The present invention relates to the technical field of nanotechnology and its applications, particularly to ligand-bound zinc sulfide nanoparticles, composition comprising the ligand-bound zinc sulfide nanoparticles, use of the ligand-bound zinc sulfide nanoparticles to prepare medications for treatment, and methods employing the ligand-bound zinc sulfide nanoparticles and composition for treatment.
Amyloid-β (Aβ) fibrosis causes or is associated with diseases, such as Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD), myositis/myopathy (MM).
Alzheimer's disease (AD) is a chronic neurodegenerative disease. Its pathological features include extracellular senile plaque deposition, intracellular neurofibrillary tangles and abnormal loss of neurons and synapses. More and more evidences show that the deposition of extracellular senile plaques and the initial accumulation of intracellular neurofibrillary tangles can trigger a series of serious pathological processes, including human astrocyte (HA) proliferative inflammation and oxidative stress. HA proliferative inflammation and oxidative stress can accelerate the accumulation of senile plaques and neurofibrillary tangles. This vicious cycle can lead to abnormal neuronal and synaptic loss. The deposition of senile plaques is caused by misfolding, abnormal aggregation and fibrosis of Aβ. Therefore, it is of great significance to develop potential drugs that can simultaneously reduce Aβ plaque and neuroinflammation.
The present invention provides ligand-bound zinc sulfide nanoparticle (R-ZnS NPs). In certain embodiments, the ligand-bound zinc sulfide nanoparticle comprises a zinc sulfide core; and a ligand (R) bound to the zinc sulfide core. In certain embodiments, the ligand (R) is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand (R) is captopril. In certain embodiments of the ligand-bound zinc sulfide nanoparticle, the diameter of the zinc sulfide core is 0.5-4.0 nm. In certain embodiments of the ligand-bound zinc sulfide nanoparticle, the diameter of the zinc sulfide core is 1.0-3.5 nm.
The present invention provides a process of preparing ligand-bound zinc sulfide nanoparticles (R-ZnS NPs). In certain embodiments, the process comprises:
In certain embodiments, the process further comprises purifying the R-ZnS NPs by centrifugation with an ultrafiltration tube; wherein the ultrafiltration tube is with a molecular weight cut-off of 5k Daltons.
The present invention provides ligand-bound zinc sulfide nanoparticles for use in treatment of a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD) or myositis/myopathy (MM). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is captopril.
The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles, where the composition is used for treatment of a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD) or myositis/myopathy (MM). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is captopril.
The present invention provides ligand-bound zinc sulfide nanoparticles for use in treatment of a subject with a condition of excessive expression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive-c-reactive-protein (Hs CRP), or tumor necrosis factor-alpha (TNFα). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is captopril.
The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles, where the composition is used for treatment of a subject with a condition of excessive expression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive-c-reactive-protein (Hs CRP), or tumor necrosis factor-alpha (TNFα). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is captopril.
The objectives and advantages of the invention will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.
Preferred embodiments according to the present invention will now be described with reference to the Figures, in which like reference numerals denote like elements.
The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.
Throughout this application, where publications are referenced, the disclosures of these publications are hereby incorporated by reference, in their entireties, into this application in order to more fully describe the state of art to which this invention pertains.
As used herein, “administering” means oral (“po”) administration, administration as a suppository, topical contact, intravenous (“iv”), intraperitoneal (“ip”), intramuscular (“im”), intralesional, intrahippocampal, intracerebroventricular, intranasal or subcutaneous (“sc”) administration, or the implantation of a slow-release device e.g., a mini-osmotic pump or erodible implant, to a subject. Administration is by any route including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
The terms “systemic administration” and “systemically administered” refer to a method of administering a compound or composition to a mammal so that the compound or composition is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (i.e. other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration, with the proviso that, as used herein, systemic administration does not include direct administration to the brain region by means other than via the circulatory system, such as intrathecal injection and intracranial administration.
As used herein, the terms “treating” and “treatment” refer to delaying the onset of, retarding or reversing the progress of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition.
The terms “patient,” “subject” or “individual” interchangeably refers to a mammal, for example, a human or a non-human mammal, including primates (e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g., felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal or rodent (e.g., rattus, murine, lagomorpha, hamster, guinea pig).
As used herein, the term “room temperature” means about 22-25 degree Celsius.
The present invention provides ligand-bound zinc sulfide nanoparticles (R-ZnS NPs).
In certain embodiments, the ligand-bound zinc sulfide nanoparticle (R-ZnS NPs) comprises a ligand (R) and a zinc sulfide core, wherein the ligand is bound to the zinc sulfide core. The ligand being bound to the zinc sulfide core means that the ligand forms a stable nanoparticle in solution with the zinc sulfide core through covalent bonds, hydrogen bonds, electrostatic force, hydrophobic force, van der Waals force, etc. In certain embodiments, the zinc sulfide core has a diameter of 0.5-4.0 nanometers (nm). In certain embodiments, the diameter of the zinc sulfide core is in the range of 1.0-3.5 nm.
In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC).
In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticle is Captopril (i.e. 1-[(S)-3-Mercapto-2-methylpropionyl]-L-proline) that is represented by formula (I):
The present invention provides a process for preparing ligand-bound zinc sulfide nanoparticles (R-ZnS NP).
In certain embodiments, the process for preparing ligand-bound zinc sulfide nanoparticles (R-ZnS NP) comprises:
In certain embodiments, the process further comprises:
The present invention provides the ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) for use in treatment of a subject with a condition of excessive expression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-1β), hypersensitive-c-reactive protein (Hs CRP), or tumor necrosis factor-alpha (TNFα). Here, the “excessive expression” means that the protein level is at least 20% higher than physiological expression level. The treatment is administration of the R-ZnS NPs or a composition containing the R-ZnS NPs. The treatment can decrease at least 50%, preferably 60%, 70%, 80%, 90% or 100%, of excess expressions of IL-6, IL-8, IL-10, Hs CRP, or TNFa, where the “excess expression” is defined as the difference between the expression level under the physiological condition and the expression level under the condition of excessive expression. In certain embodiments, the condition of excessive expression is induced by infection by microorganisms including fungi, bacteria and viruses. When a subject is infected by the pathological microorganisms, they secrete certain materials such as lipopolysaccharides (LPS) that will induce the excess expression of cytokines. LPS, also known as endotoxins, are large molecules consisting of a lipid and a polysaccharide found in the outer membrane of Gram-negative bacteria. Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the gram-staining method of bacterial differentiation. The gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter pylori, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella, cyanobacteria, spirochaetes, green sulfur, green non-sulfur bacteria,Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Salmonella enteritidis, Salmonella typhi, Acinetobacter baumannii. In certain embodiments, the condition of excessive expression is induced by autoimmune diseases or chronic inflammation disease including cancers. In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is Captopril.
The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles, where the composition is used for treatment of a subject with a condition of excessive expression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive-c-reactive-protein (Hs CRP), or tumor necrosis factor-alpha (TNFα). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyrul-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is Captopril.
The present invention provides a pharmaceutical composition for the treatment of a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD), or myositis/myopathy (MM).
In certain embodiments, the composition comprises the ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) as disclosed above and a pharmaceutically acceptable excipient. In certain embodiments, the excipient is phosphate-buffered solution, or physiological saline.
The present invention provides ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) for use in treatment of a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD) or myositis/myopathy (MM).
The present invention provides a use of the above disclosed R-ZnS NPs for treating a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD) or myositis/myopathy (MM), or a method for treating a subject with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration in glaucoma (RGCD) or myositis/myopathy (MM) using the above disclosed R-ZnS NPs. In certain embodiments, the method for treatment comprises administering a pharmaceutically effective amount of R-ZnS NPs to the subject. The pharmaceutically effective amount can be ascertained by routine in vivo studies. In certain embodiments, the pharmaceutically effective amount of R-ZnS NPs is a dosage of at least 0.001 mg/kg/day, 0.005 mg/kg/day, 0.01 mg/kg/day, 0.05 mg/kg/day, 0.1 mg/kg/day, 0.5 mg/kg/day, 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, 9 mg/kg/day, 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day, 40 mg/kg/day, 50 mg/kg/day, 60 mg/kg/day, 70 mg/kg/day, 80 mg/kg/day, 100 mg/kg/day, 200 mg/kg/day, 300 mg/kg/day, 400 mg/kg/day, 500 mg/kg/day, 600 mg/kg/day, 700 mg/kg/day, 800 mg/kg/day, 900 mg/kg/day, or 1000 mg/kg/day.
In certain embodiments, the subject is human. In certain embodiments, the subject is a pet animal such as a dog.
The following examples are provided for the sole purpose of illustrating the principles of the present invention; they are by no means intended to limit the scope of the present invention.
Currently, Aβ-induced cellular AD models and LPS-induced cellular inflammation models, as well as APP/PS1 double transgenic AD mouse models are widely used as experimental models.
2.1. Particle sizes of Cap-ZnS NPs
At room temperature, Cap-ZnS NPs were suspended in a mixture of ethanol and water at a volume ratio of 1:1, and the particle size of Cap-ZnS NPs were measured by JEM-2100F transmission electron microscope (JEOL, Japan). 100 Cap-ZnS NPs were randomly counted by Image J to calculate the particles sizes.
2.2. Infrared spectrum of Cap-ZnS NPs
At room temperature, the infrared spectra of Cap-ZnS NPs and Cap in the range of 4000-500 cm−1 were measured using the German Bruker vertex 80v FTIR. The freeze-dried samples were tested in MIR-ATR mode, and the results are shown in
The results showed that after the ligand Cap participated in the formation of Cap-ZnS NPs, the -SH stretching vibration characteristic peak (2566 cm−1) in the infrared spectrum disappeared, indicating that Cap was successfully grafted to the ZnS core through the Zn—S bond.
ESCALAB 250Xi XPS (Thermo Fisher, USA) was used to determine the elemental composition, content and binding energy of the total XPS spectra and single element spectra of C, N, O, S, and Zn. The single-element spectrum data were analyzed with XPS PEAK, and the results are shown in
The characteristic peaks of C1s, O1s, N1s, S2s, S2p and Zn2p in
The other two ligand-bound zinc sulfide nanoparticles are 4-mercaptobutyric acid (MA)-bound zinc sulfide nanoparticles (MA-ZnS NPs) and dihydrolipoic acid (DHLA)-bound zinc sulfide nanoparticles (DHLA-ZnS NPs). Their preparation methods are similar to that of Cap-ZnS NPs, except that the ligand is replaced with MA and DHLA respectively. Their size ranges are also consistent with that of Cap-ZnS NPs.
The kinetic process of Aβ40 fibrosis was studied using the Genetic Synergy™ MX microplate reader from Bio Tek, USA. The PBS buffer solution containing 40 μMAβ40 and 50 μM ThT was added to a 96-well plate with a black tube wall and a transparent glass bottom plate (final concentrations were 20 μM and 25 μM, respectively). The same volume of Cap-ZnS NPs, MA-ZnS NPs or DHLA-ZnS NPs samples with different concentrations was added to the final concentration of 0, 1, 5, 10, 20, 50 ppm respectively, and the plates with membrane seals were placed in the multi-function reading device (Synergy™ Multi-Mode MX), and the plate reading program was set up. Test conditions: scanning end-point fluorescence, excitation light wavelength is set to 445 nm, detection fluorescence emission wavelength is 485 nm. Keep the temperature at 37° C., shake the plate at a moderate intensity for 10 seconds at the end of every 10 minutes, and then measure the fluorescence emission intensity at 485 nm for continuous measurement for 60 hours. The fluorescence intensity of ThT was monitored to reflect the influence of three zinc sulfide nanoparticles bound by different ligands on the kinetics of Aβ40 fibrosis. The results are shown in
At room temperature, a FastScan atomic force microscope (Bruker, Germany) was used to study the microscopic morphology of Aβ40 fibers after 60-hours incubation in the presence of different concentrations of Cap-ZnS NPs. It adopts ScanAsyst air mode, SNL-10 pin scan, and the image resolution is 512×512.
It can be seen that when Cap-ZnS NPs (0 ppm) was not present in the reaction system, a large number of Aβ fibers appeared; when the final concentration of Cap-ZnS NPs was 1 ppm, the fibrous structure of Aβ changed to aggregates composed of small rod-like original Fiber (proto-fibril); and when the final concentration of Cap-ZnS NPs reached 5 ppm, there was almost no Aβ fiber or fibril structure in the reaction system. These results are consistent with that of the fibrosis kinetics measured by the ThT fluorescence method. It further shows that 1 ppm Cap-ZnS NPs has shown a good Aβ fibrosis inhibitory effect, while 5 ppm Cap-ZnS NPs can achieve the effects of complete suppression.
A JEM-2100F transmission electron microscope (JEOL, Japan) was used to measure the morphological changes of Af340. The results are shown in
PC-12 cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. CCK8 method was used to detect the cell viability of PC-12 cells. These cells were cultured in DMEM medium containing 10% FBS and 1% PS at a temperature of 37° C. and a CO2 concentration of 5%. After the cells have grown to a suitable number, 100 μL of cells in good condition were seeded in a 96-well plate with a density of 5×104 cells mL−1 and cultured for 24 hours (n=6). Then, removed all the media co-incubated with the cells, and added different doses of Cap-ZnS NPs, Aβ40 or their mixture to a 96-well plate (100 μL per well), and incubated for another 22 hours. Subsequently, 100 μL of DMEM solution containing 10% CCK-8 was added to each well and incubated for 2 hours. The absorbance at 450 nm with a microplate reader was measured, and the results are shown in
Test drugs: Cap-ZnS NPs, L-Cys-ZnS NPs, D-Cys-ZnS NPs, L-NIBC-ZnS NPs, D-NIBC-ZnS NPs, L-NAC-ZnS NPs, and D-NAC-ZnS NPs.
Human astroglia (HA) cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. The cell culture medium is DMEM medium containing 10% FBS and 1% PS. The culture temperature of the cell incubator is 37° C. and the CO2 concentration is 5%. HA cells in good condition were seeded in a 6-well plate at 2.4×108 cells/mL for culture. 7 groups: blank control group, LPS injury model control group, 4 test groups of Cap-ZnS NPs (final concentrations of 1 ppm, 5 ppm, 10 ppm or 20 ppm) (n=4) and 1 Cap control group (The final concentration of Cap was 20 ppm) (n=4). After the cells have grown for 24 hours, added DMEM minimal medium and Cap-ZnS NPs or Cap for pretreatment, and 2 hours later, added LPS (final concentration of 5 ppm). After culturing for another 24 hours, the culture medium and cells were collected, and the ELISA kit was used to detect the protein expression levels of inflammatory factors (IL-6, IL-8, IL-1(3, hs-CRP, TNF-α) in the cell culture medium. The specific methods are as follows: Taking 100 μL of the standard and sample diluent diluted to a specific concentration and adding it to a 96-well plate. After incubating for 90 minutes at 37° C., removing the liquid in the well, adding 100 μl of biotinylated antibody working solution and incubating for 1 hour, then washing the plate. After incubating with 100 μL of enzyme conjugate working solution for 0.5 h, washing the plate, incubating with 90 μL of chromogenic reagent (TMB) for 15 min in the dark, and stopping the reaction with 50 μL of stop solution. The microplate reader measures the absorbance at 450 nm.
The same experiments were performed using L-Cys-ZnS NPs, D-Cys-ZnS NPs, L-NIBC-ZnS NPs, D-NIBC-ZnS NPs, L-NAC-ZnS NPs, and D-NAC-ZnS NPs.
L-Cys-ZnS NPs, D-Cys-ZnS NPs, L-NIBC-ZnS NPs, D-NIBC-ZnS NPs, L-NAC-ZnS NPs, and D-NAC-ZnS NPs also showed outstanding anti-inflammatory effects, similar to Cap-ZnS NPs; for the brevity, detailed descriptions will be omitted.
6.1. Test method
Forty-two clean Kunming mice, 6-8 weeks old, weighing 25-30 kg, 21 male and female mice each, were housed in ordinary cages with 12 hours of light and darkness each day. Mice had free access to food and water. Male and female mice were randomly selected and divided into 1-6 groups (7 mice/group) for experimentation.
Group 1 and Group 2 were used for acute toxicity test in mice. The Group 1 was the drug test group, and the Group 2 was the blank control group. The drug test group was injected with 100 mg/Kg mouse body weight of Cap-ZnS NPs drug by intraperitoneal injection, and the blank control group was injected with the same volume of normal saline. The mice were sacrificed 24 hours later. After perfusion with normal saline, the heart, liver, spleen, lung, kidney, and brain were dissected out and fixed in 4% paraformaldehyde. Put the fixed animal tissue into the embedding box, and rinsed with running water for 30 minutes to remove the paraformaldehyde in the tissues. The tissues were dehydrated with an alcohol gradient to be transparent in xylene. The transparent tissues were immersed in a 1:1 mixture of paraffin wax and xylene for 90 minutes, then placed in paraffin wax for 2 hours, and immediately cooled. Used a paraffin microtome to make 51.tm continuous slices of the tissues, and baked slices at 60° C. for 2 hours. Immersed the slices in xylene for 5 minutes to deparaffinize, repeated 3 times. Then the slices were immersed in gradient ethanol (100%, 90%, 80%, and 70%) for 5 minutes each, and rinsed with running tap water for 5 minutes. Stained the section in hematoxylin staining solution for 5 minutes, then washed the excess staining solution on the slide with tap water, separated the color with 0.7% hydrochloric acid and ethanol for 10 seconds, rinsed the slide with tap water until the nucleus and nuclear chromatin are clear under the microscope. After dehydration with 70% and 90% ethanol for 10 minutes, dyed with 0.5% eosin solution for 5 minutes, and rinsed the excess dye solution with running water. The stained sections were dehydrated with 70%, 80%, 90%, and 100% ethanol for 10 seconds, soaked in xylene for 1 minute to make the tissue transparent, and air-dried naturally in a ventilated place. Quickly added proper amount of neutral gum and mount the slides. The pathological slices were observed and photographed with an optical microscope, and 2 fields of view were randomly selected for each slice to analyze all the tissues.
Groups 3 to 6 were used to explore the tissue distribution of drugs. Each group was administered Cap-ZnS NPs by intraperitoneal injection at the amount of 20 mg/Kg mouse body weight, and sacrificed after 2, 6, 12, or 24 hours. After dissection, the heart, liver, spleen, lung, kidney and brain were immediately put into liquid nitrogen and freeze-dried. After five days, it was taken out and ground into a uniform powder. Weighed 2 mg of tissue powder and digested it in a mixed solution of concentrated nitric acid and hydrogen peroxide (volume ratio 5:1) for inductive coupling Plasma emission spectroscopy tests the content of Cap-ZnS NPs in the heart, liver, spleen, lung, kidney and brain.
Acute toxicity studies found that Cap-ZnS NPs have no effect on the eyes, skin and mucous membranes of mice, as well as the breathing, food intake, exercise and excretion of mice within 24 hours. Further pathological examination found that, as shown in
Test drugs: Cap-ZnS NPs, L-Cys-ZnS NPs, D-Cys-ZnS NPs, L-NIBC-ZnS NPs, D-NIBC-ZnS NPs, L-NAC-ZnS NPs, D-NAC-ZnS NPs, MA-ZnS NP and DHLA-ZnS NP.
The test mice were 60-week-old C57BL/6 germline APP/PS1 transgenic AD model mice. The model mice were randomly divided into model control group, Cap-ZnS NPs administration group, L-Cys-ZnS NPs administration group, D-Cys-ZnS NPs administration group, L-NIBC-ZnS NPs administration group, D-NIBC-ZnS NPs administration group, L-NAC-ZnS NPs administration group, D-NAC-ZnS NPs administration group, MA-ZnS NPs administration group and DHLA-ZnS NPs administration group. At the same time, C57BL/6 wild-type mice group with the same age was set as a normal control group. 15 mice in each group. Each administration group was intraperitoneally injected with physiological saline solution of the corresponding drug once a day, the dose was 20 mg/Kg mouse body weight, and the injection volume was 100 μL. The mice of the model control group and normal control group were intraperitoneally injected with the same volume of physiological saline .
After 4 weeks of continuous administration, the Morris water maze test was used to analyze the cognitive and memory functions of all animals.
Place navigation test: The Morris water maze test system is composed of a water maze and an automatic video recording and analysis system. A camera is disposed above the water maze and connected to a computer. The water maze consists of a circular pool with a diameter of 120 cm and a height of 60 cm, and a platform with a diameter of 9 cm. The liquid level is 0.5 cm higher than the platform, and the water temperature is maintained at 22±0.5° C. Use white pigment to dye water to milky white. The place navigation test was used to measure the learning and memory abilities of mice in the water maze, which lasted 5 days. The water maze is divided into four quadrants: N, S, W, E. The platform is placed in a fixed quadrant. The position of the platform is fixed throughout the experiment. During training, the mice with head towards the pool wall were gently put into the water close to the outer wall from the ½ arc of different quadrants every day. Record the time when the mice climb on the hidden platform or stop the test when the time reaches 60 s. After the mice are on the platform, let them stay on the platform for 30 s. If the mice do not find the platform within 60 s, the experimenter guides the mouse to climb on the platform and let it stay for 30 s. The latency period in which mice seek the platform during the test was recorded by the camera tracking system. After the test is over, each animal is removed and dried gently with a hair dryer. Each animal was trained 4 times a day, with an interval of 20 minutes between trainings, for 5 consecutive days.
Space exploration test: after completing the training on the 5th day, the platform was removed on the 6th day, and the mice were gently placed in the water from the most distal point of the platform while facing the wall of the pool. Each mouse's movement trajectory within 60 seconds was recorded by the camera, and analyzed by software were the number of times the mouse crossed the platform, the stay time in the target quadrant, and the swimming time in the target quadrant.
The test results are expressed as ,C±SEM, all data are processed by SPSS software (SPSS 21), and a one-way analysis of variance (Post-Hoc Dunnett test) is used; P<0.05 indicates that the difference is statistically significant. Kruskal-Wallis H test and Mann-Whitney U test are used for statistical analysis of data that are not normally distributed.
After the behavioral tests, the mice were anesthetized by intraperitoneal injection of 7% chloral hydrate, and the cardiac perfusion connection was established, followed by rapid flushing with normal saline for 7 minutes, and then used 4% chloral hydrate to fix the tissues for 7 minutes. After the perfusion was over, the brain tissue was carefully taken and placed in 4% perfusate, and stored at room temperature for later use. Immunohistochemical methods were used to detect the expression of Aβ40 and inflammatory factors in the hippocampus and cortex: the perfused tissue was dehydrated, made transparent, waxed and embedded, and then sectioned using a paraffin microtome. A gradient of xylene and absolute ethanol was used for dewaxing. After antigen retrieval by microwave, the slices were incubated with hydrogen oxide, and the slices were blocked with serum for 30 min. Added primary antibodies against Aβ40, IL-10, TNF-α, GFAP, IL-6, or COX-2 (1:100) at room temperature, and incubated overnight (15 h) at 4° C. Discarded the primary antibody, washed the slices with PBS, ed HRP-labeled goat anti-rabbit/mouse secondary antibody, and incubated at room temperature for 30 min. After rinsing the slices with PBS, added the chromogenic reagent DAB chromogenic solution to develop the color, Harris hematoxylin counterstaining, dehydration, and mounting. A fluorescence microscope was used to take pictures, and Image J was used to quantitatively analyze the slices.
The results of the space exploration test (
For the target quadrant swimming time and target quadrant stay time (
The above data show that Cap-ZnS NPs drugs can significantly improve the cognitive and memory abilities of AD model mice, while MA-ZnS NPs and DHLA-ZnS NPs have no such effect.
The effects of L-Cys-ZnS NPs administration group, D-Cys-ZnS NPs administration group, L-NIBC-ZnS NPs administration group, D-NIBC-ZnS NPs administration group, L-NAC-ZnS NPs administration group and D-NAC-ZnS NPs administration group are similar to that of the Cap-ZnS NPs administration group. For the sake of brevity, they will not be described in details herein.
The R-ZnS NPs described in the present invention have the characteristics of simple preparation method and good biocompatibility.
R-ZnS NPs provided by the present invention have the following advantages:
First, R-ZnS NPs are significantly more effective in inhibiting Aβ fibrosis than other ligand (such as 4-Mercaptobutyric Acid (MA)- and dihydrolipoic acid, DHLA)-bound ZnS NPs; and R-ZnS-NPs at the ultra-low dose of 5 ppm can completely inhibit the fibrosis of Aβ at a concentration of 20 μM.
Second, R-ZnS NPs can greatly reduce the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α, IL-8 and hs-CRP) in LPS-treated HA cells.
Third, R-ZnS NPs significantly reduced the cytotoxicity caused by Aβ fibrosis in the Aβ-induced cell damage model experiment.
Fourth, R-ZnS NPs significantly reduced Aβ plaques in the hippocampus of AD model mice in the APP/PS1 double transgenic AD mouse model tests, and significantly reduced the level of neuroinflammatory factors in the brain of AD model mice. In the APP/PS1 double transgenic AD model mouse tests, R-ZnS NPs significantly improved the cognitive and memory behavioral obstacles of the model mice.
Fifth, R-ZnS NPs can penetrate the blood-brain barrier and enter the mouse brain.
Sixth, R-ZnS NPs have biological safety at the animal level.
The ligand-bound zinc sulfide nanoparticles with ligands of L-cysteine, D-cysteine, L-NIBC, D-NIBC), L-NAC or D-NAC have been synthesized, characterized, and tested following the same protocols as described above, and have shown the similar effects on reducing the expression of inflammatory cytokines, inhibiting Aβ fibrillation, and treating Aβ-related diseases such as AD. For the sake of brevity, they will not be described in details herein.
While the present invention has been described with reference to particular embodiments, it will be understood that the embodiments are illustrative and that the invention scope is not so limited. Alternative embodiments of the present invention will become apparent to those having ordinary skill in the art to which the present invention pertains. Such alternate embodiments are considered to be encompassed within the scope of the present invention. Accordingly, the scope of the present invention is defined by the appended claims and is supported by the foregoing description.
Filing Document | Filing Date | Country | Kind |
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PCT/CN2021/082357 | 3/23/2021 | WO |