Anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs and its applications

Abstract

The present invention belongs to the technical field of liver disease treatment drugs, and specifically relates to an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs and its applications. 5.4 O@CNDs were prepared by the following steps: dissolving CuCl2 in a carbon dot aqueous solution, reacting at 75°C to 80°C for 10 min to 12 min, then adding L-ascorbic acid aqueous solution, adjusting the pH to 7.0-8.0 with NaOH solution, and reacting at 75°C to 80°C for 10 h to 12 h. After the reaction, the precipitate was removed by centrifugation, and the supernatant was dialyzed and freeze-dried to obtain the Cu@CNDs. 5.4 O@CNDs. The research results of this invention prove that nanozyme Cu 5.4 O@CNDs has the potential to treat a variety of acute liver injury diseases and is an intervention strategy with great clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of liver disease treatment drugs, and specifically relates to an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs and their applications. Background Art

[0002] Acute liver failure (ALF) is a clinical syndrome characterized by extensive hepatocellular necrosis, hepatic hypoplasia, and multi-organ dysfunction. Clinical symptoms of ALF typically include liver dysfunction, abnormal liver biochemical parameters, and coagulopathy. Although the incidence is relatively low, up to half of cases may develop multi-organ failure and death, with a mortality rate as high as 30%. Therefore, there is an urgent need to develop effective treatment strategies targeting the causes of acute liver failure.

[0003] The causes of ALF include hepatic ischemia-reperfusion injury (HIRI), viral infection, autoimmune hepatitis and various other acute liver injury (ALI) triggers. Several recent studies have shown that high levels of oxidative stress and inflammatory response in the body play a vital role in all types of liver damage, among which reactive oxygen species (ROS) are crucial. Hepatocytes contain a large number of mitochondria. When hepatotoxic compounds and their reactive metabolites, as well as other factors (such as hypoxia and reoxidation) destroy the electron transport chain in the mitochondrial membrane, excessive ROS are generated. Due to their strong oxidative properties, ROS are important inflammatory mediators that can cause cell damage at high concentrations. The main reactive oxygen species are superoxide anion free radicals (·O2 -), hydroxyl radicals (·OH), and hydrogen peroxide (H2O2). In addition, liver damage triggers inflammation, followed by the generation of ROS, mainly by activated inflammatory cells and liver sinusoidal endothelial cells (LSECs), which further dysregulates liver redox homeostasis, forming a vicious cycle of continuous stimulation. The liver is a major organ for metabolism and detoxification. A variety of enzymes with overlapping substrate specificities are expressed in the liver and are generally classified as phase I (oxidation) and phase II (conjugation) drug metabolizing enzymes (DMEs). Approximately 90% of phase I metabolism is carried out by enzymes belonging to the cytochrome P450 (CYP) superfamily. In addition, hepatic stellate cells (HSCs) store 50%-95% of the body's vitamin A, which is composed of a series of retinol compounds, including retinol, retinoic acid, and retinal. CYP450 enzymes play a crucial role in retinol metabolism, mainly involving the CYP1, CYP2C, CYP3A, and CYP26 families. Current research indicates that regulation of retinol metabolism homeostasis is a defining characteristic of hepatic stellate cells (HSCs) in both healthy and injured livers. Therefore, scavenging excess ROS to alleviate oxidative stress, while modulating inflammatory responses, blocking persistent stimulation, and maintaining the stability of retinol metabolism will become effective therapeutic targets for acute liver injury.

[0004] It is well known that there is a powerful antioxidant enzyme system in cells that can scavenge reactive oxygen species (ROS), such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). However, this system cannot effectively resolve liver damage because it is not targeted. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs and their applications.

[0006] In the first aspect, the present invention provides an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs were prepared by the following steps:

[0007] CuCl2 was dissolved in a carbon dot aqueous solution, reacted at 75°C to 80°C for 10min to 12min, then L-ascorbic acid aqueous solution was added, the pH was adjusted to 7.0-8.0 with NaOH solution, and the reaction was carried out at 75°C to 80°C for 10h to 12h. In the reaction system, the concentration of CuCl2 was 15-20mM, the concentration of carbon dots was 3-4mg / mL, and the concentration of ascorbic acid was 300-400mM. After the reaction was completed, the precipitate was removed by centrifugation, and the supernatant was dialyzed and freeze-dried to obtain the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs.

[0008] Nanozyme Cu of the present invention 5.4 O@CNDs possessed SOD and CAT enzyme activities and ·OH free radical scavenging ability, eliminating ROS in vitro. 5.4 O@CNDs showed good targeting ability in H2O2-induced inflammatory cells and liver tissue of hepatitis mice, and had good in vitro and in vivo biocompatibility. 5.4 O@CNDs ameliorated the disruption of hepatic retinol metabolism by regulating ROS responses and the liver inflammatory network, effectively scavenged overloaded ROS, improved cell survival in vitro, and exhibited effective therapeutic effects in HIRI and LPS-ALI mouse models.

[0009] Furthermore, the carbon dots are prepared by the following steps:

[0010] The activated carbon is added to a boiling mixed acid solution, maintained at the boiling point for 1.5 hours to 2 hours, cooled to room temperature, and then neutralized with a NaHCO3 solution. The neutralized solution is filtered, dialyzed, concentrated, and freeze-dried to obtain the carbon dots, wherein the mixed acid solution is a mixture of equal volumes of an HNO3 solution with a concentration of 8 to 10 mol / L and an H2SO4 solution with a concentration of 18 to 20 mol / L, and the mass volume ratio of the activated carbon to the mixed acid solution is 0.5 g to 0.6 g:50 mL.

[0011] In the second aspect, the present invention provides the nanozyme Cu 5.4 Application of O@CNDs in the preparation of drugs for treating liver injury.

[0012] Furthermore, the liver injury includes liver ischemia-reperfusion injury and acute liver injury.

[0013] Furthermore, the drug contains the nanozyme Cu 5.4 O@CNDs, drug carriers and / or drug excipients.

[0014] In a third aspect, the present invention provides a drug for treating liver damage, comprising the nanozyme Cu 5.4 O@CNDs.

[0015] Furthermore, the drug also includes a drug carrier and / or a drug excipient.

[0016] Furthermore, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0017] Furthermore, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents.

[0018] Furthermore, the dosage form of the drug is powder, granule, capsule, tablet, pill, injection or oral solution.

[0019] Beneficial effects:

[0020] The present invention synthesized a nanozyme Cu with cascade mimetic enzyme activity. 5.4 O@CNDs, which can protect the liver from ROS-mediated stress and inflammatory response and improve vitamin A alcohol metabolism disorder, thus achieving effective therapeutic effects. 5.4 O@CNDs can improve the cell status and survival rate of THLE-2 and RAW264.7 cells under oxidative stress and inflammatory conditions in vitro. In the HIRI and LPS-induced ALI models, Cu 5.4 O@CNDs effectively scavenged ROS, reduced the expression of inflammatory cytokines, and showed significant therapeutic effects. To elucidate the potential therapeutic mechanism, transcriptome sequencing results showed that Cu 5.4 O@CNDs promote the destruction of hepatic retinol metabolism pathways by regulating ROS response and liver inflammatory network, inhibiting cell apoptosis and exerting liver protective effects. 5.4 O@CNDs have the potential to treat various acute liver injury diseases and provide a promising intervention strategy for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 C-dots, Cu 5.4 O USNPs and Cu 5.4 Synthesis and characterization of O@CNDs, Cu 5.4 O@CNDs, where:

[0022] A is C-dots, Cu 5.4 O USNPs and Cu 5.4 Synthesis route of O@CNDs.

[0023] B is the TEM image of C-dots.

[0024] C is Cu 5.4 TEM image of O USNPs.

[0025] D is Cu 5.4 TEM image of O@CNDs.

[0026] E is C-dots, Cu 5.4 O USNPs and Cu 5.4 Zeta potential of O@CNDs.

[0027] F is C-dots, Cu 5.4 O USNPs and Cu5.4 XRD patterns of O@CNDs

[0028] G is C-dots, Cu 5.4 O USNPs and Cu 5.4 FTIR spectrum of O@CNDs.

[0029] H is C-dots, Cu 5.4 O USNPs and Cu 5.4 XPS patterns of O@CNDs.

[0030] I is the C-1s peak fitting curve of C-dots.

[0031] J is Cu 5.4 C-1s peak fitting curve of O@CNDs.

[0032] K is Cu 5.4 Cu2p peak fitting curve of O USNPs.

[0033] L is Cu 5.4 Cu2p peak fitting curve of O@CNDs.

[0034] Figure 2 Cu 5.4 O USNPs, Cu 5.4 Different synthesis conditions of O@CNDs, including:

[0035] A is different Cu 2+ Cu synthesized under L-AA feed ratio (1:5, 1:10, 1:20, 1:40, 1:60) 5.4 Dissolved oxygen assay results of the hydrogen peroxide scavenging ability of O USNPs.

[0036] B is the synthesis of Cu at different C-dots feed ratios (1-4 mg / mL) 5.4 Dissolved oxygen determination results of hydrogen peroxide scavenging ability of O@CNDs.

[0037] C is Cu synthesized at different pH (pH 6-7, 7-8, 8-9, 9-10) 5.4 Dissolved oxygen determination results of hydrogen peroxide scavenging ability of O@CNDs.

[0038] D is different Cu 2+ Cu was synthesized at a concentration of 5.4 Dissolved oxygen determination of hydrogen peroxide scavenging ability of O@CNDs.

[0039] Figure 3 C-dots, Cu 5.4 O USNPs and Cu 5.4Characterization of O@CNDs, including:

[0040] A is the particle size statistics of C-dots.

[0041] B is Cu 5.4 Particle size statistics of O USNPs.

[0042] C is Cu 5.4 Particle size statistics of O@CNDs.

[0043] Figure 4 C-dots, Cu 5.4 O USNPs and Cu 5.4 Enzyme-like activity test of O@CNDs, including:

[0044] A is a SOD-like enzyme that removes O2 - Schematic diagram of .

[0045] B is C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs · O2 - Clearing ability.

[0046] C is the WST-1 kit to evaluate C-dots, Cu 5.4 O USNPs and Cu 5.4 SOD enzyme activity of O@CNDs.

[0047] D is the ESR method for the determination of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs Clear O2 - -ability.

[0048] E is a schematic diagram of CAT-like enzymes removing H2O2.

[0049] F is UV absorption test C-dots, Cu 5.4 O USNPs and Cu 5.4 The H2O2 scavenging ability of O@CNDs.

[0050] G is C-dots, Cu 5.4 O USNPs and Cu 5.4 Detection of dissolved oxygen levels by O@CNDs scavenging H2O2.

[0051] H is the ESR method for the determination of C-dots, Cu 5.4 O USNPs and Cu 5.4 The H2O2 scavenging ability of O@CNDs.

[0052] I is a schematic diagram of scavenging OH and ABTS free radicals.

[0053] J is C-dots, Cu 5.4 O USNPs and Cu 5.4 ·OH scavenging ability of O@CNDs.

[0054] K is C-dots, Cu 5.4 O USNPs and Cu 5.4 ABTS radical scavenging ability of O@CNDs.

[0055] Figure 5 Cu 5.4 Biocompatibility of O@CNDs, including:

[0056] A is Cu 5.4 THLE-2 cell viability after incubation with O@CNDs for 24 h.

[0057] B is Cu 5.4 THLE-2 cell viability after incubation with O@CNDs for 48 h.

[0058] C is the hemolysis rate of each group.

[0059] D is Cu 5.4 Body weight changes of normal mice 7 days after O@CNDs treatment.

[0060] E is a schematic diagram of the biocompatibility experiment.

[0061] F is Cu 1 day and 7 days after intravenous administration 5.4 In vivo toxicity evaluation of O@CNDs on major organs (heart, liver, spleen, lung, and spleen).

[0062] G is the level of serum liver function index and serum renal function index.

[0063] H is normal mice (control group) and intravenously injected Cu 5.4 Routine blood parameters of O@CNDs mice. Figure 6 Cu 5.4 The in vivo therapeutic effects of O@CNDs on HIRI and LPS-ALI mice:

[0064] A is a schematic diagram of the establishment and treatment plan of HIRI mice.

[0065] B is the liver tissue image.

[0066] C is H&E staining of liver tissue.

[0067] D is TUNEL, DCFH-DA, and DHE staining of liver tissue.

[0068] E: Serum ALT and AST levels in HIRI mice 24 h after different treatments.

[0069] F is the relative expression of IL-1β, IL-6, IL-12, and TNF-α cytokine mRNA.

[0070] G is a schematic diagram of the establishment and treatment plan of LPS-ALI mice.

[0071] H: H&E staining of liver tissue.

[0072] I: TUNEL, DCFH-DA, and DHE staining of liver tissue.

[0073] J: serum ALT and AST levels in LPS-ALI mice 12 h after different treatments.

[0074] K is the relative expression of IL-1β, IL-6, IL-12, and TNF-α cytokine mRNA. DETAILED DESCRIPTION

[0075] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0076] Materials and instruments involved in the following examples:

[0077] Material:

[0078] Activated carbon was purchased from Aladdin Chemical Reagent Co., Ltd. Sodium bicarbonate (NaHCO3) was purchased from Tianli Enterprise Group Co., Ltd. Sulfuric acid (H2SO4) and nitric acid (HNO3, 65%–68%) were purchased from local suppliers. Copper chloride (CuCl2) was purchased from Shanghai Macklin Co., Ltd. L-ascorbic acid (L-AA) and sodium hydroxide (NaOH) were from Sigma Aldrich Co., Ltd. in Shanghai, China. Ferrous chloride (FeCl2·4H2O) was purchased from Tianjin Bailun Biotechnology Co., Ltd., and 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Shanghai Malin Biochemical Technology Co., Ltd. Nitrotetrazolium blue chloride (NBT), l-methionine (L-met), and riboflavin were obtained from Sigma Aldrich Co., Ltd. Hydrogen peroxide (H2O2, 30%) was purchased from Tianjin Daxiong Chemical Reagent Co., Ltd., and a superoxide dismutase assay kit (S311) was purchased from Dojindo Molecular Technologies Co., Ltd. Hydrogen peroxide assay kit, ABTS total antioxidant capacity assay kit, 2′,7′-dichlorodihydrofluorescein (DCFH-DA), dihydroethidium (DHE), and animal RNA isolation kit were purchased from Beyotime Chemical Reagent Co., Ltd., and 4′,6-diaminyl-2-phenylindole (DAPI) was purchased from Roche Applied Science. All chemical reagents were used without further purification, and the detection kits were used according to the manufacturer's instructions.

[0079] instrument:

[0080] Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2F30 (FEI, USA) at an accelerating voltage of 300 kV. Powder x-ray diffraction (XRD) data were collected using a Bruker D8 ADVANCE (Germany) with a scan rate of 6° / min. FT-IR spectra were recorded by a Thermo Fisher Nicolet 5700 (USA). X-ray photoelectron spectroscopy (XPS) was recorded using a ThermoEscalab 250Xi (USA). Electron spin resonance (ESR) spectra were recorded at room temperature using a Bruker A300-9.5 / 12 (Switzerland). Flow cytometry data were collected by a FACS Calibur™, Becton Dickinson (USA). Microplate absorbance was measured using a Tecan Spark 20m multimode microplate reader (Switzerland). Fluorescence imaging was performed using an in vivo imager (IVISLumina 3, PE, USA), and images were acquired using IVIS Living Image 3.0 software (PerkinElmer, USA). Tissue sections were imaged using a Leica DM3000 microscope (Leica, Wetzlar, Germany).

[0081] Example 1: Cu 5.4 Synthesis and characterization of O@CNDs

[0082] 1. Experimental methods

[0083] (1) Synthesis of carbon dots (C-dots)

[0084] First, 0.5 g of activated carbon was added to a boiling solution of 50 mL of mixed acid (equal volumes of 8 mol / L HNO3 solution and 18 mol / L H2SO4 solution), maintained at the boiling point for 1.5 hours, cooled to room temperature, and then neutralized with NaHCO3 solution. The resulting neutralized solution was filtered through a 0.22 μm water membrane and dialyzed 4-5 times a day for one week. The dialyzed C-dots solution was filtered again through a 0.22 μm water membrane to eliminate any remaining insoluble matter. The resulting filtrate was ultrafiltered using an ultrafiltration tube with a molecular retention capacity of 100 kDa. The separated C-dots were concentrated and freeze-dried for subsequent experimental use.

[0085] (2)Cu 5.4 Synthesis of O USNPs

[0086] 10 mM CuCl2 powder was dissolved in 50 mL of deionized water and stirred in a magnetic stirring oil bath at 80°C for 10 minutes. Then, an aqueous solution of L-ascorbic acid (100 mM, 50 mL) was slowly added to the CuCl2 solution. The pH of the solution was then adjusted to 8.0-9.0 with a 1 M NaOH solution, and stirring was continued at 80°C for 12 hours. After the reaction, larger aggregates were removed by centrifugation (6577 × g, 15 minutes). The supernatant was then dialyzed against water (Mw cutoff: 10,000 Da) for 2 days to remove small molecules and lyophilized for subsequent experiments.

[0087] (3)Cu 5.4 Synthesis of O@CNDs

[0088] First, 15mM CuCl2 powder was dissolved in a C-dots aqueous solution (3mg / mL, 50mL) and stirred in an 80°C magnetic stirring oil bath for 10min. Then, L-ascorbic acid aqueous solution (300mM, 50mL) was slowly added to the above CuCl2 solution. The pH of the solution was then adjusted to 7.0-8.0 with NaOH solution (1M) and stirred continuously at 80°C for 10h. After the reaction was completed, larger aggregates were removed by centrifugation (6577×g, 15min). The supernatant (Mw cutoff: 3,500Da) was then dialyzed for 2 days to remove small molecules and lyophilized for subsequent experimental use and characterization.

[0089] 2. Experimental results

[0090] This example synthesized a Cu with cascade enzyme activity 5.4 O@CNDs. Cu 5.4 O@CNDs, C-dots and Cu 5.4 OUSNPs were synthesized using a simple and rapid one-pot method ( Figure 1 A). In order to find the best synthesis conditions, the Cu 2+ The ratio of L-ascorbic acid (AA), the concentration of C-dots, pH and Cu 2+ The final concentrations of Cu were determined to determine their effects on the particle size and catalytic activity of the resulting materials. 5.4 O USNPs in Cu 2+ The catalytic activity of CuO is basically the same when the feed ratio is 1:5, 1:10, 1:40 and 1:60. The best effect is achieved when the feed ratio is 1:20. 2+ The molar ratio of AA was fixed at 1:20, Cu 2+ The concentration is 15mM, C-dots is 3mg / ml, pH 7.0-8.0 ( Figure 2 AD).

[0091] Transmission electron microscopy (TEM) of C-dots showed that the distribution of C-dots was uniform and monodisperse, and the particle size distribution statistics showed that the average particle size was 2.39±0.50nm ( Figure 1 B and Figure 3 A). Cu 5.4 The TEM results of OUSNPs showed that the NPs were uniformly spherical, and the particle size distribution statistics showed that the average particle size was 2.06±0.50nm ( Figure 1 C and Figure 3 B). At the same time, Cu 5.4 OUSNPs and C-dots combine to form polymer Cu 5.4 O@CNDs, TEM showed that the NPs were uniformly spherical, and the particle size distribution statistics showed that the average particle size was 5.87±0.94nm ( Figure 1 D and Figure 3 The zeta potential of C-dots alone is -51.7±0.9 mV, while the addition of Cu 5.4 After O USNPs, Cu 5.4 The potential of O@CNDs reached -60.47±0.67mV. The results showed that C-dots and Cu 5.4 OUSNPs were successfully combined with ( Figure 1 E). C-dots, Cu 5.4 O USNPs and Cu 5.4The X-ray powder diffraction (XRD) results of O@CNDs showed that C-dots had obvious 2θ diffraction peaks at 25-30° and 42°, and Cu 5.4 O USNPs have obvious 2θ diffraction peaks at 25-30°, 42° and 50°. 5.4 O USNPs appeared, indicating that Cu 5.4 O@CNDs were successfully prepared ( Figure 1 F). FTIR spectrum characteristic bands prove that Cu 5.4 O@CNDs have C-dots and Cu 5.4 The characteristic peaks of O USNPs ( Figure 1 G). X-ray photoelectron spectroscopy (XPS) is commonly used to analyze the elemental bonding of composite materials. The overall XPS spectrum found elemental peaks of Na 1s, O 1s, and C1s in C-dots, and 5.4 Cu 2p and Cu LM element peaks were found in O USNPs, while 5.4 The element peaks of Na 1s, Cu 2p, Cu LM, O 1s and C1s were found in O@CNDs, which proved that C-dots and Cu 5.4 O USNPs were successfully integrated ( Figure 1 H). In C-dots and Cu 5.4 The C1s fraction peak fitting diagrams of graphite carbon at 284.8eV, alcohol carbon at 286.0eV, carbonyl carbon at 287.8eV, and carboxyl carbon at 289.0eV were detected in O@CNDs, indicating that the characteristic peaks of C-dots appear in Cu 5.4 In O@CNDs Figure 1 Cu 5.4 OUSNPs and Cu 5.4 The Cu 2p peak fitting curve of O@CNDs shows that Cu 5.4 Cu exists in O@CNDs 0 and Cu1 + ( Figure 1 The above results show that Cu 5.4 The synthesis and preparation of O@CNDs were successful, and the C-dots and Cu 5.4 O The characteristic structures and characterization of USNPs.

[0092] Example 2: C-dots, Cu 5.4 O USNPs,Cu 5.4 Enzymatic characterization of O@CNDs

[0093] 1. Experimental methods

[0094] (1)C-dots,Cu 5.4 O USNPs,Cu 5.4 SOD-like activity of O@CNDs

[0095] To evaluate SOD-like activity, a SOD assay kit was used, in which the reaction substrate WST and ·O2 - The reaction generates water-soluble formaldehyde, which exhibits a unique absorption peak at 450nm. - When dismutation is blocked, indicating the presence of SOD-like activity in the sample, the SOD enzyme activity is determined by colorimetric analysis of the WST product. The absorbance is measured at 450 nm.

[0096] (2)C-dots,Cu 5.4 O USNPs,Cu 5.4 O2 scavenging activity of O@CNDs

[0097] Using NBT reduction method to reduce O2 - The scavenging activity of the O2 - In the presence of C-dots with SOD-like activity, they competitively scavenge O2 - , resulting in a lighter or absent blue color. To this end, samples at varying concentrations (0–200 μg / mL) were mixed with NBT (0.05 mM), L-met (13 mM), and riboflavin (20 μM) in PBS buffer (pH 7.4) to a concentration of 25 mM and then illuminated with an LED for 5 minutes. Absorbance was measured at 560 nm.

[0098] The ESR method was used to determine the - 100mM DMPO, 25μM DTPA, 0.5mM HYP, 0.1U / mL XOD and 100μg / mL Cu 5.4 O@CNDs were added to PBS (pH = 7.4) and generated O2 through the hypoxanthine / xanthine oxidase (HYP / XOD) system. - , and the ESR spectrum of BMPO / ·OOH was recorded after 2 min.

[0099] (3)C-dots,Cu 5.4 O USNPs,Cu 5.4 CAT-like activity of O@CNDs

[0100] The dissolved oxygen method was used to determine the oxygen production of different materials in scavenging H2O2. The reaction system was a final volume of 15mL, containing 60μL of 30% H2O2 and ultrapure water with different concentrations of different substances for 15min.

[0101] (4)C-dots,Cu 5.4 O USNPs,Cu 5.4 H2O2 scavenging activity of O@CNDs

[0102] C-dots,Cu 5.4 O USNPs,Cu 5.4 The H2O2 scavenging ability of O@CNDs was assessed using a hydrogen peroxide detection kit (Nanjing Jiancheng Bioengineering Institute, China). H2O2 reacts with ammonium molybdate to form a stable yellow complex with an absorbance peak at 405 nm. Samples of varying concentrations (0 to 200 μg / mL) were incubated with 2 mM H2O2 at 37°C for 2 h. After the reaction, the residual H2O2 concentration was determined according to the manufacturer's instructions, and the H2O2 scavenging ability was calculated.

[0103] The H2O2 scavenging activity was determined using ESR. The different materials, at 100 μg / mL, catalyzed the degradation of H2O2 to produce O2. The O2 was captured using the ESR spin probe CTPO. The ESR spectrum of CTPO exhibited proton hyperfine structure in a control nitrogen-saturated solution. As the O2 concentration increased, the frequency of collisions between oxygen and nitroxide radicals increased, leading to a broadening of the ESR triplet spectrum and a decrease in the resolution of the proton hyperfine structure.

[0104] (5)C-dots,Cu 5.4 O USNPs,Cu 5.4 O@CNDs's OH - Scavenging activity

[0105] The ·OH scavenging activity was determined using the TMB assay. A 10μM FeCl2 solution, a 50μM H2O2 solution, and a 300μM TMB solution were prepared in ultrapure water. 100μL of each of the three solutions was then mixed. Then, 10μL of the sample solution at varying concentrations (0-200μg / mL) was mixed with 290μL of the sample solution. The mixture was reacted at room temperature for 30 minutes, and the absorbance was measured at 645nm.

[0106] The scavenging activity of ·OH was evaluated by ESR method. 100mM DMPO, 1.0mM FeSO4, 1.0mM H2O2 and 100μg / mL Cu 5.4 O@CNDs, generating ·OH. The ESR spectrum of DMPO / ·OH was recorded after 2 min.

[0107] (6)C-dots,Cu 5.4 O USNPs,Cu 5.4 ABTS free radical scavenging activity of O@CNDs

[0108] The T-AOC assay kit (S0119, Beyotime) was used to evaluate the C-dots, Cu 5.4 OUSNPs and Cu 5.4 ABTS free radical scavenging ability of O@CNDs. ABTS and oxidant solution were mixed in a 1:1 volume ratio to form a fresh working solution, which was then stored in the dark at room temperature for 12–16 hours before use. The working mixture was then diluted according to the kit instructions, and samples were analyzed at various concentrations of the various substances (0–200 μg / mL) as specified. Absorbance was measured at 734 nm.

[0109] 2. Experimental results

[0110] O2 - It is a common reactive oxygen species. The SOD-like enzyme activity level of the material can be evaluated by detecting its clearance rate ( Figure 4 A). First, NBT reduction method can indirectly detect C-dots, Cu 5.4 O USNPs and Cu 5.4 SOD-like enzyme activity of O@CNDs. In this system, xanthine and xanthine oxidase produce O2 in the presence of light. - , O2 - The NBT colorant can produce blue formaldehyde, which has a characteristic absorption peak at 560nm. Assuming that the material has SOD activity, it can remove O2 - In this case, the color of NBT will become weaker, which indirectly indicates the presence of SOD-like enzyme activity. 5.4 O@CNDs clearly outperformed Cu 5.4 O USNPs·O2 - The clearance efficiency is basically consistent with that of C-dots ( Figure 4 B). At the same time, SOD detection kit was used to detect C-dots, Cu 5.4 O USNPs and Cu 5.4 The SOD-like enzyme activity of O@CNDs was quantitatively analyzed. The results showed that the SOD-like enzyme activity of C-dots was 6055U / mg, and the Cu 5.4 O USNPs is 3063U / mg. Cu 5.4 The SOD-like enzyme activity of O@CNDs was 8557 U / mg, which was superior to the other two materials ( Figure 4 In addition, the change of free radical signal intensity was detected by ESR to evaluate the Cu 5.4 O@CNDs vs. O2 - The results show that there is no ESR signal when DMPO is alone. However, when DMPO and ·O2 - When they exist at the same time, the signal shows an obvious peak, indicating that DMPO successfully captures O2 - , showing a strong signal intensity. Adding Cu 5.4 O@CNDs, the signal peak intensity was significantly weakened, indicating that Cu 5.4 O@CNDs has excellent O2 - Cleaning ability ( Figure 4 D).

[0111] H2O2 is a product of SOD-catalyzed disproportionation and is also a toxic ROS. In order to achieve the purpose of cascade removal of ROS, H2O2 needs to be decomposed into non-toxic oxygen and water. Catalase can catalyze ( Figure 4 First, different concentrations of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs, C-dots have no H2O2 scavenging ability, Cu 5.4 O USNPs and Cu 5.4 The absorption value of O@CNDs at 240 nm decreases with the concentration. 5.4 O@CNDs is better than Cu 5.4 O USNPs have a higher H2O2 scavenging rate ( Figure 4 F). Then use a dissolved oxygen meter to detect the dissolved oxygen concentration and determine the C-dots, Cu 5.4 OUSNPs and Cu 5.4 CAT activity of O@CNDs. The results showed that the C-dots group produced almost no O2, while the Cu 5.4 O2 generated by the O@CNDs group is Cu 5.4 O USNPs group, which indicates that C-dots have no H2O2 scavenging ability. 5.4 The decomposition rate of H2O2 by O@CNDs can reach that of Cu 5.4 OUSNPs is about 2 times. 5.4 O USNPs and Cu 5.4 The oxygen production capacity of O@CNDs was dose-dependent ( Figure 4G). In addition, the generation of O2 and the catalytic degradation of H2O2 were tested using ESR test materials. Oxygen was captured using the ESR spin probe CTPO, and its ESR spectrum showed a proton hyperfine structure in a control nitrogen saturated solution. As the oxygen concentration increased, the collision frequency of oxygen and nitroxide radicals increased, the ESR triplet spectrum became broadened, and the resolution of the proton hyperfine structure decreased. Adding Cu 5.4 After O@CNDs, the CTPO fine peak weakened, indicating that it has good hydrogen peroxide scavenging ability ( Figure 4 H).

[0112] ·OH is another important ROS with oxidative properties, so ·OH scavenging can also effectively protect cells from oxidative damage ( Figure 4 First, the TMB method was used to react Fe2 + / H2O2) to generate hydroxyl radicals, and catalyze TMB to generate soluble blue products. 5.4 O USNPs and Cu 5.4 O@CNDs changed color after ionization, and the clearance rate was determined and calculated by enzyme labeling. The clearance rate of 25 μg / mL could reach more than 80%, which indirectly evaluated the clearance rate of ·OH. 5.4 O@CNDs has a significantly better hydroxyl radical scavenging ability than the other two materials mentioned above ( Figure 4 J). At the same time, the ·OH scavenging ability is detected by ESR to detect the strength of the free radical signal. In this system, ·OH is generated by the Fenton reaction, and then DMPO is used to capture ·OH to form a spin adduct (DMPO / ·OH). The change in ESR signal intensity is used to respond to the effect of ·OH removal. When the Fenton reagent is mixed with DMPO, a four-linear characteristic peak with apparent signal intensity appears, proving that a large amount of ·OH is generated in the mixed system. Adding C-dots, Cu5.4OUSNPS and Cu 5.4 O@CNDs, the intensity of the quadrilinear intrinsic peak decreases. Cu 5.4 The intensity of the quadrilinear intrinsic peak of O@CNDs is almost weakened. 5.4 The characteristic peaks of the four linear states of O@CNDs almost disappear, indicating that Cu 5.4 O@CNDs can effectively remove ·OH( Figure 4 of L).

[0113] The above experiments describe Cu 5.4 The actual scavenging ability of O@CNDs in the presence of a single free radical. Secondly, the total antioxidant capacity of Cu was determined by the ABTS method. 5.4The total free radical scavenging ability of O@CNDs was evaluated. ABTS and oxidant solution provided in the kit were mixed in a 1:1 volume ratio to prepare a fresh master solution, which was stored in the dark at room temperature for 12-16 hours before use. They diluted the master batch according to the instructions of the kit and tested samples of different concentrations as required. The absorbance at 734 nm was measured using a microplate reader. The results showed that C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs have a good total antioxidant level, and the antioxidant capacity is concentration-dependent, among which Cu 5.4 The total antioxidant capacity of O@CNDs was significantly better than that of the other two, and the clearance rate was more than 80% at 25 μg / mL ( Figure 4 of K).

[0114] The above results show that Cu 5.4 O@CNDs not only have excellent SOD and CAT activities, but also can scavenge hydroxyl radicals and have high antioxidant capacity, which indicates that Cu 5.4 O@CNDs are expected to treat ROS-related damage at the cellular and animal levels.

[0115] Example 4: Cu 5.4 Biosafety testing of O@CNDs

[0116] 1. Experimental methods

[0117] (1)Cu 5.4 In vitro biocompatibility of O@CNDs

[0118] According to the above cell culture protocol, different concentrations of Cu 5.4 After incubation with O@CNDs nanozyme solution (ranging from 0 to 100 μg / mL) for 12 and 24 h, cell viability was assessed using the MTT assay.

[0119] (4)Cu 5.4 In vivo biocompatibility of O@CNDs

[0120] To evaluate Cu 5.4 The in vivo biocompatibility of O@CNDs nanozymes was tested by 0.5 mg / kg Cu 5.4 O@CNDs were injected intravenously into 6-8 week old BALB / c mice weighing 20-25 g. A control group of mice received PBS. Blood samples were collected on day 1 and for seven consecutive days after injection for complete blood count analysis and serum biochemical analysis. Mice were then euthanized, and major organs, including the heart, liver, spleen, lungs, and kidneys, were collected for H&E staining and histological examination.

[0121] 2. Experimental results

[0122] Since biocompatibility is very important for the potential clinical application of nanomaterials, we evaluated the biocompatibility of Cu 5.4 First, the MTT assay was used to determine the toxicity of THLE-2 cells exposed to a series of Cu 5.4 The results showed that the cell viability after 24 h of incubation with O@CNDs was close to 100% even at a concentration of 100 μg / mL ( Figure 5 A). When the incubation time reached 48 hours, the cell viability was still higher than 80%, demonstrating that Cu5.4@CNDs has good safety in vitro ( Figure 5 B). Next, a hemolysis assay was performed to detect Cu 5.4 The biocompatibility of O@CNDs was studied. The results showed that when the concentration was 160 μg / mL, the hemolysis rate was still less than 5%, indicating that the nanomaterials did not cause hemolysis of blood cells and were safe for intravenous injection ( Figure 5 C).

[0123] In addition, all in vivo biocompatibility evaluation experiments were performed at a concentration of 0.5 mg / kg. 5.4 Effects of O@CNDs on blood chemistry and vital organ histopathology to reveal their in vivo biocompatibility ( Figure 5 E). Continuous injection of Cu 5.4 After 7 days of O@CNDs injection, the main organs (heart, liver, spleen, lung, kidney), serum and plasma of the rats were collected on the first and seventh days of injection, and the weight changes of the mice were followed up. 5.4 O@CNDs had no effect on the condition and body weight of mice ( Figure 5 D) H&E staining results showed that there was no obvious damage to the tissue, indicating that Cu 5.4 O@CNDs have good biocompatibility ( Figure 5 F). Serum biochemical analysis results showed that Cu 5.4 The serum concentrations of liver function indicators (AST, ALT) and renal function indicators (urea, CRE) in the O@CNDs group were comparable to those in the control group, indicating that the liver and kidney were biocompatible ( Figure 5 At the same time, the results of the complete blood cell analysis of mice showed no statistically significant changes compared with the control group ( Figure 5 All studies have shown that the synthesized Cu 5.4 O@CNDs exhibit minimal short-term and long-term toxicity in vivo.

[0124] Example 5: Cu 5.4 In vivo therapeutic effects of O@CNDs on HIRI and ALI.

[0125] 1. Experimental Animals

[0126] Female BALB / c mice (6–8 weeks, 20–25 g) were purchased from Xi’an Jiaotong University, Shaanxi Province, China. All mice were housed under standard conditions, including light, temperature, water, and food, and animal experiments were approved by the Animal Ethics Committee of Xi’an Jiaotong University.

[0127] 2. Experimental methods

[0128] (1) Construction of mouse HIRI model

[0129] Male BALB / c mice aged 6-8 weeks and weighing 20-25 g were selected and fasted for 15 hours. Anesthesia was induced using an R500 universal small animal anesthesia machine (R500IP, RWD), and then a 1.5 cm surgical incision was made along the midline of the abdomen. After exposing the liver, a non-invasive vascular clamp was used to occlude the left branch of the hepatic artery, the left hepatic duct, and the portal vein. The left and middle lobes of the liver (accounting for approximately 70% of the total volume of the liver) appeared white, indicating partial ischemia. The clamps were removed after 1 hour, and the liver tissue turned red and moist, confirming the recovery of blood circulation. Subsequently, the abdominal incision was carefully sutured in layers. 24 hours after blood flow was restored, the mice were euthanized, and blood and liver tissue were collected for analysis.

[0130] (3) Construction of mouse LPS-ALI model

[0131] Male BALB / c mice, 6-8 weeks old and weighing 20-25 g, were fasted for 15 hours and intraperitoneally injected with 30 μg / mL LPS and 200 mg / kg D-gal. Euthanasia was performed 12 hours later, and blood and liver tissue were collected for analysis.

[0132] (4) Efficacy testing

[0133] BALB / c mice with HIRI model were randomly divided into sham operation (control group), HIRI, 0.1 mg / kg dose of Cu 5.4 O@CND treatment group, 0.5 mg / kg dose of Cu 5.4 O@CND treatment group, 1.0 mg / kg dose of Cu 5.4 O@CND treatment group, 2.5 mg / kg dose of Cu 5.4 There were 6 O@CND treatment groups (n=5) in total. The body weight fluctuations of mice in each group were observed 24 hours after administration.

[0134] BALB / c mice with LPS-ALI model were randomly divided into three groups (n=5): PBS (control group), LPS-ALI, 0.5 mg / kg dose of Cu 5.4 O@CND treatment group.

[0135] 24 hours after injection, the mice were euthanized, and blood samples were collected for quantitative determination of AST and ALT levels. Liver tissue was collected to determine the transcript levels of proinflammatory factors, including IL-1β, IL-6, IL-12, and TNF-α. At the same time, part of the liver tissue was fixed with 4% paraformaldehyde and then embedded in paraffin for H&E and TUNEL staining. Another part of the liver tissue was frozen and embedded in optimal cutting temperature (OCT) sample matrix and frozen at -80°C for sectioning. The frozen liver tissue sections were stained with DAPI and DCFH / DHE at 37°C for 30 minutes. Subsequently, the sections were rinsed three times with PBS to remove excess dye. The stained sections were then observed under a fluorescence microscope to qualitatively assess the level of ROS in the liver tissue.

[0136] 3. Experimental results

[0137] Based on Cu 5.4 O@CNDs have anti-oxidative stress and anti-inflammatory effects in vitro. 5.4 Protective effect of O@CNDs on HIRI mouse model. First, the HIRI mouse model was established ( Figure 6 A). Then determine Cu 5.4 The optimal treatment dose of O@CNDs. The liver tissue was photographed, and some tissue damage was visible. The liver status of the 0.5mg / kg pretreatment group was better than that of the disease modeling group ( Figure 6 At the same time, H&E staining images of mouse liver tissue showed that the HIRI model group had punctate necrosis of liver cells, bile stasis in some capillaries, and inflammatory cell infiltration ( Figure 6 C). Pre-injection of Cu 5.4 The liver histopathological manifestations of the O@CNDs group were significantly improved, and 0.5 mg / kg Cu 5.4 In the HIRI mouse model treated with O@CNDs, the area of ​​hepatocyte necrosis and cell lysis was extremely small. Serum biochemical tests of liver function in mice were then performed, and the results showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in HIRI model mice were higher than those in the control group, while Cu 5.4 The ALT and AST levels in the O@CNDs pre-injection group were significantly reduced, indicating that Cu 5.4 O@CNDs has a significant protective effect on liver function ( Figure 6 E). When Cu 5.4 When the concentration of O@CNDs was 0.5 mg / kg, the liver function index of mice was closest to that of the control group, indicating that 0.5 mg / kg was the optimal therapeutic dose ( Figure 6 In summary, in the HIRI mouse model, Cu 5.4 O@CNDs has a protective effect on liver function, and 0.5 mg / kg was selected as the therapeutic concentration in subsequent experiments.

[0138] Frozen tissue sections were taken for TUNEL staining to evaluate the level of hepatocyte necrosis and apoptosis. The results showed that the TUNEL green fluorescence was significantly enhanced in the HIRI group, and there was no significant difference between the pretreatment group and the control group, indicating that Cu 5.4 O@CNDs has good protective effects on hepatocytes and liver function ( Figure 6 D). In order to evaluate Cu 5.4 The ability of O@CNDs to remove ROS in vivo was investigated by imaging the ROS removal effect using ROS-specific fluorescent dyes DCFH-DA and DHE. After hepatic ischemia-reperfusion, the green fluorescence of DCFH-DA in liver tissue increased significantly, and the green fluorescence of Cu@CNDs in liver tissue increased significantly. 5.4 The ROS level in the tissues after O@CNDs pre-injection and staining was reduced ( Figure 6 At the same time, DHE also enhanced the red fluorescence intensity of the modeling group and restored the staining fluorescence intensity of the pretreatment group ( Figure 6 The results showed that Cu 5.4 O@CNDs can eliminate ROS in liver ischemia-reperfusion injury and alleviate liver damage and hepatocyte apoptosis.

[0139] In addition, the liver tissue of mice was processed and relevant RNA was extracted to verify the changes in the transcription levels of common pro-inflammatory cytokines. After the model was successfully established, the four common inflammatory factors (IL-1β, IL-6, IL-12 and TNF-α) were significantly increased. 5.4 There was no significant difference in the inflammatory level between the O@CNDs group and the healthy mice in the control group ( Figure 6 F). The results showed that Cu 5.4 O@CNDs can effectively alleviate the inflammatory response by scavenging ROS in the body and prevent oxidative stress and inflammation in liver ischemia-reperfusion injury.

[0140] In order to verify Cu 5.4 O@CNDs can be applied to other acute liver injury diseases. We established a lipopolysaccharide-induced acute liver injury model (LPS-ALI). After 6 hours of LPS+D-gal induction, Cu 5.4 O@CNDs (0.5 mg / kg), mice were killed 12 hours later, and liver tissue and serum were collected for liver function test ( Figure 6 G). Similar to the HIRI model, H&E staining images of mouse liver tissue showed that obvious balloon-like lesions were observed in the liver cells of the LPS-induced hepatitis model, accompanied by a large number of inflammatory cell infiltration. 5.4 The liver pathological manifestations in the O@CNDs group were significantly improved, and the inflammatory symptoms were alleviated ( Figure 6 H). Subsequently, liver function biochemical tests were performed on mouse serum. The liver function indicators of mice with lipopolysaccharide-induced hepatitis were elevated, indicating that liver function damage was more severe, while Cu5.4 The liver function indexes of the O@CNDs treatment group decreased and were close to those of the control group, proving that the treatment effect was more obvious ( Figure 6 J).

[0141] Frozen tissue sections were taken for TUNEL staining to evaluate the level of hepatocyte necrosis and apoptosis. The results showed that the TUNEL green fluorescence of the modeling group was significantly enhanced, and there was no significant difference between the pretreatment group and the control group, suggesting that Cu 5.4 O@CNDs have good anti-inflammatory effects ( Figure 6 I). In order to evaluate Cu 5.4 The ability of O@CNDs to scavenge ROS in the liver of ALI disease was investigated. DCFH-DA and DHE were used to stain frozen sections. The green fluorescence of DCFH-DA increased significantly in the liver tissue induced by lipopolysaccharide hepatitis. 5.4 The ROS levels in O@CNDs-treated tissues were reduced ( Figure 6 At the same time, DHE observed high-intensity red fluorescence in hepatitis tissue, and the fluorescence intensity of the tissue decreased after treatment. The results showed that Cu 5.4 O@CNDs can alleviate inflammation by scavenging ROS in lipopolysaccharide-induced acute hepatitis ( Figure 6 I).

[0142] In addition, the changes in the expression levels of common pro-inflammatory cytokines were verified by extracting relevant RNA from mouse liver tissue. The results showed that after successful modeling, the expression of four common cytokines increased significantly, including Cu 5.4 There was no significant difference in the inflammatory level between the O@CNDs group and the healthy mice in the control group ( Figure 6 K). Tip Cu 5.4 O@CNDs can effectively improve oxidative stress and inflammation in acute hepatitis by scavenging ROS in the body.

[0143] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0144] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0145] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, characterized by Prepared by the following steps: CuCl2 was dissolved in a carbon dot aqueous solution, reacted at 75°C to 80°C for 10min to 12min, then L-ascorbic acid aqueous solution was added, the pH was adjusted to 7.0-8.0 with NaOH solution, and the reaction was carried out at 75°C to 80°C for 10h to 12h. In the reaction system, the concentration of CuCl2 was 15-20mM, the concentration of carbon dots was 3-4mg / mL, and the concentration of ascorbic acid was 300-400mM. After the reaction was completed, the precipitate was removed by centrifugation, and the supernatant was dialyzed and freeze-dried to obtain the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs.

2. According to claim 1, an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, characterized by The carbon dots are prepared by the following steps: The activated carbon is added to a boiling mixed acid solution, maintained at the boiling point for 1.5 hours to 2 hours, cooled to room temperature, and then neutralized with a NaHCO3 solution. The neutralized solution is filtered, dialyzed, concentrated, and freeze-dried to obtain the carbon dots, wherein the mixed acid solution is a mixture of equal volumes of an HNO3 solution with a concentration of 8 to 10 mol / L and an H2SO4 solution with a concentration of 18 to 20 mol / L, and the mass volume ratio of the activated carbon to the mixed acid solution is 0.5 g to 0.6 g:50 mL.

3. The anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity according to any one of claims 1 to 2 5.4 Application of O@CNDs in the preparation of drugs for treating liver injury.

4. The use according to claim 3, characterized in that The liver injury includes liver ischemia-reperfusion injury and acute liver injury.

5. The use according to any one of claims 3 to 4, characterized in that: The medicine contains the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, drug carriers and / or drug excipients.

6. A drug, characterized in that The drug is used to treat liver damage, and the drug comprises the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity according to any one of claims 1 to 2. 5.4 O@CNDs.

7. The drug according to claim 6, characterized in that The medicine further includes a drug carrier and / or a drug excipient.

8. The drug according to claim 7, characterized in that The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

9. The drug according to claim 7, characterized in that The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents.

10. The drug according to claim 7, characterized in that The dosage form of the medicine is powder, granule, capsule, tablet, pill, injection or oral solution.

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