Ligand-bound zinc sulfide nanoparticles, method for producing the same, and use thereof for treatment

Abstract

The present invention relates to ligand-bound zinc sulfide nanoparticles, a method for producing said ligand-bound zinc sulfide nanoparticles, a composition comprising said ligand-bound zinc sulfide nanoparticles, uses of said ligand-bound zinc sulfide nanoparticles and said composition comprising said ligand-bound zinc sulfide nanoparticles including inhibiting amyloid beta (Aβ) fibrillization, reducing expression of inflammatory factors, treating Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma or myositis-myopathy (MM) caused by / associated with Aβ fibrillization, use in the manufacture of a medicament for the treatment of AD, CAA, RGCD or MM, and a method for treating the above diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanotechnology and its applications, and particularly to ligand-bound zinc sulfide nanoparticles, a composition comprising the ligand-bound zinc sulfide nanoparticles, use in the manufacture of a medicament for treatment with the ligand-bound zinc sulfide nanoparticles, and methods of utilizing the ligand-bound zinc sulfide nanoparticles and the composition for treatment.

Background Art

[0002] The fibrillation of amyloid-β (Aβ) is a cause or related factor of diseases such as Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma, and myositis and myopathy (MM).

[0003] 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. Accumulating evidence suggests that the initial accumulation of extracellular senile plaque deposition and intracellular neurofibrillary tangles triggers a series of severe pathological processes including proliferative inflammation and oxidative stress in human astrocytes (HA). Proliferative inflammation and oxidative stress in HA accelerate the accumulation of senile plaques and neurofibrillary tangles. This vicious cycle leads to abnormal loss of neurons and synapses. The deposition of senile plaques is caused by the misfolding, abnormal aggregation, and fibrillation of Aβ. Therefore, developing potential drugs that can simultaneously suppress Aβ plaques and neuroinflammation is of great significance.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention provides ligand - bound zinc sulfide nanoparticles (R - ZnS NPs). In certain embodiments, the ligand - bound zinc sulfide nanoparticles comprise a zinc sulfide core and a ligand (R) bound to the zinc sulfide core. In certain embodiments, the ligand (R) is selected from the group consisting of L - cysteine, D - cysteine, N - isobutyryl - L - cysteine (L - NIBC), N - isobutyryl - 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 nanoparticles, the diameter of the zinc sulfide core is 0.5 - 4.0 nm. In certain embodiments of the ligand - bound zinc sulfide nanoparticles, the diameter of the zinc sulfide core is 1.0 - 3.5 nm.

[0005] The present invention provides a method for producing ligand - bound zinc sulfide nanoparticles (R - ZnS NPs). In certain embodiments, the method comprises

[0006] dissolving a ligand (R) in deionized water to obtain a ligand aqueous solution such that the concentration of the ligand in the ligand aqueous solution is 0.02 - 2.0 mol / L;

[0007] adding a zinc acetate solution to the ligand aqueous solution to obtain a zinc acetate / ligand reaction mixture, wherein the concentration of the zinc acetate aqueous solution is 0.01 - 1.0 mol / L and the molar ratio of the ligand to zinc acetate is in the range of 1:1 - 10:1;

[0008] adjusting the pH of the zinc acetate / ligand reaction mixture to the range of 7 - 10;

[0009] dropping a sodium sulfide aqueous solution into the pH - adjusted zinc acetate / ligand reaction mixture such that the molar ratio of the added sodium sulfide to zinc acetate in the zinc acetate / ligand reaction mixture is in the range of 0.1:1 - 5:1 to obtain a sodium sulfide / zinc acetate / ligand reaction mixture;

[0010] Heating the sodium sulfide / zinc acetate / ligand reaction mixture to a predetermined temperature and maintaining the reaction for a predetermined time to form R-ZnS NPs, wherein the predetermined temperature is 50-100 °C and the predetermined time is 1-5 hours; comprising.

[0011] In certain embodiments, the method further comprises purifying the R-ZnS NPs by centrifugation using an ultrafiltration tube, wherein the molecular weight cut-off of the ultrafiltration tube is 5 kDa.

[0012] The present invention provides ligand-bound zinc sulfide nanoparticles for use in the treatment of subjects having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma or myositis and myopathy (MM). In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-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.

[0013] The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles, which is used for the treatment of subjects having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma or myositis and 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-isobutyryl-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.

[0014] The present invention provides ligand-bound zinc sulfide nanoparticles for use in the treatment of subjects having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (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-isobutyryl-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.

[0015] The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles, which is used for the treatment of a subject having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (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-isobutyryl-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.

[0016] The objects and advantages of the present invention will become apparent from the following detailed description of its preferred embodiments in connection with the accompanying drawings.

Brief Description of the Drawings

[0017] Preferred embodiments according to the present invention will now be described with reference to the figures, where like reference numerals indicate like elements.

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Mode for Carrying Out the Invention

[0027] The present invention can be more easily understood by referring to the following detailed description of specific embodiments of the present invention.

[0028] Throughout this application, when publications are referenced, the disclosures of these publications are hereby incorporated by reference in their entirety to more fully describe the state of the art relevant to the present invention.

[0029] As used herein, "administer" means oral ("po") administration to a subject, administration as a suppository, topical contact, intravenous ("iv"), intraperitoneal ("ip"), intramuscular ("im"), intralesional, intrahippocampal, intracerebroventricular, intranasal or subcutaneous ("sc") administration, or implantation of a sustained release device (e.g., a mini-osmotic pump or an erodible implant). Administration can be by any route including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, and intracranial administration. Other delivery forms include, but are not limited to, the use of liposome formulations, intravenous infusion, transdermal patches, etc.

[0030] The terms "systemic administration" and "systemically administer" mean a method of administering a compound or composition to a mammal such that the compound or composition is delivered to a site within the body, including the target site of a pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (i.e., other than via the gastrointestinal tract such as intramuscular, intravenous, intraarterial, transcutaneous, and subcutaneous) administration. However, as used herein, systemic administration does not include direct administration to a brain region by means other than via the circulatory system such as intrathecal injection and intracranial administration.

[0031] As used herein, the terms "treat" and "treatment" refer to delaying the onset of a disease or condition to which the term applies, or one or more symptoms of such a disease or condition, retarding its progression or reversing it, or alleviating or preventing it.

[0032] The terms "patient", "subject" or "individual" are used interchangeably to refer to a non-human mammal including mammals such as humans, or primates (e.g., macaques, pan troglodytes, pongo), domestic mammals (e.g., felines, canines), agricultural mammals (e.g., bovines, ovines, porcines, equines), and laboratory mammals or rodents (e.g., rats, mice, rabbits, hamsters, guinea pigs).

[0033] As used herein, the term "room temperature" means about 22 to 25 °C.

[0034] The present invention provides ligand-bound zinc sulfide nanoparticles (R-ZnS NPs).

[0035] In certain embodiments, the ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) comprise a ligand (R) and a zinc sulfide core, and the ligand is bound to the zinc sulfide core. The fact that the ligand is bound to the zinc sulfide core means that the ligand forms stable nanoparticles with the zinc sulfide core in solution by covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, van der Waals forces, etc. In certain embodiments, the diameter of the zinc sulfide core is 0.5 to 4.0 nanometers (nm). In certain embodiments, the diameter of the zinc sulfide core is in the range of 1.0 to 3.5 nm.

[0036] In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine (L-NAC), and N-acetyl-D-cysteine (D-NAC).

[0037] In certain embodiments, the ligand of the ligand-bound zinc sulfide nanoparticles is captopril represented by formula (I) (i.e., 1-[(S)-3-mercapto-2-methylpropionyl]-L-proline). JPEG0007715827000001.jpg2755

[0038] The present invention provides a method for producing ligand-bound zinc sulfide nanoparticles (R-ZnS NP).

[0039] In certain embodiments, the method for producing the ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) is

[0040] dissolving the ligand in deionized water to obtain an aqueous ligand solution (in certain embodiments, the concentration of the ligand in the aqueous ligand solution is 0.02 to 2.0 mol / L, preferably 0.02 to 0.2 mol / L), and

[0041] adding a zinc acetate solution to the aqueous ligand solution to obtain a zinc acetate / ligand reaction mixture (in certain embodiments, the zinc acetate / ligand reaction mixture is stirred at room temperature for 0.1 to 3 hours, preferably 0.3 to 1.5 hours. In certain embodiments, the concentration of the aqueous zinc acetate solution is 0.01 to 1.0 mol / L, preferably 0.02 to 0.2 mol / L. In certain embodiments, the molar ratio of the ligand to zinc acetate is in the range of 1:1 to 10:1, preferably 1:1 to 5:1), and

[0042] adjusting the pH of the zinc acetate / ligand reaction mixture to a range of 7 to 10, preferably 8 to 9 (in certain embodiments, the pH-adjusted zinc acetate / ligand reaction mixture is stirred at room temperature for 0.3 to 5 hours, preferably 0.5 to 2 hours. In certain embodiments, the reagent for pH adjustment is a sodium hydroxide solution), and

[0043] dropping an aqueous sodium sulfide solution into the pH-adjusted zinc acetate / ligand reaction mixture to obtain a sodium sulfide / zinc acetate / ligand reaction mixture (in certain embodiments, the sodium sulfide / zinc acetate / ligand reaction mixture is stirred at room temperature for 1 to 5 hours, preferably 1 to 3 hours. In certain embodiments, the molar ratio of the added sodium sulfide to zinc acetate in the zinc acetate / ligand reaction mixture is in the range of 0.1:1 to 5:1, preferably 0.2:1 to 2:1), and

[0044] Heat the sodium sulfide / zinc acetate / ligand reaction mixture to a predetermined temperature and maintain the reaction for a predetermined time to form R-ZnS NPs (in certain embodiments, the predetermined temperature is 50-100 °C, preferably 50-70 °C. In certain embodiments, the predetermined time is 1-5 hours, preferably 1-2 hours.), and comprise.

[0045] In certain embodiments, the method further comprises

[0046] Purifying the R-ZnS NPs by centrifugation using an ultrafiltration tube (in certain embodiments, the conditions of the centrifugation are 5000-6000 r / min for 5 minutes. In certain embodiments, the molecular weight cut-off of the ultrafiltration tube is 5 kDa.), and

[0047] Collecting the liquid above the ultrafiltration tube to obtain purified R-ZnS NPs (in certain embodiments, the separated R-ZnS NPs are washed with ultrapure water, for example, 3 times.), and

[0048] Lyophilizing the purified L-ZnS NPs to obtain a stable powder of L-ZnS NPs, and comprise.

[0049] The present invention provides the ligand-binding zinc sulfide nanoparticles (R-ZnS NPs) for use in the treatment of a subject having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (TNFα). Here, "overexpression" means that the protein level is 20% or more higher than the physiological expression level. The treatment is administration of R-ZnS NPs or a composition containing R-ZnS NPs. The treatment can reduce the overexpression of IL-6, IL-8, IL-1β, Hs CRP, or TNFα by at least 50%, preferably 60%, 70%, 80%, 90% or 100%, where "overexpression" is defined as the difference between the expression level under physiological conditions and the expression level under the condition of overexpression. In certain embodiments, the state of overexpression is induced by infection with microorganisms such as fungi, bacteria and viruses. When a subject is infected with a pathogenic microorganism, the pathogenic microorganism secretes a specific substance such as lipopolysaccharide (LPS), which induces overexpression of cytokines. LPS, also called endotoxin, is a large molecule composed of lipids and polysaccharides present in the outer membrane of Gram-negative bacteria. Gram-negative bacteria are bacteria that do not retain crystal violet staining used in the Gram staining method, a method for differentiating bacteria.Gram-negative bacteria include Escherichia coli (E. coli), Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter pylori, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella, cyanobacteria, spirochaetes, green sulfur bacteria, 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, and Acinetobacter baumannii. In certain embodiments, the overexpressed state is induced by a chronic inflammatory disease including an autoimmune disease or cancer. In certain embodiments, the ligand of the ligand-binding zinc sulfide nanoparticles is one selected from the group consisting of L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-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.

[0050] The present invention provides a composition comprising ligand-bound zinc sulfide nanoparticles for use in the treatment of a subject having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (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-isobutyryl-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.

[0051] The present invention provides a pharmaceutical composition for the treatment of a subject having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM).

[0052] In certain embodiments, the composition comprises the above-described ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) and a pharmaceutically acceptable excipient. In certain embodiments, the excipient is phosphate buffer or physiological saline.

[0053] The present invention provides ligand-bound zinc sulfide nanoparticles (R-ZnS NPs) for use in the treatment of a subject having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM).

[0054] The present invention provides the use of the above-described R-ZnS NPs for treating a subject having Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM), or a method for treating a subject having Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM) using the above-described R-ZnS NPs. In certain embodiments, the treatment method includes administering a pharmacologically effective amount of R-ZnS NPs to the subject. The pharmacologically effective amount may be determined by conventional methods in in vivo studies. In certain embodiments, the pharmacologically effective amount of the R-ZnS NPs is 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.

[0055] In certain embodiments, the subject is a human. In certain embodiments, the subject is a pet animal such as a dog.

[0056] The following examples are provided only for the purpose of illustrating the principles of the present invention and are not intended to limit the scope of the present invention in any way.

[0057] Examples

[0058] Currently, the Aβ-induced cellular AD model, the LPS-induced cellular inflammation model, and the APP / PS1 double transgenic AD mouse model are widely used as experimental models.

[0059] Example 1. Preparation of captopril-conjugated zinc sulfide nanoparticles (Cap-ZnS NPs)

[0060] (1) Captopril (43.46 mg), a ligand, was dissolved in deionized water (10 ml) to obtain an aqueous captopril solution. The aqueous captopril solution was placed in a reaction flask, and then a zinc acetate solution (0.01 mol / L, 10 mL) was slowly added to the aqueous captopril solution to obtain a captopril / zinc acetate reaction mixture. The captopril / zinc acetate reaction mixture was stirred at room temperature for 0.5 h, and the molar ratio of captopril to zinc acetate in the captopril / zinc acetate reaction mixture was 2:1.

[0061] (2) A newly prepared sodium hydroxide solution (1 M) was added to the captopril / zinc acetate reaction mixture to adjust the pH of the captopril / zinc acetate reaction mixture to 9, obtaining a pH-adjusted captopril / zinc acetate reaction mixture, which was then stirred at room temperature for 1 h.

[0062] (3) An aqueous sodium sulfide solution (0.004 mol / L, about 10 ml) was slowly added dropwise to the pH-adjusted captopril / zinc acetate reaction mixture to obtain a sodium sulfide / captopril / zinc acetate reaction mixture. The sodium sulfide / captopril / zinc acetate reaction mixture was stirred at room temperature for 1 h, and the molar ratio of sodium sulfide to zinc acetate in the sodium sulfide / captopril / zinc acetate reaction mixture was 0.4:1.

[0063] (4) The reaction flask containing the sodium sulfide / captopril / zinc acetate reaction mixture was transferred to a 60 °C oil bath and stirred for 2 h to form Cap-ZnS NPs.

[0064] (5) The formed Cap-ZnS NPs were separated by centrifugation at 5000 - 6000 r / min for 5 minutes using an ultrafiltration tube with a molecular weight cut-off of 5k. Subsequently, the separated Cap-ZnS NPs were washed by ultrafiltration three times with ultrapure water to obtain purified Cap-ZnS NPs.

[0065] (6) The purified Cap-ZnS NPs were freeze-dried to obtain a white and stable Cap-ZnS NP powder.

[0066] Example 2. Identification of Cap-ZnS NPs

[0067] 2.1. Particle size of Cap-ZnS NPs

[0068] At room temperature, Cap-ZnS NPs were suspended in a mixture of ethanol and water with a volume ratio of 1:1, and the particle diameter of Cap-ZnS NPs was measured using a JEM-2100F transmission electron microscope (JEOL, Japan). The particle size was calculated by randomly counting 100 Cap-ZnS NPs with Image J.

[0069] Figure 1A is a representative transmission electron micrograph, indicating that the fabricated Cap-ZnS NPs had good dispersibility. Figure 1B shows the size distribution of Cap-ZnS NPs, which was mainly distributed in the range of 0.5 - 4.0 nanometers (nm).

[0070] 2.2. Infrared spectrum of Cap-ZnS NPs

[0071] At room temperature, the infrared spectra of Cap-ZnS NPs and Cap in the range of 4000 - 500 cm -1 were measured using a German Bruker vertex 80v FTIR. The freeze-dried sample was measured in the MIR-ATR mode, and the results are shown in Figure 1C.

[0072] According to the results, 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 successfully grafted onto the ZnS core via Zn-S bonds.

[0073] 2.3. X-ray Photoelectron Spectroscopy (XPS) of Cap-ZnS NPs

[0074] Using ESCALAB 250Xi XPS (Thermo Fisher, USA), the elemental composition, content, and binding energy of the full XPS spectrum and the single-element spectra of C, N, O, S, and Zn were measured. The data of the single-element spectra were analyzed by XPS PEAK, and the results are shown in Figures 1D, 1E, and 1F.

[0075] The characteristic peaks of C1s, O1s, N1s, S2s, S2p, Zn2p in Figure 1D, and the high-resolution XPS spectra of Zn2p in Figure 1E and S2p in Figure 1F are in good agreement with the theoretical predictions of Zn2p 1 / 2 , Zn2p 3 / 2 , S2p 1 / 2 and S2p 3 / 2 , indicating the presence of the ZnS core and the successful grafting (binding) of Cap on the surface of the ZnS core.

[0076] Example 3. Comparison of the anti-Aβ protein fibrillation ability of Cap-ZnS NPs with two other ligand-bound zinc sulfide nanoparticles

[0077] The other two ligand-bound zinc sulfide nanoparticles were 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 were the same as those of Cap-ZnS NPs except that the ligands were replaced with MA and DHLA respectively. Their size ranges were also consistent with those of Cap-ZnS NPs.

[0078] 3.1. Comparison of the anti-protein fibrillation abilities of Cap-ZnS NPs, MA-ZnS NPs, and DHLA-ZnS NPs by ThT fluorescence spectroscopy

[0079] Genetic Synergy from Bio Tek, USA TM The kinetic process of Aβ40 fibrillation was studied using an MX microplate reader. A PBS buffer containing 40 μM Aβ40 and 50 μM ThT was added to a 96-well plate with a black tube wall and a transparent glass bottom plate (the final concentrations were 20 μM and 25 μM, respectively). Different concentrations of Cap-ZnS NP, MA-ZnS NP, or DHLA-ZnS NP samples were each added in the same amount so that the final concentrations were 0, 1, 5, 10, 20, 50 ppm. After sealing with a membrane, the plate was placed in a multifunctional reader (Syngy TM Multi-Mode MX), and the plate reading program was set. Test conditions: scanning end-point fluorescence, excitation light wavelength of 445 nm, and detected fluorescence emission wavelength of 485 nm. The temperature was maintained at 37 °C, the plate was shaken moderately for the last 10 seconds every 10 minutes, and the fluorescence emission intensity at 485 nm was continuously measured for 60 hours. By monitoring the fluorescence intensity of ThT, the effects of three types of zinc sulfide nanoparticles bound to different ligands on the kinetics of Aβ40 fibrillation were reflected. The results are shown in Figure 2.

[0080] Figures 2A, 2B, and 2C respectively show the effects of different concentrations of Cap-ZnS NPs, MA-ZnS NPs, and DHLA-ZnS NPs on the kinetics of Aβ40 fibrillation at a concentration of 20 μM. From the results, it was found that Cap-ZnS NPs have excellent anti-Aβ protein fibrillation ability and completely suppressed Aβ fibrillation at a low concentration of 5 ppm (the kinetic curve of ThT fluorescence became completely flat). MA-ZnS NPs and DHLA-ZnS NPs also showed certain Aβ fibrillation inhibitory ability, but they could not completely suppress Aβ fibrillation even at a high concentration of 50 ppm. Comparing the kinetic curves of the three ThT fluorescences, the Aβ fibrillation inhibitory effect of 1 ppm Cap-ZnS NPs reached or exceeded that of the other two types of zinc sulfide nanoparticles with a final concentration of 50 ppm, indicating that the anti-protein fibrillation ability of Cap-ZnS NPs far exceeds that of the other two types of zinc sulfide nanoparticles.

[0081] 3.2. Study on the Effect of Cap-ZnS NPs on the Microscopic Morphology of Aβ40 Fibrillation by Atomic Force Microscopy (AFM)

[0082] At room temperature, a FastScan atomic force microscope (Bruker, Germany) was used to study the microscopic morphology of Aβ40 fibers after incubation for 60 hours in the presence of different concentrations of Cap-ZnS NPs. The ScanAsyst air mode and SNL-10 pin scan were adopted, and the image resolution was 512×512.

[0083] Figures 3A, 3B, and 3C show the results of the AFM test when the final concentrations of Cap-ZnS NPs are 0, 1, and 5 ppm, respectively.

[0084] When Cap-ZnS NPs (0 ppm) were not present in the reaction system, a large number of Aβ fibers appeared. When the final concentration of Cap-ZnS NPs reached 1 ppm, the fibrous structure of Aβ changed into an aggregate composed of small rod-shaped protofibrils. And when the final concentration of Cap-ZnS NPs reached 5 ppm, it was confirmed that there were almost no Aβ fibers or fibril structures in the reaction system. These results were consistent with the results of the fibrillation kinetics measured by the ThT fluorescence method. Furthermore, it was found that 1 ppm of Cap-ZnS NPs showed a good Aβ fibrillation inhibitory effect, and 5 ppm of Cap-ZnS NPs could achieve a complete inhibition effect.

[0085] 3.3. Transmission electron microscope

[0086] The morphological changes of Aβ40 were measured using a transmission electron microscope JEM-2100F (JEOL, Japan). The results are shown in Figures 3D, 3E, and 3F. These results were consistent with the test results of AFM.

[0087] Example 4. AD model test of Aβ-induced PC-12 cell damage

[0088] PC-12 cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. To detect the cell viability of PC-12 cells, the CCK8 method was used. These cells were cultured in DMEM medium containing 10% FBS and 1% PS under the conditions of a temperature of 37 °C and a CO2 concentration of 5%. After the cells grew to an appropriate number, 100 μL of healthy cells were seeded into a 96-well plate at 5×10 4 cells·mL -1Inoculated at a density of and cultured for 24 hours (n = 6). Next, all the medium co-cultured with the cells was removed, and different doses of Cap-ZnS NPs, Aβ40, or their mixture were added to a 96-well plate (100 μL per well), and further incubated for 22 hours. Then, 100 μL of DMEM solution containing 10% CCK-8 was added to each well and incubated for 2 hours. The absorbance at 450 nm was measured with a microplate reader, and the results are shown in Figure 4.

[0089] Figure 4A shows the effect of different concentrations of Cap-ZnS NPs on the viability of PC-12 cells. When the final concentration of Cap-ZnS NPs reached 100 ppm, the cell viability still maintained above 92%, indicating that Cap-ZnS NPs have good safety at the cellular level. Figure 4B shows the effect of different concentrations of Cap-ZnS NPs on the cell viability of PC-12 cells in the presence of Aβ40 (final concentration 25 μM). Aβ40 significantly decreased the viability of PC-12 cells (from 100% to 68.5 ± 5.2%), but the addition of Cap-ZnS NPs significantly restored the cell viability, and this effect significantly increased with the increase in the concentration of Cap-ZnS NPs. When the final concentration of Cap-ZnS NPs reached 100 ppm, the cell viability recovered to more than 90%. These results indicate that Cap-ZnS NPs can significantly reduce the damage of PC-12 cells induced by Aβ40, demonstrating the neuroprotective effect of Cap-ZnS NPs.

[0090] Example 5. LPS-induced cell inflammation experiment

[0091] 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.

[0092] Human astrocytes (HA) cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. The cell culture medium was DMEM medium containing 10% FBS and 1% PS. The culture temperature of the cell incubator was 37°C and the CO2 concentration was 5%. Healthy HA cells were seeded in a 6-well plate at 2.4×10 8 cells / mL and cultured. There were a total of seven groups: a blank control group, a LPS injury model control group, four test groups of Cap-ZnS NPs (final concentrations of 1 ppm, 5 ppm, 10 ppm, or 20 ppm) (n = 4), and one group of Cap control group (the final concentration of Cap was 20 ppm) (n = 4). After 24 hours of culture, for pretreatment, DMEM minimum medium and Cap-ZnS NP or Cap were added, and after 2 hours, LPS (final concentration 5 ppm) was added. After further culturing for 24 hours, the culture medium and cells were collected, and an ELISA kit was used to detect the protein expression levels of inflammatory factors (IL-6, IL-8, IL-1β, hs-CRP, TNF-α) in the cell culture medium. The specific method was as follows: 100 μL of standard solution and sample diluent diluted to a specific concentration were taken and added to a 96-well plate. After incubating at 37°C for 90 minutes, the liquid in the wells was removed, 100 μL of biotinylated antibody working solution was added, incubated for 1 hour, and then the plate was washed. After incubating with 100 μL of enzyme conjugate working solution for 0.5 hour, the plate was washed, 90 μL of chromogenic reagent (TMB) was added, incubated in the dark for 15 minutes, and the reaction was stopped with 50 μL of stop solution. The microplate reader measured the absorbance at 450 nm.

[0093] Similar experiments were carried out 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.

[0094] Figures 5A, 5B, 5C, 5D, and 5E respectively show the protein expression levels of five inflammatory factors, IL-6, IL-8, IL-1β, hs-CRP, and TNF-α. The five inflammatory factors were significantly increased by LPS (compared with the blank control group, all P values were less than 0.001, ##), indicating that the model was successfully established. The addition of Cap-ZnS NPs significantly inhibited the increase of the five inflammatory factors (compared with the LPS model control group, all P values were less than 0.05, *, less than 0.01, **, or less than 0.001, ***), and with the increase in concentration, this effect showed an obvious increasing trend. When the concentration of Cap-ZnS NPs reached 100 ppm, the levels of the five inflammatory factors decreased to almost the same levels as the normal control group. However, the levels of inflammatory factors in the Cap control group did not significantly decrease compared with the LPS model control group. From the above results, it was found that Cap-ZnS NPs showed excellent anti-inflammatory effects in cell experiments.

[0095] 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 excellent anti-inflammatory effects similar to Cap-ZnS NPs. For the sake of brevity, detailed descriptions are omitted.

[0096] Example 6. Acute Toxicity, Tissue Distribution, and Metabolism Tests in Mice

[0097] 6.1. Test Methods

[0098] (1) Maintenance and Management of Mice

[0099] Forty-two clean Kunming mice (21 males and 21 females) aged 6 - 8 weeks and weighing 25 - 30 kg were housed in ordinary cages under a 12-hour light:12-hour dark cycle. The 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 the experiment.

[0100] (2) Tissue Processing

[0101] For the acute toxicity test on mice, Groups 1 and 2 were used. Group 1 was the drug test group, and Group 2 was the blank control group. In the drug test group, Cap-ZnS NPs agent was intraperitoneally injected at 100 mg / Kg mouse body weight, and in the blank control group, the same amount of normal saline was injected. The mice were sacrificed after 24 hours. After perfusion with normal saline, the heart, liver, spleen, lungs, kidneys, and brain were dissected and fixed with 4% paraformaldehyde. The fixed animal tissues were placed in an embedding box and washed with running water for 30 minutes to remove paraformaldehyde in the tissues. The tissues were dehydrated with an alcohol gradient and made transparent with xylene. The transparent tissues were immersed in a 1:1 mixture of paraffin wax and xylene for 90 minutes, placed in paraffin wax for 2 hours, and immediately cooled. 5-μm serial sections of the tissues were prepared using a paraffin microtome, and the sections were baked at 60 °C for 2 hours. The sections were immersed in xylene for 5 minutes to deparaffinize, and this was repeated 3 times. Then, the sections were immersed in gradient ethanol (100%, 90%, 80%, 70%) for 5 minutes each and rinsed with running tap water for 5 minutes. After staining the sections with hematoxylin staining solution for 5 minutes, the excess staining solution on the slides was washed with tap water, color-separated with 0.7% hydrochloric acid and ethanol for 10 seconds, and the slides were rinsed with tap water until the nuclei and nuclear chromatin were clear under the microscope. After dehydration with 70% and 90% ethanol for 10 minutes, the sections were stained with 0.5% eosin solution for 5 minutes, and the excess dye solution was rinsed with running water. The stained sections were dehydrated with 70%, 80%, 90%, and 100% ethanol for 10 seconds each, immersed in xylene for 1 minute to make the tissues transparent, and then air-dried in a ventilated place. An appropriate amount of neutral gum was added and mounted on the slides. These pathological sections were observed and photographed with an optical microscope. For each section, 2 fields of view were randomly selected and the whole tissue was analyzed.

[0102] Groups 3 to 6 were used to investigate the tissue distribution of the drug. Cap-ZnS NPs were intraperitoneally injected into each group at a dose of 20 mg / Kg of mouse body weight, and the mice were sacrificed after 2, 6, 12, or 24 hours. After dissection, the heart, liver, spleen, lung, kidney, and brain were immediately placed in liquid nitrogen and freeze-dried. Five days later, they were taken out and ground into a uniform powder. 2 mg of the tissue powder was weighed, digested with a mixed solution of concentrated nitric acid and hydrogen peroxide (volume ratio 5:1), and the content of Cap-ZnS NPs in the heart, liver, spleen, lung, kidney, and brain was measured by inductively coupled plasma optical emission spectrometry.

[0103] 6.2. Test Results

[0104] As a result of the acute toxicity test, it was found that Cap-ZnS NPs did not affect the eyes, skin, mucosa of the mice, and respiration, food intake, movement, and excretion within 24 hours. Furthermore, upon performing pathological examinations, as shown in Figure 6, compared with the blank control group (upper), the major organs such as the heart, liver, spleen, lung, and brain tissues of the mice in the Cap-ZnS NPs test group were normally arranged, and no infiltration of inflammatory cells was observed. From the above research, it was found that Cap-ZnS NPs do not cause obvious toxicity or side effects to normal tissues and organs and have good biological safety.

[0105] Figure 7 shows the content of Cap-ZnS NPs in the tissues of the heart, liver, spleen, lung, kidney, and brain. As a result, it was confirmed that the drug content in each organ reached its maximum at about 6 hours and then gradually decreased over time. Also, a significant amount of the drug was observed in the brain, indicating that the drug passed through the blood-brain barrier and entered the brain.

[0106] Example 7. Test Using APP / PS1 Double Transgenic AD Model Mice

[0107] 7.1. Test Method

[0108] 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.

[0109] As test mice, 60-week-old C57BL / 6 germline APP / PS1 transgenic AD model mice were used. These model mice were randomly divided into a model control group, a Cap-ZnS NPs administration group, an L-Cys-ZnS NPs administration group, a D-Cys-ZnS NPs administration group, an L-NIBC-ZnS NPs administration group, a D-NIBC-ZnS NPs administration group, an L-NAC-ZnS NPs administration group, a D-NAC-ZnS NPs administration group, an MA-ZnS NPs administration group and a DHLA-ZnS NPs administration group. At the same time, a group of C57BL / 6 wild-type mice of the same age was set as a normal control group. There were 15 mice in each group. In each administration group, a physiological saline solution of the corresponding drug was intraperitoneally injected once a day, the dosage was 20 mg / Kg mouse body weight, and the injection volume was 100 μL. Mice in the model control group and the normal control group were intraperitoneally injected with the same amount of physiological saline.

[0110] After 4 weeks of continuous administration, the cognitive and memory functions of all animals were analyzed by the Morris water maze test.

[0111] Spatial Navigation Test: The Morris water maze test system consists of a water maze and an automatic video recording and analysis system. The camera is placed above the water maze and connected to a computer. The water maze is composed 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 was 0.5 cm higher than the platform, and the water temperature was maintained at 22 ± 0.5 °C. White pigment was used to dye the water milky white. To measure the learning and memory ability of mice in the water maze, a 5-day spatial navigation test was conducted. The water maze was divided into four quadrants: N, S, W, and E, and the platform was placed in a fixed quadrant. The position of the platform was fixed during the experiment. In training, every day, a mouse with its head facing the wall of the pool was gently placed into the water near the outer wall from a 1 / 2 arc in a different quadrant. The time it took for the mouse to climb onto the hidden platform was recorded, or the test was terminated when 60 seconds had elapsed. If the platform was not found within 60 seconds, the experimenter guided the mouse and made it stay for 30 seconds. During the test, the latency time until the mouse searched for the platform was recorded by the camera tracking system. After the test, each animal was taken out and gently dried with a hair dryer. Each animal was trained continuously for 5 days, 4 times a day, with a 20-minute interval between trainings.

[0112] Spatial Exploration Test: After the training on the 5th day ended, on the 6th day, the platform was removed, and the mouse was gently placed into the water from the farthest point of the platform while facing the wall of the pool. The movement trajectory of each mouse within 60 seconds was recorded by the camera, and the number of times the mouse crossed the platform, the staying time in the target quadrant, and the swimming time in the target quadrant were analyzed by software.

[0113] JPEG0007715827000002.jpg34166

[0114] After the behavioral test, the mice were anesthetized by intraperitoneal injection of 7% chloral hydrate. After establishing the cardiac perfusion connection, normal saline was rapidly flushed for 7 minutes, and the tissues were fixed for 7 minutes using 4% chloral hydrate. After the perfusion was completed, the brain tissues were carefully collected, placed in 4% perfusion fluid, and stored at room temperature for later use. To detect the expression of Aβ40 and inflammatory factors in the hippocampus and cerebral cortex, immunohistochemical methods were used: the perfused tissues were dehydrated and cleared, embedded in wax, and 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 peroxide and blocked with serum for 30 minutes. The primary antibodies (1:100) against Aβ40, IL-1β, TNF-α, GFAP, IL-6, or COX-2 were added at room temperature and incubated at 4°C overnight (15 hours). The primary antibodies were discarded, the slices were washed with PBS, and HRP-labeled goat anti-rabbit / mouse secondary antibodies were used and incubated at room temperature for 30 minutes. After washing the slices with PBS, the chromogenic reagent DAB chromogenic solution was added for color development, followed by Harris hematoxylin counterstaining, dehydration, and mounting. A fluorescence microscope was used for photography, and Image J was used for quantitative analysis of the slices.

[0115] 2. Test Results

[0116] Figure 8 shows the effects of continuous daily administration of Cap-ZnS NPs, MA-ZnS NPs, and DHLA-ZnS NPs for 4 weeks on the performance of male mice in the Morris water maze. During the training process of the place navigation test (Figure 8A), compared with the normal control group (●), from the 2nd to the 5th day of training, the latency of the mice in the model control group (■) was significantly higher than that of normal mice (P<0.05, #; P<0.01, #). Compared with the model control group, the Cap-ZnS NPs administration group (△) could significantly shorten the latency of the mice, and significant differences were observed compared with the model control group after the 3rd day (the P values on the 3rd, 4th, and 5th days were all less than 0.05, *). However, no significant effect on shortening the latency was observed in the MA-ZnS NPs (□) administration group and the DHLA-ZnS NPs (○) administration group.

[0117] Results of the space exploration test (Figs. 8B - D). Compared with the normal control group, in the model control group, the number of platform crossings (Fig. 8B) (P < 0.05, #), swimming time in the target quadrant (Fig. 8C) (#), and residence time in the target quadrant (Fig. 8D) (P < 0.05, #) were significantly decreased. Compared with the model control group, MA-ZnS NPs and DHLA-ZnS NPs could not increase the number of platform crossings in mice (Fig. 8B), and the number of platform crossings was significantly increased in the Cap-ZnS NPs administration group, but there was no statistical difference (P > 0.05).

[0118] Regarding the swimming time in the target quadrant and the residence time in the target quadrant (Figs. 8C and 8D), the Cap-ZnS NPs agent significantly increased these two values (P < 0.05, *), while neither MA-ZnS NPs nor DHLA-ZnS NPs could increase these two values.

[0119] The above data indicate that the Cap-ZnS NPs agent can significantly improve the cognitive and memory abilities of AD model mice, while MA-ZnS NPs and DHLA-ZnS NPs do not have such effects.

[0120] The effects of the 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 the same as those of the Cap-ZnS NPs administration group. For the sake of brevity, the detailed descriptions of these are omitted in this specification.

[0121] Figure 9 is a representative photograph of the results of immunohistochemistry. As shown in Figure 9, no Aβ40 plaques were observed in the hippocampus of wild-type mice, and no significant expression of inflammatory factors including IL-1β, TNF-α, and GFAP was observed. Compared with wild-type mice in the normal control group, a large number of Aβ40 plaques were observed in the hippocampus of the model control group, and the expression of IL-1β, TNF-α, and GFAP was also observed. In addition, in the hippocampus of the Cap-ZnS NPs administration group, there were a small number of Aβ40 plaques, and the expression of TNF-α, IL-1β, and GFAP was slight, being in a state close to that of the normal control group. Moreover, as a result of statistical analysis, the number of Aβ40 plaques in the hippocampus of the Cap-ZnS NPs administration group decreased by 71.8% compared with the model control group, showing a statistically significant difference (P<0.01), and the expression of TNF-α, IL-1β, and GFAP decreased by more than 70% (P<0.01). From the above, it was found that administration of Cap-ZnS NPs can significantly reduce Aβ plaques in the brains of AD model mice and greatly suppress central inflammation in AD model mice, thereby reducing neuronal damage and curing AD. The AD model mice used in these tests were 60-week-old aged mice, and the administration cycle was also as short as 4 weeks. The fact that such good effects can be obtained indicates that Cap-ZnS NPs pharmaceuticals have excellent applicability in the treatment of AD.

[0122] The R-ZnS NPs described in the present invention have the characteristics of a simple manufacturing method and good biocompatibility.

[0123] The R-ZnS NPs provided by the present invention have the following advantages:

[0124] First, the R-ZnS NPs have a significantly higher effect of inhibiting the fibrillation of Aβ than other ligand (such as 4-mercaptobutyric acid (MA) and dihydrolipoic acid (DHLA))-bound ZnS NPs. The ultra-low dose of R-ZnS-NPs at 5 ppm can completely inhibit the fibrillation of Aβ at a concentration of 20 μM.

[0125] Second, R-ZnS NPs can significantly reduce the expression of inflammatory factors (IL-1β, IL-6, TNF-α, IL-8, and hs-CRP) in LPS-treated HA cells.

[0126] Third, in the Aβ-induced cell damage model experiment, R-ZnS NPs significantly reduced the cytotoxicity caused by Aβ fibrillation.

[0127] Fourth, in the APP / PS1 double transgenic AD model mouse test, R-ZnS NPs significantly reduced Aβ plaques in the hippocampus of AD model mice and significantly reduced the levels of neuroinflammatory factors in the brains of AD model mice. In the APP / PS1 double transgenic AD model mouse test, R-ZnS NPs significantly improved the cognitive and memory behavioral impairments of model mice.

[0128] Fifth, R-ZnS NPs can cross the blood-brain barrier and enter the brains of mice.

[0129] Sixth, R-ZnS NPs have biological safety at the animal level.

[0130] Ligand-bound zinc sulfide nanoparticles with L-cysteine, D-cysteine, L-NIBC, D-NIBC, L-NAC, or D-NAC as ligands have been synthesized, characterized, and tested according to the same protocol as above, and have shown similar effects in reducing the expression of inflammatory cytokines, inhibiting Aβ fibrillation, and treating Aβ-related diseases such as AD. For the sake of brevity, their detailed descriptions are omitted in this specification.

[0131] Although the present invention has been described with reference to specific embodiments, it is understood that the embodiments are illustrative and that the scope of the present invention is not so limited. Alternative embodiments of the present invention will be apparent to those of ordinary skill in the art to which the present invention pertains. Such alternative embodiments are considered to be 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.

Claims

1. A zinc sulfide nanoparticle comprising a zinc sulfide core consisting only of zinc sulfide and a ligand bound to the zinc sulfide core, wherein the diameter of the zinc sulfide core is 0.5 to 4.0 nm, wherein the ligand is captopril, A ligand-bound zinc sulfide nanoparticle.

2. The ligand-bound zinc sulfide nanoparticle according to claim 1, wherein the diameter of the zinc sulfide core is 1.0 to 3.5 nm.

3. A method for producing the ligand-bound zinc sulfide nanoparticle (R-ZnS NPs) according to claim 1 or 2, comprising: dissolving a ligand in deionized water to obtain a ligand aqueous solution such that the concentration of the ligand in the ligand aqueous solution is 0.02 to 2.0 mol / L; adding a zinc acetate solution to the ligand aqueous solution to obtain a zinc acetate / ligand reaction mixture, wherein the concentration of the zinc acetate aqueous solution is 0.01 to 1.0 mol / L and the molar ratio of the ligand to zinc acetate is in the range of 1:1 to 10:1; adjusting the pH of the zinc acetate / ligand reaction mixture to a range of 7 to 10; dropping a sodium sulfide aqueous solution into the pH-adjusted zinc acetate / ligand reaction mixture such that the molar ratio of the added sodium sulfide to zinc acetate in the zinc acetate / ligand reaction mixture is in the range of 0.1:1 to 5:1 to obtain a sodium sulfide / zinc acetate / ligand reaction mixture; heating the sodium sulfide / zinc acetate / ligand reaction mixture to a predetermined temperature and maintaining the reaction for a predetermined time to form R-ZnS NPs, wherein the predetermined temperature is 50 to 100 °C and the predetermined time is 1 to 5 hours. A method comprising the above steps.

4. The method further comprises: purifying the R-ZnS NPs by centrifugation using an ultrafiltration tube, wherein the molecular weight cut-off of the ultrafiltration tube is 5 kDa. The method according to claim 3.

5. A ligand-bound zinc sulfide nanoparticle for treating a subject having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) caused by or associated with the fibrillation of amyloid-β (Aβ), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM), comprising a zinc sulfide core consisting only of zinc sulfide and a ligand bound to the zinc sulfide core, wherein the diameter of the zinc sulfide core is 0.5 to 4.0 nm, The ligand is captopril. Ligand-conjugated zinc sulfide nanoparticles.

6. A composition comprising the ligand-conjugated zinc sulfide nanoparticles according to claim 5 and used for the treatment of a subject having Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) caused by or related to the fibrillation of amyloid β (Aβ), retinal ganglion cell degeneration (RGCD) in glaucoma, or myositis and myopathy (MM).

7. Ligand-conjugated zinc sulfide nanoparticles for the treatment of a subject having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (TNFα), comprising a zinc sulfide core consisting only of zinc sulfide and a ligand conjugated to the zinc sulfide core, wherein the diameter of the zinc sulfide core is 0.5 to 4.0 nm, the ligand is captopril, Ligand-conjugated zinc sulfide nanoparticles.

8. A composition comprising the ligand-conjugated zinc sulfide nanoparticles according to claim 7 and used for the treatment of a subject having a state of overexpression of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), hypersensitive C-reactive protein (Hs CRP), or tumor necrosis factor-α (TNFα).

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