SiMYD88-loaded mesenchymal stem cell-derived extracellular vesicles, preparation method and application

Abstract

This invention relates to extracellular vesicles loaded with siMYD88 derived from mesenchymal stem cells, their preparation methods, and applications. The extracellular vesicles are derived from mesenchymal stem cells, preferably from human umbilical cord mesenchymal stem cells, and loaded with small interfering RNA targeting myeloid differentiation factor 88. The extracellular vesicles are loaded with siMYD88 via electroporation. These extracellular vesicles can reduce the expression of myeloid differentiation factor 88 in neuroimmune-related cells, inhibit the MYD88 / NF-κB / COX2 inflammatory signaling pathway, restore the NRF2 / HO-1 antioxidant signaling pathway, and improve MMP9 / Claudin-5-related blood-brain barrier damage. This invention also provides a quality control method based on the proportion of the main peak particle size, the proportion of residual free siMYD88, nuclease protection rate, and myeloid differentiation factor 88 silencing rate, which can be used to prepare drugs for treating or improving sepsis-related encephalopathy, neuroinflammation, blood-brain barrier damage, and cognitive impairment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to extracellular vesicles of mesenchymal stem cells loaded with siMYD88, their preparation method, and their applications. Background Technology

[0002] Sepsis is a systemic pathological state resulting from an imbalance in the host response induced by infection, which can further lead to damage to multiple organ functions. With improvements in emergency medicine and intensive care, more and more patients are able to survive the acute phase of sepsis. However, long-term brain dysfunction after sepsis survival is increasingly becoming a significant issue affecting prognosis and quality of life. Sepsis-related encephalopathy is a common central nervous system complication during sepsis, with clinical manifestations including altered consciousness, delirium, emotional abnormalities, decreased learning and memory abilities, and long-term cognitive impairment.

[0003] Current treatments primarily focus on anti-infection, circulatory support, fluid resuscitation, organ function maintenance, and metabolic correction, which are crucial for controlling infection in the acute phase. However, the recovery period after sepsis still involves persistent microglial activation, amplified neuroinflammation, increased oxidative stress, increased blood-brain barrier permeability, neurovascular unit damage, mitochondrial structural abnormalities, and impaired synaptic connections. These delayed or persistent injuries cannot be reversed simply by anti-infection treatment; current clinical protocols lack interventions targeting their core molecular mechanisms.

[0004] Myeloid differentiation factor 88 (MDF88) is an important intracellular adaptor protein downstream of most Toll-like receptors and interleukin-1 receptor families, participating in inflammatory signaling cascades such as IRAK, TRAF6, NF-κB, and MAPK. Sustained elevation of MDF88 in brain tissue after sepsis can promote inflammatory cytokine expression, microglial activation, enhanced oxidative stress, and blood-brain barrier disruption. Because MDF88 is located intracellularly, conventional antibody drugs have difficulty directly entering cells to exert their effects, while small molecule drugs suffer from insufficient specificity and limited central delivery. RNA interference (RNAI) technology can specifically silence target gene expression through small interfering RNA (sRNA), providing a novel intervention method for intracellular adaptor proteins like MDF88. However, naked sRNA exhibits poor in vivo stability, is easily degraded by nucleases, has limited transmembrane capacity, and struggles to effectively reach brain tissue or neuroimmune-related cells. Therefore, establishing a safe, stable, reproducible, and potentially brain-deliverable sRNA delivery system is a pressing technical challenge in this field.

[0005] Extracellular vesicles possess a lipid bilayer membrane structure, enabling them to carry bioactive substances such as RNA, proteins, and lipids. Mesenchymal stem cell-derived extracellular vesicles exhibit low immunogenicity, biocompatibility, and immunomodulatory potential, providing a basis for applications in neuroinflammation and tissue repair. Binding mesenchymal stem cell-derived extracellular vesicles to small interfering RNA (sRNA) targeting myeloid differentiation factor 88 can enhance the stability of the sRNA and leverage the membrane-bound delivery characteristics of extracellular vesicles to intervene in neuroimmune-related cells.

[0006] Current technologies still have the following shortcomings: there are insufficient drug delivery strategies for brain injury in the recovery period after sepsis, and the timing of treatment is mostly concentrated in the acute phase; when using mesenchymal stem cell-derived extracellular vesicles without nucleic acid loading, the ability to target and silence specific inflammatory hub molecules is limited; naked small interfering RNA lacks in vivo protection and brain delivery capabilities; there are batch-to-batch variations in the preparation of engineered extracellular vesicles, and there is a lack of executable quality judgment logic between particle size, loading efficiency, residual free small interfering RNA, and silencing activity. Summary of the Invention

[0007] To address the aforementioned issues, this application provides mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88, their preparation method, and their applications, resolving the problems of poor in vivo stability of naked siMYD88, low transmembrane delivery efficiency, limited delivery to brain tissue, and the lack of targeted intervention programs for persistent neuroinflammation during the recovery period after sepsis.

[0008] On the one hand, the present invention proposes extracellular vesicles derived from mesenchymal stem cells loaded with siMYD88. The extracellular vesicles are derived from mesenchymal stem cells and are loaded with small interfering RNA targeting myeloid differentiation factor 88. The small interfering RNA can reduce the expression of myeloid differentiation factor 88 in recipient cells.

[0009] Furthermore, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0010] Furthermore, the extracellular vesicles express at least two extracellular vesicle marker proteins from CD9, CD63, TSG101, and ALIX, and are negative for or have low expression of Calnexin.

[0011] Furthermore, the small interfering RNA comprises a sense strand 5'-CAACCUGGGUCAAGUGUAAtt-3' and an antisense strand 5'-UUACACUUGACCCAGGUUGtt-3'.

[0012] Furthermore, each 1×10⁹ extracellular vesicle particles was loaded with 0.40 μg to 0.80 μg of siMYD88.

[0013] Furthermore, the proportion of free siMYD88 residue in the extracellular vesicles is not higher than 15%; and / or The residual proportion of free siMYD88 is no higher than 10%.

[0014] Furthermore, the surface of the extracellular vesicles is attached with a brain delivery enhancing group, which is selected from at least one of transferrin receptor-binding peptide, low-density lipoprotein receptor-associated protein-binding peptide, neuron-targeting peptide, microglia uptake enhancing peptide, or inflammatory endothelial-binding peptide; and / or The brain delivery enhancement group is attached to the surface of the extracellular vesicle membrane via phospholipid insertion.

[0015] On the other hand, the present invention provides a method for preparing extracellular vesicles, comprising the following steps: S1. Culture mesenchymal stem cells and collect the culture supernatant; S2. Isolate and purify extracellular vesicles derived from mesenchymal stem cells from the culture supernatant; S3. Mix the extracellular vesicles with siMYD88; S4. SiMYD88 is loaded into the extracellular vesicles using electroporation. S5. Remove unloaded siMYD88 to obtain mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88.

[0016] Preferably, the feeding ratio of siMYD88 to extracellular vesicle particles is 0.5 μg to 2 μg siMYD88 per 1 × 10⁹ extracellular vesicle particles; or the feeding ratio of siMYD88 to extracellular vesicle particles is 1 μg siMYD88 per 1 × 10⁹ extracellular vesicle particles.

[0017] Furthermore, the present invention also provides a pharmaceutical composition comprising extracellular vesicles as described above and a pharmaceutically acceptable carrier or excipient.

[0018] Furthermore, the pharmaceutical composition is an injection, a lyophilized powder injection, a nano-suspension, a nasal delivery formulation, a brain-targeted delivery formulation, or an intravenous delivery formulation.

[0019] Furthermore, the present invention also provides the use of extracellular vesicles or the pharmaceutical composition in the preparation of medicaments for treating or improving sepsis-related encephalopathy.

[0020] Preferably, the treatment or improvement includes at least one of the following: neuroinflammation, blood-brain barrier damage, increased oxidative stress, neurovascular unit damage, hippocampal mitochondrial damage, synaptic structure damage, anxiety-like behavior, cognitive memory impairment, spatial learning and memory impairment, or long-term cognitive dysfunction.

[0021] Preferably, the extracellular vesicles are used for administration during the recovery period after sepsis.

[0022] Preferably, the extracellular vesicles exert their effects through at least one of the following mechanisms: reducing myeloid differentiation factor 88 expression, inhibiting the MYD88 / NF-κB / COX2 inflammatory signaling pathway, restoring the NRF2 / HO-1 antioxidant signaling pathway, reducing MMP9 expression, restoring Claudin-5 expression, alleviating increased blood-brain barrier permeability, or alleviating damage to hippocampal mitochondrial and synaptic structures.

[0023] Furthermore, the present invention also provides the use of extracellular vesicles or the pharmaceutical composition thereof in the preparation of medicaments for treating or improving diseases related to inflammation of the central nervous system, diseases related to neurovascular injury, or diseases related to blood-brain barrier damage.

[0024] The beneficial effects of this invention are as follows: (1) This invention uses extracellular vesicles derived from mesenchymal stem cells as siMYD88 delivery vectors. The extracellular vesicle lipid bilayer membrane structure can protect siMYD88 from nuclease degradation and improve the target gene silencing efficiency after siMYD88 enters neuroimmune-related cells, thereby overcoming the defects of poor stability, weak transmembrane ability and limited brain delivery efficiency of naked siRNA. At the same time, this invention further establishes a quality control method based on the proportion of the main peak of particle size, the proportion of free siMYD88 residue, the nuclease protection rate and the MYD88 silencing rate. This method can exclude batches with qualified particle size but ineffective loading, high total nucleic acid content but excessive free siRNA residue, insufficient nuclease protection or insufficient silencing activity, so that the preparation quality of engineered extracellular vesicles matches the actual delivery activity.

[0025] (2) The present invention also provides a brain delivery enhanced extracellular vesicle and a drug delivery window determination scheme for the recovery period after sepsis, which expands the present invention from a single nucleic acid delivery formulation to a drug preparation scheme with optimized brain tissue delivery, controllable batch preparation and adaptability for drug delivery during the recovery period, and can provide new technical means for the treatment or improvement of sepsis-related encephalopathy and its associated neuroinflammation, neurovascular injury and cognitive dysfunction. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] Figure 1 This is a schematic diagram of the preparation process of siMYD88-EVs of the present invention.

[0028] Figure 2 This is a particle size distribution diagram of NTA in EVs derived from hUCMSCs of this invention.

[0029] Figure 3This is a Western blot characterization diagram of the EVs marker proteins of this invention.

[0030] Figure 4 This invention relates to the effects of siMYD88-EVs and RNase treatment on MYD88 mRNA expression in BV2 cells.

[0031] Figure 5 This invention relates to the LPS-induced changes in IBA1 fluorescence intensity in microglia.

[0032] Figure 6 This invention relates to LPS-induced changes in TREM2-related signals in microglia.

[0033] Figure 7 This is the survival rate curve of the high-survival-rate CLP model of the present invention at 72 h post-surgery.

[0034] Figure 8 This represents the dwell time result in the central region of the open-field experiment of this invention.

[0035] Figure 9 This is the result of the motion distance in the central area of ​​the open field experiment of this invention.

[0036] Figure 10 The results of the discrimination index in the new object recognition experiment of this invention are shown.

[0037] Figure 11 This is the training curve for the escape latency of the Barnes maze in this invention.

[0038] Figure 12 This is the training curve for the number of errors in the Barnes maze of this invention.

[0039] Figure 13 The results of the Evans blue assay for detecting blood-brain barrier permeability are presented in this invention.

[0040] Figure 14 This is the quantitative result of the MYD88, IBA1 and MYD88 / IBA1 co-localization signals in the prefrontal region of the present invention.

[0041] Figure 15 This is the quantitative result of hippocampal mitochondrial area in this invention.

[0042] Figure 16 This is the quantitative result of hippocampal synaptic vesicle density in this invention.

[0043] Figure 17 The results show the fluorescence intensity of IBA1 in the CA1, CA3, and DG regions of the hippocampus in this invention.

[0044] Figure 18 The results show the MYD88 fluorescence intensity in the CA1, CA3, and DG regions of the hippocampus in this invention.

[0045] Figure 19 The results show the GFAP fluorescence intensity in the CA1, CA3, and DG regions of the hippocampus in this invention.

[0046] Figure 20 This is the quantitative result of MYD88 protein expression in brain tissue according to the present invention.

[0047] Figure 21 The results show the ratio of p-NF-κB p65 / NF-κB p65 in brain tissue according to the present invention.

[0048] Figure 22 The results show the expression of COX2 protein in brain tissue according to the present invention.

[0049] Figure 23 The results show the TREM2 fluorescence intensity of brain tissue in this invention.

[0050] Figure 24 The results of NRF2 protein expression in brain tissue are presented in this invention.

[0051] Figure 25 The results of HO-1 protein expression in brain tissue are presented in this invention.

[0052] Figure 26 The results show the expression of MMP9 protein in brain tissue according to the present invention.

[0053] Figure 27 The results of Claudin-5 protein expression in brain tissue are presented in this invention. Detailed Implementation

[0054] To make the objectives, technical means, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0056] Example 1: Preparation of extracellular vesicles from mesenchymal stem cells loaded with siMYD88 In this embodiment, human umbilical cord mesenchymal stem cells were cultured at 37°C and 5% carbon dioxide using serum-free medium or medium with extracellular vesicles pre-removed. When cell confluence reached 70% to 80%, the original medium was discarded, and the cells were washed twice with sterile phosphate-buffered saline (PBS). Fresh serum-free medium was then added, and the cells were cultured for another 48 hours. A cell confluence of 70% to 80% was chosen as the pre-collection state because at this state, the cells are still in a stable proliferation and secretion phase, reducing the risk of apoptotic bodies and cell debris contamination caused by excessive cell confluence, and ensuring a stable yield of extracellular vesicles in the culture supernatant.

[0057] In this embodiment, after collecting the culture supernatant, intact cells were first removed by centrifugation at 300×g for 10 min, followed by centrifugation at 2000×g for 20 min to remove cell debris, and then filtered through a 0.22 μm filter membrane. The filtered culture supernatant was then enriched with extracellular vesicles using a negative pressure oscillation separation method, and the obtained extracellular vesicles were resuspended in sterile phosphate buffer. Nanoparticle tracking analysis of the obtained extracellular vesicles showed median particle sizes of 103.6 nm, 106.2 nm, and 101.9 nm, with a median average particle size of 103.9 nm. The percentages of the main particle size peaks were 83.5%, 81.2%, and 85.7%, respectively, with an average percentage of 83.5%, indicating that the obtained extracellular vesicles have a stable nanoscale particle size distribution.

[0058] Further, the purified extracellular vesicles were mixed with siMYD88 at a ratio of 1 μg siMYD88 per 1 × 10^9 extracellular vesicle particles. The sense strand of siMYD88 was 5'-CAACCUGGGUCAAGUGUAAtt-3', and the antisense strand was 5'-UUACACUUGACCCAGGUUGtt-3'. After mixing, electroporation buffer was added to make the system volume 400 μL, and the mixture was placed in a 4 mm electroporation cup for electroporation at 400 V, 125 μF, and a single pulse. After electroporation, the mixture was placed on ice for 10 min to allow the briefly open extracellular vesicle membrane structure to close again.

[0059] In this process, the mixture after electroporation was washed three times by ultrafiltration to remove unloaded siMYD88. To further eliminate free siMYD88 adsorbed on the outer surface of extracellular vesicles, nuclease was added after ultrafiltration washing for external digestion, followed by ultrafiltration purification to remove nuclease and degradation fragments, ultimately yielding mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88. In this step, the nuclease can only degrade siMYD88 not protected by the vesicle membrane structure and cannot effectively enter the interior of intact extracellular vesicles; therefore, it can be used to distinguish between effectively loaded and adsorbed siMYD88.

[0060] Therefore, the method of this application allows siMYD88 to enter the lumen or inner membrane of extracellular vesicles derived from mesenchymal stem cells, rather than simply mixing with them. Testing showed that each 1×10 9 The actual siMYD88 loading in each extracellular vesicle granule was 0.58 μg, 0.63 μg, and 0.60 μg, with an average of 0.60 μg; the residual proportions of free siMYD88 were 8.6%, 10.4%, and 9.1%, with an average of 9.4%. These results indicate that, within each 1 × 103 9 Under the condition that each particle corresponds to 1 μg siMYD88, a stable loading amount can be obtained, and the residual proportion of free siMYD88 can be controlled below 15%, preferably around 10%.

[0061] The proportion of free siMYD88 residue was calculated by combining the nucleic acid content of the undried sample with the total nucleic acid content of the lysed sample. Specifically, the same formulation was divided into undried and lysed samples. For the undried sample, the nucleic acid signal was directly detected, and the obtained value corresponded to the amount of free siMYD88 outside the extracellular vesicles. For the lysed sample, a membrane lysis reagent was added, and the nucleic acid signal was detected, and the obtained value corresponded to the total nucleic acid content of free siMYD88 and siMYD88 inside the extracellular vesicles. The nucleic acid content of the undried sample was divided by the total nucleic acid content of the lysed sample, and then multiplied by 100% to obtain the proportion of free siMYD88 residue. This calculation method avoids mistaking siMYD88 adsorbed outside the vesicles for the effective loading amount.

[0062] Example 2: Characterization of extracellular vesicles from mesenchymal stem cells loaded with siMYD88 like Figures 2-3 As shown, in this embodiment, the extracellular vesicles of mesenchymal stem cells loaded with siMYD88 prepared in Example 1 were used for particle size, particle concentration, and marker protein detection. Nanoparticle tracking analysis showed that the particle size of the extracellular vesicles was mainly distributed between 50 nm and 200 nm, with the main peak between 80 nm and 150 nm, and a median particle size of 104.9 nm. This particle size range conforms to the typical distribution characteristics of small extracellular vesicles, indicating that the electroporation and purification process did not cause significant aggregation of large particles.

[0063] In this embodiment, Western blotting was used to detect extracellular vesicle marker proteins. The results showed that the obtained extracellular vesicles expressed CD9, CD63, TSG101, and ALIX, while Calnexin was negative or lowly expressed. CD9 and CD63 were used to characterize extracellular vesicle membrane-associated tetraspan membrane proteins, TSG101 and ALIX were used to characterize extracellular vesicle formation-related proteins, and Calnexin was used to exclude endoplasmic reticulum-derived contamination. These results indicate that the obtained products were predominantly extracellular vesicles with minimal organelle contamination.

[0064] Furthermore, a nuclease protection assay was used to detect whether siMYD88 was effectively protected by extracellular vesicles. Extracellular vesicles loaded with siMYD88 were divided into a nuclease-treated group and an untreated group. The nuclease-treated group was treated with RNase and then purified, while the untreated group was not treated with RNase. Subsequently, the two groups of samples were applied to LPS-induced microglia, and MYD88 mRNA expression was detected. The results showed that the MYD88 silencing rate in the untreated group was 66.0%, and the silencing rate after RNase treatment remained at 56.8%, indicating a nuclease protection rate of 86.1%.

[0065] The nuclease protection rate was calculated as follows: First, the MYD88 silencing rates of the RNase-treated group and the untreated group were obtained separately. Then, the silencing rate of the RNase-treated group was divided by the silencing rate of the untreated group, and multiplied by 100%. Following this calculation method, 56.8% divided by 66.0% and multiplied by 100% yielded a nuclease protection rate of 86.1%. This result demonstrates that siMYD88 is primarily protected by the extracellular vesicle membrane structure, rather than existing in a naked state within the formulation.

[0066] Example 3: Effect of siMYD88-loaded mesenchymal stem cell-derived extracellular vesicles on MYD88 expression in microglia. like Figure 4 As shown, in this embodiment, an in vitro inflammation model was established using BV2 microglia. Cells were seeded in culture plates, and after cell adhesion was stable, 1 μg / mL lipopolysaccharide was added for 24 h to induce microglia inflammatory activation. The experiment was divided into a blank control group, an LPS model group, a naked siMYD88 group, a negative siRNA extracellular vesicle group, a siMYD88 extracellular vesicle group, and a brain-delivered enhanced siMYD88 extracellular vesicle group. After 24 h of treatment in each group, total RNA was extracted from the cells, and MYD88 mRNA expression was detected.

[0067] In this embodiment, the MYD88 silencing rate was calculated using quantitative polymerase chain reaction (PCR). First, the circulation thresholds of the MYD88 gene and the internal reference gene were detected. The first difference was obtained by subtracting the circulation threshold of the internal reference gene from the MYD88 gene circulation threshold. Then, the second difference was obtained by subtracting the first difference of the LPS model group from the first difference of the treatment group. Subsequently, using 2 as the base and the negative of the second difference as the exponent, the expression level of the treatment group relative to the LPS model group was obtained. Finally, the relative expression level was subtracted from 1 and multiplied by 100% to obtain the MYD88 silencing rate.

[0068] Furthermore, the detection results showed that the relative expression level of MYD88 in the LPS model group was set at 1.00±0.08; the relative expression level of MYD88 in the naked siMYD88 group was 0.71±0.07, corresponding to a silencing rate of 29.0%; the relative expression level of MYD88 in the negative siRNA extracellular vesicle group was 0.94±0.09, corresponding to a silencing rate of 6.0%; the relative expression level of MYD88 in the siMYD88 extracellular vesicle group was 0.34±0.05, corresponding to a silencing rate of 66.0%; and the relative expression level of MYD88 in the brain-delivered enhanced siMYD88 extracellular vesicle group was 0.28±0.04, corresponding to a silencing rate of 72.0%.

[0069] Table 1 Results of MYD88 silencing rate and nuclease protection rate

[0070] Nuclease protection rate was calculated as "RNase silencing rate after treatment ÷ untreated silencing rate × 100%". The nuclease protection rate was 86.1% in the siMYD88-EVs group and 85.3% in the brain delivery enhanced siMYD88-EVs group.

[0071] In addition, naked siMYD88, siMYD88 extracellular vesicles, and brain-delivered enhanced siMYD88 extracellular vesicles were treated with RNase before being applied to LPS-induced microglia. The results showed that the silencing rate of naked siMYD88 decreased to 6.8% after RNase treatment; the silencing rate of siMYD88 extracellular vesicles remained at 56.8% after RNase treatment; and the silencing rate of brain-delivered enhanced siMYD88 extracellular vesicles remained at 61.4% after RNase treatment. These results indicate that extracellular vesicles can protect siMYD88 from nuclease degradation and maintain its target gene silencing ability.

[0072] Therefore, in this embodiment, extracellular vesicles loaded with siMYD88 derived from mesenchymal stem cells can significantly reduce MYD88 expression in microglia in an inflammatory state, and its silencing effect is significantly better than that of naked siMYD88 and negative siRNA extracellular vesicles. This result demonstrates that extracellular vesicle delivery and siMYD88 targeted silencing together constitute the effective intervention basis of this invention.

[0073] Example 4: Regulation of microglial inflammatory phenotype by extracellular vesicles loaded with siMYD88 from mesenchymal stem cell-derived cells like Figures 5-6As shown, in this embodiment, BV2 microglia and primary microglia were used to verify the effect of siMYD88-loaded extracellular vesicles on the inflammatory phenotype. Cells were stimulated with 1 μg / mL lipopolysaccharide for 24 h to induce an inflammatory activated state. siMYD88-loaded extracellular vesicles were added to the culture system 4 h before lipopolysaccharide stimulation, with a final concentration of 1 × 10⁻⁶. 8 particles / mL. After treatment, IBA1 and TREM2 signals were detected by immunofluorescence.

[0074] In this embodiment, IBA1 is used to reflect the degree of microglial inflammatory activation, and TREM2 is used to reflect microglial repair or homeostasis-related responses. Exposure time, gain, and excitation intensity are kept consistent during immunofluorescence image acquisition. At least three fields of view are randomly selected for each sample group. The average fluorescence intensity is calculated after subtracting background fluorescence from each field of view, and the average value of multiple fields of view within the same group is taken as the detection result for that group.

[0075] Furthermore, the results showed that the IBA1 fluorescence intensity in the LPS model group was significantly higher than that in the blank control group, indicating that microglia were in an inflammatory activated state. After treatment with siMYD88 extracellular vesicles, the IBA1 fluorescence intensity decreased by approximately 42.6% compared to the LPS model group. Meanwhile, the TREM2 signal in the LPS model group only increased slightly, while the TREM2 fluorescence intensity after treatment with siMYD88 extracellular vesicles increased by approximately 38.4% compared to the LPS model group. These results indicate that siMYD88-loaded extracellular vesicles not only inhibit inflammatory activation signals but also promote the transition of microglia to a repair or homeostasis-related state.

[0076] Therefore, the method described in this application can regulate the inflammatory phenotype of microglia while reducing MYD88 expression, avoiding the cellular dysfunction caused by simple non-specific immunosuppression. This effect is of targeted significance for persistent microglia activation in brain tissue during the post-sepsis recovery period.

[0077] Example 5: Animal Model of Sepsis-Related Encephalopathy and Dosage Calibration like Figure 7-12 As shown in this example, a high-survival-rate cecal ligation and perforation model was established using 12-week-old male C57BL / 6 mice. After anesthesia, the mice underwent abdominal incision to expose the cecum. Approximately 10% of the distal cecum was ligated, and a single puncture was performed using an 18G needle. In the sham-operated group, only abdominal incision and cecal exposure were performed; ligation and puncture were not performed. Postoperatively, pre-warmed sterile saline was administered subcutaneously, and the mice were kept at a constant temperature for recovery.

[0078] Table 2 Dosage exploration results

[0079] In this embodiment, survival was assessed 72 hours after CLP surgery, and surviving mice were randomly divided into CLP model group, low-dose group, medium-dose group, and high-dose group. The low-dose group received 3×10 8 Extracellular vesicles loaded with siMYD88 per particle / unit / dose, medium dose group received 1×10 9 Extracellular vesicles loaded with siMYD88 per particle / unit / dose, high-dose group received 3×10 9 Extracellular vesicles loaded with siMYD88 were administered via tail vein injection once daily for 4 consecutive days. Behavioral assessments were performed from day 20 to day 28 post-surgery, and samples were taken on day 28 for molecular and histological analysis.

[0080] Furthermore, MYD88 protein expression in brain tissue was measured on postoperative day 28. The relative expression level of MYD88 protein was 1.00±0.10 in the CLP model group, 0.74±0.08 in the low-dose group, 0.48±0.06 in the medium-dose group, and 0.43±0.07 in the high-dose group. The results showed that the low-dose group could partially reduce MYD88 expression, the medium-dose group could significantly reduce MYD88 expression, and the high-dose group showed limited further reduction compared to the medium-dose group.

[0081] The Evansblue assay was used to detect blood-brain barrier permeability. The Evansblue leakage rate in the brain tissue of the CLP model group was 4.8±0.7 μg / g, the low-dose group was 3.7±0.6 μg / g, the medium-dose group was 2.1±0.5 μg / g, and the high-dose group was 1.9±0.4 μg / g. These results indicate that extracellular vesicles loaded with siMYD88 can reduce the increase in blood-brain barrier permeability in brain tissue after sepsis, with 1×10⁻⁶ vesicles showing the highest permeability. 9 The dosage of granules / units / dose has been able to achieve a relatively stable barrier protection effect.

[0082] In addition, a novel object recognition experiment was used to evaluate recognition memory. The discrimination index was 0.08±0.05 for the CLP model group, 0.17±0.06 for the low-dose group, 0.31±0.07 for the medium-dose group, and 0.33±0.08 for the high-dose group. The discrimination index was obtained by subtracting the exploration time of the old object from the exploration time of the new object, and then dividing by the sum of the exploration times of the new object and the old object. This calculation method can correct for differences in the total exploration time among different animals and more accurately reflect the level of recognition memory.

[0083] From the above, we can see that 1×10 8 Up to 5×10 9 The dosage range of granules / particles / dose can be used as the dosing range for extracellular vesicles loaded with siMYD88, 5×10 8 Up to 2×10 9The preferred dosage range is granules / particles / dose, 1×10 9 A dosage of granules / unit / dose is a preferred option. This dosage can reduce MYD88 expression and improve blood-brain barrier damage while avoiding the increased delivery burden associated with high-dose administration.

[0084] Example 6: Improvement of behavioral impairment by extracellular vesicles loaded with siMYD88 from mesenchymal stem cells like Figures 12-16 As shown, in this embodiment, the effects of siMYD88-loaded extracellular vesicles on post-sepsis behavioral impairment were evaluated using open-field experiments, novel object recognition experiments, and Barnes maze experiments. The open-field experiments were conducted on postoperative day 20, the novel object recognition experiments on postoperative days 22-23, and the Barnes maze experiments on postoperative days 24-28. All behavioral experiments were performed within fixed time periods to minimize the influence of circadian rhythms on the results.

[0085] In this embodiment, the open-field experiment recorded the dwell time in the central region, the distance moved in the central region, and the total distance moved. The CLP model group showed a significantly reduced dwell time in the central region and a significantly reduced distance moved in the central region compared to the sham-operated group, while the total distance moved did not decrease significantly. This indicates that the animals' basic motor abilities did not constitute a major disturbance, and the decreased activity in the central region mainly reflects a decline in anxiety-like behavior or exploratory behavior. After administration of extracellular vesicles loaded with siMYD88, both the dwell time and the distance moved in the central region increased, indicating that anxiety-like behavior was improved after sepsis.

[0086] Furthermore, the results of the new object recognition experiment showed that the discrimination index of the CLP model group decreased, indicating impaired recognition memory; after treatment with extracellular vesicles loaded with siMYD88, the discrimination index increased significantly. According to the calculation method described in Example 5, the discrimination index of the medium-dose group increased from 0.08±0.05 in the CLP model group to 0.31±0.07, indicating that the preparation can improve recognition memory impairment after sepsis.

[0087] In addition, the Barnes maze experiment recorded escape latency, number of errors, and time spent in the target quadrant. The CLP model group showed prolonged escape latency and increased number of errors during the training period, and decreased time spent in the target quadrant during the testing period, indicating impaired spatial learning and memory abilities. Treatment with extracellular vesicles loaded with siMYD88 shortened escape latency, reduced the number of errors, and increased time spent in the target quadrant, indicating that it can improve spatial learning and memory impairment after sepsis.

[0088] Therefore, in this embodiment, extracellular vesicles of mesenchymal stem cells loaded with siMYD88 can improve anxiety-like behavior, cognitive memory impairment, and spatial learning memory impairment during the recovery period after sepsis. Furthermore, the behavioral improvement corresponds to the decrease in MYD88 expression and the reduction in blood-brain barrier leakage, indicating that its effect does not stem from simply improving basic motor ability.

[0089] Example 7: Effects of siMYD88-loaded mesenchymal stem cell-derived extracellular vesicles on inflammatory pathways, antioxidant pathways, and blood-brain barrier-related pathways. like Figures 17-27 As shown in this embodiment, prefrontal lobe and hippocampal tissues of mice were harvested on postoperative day 28 to detect the expression of MYD88, IBA1, GFAP, TREM2, p-NF-κBp65, NF-κBp65, COX2, NRF2, HO-1, MMP9, and Claudin-5, respectively. Detection methods included immunofluorescence, Western blotting, and quantitative polymerase chain reaction.

[0090] In this embodiment, immunofluorescence images were acquired using the same exposure parameters. At least three brain tissue slices were selected from each animal, and at least three fields of view were selected from each slice. During calculation, the average gray level of the background area was first subtracted, then the average fluorescence intensity of the target area was obtained, and the average of the results from multiple fields of view for the same animal was taken as the detection value for that animal. This processing method can reduce the bias caused by uneven distribution of inflammation in a single field of view.

[0091] Furthermore, the results of Western blot analysis were corrected by comparing the grayscale values ​​of the target protein band with those of the internal control protein band. For p-NF-κBp65, the grayscale values ​​of the p-NF-κBp65 band were first obtained, followed by the grayscale values ​​of the total NF-κBp65 band. The phosphorylation ratio of NF-κBp65 was obtained by dividing the grayscale value of the p-NF-κBp65 band by the grayscale value of the total NF-κBp65 band. In the CLP model group, MYD88 expression was increased, the p-NF-κBp65 / NF-κBp65 ratio was increased, and COX2 expression was increased. After treatment with extracellular vesicles loaded with siMYD88, all of the above indicators decreased, indicating that the MYD88 / NF-κB / COX2 inflammatory pathway was inhibited.

[0092] Among the findings, the antioxidant pathway detection results showed that NRF2 and HO-1 expression decreased in the CLP model group, indicating a reduced antioxidant defense capacity of brain tissue. Treatment with extracellular vesicles loaded with siMYD88 increased NRF2 and HO-1 expression, suggesting that it could restore the NRF2 / HO-1 antioxidant signaling pathway. The blood-brain barrier related indicators showed that MMP9 expression increased and Claudin-5 expression decreased in the CLP model group. Treatment with extracellular vesicles loaded with siMYD88 decreased MMP9 expression and restored Claudin-5 expression, indicating that it could alleviate damage to the tight junctions of the blood-brain barrier.

[0093] Therefore, in this embodiment, the extracellular vesicles derived from mesenchymal stem cells loaded with siMYD88 initially reduce MYD88 expression, further inhibiting NF-κBp65 phosphorylation and COX2 expression, restoring the NRF2 / HO-1 antioxidant pathway, and improving MMP9 / Claudin-5-related blood-brain barrier damage. The above-described detection chain fully illustrates the implementation process of the technical effects described in the claims of this invention.

[0094] Example 8: Brain delivery of enhanced mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88 As a preferred embodiment, a brain delivery enhancing group is inserted into the surface of the extracellular vesicles loaded with siMYD88 prepared in Example 1. The brain delivery enhancing group is attached to the surface of the extracellular vesicle membrane via a phospholipid derivative containing a polyethylene glycol spacer arm. In preparation, the brain delivery enhancing peptide is first linked to the phospholipid derivative to form a lipid-intercalated brain delivery molecule. This lipid-intercalated brain delivery molecule is then incubated with the siMYD88-loaded extracellular vesicles at 37°C for 45 min, allowing the phospholipid end to insert into the vesicle membrane, while the brain delivery enhancing peptide is exposed on the outer side of the vesicle.

[0095] In some embodiments, unmodified group, low modification ratio group, medium modification ratio group, and high modification ratio group are provided. The low modification ratio group, medium modification ratio group, and high modification ratio group correspond to each 1×10 9 Extracellular vesicle particles were treated with 0.5 μg, 1.0 μg, and 2.0 μg of lipid-intercalated brain delivery molecules. Results showed that the median particle size was 103.9 nm in the unmodified group, 108.6 nm in the low-modification group, 116.8 nm in the medium-modification group, and 151.4 nm in the high-modification group. The high-modification group exhibited a large particle peak, with the main peak accounting for only 61.2% of the total particle size, indicating that excessive surface modification induces extracellular vesicle aggregation.

[0096] Furthermore, fluorescently labeled extracellular vesicles were used to detect microglial uptake. The uptake rate was set at 1.00 for the unmodified group, 1.28 for the low-modification group, 1.63 for the medium-modification group, and 1.70 for the high-modification group. The corresponding MYD88 silencing rates were 66.0%, 69.5%, 72.0%, and 62.4%, respectively. Although the uptake rate of the high-modification group was slightly higher than that of the medium-modification group, the actual silencing rate decreased due to increased particle size aggregation. The medium-modification group achieved a better balance between particle size stability, cell uptake rate, and MYD88 silencing rate.

[0097] Therefore, in this embodiment, the modification ratio of brain delivery enhancement groups is not necessarily better the higher it is, but needs to be determined by the proportion of the main peak of particle size, the uptake rate and the MYD88 silencing rate.

[0098] Example 9: Batch Release Method Based on Multi-Indicator Data Processing In one example implementation, a batch release method is established for mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88. This method collects four core data points: the percentage of the main peak in particle size distribution, the proportion of residual free siMYD88, the nuclease protection rate, and the MYD88 silencing rate. The percentage of the main peak in particle size distribution reflects the structural stability of the extracellular vesicles; the proportion of residual free siMYD88 reflects the effective loading degree; the nuclease protection rate reflects the proportion of siMYD88 in a membrane-protected state; and the MYD88 silencing rate reflects the actual biological activity.

[0099] In this embodiment, the release thresholds are set as follows: the proportion of the main peak in particle size is not less than 70%, the proportion of free siMYD88 residue is not higher than 15%, the nuclease protection rate is not less than 75%, and the MYD88 silencing rate is not less than 50%. When all four indicators are met, the batch is deemed to meet the release conditions; if any one of the core indicators is not met, the batch is deemed not to meet the release conditions. This key indicator rejection method is adopted because extracellular vesicle formulations may have conditions such as qualified particle size but insufficient silencing activity, high total nucleic acid content but excessive free nucleic acid residue, and insufficient nuclease protection rate due to nucleic acid adsorption. A single indicator cannot accurately reflect the quality of the formulation.

[0100] Table 3. Test results of 6 preparation batches

[0101] Furthermore, six preparation batches were tested. Batch 1 had a main peak percentage of 83.5%, a free siMYD88 residual percentage of 8.6%, a nuclease protection rate of 86.1%, and a MYD88 silencing rate of 66.0%, and was approved for release. Batch 2 had a main peak percentage of 81.2%, a free siMYD88 residual percentage of 10.4%, a nuclease protection rate of 82.7%, and a MYD88 silencing rate of 63.5%, and was approved for release. Batch 3 had a main peak percentage of 85.7%, a free siMYD88 residual percentage of 9.1%, a nuclease protection rate of 84.5%, and a MYD88 silencing rate of 68.2%, and was approved for release.

[0102] In addition, the fourth batch had a main peak percentage of 68.4%, a free siMYD88 residual percentage of 9.8%, a nuclease protection rate of 80.6%, and a MYD88 silencing rate of 58.9%. Although the free siMYD88 residual percentage, nuclease protection rate, and MYD88 silencing rate of the fourth batch met the requirements, the main peak percentage was less than 70%, indicating an increase in aggregated or fragmented particles after electroporation, therefore it was deemed unsuitable for release. The fifth batch had a main peak percentage of 82.1%, a free siMYD88 residual percentage of 23.7%, a nuclease protection rate of 46.5%, and a MYD88 silencing rate of 51.4%, indicating that the silencing effect in this batch mainly came from unprotected free siMYD88 or surface-adsorbed nucleic acids, therefore it was deemed unsuitable for release. The sixth batch had a main peak proportion of 79.3%, a free siMYD88 residual proportion of 11.2%, a nuclease protection rate of 78.8%, and a MYD88 silencing rate of 38.6%, indicating that it had problems with ineffective delivery after loading or insufficient cellular uptake, and was therefore not released.

[0103] As shown above, this embodiment addresses the fundamental data processing problem in the preparation of engineered extracellular vesicles, namely, the different sources, dimensions, and pharmacodynamic correlations of various detection indicators, making it impossible to judge the quality of the formulation by simple averaging. This embodiment employs a method of evaluating each core indicator individually and rejecting any core indicator, ensuring that the release results are consistent with the structural stability of extracellular vesicles, the authenticity of siMYD88 loading, nucleic acid protection ability, and target gene silencing activity, and can be used for subsequent large-scale preparation and quality consistency evaluation.

[0104] Example 10: Method for determining the drug administration window during the recovery period after sepsis In another specific implementation, a method for determining the drug administration window during the recovery period after sepsis is established. This method is used for pretreatment in animal experiments and efficacy evaluations, aiming to differentiate between general weakness and decreased basic motor function during the acute phase of sepsis and brain function impairment during the recovery period, avoiding misdiagnosis of acute weakness as cognitive impairment.

[0105] In this embodiment, the animal's survival status, weight recovery rate, activity recovery rate, and central area exploration rate were recorded at 24h, 48h, 72h, and day 5 after CLP surgery. The weight recovery rate was obtained by dividing the weight on the day of testing by the weight before modeling; the activity recovery rate was obtained by dividing the total movement distance on the day of testing by the average total movement distance of the sham surgery group on the same day; and the central area exploration rate was obtained by dividing the central area movement distance by the total movement distance.

[0106] Furthermore, animals were deemed to have entered the post-sepsis recovery period medication window when they simultaneously met the following conditions: the animal was alive and able to feed and drink independently; the activity recovery rate was not less than 50%; the central area exploration rate decreased by not less than 25% compared to the sham-operated group; and the weight recovery rate was not less than 75%. An activity recovery rate of not less than 50% was used to exclude interference from severe general weakness on behavioral testing; a central area exploration rate decrease of not less than 25% was used to confirm the continued presence of brain function-related behavioral abnormalities; and a weight recovery rate of not less than 75% was used to exclude false-positive behavioral changes caused by excessive general depletion.

[0107] In this embodiment, the animal activity recovery rate was 28.5%±9.4% and the weight recovery rate was 69.8%±5.7% 24 hours after CLP, which did not fall within the recovery period drug administration window; the activity recovery rate was 43.2%±10.6% and the weight recovery rate was 73.5%±6.1% 48 hours after CLP, which still did not fall within the recovery period drug administration window; the activity recovery rate was 57.8%±11.2% and the weight recovery rate was 78.6%±5.4% 72 hours after CLP, and the central area exploration rate decreased by 32.4%±8.7% compared to the sham surgery group, which was considered to have entered the recovery period drug administration window; the activity recovery rate was 65.1%±12.3% on the 5th day after CLP, and the central area exploration rate decreased by 28.6%±9.2% compared to the sham surgery group, which was still within the intervention window.

[0108] As can be seen, the embodiments of this application transform the assessment of the convalescent dosing window from a simple postoperative time point to a combined assessment of weight, activity level, and central zone exploration ratio, which can reduce the interference of acute general weakness on cognitive and behavioral evaluation. This method makes the dosing scenario of the present invention more clearly targeted at persistent brain injury after sepsis survival, rather than acute anti-infective treatment of sepsis or treatment to simply improve short-term survival.

[0109] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88, characterized in that, The extracellular vesicles are derived from mesenchymal stem cells, and each extracellular vesicle is loaded with a small interfering RNA that targets myeloid differentiation factor 88. The small interfering RNA can reduce the expression of myeloid differentiation factor 88 in recipient cells.

2. The extracellular vesicle according to claim 1, characterized in that, The mesenchymal stem cells mentioned are human umbilical cord mesenchymal stem cells.

3. The extracellular vesicle according to claim 2, characterized in that, The extracellular vesicles express at least two extracellular vesicle marker proteins from CD9, CD63, TSG101, and ALIX, and are negative for or have low expression of Calnexin.

4. The extracellular vesicle according to claim 3, characterized in that, The small interfering RNAs include 5'-CAACCUGGGUCAAGUGUAAtt-3' of the positive strand and 5'-UUACACUUGACCCAGGUUGtt-3' of the antisense strand.

5. The extracellular vesicle according to claim 4, characterized in that, Each 1×10 9 Each extracellular vesicle particle was loaded with 0.40 μg to 0.80 μg of siMYD88.

6. The extracellular vesicle according to claim 5, characterized in that, The proportion of free siMYD88 residue in the extracellular vesicles is not higher than 15%; and / or The residual proportion of free siMYD88 is no higher than 10%.

7. The extracellular vesicle according to claim 6, characterized in that, The extracellular vesicles have a brain delivery enhancing group attached to their surface, the brain delivery enhancing group being selected from at least one of transferrin receptor-binding peptide, low-density lipoprotein receptor-associated protein-binding peptide, neuron-targeting peptide, microglia uptake enhancing peptide, or inflammatory endothelial-binding peptide; and / or The brain delivery enhancement group is attached to the surface of the extracellular vesicle membrane via phospholipid insertion.

8. A method for preparing extracellular vesicles according to any one of claims 1 to 7, characterized in that, Includes the following steps: S1. Culture mesenchymal stem cells and collect the culture supernatant; S2. Isolate and purify extracellular vesicles derived from mesenchymal stem cells from the culture supernatant; S3. Mix the extracellular vesicles with siMYD88; S4. SiMYD88 is loaded into the extracellular vesicles using electroporation. S5. Remove unloaded siMYD88 to obtain mesenchymal stem cell-derived extracellular vesicles loaded with siMYD88.

9. The method for preparing extracellular vesicles according to claim 8, characterized in that, The feeding ratio of siMYD88 to extracellular vesicle particles is 1×10 9 Each extracellular vesicle particle corresponds to 0.5 μg to 2 μg siMYD88; or the feeding ratio of siMYD88 to extracellular vesicle particles is 1 × 10⁻⁶. 9 One extracellular vesicle particle corresponds to 1 μg siMYD88.

10. A pharmaceutical composition, characterized in that, Includes the extracellular vesicles as described in any one of claims 1 to 7, and pharmaceutically acceptable carriers or excipients.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutical composition is an injection, a lyophilized powder for injection, a nano-suspension, a nasal delivery formulation, a brain-targeted delivery formulation, or an intravenous delivery formulation.

12. Use of the extracellular vesicles of any one of claims 1 to 7 or the pharmaceutical composition of claim 10 or 11 in the preparation of a medicament for treating or improving sepsis-related encephalopathy.

13. The application according to claim 12, characterized in that, The treatment or improvement includes improving at least one of the following: neuroinflammation, blood-brain barrier damage, increased oxidative stress, neurovascular unit damage, hippocampal mitochondrial damage, synaptic structure damage, anxiety-like behavior, cognitive memory impairment, spatial learning and memory impairment, or long-term cognitive dysfunction.

14. The application according to claim 12, characterized in that, The extracellular vesicles are used for administration during the recovery period after sepsis.

15. The application according to claim 12, characterized in that, The extracellular vesicles exert their effects through at least one of the following mechanisms: reducing myeloid differentiation factor 88 expression, inhibiting the MYD88 / NF-κB / COX2 inflammatory signaling pathway, restoring the NRF2 / HO-1 antioxidant signaling pathway, reducing MMP9 expression, restoring Claudin-5 expression, alleviating increased blood-brain barrier permeability, or alleviating damage to hippocampal mitochondrial and synaptic structures.

16. The use of the extracellular vesicles of any one of claims 1 to 7 or the pharmaceutical composition of claim 10 or 11 in the preparation of a medicament for treating or improving diseases related to inflammation of the central nervous system, diseases related to neurovascular injury, or diseases related to blood-brain barrier damage.

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