Nasal administration three-stage recursion type targeting hydrogel microsphere as well as preparation method and application of nasal administration three-stage recursion type targeting hydrogel microsphere

Abstract

The invention provides a nasal drug delivery three-stage recursion type targeting hydrogel microsphere as well as a preparation method and application thereof, and belongs to the technical field of drug delivery. The invention develops a three-stage recursive targeting nasal drop, and the first-stage nasal mucosa targeting is realized by non-invasive drug delivery into the brain through the charge modified hydrogel microsphere nasal cavity; a targeting liposome carrying CTLA-4 is combined with the activated microglial cells, so that second-stage cell targeting is realized; mitochondrial regulation and control are carried out by releasing DHEA to complete three-stage targeting, and finally, multi-stage precise targeting and intracerebral regulation and control are realized. The three-stage recursion type targeted nasal drops can break through the complex physiological and pathological microenvironment of a brain body layer by layer to directly reach lesion core mitochondria of microglia cells, the Drp-1 pathway is regulated and controlled through DHEA, the abnormal mitochondrial division is remarkably inhibited, the mitochondrial morphological function is stabilized, microglia cell activation is inhibited, and cognitive impairment caused by anesthesia operation is relieved. In conclusion, the research provides a new method for treating various central nervous system diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery, and in particular relates to a three-stage recursive targeting hydrogel microsphere for nasal administration, and a preparation method and application thereof. Background Art

[0002] Due to the specificity and complexity of the brain's structure and function, the treatment of central nervous system diseases has always been faced with the following difficulties: (1) Due to the existence of the blood-brain barrier, the efficiency of peripheral drug delivery to the brain is low and it is difficult for drugs to enter the brain; (2) The microenvironment in the brain is complex, with multiple interacting cell types, making it difficult to locally enrich drugs in pathological cells; (3) How to enable drugs to enter cells and precisely target and regulate organelles.

[0003] As the only resident immune cells in the central nervous system, microglia play a crucial role in maintaining neural homeostasis, clearing damaged cellular debris, and regulating inflammatory responses. Microglia are implicated in a range of central nervous system diseases, such as Alzheimer's disease, Parkinson's disease, cerebral ischemia, and septic encephalopathy. Under pathological conditions, microglia become overactivated and release proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, as well as harmful metabolites such as reactive oxygen species (ROS) and lactate. These proinflammatory substances further trigger an inflammatory cascade, leading to neural damage and, subsequently, abnormal brain function. Therefore, targeting microglia to regulate central inflammatory responses is expected to become a potential therapeutic target for numerous central nervous system diseases.

[0004] DHEA is the most abundant steroid hormone in the body, with anti-inflammatory and antioxidant properties. Studies have shown that DHEA levels in the brains of elderly patients decrease with age, which may be a major cause of brain inflammation and central inflammatory diseases such as AD. DHEA supplementation can improve cognitive function in elderly patients. Therefore, targeting microglia to deliver drugs such as DHEA is an important therapeutic strategy for treating central nervous system diseases. CD80 / 86 is a lymphocyte co-stimulatory molecule that is primarily expressed in immune cells such as monocytes and macrophages. Microglia, as the only resident immune cells in the brain, specifically express CD80 / 86 in the brain. In neuroinflammatory states, the expression of CD80 / 86 in activated microglia is further upregulated. Therefore, using the CD80 / 86 ligand CTLA-4 as a targeting protein is expected to achieve precise microglial targeting to regulate inflammation. However, more than 99% of DHEA in the circulation exists in the form of the low-activity sulfide DHEAS. Most of the DHEA administered peripherally will be converted into DHEAS by the sulfotransferase SULT2A1, resulting in its inability to cross the blood-brain barrier.

[0005] Inflammatory activation of microglia is closely linked to metabolic reprogramming involving mitochondria. As the center of energy metabolism, mitochondria play a central role in cellular homeostasis. Mitochondria continuously fission and fusion, altering their morphology, structure, and function. When mitochondrial fusion is impaired or fission is hyperactive, excessive mitochondrial fragmentation can lead to disease. Therefore, regulating abnormal mitochondrial fission in microglia is crucial for the treatment of central nervous system diseases.

[0006] Drp-1 is a key protein in maintaining mitochondrial morphology and the only mitochondrial fission protein in mammals. Drugs that directly regulate Drp-1, a regulatory protein in the mitochondrial outer membrane, can precisely regulate mitochondria. However, existing mitochondrial-targeting materials suffer from numerous issues, such as low targeting efficiency, drug and material toxicity, and uncontrollable targeted drug activity, which hinder their efficacy.

[0007] Therefore, whether DHEA can be used as an important neurotransmitter to target the regulation of mitochondrial membrane remodeling protein Drp-1, in order to achieve mitochondrial structure regulation by stabilizing microglial Drp-1, there is currently no relevant research, and how to avoid the problem of DHEA existing in the form of low-activity sulfide DHEAS and being unable to pass through the blood-brain barrier still needs to be further resolved. On the other hand, affected by the complex physiological and pathological microenvironment of the brain, conventional drug administration methods make it difficult to locally enrich effective drug concentrations and target them to the mitochondria of microglia to exert their efficacy. Therefore, how to develop a corresponding targeted delivery system to achieve the smooth delivery of DHEA to the mitochondria of glial cells is crucial.

[0008] In recent years, nasal administration has rapidly developed as a new method for brain drug delivery. Because the nasal cavity is rich in blood vessels and nerve plexuses, drugs can be transported through a combination of multiple pathways, avoiding digestive tract damage and first-pass effects, and bypassing the blood-brain barrier (BBB) ​​and blood-cerebrospinal fluid barrier (BCSFB). Secondly, the nasal cavity is close to the brain, which can rapidly increase drug concentrations in the brain and spinal cord, facilitating targeted therapy and reducing side effects. Compared with the low brain entry efficiency of peripherally administered drugs and the greater trauma of intracranial and intrathecal injections, nasal administration is more non-invasive and convenient, with good patient compliance. Therefore, the "nose-to-brain route" of drug delivery holds great promise as a potential new treatment for brain diseases. However, the rapid clearance of nasal mucociliary fluid, the degradation of nasal drops by multiple enzymes in the nose, and the small single-dose dosage limit the clinical translation and application of nasal administration.

[0009] On the other hand, existing brain-targeted nasal drops primarily target the nose and brain, using materials like liposomes and chitosan to facilitate drug delivery through the nasal mucosa. These solutions lack directional targeting and regulatory functions. Consequently, they are unable to target diseased microglia or achieve targeted regulation of mitochondrial function, resulting in low drug utilization. Consequently, existing nasal drops are generally ineffective in treating brain diseases.

[0010] Therefore, developing a drug carrier that can effectively adhere to the nasal mucosa, increase residence time, and avoid rapid clearance by the nasal mucociliary system, allowing drugs to enter the brain more efficiently, has become a technical problem that needs to be solved urgently. Although existing research has involved delivering drugs to the brain through the nasal cavity, the relevant materials can only deliver drugs to the brain and cannot locate specific cells and regulate organelles. At the same time, how to develop a drug carrier that can successfully carry DHEA, so as to realize the use of DHEA as an important neurotransmitter that targets the mitochondrial membrane remodeling protein Drp-1 and exerts its mitochondrial regulatory function, has become another technical problem that needs to be solved urgently. Summary of the Invention

[0011] The present invention aims to address the aforementioned technical issues, thereby providing a three-stage recursive targeted hydrogel microsphere for nasal drug delivery, as well as its preparation method and application. The technical objectives of the present invention are to develop a drug carrier that effectively adheres to the nasal mucosa, increases residence time, and avoids rapid clearance by the nasal mucociliary system, enabling more efficient drug delivery to the brain. Furthermore, the invention aims to successfully deliver DHEA across the blood-brain barrier into the brain, thereby utilizing DHEA as an important neurotransmitter regulated by the mitochondrial membrane remodeling protein Drp-1, thereby exerting mitochondrial regulatory functions and effectively treating central nervous system diseases.

[0012] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0013] The present invention first provides a method for preparing three-stage recursive targeting hydrogel microspheres for nasal administration, comprising the following steps:

[0014] (1) Using DOPE, DOTAP, and cholesterol as lipid raw materials, DHEA-loaded liposomes were prepared, and CTLA-4 was physically modified on the liposome phospholipid membrane by co-extrusion;

[0015] (2) Using hyaluronic acid and methacrylic anhydride as raw materials, the monomer ratio of the raw materials was controlled at 0.5:1, and methacrylated hyaluronic acid hydrogel microspheres were prepared by microfluidic method;

[0016] (3) The liposome solution obtained in step (1) is mixed with the methacrylated hyaluronic acid hydrogel microspheres obtained in step (2) to prepare drug-loaded hydrogel microspheres.

[0017] The above method of the present invention has successfully developed a "three-stage recursive targeted drug-loaded hydrogel microsphere", which can be non-invasively administered into the brain through the nasal cavity - microglial targeting - mitochondrial regulation, to achieve multi-stage targeting and precise delivery of drugs to the core of brain lesions. The method of the present invention first uses microfluidics technology and chemical functional modification to prepare hydrogel microspheres with positive surface charges, which effectively adhere to the nasal mucosa and avoid rapid cilia clearance of nasal mucus, thereby achieving the first stage of nasal mucosa targeting; then, a targeted liposome carrying CTLA-4 is constructed and modified in the microspheres. The microspheres swim with the cilia, releasing the liposomes into the brain through the nasal cavity, and precisely targeting microglia by binding to CD80 / 86 on the surface of activated microglia, thereby achieving the second stage of cell targeting; by responding to the microenvironment to release the endogenous neurotransmitter DHEA, the key protein of mitochondrial membrane remodeling Drp-1 is precisely regulated to inhibit the abnormal mitochondrial fission caused by its activation, thereby achieving the third stage of mitochondrial targeting. Studies have shown that this "three-stage recursive targeted drug-loaded hydrogel microsphere" can enable DHEA to directly reach the mitochondria in the microglial cell core, significantly inhibiting Drp-1 phosphorylation on the mitochondrial outer membrane, stabilizing mitochondrial morphology and function, inhibiting microglial activation, and alleviating cognitive dysfunction caused by anesthesia and surgery. Therefore, the method of this invention provides a potential new treatment strategy for central nervous system diseases.

[0018] The technical difficulty of the present invention lies in the fact that nasal mucosal adhesion requires a balance between positive charge density and biocompatibility. A carrier with too strong a charge can easily cause nasal mucosal inflammation, while a carrier with too weak a charge cannot resist ciliary clearance (normal clearance time is <15 minutes, but the present invention can extend this to several hours). Therefore, the present invention's solution experienced many setbacks during the trial and error process before finally successfully preparing the present invention's three-stage recursive targeted hydrogel microspheres for nasal administration.

[0019] As shown in the comparative example, firstly, the monomer ratio needs to be precisely controlled in the microfluidic preparation of the present invention, otherwise the microsphere particle size will be uneven, affecting the ciliary swimming efficiency.

[0020] Secondly, the raw materials of the present invention are irreplaceable: if natural positively charged materials such as chitosan are used instead, the adhesion time of the microspheres will be shortened by more than 60% due to enzymatic hydrolysis; if negatively charged materials (such as sodium alginate) are used instead, the mucosal adhesion rate of the microspheres will drop by 90%.

[0021] Third, the modification rate of CTLA-4 needs to be controlled at 8-12 molecules / liposome. If it is too low, it cannot effectively bind to CD80 / 86, and if it is too high, it will cause liposome aggregation. The liposome particle size must strictly match the microsphere pore size, otherwise it will cause drug burst release or retention. In addition, compared with the FITC-modified control group, the uptake efficiency of CTLA-4 liposomes into microglia is increased by more than 200 times (flow cytometry data), demonstrating the accuracy of targeting.

[0022] The targeted hydrogel microspheres constructed by the present invention, when the positively charged hydrogel microspheres are used alone, the DHEA drug is released suddenly and cannot be delivered to the center of brain disease; when the CTLA-4 liposomes are used alone for nasal administration, only 0.5% of the DHEA drug enters the brain. The three-level recursive system of the present invention increases the brain targeting efficiency to 40 times that of conventional nasal drops, and the targeting efficiency is extremely high. It has been verified by animal models: in PND model mice, the three-level recursive system group of the present invention has a 40% increase in the degree of reduction of brain inflammation (TNF-α) compared with the group with the CTLA-4 liposome system alone (p<0.01), and the improvement of cognitive behavior has increased by 50% (p<0.01), and there is no damage to the nasal ciliary structure.

[0023] Furthermore, the weight ratio of DOPE, DOTAP and cholesterol in step (1) is 2:2:1.

[0024] Furthermore, the liposomes described in step (1) are prepared by dissolving DOPE, DOTAP, cholesterol, and DHEA in a mixed solvent of chloroform and methanol (1 / 1, v / v), ultrasonically mixing, and drying on a rotary evaporator to a lipid film. PBS is added, and after hydration at 40°C for 20 minutes, the liposomes are co-extruded with CTLA-4 through a 0.22 μm film extruder to obtain liposomes.

[0025] Furthermore, in the microfluidic method described in step (2), the flow rate ratio of the aqueous phase to the oil phase is controlled to be 1:5.

[0026] Furthermore, the concentration of the liposome solution in step (3) is 2.5 wt %, and the concentration of the methacrylated hyaluronic acid hydrogel microspheres is 4 wt %.

[0027] Furthermore, the reaction conditions in step (3) are: 4 degrees Celsius, 90 minutes of gentle mixing.

[0028] Furthermore, the concentration of the polylysine solution in step (4) is 0.005-5 mg / mL, and the weight ratio of the microspheres to polylysine is 3:1-55:1.

[0029] The second object of the present invention is to provide three-stage recursive targeting hydrogel microspheres for nasal administration prepared by the above method.

[0030] Furthermore, the diameter of the three-stage recursive targeting hydrogel microspheres for nasal administration is about 195 to 200 μm.

[0031] The third object of the present invention is to provide the use of the three-stage recursive targeting hydrogel microspheres for nasal administration as described above in the preparation of drugs for treating central nervous system diseases.

[0032] Furthermore, the above-mentioned three-stage recursive targeted hydrogel microspheres for nasal administration are prepared into a nasal drop preparation for correcting abnormal mitochondrial fission of microglia in the brain.

[0033] The beneficial effects of the present invention are as follows:

[0034] This study successfully developed a "three-step recursive targeted nasal drop" capable of targeting the brain's core and microenvironmental response pathways. By establishing a POCD mouse model, transcriptomic analysis focused on microglia involved in POCD and mitochondria, key organelles in the regulation of neuroinflammation within the brain. Clinical data analysis identified the endogenous neurotransmitter DHEA as a regulator of mitochondrial morphology. This represents the first development of a three-step recursive targeted nasal drop, combining non-invasive brain delivery, microglial targeting, and mitochondrial regulation. In vitro experiments evaluated the targeting efficiency of CTLA-4 on microglia and the regulatory effect of DHEA on mitochondria using immunofluorescence and flow cytometry. In vivo, by establishing a POCD model, behavioral and other analytical methods systematically evaluated the three-step targeted regulation efficiency of the nasal drop and its therapeutic effects on cognitive behavior in POCD mice. Finally, transcriptomic analysis systematically elucidated the underlying mechanism of action of this "three-step recursive targeted nasal drop" for brain treatment, providing a new approach and strategy for the treatment of brain diseases with promising clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The "three-level recursive targeted nasal drops" regulate the phosphorylation level of Drp-1 on the mitochondrial outer membrane through hydrogel microsphere nasal mucosa targeting - microglia specific targeting - DHEA endogenous regulation of mitochondria, thereby maintaining the morphological function of microglial mitochondria and alleviating POCD.

[0036] Figure 2 To investigate the involvement of DHEA in the development of POCD; A. Experimental flow chart: peripheral blood extraction and plasma DHEA detection in patients undergoing hip surgery 1 day before and 7 days after surgery; B. Preoperative plasma DHEA levels in patients with POCD and those without POCD; C. Changes in plasma DHEA levels before and after surgery in all surgical patients; D. Correlation analysis between preoperative DHEA levels and MMSE scores in all surgical patients; E. Experimental flow chart: mice underwent anesthesia and surgical modeling, and peripheral blood and hippocampus were extracted from mice for related tests 3 days after surgery; F. Changes in peripheral blood and hippocampus DHEA levels in mice with POCD.

[0037] Figure 3DHEA participates in the regulation of POCD microglia by stabilizing mitochondrial morphology and function; A, Transcriptome analysis of the hippocampus of POCD mice showed that there were 68 differentially expressed genes related to mitochondria in the POCD group of mice; B, The main biological functions of the 68 differentially expressed mitochondrial genes were respectively; C, MDS analysis of the differentially expressed mitochondrial-related genes can clearly distinguish the Control and POCD groups; D, GO function enrichment results showed that the differentially expressed mitochondrial-related genes are mainly involved in metabolic processes, biological regulation, stress and other biological functions; E, 1 μL (500 μg / mL) of DHEA was stereotactically injected into the mouse brain, and anesthesia and surgery were performed 1 hour later. The production of lactate and ROS in the mouse brain was detected 24 hours later; F, Electron microscopy was used to detect the changes in mitochondrial morphology of hippocampal microglia in the three groups of mice; G, Microglial activation in the hippocampus of the three groups of mice.

[0038] Figure 4 DHEA regulates abnormal mitochondrial fission in microglia by inhibiting DRP-1 phosphorylation; A, primary cultured microglia, LPS-stimulated microglia, and rescued with 10 μM DHEA in advance, with changes in microglial lactate and ROS production among different groups; B, changes in microglial ATP among different groups; CD, JC-1 detection of mitochondrial membrane potential changes; EF, WB detection of Drp-1 and P-Drp-1 in microglia; G, changes in microglial mitochondria among different groups; H, effects of DHEA on microglial inflammatory factors TNF-α, IL-6, and IL-1β.

[0039] Figure 5 Preparation and characterization of three-stage recursive targeted nasal drops; A. Experimental flow chart: preparation of liposomal CTLA-4 / Lipo / DHEA; B. Experimental flow chart: preparation of HAMA / CTLA-4 / Lipo / DHEA; C. Transmission electron micrograph of liposomal CTLA-4 / Lipo / DHEA; D. Particle size graph of liposomal CTLA-4 / Lipo / DHEA; E. Drug release graph of liposomal CTLA-4 / Lipo / DHEA; F. Light micrograph of HAMA and HAMA / CTLA-4 / Lipo / DHEA; G. Electron micrograph of HAMA and HAMA / CTLA-4 / Lipo / DHEA; H. Fluorescence graph and fluorescence statistics of microsphere HAMA / CTLA-4 / Lipo / FITC.

[0040] Figure 6Figure 3 shows the uptake of CTLA-4 / Lipo / DHEA by microglia in vitro; A, FITC, Lipo / FITC, and CTLA-4 / Lipo / FITC were added into microglia, respectively, and the uptake of FITC in microglia was detected by immunofluorescence; B, Flow cytometry was used to detect the fluorescence uptake of microglia in different groups.

[0041] Figure 7 HAMA / CTLA-4 / Lipo / DHEA stabilizes the morphology and function of microglial mitochondria in vitro; A, Experimental flow chart; B, LPS-stimulated microglia, PBS, DHEA, L-DHEA, and CL-DHEA were given in advance, and the secretion of inflammatory factors TNF-α, IL-6, and IL-1β by microglia in different groups; C, Changes in the morphological structure of microglial mitochondria in different groups; D, The mitochondrial-related metabolites lactate and ROS in different groups; E, The mitochondrial-related metabolite ATP in different groups; F, JC-1 was used to detect the mitochondrial membrane potential of microglial cells in different groups; GH and WB were used to detect the changes in intracellular Drp-1 and P-Drp-1.

[0042] Figure 8 To detect the fluorescence uptake of microglia in the brain in vivo; AB mice were nasally instilled with FITC, Lipo / FITC, CTLA-4 / Lipo / FITC, HAMA / CTLA-4 / Lipo / FITC (20μl), and the fluorescence uptake of microglia in the brain was detected; CD, FITC, Lipo / FITC, CTLA-4 / Lipo / FITC, HAMA / CTLA-4 / Lipo / FITC (20μl), and the fluorescence uptake of microglia in the brain was detected

[0043] Figure 9 Effects of HAMA / CTLA-4 / Lipo / DHEA on microglia in POCD mice; A. POCD model was established, and PBS, DHEA, CTLA-4 / Lipo / DHEA, and HAMA / CTLA-4 / Lipo / DHEA were instilled into the nasal cavity, respectively, and the expression of P-Drp-1 in the hippocampus of the five groups of mice was compared; B. Lactic acid and ROS production in the hippocampus; C. Abnormal mitochondrial fission of microglia in the hippocampus of mice; D. Activation of microglia in the hippocampus of the five groups of mice; Expression and grayscale value analysis of inflammatory mediators TNF and COX-2 in the hippocampus of EF and hippocampus.

[0044] Figure 10Figure 3. Effects of HAMA / CTLA-4 / Lipo / DHEA on the behavior of POCD mice. A. Experimental flow chart: Anesthesia and surgical modeling were performed 1 hour after intranasal administration. B. Experimental schedule, NOR, novel object recognition test. C. Open field test results: total distance traveled in the open field, distance traveled in the center zone, number of entries into the center zone, and time spent in the center zone. D. Activity trajectories of mice in each group during the open field test. E. Schematic diagram of conditioned fear duration. F. Statistics of freezing time of mice in each conditioned fear group.

[0045] Figure 11 Effects of HAMA / CTLA-4 / Lipo / DHEA on the transcriptome of the hippocampus of POCD mice; A. Transcriptomic analysis found that 433 genes were differentially expressed in the hippocampus tissues of POCD mice and normal mice, of which 234 genes were overexpressed in the POCD group and 208 were underexpressed; B. Differentially expressed genes were significantly enriched in metabolic regulation and steroid synthesis pathways; C. Differentially expressed genes were enriched in the steroid hormone biosynthesis pathway; D. Differential expression changes between the HAMA / CTLA-4 / Lipo / DHEA treatment group and POCD; E. Changes in mitochondrial-related differentially expressed genes between POCD mice and normal mice; F. Changes in the expression of Cyp51, Dhcr7, Lss, Msmo, and Nsdh1 genes in the hippocampus of mice in the three groups; G. Changes in mitochondrial-related differentially expressed genes in the hippocampus of mice in the three groups. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0047] Example 1

[0048] 1. Experimental Materials and Methods

[0049] 1. Ethics Statement

[0050] The animal protocol for this experiment was approved by the Tongji University Animal Standing Committee (protocol number: TJBH07023101). Human blood collection and related experimental protocols were approved by the Institutional Research Ethics Committee of Shanghai Fourth People's Hospital (protocol number: 2021119-001).

[0051] 2. Preparation and Characterization of Liposomes

[0052] Targeted drug-loaded liposomes were prepared using ultrasonic dispersion. 40 mg of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 40 mg of DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride), and 20 mg of cholesterol (Macklin, China) were dissolved in 50 mL of organic solvent (chloroform:methanol = 1:1, v / v). Subsequently, 10 mg of DHEA (dehydroepiandrosterone) powder was added until completely dissolved and the mixture was dried on a rotary evaporator to form a thin lipid film. Subsequently, 10 mL of deionized water was added and the mixture was hydrated at 40°C for 20 minutes to prepare a crude liposome suspension. Finally, 10 μg of CTLA-4 peptide was added to the suspension and co-extruded 11 times through a liposome extruder containing a 0.22 μm polyethersulfone membrane (Avanti Polar Lipids) to uniformly insert CTLA-4 into the lipid bilayer surface, forming modified liposomes. The particle size, polydispersity index (PDI) and zeta potential of the final liposomes were characterized using a dynamic light scattering instrument (Nano-ZS, Malvern, UK), and the particle size was controlled in the range of 120-150 nm. The liposome morphology was observed by transmission electron microscopy (TEM).

[0053] 3. Synthesis and characterization of hydrogel microspheres

[0054] 2wt% HA (hyaluronic acid, M w =100 kDa; Bloom Chifreda Biological Products Co., Ltd., China) was dissolved in phosphate-buffered saline (PBS) at room temperature. The HA solution was mixed with methacrylic anhydride (molar ratio of 0.5:1; Aladdin, China) in an ice bath at pH 8.0 and stirred continuously for 12 hours. The reaction solution was then purified by dialysis (molecular weight cutoff (MWCO), 8000 kDa) against deionized water for three days. HAMA was extracted from the purified solution as a white powder. The prepared microspheres were mixed with previously prepared CTLA-4-modified liposomes (loaded with DHEA) in a 1:1 volume ratio and gently rotated at pH 7.4 and 4°C for 1 hour to form a liposome-microsphere composite. A polylysine (PLL) solution was then added to the mixture at a final concentration of 0.01 mg / mL and incubated for an additional 15 minutes to achieve surface positive charge modification of the microspheres. After modification, the mixture was washed three times with PBS to remove unbound liposomes and PLL, resulting in the final three-stage recursive targeting composite hydrogel microspheres.

[0055] 4. Patient sample collection

[0056] A total of 56 patients who underwent femoral neck fracture surgery were included in this study. Patients with major heart, lung, and brain diseases, malignant tumors, preoperative cognitive dysfunction, confusion, or a history of severe stroke were excluded. Gender, age, body mass index (BMI), American Society of Anesthesiologists (ASA) classification, operative time, fluid volume, recovery time, and educational level were recorded, and the above indicators were statistically analyzed, as shown in Table 1. The MMSE scale was used to evaluate the patients' cognitive function 1 day before surgery and 3 days after surgery. According to the MMSE scale, the patients were divided into a POCD group and a normal group. There were 42 patients in the normal group and 14 patients in the POCD group. Peripheral blood was collected from all enrolled patients 1 day before surgery and 3 days after surgery.

[0057] Table 1

[0058]

[0059] 5. Construction of POCD (postoperative cognitive dysfunction) model

[0060] 18-week-old male mice were purchased from Jihui Company and raised at the Experimental Animal Center of Tongji University. All animals were adapted to a 12-hour light / 12-hour dark cycle for one week before the experiment. After general anesthesia (1.4% isoflurane + 40% oxygen, 2L / min), a 2cm abdominal incision was made, and the abdominal skin, muscle and peritoneum were cut in turn, and the intestine was explored with an intestinal probe. After no abnormal intestinal peristalsis was observed, the incision was sutured in layers. Then, isoflurane inhalation was continued until the total anesthesia time was 2 hours. The hippocampus on both sides of the mouse was taken 3 days after the operation.

[0061] 6. Cell Processing

[0062] Mouse primary microglia were isolated from the hippocampus of C57BL / 6 mice 24 hours after birth. These mice were sacrificed to dissect the hippocampus. The brain of each mouse was digested in 0.25% trypsin solution (SSA) with 0.05% DNase I (Merck, Germany) at 37°C for 30 minutes and then incubated with 10% fetal bovine serum (FBS) (Lonsera, Uruguay). The tissue pellet was centrifuged at 1000 rpm for 5 minutes at 4°C and washed twice with HBSS (Merck, Germany). After cultivation, the cells were cultured in DMEM (Gibco, USA) with 10 ng / mL GM-CSF (MCE, USA) supplemented with 10% fetal bovine serum, 50 U penicillin, and 50 mg / mL streptomycin (Gibco, USA).

[0063] 7. Stereotactic injection

[0064] Mice were anesthetized with 1.4% isoflurane and placed in a stereotactic frame. After exposing the skull surface, the dorsal hippocampal target was determined using stereotactic coordinates (anteroposterior -1.5 mm; mediolateral ±1.0 mm; dorsoventral -2.0 mm). Mice were injected with 1 μL of DHEA (500 μg / mL) or PBS at a rate of 0.25 μL / min into both hippocampi. The needle was gently withdrawn 10 minutes after injection.

[0065] 8. Nasal administration

[0066] Mice were anesthetized with 1.4% isoflurane. The nostrils were opened and a pipette was used to draw up liquid. A small amount of liquid was then pushed out, forming small droplets at the tip of the pipette. These drops were placed directly in the middle of the nostril and the mouse was allowed to inhale during breathing. After observing the mouse's inhalation of the drug solution, small, multiple doses were administered, totaling 10 mg / kg.

[0067] 9. DHEA measurement

[0068] Peripheral blood was collected from clinical patients and centrifuged at 12,000 rpm for 10 minutes to obtain serum. Plasma DHEA concentrations were measured using an ELISA (Enzo, USA). Cardiac blood was collected from mice under anesthesia and centrifuged to obtain the peripheral blood supernatant. Immediately after death, hippocampal homogenates were obtained and DHEA levels in plasma and brain tissue were measured using an ELISA kit.

[0069] 10. Enzyme-linked immunosorbent assay (ELISA) analysis

[0070] Elisa assay kits for mouse TNF-α (R&D, USA), IL-6 (R&D, USA), and IL-1β (Elabscience, China) were used to detect TNF-α, IL-6, and IL-1β levels in mice and cell culture, respectively. Spectrophotometric readings were obtained using a spectrophotometer.

[0071] 11. Lactic acid testing

[0072] Tissue homogenates or cell supernatants were collected and centrifuged at 12,000 rpm for 10 min at 4°C. Samples (50 μL) were analyzed using a lactate assay kit (Elabscience, China) according to the manufacturer's instructions. Absorbance was measured at 530 nm using a microplate reader, and different lactate concentrations were calculated based on a standard curve.

[0073] 12. ROS detection

[0074] Single-cell suspensions of brain tissue or cultured microglia were prepared by adding a probe (Elabscience, China) and incubating the cells. After centrifugation and washing, the cells were resuspended. The absorbance at 530 nm was measured using an enzyme reader, and ROS levels were calculated.

[0075] 13. Western blot (WB) detection

[0076] Tissues and cells from different treatment groups were lysed with protein lysis buffer (Thermo Fisher, USA). The whole proteins were transferred to PVDF membranes (Bio-Rad, USA). Primary antibodies included β-actin antibody (1:1000 dilution, Cell Signaling Technology, USA), P-Drp-1 antibody (1:1000 dilution, Cell Signaling Technology, USA), Drp-1 antibody (1:1000 dilution, Cell Signaling Technology, USA), TNF-α antibody (1:1000 dilution, Cell Signaling Technology, USA), and COX-2 antibody (1:1000 dilution, Cell Signaling Technology, USA). Secondary antibodies were mouse IgG HRP-linked antibody and rabbit IgG HRP-linked antibody purchased from Cell Signaling Technology. Bands were visualized using a Western chemiluminescence developer (Bio-Rad, USA), and then visualized using a chemical Doc. Quantification was performed using the XRS+ system (Bio-Rad, USA). β-actin was used to normalize the target protein levels and to control for differences in total protein loading.

[0077] 14. Electron microscope detection

[0078] Mouse hippocampal tissue or isolated cells were collected, fixed with electron microscopy fixative (Servicebio, China, G1102), and stored at 4°C overnight. After rinsing with 0.1M phosphate buffer PB (pH 7.4), centrifugation was performed and 1% agarose solution was added. Tissues and cells were fixed with light-free osmium for 2 hours at room temperature, then rinsed three times with 0.1M phosphate buffer PB (pH 7.4), and then dehydrated with alcohol and acetone. Then, acetone and embedding agent were used for infiltration and embedding at 37°C overnight. After incubation at 60°C for 48 hours, the embedding plate was cut into 6-8nm slices. The slices were stained with 2.6% citric acid. A transmission electron microscope (Hitachi, Japan) was used to take micrographs.

[0079] 15. Immunofluorescence detection

[0080] Mice were sacrificed 3 days after anesthesia. Both the treated and control groups were deeply anesthetized and perfused transcardially with 20 ml of 0.01 M PBS (pH 7.4), followed by an injection of 20 ml of 4% (w / v) paraformaldehyde (PFA). Brain tissue was isolated and fixed with 4% PFA for 4 h at 4°C. After cryoprotection overnight in 0.1 M phosphate buffered saline (PBS) and 30% (w / v) phosphate buffered saline at 4°C, the brains were cut into 30 μm thick frontal lobe sections using a freezing microtome. Sections were incubated with mouse Iba1 antibody (1:200, Synaptic Systems, Japan) overnight at 4°C. Sections were washed and incubated with anti-rabbit IgG (1:2000, CST, USA) for 2 h. Images were acquired using a confocal microscope (Olympus, FV3000, Japan).

[0081] 16. Open field test (OFT)

[0082] Mice were placed in the center of a square chamber (50 cm × 50 cm × 40 cm), and their movements were recorded for 10 minutes using a video tracking system. The total distance and time the animals spent in the central square (25 cm × 25 cm) were recorded and analyzed. During the experiment, olfactory cues were removed by wiping with 75% alcohol.

[0083] 17. Fear Conditioning (FC)

[0084] Mice underwent a contextual fear conditioning paradigm in a chamber (32 cm × 25 cm × 25 cm). Baseline freezing was measured during the first 2 min. A total of 5 foot shocks were delivered (2:10 min, first foot shock: 0.7 mA, 2 s; inter-foot shock interval 35-60 s). Freezing between foot shocks was measured within 10 s before each foot shock. The conditioning phase ended 30 s after the last foot shock. One day later, mice were returned to the original chamber for 5 min without foot shock. Freezing behavior, defined as the absence of visible movement other than breathing, was recorded and scored.

[0085] 18. Microglia targeting ability of CTLA-4 modified liposomes

[0086] 1×10 5BV2 cells were cultured in 6-well plates and then treated with PBS, FITC, Lipo / FITC, or CTLA-4 / Lipo / FITC for 3 hours. The cells were collected and analyzed using a BD-LSRFortessa flow cytometer (BD, USA). The data were analyzed using FlowJo software. BV2 cells were seeded onto glass cups and co-cultured with PBS, FITC, Lipo / FITC, or CTLA-4 / Lipo / FITC. The cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.2% TritonX-100 for 10 minutes, incubated with 2% BSA for 1 hour, incubated with Iba1 antibody and anti-rabbit IgG, and fluorescence images were captured using a confocal microscope.

[0087] We also tested the microglial targeting ability of HAMA / CTLA-4 / Lipo in vivo. Mice were randomly divided into four groups: FITC, L / FITC, CL-FITC, and HL-FITC. Mice in each group were instilled with FITC, Lipo / FITC, CTLA-4 / Lipo / FITC, and HAMA / CTLA / 4 / Lipo / FITC (0.1 μg / μL, 200 μL), respectively. Bama miniature piglets were intranasally instilled with PBS or HAMA / CTLA-4 / Lipo / FITC (0.1 μg / μL, 1 L). Brain sections of mice and piglets were incubated with Iba1 antibody overnight at 4°C, and images were acquired using a confocal microscope.

[0088] 19. Screening of differentially expressed genes

[0089] After RNA extraction and library construction, RNA-seq and sequencing data quality assessment were performed using an Illumina NovaSeq 6000 and Fastp; the cleaned data were aligned to the mouse genome (GRCm38 / mm10) using Hist2. The number of reads per gene was counted using RSEM, and the FPKM for each gene was calculated. Differentially expressed genes (DEGs) were determined using DESeq2 and defined by a P value less than 0.05. Based on the expression of DEGs, dimensionality reduction analysis was performed using multidimensional scaling (MDS) analysis.

[0090] 20. Functional enrichment analysis of differentially expressed genes

[0091] DEG enrichment analysis was performed using the Metascape web platform (http: / / metascape.org). The enrichment of DEGs in Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was investigated to gain insight into potential biological changes associated with anesthesia. Data visualization and analysis were performed using R 4.2.2.

[0092] 21. Statistical Analysis

[0093] Normally distributed data were analyzed using the t-test or one-way analysis of variance (ANOVA), and the results are expressed as mean ± SD. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software (version 8.0; GraphPad Software, USA).

[0094] 2. Experimental Results and Discussion

[0095] 1. Decreased DHEA levels are involved in the occurrence of POCD

[0096] DHEA is an important neurosteroid hormone in the brain, which plays an important role in brain immune regulation and the maintenance of neurological function. In order to study whether DHEA is involved in POCD in elderly patients, we collected 56 patients who underwent hip surgery and divided them into POCD group and normal group according to MMSE score. There were no significant differences between the two groups of patients in terms of gender, age, weight, height, etc. Through analysis, we found that the serum DHEA level of patients who underwent general anesthesia for hip joint was significantly lower on the third day after surgery than before surgery. The serum DHEA level of patients in the POCD group was significantly lower than that of patients in the non-POCD group before surgery, and the DHEA level was significantly correlated with the patient's MMSE score ( Figure 2 Chinese BD).

[0097] Since it is impossible to detect the DHEA content in the brain of clinical patients, we used animal experiments to verify the effect of anesthesia and surgery on DHEA in the brain. 18-month-old C57 / BL mice underwent laparotomy under isoflurane anesthesia. 3 days after the anesthesia, the DHEA content in the peripheral blood and hippocampus was measured. The results showed that the DHEA content in the peripheral blood and hippocampus of POCD mice was significantly lower than that of the control group. These results indicate that patients with low DHEA may have a higher incidence of POCD ( Figure 2 F), and anesthesia surgery may cause a further decrease in DHEA.

[0098] 2. DHEA participates in the metabolic regulation of POCD by stabilizing mitochondria

[0099] Studies have shown that DHEA can treat cerebral ischemia and liver disease in rats by inhibiting inflammatory responses and reducing oxidative stress. In order to explore the biological activities that DHEA may be involved in in POCD, we detected the changes in the transcriptome of the hippocampus of mice in anesthesia surgery model. The results showed that the expression of mitochondrial-related genes in POCD mice was significantly changed. Using MDS analysis of differentially expressed mitochondrial-related genes, POCD mice could be clearly distinguished. GO functional enrichment results showed that differentially expressed mitochondrial-related genes were mainly involved in metabolic processes, biological regulation, stress and other biological functions, and were related to mitochondrial assembly, assembly regulation and mitochondrial transport ( Figure 2 (in AD).

[0100] Based on transcriptomic results, we speculate that DHEA may inhibit brain inflammation by regulating mitochondrial metabolic regulation, thereby protecting neurological function. To this end, we conducted the following experiments. First, we found significant metabolic abnormalities in the brains of POCD mice, as evidenced by increased levels of ROS and lactate in the brain ( Figure 3 Electron microscopy showed that mitochondrial morphology changed (mitochondrial cristae disappeared and became vacuolated) and division increased ( Figure 3 Immunofluorescence results showed a significant increase in the intensity of Iba1, a well-known marker of microglia, suggesting a significant increase in microglial activation ( Figure 3 Middle G). Injecting DHEA (1 μL, 500 μg / mL) into the dorsal hippocampus of mice 1 hour before surgery through intracerebral stereotaxic can reduce the production of ROS and lactic acid, improve abnormal mitochondrial fission, inhibit microglial activation, and improve the behavioral cognition of mice ( Figure 3 (EG).

[0101] We further verified the specific mechanism by which DHEA improves microglial mitochondrial morphology and metabolism through in vitro experiments. Studies have shown that lipopolysaccharide-stimulated microglia can well mimic the POCD model as a classic model of neuroinflammation in vitro. Microglia were co-cultured with DHEA (10 μM) for 1 hour and then co-cultured with LPS (100 ng / mL) or PBS to determine whether DHEA pretreatment would reverse LPS-induced microglial activation. After 24 hours, cells were collected to detect changes in mitochondrial morphology, metabolites, and inflammatory factors. The results showed that LPS stimulation-induced ROS lactate production increased ( Figure 4 A), ATP content decreased ( Figure 4 Middle B), the cell membrane potential decreases ( Figure 4 C, D), increased mitochondrial fission in microglia ( Figure 4 Middle G), the inflammatory factors TNF-α, IL-6 and IL-1β secreted by cells increased ( Figure 4H). DHEA pretreatment can reverse the above changes. Since Drp-1 is a key protein in maintaining mitochondrial morphology, we tested whether DHEA has an effect on Drp-1. WB results showed that LPS stimulation increased the phosphorylation level of Drp-1 in microglia, while DHEA pretreatment reduced P-Drp-1 ( Figure 4 These results suggest that decreased DHEA levels caused by anesthesia and surgery are a key factor in POCD. DHEA stabilizes Drp-1 on mitochondria, reduces abnormal mitochondrial fission caused by P-Drp-1 activation, stabilizes mitochondrial metabolic function, reduces the formation of harmful metabolites, and inhibits excessive microglial activation, thereby improving cognitive function in POCD mice.

[0102] 3. CTLA-4 / Lipo / FITC targeting efficiency of microglia in vitro

[0103] DHEA has been widely used in clinical practice, but because peripheral DHEA is converted into DHEAS by sulfidase, it cannot pass through the blood-brain barrier, which greatly affects the treatment of cognitive impairment caused by abnormal mitochondria in the brain. In order to solve the problem of clinical medication, we designed and developed a hydrogel microsphere liposome material ( Figure 5 ) to conduct further experiments. The first is to address the issue of microglial targeting. CD80 / 86 is mainly expressed in microglial cells in the brain. The activation of microglial cells will lead to upregulation of CD80 / 86 expression, and CTLA-4 is an important ligand for the above two. Therefore, we modified liposomes with CTLA-4, and the liposome material specifically targeted microglia to deliver DHEA through CD80 / 86. In order to verify the microglial localization characteristics of CTLA-4 liposomes, we labeled the liposomes with FITC fluorescent dye and then performed immunofluorescence detection. BV2 cells were treated with 10μM PBS, FITC, Lipo / FITC and CTLA-4 / Lipo / FITC at 37°C for 3 hours. As Figure 6 As shown in A, the fluorescence uptake in BV2 cells of the CL-FITC group was significantly higher than that of the other groups. Further analysis by flow cytometry showed that the results were consistent with the fluorescence results ( Figure 6 In middle B), the uptake rate of fluorescence by BV2 cells in the CL-FITC group was as high as 70% ( Figure 6 The above results indicate that CTLA-4 modification promotes the targeting ability of liposomes to microglia.

[0104] 4. CTLA-4 / Lipo / DHEA alleviates inflammatory activation caused by microglial mitochondrial fission

[0105] To further determine whether targeting microglia with CTLA-4 to increase drug uptake can better enhance DHEA's regulation of mitochondrial membrane fission proteins and stabilize mitochondrial structure and function, we pretreated microglia with CTLA-4 / Lipo / DHEA, Lipo / DHEA, DHEA, and PBS, and determined the effect of DHEA on mitochondria 24 hours after LPS stimulation. LPS stimulation of microglia significantly increased the secretion of proinflammatory cytokines TNF-α, IL-1β, and IL-6, while the secretion of proinflammatory cytokines by microglia given CTLA-4 / Lipo / DHEA, Lipo / DHEA, and DHEA in advance was significantly reduced, and the anti-inflammatory ability of CTLA-4 / Lipo / DHEA was higher than that of Lipo / DHEA and DHEA alone ( Figure 7 (B). WB and electron microscopy results showed that P-Drp-1 and mitochondrial fission were significantly increased in the LPS-stimulated group compared with the control group. CTLA-4 / Lipo / DHEA, Lipo / DHEA, and DHEA pretreatment reversed these changes, with CTLA-4 / Lipo / DHEA pretreatment having the most significant effect ( Figure 7 Next, we measured the levels of metabolites ROS, lactate, ATP, and mitochondrial membrane potential. Compared with the control group, LPS treatment showed an increase in ROS and lactate, and a decrease in ATP and membrane potential. CTLA-4 / Lipo / DHEA, Lipo / DHEA, and DHEA pretreatment reduced metabolite levels and increased ATP and mitochondrial membrane potential, with the CTLA-4 / Lipo / DHEA group being superior to the other groups ( Figure 7 (D, E, F).

[0106] 5. In vivo microglial cell HAMA / CTLA-4 / Lipo / FITC targeting efficiency

[0107] Next, we used mice and Bama pigs to test the efficiency of nasal administration and the ability of brain microglia to absorb FITC to evaluate the important role of hydrogel microsphere liposome materials and targeting proteins. 18-month-old C57BL / 6 mice were given 100μM HAMA / CTLA-4 / Lipo / FITC, CTLA-4 / Lipo / FITC, Lipo / FITC, and FITC through the nasal cavity. After 4 hours, the mouse brains were harvested and the slices were fluorescently stained to detect the ability of hippocampal microglia to absorb FITC in different groups of mice. It can be seen that the content of fluorescence absorbed by microglia in the hippocampus of the HL-FITC and CL-FITC groups was significantly higher than that of the L-FITC and FITC groups ( Figure 8 (A, B) in the middle, indicating that CTLA-4 promotes the uptake of fluorescence by microglia, and the use of hydrogel microspheres can increase nasal adhesion and slowly release liposomes, which is more conducive to the delivery of drugs in the brain.

[0108] In addition, we conducted experiments on 6-month-old Bama pigs. 1 mL (100 μM) of HAMA / CTLA-4 / Lipo / FITC, CTLA-4 / Lipo / FITC, Lipo / FITC, and FITC were administered intranasally. Four hours later, brain tissue was collected from Bama pigs and sections were sectioned for fluorescence staining. The results showed that HAMA / CTLA-4 / Lipo / FITC could successfully enter the piglet brain and be absorbed by microglia after intranasal administration ( Figure 8 These results indicate that the nose-to-brain route is a promising approach for intracranial drug delivery, and that the use of HAMA microspheres can enhance the efficiency of liposome delivery into the brain.

[0109] 6. Effects of HAMA / CTLA-4 / Lipo / DHEA on mitochondrial fission and brain inflammation in mice

[0110] Based on the results of in vitro experiments and microglial uptake of fluorescence, we continued to verify the effects of hydrogel microsphere liposome targeting materials on abnormal mitochondrial fission and microglial activation-induced brain inflammation in POCD mice through in vivo experiments. 18-month-old female C57 mice were randomly divided into five groups: (1) Control group (no anesthesia, surgery, or any drug treatment); (2) POCD group (1 hour after nasal instillation of PBS, followed by laparotomy under 2% isoflurane anesthesia); (3) POCD+DHEA group (20 μl of DHEA was administered intranasally 1 hour before surgery); (4) POCD+L-DHEA group (CTLA-4 / Lipo / DHEA was administered intranasally 1 hour before surgery); and (5) POCD+HL-DHEA group (HAMA / CTLA-4 / Lipo / DHEA was administered intranasally 1 hour before surgery). Three days after anesthesia and surgery, the hippocampus of mice in each group was harvested for the detection of relevant biomarkers. WB results showed that compared with the control group, the phosphorylation level of Drp-1 in the hippocampus of POCD mice was significantly increased ( Figure 9 Middle A); Electron microscopy results showed that the mitochondrial structure in hippocampal microglia changed, and the mitochondrial cristae disappeared. HAMA / CTLA-4 / Lipo / DHEA pretreatment significantly reversed the above changes, but there was no significant statistical difference between the POCD+L-DHEA, POCD+DHEA, and POCD groups ( Figure 9 Middle B). Metabolites ROS and lactate water in the hippocampus of POCD mice were further detected. Similarly, compared with the control group, the ROS and lactate levels in the POCD group showed an increased trend, while HAMA / CTLA-4 / Lipo / DHEA and CTLA-4 / Lipo / DHEA pretreatment reversed the increase in ROS and lactate levels, and the effect of HAMA / CTLA-4 / Lipo / DHEA was significantly better than that of CTLA-4 / Lipo / DHEA ( Figure 9 Middle C).

[0111] In addition, brain inflammation is a key pathological change in POCD. We further studied the effect of the microsphere material on hippocampal microglial activation and brain inflammation in POCD mice. Confocal fluorescence detection showed that the intensity of Iba-1 increased in the POCD group. HAMA / CTLA-4 / Lipo / DHEA could significantly reduce Iba-1 in the hippocampus of POCD mice. The number of activated microglia in the hippocampus of mice in the POCD+L-DHEA group also decreased. However, DHEA alone had no significant effect on brain microglia. Figure 9 WB detection of the protein expression of pro-inflammatory substances TNF-α and COX-2 in the hippocampus showed that the protein expression of TNF-α and COX-2 in the hippocampus of mice in the POCD group was significantly increased, while the protein expression of TNF-α and COX-2 in the hippocampus of mice given HAMA / CTLA-4 / Lipo / DHEA was significantly decreased. However, the improvement in the POCD+L-DHEA group and the POCD+DHEA group was very small ( Figure 9 These results indicate that both HAMA / CTLA-4 / Lipo / DHEA and CTLA-4 / Lipo / DHEA can improve abnormal mitochondrial fission and microglial activation in the mouse brain to varying degrees. Furthermore, the use of hydrogel microspheres can increase adhesion to the nasal cavity and have a sustained-release function, which may promote better brain uptake of DHEA and its effects on brain mitochondria. The ineffectiveness of nasal administration of DHEA alone may be due to the low concentration of DHEA entering the brain, which cannot significantly affect brain microglia.

[0112] 7. Hydrogel microspheres HAMA / CTLA-4 / Lipo / FITC improve cognitive function in POCD mice

[0113] Next, we used behavioral methods to detect the improvement of the cognitive function of mice by microsphere materials. First, the open field test was used to evaluate the exploratory behavior of mice. The results showed that there was no significant difference in the total activity distance of the four groups of mice, indicating that anesthesia surgery had no significant effect on the motor ability of mice; the activity of POCD mice in the central area (distance, time and number of entries into the central area) was significantly reduced, indicating that the POCD model constructed by surgical anesthesia affected the exploratory behavior of mice. HAMA / CTLA-4 / Lipo / DHEA pretreatment can significantly reverse the above changes. Compared with POCD mice, CTLA-4 / Lipo / DHEA pretreatment improved the distance obtained and the number of entries into the central area ( Figure 10(C and D). However, no improvement in exploratory behavior was observed in mice receiving intranasal administration of DEHA alone. These results suggest that anesthesia and surgery significantly negatively impact exploratory behavior in mice but have no significant effect on their motor function. HAMA / CTLA-4 / Lipo / DHEA is more effective than CTLA-4 / Lipo / DHEA in treating POCD mice, suggesting that intranasal administration of DHEA alone may be ineffective.

[0114] Conditioned fear experiments have been primarily used to examine hippocampal-dependent learning. We found through experiments that the freezing time of the contextual fear conditioning test in the POCD group of mice was significantly shortened, and HAMA / CTLA-4 / Lipo / DHEA treatment rescued this reduction. There was no significant difference between mice given CTLA-4 / Lipo / DHEA or DHEA intranasally and mice in the anesthesia surgery group. The above research results indicate that HAMA / CTLA-4 / Lipo / DHEA can significantly improve cognitive impairment caused by surgery and anesthesia, while CTLA-4 / Lipo / DHEA or DHEA has no significant improvement on memory learning ( Figure 10 (F) This difference may be due to the sustained release of hydrogel microspheres, which allows DHEA to enter the brain at a higher concentration and exert specific and long-term functions.

[0115] 8. HAMA / CTLA-4 / Lipo / DHEA improves transcriptional levels in POCD mice

[0116] Finally, we used transcriptomics to investigate whether the mechanism by which hydrogel microspheres alleviate POCD is related to mitochondrial regulation involving steroid hormones. We found that 433 genes were differentially expressed in the hippocampus tissues of POCD mice and normal mice, of which 234 genes were highly expressed in the POCD group and 208 were underexpressed. These differentially expressed genes were mainly enriched in the steroid synthesis pathway ( Figure 11 KEGG functional enrichment analysis showed that six DEGs (Nsdh1, Msmo, Lss, Fdft1, Dhcr7, and Cyp51) were significantly enriched in the steroid hormone biosynthesis pathway, and these genes were down-expressed in the POCD group, suggesting that steroid hormone biosynthesis is impaired in the hippocampus of POCD mice ( Figure 11 In the hippocampus of HAMA / CTLA-4 / Lipo / DHEA-treated mice and normal mice, 2651 genes were differentially expressed, of which 1223 genes were highly expressed in the HAMA / CTLA-4 / Lipo / DHEA-treated group and 1428 genes were lowly expressed ( Figure 11Five genes enriched in the steroid hormone biosynthesis pathway (Fdft1 gene was not detected in the DHEA group) were relieved after treatment with HAMA / CTLA-4 / Lipo / DHEA, and there was no statistical difference between the mice in the normal group ( Figure 11 F). There were significant differences in the expression of 53 mitochondrial-related genes between the POCD group and the normal group, and between the HL-DHEA group and the POCD group. The expression of related genes was significantly improved after treatment with HAMA / CTLA-4 / Lipo / DHEA ( Figure 11 These results indicate that the administration of HAMA / CTLA-4 / Lipo / DHEA can significantly improve the synthesis of steroid hormones and alleviate the differential expression of mitochondrial-related genes in POCD mice.

[0117] 3. Conclusion

[0118] Clinically, DHEA has been used as a drug for anti-aging and treatment of reproductive system diseases, but there are few studies on neurological diseases. This is mainly due to the particularity of DHEA's existence form. 99% of DHEA in the circulation exists in the form of low-activity sulfide DHEAS. Most of the peripherally administered DHEA is converted into DHEAS by the sulfotransferase SULT2A1, which cannot pass through the blood-brain barrier, so it is difficult for it to enter the brain. In addition, large-dose peripheral administration of DHEA may cause an imbalance in hormone levels, leading to a series of adverse consequences. The inventors discovered the correlation between DHEA and perioperative cognitive dysfunction through clinical patient data analysis and transcriptomics testing, and verified the specific mechanism by which DHEA regulates microglial mitochondria through experiments. And by constructing a three-level targeted nasal drop, the problem that peripheral administration cannot enter the brain to cascade target microglial mitochondria was solved, the DHEA content in the brain was increased, and the loading rate was as high as 98.3%, which improved the cognitive impairment caused by anesthesia surgery ( Figure 1 ).

[0119] The present invention has successfully developed a "three-level recursive targeted nasal drop" that is delivered to the brain through the nasal cavity non-invasively via hydrogel microspheres, achieving first-stage nasal mucosa targeting; second-stage cell targeting is achieved by binding to activated microglia with targeted liposomes carrying CTLA-4; and three-level targeting is achieved by regulating mitochondria through the release of DHEA, ultimately achieving multi-stage precise targeting and brain regulation. This "three-level recursive targeted nasal drop" can break through the complex physiological and pathological microenvironment of the brain layer by layer to reach the diseased core mitochondria of microglia, and regulate the Drp-1 pathway through DHEA to significantly inhibit abnormal mitochondrial fission, stabilize mitochondrial morphology and function, inhibit microglial activation, and alleviate cognitive dysfunction caused by anesthesia surgery. In summary, this study provides a new method for the treatment of various central nervous system diseases.

[0120] Comparative Example 1

[0121] Based on Example 1, the effects of different charge intensities on microsphere delivery were investigated. Specifically, hydrogel microspheres were modified with either quaternized chitosan HTCC or branched polyethyleneimine (BPEI). The results showed that while both quaternized chitosan HTCC and BPEI-modified microspheres had high charge densities and strong adhesion, they were susceptible to inducing significant nasal mucosal inflammation. In this example, PLL-modified microspheres maintained good biocompatibility while increasing their nasal retention time to over 60 minutes.

[0122] Comparative Example 2

[0123] Based on Example 1, the effect of the monomer ratio of hydrogel microspheres prepared by the microfluidic method on microsphere delivery was studied. The specific method was as follows: HAMA hydrogel microspheres were prepared with a molar ratio of HA:MA of 0.2:1, 0.5:1, 1:1, and 2:1, respectively. The particle size distribution, surface morphology, and stability of the loaded liposomes of the obtained microspheres were analyzed, and their nasal retention time and brain drug delivery efficiency were compared in the PND model. When HA:MA was 0.5:1, the microsphere particle size distribution was uniform, the structure was stable, the liposome loading rate was high, and the drug delivery efficiency was optimal. When the ratio was too low (0.2:1), the cross-linking density was too strong, resulting in excessive rigidity of the microsphere structure, which easily hindered the embedding of liposomes and reduced the drug release rate; when the ratio was too high (>1:1), the microspheres were insufficiently cross-linked and easily deformed and collapsed.

[0124] Comparative Example 3

[0125] Based on Example 1, the effects of different charged materials on microsphere delivery were investigated. Specifically, chitosan and sodium alginate were used in place of HAMA in the microsphere preparation, maintaining consistent liposome and DHEA loading conditions. The results showed that while chitosan microspheres had a high initial delivery efficiency, nasal enzymatic hydrolysis shortened retention time by approximately 60%, resulting in nearly complete drug release within 24 hours. Alginate microspheres, on the other hand, exhibited low drug loading and poor nasal retention, with drug adhesion reduced by over 90%.

[0126] Comparative Example 4

[0127] According to the method of Example 1, positively charged hydrogel microspheres were used alone to prepare nasal drops for nasal administration. The results showed that DHEA was released suddenly and could not be delivered to the center of brain disease.

[0128] Comparative Example 5

[0129] According to the method of Example 1, CTLA-4 liposomes were used alone to prepare nasal drops for nasal administration. The results showed that only 0.5% of the DHEA drug entered the brain.

Claims

1. A method for preparing three-stage recursive targeting hydrogel microspheres for nasal administration, characterized in that: The following steps are involved: (1) Using DOPE, DOTAP, and cholesterol as lipid raw materials, DHEA-loaded liposomes were prepared, and CTLA-4 was physically modified on the liposome phospholipid membrane by co-extrusion; (2) Using hyaluronic acid and methacrylic anhydride as raw materials, the monomer ratio of the raw materials was controlled at 0.5:1, and methacrylated hyaluronic acid hydrogel microspheres were prepared by microfluidic method; (3) mixing the liposome solution obtained in step (1) with the methacrylated hyaluronic acid hydrogel microspheres obtained in step (2), and finally preparing drug-loaded hydrogel microspheres by functional modification with polylysine.

2. The method according to claim 1, characterized in that The weight ratio of DOPE, DOTAP and cholesterol in step (1) is 2:2:

1.

3. The method according to claim 1, characterized in that The liposomes described in step (1) are prepared by dissolving DOPE, DOTAP, cholesterol, and DHEA in a 1 / 1, v / v, chloroform and methanol mixture, ultrasonically mixing, and drying on a rotary evaporator to a thin lipid film. PBS is added, and after hydration at 40°C for 20 minutes, the mixture is co-extruded with CTLA-4 through a 0.22 μm film extruder to obtain liposomes.

4. The method according to claim 1, wherein In the microfluidic method described in step (2), the flow rate ratio of the aqueous phase to the oil phase is controlled to be 1:

5.

5. The method according to claim 1, wherein The concentration of the liposome solution in step (3) is 2.5 wt %, and the concentration of the methacrylated hyaluronic acid hydrogel microspheres is 4 wt %. Preferably, the reaction conditions in step (3) are: 4° C., 90 min, and gentle mixing.

6. The method according to claim 1, characterized in that The concentration of the polylysine solution in step (3) is 0.005-5 mg / mL, and the weight ratio of the microspheres to polylysine is 3:1-55:

1.

7. Three-stage recursive targeting hydrogel microspheres for nasal administration prepared by the method according to any one of claims 1 to 6.

8. The three-stage recursive targeting hydrogel microspheres for nasal administration according to claim 7, characterized in that: The diameter of the microspheres is 200 μm.

9. Use of the three-stage recursive targeting hydrogel microspheres for nasal administration according to claim 7 or 8 in the preparation of drugs for treating central nervous system diseases.

10. Use of the three-stage recursive targeting hydrogel microspheres for nasal administration according to claim 7 or 8 in the preparation of nasal drops.

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