Nanogold loaded ns9283 targeted long-acting analgesic drug and preparation method thereof

Abstract

The present application relates to nano gold loaded NS9283 targeted long-acting analgesic drug and preparation method. NS9283 is loaded on the surface of sodium citrate coated gold nanoparticles by ligand exchange method, and then Pickering emulsion method is used to construct amphiphilic Janus AuNPs self-assembly microcapsules. The drug has the following advantages: stability is improved, long-term activity is maintained at room temperature; targeted controlled release, in vivo fluorescence imaging shows that the drug is continuously enriched at the nerve injury site for more than 7 days; long-acting analgesia: analgesic effect lasts for 28 days after local administration, which is at least 4 times higher than that of free NS9283; safety guarantee: no neurotoxicity is caused after continuous administration for 14 days, and there is no significant difference in mouse weight, motor coordination and main organ function compared with the control group. The drug provides a safe and efficient treatment scheme for chronic pain.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a long-acting analgesic drug for targeting NS9283 supported by gold nanoparticles and its preparation method. Background Technology

[0002] Pain is the third leading health problem after cardiovascular disease and cancer. Postoperative neuropathic pain (PNP) is an extremely common form of chronic pain. Even with successful surgery and wound healing, patients may still suffer from long-term pain, significantly reducing their quality of life. PNP can not only lead to loss of work and self-care abilities but also trigger anxiety and depression, placing a heavy burden on families and society. Although perioperative analgesia regimens (such as lidocaine and gabapentin) can partially relieve acute pain, their analgesic effect on chronic PNP is limited, and long-term use may cause side effects such as central nervous system depression and metabolic disorders. Therefore, existing analgesics suffer from short duration of action and significant side effects, becoming a bottleneck in the long-term pain management of PNP patients.

[0003] The α4β2 nAChR ion channel participates in pain signal transduction by regulating the transmembrane flow of sodium, potassium, and calcium ions. Nature first reported the three-dimensional structure of α4β2 nAChR in 2016, laying the foundation for the development of highly selective compounds. Natural compounds that selectively activate α4β2 nAChR extracted from plants and animals (such as thorny-smelling toxin and cytisine) and compounds designed and synthesized based on natural ligand structures, such as the thorny-smelling toxin analog ABT-594, have shown better analgesic activity in rodent neuropathic pain models. dFBr is currently the most studied highly selective natural positive allosteric modulator of α4β2 nAChR. Studies have shown that dFBr does not activate α4β2 nAChR alone, but it can enhance acetylcholine potency, regulate receptor desensitization, optimize the safety window and decouple dose dependence, amplify the analgesic effect of endogenous ACh at extremely low concentrations, significantly improve drug safety, and reduce the incidence of side effects.

[0004] NS9283 (Chemical formula: C 14H8N4O; 3-(3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)benzonitrile) is a compound designed and synthesized based on the structure of the natural positive allosteric modulator dFBr. The binding site of NS9283 is located between amino acids L256 and F316 at the α4 / α4 interface, meaning that NS9283 acts only on the highly selective α4β2nAChR containing three α4 subunits and two β2 subunits. This compound significantly enhances the response of α4β2nAChR to endogenous acetylcholine and exhibits excellent analgesic effects in a mouse model of neuropathic pain. However, NS9283 has relatively poor solubility and chemical stability (solid powder needs to be stored at -20℃, and solution needs to be stored at -80℃ protected from light), and is easily decomposed in the in vivo environment.

[0005] Nanoparticle-based targeted drug delivery technology offers a new approach to long-term pain management for patients with polycystic angina (PNP). Targeted drug delivery ensures both local drug concentration and duration of action while avoiding the impact of in vivo metabolism on the stability of NS9283. Gold nanoparticles (AuNPs) possess excellent biocompatibility, high drug loading capacity, and high chemical stability, making them a currently popular targeted delivery method. The drug molecule NS9283 can be chemically modified to bind with AuNPs and delivered to the affected area, thereby increasing local drug concentration and prolonging the duration of action. This method is simple and safe, providing a new option for PNP treatment.

[0006] Microencapsulation (including nanocapsules with a particle size of less than 100 nm) technology plays a crucial role in improving the bioavailability, stability, controlled release, and targeted delivery of active pharmaceutical ingredients, thereby enhancing efficacy and reducing side effects. By modifying the surface asymmetrically, nanoparticles can form hydrophilic and hydrophobic ligand partitions, resulting in amphiphilic Janus nanoparticles. These amphiphilic Janus nanoparticles exhibit surfactant-like properties, self-assembling into hollow microspheres (microcapsules) in a single aqueous or oil phase to reduce surface tension. Preparation methods for amphiphilic Janus nanoparticles include masking, block polymer self-assembly, and Pickering emulsion methods. Currently, new microcapsule research continues to emerge, showing a trend from single-chamber microcapsules to multi-chamber intelligent microcapsules.

[0007] Therefore, there is an urgent need for a stable and efficient formulation for delivering NS9283. This invention encapsulates NS9283 in amphiphilic Janus AuNPs microcapsules (Janus AuNPs-NS9283), constructing a novel and highly efficient targeted drug delivery system. This increases the targeted drug loading of NS9283, achieving long-term controlled drug release and overcoming the technical bottlenecks of poor stability and short analgesic duration of NS9283 injection. This enables pre-analgesia and long-term safety management of PNPs. Summary of the Invention

[0008] This invention provides a nanocomposite comprising nanoparticles and a functional molecule; the functional molecule is a positive allosteric modulator of α4β2 nicotinic acetylcholine receptors.

[0009] The functional molecule is loaded onto nanoparticles. According to an embodiment of the present invention, the functional molecule is compound NS9283.

[0010] According to an embodiment of the present invention, the nanoparticles are selected from any one, two or more of the following: gold nanoparticles, silver nanoparticles, platinum nanoparticles, etc.; preferably, the nanoparticles are gold nanoparticles.

[0011] Preparation of AuNPs: Take 200 mL of deionized water in an Erlenmeyer flask and add 2–5 mL of 0.1 mol / L sodium citrate solution. Heat on a magnetic stirrer for 80–100 °C. o C. Set the stirring speed to 300-500 rpm, add 0.5-2.0 ml of 0.1 mol / L chloroauric acid solution, and react for 1-3 h. After stopping heating, continue stirring for 4-9 h. Centrifuge at 10000-12000 rpm to obtain Au NPs protected by sodium citrate ligands, and wash with physiological saline 3-5 times.

[0012] Preparation of AuNPs-NS9283: Dissolve 3–6 mg of NS9283 in 30–50 mL of N,N-dimethylformamide (DMF) and stir at 400 rpm at room temperature using a magnetic stirrer. Then add Au NPs protected by sodium citrate ligands (at a concentration of 0.5–1.5 mg / mL) and stir for 4–8 h to complete the ligand exchange on the AuNPs surface. After the reaction solution has stood for 24 h, centrifuge at 10,000–12,000 rpm to obtain NS9283-protected Au NPs, and wash with physiological saline by centrifugation 3–5 times.

[0013] In vitro construction study of Janus AuNPs-NS9283 self-assembled microcapsule targeted delivery system

[0014] (1) A method for small-batch synthesis of amphiphilic Janus AuNPs using the Pickering emulsion method: Utilizing the strong binding affinity of gold to elements such as sulfur and phosphorus, ligand exchange can be performed on AuNPs, the precursors of chloroauric acid reduction, to obtain amphiphilic Janus gold nanoparticles with the desired structure. The inventors intend to design and synthesize amphiphilic Janus AuNPs with DL-mercaptosuccinic acid (MSA) and benzyl mercaptan (BM) ligand molecules on their surface, using a Pickering emulsion "one-step" reduction method to synthesize chloroauric acid. 45 mL of 1.0 mM BM toluene solution and 30 mL of 1.5 mM MSA aqueous solution are mixed and placed in an Erlenmeyer flask, and stirred evenly using a high-speed homogenizer at a speed of 4000 rpm to form an emulsion. Subsequently, 10 mL of 4.5 mM chloroauric acid aqueous solution is added. Two minutes later, emulsification and stirring continued, and 5 mL of freshly prepared aqueous solution containing 17.0 mg of sodium borohydride was added dropwise to the conical flask to trigger the Pickering emulsification reaction, which gradually turned dark brown as the reaction proceeded. To ensure complete reaction, stirring was continued for 2 hours. After the reaction, the Pickering emulsion was allowed to stand overnight, after which the product accumulated at the toluene-water immiscible liquid-liquid interface. The product was removed using a separatory funnel and washed three times by centrifugation with toluene and water, respectively. The sample was then freeze-dried under vacuum to obtain the final sample.

[0015] (2) Formation of amphiphilic Janus AuNPs microcapsules and the encapsulation method of NS9283: 80 mg of amphiphilic Janus AuNPs were added to 50 mL of deionized water and stirred at 400 rpm at room temperature on a magnetic stirrer to prepare an amphiphilic Janus AuNPs-stabilized Pickering emulsion. 5 mg of NS9283 was dissolved in 50 mL of N,N-dimethylformamide (DMF) solution, and then the DMF solution was added dropwise to the Pickering emulsion. The mixture was stirred continuously for 2 hours and then allowed to stand overnight. The amphiphilic Janus AuNPs-stabilized Pickering droplets encapsulated with NS9283 were separated by filtration and then dispersed in physiological saline.

[0016] (3) Construction method of Janus AuNPs-NS9283 reference targeted drug delivery system:

[0017] i. Preparation of AuNPs precursors: Take 200 mL of deionized water in an Erlenmeyer flask and add 2 mL of 0.1 mol / L sodium citrate solution. Heat to 95°C on a magnetic stirrer, set the stirring speed to 400 rpm, add 0.5 mL of 0.1 mol / L chloroauric acid solution and react for 1 hour. Turn off the heating and continue stirring for 6-8 hours. Centrifuge at 12000 rpm to obtain AuNPs protected by sodium citrate ligands, and wash 2-3 times with physiological saline.

[0018] ii. Preparation of AuNPs-NS9283: Dissolve 5 mg of NS9283 in 50 mL of DMF solution. Set the stirring speed to 400 rpm at room temperature on a magnetic stirrer. Add AuNPs protected by sodium citrate ligands after centrifugation. Stir for ligand exchange for 4 hours and let stand for 24 hours. Centrifuge at 12000 rpm to obtain AuNPs protected by NS9283 ligands. Wash with physiological saline 2-3 times.

[0019] According to an embodiment of the present invention, the particle size of the nanoparticles is 10nm-20nm, for example 10nm, 10.5nm, 11nm, 11.5nm, 12nm, 12.5nm, 13nm, 13.5nm, 14nm, 14.5nm, 14.6nm, 15nm, 15.5nm, 16nm, 16.5nm, 17nm, 17.5nm, 18nm, 18.5nm, 19nm, 19.5nm, and 20nm.

[0020] According to an embodiment of the present invention, the ligand is selected from any one, two or more of the following: sodium citrate ligand, thioglycolic acid ligand, dodecyl mercaptan ligand; preferably, the ligand is sodium citrate ligand.

[0021] According to an embodiment of the present invention, the nanoparticles are selected from any one, two or more of the following: gold nanoparticles, silver nanoparticles, platinum nanoparticles, etc.; preferably, the nanoparticles are gold nanoparticles.

[0022] According to an embodiment of the present invention, the ligand-protected nanoparticles are nanoparticles protected by sodium citrate ligands, preferably gold nanoparticles protected by sodium citrate ligands.

[0023] According to an embodiment of the present invention, the preparation process of sodium citrate ligand-protected gold nanoparticles is as follows: sodium citrate solution is reacted with chloroauric acid solution to obtain sodium citrate ligand-protected gold nanoparticles.

[0024] According to an embodiment of the present invention, the concentration of the sodium citrate solution is 0.01 mol / L to 0.5 mol / L, for example 0.01 mol / L, 0.03 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, and 0.5 mol / L. According to an embodiment of the present invention, the concentration of the chloroauric acid solution is 0.01 mol / L to 0.5 mol / L, for example, 0.01 mol / L, 0.03 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, and 0.5 mol / L. According to an embodiment of the present invention, the molar ratio of sodium citrate to chloroauric acid is 1-10:1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1. According to an embodiment of the present invention, the reaction temperature is 80-100°C. According to an embodiment of the present invention, the reaction time is 1-15 hours, for example 1-3 hours, 4-9 hours, 2 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours. According to an embodiment of the present invention, after the reaction is completed, optionally, centrifugation, washing, or other steps are performed.

[0025] According to embodiments of the present invention, the concentration of ligand-protected nanoparticles is 0.1 mg / mL to 3 mg / mL, preferably 0.5 mg / mL to 1.5 mg / mL. According to embodiments of the present invention, the concentration of the functional molecule is 0.01 mg / mL to 0.5 mg / mL, for example 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, and 0.5 mg / mL.

[0026] According to an embodiment of the present invention, mixing is carried out by stirring; the stirring time is preferably 4 to 8 hours.

[0027] According to an embodiment of the present invention, after mixing, the mixture is allowed to stand and then centrifuged to obtain the nanocomposite.

[0028] The present invention also provides nanocomposites prepared according to the above preparation method.

[0029] The present invention also provides the use of the above-described nanocomposite in the preparation of medicaments for treating and / or preventing pain.

[0030] According to an embodiment of the present invention, the pain is neuropathic pain, such as neuropathic pain or postoperative neuralgia.

[0031] The present invention also provides a method for treating and / or preventing pain, the method comprising administering a therapeutically effective amount of the above-described nanocomposite to a pain patient.

[0032] According to an embodiment of the present invention, the pain is neuropathic pain, such as neuropathic pain or postoperative neuralgia.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In the nanocomposite provided by this invention, the drug molecule NS9283 is combined with AuNPs and delivered to the affected area, which can increase the local drug concentration and prolong the duration of action. The method is simple and safe, providing a new option for the treatment of neuropathic pain.

[0035] Compared with existing technologies, the nanocomposite provided by this invention has the following advantages:

[0036] (1) Long-acting controlled release: Janus AuNPs-NS9283 is used to extend the duration of drug action and reduce the frequency of administration.

[0037] (2) High targeting: Using amphiphilic Janus AuNPs microcapsules as carriers, NS9283 can be precisely delivered to the site of nerve injury, increasing the local drug concentration and reducing the impact on non-target tissues;

[0038] (3) High stability: The high chemical stability of Janus AuNPs-NS9283 effectively protects NS9283 from degradation by the complex environment in the body, thus improving the stability of the drug;

[0039] (4) Low toxicity: AuNPs have good biocompatibility, and experiments have shown that no obvious neurotoxicity or systemic toxicity has been observed. Attached Figure Description

[0040] Figure 1 Pharmacological characteristics of NS9283. Figure 1 In this context, A represents the stronger positive regulatory effect of NS9283 on (α4)3(β2)2nAChR than that of (α4)2(β2)3nAChR. Figure 1 In the figure, B represents the AI ​​model's prediction that α4L256 is the major binding site for NS9283; Figure 1C in the cluster represents the most common binding posture of NS9283 at the α4α4δ binding site; Figure 1 D in the cluster represents the most common binding posture of NS9283 at the α4α4ε binding site.

[0041] Figure 2 NS9283 binding site map. The regulatory effects of mutation sites α4L256 and α4F316 on NS9283 are significantly weakened and... EC 50 Move to the right.

[0042] Figure 3 NS9283 significantly alleviated mechanical pain in CINP model mice. Figure 3 In the figure, A represents the change in pain threshold of mice in each group at 30 min, 60 min, 90 min, 120 min, and 180 min after drug administration; Figure 3 B in the figure represents the change in mechanical pain threshold on days 1, 3, 5, 7, and 14 after drug administration; Figure 3 In the figure, C represents different time points after drug administration, and the effect of each intervention on the pain threshold.

[0043] Figure 4 NS9283 significantly alleviated mechanical pain in SNI model mice. Figure 4 In the figure, A represents the change in pain threshold of mice in each group at 30 min, 60 min, 90 min, 120 min, and 180 min after drug administration; Figure 4 B in the figure represents the change in mechanical pain threshold on days 1, 3, 5, 7, and 14 after drug administration; Figure 4 In the figure, C represents different time points after drug administration, and the effect of each intervention on the pain threshold.

[0044] Figure 5 The ligand substitution loading of NS9283 onto surface-modified citrate AuNPs was performed using the sodium citrate reduction method. Morphological characterization of the sodium citrate gold nanoparticles: Figure 5 In the image, 'a' represents a transmission electron microscope image, and the inset is a particle size distribution map. Figure 5 In this context, 'b' represents HRTEM; morphology characterization (CD) of NS9283 gold nanoparticles: Figure 5 In this context, 'c' represents a transmission electron microscope image; Figure 5 In this context, d stands for HRTEM.

[0045] Figure 6 XPS peak fitting analysis before and after ligand replacement. Figure 6 In the diagram, 'a' represents the ligand exchange from sodium citrate to NS9283 on the gold nanoparticles. Figure 6 In the figure, b represents the XPS C1s peak of sodium citrate ligand gold nanoparticles. Figure 6In the figure, c represents the XPS Au 4f peak of sodium citrate ligand gold nanoparticles; Figure 6 In the figure, d represents the XPS O1s peak of the sodium citrate ligand gold nanoparticles; Figure 6 In the figure, e represents the XPS N 1s peak of the NS9283 ligand gold nanoparticles. Figure 6 f in the figure represents the XPS C 1s peak of NS9283 ligand gold nanoparticles; Figure 6 In the figure, g represents the XPS Au 4f peak of NS9283 ligand gold nanoparticles; Figure 6 h in the figure represents the XPS O 1s peak of NS9283 ligand gold nanoparticles.

[0046] Figure 7 Comparison of the effects of AuNPs-NS9283 in preventing pain in a mouse SNI model. Figure 7 In the diagram, 'a' represents a schematic diagram of local drug delivery. Figure 7 In this context, b represents the change in the von-Frey mechanical pain threshold.

[0047] Figure 8 No significant neurotoxicity was observed in local nerves after local administration, intraperitoneal injection, or oral administration of AuNPs-NS9283, NS9283, and AuNPs.

[0048] Figure 9 Figure showing changes in mouse body weight and diet after 14 days of continuous intraperitoneal injection. Figure 9 In this context, A represents the change in the average weight of the mice before and after the change. Figure 9 In this context, B represents the average daily water intake of each mouse. Figure 9 In the figure, C represents the average daily food intake per mouse. Data are expressed as MEAN±SEM. Compared with the control group, mice in the Nicotine group had reduced food intake, increased water intake, and a slight decrease in body weight. ns indicates no significant difference, n=4.

[0049] Figure 10 .Graph of blood biochemistry results. Figure 10 The AF values ​​represent the changes in glucose, total cholesterol, triglycerides, calcium, phosphorus, and magnesium at 6, 12, and 24 hours after intraperitoneal injection of NS9283. Figure 10 In the GL group, the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase isoenzyme, lactate dehydrogenase (LDH), creatinine, and uric acid were observed on days 3, 5, and 7 after intraperitoneal injection in the Control group, Nicotine group, and NS9283 group.

[0050] Figure 11 Open field test diagram 2 hours after drug administration. Note: Figure 11 AB in the figure represents the open field test trajectory diagram and heat map of the three groups of mice 2 hours after intraperitoneal administration; Figure 11In this context, C represents the total distance the mouse traveled in 10 minutes during the open field test. Figure 11 In this context, D represents the average speed of the mouse's movement. Figure 11 In this context, E represents the time the mouse remains stationary in the open field. Figure 11 F in the table represents the number of times a mouse crossed the central region. Data are expressed as MEAN±SEM. Compared with the Control group, mice in the Nicotine group had a decreased total mileage and number of times they crossed the central region, but an increased resting time (*p<0.05, n=6). Compared with the Control group, mice in the NS9283 group showed no significant differences in total mileage, average speed, number of times they entered the central region, and resting time (ns indicates no significant difference, n=6).

[0051] Figure 12 Open field test diagram after 7 days of daily intraperitoneal administration. Figure 12 AB in the figure represents the trajectory diagram and heat map of the open field test after a single test 7 days in the three groups of mice; Figure 12 In this context, C represents the total distance the mouse traveled in 10 minutes during the open field test. Figure 12 In this context, D represents the average speed of the mouse's movement. Figure 12 In this context, E represents the time the mouse remains stationary in the open field. Figure 12 In the figure, F represents the number of times the mouse crossed the central region. Compared with the control group, there were no significant differences in total distance traveled, average speed of movement, number of times the mouse entered the central region, and resting time between the Nicotine group and the NS9283 group. ns indicates no significant difference, n=6.

[0052] Figure 13 Conditioned position preference experiment diagrams for the Nicotine and NS9283 groups. Note: In the conditioned position preference experiment, the left side of the box represents the saline tank, the right side represents the medication tank, and the time and distance in the middle box are not included in the statistics. Mice in the Nicotine group showed a significant position preference for the medication tank, while mice in the NS9283 group showed comparable preferences for both boxes. Figure 13 In this context, A represents the time the mouse spends in the medication box. Figure 13 In this context, B represents the number of times the mouse traversed between the medication tank and the saline tank. Figure 13 In this context, C represents the CPP value, which is the difference between the time the mouse spends in the drug-treated box and the time spent in the saline box. Figure 13 In this context, D represents the percentage of time the mouse spends in the medication box, i.e., the preference index. Figure 13 E and Figure 13In the figure, F represents the trajectory and heatmap of Nicotine group mice 15 minutes after training in the CPP test chamber, with the saline chamber on the left and the drug-treated chamber on the right. (G~H) represent the trajectory and heatmap of NS9283 group mice in the conditional position preference test chamber, with the saline chamber on the left and the drug-treated chamber on the right. Data are expressed as MEAN±SEM. Nicotine group mice had significantly higher CPP values, longer time in the drug-treated chamber, and higher preference index than NS9283 group mice. NS9283 group mice did not show a significant preference for either chamber during the experiment, with a mean CPP value closer to 0 and a preference index closer to 50%. *p<0.05, **p<0.01, ***p<0.001, n=10.

[0053] Figure 14 Forced swimming test after 7 days of daily intraperitoneal administration. Note: Figure 14 In this context, A represents the total resting time of the mice within 4 minutes after forced swimming; Figure 14 B in the table represents the total struggling time of mice within 4 minutes after forced swimming. Data are expressed as MEAN±SEM. Compared with the Control group, mice in the Nicotine group had increased struggling time and decreased resting time (*p<0.05). Mice in the NS9283 group did not show significant differences in these key behavioral indicators compared with the Control group (ns indicates no statistical difference, n=6).

[0054] Figure 15 Rotor fatigue test images 2 hours after drug administration and after 7 days of daily administration. Note: Figure 15 In this context, A represents the total crawling time of the mice 2 hours after drug administration; Figure 15 B in the table represents the total crawling time of mice after 7 days of daily administration. Data are expressed as MEAN±SEM. Compared with the Control group, mice in the Nicotine group and the NS9283 group did not show significant differences in crawling time and fatigue time. ns indicates no significant difference, n=6.

[0055] Figure 16Pathological changes in major organs after long-term drug exposure. Note: HE staining of liver, kidney, heart, and brain tissues; overview of tissue and organ sections; and observation under a 200x microscope. In liver tissue, mild edema of hepatocytes was observed under the microscope 3 days after administration in the Nicotine group, with cell swelling and loose, pale cytoplasm. Severe edema of hepatocytes was widely observed under the microscope 7 days after administration, with cell swelling and loose, pale cytoplasm. In contrast, only a small amount of mild edema was observed under the microscope in the Control and NS9283 groups. In kidney tissue, no significant edema was observed in the renal tubules in all groups, and the morphology of the renal tubular epithelial cells was normal, with no obvious damage. In heart tissue, cardiomyocytes were spindle-shaped, with the nucleus located on one side of the cell, and the cells were neatly arranged and aligned, with no obvious abnormalities. In spleen tissue, there was no significant difference in the number of marginal lymphocytes and the number of erythrocytes in the red pulp and splenic sinusoids among all groups. Black arrows indicate cell edema.

[0056] Figure 17 LFB staining images of mouse spinal cord and brain. Note: Spinal cord sections were taken along the longitudinal axis of the lumbar enlargement, and brain tissue was observed at the hippocampal-thalamic level. Each group was scanned for an overview or observed and photographed at 200x magnification. Microscopic examination of the brain and spinal cord of all three groups of mice revealed abundant longitudinal nerve fibers in the spinal cord white matter. The myelin sheath was blue, evenly distributed, densely arranged, and of normal morphology; no obvious demyelination was observed.

[0057] Figure 18 Open field test diagram for acute toxicity testing of NS9283. Note: Figure 18 A and Figure 18 B in the figure shows the open field test trajectory and heat map of the three groups of mice 2 hours and 24 hours after intraperitoneal administration; Figure 18 In this context, C represents the total distance the mouse traveled in 10 minutes during the open field test. Figure 18 In this context, D represents the number of times the mouse traversed the central region; Figure 18 In this context, E represents the average speed of the mouse's movement; Figure 18 F represents the number of times mice entered the central zone. Compared with the control group, 2 hours after NS9283 administration, mice in the 10 mg / kg group showed significantly reduced distance traveled in the open field and the number of times they entered the central zone, while the average speed showed no significant difference. The 5 mg / kg group showed no significant difference in distance traveled in the open field, average speed, or number of times they entered the central zone compared to the control group. 24 hours after administration, compared with the control group, the 5 mg / kg and 10 mg / kg groups showed no significant difference in distance traveled and average speed, but relatively reduced numbers of times they entered and crossed the central zone. *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no statistical difference, n = 6.

[0058] Figure 19Pathological staining image of NS9283 acute toxicity test. Note: Figure 19 A in the image represents LFB staining of spinal cord and brain tissue. Numerous longitudinal nerve fibers are visible in the spinal cord white matter and brain medulla. The myelin sheath is blue, evenly distributed, densely arranged, and has a normal morphology. No obvious demyelination is observed. Figure 19 B in the diagram represents HE staining of major organs. No obvious necrosis was observed in hepatocytes in the 5 mg / kg and 10 mg / kg groups, but severe edema was observed in a small number of hepatocytes. Cardiac cells were neatly arranged and aligned. There were no significant differences in the number of lymphocytes at the spleen margin and the number of erythrocytes in the red pulp splenic sinusoids. Black arrows represent cellular edema.

[0059] Figure 20 . Janus AuNPs microcapsule encapsulation method for NS9283. Figure 20 In this context, A represents the structure of the amphipathic Janus AuNPs; Figure 20 B in the diagram is a schematic diagram of the self-assembly method for encapsulating NS9283 drug.

[0060] Figure 21 A research flowchart illustrating the approach to optimizing the treatment of PNPs caused by iatrogenic neurological injury using targeted controlled release of Janus AuNPs-NS9283. Detailed Implementation

[0061] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0062] Example 1: NS9283 selectively and positively allosterically regulates (α4)3(β2)2nAChR, with α4L256 and α4F316 being its binding sites.

[0063] (1) Using the dual-voltage electrode patch-clamp technique, the pharmacological properties of NS9283 in positive allosteric regulation of α4β2 nAChR were confirmed.

[0064] Injected at ratios of 10:1 and 1:4 respectively. CHRNA4 and CHRNB2 To obtain single-type receptors expressing (α4)3(β2)2nAChR or (α4)2(β2)3nAChR in oocytes, respectively. After the addition of NS9283, the peak response heights were significantly increased in both (α4)3(β2)2nAChR and (α4)2(β2)3nAChR [(α4)3(β2)2: 2.04 ± 0.64 vs 0.88 ± 0.13 and (α4)2(β2)3: 2.61 ± 0.42 vs 0.96 ± 0.03] P <0.05), no ACh concentration-response curve was observed. EC50 Changes ( Figure 1 (A in the middle).

[0065] (2) The binding mode of NS9283 and α4β2 nAChR was successfully predicted by computer simulation.

[0066] The binding mode of NS9283 and α4β2 nAChR was predicted using the open-source AI model GNINA 1.0.3. The binding energy fraction of the original pocket and NS9283 was -9.98 ± 1.96. After mutating α4L256 to α4A256, the binding energy fraction changed to -8.26 ± 1.89, indicating a decrease in affinity. The binding modes of NS9283, the original pocket, and the mutated pocket were all generated using GNINA 1.0.3, which uses the Monte Carlo Metropolis algorithm for pose sampling and a convolutional neural network for scoring. After obtaining the initial structure, energy minimization was performed using the MMFF94 force field in Openbabel to generate the lowest energy conformation. Figure 1 In addition, AI calculations clarified the role of NS9283 in α4α4δ (B). Figure 1 C) and α4α4ε ( Figure 1 The most common binding postures of the D) binding sites are clustered.

[0067] (3) The binding site of NS9283 was identified by applying cDNA point mutation and dual-voltage electrode patch-clamp techniques.

[0068] Furthermore, using site-directed mutagenesis, cDNA cloning and in vitro transcription techniques, and two-electrode voltage-clamp electrophysiological recording, amino acid substitutions were performed on the transmembrane binding domains α4L256 and α4F316 of the α4 subunit. Compared with wild-type (α4)3(β2)2 and (α4)2(β2)3nAChR, the amino acid mutant receptors α4L256F and α4F316L significantly reduced the positive regulatory effect of NS206 on ACh-induced current [(α4)3(β2)2: 6.91 ± 0.57 vs 6.23 ± 4.35 and 0.84 ± 1.18; (α4)2(β2)3: 4.14 ± 0.38 vs 2.37 ± 0.1 and 0.94 ± 0.14], and preliminary observations were made on the effects of NS9283 on ACh-induced current. EC 50 The activity of NS9283 shifts to the right in cases of nAChR mutations. Figure 2 These results indicate that α4L256 and α4F316 of α4β2 nAChR are binding sites for NS9283.

[0069] Example 2: NS9283 has a significant analgesic effect on neuropathic pain.

[0070] (1) NS9283 has a significant analgesic effect on chemotherapy-induced neuropathic pain (CINP) in C57BL / 6 mice.

[0071] A wild-type C57BL / 6 mouse model of CINP was established by intraperitoneal injection of 3 mg / kg oxaliplatin (3 times / week for 3 consecutive weeks). Mice were also intraperitoneally injected with 0.6 mg / kg NS9283, 1 mg / kg NS9283, 3 mg / kg NS9283, 0.5 mg / kg nicotine, and saline, respectively. The mechanical pain threshold was measured using von-Frey fibers before administration (baseline), on the day of administration (30 min, 60 min, 90 min, 120 min, 180 min), and at 1, 3, 5, 7, and 14 days after administration.

[0072] The results showed that there was no significant difference in analgesic effect among 0.6 mg / kg, 1 mg / kg, and 3 mg / kg NS9283, but all were significantly better than the 0.5 mg / kg nicotine group (0.5 mg / kg Nicotine group) and the saline group (Control group), and the pain threshold was close to that of unmodeled healthy mice (Naive group). Figure 3 A and Figure 3 (B in the text). Furthermore, at all time points after administration (30 min, 60 min, 90 min, 120 min, 180 min, 3, 5, 7, and 14 days), the analgesic effect of the NS9283 group was significantly better than that of the 0.5 mg / kg Nicotine group and the Control group, and the pain threshold was close to that of the Naive group. Figure 3 (C in the middle).

[0073] (2) NS9283 has a significant analgesic effect on the SNI model in C57BL / 6 mice.

[0074] To further verify the analgesic effect of small molecule NS9283 on neuropathic pain, a spontaneous intraperitoneal (SNI) model was constructed by severing and ligating the common peroneal nerve. Mice were intraperitoneally injected with 0.6 mg / kg NS9283, 1 mg / kg NS9283, 3 mg / kg NS9283, 0.5 mg / kg nicotine, and saline, respectively. The mechanical pain threshold of each group of mice was measured using von-Frey fibers before administration (baseline), on the day of administration (30 min, 60 min, 90 min, 120 min, 180 min), and at 1, 3, 5, 7, and 14 days after administration.

[0075] The results were consistent with the CINP model. There was no significant difference in analgesic effect among 0.6 mg / kg, 1 mg / kg, and 3 mg / kg NS9283, but all were significantly better than the 0.5 mg / kg Nicotine group and the Control group, and the pain threshold was close to that of unmodeled healthy mice (Naive group). Figure 4 A and Figure 4 (B in the text). Furthermore, at all time points after administration (30 min, 60 min, 90 min, 120 min, 180 min, 3, 5, 7, and 14 days), the analgesic effect of the NS9283 group was significantly better than that of the 0.5 mg / kg Nicotine group and the Control group, and the pain threshold was close to that of the Naive group (…). Figure 4 (C in the middle).

[0076] Example 3: Preparation and Characterization of AuNPs-NS9283

[0077] 1. Preparation of AuNPs:

[0078] Take 200 mL of deionized water into an Erlenmeyer flask and add 2 mL of 0.1 mol / L sodium citrate solution. Heat on a magnetic stirrer at 95°C. o C. Set the stirring speed to 400 rpm, add 0.5 ml of 0.1 mol / L chloroauric acid solution and react for 1 h. After stopping heating, continue stirring for 8 h. Centrifuge at 12000 rpm to obtain Au NPs protected by sodium citrate ligands, and wash three times with physiological saline.

[0079] 2. Preparation of AuNPs-NS9283

[0080] Dissolve 5 mg of NS9283 in 50 mL of N,N-dimethylformamide (DMF) and stir at 400 rpm at room temperature using a magnetic stirrer. Then add Au NPs protected by sodium citrate ligands (4.3 mg / mL). Stir for 4 h to complete the ligand exchange on the Au NPs surface. After the reaction solution has stood for 24 h, centrifuge at 12000 rpm to obtain NS9283-protected Au NPs, and wash with physiological saline 3–5 times.

[0081] 3. Characterization of AuNPs-NS9283

[0082] Figure 5 Figure 'a' shows a transmission electron microscope image and particle size distribution of precursor gold nanoparticles reduced by sodium citrate, with an average particle size of 12.6 nm. Figure 5In the high-resolution transmission electron microscope (HRTEM) image shown in b, diffraction fringes can be seen on the particles, and the lattice spacing measured is 0.235 nm, corresponding to the gold (111) crystal plane. Figure 5 The transmission electron microscope image shown in c reveals that the AuNPs-NS9283 nanoparticles also exhibit a spherical morphology with uniform size and an average particle size of 14.6 nm. The slight increase in particle size is due to ligand exchange between the surface and NS9283. Figure 5 HRTEM in d further revealed the crystal properties of AuNPs-NS9283, in which the lattice fringes were determined to be 0.235 nm, corresponding to the gold (111) crystal plane.

[0083] Figure 6 The 'a' in the diagram represents a schematic representation of the ligand exchange process. XPS peak fitting analysis was performed to examine the ligand composition on the surface of the gold nanoparticles before and after the ligand exchange reaction. Gold, carbon, and oxygen were detected in both AuNPs-sodium citrate and AuNPs-NS9283. The presence of 3.96% nitrogen, unique to AuNPs-NS9283, indicates that NS9283 successfully replaced the sodium citrate ligand, as sodium citrate does not contain nitrogen.

[0084] For AuNPs-sodium citrate Figure 6 The b in the figure shows that the C1s orbital peaks are located at 284.8 eV, 286.6 eV, and 288.5 eV, which correspond to the C-C, CO, and O=CO bonds in sodium citrate, respectively. Figure 6 c in the figure shows the fitting of the Au4f orbital peaks, with two peaks appearing at 83.9 eV and 87.6 eV, respectively, corresponding to the 4f 7 / 2 and 4f 5 / 2 energy levels of metallic gold. Figure 6 The 'd' in the figure shows the fitted O1s signal, where the peak at 532.1 eV is attributed to the OC bond in sodium citrate, while the peak at 535.4 eV corresponds to adsorbed water. As for AuNPs-NS9283, Figure 6 The 'e' in the figure shows the fitted N1s orbital peak, where the peak at 399.8 eV is attributed to the NC bond in NS9283. Figure 6 f in the figure shows the analyzed C1s signal, which is fitted with three peaks at 284.8 eV, 286.6 eV and 288.5 eV, respectively, which are attributed to the CC / CN, CO and O=CO bonds in NS9283. Figure 6 The g in the figure shows the fitted Au4f peaks, located at 83.9 eV and 87.6 eV, respectively, corresponding to the 4f 7 / 2 and 4f 5 / 2 energy levels of metallic gold. Figure 6The 'h' in the figure shows the fitting of the analytical O1s signal, where the peak at 532.1 eV corresponds to the peak of the OC bond in NS9283.

[0085] Example 4: AuNPs-NS9283 targeted drug delivery provides prolonged analgesia without producing significant neurotoxicity.

[0086] To verify the preventive, analgesic, and long-term analgesic effects of AuNPs-NS9283 on neuropathic pain, a spontaneous neuropathic pain (SNI) model was established by severing the common peroneal nerve. Simultaneously, AuNPs-NS9283 was administered locally, NS9283 was administered locally, NS9283 was administered intraperitoneally, and AuNPs were administered locally (the NS9283 dosage was the same in all groups). The mechanical pain threshold of mice in each group was measured using von-Frey fibers before administration (baseline) and at 1, 3, 5, 7, 14, and 28 days after administration. The results indicated that local administration of AuNPs-NS9283 had a significantly better analgesic effect than local administration or intraperitoneal injection of NS9283, and the duration of the analgesic effect was also significantly prolonged. Figure 7 ).

[0087] Furthermore, when HE staining was applied to the nerve sites, no significant nerve damage was observed in any of the groups. Figure 8 These preliminary results suggest the importance of AuNPs-NS9283 as a potential preventative treatment strategy in the management of neuropathic pain.

[0088] Example 5: Compared to nicotine, a common agonist of nAChR, Janus AuNPs-NS9283 exhibits good safety.

[0089] Amphiphilic Janus nanoparticles exhibit surfactant-like properties, self-assembling into hollow microspheres (microcapsules) in a single aqueous or oil phase to reduce surface tension. Preparation methods for amphiphilic Janus nanoparticles include masking, block polymer self-assembly, and Pickering emulsion methods. The smaller the nanoparticle size, the more difficult it is to modify the surface asymmetry. In previous research, the inventors innovatively synthesized a series of amphiphilic Janus AuNPs (with particle sizes as low as 2 nm) using a liquid / liquid interface ligand exchange method. These Janus AuNPs can be self-assembled into microcapsules by encapsulating the oil phase using the Pickering emulsion method. These microcapsules can be used for encapsulating the hydrophobic targeted drug NS9283. The self-organization rules and mechanisms of Janus AuNPs under different environmental conditions were analyzed using the Monte Carlo method. The method for encapsulating NS9283 with Janus AuNPs microcapsules is as follows: Figure 20As shown, the research approach for optimizing the treatment of PNP caused by iatrogenic neurological injury with targeted controlled release of Janus AuNPs-NS9283 is as follows: Figure 21 As shown.

[0090] 1. In vitro construction of a Janus AuNPs-NS9283 self-assembled microcapsule targeted delivery system

[0091] (1) A method for small-batch synthesis of amphiphilic Janus AuNPs using the Pickering emulsion method: Taking advantage of the strong binding affinity of gold to elements such as sulfur and phosphorus, ligand exchange can be performed on AuNPs, the precursors of chloroauric acid, to obtain amphiphilic Janus gold nanoparticles with the desired structure. The inventors intend to design and synthesize an amphiphilic Janus AuNP with DL-mercaptosuccinic acid (MSA) and benzyl mercaptan (BM) ligand molecules on its surface, using the Pickering emulsion "one-step" reduction of chloroauric acid.

[0092] 45 mL of a 1.0 mM BM toluene solution and 30 mL of a 1.5 mM MSA aqueous solution were mixed in an Erlenmeyer flask and homogenized using a high-speed homogenizer at 4000 rpm to form an emulsion. Then, 10 mL of a 4.5 mM chloroauric acid aqueous solution was added. After 2 minutes, emulsification and stirring continued, and 5 mL of freshly prepared aqueous solution containing 17.0 mg of sodium borohydride was added dropwise to the Erlenmeyer flask to trigger the Pickering emulsification reaction, which gradually turned dark brown as the reaction proceeded. Stirring was continued for 2 hours to ensure complete reaction. After the reaction, the Pickering emulsion was allowed to stand overnight, after which the product accumulated at the toluene-water immiscible liquid-liquid interface. The product was removed using a separatory funnel and washed three times by centrifugation with toluene and water, respectively. The sample was then freeze-dried under vacuum to obtain the final sample.

[0093] (2) Formation of amphiphilic Janus AuNPs microcapsules and the encapsulation method of NS9283: 80 mg of amphiphilic Janus AuNPs were added to 50 mL of deionized water and stirred at 400 rpm at room temperature on a magnetic stirrer to prepare an amphiphilic Janus AuNPs-stabilized Pickering emulsion. 5 mg of NS9283 was dissolved in 50 mL of N,N-dimethylformamide (DMF) solution, and then the DMF solution was added dropwise to the Pickering emulsion. The mixture was stirred continuously for 2 hours and then allowed to stand overnight. The amphiphilic Janus AuNPs-stabilized Pickering droplets encapsulated with NS9283 were separated by filtration and then dispersed in physiological saline.

[0094] (3) Construction method of Janus AuNPs-NS9283 reference targeted drug delivery system:

[0095] i. Preparation of AuNPs precursors: Take 200 mL of deionized water in an Erlenmeyer flask and add 2 mL of 0.1 mol / L sodium citrate solution. Heat to 95°C on a magnetic stirrer, set the stirring speed to 400 rpm, add 0.5 mL of 0.1 mol / L chloroauric acid solution and react for 1 hour. Turn off the heating and continue stirring for 6-8 hours. Centrifuge at 12000 rpm to obtain AuNPs protected by sodium citrate ligands, and wash 2-3 times with physiological saline.

[0096] ii. Preparation of AuNPs-NS9283: Dissolve 5 mg of NS9283 in 50 mL of DMF solution. Set the stirring speed to 400 rpm at room temperature on a magnetic stirrer. Add AuNPs protected by sodium citrate ligands after centrifugation. Stir for ligand exchange for 4 hours and let stand for 24 hours. Centrifuge at 12000 rpm to obtain AuNPs protected by NS9283 ligands. Wash with physiological saline 2-3 times.

[0097] 2. Identification methods for Janus AuNPs

[0098] NOESY (Nuclear Overhauser Effect Spectroscopy) is an important two-dimensional experimental technique in nuclear magnetic resonance (NMR). NOESY utilizes the distance-dependent NOE (Nuclear Overhauser Effect) between proton spins to detect phase separation. The points on the NOESY spectrum are mainly arranged along the diagonal, and there is no signal at the intersection of the proton chemical shifts of BM and MSA molecules. This indicates that the protons of BM and MSA molecules are far apart, exhibiting a separated state. Therefore, it can be deduced that BM and MSA molecules exist in a Janus distribution on the surface of AuNPs.

[0099] Furthermore, due to the amphiphilic nature of Janus AuNPs, if the characteristics of Janus AuNPs are reassembled to form a thin film, different wettability will inevitably be exhibited on both sides of the film. In the experiment, 1.0 mg of sample was dispersed in 10 mL of DMF solution, and then 5 mL of solution was injected into the surface of ultrapure water in a Langmuir tank. After the sample was spread out on the water surface, its surface pressure-surface area was measured. As the surface film was compressed, the film pressure gradually increased, exhibiting characteristics of a gaseous film (G), a liquid film (L), and a solid film (S), respectively. Under a surface pressure of 10 mN / m, the reassembled film sample was collected by lifting a quartz glass slide upward (A surface, oleophilic side facing up) or downward (B surface, hydrophilic side facing up). The obtained Janus AuNPs film showed hydrophilicity on one side and oleophilicity on the other side, proving the successful construction of Janus AuNPs-NS9283 of the present invention.

[0100] In the nicotine group (n = 20) mice, the following results were observed: 4 mice died after continuous nicotine administration. No obvious external injury or infection was observed at the time of death. The deaths occurred on days 3, 5, 6 and 10, respectively, with an overall mortality rate of 20%. In contrast, no deaths were observed in the Janus AuNPs-NS9283 group (NS9283) mice during the 14-day observation period.

[0101] Each group consisted of 20 mice, with 5 mice per cage. Food intake was recorded (n = 4), and body weight was recorded for each mouse before and after treatment (n = 20). Compared to the Control group, we observed a decrease in food intake, an increase in water intake, and a slight decrease in body weight in the Nicotine group; these differences were not statistically significant. Figure 9 (P>0.05). Compared with the Control group, the NS9283 group mice showed no trend in food intake and body weight, and the difference was not statistically significant (P>0.05).

[0102] We performed biochemical tests on plasma from three groups of mice, divided into short-term and long-term observation groups. The short-term observation group was further divided into 6-hour, 12-hour, and 24-hour observation groups (n=5), while the long-term observation group was divided into 3-day, 5-day, and 7-day observation groups (n=5). During the short-term observation, we mainly focused on changes in glucose metabolism, lipid metabolism, and electrolytes, specifically measuring plasma levels of glucose (GLU), triglycerides (TG), total cholesterol (TC), calcium (Ca), magnesium (Mg), and phosphorus (P). The results showed no significant changes in metabolic indicators among the groups, and no significant differences in metabolism were observed. Figure 10 , P >0.05), indicating that the metabolic status of mice in each group did not change significantly during the acute phase.

[0103] In the long-term observation group, in addition to detecting the aforementioned metabolic-related indicators, we further focused on changes in liver function, kidney function, cardiac function, and inflammatory markers. Liver function was assessed by detecting indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), and gamma-glutamyl transferase (γ-GT); kidney function was reflected by serum uric acid (UA) and creatinine (CREA) levels; and cardiac function was assessed by indicators such as creatine kinase isoenzyme (CKMB) and lactate dehydrogenase (LDH). The results showed that no significant changes were observed in these key biochemical indicators in the three groups of mice, further demonstrating that no significant abnormalities occurred in liver, kidney, and cardiac function in the three groups of mice during the subacute observation period.

[0104] In an open field test 2 hours after intraperitoneal injection of the drug, mice in the Nicotine group exhibited significant behavioral changes. Compared with the control group, mice in the Nicotine group showed a decrease in total traverse distance and number of crossings of the central zone, and an increase in resting time, with statistically significant differences. Figure 11 (P<0.05), indicating that intraperitoneal injection of nicotine resulted in mice exhibiting significantly increased respiration and reduced spontaneous activity. However, the NS9283 group showed no significant differences compared to the control group in total traverse distance, traverse speed, number of times entering the central zone, and resting time, suggesting that NS9283 does not produce nicotine-like behavioral changes.

[0105] Mice were administered the drug intraperitoneally daily for 7 days. An open field test was performed 24 hours after the last administration to rule out short-term effects. Results showed no significant differences between the Nicotine and NS9283 groups in total traverse distance, average traverse speed, number of central zone entries, and resting time compared to the Control group. Figure 12 (P>0.05), indicating that continuous administration of NS9283 does not cause lasting behavioral changes or excitatory responses, and does not significantly affect motor behavior and anxiety levels in mice.

[0106] We trained two groups of mice (n = 10) for one week, and then placed them in a conditional place preference test chamber for random movement. The results showed that the Nicotine group mice had significantly higher CPP values, time spent in the drug-accompanied chamber, and preference index than the NS9283 group ( Figure 13 (P<0.05). This result indicates that intraperitoneal injection of nicotine significantly increased the time mice spent in the drug-filled box and their activity level, suggesting that nicotine induces a preference for specific environments in mice.

[0107] Conversely, mice in the NS9283 group did not show a clear preference for either box in the experiment, with mean CPP values ​​closer to 0 and a preference index closer to 50%. This indicates that NS9283 treatment failed to significantly alter the mice's environmental preferences or induce drug-related behavioral changes, and there was no obvious drug dependence response.

[0108] In a forced swimming test after 7 days of continuous drug administration, we evaluated the behavioral performance of three groups of mice (n=6), including two key indicators: resting time and struggling time. The results showed that compared with the control group, the Nicotine group mice had increased struggling time and decreased resting time, and the differences were statistically significant. Figure 14 (P<0.05). However, the NS9283 group showed no significant differences in these key behavioral indicators compared to the Control group (P>0.05). This indicates that NS9283 did not induce depressive-like behavior after one week of continuous intraperitoneal administration, nor did it increase stress response or inhibit behavior in animals.

[0109] We evaluated the performance of three groups of mice (n = 6) in endurance tests, measuring total crawling time 2 hours after drug administration and 7 days of daily administration as observation indicators. The results showed that, compared with the Control group, mice in the Nicotine and NS9283 groups did not show significant differences in crawling time and fatigue time 2 hours after drug administration and 7 days of daily administration. Figure 15 (P>0.05). This indicates that neither NS9283 nor Nicotine treatment had a significant effect on the physical exertion of mice, further demonstrating that these two drugs had no significant effect on the endurance or fatigue perception of mice under the experimental conditions.

[0110] In this experiment, major organs of mice, including heart, liver, spleen, and kidney, were collected on days 3 and 7 after drug injection for histological staining and pathological evaluation. After HE staining, observation under a 200x microscope revealed that in the liver tissue, mild edema, cell swelling, and loose, lightly stained cytoplasm were commonly observed in the Nicotine group 3 days after administration. On day 7, widespread to severe edema, cell swelling, and loose, lightly stained cytoplasm were observed in the hepatocytes. In contrast, only a small amount of mild edema was observed in the Control and NS9283 groups. In the spleen tissue, there was no significant difference in the number of marginal lymphocytes and erythrocytes in the red pulp splenic sinusoids among all groups. In the kidney tissue, no significant edema was observed in the renal tubules in all groups, and the morphology of the renal tubular epithelial cells was normal, with no obvious damage. In the heart tissue, the cardiomyocytes were spindle-shaped, with the nucleus located on one side of the cell, and the cells were neatly arranged and aligned, with no obvious abnormalities. Figure 16 ).

[0111] At both time points mentioned above, mice in the Nicotine and NS9283 groups showed no significant pathological changes in the heart, liver, spleen, and kidneys compared to the Control group. The tissue structure of each organ remained intact, with no obvious cell swelling, necrosis, inflammatory infiltration, or other pathological damage. The morphology and distribution of blood vessels within the organs also showed no significant abnormalities. This indicates that NS9283 does not cause significant pathological damage to the major organs of mice under long-term drug exposure.

[0112] The experimental groups in this section were the same as those for the pathological staining of major tissues and organs. On days 3 and 7 after drug injection, the brains and spinal cords of mice were collected for LFB histochemical staining to observe whether demyelination occurred in the central nervous system. Brain tissue was analyzed using coronal sections of the hippocampus, thalamus, and amygdala; spinal cord tissue was analyzed using coronal sections of the lumbar enlargement. Results showed that under the microscope, the brains and spinal cords of all three groups of mice exhibited abundant longitudinal nerve fibers in the spinal cord white matter. The myelin sheath was blue, evenly distributed, densely arranged, and morphologically normal, with no obvious demyelination observed in any group. Figure 17 ).

[0113] In the nicotine median lethal dose (LD50) experiment, we evaluated the acute toxicity of nicotine in mice by intraperitoneal injection of different doses. Multiple dose groups (n=6) were set up: 10 mg / kg, 8 mg / kg, 5 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg. Physiological changes and mortality were observed in each group. The results showed that with increasing nicotine dosage, the mortality rate of mice gradually increased, exhibiting obvious symptoms of poisoning, including rapid breathing, muscle twitching, ataxia, coma, and ultimately death, as detailed in the table below.

[0114] Based on experimental data, statistical analysis, and Probit regression calculations, the LD50 of nicotine is approximately 4.03 mg / kg. This means that at this dose, 50% of the experimental mice would die. Analysis of this data indicates that nicotine has significant acute toxicity, and its toxic effects are positively correlated with dose.

[0115] Table 1. LD50 of nicotine administered intraperitoneally in mice

[0116]

[0117] Note: The mortality rate and physiological changes of mice after a single intraperitoneal injection of gradient doses of nicotine were observed for 14 consecutive days.

[0118] Since no mouse deaths were observed even after intraperitoneal injection at more than twice the LD50 of nicotine, and no behavioral changes such as respiratory distress, muscle twitching, or vomiting induced by nicotine were observed, we selected three groups (n=6) for the experiment to investigate the acute toxicity of NS9283, considering its low solubility in solvents. These groups included a solvent control, 5 mg / kg, and 10 mg / kg.

[0119] Open field tests showed that, compared to the control group, mice in the 10 mg / kg group had significantly reduced movement distance and number of entries into the central area within the open field 2 hours after NS9283 administration. Figure 18 (P<0.05) There was no significant difference in average movement speed; the 5 mg / kg group showed no significant difference in movement distance, average movement speed, and number of times entering the central zone compared to the Control group. 24 hours after administration, compared to the Control group, the 5 mg / kg and 10 mg / kg groups showed no significant difference in movement distance and average speed, but relatively fewer times entering and crossing the central zone.

[0120] In terms of histopathology, HE staining of major organs showed that, compared with the control group, no obvious necrosis of hepatocytes was observed in the 5 mg / kg and 10 mg / kg groups, but severe edema was observed in a small number of hepatocytes. Figure 19 The cardiomyocytes were neatly arranged and aligned. There were no significant differences in the number of marginal lymphocytes in the spleen and the number of erythrocytes in the splenic sinuses. LFB staining of the spinal cord and brain tissue revealed abundant longitudinal nerve fibers in the spinal cord white matter and brain medulla. The myelin sheath was blue, evenly distributed, densely arranged, and morphologically normal, with no obvious demyelination. This indicates that even under high-dose NS9283 loading, no significant tissue damage or pathological changes were observed, suggesting that the drug has low toxicity in the liver, heart, spleen, spinal cord, and brain tissue, and that high-dose, short-term administration will not cause significant structural changes or nerve damage.

[0121] In summary, by encapsulating NS9283 with amphiphilic Janus AuNPs microcapsules (Janus AuNPs-NS9283), a novel and highly efficient targeted drug delivery system was constructed, increasing the targeted drug loading capacity of NS9283 and achieving long-term controlled drug release. This overcomes the technical bottlenecks of poor stability and short analgesic duration of NS9283 after injection, enabling pre-analgesia and long-term safety management of PNPs, and promoting the clinical translation of nanomedicine delivery systems.

Claims

1. A nano-gold-supported NS9283-targeted long-acting analgesic drug, characterized in that: Janus-type amphiphilic microcapsules were assembled from gold nanoparticles with an average particle size of 12-18 nm by ligand exchange loading of NS9283 molecules on their surface and then assembled using the Pickering emulsion method. The Janus-type amphiphilic microcapsules were formed from gold nanoparticles with DL-mercaptosuccinic acid and benzyl thiol ligands on their surface, with an NS9283 loading of 3.5-5.2 wt% and a microcapsule particle size distribution of 50-200 nm. The gold nanoparticles were prepared by reducing chloroauric acid with sodium citrate. The reaction conditions were: sodium citrate concentration 0.01-0.05 mol / L, chloroauric acid concentration 0.005-0.02 mol / L, reaction temperature 80-100℃, and stirring speed 300-500 rpm. The method for constructing the Janus-type amphiphilic microcapsules is as follows: NS9283 is dissolved in DMF solution, and added to the ligand exchange reaction system at a mass ratio of NS9283:AuNPs of 1:8-12. The reaction time is 4-8 hours, and after centrifugation and washing, the microcapsules are dispersed in physiological saline to form a stable colloid.

2. The use of the drug according to claim 1 in the preparation of a drug for treating postoperative neuralgia, characterized in that: Administered via local injection at a dose of 0.5-2 mg / kg, with an analgesic effect lasting up to 28 days.

3. The use of the drug according to claim 1 in the preparation of a drug for treating chemotherapy-related neuropathic pain, characterized in that: Continuous administration for 7 days can completely suppress oxaliplatin-induced mechanodysia.

4. A method for preparing the drug according to claim 1, characterized in that... Includes the following steps: (a) Preparation of gold nanoparticles by sodium citrate reduction of chloroauric acid; (b) DMF solution with ligand exchange loading of NS9283; (c) Construction of Janus microcapsules using the Pickering emulsion method; (d) Wash and purify with physiological saline.

5. The method according to claim 4, characterized in that: In step (a), the molar ratio of sodium citrate to chloroauric acid is 2-5:1, and the pH of the reaction system is controlled at 3.5-4.5.

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