Preparation method of nanomaterial of salidroside loaded by dendrimer and application of blood-brain barrier targeted penetration thereof

Abstract

The application relates to a preparation method of a dendrimer-loaded salidroside nanomaterial and an application of the dendrimer-loaded salidroside nanomaterial in blood-brain barrier targeted penetration, wherein salidroside is reacted with trimethylchlorosilane and hexamethyldisilazane in anhydrous pyridine; the product is dissolved in dichloromethane, and an ammonium acetate methanol solution is added dropwise; after reaction, the obtained product is dissolved in anhydrous dichloromethane, triethylamine and succinic acid monobenzyl ester acid chloride are added, the obtained product is dissolved in chromatographic grade methanol, and palladium-carbon catalyst is added; after reaction, the obtained product is dissolved in N,N-dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and 4-dimethylamino are added, and activation is carried out; and hydroxyl-terminated polyamide-amine type dendrimers are added dropwise to obtain the final product. Advantages are as follows: the structure is clear, and the drug loading amount is high. The salidroside loaded by the dendrimer nanosystem can effectively inhibit the release of neuroinflammatory factors, reduce neuron damage, and further improve diabetic cognitive dysfunction.

Description

Technical Field

[0001] This invention relates to a method for preparing dendritic molecularly loaded rhodioloside nanomaterials and their application in targeted penetration of the blood-brain barrier. Background Technology

[0002] Diabetic cognitive impairment (DCD) is a common but often overlooked serious complication in patients with diabetes mellitus (DM), primarily manifesting as decreased learning ability, memory loss, slowed reaction time, and executive dysfunction. With the continued rise in global diabetes prevalence, the number of people suffering from this complication has increased significantly. Studies have shown that the risk of cognitive impairment in elderly patients with type 2 diabetes is far higher than in their age-matched non-diabetic populations, and the longer the history of diabetes and the greater the fluctuations in blood glucose, the higher the risk of cognitive impairment. Its pathogenesis is complex, mainly involving factors such as oxidative stress induced by chronic hyperglycemia, deposition of advanced glycation end products (AGEs), cerebrovascular disease, and insulin resistance in the brain. These pathological processes collectively lead to damage and apoptosis of neurons in key brain regions such as the hippocampus. Currently, clinical treatment options for diabetic cognitive impairment are very limited. Existing strategies mainly include lifestyle interventions, strict blood glucose control, and symptomatic treatment. Clinically, cholinesterase inhibitors (such as donepezil), glutamate receptor antagonists (such as memantine), or nootropic drugs such as ginkgo biloba preparations may be used as appropriate. However, these drugs primarily target the pathological features of Alzheimer's disease (AD), and their effectiveness in improving diabetes-specific cognitive decline is unclear. Furthermore, long-term use can lead to side effects such as gastrointestinal reactions and liver and kidney toxicity.

[0003] Due to the presence of the blood-brain barrier (BBB), over 98% of small molecule drugs and almost all large molecule drugs struggle to effectively penetrate the brain parenchyma. This prevents most drugs with neuroprotective potential from reaching effective therapeutic concentrations at the site of brain lesions, resulting in extremely low bioavailability and severely limiting their clinical translation and application.

[0004] Rhodioloside, the main active ingredient derived from the high-altitude plant Rhodiola rosea, has been confirmed by modern pharmacological studies to possess significant neuroprotective effects. Its mechanisms of action are broad, including anti-inflammatory, antioxidant, anti-apoptotic, inhibition of glutamate neurotoxicity, and improvement of mitochondrial function. Studies have shown that rhodioloside has protective effects against various central nervous system injuries, such as ischemic brain injury and traumatic brain injury, and holds promise as an ideal neuroprotective agent. However, rhodioloside faces severe technical bottlenecks in clinical application: it is highly water-soluble, rapidly metabolized in vivo, has an extremely short half-life, and lacks the ability to specifically recognize the blood-brain barrier. Therefore, when using conventional intravenous or oral administration, rhodioloside struggles to penetrate the blood-brain barrier and enter brain tissue, resulting in extremely low bioavailability in the brain. This prevents its inherent neuroprotective value from being fully utilized in the treatment of brain diseases such as diabetic cognitive impairment. Therefore, developing a carrier system capable of efficiently loading rhodioloside, enhancing its brain-targeted delivery capability, and improving its bioavailability has become an urgent technical challenge in this field. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for preparing dendritic molecularly loaded rhodioloside nanomaterials and their blood-brain barrier targeted penetration application.

[0006] The technical solution of this invention is: A method for preparing dendritic molecularly loaded rhodioloside nanomaterials includes the following steps: (1) Rhodioloside was reacted with trimethylchlorosilane and hexamethyldisilazane in anhydrous pyridine and stirred at room temperature for 24 h. After washing, the mixture was purified by silica gel column chromatography to obtain compound 1. (2) Compound 1 was dissolved in dichloromethane to prepare a compound 1 solution with a molar concentration of 0.25 mol / mL; ammonium acetate was dissolved in methanol to prepare an ammonium acetate solution with a molar concentration of 0.5 mol / mL; the ammonium acetate solution was slowly added dropwise to the compound 1 solution under stirring, and the reaction was carried out at -20℃ for 24 h; after washing, the compound 2 was obtained by silica gel column purification. (3) Compound 2 was dissolved in anhydrous dichloromethane to prepare a compound 2 solution with a molar concentration of 0.135 mol / mL; the compound 2 solution was cooled to 0℃, and triethylamine and succinic acid monobenzyl ester chloride were added sequentially at 0℃ with stirring. After stirring evenly, the reaction was continued at room temperature for 2 h; the compound 3 was obtained by silica gel column purification. (4) Compound 3 was dissolved in chromatographic grade methanol to prepare a compound 3 solution with a molar concentration of 0.1 mol / mL, and palladium on carbon catalyst was added; the reaction was stirred at room temperature for 4 h under hydrogen atmosphere; the catalyst was removed by filtration, the solvent was removed by concentration under reduced pressure, and the compound 4 was obtained by silica gel column purification. (5) Dissolve the 64 hydroxyl-terminated polyamide-amine dendritic molecules in N,N-dimethylformamide to obtain a N,N-dimethylformamide solution of hydroxyl-terminated polyamide-amine dendritic molecules with a molar concentration of 0.0033 mmol / mL; Compound 4 was dissolved in N,N-dimethylformamide to obtain a solution of compound 4 with a molar concentration of 0.134 mmol / mL; According to the molar ratio of compound 4 to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine of 0.67:1.035:0.242, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added sequentially to the solution of compound 4. After stirring and activating at room temperature for 10 min, a solution of N,N-dimethylformamide containing 64 hydroxyl-terminated polyamide-amine dendritic molecules was added dropwise. The molar ratio of the 64 hydroxyl-terminated polyamide-amine dendritic molecules to compound 4 was 0.033:0.67. The reaction was continued at room temperature for 48 h. After the reaction was completed, the reaction solution was diluted with deionized water, dialyzed in deionized water for 12 h, and freeze-dried to obtain compound 5. (6) The compound 5 obtained in step (5) was subjected to a reaction to remove the trimethylsilyl protecting group in N,N-dimethylformamide in the presence of trifluoroacetic acid. After neutralization, dialysis and drying, the rhodioloside-hydroxy-terminated polyamide-amine dendritic molecule (SAL-PAMAM) was obtained.

[0007] Furthermore, in step (1), the molar ratio of rhodioloside, trimethylchlorosilane and hexamethyldisilazane is 1:6:2.

[0008] Furthermore, in step (2), the molar ratio of compound 1 to ammonium acetate is 1:2.

[0009] Furthermore, in step (3), the molar ratio of compound 2, triethylamine and succinic acid monobenzyl chloride is 2.7:16.1:4.1.

[0010] Furthermore, in step (4), the mass ratio of compound 3 to palladium on carbon catalyst is 10:1.

[0011] Furthermore, in steps (5) and (6), dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da.

[0012] Furthermore, in step (6), the amount of trifluoroacetic acid used is 0.12 mL of trifluoroacetic acid per 100 mg of compound 5; the reaction to remove the trimethylsilyl protecting group is carried out at 0°C to room temperature for 1 hour; and sodium bicarbonate aqueous solution is used for neutralization.

[0013] The dendritic molecularly loaded rhodioloside nanomaterials were prepared by the above method.

[0014] The application of dendritic molecularly loaded rhodioloside nanomaterials prepared by the above method in drugs that target and penetrate the blood-brain barrier to treat cognitive impairment in diabetes.

[0015] This invention provides a nanoparticle drug delivery system based on 64 hydroxyl-terminated polyamide-amine dendritic molecules (PAMAM). Polyamide-amine dendritic molecules are mostly amino-terminated (PAMAM-NH2), possessing advantages such as well-defined structure and high drug loading capacity. The hydroxyl-terminated dendritic molecules are used as drug carriers. The electrically neutral nature of the hydroxyl groups fundamentally eliminates the risk of cell membrane damage caused by positive charges (on the one hand, positive charges can non-specifically damage cell membrane integrity, leading to significant cytotoxicity and hemolytic effects, making it difficult to meet the safety requirements for long-term use in chronic diseases; on the other hand, this strong positive charge makes it easy to non-specifically bind to blood proteins, be rapidly cleared by the reticuloendothelial system, have a short in vivo circulation time, and lack the ability to actively recognize lesions in the brain; even if a small amount enters brain tissue, it is difficult to accurately accumulate in areas of neuroinflammation). This endows the carrier system with excellent biocompatibility and blood safety. Simultaneously, the hydrophilicity and electrical neutrality of the hydroxyl surface effectively reduce plasma protein adsorption, significantly prolonging the in vivo circulating half-life of the nanoparticles, enabling them to efficiently cross the blood-brain barrier. Upon entering brain tissue, this nanosystem, with its suitable nanoscale size, specifically accumulates in inflammatory sites of the brain with increased vascular permeability and inflammatory cell infiltration through high permeability and retention effect (EPR effect). This achieves precise brain delivery of rhodioloside, significantly improving its bioavailability in the brain and providing a safe, effective, and innovative solution for improving cognitive impairment in diabetes. Its beneficial effects are: (I) Synthesis and characterization of SAL-PAMAM nano-drug delivery system.

[0016] 1. 1 H NMR and 13 Both C NMR and HRMS confirmed the successful synthesis of each product and the final product.

[0017] 2. SAL-PAMAM exhibits a regular, uniformly dispersed spherical or near-spherical structure, with its core particle size mainly distributed around 100nm, and the system has good dispersibility.

[0018] 3. SAL was successfully loaded onto PAMAM with a drug loading of approximately 33%; compared with free SAL, SAL-PAMAM exhibits good sustained-release properties.

[0019] 4. This nanosystem can significantly alter the pharmacokinetic characteristics of SAL, including increasing systemic exposure and significantly prolonging drug circulation time in vivo. Brain distribution studies confirmed that the intracranial drug concentration of SAL-PAMAM was significantly higher than that of the free drug group at all time points, indicating that the system has good brain-targeted delivery capabilities.

[0020] 5. SAL-PAMAM has good in vitro safety.

[0021] (II) SAL-PAMAM's penetration of the blood-brain barrier and its uptake by BV-2 cells and its therapeutic effects.

[0022] 1. SAL-PAMAM can effectively penetrate the bEnd.3 cell monolayer and be taken up by BV-2 cells in the lower chamber in a time-dependent manner.

[0023] 2. SAL-PAMAM can significantly reduce the expression of M1 microglial markers and pro-inflammatory factors, while increasing the expression of M2 microglial markers and anti-inflammatory factors, with better effects than SAL monotherapy.

[0024] (III) Application of SAL-PAMAM in diabetic rats with cognitive impairment.

[0025] 1. SAL-PAMAM can penetrate the blood-brain barrier and be taken up by brain cells to exert its effects.

[0026] 2. Compared with the CON group, DM rats showed reduced learning and memory abilities and decreased spatial exploration abilities. After treatment with SAL-PAMAM, the rats' spatial exploration abilities were enhanced, and their learning and memory abilities were significantly improved compared with the SAL group.

[0027] 3. Compared with the CON group, the DM group showed a decrease in the number and length of microglia axons, and an increase in cell body size in the brains of rats with microglia, indicating that the microglia were in an activated state. Rats treated with SAL-PAMAM showed a significant increase in the number of microglia axons, a marked increase in length, and a significant decrease in cell body area, with better results than the SAL group.

[0028] 4. SAL-PAMAM can more effectively inhibit the transformation of microglia into M1 type and regulate the balance of inflammatory factors, and its therapeutic effect is better than that of SAL alone.

[0029] 5. Significant neuronal damage was observed in the CA1 region of the hippocampus in the DM group, and both SAL and SAL-PAMAM treatments were effective. Compared to SAL, SAL-PAMAM significantly increased the number of neurons and improved cell morphology, demonstrating a better neuroprotective effect.

[0030] 6. SAL-PAMAM has good in vivo biocompatibility.

[0031] This study successfully prepared a safe and efficient nanoparticle-loaded drug delivery system (dendritic nanoparticle-loaded rhodioloside, SAL-PAMAM), which can effectively load rhodioloside. Pharmacokinetic and in vivo imaging results showed that the system can efficiently cross the blood-brain barrier after intravenous administration and target the site of inflammation in the brain. Compared with free rhodioloside, rhodioloside loaded via the dendritic nanoparticle system can more effectively inhibit the release of neuroinflammatory factors and reduce neuronal damage, thus exhibiting superior effects in improving cognitive function in a rat model of diabetic cognitive impairment. Combining the inherent neuroprotective effects of rhodioloside with the targeted delivery advantages of dendritic nanoparticle carriers, this strategy provides a new potential therapeutic approach for delaying diabetic cognitive impairment and offers an innovative technical reference for brain-targeted delivery research of rhodioloside and other neuroactive drugs, which is of great value for developing therapeutic strategies for chronic neurodegenerative diseases. Attached Figure Description

[0032] Figure 1 This is the proton spectrum image of compound 1 of the present invention; Figure 2 This is a carbon spectrum image of compound 1 of the present invention; Figure 3 This is the proton NMR spectrum of compound 2 of the present invention; Figure 4 This is a carbon spectrum image of compound 2 of the present invention; Figure 5 This is a mass spectrum image of compound 2 of the present invention; Figure 6 This is the proton NMR spectrum of compound 3 of the present invention; Figure 7 This is the carbon spectrum image of compound 3 of the present invention; Figure 8 This is a mass spectrum image of compound 3 of the present invention; Figure 9 This is the proton NMR spectrum of compound 4 of the present invention; Figure 10 This is the proton NMR spectrum of compound 5 of the present invention; Figure 11 This is the proton spectrum image of the SAL-PAMAM of this invention; Figure 12 This is the synthetic route of SAL-PAMAM in this invention; In the figure: A: Synthetic route of SAL complex, (I) TMSCl, HMDS, Pyridine, rt, 24h; (II) NH4OAc, -20℃, 24h; (III) SOCl2, 0℃-rt; (IV) NET3, DCM, 0℃-rt, 2h; (V) Pd / C, H2, NET3, rt, 4h; B: Synthetic route of SAL-PAMAMA, (VI) Compound 4, EDCl, DMAP, DMF, rt, 48h; (VII) TFA, DMF, NaHCO3, rt, 1h; Figure 13 This is the synthetic route of SAL-PAMAM-Ro of the present invention; in the figure: (I) HATU, DIPEA, DMF, rt, 48h; Figure 14 This is a characterization experiment diagram of SAL-PAMAM of the present invention; in the diagram: A: Transmission electron microscopy image of SAL-PAMAMA; B: Statistical diagram of particle size distribution of SAL-PAMAMA and PAMAM; C: Zeta potential of each group; D: PDI dispersion index of each group; data are expressed as mean ± SEM (n=3). Figure 15 The figure shows the physicochemical properties of SAL-PAMAM of this invention; in the figure: A: 1H NMR spectra of SAL, SAL-PAMAMA and PAMAM; B: Fourier transform infrared spectra of SAL, SAL-PAMAMA and PAMAM; C: Drug release results of SAL-PAMAMA in a neutral environment (pH=7.4); data are expressed as mean ± SEM (n=3). Figure 16 The pharmacokinetic results of this invention are shown in the figure; in the figure: A: pharmacokinetic results of SAL and SAL-PAMAM; B: calculated results of the half-life (t1 / 2) and area under the curve (AUC) of SAL and SAL-PAMAM; C: biodistribution of SAL and SAL-PAMAM in brain tissue at each time point; data are expressed as mean ± SEM (n=3). Figure 17 This is a figure showing the cell safety and hemolysis test results of SAL-PAMAM of the present invention; in the figure: AB: comparative images and absorbance analysis of different concentrations of SAL-PAMAM incubated with red blood cells for 30 min; C: cell viability of different concentrations of SAL-PAMAM incubated with BV-2 cells for 48 h as detected by CCK8; data are expressed as mean ± SEM (n=3). Figure 18This is a diagram showing the Transwell experimental results of this invention; in the diagram: A: Schematic diagram of the in vitro blood-brain barrier Transwell co-culture model; B, C: Statistical graphs of intracellular fluorescence intensity and mean fluorescence intensity of BV-2 cells at different time points (0, 0.5, 1 and 4 h) detected by flow cytometry, with data expressed as mean ± SEM (n=3); D, E: Representative images and semi-quantitative analysis results of SAL-PAMAM-Ro uptake by BV-2 cells at different time points (0, 0.5, 1 and 4 h) observed by laser confocal microscopy; red light represents SAL-PAMAM-Ro, green light represents the BV-2 marker IBA-1, and blue light represents DAPI; Scale bar = 25µm; Figure 19 This is a graph showing the effect of SAL-PAMAM of the present invention on the release of inflammatory factors in BV-2 cells; in the graph: AL: representative Western blotting images and semi-quantitative analysis of protein expression of TNF-α, IL-6, IL-1β, IL-4, IL-10, CD86, CD206, INOS and Arg1 in each group; data are expressed as Mean±SEM (n=5), and one-way ANOVA was used to perform Tukey's test; *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference; Figure 20 This is a diagram showing the distribution of SAL-PAMAM in an in vivo imaging experiment according to the present invention. In the diagram: AG: Comparative images and fluorescence intensity statistics of brain, liver, kidney, spleen, lung, and heart at various time points after injection of SAL-PAMAM-Ro in rats; HI: Comparative images and fluorescence intensity statistics of brain in CON rats and DM rats 2 hours after injection of 0.9% NaCl and SAL-PAMAM-Ro; JK: Comparative images and fluorescence intensity statistics of brain, liver, kidney, spleen, lung, and heart in DM rats 2 hours after injection of SAL-PAMAM-Ro. The data are expressed as mean ± SEM (n=3). Figure 21 This is a diagram showing the results of the brain-targeting experiment of this invention; in the diagram: A: Immunofluorescence staining images of IBA-1, GFAP, and NeuN with SAL-PAMAM-Ro taken using a confocal fluorescence microscope; Scale bar=25μm; B: Statistical analysis of the area of ​​SAL-PAMAM-Ro taken up by IBA-1, GFAP, and NeuN; data are expressed as mean ± SEM (n=3). Figure 22This invention relates to a water maze experiment used to assess the cognitive function of rats. The diagram shows: A: Rat's trajectory onto the platform; B: Rat's trajectory away from the platform; C: Analysis of the rat's escape latency; D: Time spent by the rat in the target area; E: Number of times the mouse traversed the target platform; F: Swimming speed of each group of animals. Data are expressed as mean ± SEM (n=5). Analysis of variance (C) was performed using two-way ANOVA, and analysis of variance (DF) was performed using one-way ANOVA, with Tukey's test applied. *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference. Figure 23 This invention presents morphological images of microglia detected by immunohistochemistry. In the images: A: Representative images of IBA-1 immunohistochemistry, local magnification, and skeletal formation in the CA1 region of the hippocampus of each group of rats. Scale bar = 50 μm; B: Cell body area of ​​microglia in each group; C: Maximum branch length of microglia in each group; D: Number of terminal branches of microglia in each group. Data are expressed as Mean ± SEM (n=5), and one-way ANOVA was performed using Tukey's test. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference; Figure 24 This invention presents images of microglial marker expression detected by immunofluorescence and Western blotting experiments. In the images: A and C: CD86 immunofluorescence staining images and quantitative analysis of the results. Data are expressed as Mean ± SEM (n=3); B and DG: Representative Western blotting images and semi-quantitative analysis of CD86, CD206, INOS, and Arg1 expression in the hippocampus of each group. Data are expressed as Mean ± SEM (n=5). One-way ANOVA was used, and Tukey's test was performed. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference; Figure 25 This is a Western blotting experiment of the present invention to detect the effect of SAL-PAMAM on the release of inflammatory factors; in the figure: AF: representative Western blotting images and semi-quantitative analysis of the expression of TNF-α, IL-6, IL-1β, IL-4 and IL-10 in the hippocampus of each group. Data are expressed as Mean±SEM (n=5), and one-way ANOVA was used to perform Tukey's test; *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference; Figure 26Images show the statistical results of complete blood count and blood biochemistry in rats according to this invention. In the images: blood parameters were measured in each group of rats, with A and B representing white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CR), and serum albumin (ALB), respectively. Data are expressed as mean ± SEM (n=3), and ns indicates no significant difference. Figure 27 These are HE staining images of rat liver, heart, spleen, lung, and kidney from this invention; in the figure: schematic diagram of HE staining of heart, liver, spleen, lung, and kidney of rats in each group (n=3), scale bar=100µm. Detailed Implementation

[0033] Example 1 (I) Preparation of SAL-PAMAM 1. Preparation of Compound 1 Rhodioloside SAL (3 g, 9.99 mmol), trimethylchlorosilane (TMSCl, 7.62 mL, 60 mmol), and hexamethyldisilazane (HMDS, 4.20 mL, 20 mmol) were dissolved in 4 mL of anhydrous pyridine and stirred in a round-bottom flask at room temperature for 24 h. After the reaction was complete, dichloromethane was added for dilution, the mixture was filtered, and the filtrate was collected. The solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate, transferred to a separatory funnel, and washed three times with saturated NaHCO3 solution, followed by three times with saturated NaCl solution. The organic phase was collected in a flask, dried over anhydrous Na2SO4, filtered, and then evaporated in toluene by rotary evaporation to remove residual pyridine via azeotropic distillation. Subsequently, the mixture was concentrated to dryness under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1). v / v ), yielding 5.42 g of clear liquid, with a yield of 82.17%; 1 H NMR (400MHz, Chloroform- d ) δ 6.90 (dd, J =8.5, 3.3 Hz, 2H), 6.65 – 6.46 (m, 2H), 4.85 (s, 1H), 4.04 (d, J = 7.5 Hz, 1H), 3.84 (tt, J = 9.3, 7.0 Hz, 1H), 3.64 (dt, J= 11.3, 1.5 Hz, 1H), 3.59 – 3.36 (m,2H), 3.36 – 3.21 (m, 2H), 3.21 – 3.07 (m, 1H), 3.02 (dtt, J = 7.0, 5.1, 2.0 Hz,1H), 2.71 (ht, J = 6.6, 3.7 Hz, 2H), 0.01 (d, J = 58.4 Hz, 41H). 13 C NMR (101 MHz, Chloroform- d ) δ 153.19, 152.65, 130.56, 129.72, 129.13, 128.97, 119.05,114.35, 102.10, 77.88, 77.84, 75.68, 75.25, 70.81, 69.55, 69.44, 61.39,34.61,34.52, 28.83, -1.09, -1.11. 2. Preparation of Compound 2 Compound 1 (3.30 g, 5 mmol) was dissolved in 20 mL of dichloromethane (DCM) to prepare a compound 1 solution; ammonium acetate (NH4OAc, 0.771 g, 10 mmol) was dissolved in 20 mL of methanol (MeOH) to prepare an ammonium acetate solution; the ammonium acetate solution was slowly added dropwise to the compound 1 solution with stirring, and the reaction was carried out at -20 °C for 24 h; after the reaction was completed, the reaction solution was transferred to a separatory funnel, washed three times with saturated NaHCO3 solution, the organic phase was collected in a flask, dried with anhydrous Na2SO4, filtered, and concentrated to dryness under vacuum; the crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (10:1, v / v) as eluent to give 2.45 g of white solid, yield 83.33%; 1 H NMR (400MHz, Chloroform- d )δ 6.98 – 6.72(m, 2H), 6.69 – 6.44 (m, 2H), 5.30 (s, 1H), 4.05 (d, J = 7.5 Hz, 1H), 3.83 (td, J = 9.0, 7.0 Hz, 1H), 3.66 (dd, J = 11.3, 2.1 Hz, 1H), 3.51 (dd, J = 11.3, 5.6 Hz, 1H), 3.43 (td,J = 8.9, 6.8 Hz, 1H), 3.38 – 3.22 (m, 2H), 3.22 – 3.12 (m, 1H), 3.05 (ddt, J = 7.5, 3.7, 2.0 Hz, 1H), 2.69 (ddd, J = 9.0, 6.8, 2.0 Hz, 2H), -0.06(s, 38H). 13 C NMR (101 MHz, Chloroform- d ) δ 153.29, 129.47, 129.08, 114.39,102.10,77.74, 75.67, 75.20, 70.88, 69.64, 61.57, 34.48, -1.16. HRMS (ESI + , m / z ): (C 26 H 52 O7Si4, [M+Na] + ]),calcd 611.27901, found 611.2687. 3. Preparation of Compound 3 Monobenzyl succinate (1.5 g, 7.20 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and 10 μL of N,N-dimethylformamide (DMF) was added as a catalyst. Then, thionyl chloride (SOCl2, 5 mL, 39.9 mmol) was slowly added dropwise. The mixture was heated under reflux for 5 h. After the reaction was completed, the solvent and excess thionyl chloride were removed by concentration under reduced pressure to obtain monobenzyl succinate chloride. Compound 2 (1.6 g, 2.70 mmol) was dissolved in 20 mL of anhydrous dichloromethane and cooled to 0 °C. Triethylamine (NET3, 2.24 mL, 16.1 mmol) and the prepared monobenzyl succinate chloride (0.9235 g, 4.086 mmol) were added sequentially at 0 °C with stirring. The mixture was then allowed to return to room temperature and the reaction was continued with stirring for 2 h. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1, v / v) to give 1.55 g of an oily liquid product, with a yield of 73.8%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.27 – 7.11 (m, 5H), 7.08 – 6.90 (m, 2H), 6.85 – 6.69 (m,2H), 4.99 (s, 2H), 4.05 (d, J= 7.5 Hz, 1H), 3.87 (td, J = 9.0, 6.8 Hz, 1H), 3.63(dd, J = 11.4, 2.0 Hz, 1H), 3.51 (ddd, J = 12.3, 10.3, 6.6 Hz, 2H), 3.36 – 3.21(m, 2H), 3.21 – 3.07 (m, 1H), 3.01 (ddd, J = 9.0, 5.0, 2.0 Hz, 1H), 2.88 – 2.67(m, 4H), 2.67 – 2.56 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 171.00, 169.99,148.19, 135.33, 134.86, 128.93, 127.71, 127.43, 127.39, 120.43, 102.15,77.86, 75.70, 75.23, 70.72, 69.08, 65.80,61.33, 34.82, 28.45, 28.34, -1.08.HRMS (ESI + , m / z ): (C 37 H 62 O 10 Si4, [M+Na] + ]),calcd 801.34200, found .801.3303. 4. Preparation of Compound 4 Compound 3 (0.46 g, 0.6 mmol) was dissolved in 6 mL of chromatographic grade methanol, and 46 mg of palladium on carbon (Pd / C) catalyst was added. The mixture was stirred at room temperature for 4 h under a hydrogen atmosphere. After the reaction was completed, 50 μL of triethylamine was added to prevent the product from decomposing under acidic conditions in subsequent processing. The reaction solution was filtered through a diatomaceous earth filter to remove the catalyst, and the filtrate was concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1, v / v) to give 0.38 g of white colloid, with a yield of 92%. 1 H NMR (400 MHz, Chloroform- d) δ 7.03 (d, J = 7.9 Hz, 1H), 6.97 –6.86 (m, 1H), 6.83 (dd, J = 8.4, 2.2 Hz, 1H), 6.58 (d, J = 8.5 Hz, 1H), 4.10(dd, J = 7.7, 5.6 Hz, 1H), 3.95 – 3.73 (m, 1H), 3.62 (dt, J = 11.6, 2.7 Hz,1H), 3.58 – 3.40 (m, 2H), 3.40 – 3.19 (m, 2H), 3.19 – 2.99 (m, 2H), 2.82 –2.37(m, 4H), 0.11 – -0.16 (m, 20H). 5. Preparation of Compound 5 0.4924 g, 0.033 mmol) of polyamide-amine dendritic molecules (PAMAM) with 64 hydroxyl-terminated ends were dissolved in 10 mL of N,N-dimethylformamide (DMF) to obtain a DMF solution of PAMAM for later use. Compound 4 (0.46 g, 0.67 mmol) was dissolved in 5 mL of N,N-dimethylformamide (DMF), followed by the sequential addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.1984 g, 1.035 mmol) and 4-dimethylaminopyridine (DMAP) (0.0295 g, 0.2415 mmol). After activation by stirring at room temperature for 10 min, the aforementioned PAMAM DMF solution was slowly added dropwise, and the reaction was continued at room temperature for 48 h. After the reaction was complete, the reaction solution was diluted with deionized water and transferred to a dialysis bag (molecular weight cutoff: 1000 Da). Dialysis was performed in deionized water for 12 h, with the deionized water changed every 2 h. Finally, the dialysate was freeze-dried to obtain 0.61 g of a white gel-like substance, with a yield of 75.3%. 1 H NMR (400 MHz, DCDSO-) d 6) δ 7.81 (s, 2H), 7.68 (d, J = 14.4 Hz, 2H), 7.16 (d, J = 8.2 Hz, 1H), 6.89 (d, J = 7.9Hz, 2H), 6.55 (d, J = 8.0 Hz, 1H), 4.46 (s, 1H), 4.15 (t, J= 8.4 Hz, 1H), 3.88(s, 1H), 3.53 – 3.45 (m, 4H), 3.44 (s, 1H), 3.32 (s, 2H), 3.17 (s, 2H), 3.08 –2.98 (m, 8H), 2.97 (d, J = 6.4 Hz, 7H), 2.73 (dt, J = 20.5, 7.0 Hz, 3H), 2.62 (t, J = 7.5 Hz, 3H), 2.53 (t, J = 7.5 Hz, 12H), 2.44 (s, 2H), 2.38 (s, 5H), 2.31 (t, J = 7.2 Hz, 7H), 2.08 (t, J = 7.0 Hz, 11H), 1.08 (s, 1H), 0.65 (s, 1H), -0.06(s, 4H). 6. Preparation of SAL-PAMAM Compound 5 (100 mg, 0.00407 mmol) was dissolved in 5 mL of DMF, cooled to 0 °C, and then trifluoroacetic acid (TFA, 0.12 mL, 1.57 mmol) was slowly added. The mixture was brought back to room temperature and stirred for 1 h. The system was then cooled to 0 °C again, and an aqueous solution of sodium bicarbonate (0.135 g, 1.607 mmol) was slowly added dropwise for neutralization. After the reaction system no longer produced bubbles, the mixture was diluted with deionized water. The resulting solution was transferred to a dialysis bag (molecular weight cutoff: 1000 Da) and dialyzed with deionized water for 12 h, with the dialysate changed every 2 h. Finally, the dialysate was collected and freeze-dried to obtain 65 mg of white solid powder as the final product, rhodioloside-hydroxy-terminated dendritic molecule (SAL-PAMAM), with a yield of 91.83%. 1 H NMR (400 MHz, Deuterium Oxide) δ 7.09 (d, J= 8.3 Hz, 1H), 6.77 – 6.65 (m, 1H), 3.83 – 3.69(m, 1H), 3.63 – 3.48 (m, 1H), 3.49 (s, 2H), 3.40 – 3.21 (m, 4H), 3.18 (s, 3H), 2.80 – 2.71 (m, 1H), 2.69 (s, 6H), 2.66 – 2.55 (m, 5H), 2.50 (s, 1H), 2.34 – 2.27 (m, 1H), 2.23 (s, 0H). 7. Preparation of SAL-PAMAM-Ro Rhodamine B (30 mg, 0.0626 mmol) was dissolved in 4 mL of DMF and set aside. Then, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 30 mg, 0.0789 mmol) was added, and the mixture was stirred until homogeneous. N,N-diisopropylethylamine (DIPEA, 12 μl, 0.069 mmol) was added to the solution and reacted for 1 h. SAL-PAMAM (1.06 g, 0.061 mmol) was dissolved in 5 mL of DMF. In DMF, the solution was slowly added dropwise to the above Rhodamine B solution, and the reaction was continued at room temperature for 48 h. After the reaction was completed, the reaction solution was diluted with deionized water and transferred to a dialysis bag (molecular weight cutoff: 1000 Da). Dialysis was performed in deionized water for 12 h, with the deionized water being replaced every 2 h. Finally, the dialysate was freeze-dried to obtain 0.94 g of red solid powder, namely SAL-PAMAM-Ro, with a yield of 85.61%.

[0034] I. Product Characterization of the Invention 1. Transmission electron microscopy (TEM): PAMAM and SAL-PAMAM solutions were dropped onto a special copper grid, dried, and then tested.

[0035] Transmission electron microscopy (TEM) Figure 14 A) Observations show that SAL-PAMAM nanoparticles have a regular, uniformly dispersed spherical or near-spherical structure, with the core particle size mainly distributed at around 100 nm.

[0036] 2. Dynamic Light Scattering (DLS): PAMAM and SAL-PAMAM were prepared as 1 mg / mL aqueous solutions, filtered through a 0.22 μm microporous membrane, and their particle size, zeta potential, and PDI index were measured. Each experiment was repeated three times.

[0037] Dynamic light scattering (DLS) and zeta potential results further revealed the hydrodynamic properties and surface charge of SAL-PAMAM. Figure 14 (BD). DLS measurements showed that the hydrodynamic diameter of PAMAM before SAL loading was approximately 10 nm, and after SAL loading, the hydrodynamic diameter of SAL-PAMAM was approximately 100 nm, still consistent with the properties of nanoparticles. Zeta potential measurements showed that the blank PAMAM support carried a negative charge (approximately -8 mV) due to its rich hydroxyl content. After SAL loading, the potential of the formed SAL-PAMAM complex decreased to approximately -20 mV. This significant decrease in potential strongly demonstrates that SAL was successfully loaded onto the surface of the PAMAM support through interaction, enhancing the stability of the system. The polydispersity index (PDI) of SAL-PAMAM was less than 0.2, indicating good dispersibility of the system. All these results indicate the successful construction of a uniformly morphologically uniform nanoparticle drug delivery system.

[0038] II. Physicochemical Properties of the Product of this Invention To verify the successful loading of SAL onto the PAMAM surface, the synthesized SAL-PAMAM was validated using ¹H NMR and Fourier transform infrared (FTIR) spectroscopy. In vitro drug release experiments were also conducted.

[0039] 1H NMR spectroscopy results ¹H NMR spectrum as shown Figure 15 As shown in Figure A, in the spectrum of pure SAL, the characteristic hydrogen signals of the benzene ring (δ 6.5-7.5 ppm, peaks a and b) and the characteristic hydrogen signals of the glucose unit (δ 3.0-5.5 ppm, peak ce) can be clearly identified. The spectrum of the 64-hydroxyl-terminated polyamide-amine dendritic molecule (PAMAM) contains the following structure, mainly including the internal repeating unit: the broad peak (peak P1) at δ 2.20-2.50 ppm is mainly attributed to the amide bond ortho position inside the dendritic molecule and the methylene proton (-N-CH2-CH) in the backbone. 22 -CO-, -N-CH2-CH2-N-). Terminal hydroxyl unit: The broad peak (peak P2) at δ 2.50-2.80 ppm corresponds to the methylene proton (-CH2-OH) directly attached to the terminal hydroxyl unit. The broad peak (peak P3) at δ 3.30-3.60 ppm is the signal of the terminal hydroxyl (-OH) proton.

[0040] In the ¹H NMR spectrum of the SAL-PAMAM complex, a series of decisive changes were observed. The characteristic signals of the SAL benzene ring (peaks a, b) and the proton signals of the sugar ring (peak ce) remained clearly discernible, demonstrating that the structural integrity of SAL was maintained during loading. Key evidence lies in the systematic chemical shifts (Δδ approximately -0.05 to -0.15 ppm) of these signals, with a slight overall shift to higher fields. This phenomenon clearly indicates a change in the local electronic environment of the SAL molecule, with its protons affected by the shielding effect of the PAMAM molecule. This rules out the possibility of mere physical mixing and confirms a tight bond at the molecular level. Furthermore, the terminal hydroxyl signal of PAMAM also showed changes; the broad hydroxyl proton peak associated with the terminal -CH₂-OH (peak P3) was significantly weakened and shifted to lower fields (approximately Δδ +0.10 ppm) in the complex spectrum. This change strongly indicates that the numerous terminal hydroxyl groups of PAMAM participate in forming an intermolecular hydrogen bond network with hydrogen-donating groups (mainly phenolic and glycocyclic hydroxyl groups) in the SAL molecule, leading to a decrease in the electron cloud density of hydroxyl protons and a lower field shift in the resonance signal. Simultaneously, the dynamic and diverse nature of hydrogen bonding results in signal broadening and a decrease in intensity. The methylene proton signal (peak P2) connected to the hydroxyl groups also shows significant broadening and a slight shift, further confirming that the terminal -CH2-OH group is the main active site for the loading effect. In contrast, the methylene signal (peak P1) of the PAMAM internal framework shows very little shift change in the composite spectrum, indicating that the loading effect mainly occurs on the surface and ends of PAMAM, and its internal structure remains relatively stable during the loading process. This is consistent with the structural characteristics of PAMAM as a nanocarrier. The composite spectrum has a smooth baseline and no unknown sharp impurity peaks, indicating that the loading process was clean and no significant side reaction products were introduced, successfully obtaining a high-purity SAL-PAMAM composite.

[0041] Fourier transform infrared spectroscopy results Fourier Transform Infrared Spectroscopy (FTIR): (1) Weigh 200 mg of KBr, dry and grind it, and transfer it to a tablet mold to make a blank tablet. (2) Weigh 200 mg of each of three KBr samples; weigh 1 mg each of SAL-PAMAM sample, SAL sample, and PAMAM sample, mix them separately with the three KBr samples, mix well, grind and dry them, and transfer them to a tablet mold to make sample tablets. Place the blank KBr tablet in the optical path of the infrared spectrophotometer and perform background scanning. (3) Place the sample tablet in the optical path of the infrared spectrophotometer and perform sample scanning. After automatically subtracting the background, the FTIR spectrum of the sample is obtained.

[0042] FTIR results as follows Figure 15As shown in B, the infrared spectrum of the PAMAM raw material exhibits its typical molecular structure characteristics: at 3313 cm⁻¹... -1 The broad and strong absorption peak at 1645 cm⁻¹ is attributed to the superposition of stretching vibrations of numerous hydroxyl groups (-OH) and secondary amine groups (-NH₃) within the molecule; more importantly, the peak at 1645 cm⁻¹... -1 With 1550cm -1 The two strong characteristic absorption peaks appearing at 3313 cm⁻¹ correspond to the amide I band (C=O stretching vibration) and the amide II band (coupling of NH in-plane bending and CN stretching vibration), respectively, clearly demonstrating the existence of the PAMAM dendritic molecular framework structure. The infrared spectrum of the SAL standard reveals its unique functional group information: at 3313 cm⁻¹... -1 The broad absorption peak at 1516 cm⁻¹ is attributed to the OH stretching vibration of its sugar ring and phenolic hydroxyl groups; -1 The absorption peak appearing at 1300-1000 cm⁻¹ is due to the stretching vibration of the benzene ring skeleton (C=C), a characteristic signal of the tyrosol segment in the SAL molecule; -1 The nearby broad and strong absorption peak originates from the stretching vibration of COC and CO in the glucose syl group (the carbohydrate fingerprint region); in addition, at 821 cm⁻¹... -1 The absorption peaks appearing at the specified position clearly indicate that the benzene ring is in a para-disubstituted mode, which is completely consistent with the chemical structure of SAL.

[0043] In the spectrum of SAL-PAMAM, the superposition and variation of the PAMAM main framework and SAL characteristic structures can be observed. Firstly, the characteristic peak of the amide bond in PAMAM (1645 cm⁻¹) is observed. -1 With 1550cm -1 The characteristic absorption peak of SAL remains clearly visible, indicating that the core architecture of PAMAM was completely preserved during the modification process. The most crucial evidence is the presence of the characteristic absorption peak of SAL in the complex spectrum: at 1516 cm⁻¹. -1 A new absorption peak appeared at 821 cm⁻¹; simultaneously, a new absorption peak appeared at 821 cm⁻¹. -1 A new, clear absorption peak was observed at [location missing]. The appearance of these two characteristic peaks confirms the existence of the benzene ring structure and its para-disubstituted mode, respectively, and is direct evidence that the SAL molecule was successfully introduced into the complex. Furthermore, at 1300-1000 cm⁻¹... -1 Within the fingerprint region, the absorption bands of the complex showed significant deformation and enhancement compared to the PAMAM raw material. This is due to the superposition of the strong CO / COC vibrations of the sugar ring in the SAL molecule and the CO vibrations of PAMAM itself, further supporting the incorporation of SAL. The complex showed an absorption band at 3313 cm⁻¹. -1The OH stretching vibration peak at [a specific point] remains broad, indicating the presence of a large number of unreacted hydroxyl groups in the system. FTIR analysis shows that the characteristic amide peak of the PAMAM dendritic molecule is retained after modification, while the characteristic benzene ring skeletal vibration (1516 cm⁻¹) of SAL is clearly visible in the spectrum. -1 ) and the out-of-plane bending vibration of the para-substituted benzene ring CH (821 cm) -1 Characteristic absorption peaks such as )

[0044] These data strongly confirm that SAL has been successfully grafted onto the surface of PAMAM dendritic molecules through chemical bonding, and SAL-PAMAM has been successfully prepared.

[0045] Results of in vitro drug release experiments Experimental methods: (1) Construction of SAL standard curve: ① Preparation of stock solution: Prepare 1 mg / mL SAL stock solution. ② Preparation of series of standard solutions: Dilute the stock solution stepwise to prepare standard solutions of different concentrations (1, 50, 100, 200, 400, 600, 800, 1000 μg / mL). ③ Injection analysis. ④ Construction of standard curve: Plot linear regression with SAL concentration (X) as the abscissa and peak area (Y) as the ordinate to obtain the standard curve equation Y=aX+b and the correlation coefficient R. 2 (2) Recording of in vitro drug release: ① PBS (pH=7.4) was selected as the release medium to simulate the environment of human blood and extracellular fluid. ② 33 mg SAL and 100 mg SAL-PAMAM were dissolved in 4 mL of ultrapure water respectively, transferred to two dialysis bags, and the dialysis bags were tied and placed in two beakers containing 200 mL of PBS (pH=7.4), ensuring that the liquid level in the bag was lower than the liquid level of the release medium. ③ The beakers were placed in a 37℃ water bath and magnetically stirred. ④ Samples were taken at preset times (0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h). ⑤ 5 mL of sample was taken each time, and 5 mL of fresh release medium was added to maintain the total volume and trough conditions. ⑥ The taken samples were filtered through a 0.22 μm microporous membrane and analyzed by HPLC. ⑦ The HPLC analysis conditions were as follows: C18 reverse-phase column, mobile phase (acetonitrile:water = 12.5:87.5, v / v), flow rate (1.0 mL / min), detection wavelength 275 nm. After detection, the peak area was calculated, and the drug concentration (C0) was recorded by substituting it into the standard curve. The release percentage (Q%) was then calculated. The calculation formula is: Q% = [(V0 × C0) / ( ... n )+V×∑(C i )] / W total_drug ×100%; Q%: Cumulative release percentage at time point n; V0: Total volume of the release medium (mL); C n: Drug concentration measured at the i-th time point (μg / mL); V: Volume of each sample taken (mL); ∑(C i W: The sum of drug concentrations (μg / mL) measured from time point 1 to time point n-1; total_drug Total amount of drug (μg) contained in the drug delivery system inside the dialysis bag.

[0046] Test result: Pass 1 ¹H NMR calculations showed that the drug loading of SAL-PAMAM was 33.3%. To evaluate the in vitro release characteristics of the SAL-PAMAM nanoformulation, the release behavior of free SAL and SAL-PAMAM was investigated in PBS buffer at pH 7.4. The results are as follows: Figure 15 As shown in Figure C, the release of free SAL exhibits typical rapid release characteristics. Within the first 2 hours of the release experiment, the cumulative release rate of free SAL rapidly reached 80%, indicating that the free drug readily diffuses through the dialysis membrane into the medium. Subsequently, the release leveled off, and by approximately 3 hours, the cumulative release rate had reached over 95%, indicating near-complete release. In stark contrast, the SAL-PAMAM nanoformulation exhibited significant sustained-release properties. Within the first 24 hours of release, the immediate release rate of SAL reached approximately 80%. After 24 hours, the release rate slowed significantly, entering a stable and sustained release period, reaching approximately 90% by 48 hours.

[0047] The results indicate that loading SAL onto PAMAM significantly slows down the drug release rate. This sustained-release behavior facilitates long-term drug circulation in the body, reduces dosing frequency, and may promote drug accumulation at the lesion site through enhanced penetration and retention effects.

[0048] III. Pharmacokinetic Experimental Results of the Product of this Invention Experimental methods: (1) Blood drug concentration test: ① After successful modeling of male SD rats, 6 rats were randomly divided into 2 groups (n=3). SAL and SAL-PAMAM were administered by tail vein injection, respectively. Blood was collected at different time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h). Whole blood was placed in centrifuge tubes containing heparin anticoagulant and plasma was collected by centrifugation. ② Plotting standard curve: Untreated rat fundus blood was taken to prepare plasma samples. 500 μL of SAL methanol solution of each concentration was added to every 500 μL of plasma. Acetaminophen (10 μg / mL) was used as an internal standard. After vortexing for 2 min, the supernatant was collected by centrifugation (4℃, 12000r / min, 20 min). The sample was dried with nitrogen. ③ The sample was reconstituted using 500 μL of mobile phase (methanol:water = 2:8, v:v). The sample was filtered through a 0.22 μm microporous membrane and analyzed by HPLC. The HPLC analysis conditions were: C18 reverse-phase column, mobile phase (methanol:water = 2:8, v:v), flow rate (1.0 mL / min), and detection wavelength of 275 nm. The peak area was calculated after detection. ④ Linear regression was performed with the concentration of SAL (X) as the abscissa and the peak area (Y) as the ordinate to obtain the standard curve equation Y = aX + b and the correlation coefficient R². ⑤ The processed plasma samples were injected sequentially, and the peak area was calculated after detection. The peak area was then substituted into the standard curve to record the drug concentration (Ct) of the sample taken at each time point. A drug-time curve was plotted with time (t) as the abscissa and blood drug concentration (Ct) as the ordinate.

[0049] (2) Brain tissue biodistribution test: ① After successful modeling in male SD rats, 36 rats were randomly divided into two groups (n=3). SAL and SAL-PAMAM were administered via tail vein injection, respectively. Brain tissue was collected at different time points (1h, 2h, 4h, 8h, 12h, 24h). ② Standard curve plotting: Untreated rat brain tissue was taken, the surface blood was rinsed with physiological saline, dried with filter paper, accurately weighed, and recorded. The weighed brain tissue was placed in a homogenization tube, and 5mL of physiological saline was added to prepare a homogenized tissue homogenate. ③ Take 500 μL of homogenate into a centrifuge tube, add 500 μL of SAL standard solution of known concentration to prepare a series of concentrations (10, 20, 50, 100, 200, 600, 800, 1000, 2000 μg / g tissue), add acetaminophen (10 μg / mL) as an internal standard, vortex for 2 min, centrifuge and collect the supernatant, and dry the sample with nitrogen. ④ Reconstitute the sample with 500 μL of mobile phase (methanol:water = 2:8, v:v), filter the sample through a 0.22 μm microporous membrane, and perform HPLC analysis. The HPLC analysis conditions are: C18 reverse-phase column, mobile phase (methanol:water = 2:8, v:v), flow rate (1.0 mL / min), detection wavelength 275 nm, and calculate the peak area after detection. ⑤ Using the concentration (X) of SAL as the x-axis and the peak area (Y) as the y-axis, a linear regression was performed to obtain the standard curve equation Y=aX+b and the correlation coefficient R². ⑥ The processed brain tissue homogenate samples were sequentially injected, and after detection, the peak area was calculated and substituted into the standard curve to record the drug concentration (C) in the brain tissue at each time point. n ). With time (t) as the x-axis, the drug concentration in brain tissue (C) n Plot a curve with y as the ordinate.

[0050] Test Results: To evaluate the ameliorative effect of PAMAM-loaded SAL on the in vivo behavior of SAL, the pharmacokinetic parameters of the SAL-PAMAM group and the SAL solution group were compared. Blood samples were collected from DM rats at different time points, and the plasma concentration-time curves between the two groups are shown below. Figure 16 As shown in Figure A, the area under the drug-time curve (AUC(0-t)) of SAL in the SAL-PAMAM group was significantly increased by 2.5 times compared to the free SAL group, indicating that the nano-formulation greatly improved the in vivo systemic exposure of SAL. The trend of AUC(0-∞) was completely consistent with this. Compared to the elimination half-life (t) of free SAL... 1 / 2 The mean t in the SAL-PAMAM group was 1.057 ± 0.008 h. 1 / 2 The time to elimination from the body increased to 3.284 ± 0.077 h, indicating that PAMAM effectively delayed the elimination process of the drug from the body. This suggests that SAL-PAMAM can improve the bioavailability of SAL in vivo, which is beneficial for enhancing therapeutic effects.

[0051] This study further investigated the biodistribution characteristics of free SAL and the SAL-PAMAM nanocarrier system in diabetic rats at different time points (1, 2, 4, 8, 12, 24 h). Figure 16 (B) Considering that the blood-brain barrier may be disrupted or its permeability altered due to pathological conditions in this disease model, the experimental results are of great significance for evaluating the brain targeting ability of nanomedicines. The results showed that at all detection time points, the SAL concentration in the brain tissue of the SAL-PAMAM group was significantly higher than that of the free drug group. This result suggests that the SAL-PAMAM nanosystem can not only effectively deliver higher doses of SAL to the brain, but also demonstrates good brain targeting ability and selective release capability at lesion sites even in pathological environments where the blood-brain barrier may be impaired.

[0052] IV. In vitro safety of the product of this invention—hemolysis test and CCK8 cell safety test of SAL-PAMAM Hemolysis test: (1) 1 mL of blood was collected from the orbital venous plexus of normal SD rats and placed in an anticoagulant tube. The blood was centrifuged (3000 r / min, 30 min) to separate plasma and red blood cells. The plasma was discarded and the red blood cells were left. 0.9% physiological saline was added for washing and centrifuged three times (3000 r / min, 30 min) until the supernatant was colorless. (2) The red blood cells were diluted with 0.9% physiological saline to a 2% concentration red blood cell suspension. (3) 2% red blood cell suspensions were placed in several EP tubes and centrifuged (3000 r / min, 30 min). The supernatant was discarded. (4) Different concentrations (50, 100, 200, 400, 800 μg / ml) of the test drug were dissolved in 0.9% physiological saline and added to the red blood cell suspension prepared in step 4. At the same time, 1 mL of 0.9% physiological saline was used as a negative control and 1 mL as a positive control. (5) Mix the solution, incubate at 37℃ for 30 min, then centrifuge (3000 r / min, 20 min) and record the color. (6) Take 100 μL of each sample into a 96-well plate and measure the OD value at 540 nm. The calculation formula is as follows: Hemolysis rate % = [(sample to be tested - negative control) / (positive control - negative control)] × 100%. A hemolysis rate exceeding 5% is considered hemolysis.

[0053] SAL-PAMAM CCK8 cell safety experiment: (1) BV-2 cell suspension was seeded into culture flasks and cultured. (2) When the cell density was moderate, BV-2 cells were digested with trypsin. (3) The supernatant was collected, centrifuged (1200 r / min, 5 min), the supernatant was discarded, and the cells were resuspended in culture medium. (4) Cells were counted using a hemocytometer, and then diluted to 5 × 10⁻⁶. 3 Up to 5×10 4Single-cell suspension of cells / mL. Seed cells in 96-well plates. Five replicate wells were designed for each concentration group, and blank and control groups were set up. Pre-cultured in an incubator for 24h. Add different concentrations of SAL-PAMAM to each well and continue incubation for 24h. Add 10μL CCK8 solution. Continue incubation for 2h. Measure the OD value at 490nm. (5) Data analysis, cell viability calculation formula: survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%.

[0054] Test Results: The biocompatibility of SAL-PAMAM was assessed using cell safety and hemolysis assays. CCK8 results are as follows: Figure 17 As shown in Figure A, the results indicate that within the drug concentration range of 0-800 μg / mL, cell viability decreased in a dependent manner with increasing SAL-PAMAM concentration, but overall viability remained above 80%, demonstrating that SAL-PAMAM exhibits low cytotoxicity within a certain range. Hemolysis experiments are as follows... Figure 17 As shown in B and C, SAL-PAMAM showed no significant hemolysis at a concentration of 1600 μg / mL, and the hemolysis rate was less than 5%, demonstrating that the material has good in vitro safety.

[0055] V. In vitro experimental verification of the cross-border movement of the blood-brain barrier and uptake by microglia of the product of this invention, SAL-PAMAM-Ro. Experimental methods: (1) Establishment of cell culture and Transwell co-culture model: ① Cell culture: Mouse brain microvascular endothelial cells bEnd.3 and mouse microglial cell line BV-2 were cultured in DMEM complete medium. ② Model construction: BV-2 cells were digested and resuspended, and seeded in 24-well plates (lower chamber) and cultured overnight to allow the cells to adhere. bEnd.3 cells were digested and resuspended, and seeded on the inner side of the upper chamber membrane of the Transwell chamber. The chamber seeded with bEnd.3 was placed back into the 24-well plate containing BV-2 cells to form a co-culture system. Until bEnd.3 fused to form a tight monolayer, simulating the complete blood-brain barrier. (2) SAL / PAMAM-Ro cross-barrier transport experiment: ① After the bEnd.3 cell monolayer was formed, the old medium in the upper chamber was removed. Fresh medium containing SAL / PAMAM-Ro (100µg / mL) was added to the upper chamber. At the same time, medium without nanoparticles was added to the control well. ② Place the Transwell plate back into the incubator and continue incubation for 0.5h, 1h and 4h respectively. (3) Sample collection for detection and analysis: ① After reaching each preset time point, remove the Transwell chamber. Aspirate the liquid in the upper chamber and wash the chamber 3 times with PBS. Digest the BV-2 cells in the lower chamber with trypsin and collect them in a flow cytometer. Wash with PBS, centrifuge and discard the supernatant. Resuspend the cells with PBS and detect them using a flow cytometer. Use the untreated group of cells as a negative control for testing. ② Take another cell, fix it with 4% PFA for 15min and wash it with PBS. Add 0.5% Triton X-100 and incubate at room temperature for 30min, and wash the cells three times with PBS. Block the tissue with goat serum for 30min. Then add the primary antibody to the cells and incubate overnight at 4℃. Take out the cells and place them at room temperature for 30min, and then wash the cells three times with PBS. Incubate the fluorescent secondary antibody in the dark for 2h and wash with PBS. Add DAPI to the cells, incubate in the dark for 5 minutes, mount the slide, and photograph it using a laser confocal microscope.

[0056] Results: To investigate the ability of fluorescently labeled SAL-PAMAM to penetrate the blood-brain barrier and its kinetics of uptake by microglia, we constructed an in vitro blood-brain barrier model using a Transwell co-culture system and synthesized SAL-PAMAM (SAL-PAMAM-Ro) with the Rhodamine B fluorescent group without disrupting the original structure or altering the properties of the compounds. bEnd.3 cells were seeded in the upper chamber of the Transwell to simulate the endothelial layer of the blood-brain barrier, and BV-2 mouse microglia were seeded in the lower chamber to simulate target cells in the brain parenchyma. Before drug administration, the integrity of the in vitro blood-brain barrier model was verified. The barrier density of the bEnd.3 cell monolayer was assessed by detecting the transendothelial permeability of sodium fluorescein (a small molecule permeability tracer). The results showed that by day 6 of culture, the apparent permeability coefficient (Papp) of sodium fluorescein was significantly lower than that of the blank control wells, indicating that dense tight junctions had formed between bEnd.3 cells, effectively limiting the free diffusion of small molecules and confirming the successful construction of the in vitro blood-brain barrier model. After bEnd.3 cells formed a tight monolayer, SAL-PAMAM-Ro (…) was added to the upper chamber. Figure 18 A) cells were co-incubated for 0.5, 1, and 4 hours, respectively. The fluorescence intensity of lower chamber BV-2 cells was then quantitatively detected by flow cytometry, and changes in intracellular fluorescence intensity were observed using laser confocal microscopy. Flow cytometry results showed that the proportion of positive results for lower chamber BV-2 cells increased in a time-dependent manner with prolonged incubation time (A). Figure 18 (B, C) Fluorescence signal enhancement could be detected at 0.5h, and the signal significantly increased from 1 to 4h; laser confocal microscopy further confirmed this result, showing that the fluorescence signal in the cytoplasm of BV-2 cells gradually increased over time, and was mainly located in the cytoplasm ( Figure 18 (D, E). In summary, this study successfully constructed a Transwell blood-brain barrier model, demonstrating that SAL-PAMAM can effectively penetrate the bEnd.3 cell monolayer and be taken up by BV-2 cells in the lower chamber in a time-dependent manner. These results provide experimental evidence for the nanoparticles as a potential drug delivery carrier for the central nervous system.

[0057] To investigate the inhibitory effect of SAL-PAMAM on high glucose-induced BV-2 microglia activation, this study used Western blotting to detect the expression levels of inflammation-related proteins. The HG group was used to simulate the activation state of BV-2 cells under the high glucose environment of diabetes, the MA group was used to exclude the influence of osmotic pressure changes, and the SAL, SAL-PAMAM, and PAMAM groups served as treatment groups. Results are as follows: Figure 19As shown, under high glucose stimulation, the protein expression of pro-inflammatory factors IL-1β, IL-6, and TNFα in BV-2 cells increased (P<0.001), while the protein expression of anti-inflammatory factors IL-10 and IL-4 decreased (P<0.001). The protein expression levels of M1 markers CD86 and iNOS significantly increased (P<0.001), while the protein expression levels of M2 markers CD206 and Arg1 significantly decreased (P<0.001). After treatment with SAL and SAL-PAMAM, the protein expression of pro-inflammatory and anti-inflammatory factors and markers reversed, with the most significant changes observed in the SAL-PAMAM group (P<0.01). These results indicate that, compared with the SAL group, SAL loaded with PAMAM more effectively inhibited high glucose-induced BV-2 cell activation, exhibiting a stronger anti-neuroinflammatory effect.

[0058] VI. The product SAL-PAMAM of this invention has excellent brain-targeting ability. Experimental methods: (1) SD rats were injected with 303 μg / mL SAL-PAMAM-Ro via tail vein. Brain tissue, heart, liver, spleen, lung, and kidney of the rats were collected at various time points (1h, 2h, 3h, 4h, 6h, 12h, 24h). Imaging was performed using a small animal in vivo imaging system. (2) Rats were divided into CON group and DM group, and injected with 0.9% NaCl solution and 303 μg / mL SAL-PAMAM-Ro via tail vein, respectively. The brain tissue of the rats was imaged using a small animal in vivo imaging system 2h after administration. λex=488nm, λem=500-540nm.

[0059] Test Results: To investigate the ability of PAMAM-loaded SAL to penetrate the blood-brain barrier and its distribution characteristics in vivo, we performed in vivo imaging analysis on various tissues of DM rats at different time points after tail vein injection of SAL-PAMAM-Ro. Figure 20 (AG). Quantitative fluorescence intensity results showed that fluorescence signals could be detected in various tissues 1 hour after administration; the fluorescence signal in brain tissue significantly increased after 2 hours, indicating that SAL-PAMAM rapidly accumulated in the brain and reached peak concentration at this time point; the signal in the brain gradually weakened after 4 hours, and was basically metabolized by 24 hours. In addition, organ imaging results showed that the fluorescence intensity in the liver and kidneys was significantly higher than that in other organs, suggesting that SAL-PAMAM is mainly metabolized through the liver and kidneys.

[0060] We then conducted further intergroup comparisons, and the results showed that no fluorescent signal was observed in the brains of rats in the control group injected with saline. Compared with DM rats, the uptake of SAL-PAMAM-Ro in the brain tissue of DM rats was significantly increased 2 hours after drug administration. Figure 20HI). Our analysis suggests that this is primarily related to the disruption of blood-brain barrier integrity and increased permeability in diabetic patients. This result directly confirms that SAL-PAMAM possesses highly efficient brain-targeted delivery capabilities, and that it is even more advantageous in pathological states. Figure 20 JK).

[0061] To investigate the cellular uptake of SAL-PAMAM in brain tissue, we used immunofluorescence co-localization technology for analysis. Figure 21 The results showed that the fluorescence signal of SAL-PAMAM-Ro was significantly colocalized with microglia marker IBA-1, astrocyte marker GFAP, and neuronal marker NeuN, indicating that the delivery system can be widely taken up by neurons, astrocytes, and microglia after entering the brain, and does not have strict cell selectivity.

[0062] Given that microglia, as innate immune cells of the central nervous system, play a crucial role in driving neuroinflammation in the progression of diabetes-related cognitive impairment, and considering that this study primarily focuses on exploring the regulatory effects and mechanisms of SAL-PAMAM on neuroinflammation, subsequent analyses will concentrate on the regulatory effects of SAL-PAMAM on microglia function. These findings provide an important cellular basis for understanding that SAL-PAMAM may exert its anti-inflammatory effects through its action on microglia.

[0063] VII. The product SAL-PAMAM of this invention can effectively improve cognitive dysfunction in rats. Experimental Methods: Each rat entered the water from a different quadrant and had 60 seconds to search for the platform. If the rat found the platform within 60 seconds, the time was recorded and the rat was removed. If it did not find the platform, it was placed on the platform for 10 seconds to enhance learning and memory; this time was recorded as 60 seconds. Days 1 to 5 were the learning and memory phase for the rats. On days 6 and 7, the platform was removed, and the rats were placed into the pool from the opposite side of quadrant 4, facing the pool wall. The EthoVision tracking system was used to record the escape latency, the number of times the rats crossed the platform, and the percentage of time spent in the target quadrant within 60 seconds.

[0064] We used the Morris water maze test to assess the spatial cognition and memory abilities of rats in each group. Figure 22As shown, compared with the CON group, the DM group rats exhibited significant impairments in spatial cognition and memory, mainly manifested as a significantly prolonged escape latency (P<0.001) and a reduced number of platform crossings in the spatial exploration experiment, with a significantly shortened time spent in the target quadrant. These findings indicate that the DM group rats had significantly impaired spatial cognition and memory. This comprehensive deterioration of these classic behavioral indicators confirms the successful establishment of the DM animal model and provides a clear pathological benchmark for subsequent evaluation of the intervention efficacy of the SAL-PAMAM nanodelivery system. The positioning and navigation results showed ( Figure 22 C), after treatment with SAL and SAL-PAMAM, the escape latency time of rats was significantly reduced (P<0.001), with the SAL-PAMAM group showing a significantly shorter latency; in the space exploration experiment ( Figure 22 Rats treated with SAL-PAMAM exhibited improved spatial orientation abilities, primarily reflected in the higher number of platform crossings and the longer time spent in the target quadrant compared to the DM group (P<0.001), with SAL-PAMAM showing a more significant therapeutic effect. There were no statistically significant differences in swimming speed among the groups. Behavioral experiments confirmed that SAL-PAMAM treatment effectively improved the spatial learning, memory, and cognitive abilities of DM rats, and that SAL-PAMAM-loaded treatment was significantly superior to SAL-PAMAM alone, demonstrating a major therapeutic advantage of SAL-PAMAM.

[0065] 8. SAL-PAMAM inhibits the activation of rat hippocampal microglia and downregulates the expression of inflammatory cytokine proteins. To further explore the effects of SAL-PAMAM on microglial activation and its potential mechanisms, we treated diabetic rats with SAL-PAMAM and systematically analyzed the changes in microglial morphology, phenotypic transformation, and expression of inflammatory factor proteins using various experimental methods.

[0066] (I) Effects of SAL-PAMAM on microglia activation Immunohistochemical staining results showed ( Figure 23 A) Compared with the CON group, the morphology of microglia in the hippocampus of rats in the DM group was significantly altered, specifically manifested as a decrease in cell processes, a shortening in length, and an enlarged cell body (P<0.001). Clear cytoskeleton remodeling revealed these structural changes, suggesting that the microglia were in an activated state. Statistical analysis further confirmed the above observations. Figure 23(BD), all morphological indicators in the DM group were statistically different from those in the control group. After intervention with SAL and SAL-PAMAM, the number of microglia in the hippocampus of DM rats increased significantly, the length of the microglia increased significantly, and the cell body area decreased significantly. The improvement effect was more significant in the SAL-PAMAM group (P<0.001), indicating that SAL-PAMAM can more effectively reverse the activated phenotype of microglia.

[0067] Immunofluorescence results showed that, compared with the CON group, the fluorescence intensity of CD86 in the brain tissue of DM rats was significantly enhanced (P<0.001). After treatment, compared with the DM group, the fluorescence intensity of the SAL group and the SAL-PAMAM group was significantly reduced, and the SAL-PAMAM group showed the most significant effect (P<0.01). Figure 24 A, C). This indicates that SAL-PAMAM can effectively inhibit the activation of M1 microglia in the brain. Western blotting results showed that, compared with the CON group, the protein expression levels of M1 microglia markers CD86 and INOS were significantly upregulated in the DM group, while the protein expression levels of M2 microglia markers CD206 and Arg1 were significantly downregulated (P<0.001). Figure 24 B, DG). After SAL-PAMAM and SAL treatment, the protein expression levels of M1 markers CD86 and iNOS were significantly reduced, while the protein expression levels of M2 markers CD206 and Arg1 were significantly increased. The therapeutic effect of SAL-PAMAM was more significant (P<0.05).

[0068] (II) Effects of SAL-PAMAM on the expression of inflammatory cytokine proteins To further confirm the effect of SAL-PAMAM on the inflammatory response of hippocampal microglia, we used Western blotting to detect the protein expression levels of inflammatory factors in hippocampal tissue. Figure 25 The experimental results showed that, compared with the CON group, the protein expression of inflammatory factors TNF-α, IL-1β, and IL-6 in the hippocampus of rats in the DM group was increased, while the protein expression levels of anti-inflammatory factors IL-10 and IL-4 were significantly decreased (P<0.001). However, after treatment with SAL and SAL-PAMAM, the protein expression of pro-inflammatory and anti-inflammatory factors was reversed, with the most significant change observed in the SAL-PAMAM group (P<0.05). In conclusion, SAL-PAMAM exhibits superior anti-neuroinflammatory activity compared to the SAL group by effectively inhibiting excessive microglial activation.

[0069] IX. SAL-PAMAM In Vivo Safety Testing Blood routine and biochemical index analysis experiment of SD rats: After acclimatization, rats were administered SAL-PAMAM at a dose of 303 mg / kg via tail vein injection, while the control group received an equal volume of the solvent. Treatment was administered twice weekly for 8 weeks, with rats fasted but allowed free water for 12 hours. Blood was collected from the retro-orbital venous plexus in procoagulant tubes for blood routine analysis.

[0070] Blood routine and blood biochemical index analysis results The in vivo safety of SAL-PAMAM was evaluated. The primary method involved collecting blood samples from normal rats 8 weeks after administration for complete blood count and blood biochemistry analysis. Results are as follows: Figure 26 As shown, compared with the CON group, no significant abnormalities were observed in the blood routine indicators of rats injected with SAL-PAMAM, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), kidney function indicators such as blood urea nitrogen (BUN) and creatinine (CR), and serum albumin (ALB). This indicates that SAL-PAMAM does not affect the normal function of the tested rats.

[0071] HE staining results of various organs of rats like Figure 27 As shown, after intravenous injection of SAL-PAMAM, no obvious pathological changes or signs of damage were observed in the liver, heart, spleen, lungs, and kidneys of rats; cell structure and morphology remained normal, proving that it does not cause systemic toxicity at the current dose. This good safety profile may stem from the material's biodegradable chemical structure, biofriendly composition, optimized nanoscale size, and surface charge, among other advantages. Biosafety assessments of SAL-PAMAM indicate that the material exhibits good biocompatibility and low toxicity both in vivo and in vitro.

[0072] The above are merely specific embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing dendritic molecularly loaded rhodioloside nanomaterials, characterized in that: Includes the following steps: (1) Rhodioloside was reacted with trimethylchlorosilane and hexamethyldisilazane in anhydrous pyridine and stirred at room temperature for 24 h. After washing, the mixture was purified by silica gel column chromatography to obtain compound 1. (2) Dissolve compound 1 in dichloromethane to prepare a compound 1 solution with a molar concentration of 0.25 mol / mL; dissolve ammonium acetate in methanol to prepare an ammonium acetate solution with a molar concentration of 0.5 mol / mL; slowly add the ammonium acetate solution dropwise to the compound 1 solution under stirring, and react at -20℃ for 24 h; After washing, the compound was purified by silica gel column chromatography to obtain compound 2; (3) Compound 2 was dissolved in anhydrous dichloromethane to prepare a compound 2 solution with a molar concentration of 0.135 mol / mL; the compound 2 solution was cooled to 0℃, and triethylamine and succinic acid monobenzyl ester chloride were added sequentially at 0℃ with stirring. After stirring evenly, the reaction was continued at room temperature for 2 h; the compound 3 was obtained by silica gel column purification. (4) Compound 3 was dissolved in chromatographic grade methanol to prepare a compound 3 solution with a molar concentration of 0.1 mol / mL, and palladium catalyst on carbon was added; the reaction was stirred at room temperature for 4 h under hydrogen atmosphere; the catalyst was removed by filtration, the solvent was removed by concentration under reduced pressure, and the compound 4 was obtained by silica gel column purification. (5) Dissolve the 64 hydroxyl-terminated polyamide-amine dendritic molecules in N,N-dimethylformamide to obtain a N,N-dimethylformamide solution of hydroxyl-terminated polyamide-amine dendritic molecules with a molar concentration of 0.0033 mmol / mL; Compound 4 was dissolved in N,N-dimethylformamide to obtain a solution of compound 4 with a molar concentration of 0.134 mmol / mL; According to the molar ratio of compound 4 to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine of 0.67:1.035:0.242, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added sequentially to the solution of compound 4. After stirring and activating at room temperature for 10 min, a solution of N,N-dimethylformamide containing 64 hydroxyl-terminated polyamide-amine dendritic molecules was added dropwise. The molar ratio of the 64 hydroxyl-terminated polyamide-amine dendritic molecules to compound 4 was 0.033:0.

67. The reaction was continued at room temperature for 48 h. After the reaction was completed, the reaction solution was diluted with deionized water, dialyzed in deionized water for 12 h, and freeze-dried to obtain compound 5. (6) The compound 5 obtained in step (5) was subjected to a reaction to remove the trimethylsilyl protecting group in N,N-dimethylformamide in the presence of trifluoroacetic acid. After neutralization, dialysis and drying, the rhodioloside-hydroxy-terminated polyamide-amine dendritic molecule (SAL-PAMAM) was obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of rhodioloside, trimethylchlorosilane and hexamethyldisilazane is 1:6:

2.

3. The method for preparing dendritic molecularly supported rhodioloside nanomaterials according to claim 1, characterized in that: In step (2), the molar ratio of compound 1 to ammonium acetate is 1:

2.

4. The method for preparing dendritic molecularly supported rhodioloside nanomaterials according to claim 1, characterized in that: In step (3), the molar ratio of compound 2, triethylamine and succinic acid monobenzyl chloride is 2.7:16.1:4.

1.

5. The method for preparing dendritic molecularly supported rhodioloside nanomaterials according to claim 1, characterized in that: In step (4), the mass ratio of compound 3 to palladium on carbon catalyst is 10:

1.

6. The method for preparing dendritic molecularly supported rhodioloside nanomaterials according to claim 1, characterized in that: In steps (5) and (6), dialysis is performed using dialysis bags with a molecular weight cutoff of 1000 Da.

7. The method for preparing dendritic molecularly supported rhodioloside nanomaterials according to claim 1, characterized in that: In step (6), the amount of trifluoroacetic acid used is 0.12 mL of trifluoroacetic acid per 100 mg of compound 5; the reaction to remove the trimethylsilyl protecting group is carried out at 0°C to room temperature for 1 hour; and sodium bicarbonate aqueous solution is used for neutralization.

8. Dendritic molecularly loaded rhodioloside nanomaterials prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the dendritic molecularly loaded rhodioloside nanomaterials prepared by the method according to claim 1 in drugs for targeted penetration of the blood-brain barrier to treat cognitive impairment in diabetes.

Images