiotic and/or anti-inflammatory agent, and may further contain a suitable carrier, excipient or diluent commonly used for preparation of a pharmaceutical composition.
In the present disclosure, the “carrier”, which is also called a vehicle, refers to a compound which facilitates the introduction of a protein or a peptide into a cell or a tissue. For example, dimethyl sulfoxide (DMSO) is a carrier commonly used to facilitate the introduction of many organic substances into the cells or tissues of an organism.
In the present disclosure, the “diluent” is defined as a compound which stabilizes the biologically active form of a target protein or peptide and is diluted in water wherein the protein or peptide is dissolved. A salt dissolved in a buffer solution is used as a diluent. Phosphate-buffered saline, which mimics the salt form of human body fluid, is a commonly used buffer solution. A buffer diluent rarely alters the biological activity of a compound because a buffer salt can control the solution pH at low concentration. The compound used in the present disclosure can be administered to a human patient either alone or in combination with another ingredient in combination therapy or can be administered as being mixed with a suitable carrier or excipient in a pharmaceutical composition.
In addition, the pharmaceutical composition according to the present disclosure may be formulated into a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, a sterile injection solution, etc. according to common methods and, depending on purposes, the composition of the present disclosure may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically), specifically parenterally, more specifically intravenously. The administration dosage may be determined adequately by those skilled in the art although it may vary depending on the physical condition and body weight of a patient, the severity of a disease, drug type, administration route and administration period. For example, about 0.001-1000 mg may be administered as a mixture with a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure may be administered once or several times a day as necessary, and may be administered alone or in combination with surgery, hormone therapy, drug therapy or a biological response modulator.
The functionalized 2D TMD nanosheet of the present disclosure may be provided as a quasi-drug composition for the purpose of preventing or improving a microbial infection disease inducing sepsis. The quasi-drug composition of the present disclosure may be used together with another quasi-drug or quasi-drug ingredient according to a common method. The quasi-drug composition may be used in an antibacterial cleanser, a shower foam, a gargle, a wet tissue, a laundry soap, a hand sanitizer, a humidifier filler, a mask, an ointment, a filter filler, etc., although not being limited thereto.
The present disclosure may be changed variously and may have various exemplary embodiments. Hereinafter, specific exemplary embodiments will be described in detail referring to drawings. However, it is not intended to limit the present disclosure to those specific exemplary embodiments, and it should be understood that all modifications, equivalents and substitutes thereof are included in the scope of the present disclosure. When describing the present disclosure, detailed description of the known art will be omitted if it is determined that it may unnecessarily obscure the subject matter of the present disclosure
[Experimental Method]
1. Preparation of TMD Nanosheets
Bulk TMDs (0.6 g of WS2, 0.61 g of MoSe2 and 0.83 g of WSe2) were added to 20 mL of a PCL460-b-PEG5000 solution (2 mg/mL). Then, the mixture was sonicated for 1 hour (pulse-on for 6 sec, pulse-off for 2 sec). The solution was centrifuged at 700 g for 1 hour. After centrifuging the obtained supernatant at 14,500 g for 1 hour, the produced precipitate was centrifuged again after adding water and then washed after centrifuging again. The precipitate was dispersed in 8.5 mL of water and the solution was centrifuged at 2,000 g for 30 minutes. TMD nanosheets were obtained from the supernatant.
2. Measurement of ROS and RNS Scavenging Activity of TMD Nanosheets
2-1. Measurement of H2O2 Scavenging Activity
After reacting 150 μL of H2O2 (3 mM) and 150 μL of the TMD nanosheet (54 nM) for 8 hours, the TMD nanosheet was removed by centrifugation. Then, after mixing with 100 μL of a FeSO4 (120 μM) solution and leaving alone for 1 hour, 50 μL of terephthalic acid (TA, 22.5 mM in 0.1 M NaOH) was added and fluorescence emission was measured at 45 minutes with a spectrofluorometer.
2-2. Measurement of Hydroxyl Radical Scavenging Activity
125 μL of H2O2 (6 mM) and 125 μL of FeSO4 (60 μM) were mixed for 30 minutes to generate hydroxyl radicals. After adding 200 μL of the TMD nanosheet (60 nM) to the hydroxyl radical solution, the mixture was stirred at 25° C. for 90 minutes. After adding to TA (50 μL, 30 mM, 0.1 M NaOH), the mixture was agitated for 45 minutes. Then, after removing the TMD nanosheet by centrifugation, fluorescence emission was measured with a spectrofluorometer.
2-3. Measurement of Superoxide Scavenging Activity
Superoxide was generated by reacting xanthine (XAN) with xanthine oxidase (XOD). After mixing 30 μL of XAN (1 mM), 125 μL of the TMD nanosheet (54 nM) and 15 μL of WST-8 (5-(2,4-disulfophenyl)-3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazolium), 30 μL of XOD (0.05 U mL−1) was added. After stirring the mixture at 25° C. for 2 hours and removing the TMD nanosheet through centrifugation, the absorbance of the supernatant was measured using a microplate reader and a UV-Vis spectrometer.
2-4. Measurement of Nitric Oxide Scavenging Activity
NO (nitric oxide) was generated using sodium nitroprusside (SNP). The amount of NO after scavenging by the TMD was measured using an analysis kit (Abcam). Specifically, 150 μL of SNP (20 mM) and 150 μL of the TMD nanosheet (54 nM) were mixed with a 0.1 M PBS solution (pH 7.4) and then reacted by shaking for 2 hours. 7.5 μL of nitrate reductase and 7.5 μL of enzyme cofactor were added to the mixture and reaction was conducted for 1 hour. Then, after conducting reaction with nitrite for 20 minutes by adding 50 μL of Griess reagents R1 and R2, the TMD nanosheet was removed. Then, the absorbance of the supernatant was measured with a microplate reader and a UV-Vis spectrometer.
3. Flow Cytometry of ROS and RNS Production
Cells cultured in SFM (serum-free medium) supplemented with a redox-sensitive dye (2 μM dihydroethidium (DHE for 02 Calbiochem), 10 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA for H2O2; Calbiochem), 10 μM 4,5-diaminofluorescein diacetate (DAF-2DA for NO; Calbiochem) or 5 μM MitoSOX™ Red mitochondrial superoxide indicator (for mitochondrial ROS; Molecular Probes)) were subjected to flow cytometry for measurement of intracellular ROS. The cells were washed quickly with pulse spin and then analyzed immediately using FACSCalibur (BD Biosciences, San Jose, Calif.). Data were quantified using the CellQuest software (BD Biosciences).
4. Measurement of NADPH Oxidase Activity
The production of peroxide in cells was measured by the lucigenin (bis-N-methylacridinium nitrate)-ECL method. Specifically, a cell lysate was mixed with 50 mM PBS (pH 7.0), 1 mM EGTA, 150 mM sucrose and a protease inhibitor. After reaction at 37° C. for 30 minutes, a Krebs-HEPES buffer containing lucigenin (5 μM) and NADPH (100 μM) was added. All data were expressed as relative light units per 1×105 cells.
5. Preparation of Animal Models
CLP (cecal ligation and puncture)- or bacteria-induced sepsis models were prepared using 6-week-old C57BL/6 female mice (Samtako Bio, Gyeonggi-do, Korea).
The CLP-induced mouse model was prepared by intravenously injecting pentothal sodium (50 mg/kg) to the mice, exposing the cecum by preparing a small incision at the center of the abdomen, ligating the cecum below the serosa, preparing two openings using a 22-gauge needle and then closing the abdomen. The survival rate of the mice was monitored for 7 days.
The bacterial infection-induced sepsis model was prepared by culturing E. coli (serotype 086: K61 (B7), ATCC 12701), S. aureus (ATCC 6538) or P. aeruginosa (ATCC 10145) at 37° C. in BHI (brain-heart-infusion) BD (broth medium). Mid-log-phase bacteria (absorbance 0.5) were used. The bacterial culture was isolated and stored at −80° C. until use. The effective concentration of LPS was 50 pg/mL or lower. All the procedures were approved by the Hanyang University Biosafety Committee (Protocol 2014-01).
In order to exfoliate ultrathin functionalized 2D TMD nanosheets in aqueous solutions, an amphiphilic poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) diblock copolymer was used for liquid-phase exfoliation of bulk (3D) WS2, MoSe2 or WSe2 (
It was confirmed from the TEM (transmission electron microscopy) and AFM (atomic force microscopy) images of the WS2, MoSe2 and WSe2 nanosheets that each nanosheet has an average lateral length of 37.5 nm and an average thickness of 4 nm (
The functionalization of the 2D TMD nanosheet with PCL-b-PEG was identified clearly from FT-IR (Fourier transform infrared) spectra (
The chemical structure of the 2D TMD nanosheets functionalized with PCL-b-PEG was analyzed by XPS (X-ray photoelectron spectroscopy) (
Unlike the TMD with an octahedral (1T) structure exhibiting cytotoxicity, the TMD nanosheet of the 2H phase has superior biocompatibility in vitro and in vivo. Accordingly, it can be seen that all the three TMD nanosheets functionalized with PCL-b-PEG and exfoliated in aqueous solutions have 2H structures and have superior biocompatibility.
In addition, the 2H structure of the exfoliated TMD nanosheets could be identified from optical properties. The characteristic A excitonic absorption peaks of the WS2, MoSe2 and WSe2 nanosheets were observed at 623, 792 and 749 nm, respectively. Through this, it was clearly confirmed that the exfoliated TMD nanosheets have 2H phases. In addition, the structure of the exfoliated TMD nanosheets was confirmed again by Raman spectroscopy. The intensity ratio of the longitudinal acoustic mode [2LA(M)] peak at 350 cm−1 to the out-of-plane vibrational mode (A1g) peak at 418 cm−1 in the Raman spectrum was larger than 6.7 for the WS2 nanosheet, indicating that WS2 was exfoliated very thinly. The peak position in the A1g mode is indicative of the number of MoSe2 layers. The MoSe2 nanosheet showed an A1g mode peak at 239 cm−1, which corresponds to MoSe2 having thin layers. In addition, the WSe2 nanosheet showed a single peak (248 cm−1) due to degeneration of A1g and E12g modes. This means that the WSe2 nanosheet has been exfoliated well as thin layers (
The sustainability of the ROS scavenging activity of the three TMD nanosheets functionalized with PCL-b-PEG was investigated by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay. As can be seen from
Then, the ROS scavenging activity of the three TMD nanosheets for hydrogen peroxide was investigated by TA (terephthalic acid) assay. As can be seen from
Then, the scavenging activity of the three TMD nanosheets for hydroxyl radicals was investigated by TA assay. As can be seen from
The scavenging activity of the three TMD nanosheets for superoxide was investigated by WST-8 (tetrazolium salt) assay. Superoxide radicals generated by the reaction between xanthine and xanthine oxidase convert WST-8 tetrazolium into WST-8 formazan, and the WST-8 formazan exhibits strong absorbance at 460 nm. As can be seen from
Nitric oxide (NO) can be produced spontaneously from sodium nitroprusside (SNP) at physiological pH. Nitric oxide can be quantitated by Griess reaction after converting all nitrate (NO3−) to nitrite (NO2−) with nitrate reductase. In the presence of NO in solution, strong absorption at 540 nm was observed after the reaction of the Griess reagent with nitrite converted from NO (
Subsequently, the oxidation state of the WS2 nanosheets was investigated by XPS analysis and the mechanism responsible for the ROS and RNS scavenging of the TMD nanosheets was investigated. As can be seen from the XPS spectra (
The ROS and RNS scavenging mechanism of the TMDs could be understood from these results (
First, the cytotoxicity of WS2, MoSe2 and WSe2 was investigated in macrophages. As can be seen from
Then, the ROS scavenging activity of the TMD nanosheets in mitochondria was measured using LPS-induced inflammatory cells. As can be seen from
Then, it was investigated by measuring the activity of enzymes involved in the production of ROS in macrophages stimulated with LPS whether the TMDs inhibit the activity of the enzymes. Specifically, the activity of NADPH oxidase (NOX) involved in the formation of intracellular ROS and OXPHOS (mitochondrial oxidative phosphorylation) complexes I & III involved in the formation of mitochondrial ROS was investigated. As can be seen from
From these result, it can be seen that, although the TMD nanosheets effectively remove ROS and RNS in cells and mitochondria, they have no effect on the activity and expression of ROS-producing enzymes in macrophages stimulated with LPS.
The increased level of ROS and RNS activates immune cells in sepsis, TLR4 activation of immune cells leads to organ damage. Therefore, it was investigated whether the TMD nanosheets affect the inflammatory response regulated by ROS. It was confirmed that the TMD nanosheets functionalized with PCL-b-PEG effectively inhibits the excessive secretion of acute inflammatory cytokines including TNF-α, IL-1β and IL-18 in a concentration-dependent manner (
In order to further understand the intracellular regulatory mechanism of the TMD nanosheets for scavenging of ROS and RNS and inhibition of inflammation in macrophages, Raw264.7 cells were pre-incubated with cytochalasin D. Cytochalasin D can completely block the introduction of the TMD nanosheets into cells by inhibiting the polymerization of actin. As can be seen from
Then, the WS2 nanosheet showing the best activity among the three TMD nanosheets was applied to human monocytes and its activity of scavenging ROS and RNS and inhibiting inflammatory cytokines was investigated. Specifically, monocytes were infected with common bacteria such as E. coli, P. aeruginosa, S. aureus, etc. or stimulated with LPS. As can be seen from
From these results, it can be seen that, since the WS2 nanosheet decreases the excessive secretion of inflammatory cytokines and scavenges intracellular and mitochondrial ROS and RNS in sepsis, it can be used as an effective antioxidant for treating sepsis by inhibiting inflammatory response.
Then, the therapeutic effect of the WS2 nanosheet for sepsis was investigated for an animal model of severe sepsis. A CLP (cecal ligation and puncture)-induced bacteremic mouse model, which is the most similar to human microbial infection, was used, and the administration dose, administration route and administration period of the WS2 nanosheet were varied (
The therapeutic effect of the WS2 nanosheet antioxidant was similarly manifested in the animal model of severe sepsis. Additionally, the number of bacteria in the septic mice was counted after the treatment with the WS2 nanosheet because there is a positive correlation between CLP-induced lethality and the number of bacterial colonies in peripheral blood and peritoneal fluid. As can be seen from
Furthermore, histological analysis was performed on the liver, lung and spleen tissues. Specifically, the thickness of the alveolar wall and the necrosis of liver and spleen cells was compared with normal tissues by staining the tissues of the CLP-induced bacteremic mice with H&E (hematoxylin & eosin) (
Finally, the biodistribution and pharmacokinetics of the WS2 nanosheet were investigated using a CT (IVIS spectrum-chromatography) system. The WS2 nanosheet was functionalized with a fluorescent dye (IR783,
While the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are merely preferred exemplary embodiments and the scope of the present disclosure is not limited by them. Accordingly, it is to be appreciated that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0077609 | Jun 2020 | KR | national |
| 10-2021-0081531 | Jun 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/007973 | 6/24/2021 | WO |
