Application of placenta-targeted liposome TLR4 siRNA in improvement of pregnancy complications

Abstract

The invention relates to an application of TLR < 4 > siRNA (TLR < 4 > siRNA (at) LNP-CSA-BP) coated with TLR < 4 > siRNA (TLR < 4 > siRNA) connected with placental Chondroitin sulfate A binding peptide (Placental Chondroitin sulfate A binding peptide, CSA-BP) injected in a maternal pregnancy period, in improvement of pregnancy complications and prevention of progeny neurodevelopment disorder diseases, in particular to an application of TLR < 4 > siRNA coated with LNP-CSA-BP coated with TLR < 4 > siRNA coated with LNP-CSA-BP. According to the invention, TLR4siRNA (at) LNP-CSA-BP is used for carrying out caudal vein injection on a pregnant mouse, so that a placenta and fetal brain inflammation microenvironment (induced by toxoplasma gondii STAg) maternal immune activation can be inhibited. Behavior experiments further prove that under a maternal immune activation model, TLR4siRNA (at) LNP-CSA-BP caudal vein injection pregnant mice can significantly improve phenotypes of neurodevelopmental disorder diseases of filial generation mice, such as core symptoms (social ability and repeated engraving behaviors) of autism behaviors, and the promising transformation prospect is proved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and specifically relates to application of a liposome TLR4 siRNA targeting placenta in improving pregnancy complications, including a liposome delivery system targeting placenta and application thereof, aiming to improve pregnancy complications and prevent neurodevelopmental disorders (NDDs) of offspring by specifically targeting placental tissue. BACKGROUND

[0002] Maternal immune activation is an important risk factor for the occurrence of neurological diseases in offspring. The placenta, as a key hub of maternal-fetal circulation, plays a crucial role in regulating fetal development, maintaining maternal-fetal immune tolerance, and ensuring pregnancy homeostasis. Inflammatory cytokines and chemokines released by the activation of the systemic immune system during pregnancy exposure to pathogens enter the placenta through the bloodstream, causing placental immune microenvironment disorder, and then inducing a series of pregnancy complications, and ultimately affecting early brain development of the fetus, increasing the risk of NDDs in offspring, so appropriate regulation of placental inflammation is considered a promising therapeutic strategy for pathological pregnancy.

[0003] Lipid nanoparticles (LNP) as one of the most promising drug carriers have attracted much attention due to their excellent biocompatibility, low immunogenicity and high drug loading efficiency. They can precisely control drug release, enhance in vivo targeting, and thus reduce toxicity and improve efficacy.

[0004] However, unmodified LNPs have a significant liver targeting tendency, making it difficult to achieve specific targeting and efficient antigen presentation. Therefore, improving the active targeting, stability and prolonging the circulation time of LNPs has become the core direction of current drug delivery system research.

[0005] Given the high similarity in physiological characteristics between the placenta and the liver, such as high blood flow and fenestrated endothelial structure, it suggests that the placenta may have a potential structural basis for mediating LNP targeted delivery of nucleic acids. Studies have shown that direct delivery of mRNA encoding vascular endothelial growth factor (VEGF) to the placenta by specific formulation of LNPs can alleviate the pathological symptoms of preeclampsia in mouse models without any adverse effects on fetal mice.

[0006] However, although LNPs have been used to explore the therapeutic potential of placental dysfunction and related diseases during pregnancy, there is currently no research on their delivery of nucleic acids through the placenta to improve neurobehavioral aspects of offspring. The main reason is that:

[0007] Firstly, at the molecular level, although the placenta has been proven to highly express the TLR4 receptor, the prior art lacks delivery tools that can specifically recognize and target this receptor, resulting in the inability to achieve precise intervention on the placental inflammatory pathway; secondly, in terms of physiological barriers, the special anatomical structure formed by the placental sinus endothelium makes it difficult for traditional anti-inflammatory drugs to effectively penetrate, severely limiting the therapeutic effect; thirdly, in terms of delivery system design, due to the high similarity between the liver and the placenta in terms of vascular richness and endothelial permeability, unmodified lipid nanoparticles are often preferentially taken up by the liver, resulting in low placental targeting efficiency; finally, at the level of treatment concept, although a large number of studies have confirmed that the placental immune microenvironment disorder is clearly associated with offspring neurodevelopmental disorders, this important pathological mechanism has not been effectively translated into a clinically valuable treatment target and design scheme. These interrelated technical defects collectively constitute the main obstacle to the development of a safe and effective placenta-targeted therapeutic system.

[0008] More specifically, in the prior art, the treatment scheme for placenta-related diseases mainly includes the use of non-targeted lipid nanoparticles to deliver VEGF mRNA technology, which has been proven to have therapeutic effects on preeclampsia in mouse models, but this technology only focuses on the short-term improvement of pregnancy complications and fails to explore its long-term impact on fetal development. This technology ignores the integrity of the maternal-placenta-fetus biological axis, especially lacking a systematic evaluation of the potential impact on the offspring's nervous system development. The limitations of this treatment strategy make it unable to meet the actual needs of clinical intervention on pregnancy complications and the neurodevelopmental disorders they may cause. The "treat the symptoms but not the root cause" nature of the prior art highlights the urgency of developing a new targeted treatment scheme that can not only improve maternal pregnancy complications but also prevent offspring neurological diseases. SUMMARY

[0009] In view of the above technical problems existing in the prior art, the present application not only designs a variety of formulations of LNPs, but also innovatively uses placental chondroitin sulfate A-binding peptide (CSA-BP) to modify LNPs and load TLR4 siRNA, and then proposes a potential strategy for early intervention in offspring NDDs.

[0010] The technical scheme of the present application is:

[0011] The first object of the present application is to provide a placenta-targeted liposome TLR4 siRNA delivery system, which encapsulates TLR4 siRNA in the targeted LNP and connects placental chondroitin sulfate A-binding peptide CSA-BP on the surface of the LNP;

[0012] The targeting LNP comprises DSPC, cholesterol, DSPE-PEG2000-NHS and cationic lipid DLinDMA in a molar ratio of (10:40:0:50)~(10:40:45:5);

[0013] The TLR4 siRNA nucleotide sequence is:

[0014] The sense strand shown in SEQ ID NO. 1 CUUCUUCAACCAAGAACAU(dT)(dT),

[0015] The antisense strand shown in SEQ ID NO. 2 AUGUUCUUGGUUGAAGAAG(dT)(dT);

[0016] The mass ratio of the TLR4 siRNA to the targeting LNP is 1:10~1:20;

[0017] The placenta chondroitin sulfate A binding peptide CSA-BP amino acid sequence is

[0018] EDVKDINFDTKEKFLAGCLIVSFHEGKC;

[0019] The molar ratio of the placenta chondroitin sulfate A binding peptide CSA-BP targeting placenta to DSPE-PEG2000-NHS in the liposome is 1:1~1:5.

[0020] A second object of the present application is to provide a construction method of the aforementioned placenta-targeting liposome TLR4 siRNA delivery system, which comprises the following steps:

[0021] S1, four kinds of lipids are mixed in a ratio according to the aforementioned amount ratio and dissolved in chloroform to obtain a lipid solution;

[0022] S2, the solvent of the lipid solution obtained in S1 is removed by rotary evaporation, and the lipid film after rotary evaporation is reserved for standby;

[0023] S3, the aforementioned TLR4 siRNA is added to 10mM citric acid buffer with pH=4 to obtain a TLR4 siRNA solution;

[0024] S4, the lipid film obtained in S2 is mixed with the TLR4 siRNA solution obtained in S3, and is placed at room temperature for hydration, and is ultrasonicated until the lipid film is dispersed into liposomes, and a liposome extruder (Avanti Polar Lipids) is used to cooperate with a 100nm polycarbonate membrane for extrusion to form liposomes with uniform size;

[0025] S5, mixing the placenta chondroitin sulfate A binding peptide CSA-BP and the liposome obtained in S4, and vortexing to prepare the placenta-targeted liposome TLR4 siRNA delivery system.

[0026] Further, the ratio of the four kinds of lipids in chloroform in S1 is 1.5-2.0 mg / mL.

[0027] Further, the rotary evaporation condition in S2 is rotary evaporation at 40°C for 5-10 min.

[0028] Further, the mass ratio of TLR4 siRNA to the four kinds of lipids in S3 is 1:5-1:10, and the concentration of TLR4 siRNA in the citric acid buffer is 75-100 μg / mL.

[0029] Further, the standing hydration time in S4 is 30-40 min, and the ultrasonic time is 10-30 min.

[0030] Further, the molar ratio of the placenta chondroitin sulfate A binding peptide CSA-BP to DSPE-PEG2000-NHS in the liposome in S5 is 1:1-1:5, and the vortexing time is 60-90 min.

[0031] A third object of the present application is to provide the use of the aforementioned placenta-targeted liposome TLR4 siRNA delivery system in the preparation of a medicament for preventing and / or treating maternal pregnancy complications and preventing neurological developmental disorders in offspring.

[0032] Further, the medicament specifically targets placental tissue.

[0033] The beneficial effects of the technical scheme of the present application are:

[0034] The present application focuses on the innovative design and construction of a placenta-targeted liposome delivery system, and solves three key technical problems: first, traditional lipid nanoparticles lack specific targeting ligand modification, showing a significant liver enrichment tendency, resulting in low efficiency of nucleic acid drugs delivered to placental tissue.

[0035] Second, for the placental inflammatory response triggered by maternal immune activation, existing treatment methods have the dual limitations of insufficient delivery efficiency and fetal safety risks, and traditional anti-inflammatory drugs are difficult to effectively penetrate the placental barrier and may produce developmental toxicity.

[0036] Finally, there is currently a lack of targeted delivery strategies that can improve abnormal neurological development in offspring by precisely regulating placental TLR4 signals.

[0037] The patent successfully constructs a novel delivery system with placenta-specific targeting by innovatively optimizing the liposome formula composition and surface modification process. Through systematic in vitro and in vivo experimental verification, this technology not only significantly improves the enrichment efficiency of therapeutic nucleic acids in placental tissue, but also ensures good pregnancy safety, providing an important technical breakthrough for the development of a new generation of safe and effective intervention programs for pregnancy complications.

[0038] The innovation of the patent mainly lies in the following two key aspects:

[0039] Firstly, the surface of the lipid nanoparticle is modified by CSA-BP, and through a precisely designed coupling process, a nucleic acid delivery system with high placental targeting is successfully constructed. Experimental data show that the placental tissue enrichment efficiency of this system is significantly higher than that of conventional LNPs, and the active targeting delivery of TLR4 siRNA in placental tissue is realized for the first time.

[0040] Secondly, the patent innovatively proposes a new strategy for cross-generation intervention of the "placental inflammation-offspring neural development" axis. By targeting and silencing the expression of TLR4 gene in placenta, not only the placental inflammatory microenvironment is significantly improved, but also the prevention effect on offspring autism-like behavior in animal models is confirmed. This systematic treatment program fills the gap in the field of cross-generation neuroprotection in the prior art.

[0041] Mechanism: Since siRNA is negatively charged, and the ionizable cationic lipid in LNP can be protonated in acidic solution, it can encapsulate siRNA to form a stable complex through electrostatic interaction. At the same time, the lipid bilayer structure of LNP can shield siRNA, avoiding degradation by serum nucleases and rapid clearance by the kidney, prolonging the in vivo half-life. On the one hand, LNP can achieve passive targeting of the placenta with rich blood supply through the vascular leakage retention effect (EPR effect); on the other hand, the PEG lipid with NHS group in LNP can be covalently coupled with the targeting polypeptide with amino group to achieve active targeting of the placenta. The CSA applied in the patent is a key molecule for maintaining placental function, which is highly expressed on the surface of syncytiotrophoblast cells, involved in the integrity of placental barrier, immune regulation and nutrient transport process. CSA can mediate specific endocytosis of LNP by binding with endogenous ligands (such as malaria parasite VAR2CSA protein) or exogenous targeting molecules, thereby enhancing the intracellular delivery efficiency of siRNA. In addition, CSA is abnormally expressed in pathological pregnancy (such as preeclampsia), and targeted intervention of CSA-related pathways can indirectly regulate TLR4-mediated inflammatory response, providing a new treatment strategy for placental dysfunction diseases. Based on these technical principles, the final realization of siRNA precise delivery to the placenta and TLR4 gene silencing. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1Example 1 Evaluation of in vivo delivery efficiency of targeting placenta liposome. Mice were injected with LNPs via tail vein at embryonic day 17.5 (E15.5), and the distribution of Cy5.5-labeled LNPs was visualized by in vivo imaging system (IVIS) 6 hours later, wherein:

[0043] Figure 1 A is a representative graph of the distribution of fetal mouse, placenta imaging;

[0044] Figure 1 B is a statistical graph of the distribution of fetal mouse, placenta imaging.

[0045] Figure 2 Example 2 Experimental design of injecting NS siRNA@LNP-5 and TLR4 siRNA@LNP-5 via tail vein of pregnant mice.

[0046] Figure 3 Example 3 Injecting TLR4 siRNA@LNP-5 via tail vein of pregnant mice can significantly reduce the level of placental inflammatory factors of STAg-MIA pregnant mice. The placenta of pregnant mice was taken at E18.5, wherein:

[0047] Figure 3 A is the mRNA level of placental inflammatory factor Il6 detected by RT-PCR;

[0048] Figure 3 B is the mRNA level of placental Tnfα detected by RT-PCR;

[0049] Figure 3 C is the mRNA level of placental Il1β detected by RT-PCR.

[0050] Figure 4 Example 4 Injecting TLR4 siRNA@LNP-5 via tail vein of pregnant mice can significantly reduce the level of inflammatory factors of fetal brain of STAg-MIA offspring. The fetal brain was taken at E18.5, wherein:

[0051] Figure 4 A is the mRNA level of fetal brain Il6 detected by RT-PCR;

[0052] Figure 4 B is the mRNA level of fetal brain Tnfα detected by RT-PCR;

[0053] Figure 4 C is the mRNA level of fetal brain Il1β detected by RT-PCR.

[0054] Figure 5Example 5: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice significantly inhibited the activation of placental NF-κB signaling in STAg-MIA pregnant mice, wherein:

[0055] Figure 5 A and B represent mouse placentas taken at 18.5 days of gestation, respectively. Protein imprinting was used to detect the expression levels of p-p65 and T-p65 in the placenta.

[0056] Figure 6 Example 6: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice significantly alleviated anxiety-like behavior in STAg-MIA offspring mice, wherein:

[0057] Figure 6 A and B are statistical graphs showing the total distance traveled and the average speed of the mice in the open field test.

[0058] Figure 6 CE is a statistical graph showing the number of times, time, and distance of people entering the center of the open field;

[0059] Figure 6 FH is a statistical graph showing the number of times the tested mice entered the open arm, the time taken, and the proportion of the total time taken in the elevated plus maze test.

[0060] All of the above experiments were used to detect anxiety-like behaviors.

[0061] Figure 7 Example 7: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice significantly alleviated the social abilities of STAg-MIA offspring mice, wherein:

[0062] Figure 7 A represents the social ability of the tested mice in the Three-chamber social interaction test;

[0063] Figure 7 B is a statistical graph showing the time spent interacting between the tested mice and unfamiliar mice;

[0064] This experiment was used to assess the social abilities of mice.

[0065] Figure 8 Example 8: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice significantly alleviated repetitive stereotyped behaviors in STAg-MIA offspring mice; the results of the marble burying test were used to detect repetitive stereotyped behaviors in mice. Detailed Implementation

[0066] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0067] Example 1: Preparation of the Delivery System

[0068] (1) According to the molar ratio of four lipids (2-disteaaroyl-sn-glycero-3-phosphocholin (DSPC), cholesterol, DSPE-PEG2000-NHS and cationic lipid DLinDMA), the liposomes were divided into the following four groups: LNP-1 (10:40:0:50), LNP-2 (10:40:5:45), LNP-3 (10:40:10:40), and LNP-4 (10:40:20:30).

[0069] (2) Based on the above grouping, take different amounts of the four lipids (total lipid mass controlled at 1.5 mg) and dissolve them in 1 mL of chloroform, and place them in a 5 mL round bottom flask.

[0070] (3) Evaporate the lipid solution at 40°C for 5-10 min (400 mbar, 90 rpm) to remove chloroform and form an attached lipid film on the bottom of the round-bottom flask for later use.

[0071] (4) Prepare 1 mL of 10 mM citrate buffer (pH=4) containing 75 μg / mL Cy5.5 at a mass ratio of Cy5.5 to total lipids of 5:100.

[0072] (5) Transfer the Cy5.5 solution from step 4 to the flask containing lipids from step 3, let it stand at room temperature for 30 min to hydrate, then sonicate the flask in a water bath for 10 min to disperse the lipid membrane to form liposomes, and use a liposome extruder with a 100 nm polycarbonate membrane to extrude the liposomes to form liposomes of uniform size (LNP-1 to LNP-4).

[0073] (6) Subsequently, LNP-4 was mixed with CSA-BP solution at a molar ratio of 1:1 between placental chondroitin sulfate A binding peptide (CSA-BP) and DSPE-PEG2000-NHS in liposomes, and vortexed for 60 min to obtain LNP-5.

[0074] Example 2: Evaluation of in vivo delivery efficiency of placental liposomes.

[0075] Pregnant mice were injected with LNPs via the tail vein on day E17.5. The experiment was divided into five groups: Ctrl group, LNP-1 group, LNP-2 group, LNP-3 group, LNP-4 group, and LNP-CSA-BP (LNP-5) group. The distribution of Cy5.5-labeled LNPs was visualized by IVIS 6 hours after injection.

[0076] The treatment for the Ctrl group was: a tail vein injection of the same volume of PBS as the LNP-1 to LNP-5 groups;

[0077] The LNP-1 group was treated by tail vein injection of LNP-1 (dissolved in PBS) prepared in Example 1 at a dose of 0.6 mg / kg.

[0078] The LNP-2 group was treated by tail vein injection of LNP-2 (dissolved in PBS) prepared in Example 1 at a dose of 0.6 mg / kg.

[0079] The LNP-3 group was treated by tail vein injection of LNP-3 (dissolved in PBS) prepared in Example 1 at a dose of 0.6 mg / kg.

[0080] The LNP-4 group was treated by tail vein injection of LNP-4 (dissolved in PBS) prepared in Example 1 at a dose of 0.6 mg / kg.

[0081] The treatment method for the LNP-CSA-BP (LNP-5) group was as follows: LNP-5 (dissolved in PBS) prepared in Example 1 was injected via tail vein at a dose of 0.6 mg / kg.

[0082] The results showed that, compared with the Ctrl group, LNP-5 exhibited the highest specificity for targeting the placenta and less fetal fluorescence enrichment, thus it was used for subsequent experiments. Meanwhile, the results of experiments with LNP-1 to LNP-4 showed that, although they lacked only CSA-BP compared to LNP-5, their specificity for targeting the placenta was significantly weaker than that of LNP-5. Figure 1 ).

[0083] Example 3: Pregnant rats were injected via tail vein with NS siRNA@LNP-5 and TLR4 siRNA@LNP-5.

[0084] Pregnant mice were intraperitoneally injected with soluble tachyzoite antigen (STAg) on ​​day E12.5, and intravenously injected with NS siRNA@LNP-5 and TLR4 siRNA@LNP-5 on day E15.5. The experiment was divided into six groups:

[0085] To synthesize liposomes encapsulated with siRNA, the Cy5.5 in the LNP-5 synthesis process of Example 1 was replaced with NS siRNA (sense strand as shown in SEQ ID NO.3, antisense strand as shown in SEQ ID NO.4) and TLR4 siRNA (sense strand as shown in SEQ ID NO.1, antisense strand as shown in SEQ ID NO.2) according to the above steps. NS siRNA@LNP-5 and TLR4 siRNA@LNP-5 were synthesized and prepared, with NS siRNA@LNP-5 serving as an empty vector control.

[0086] SEQ ID NO.3: UUCUCCGAACGUGUCACGU(dT)(dT);

[0087] SEQ ID NO. 4: ACGUGACACGUUCGGAGAA(dT)(dT).

[0088] (1) The treatment method for the Ctrl group was: E12.5 injection and 90μg STAg of PBS of equal volume. This group was the blank control group (mock group) for PBS injection.

[0089] (2) The treatment for the STAG group was: 90 μg STAg was injected into E12.5. This group served as the blank control group (mock group) for STAG injection.

[0090] (3) The treatment method for the Ctrl+NS siRNA@LNP-5 group was as follows: on day E12.5, the same volume of PBS as the blank control group (STAg) was injected, and on day E15.5, 0.6 mg / kg NS siRNA@LNP-5 was injected.

[0091] (4) The treatment method for the STAg+NS siRNA@LNP-5 group was: 90μg STAg was injected on day E12.5, and 0.6mg / kg NS siRNA@LNP-5 was injected on day E15.5;

[0092] (5) The treatment method for the Ctrl+TLR4 siRNA@LNP-5 group was: on E12.5, the same volume of PBS as STAG in group (4) was injected, and on E15.5, 0.6 mg / kg TLR4 siRNA@LNP-5 was injected;

[0093] (6) The treatment method for the STAG+TLR4 siRNA@LNP-5 group was: 90 μg STAg was injected on day E12.5, and 0.6 mg / kg TLR4 siRNA@LNP-5 was injected on day E15.5.

[0094] E18.5 days after birth, the mice were euthanized by cervical dislocation, and fetal brain, placenta, and other tissues were collected. Another batch of pregnant mice were kept to produce offspring, and behavioral tests were performed at 6-8 weeks of age.

[0095] Example 4: TLR4 siRNA@LNP-5 was injected into the tail vein of pregnant mice to inhibit the level of placental pro-inflammatory factors in STAG-MIA pregnant mice.

[0096] Placental tissue was collected from pregnant mice treated in Example 3 at day 18.5. The levels of placental inflammatory factors (Il6, Tnfα, and Il1β) were detected by RT-PCR. The experimental results for the Ctrl group, STAG group, Ctrl+NS siRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4 siRNA@LNP-5 group, and STAG+TLR4 siRNA@LNP-5 group were as follows: Figure 3 The mRNA level of placental Il6 in A Figure 3 B's placental Tnfα mRNA level, Figure 3 mRNA levels of placental Il1β in C.

[0097] The results showed that, compared with the PBS-injected blank control group and the NS siRNA@LNP-5 group, STAg-MIA significantly induced the mRNA levels of placental pro-inflammatory factors Il6, Tnfα, and Il1β in pregnant mice in both the blank control group and the NS siRNA@LNP-5 group; compared with the STAg-MIA blank control group and the NS siRNA@LNP-5 group, TLR4 siRNA@LNP-5 significantly inhibited the mRNA levels of placental Il6, Tnfα, and Il1β in STAg-MIA-injected pregnant mice. Figure 3 AC).

[0098] Example 5: TLR4 siRNA@LNP-5 was injected into the tail vein of pregnant rats to inhibit the inflammatory response of the fetal brain in STAG-MIA offspring.

[0099] Fetal brain tissue was collected from pregnant mice treated in Example 3 at day 18.5. The mRNA levels of fetal brain inflammatory factors (Il6, Tnfα, and Il1β) were detected by RT-PCR. The experimental results for the Ctrl group, STAG group, Ctrl+NS siRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4 siRNA@LNP-5 group, and STAG+TLR4 siRNA@LNP-5 group were as follows: Figure 4 The mRNA level of fetal brain Il6 in A, Figure 4 B's fetal brain Tnfα mRNA level, Figure 4 C's fetal brain Il1β mRNA level.

[0100] The results showed that, compared with the PBS-injected blank control group and the NS siRNA@LNP-5 group, STAg-MIA significantly induced the mRNA levels of pro-inflammatory factors Il6, Tnfα, and Il1β in the fetal brain of pregnant mice in the blank control group and the NS siRNA@LNP-5 group; compared with the STAg-MIA blank control group and the NS siRNA@LNP-5 group, TLR4 siRNA@LNP-5 significantly inhibited the mRNA levels of Il6, Tnfα, and Il1β in the fetal brain of STAg-MIA-injected pregnant mice. Figure 4 AC).

[0101] Example 6: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice inhibited the activation of NF-κB signaling in the placenta of STAG-MIA.

[0102] Placental tissue was collected from pregnant mice treated in Example 3 at day 18.5. Protein blot analysis was performed to detect proteins related to the placental NF-κB signaling pathway. The experimental results for the Ctrl group, STAG group, Ctrl+NS siRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4 siRNA@LNP-5 group, and STAG+TLR4 siRNA@LNP-5 group were as follows: Figure 5 The expression level of placental p-p65 in A, Figure 5 The expression level of placental T-p65 in B.

[0103] Figure 5 The results showed that, compared with the PBS-injected blank control group and the NS siRNA@LNP-5 group, STAg-MIA significantly induced placental p-p65 expression in pregnant mice in both groups; and compared with the STAg-MIA blank control group and the NS siRNA@LNP-5 group, TLR4 siRNA@LNP-5 significantly inhibited placental p-p65 expression in STAg-MIA-injected pregnant mice. Figure 5 AB).

[0104] Example 7: TLR4 siRNA@LNP-5 was injected into the tail vein of pregnant mice to alleviate anxiety-like behavior in STAG-MIA offspring mice.

[0105] The anxiety-like behavior of adult offspring mice (6-8 weeks old) was detected using the open field test and the elevated cruciate maze test. The experimental groups were Ctrl group, STAG group, Ctrl+NS siRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4siRNA@LNP-5 group, and STAG+TLR4 siRNA@LNP-5 group. All offspring mice in each group were bred from pregnant mice obtained by the same treatment method as in Example 3 and were routinely fed to adulthood.

[0106] Open field experiment: The apparatus was a square open field (50×50×50 cm) with opaque plastic walls on all sides. Each mouse was placed individually in the field and allowed to move freely for 10 minutes. The field was divided into a central area (25×25 cm) and an outer area. The number of times and duration of entry into the central area were analyzed and recorded using SuperMaze high-throughput animal behavior analysis software. Figure 6 The AE display shows the results of the open field experiment. Figure 6 A and B show the statistics of the total movement distance and average speed of the tested mice in the open field test. The results show that there are no significant differences in these two indicators among the groups, indicating that the movement ability of the mice in each group is normal. Figure 6 CE represents the statistical analysis of the number of times, time, and distance of mice entering the center of the open field. The results showed that, compared with the blank control group injected with PBS and the Ns-siRNA@LNP-5 group of pregnant mice, STAg-MIA significantly inhibited the number of times, time, and distance of mice entering the center of the open field in the blank control group and the Ns-siRNA@LNP-5 group of pregnant mice; while compared with the blank control group of STAg-MIA and the Ns-siRNA@LNP-5 group of pregnant mice, TLR4 siRNA@LNP-5 significantly induced the number of times, time, and distance of mice entering the center of the open field in the STAg-MIA group of pregnant mice.

[0107] Elevated Cross Maze Experiment: The elevated cross maze consisted of two opposing open arms (35×5 cm) and two identical closed arms (15 cm high walls), with a central platform (5×5 cm) located 60 cm above the ground. Each mouse was placed on the central platform, facing one of the open arms, and allowed to move freely within the elevated cross maze for 10 minutes. The time and trajectory of the mice during opening and closing were recorded using SuperMaze high-throughput animal behavior analysis software. Figure 6FH represents the number of times mice entered the elevated cross maze, the time taken, and the proportion of the total time spent in the experiment. The results showed that, compared with the blank control group injected with PBS and the offspring of pregnant mice in the NS siRNA@LNP-5 group, STAg-MIA significantly inhibited the number of times mice entered the elevated cross maze, the time taken, and the proportion of the total time spent in the offspring of pregnant mice in the blank control group and the NS siRNA@LNP-5 group; while compared with the blank control group of STAg-MIA and the offspring of pregnant mice in the NS siRNA@LNP-5 group, TLR4 siRNA@LNP-5 significantly induced the number of times mice entered the elevated cross maze, the time taken, and the proportion of the total time spent in the offspring of pregnant mice in the STAg-MIA group.

[0108] Given that both the open field test and the elevated cross maze test reflect anxiety-like behavior in mice, the above results suggest that anxiety-like behavior in the STAg-MIA group of mice injected with TLR4 siRNA@LNP-5 via the tail vein was significantly alleviated compared with that in the STAg-MIA group of pregnant mice injected with NS siRNA@LNP-5 via the tail vein.

[0109] Example 8: Intravenous injection of TLR4 siRNA@LNP-5 into the tail vein of pregnant mice improved social deficits in STAG-MIA offspring mice.

[0110] The social abilities of mice were assessed using a three-box socialization test. The experimental groups were Ctrl group, STAG group, Ctrl+NSsiRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4 siRNA@LNP-5 group, and STAG+TLR4siRNA@LNP-5 group. All offspring mice in each group were bred from pregnant mice obtained using the same treatment method as in Example 3 and were routinely fed to adulthood.

[0111] The Three-Box Social Experiment: The three-box social experiment setup consisted of three areas (20×40×22 cm), divided into three interconnected chambers (left, center, and right), referred to as the three boxes. In the first stage, the experimental mouse was placed in the center box and allowed to freely explore each chamber for 10 minutes. In the second stage, the time spent in the left and right chambers was checked. The mouse was confined to the center chamber, and a strange male C57BL / 6 mouse (Stranger 1) was placed in one of two wire cups (the side the tested mouse spent less time on). Obstacles in adjacent chambers were removed, and the experimental mouse was allowed to freely move between the three chambers for 10 minutes. The interaction time with Stranger 1 was measured. In the third stage, the tested mouse was confined to the center box, not allowed to go to the left or right sides, and the time was recorded for 10 minutes. In the fourth stage, a second strange male C57BL / 6 mouse (Stranger 2) was placed in another empty wire cup, and the experimental mouse was allowed to freely move between the three chambers for 10 minutes. The SuperMaze high-throughput animal behavior analysis software was used to analyze and record the sniffing (social) time and total distance traveled by the experimental mice. Figure 7 A represents the time taken for each group of offspring mice to sniff the empty cage and Stranger 1 in the three-box socialization experiment. Figure 7 B represents the time it takes for each group of offspring mice to sniff Stranger 1 and Stranger 2.

[0112] The results showed that when Stranger 1 was introduced, the interaction time between offspring mice and Stranger 1 was significantly longer in all groups than in the empty control group (Empty). Figure 7 A). After the introduction of Stranger 2, the sniffing time of the offspring of pregnant mice in the PBS-injected blank control group and the NS siRNA@LNP-5 group was significantly longer than that of Stranger 1, while there was no significant difference in the communication time of the offspring of pregnant mice in the STAg-MIA blank control group and the NS siRNA@LNP-5 group sniffing Stranger 1 and Stranger 2, suggesting a deficiency in the social ability of STAg-MIA offspring mice; compared with the offspring of pregnant mice in the STAg-MIA blank control group and the NS siRNA@LNP-5 group, TLR4 siRNA@LNP-5 significantly induced the sniffing time of STAg-MIA pregnant mice offspring to Stranger 2, and the social impairment of the offspring mice was alleviated. Figure 7 B).

[0113] Example 9: TLR4 siRNA@LNP-5 was injected into the tail vein of pregnant mice to alleviate repetitive stereotyped behaviors in STAG-MIA offspring mice.

[0114] The bead embedding experiment was used to detect repetitive stereotyped behaviors in mice. The experimental groups were Ctrl group, STAG group, Ctrl+NSsiRNA@LNP-5 group, STAG+NS siRNA@LNP-5 group, Ctrl+TLR4 siRNA@LNP-5 group and STAG+TLR4siRNA@LNP-5 group. All mice in each group were offspring of pregnant mice obtained by the same treatment method as in Example 3 and were routinely fed to adulthood.

[0115] The bead embedding test was used to detect repetitive stereotyped behaviors in offspring mice. Each mouse was placed in a test area (36×20×13 cm) with 12 transparent glass marbles (15 mm in diameter) evenly distributed in a 3×5 pattern on the surface of a 5 cm layer of corn bedding. The mice were allowed to explore freely for 30 minutes, after which they were gently removed from the test cage, and the number of marbles buried under the corn bedding was recorded (successful embedding was defined as the corn bedding covering more than two-thirds of the surface). Figure 8 Statistics on the number of marbles buried in mice.

[0116] The results showed that, compared with the blank control group injected with PBS and the offspring of pregnant mice in the NS siRNA@LNP-5 group, STAg-MIA significantly induced the number of embedded beads in the blank control group and the offspring of pregnant mice in the NS siRNA@LNP-5 group, suggesting repetitive stereotyped behaviors in mice; while compared with the blank control group injected with STAg-MIA and the offspring of pregnant mice in the NS siRNA@LNP-5 group, TLR4siRNA@LNP-5 significantly inhibited the number of embedded beads in the offspring of pregnant mice in the STAg-MIA group, suggesting the alleviation of repetitive stereotyped behaviors in mice.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A liposomal TLR4 siRNA delivery system targeting the placenta, characterized in that, The delivery system encapsulates TLR4 siRNA in a targeted LNP and attaches placental chondroitin sulfate A-binding peptide CSA-BP targeting the placenta to the surface of the LNP; the targeted LNP comprises DSPC, cholesterol, and other components in a molar ratio of (10:40:0:50) to (10:40:45:5). DSPE-PEG2000-NHS and cationic lipid DLinDMA; The TLR4 siRNA nucleotide sequence is as follows: The justice chain shown in SEQ ID NO.1 is CUUCUUCAACCAAGAACAU(dT)(dT). The antisense chain shown in SEQ ID NO.2 is AUGUUCUUGGUUGAAGAAG(dT)(dT); The mass ratio of the TLR4 siRNA to the targeted LNP is 1:10 to 1:20; The amino acid sequence of placental chondroitin sulfate A-binding peptide CSA-BP is EDVKDINFDTKEKFLAGCLIVSFHEGKC; The molar ratio of the placental chondroitin sulfate A-binding peptide CSA-BP targeting the placenta to DSPE-PEG2000-NHS in liposomes is 1:1 to 1:

5.

2. The method for constructing the placental-targeting liposome TLR4 siRNA delivery system according to claim 1, characterized in that, The construction method includes the following steps: S1. The four lipids are mixed according to the proportions described in claim 1 and then dissolved in chloroform to obtain a lipid solution; S2. The lipid solution obtained in S1 is removed by rotary evaporation to remove the solvent, and the lipid membrane after rotary evaporation is retained for later use. S3. Add the TLR4 siRNA described in claim 1 to a 10mM citrate buffer solution at pH=4 to obtain a TLR4 siRNA solution; S4. Mix the lipid membrane obtained in S2 with the TLR4 siRNA solution obtained in S3, allow it to stand at room temperature for hydration, and sonicate until the lipid membrane is dispersed into liposomes. Use a liposome extruder (Avanti Polar Lipids) with a 100nm polycarbonate membrane to extrude the liposomes to form liposomes of uniform size. S5. The placental chondroitin sulfate A-binding peptide CSA-BP described in claim 1 is mixed with the liposomes obtained in S4 and vortexed to prepare the placental-targeting liposome TLR4 siRNA delivery system.

3. The construction method according to claim 2, characterized in that, The ratio of the four lipids in S1 to chloroform is 1.5–2.0 mg / mL.

4. The construction method according to claim 2, characterized in that, The rotary evaporation conditions described in S2 are rotary evaporation at 40°C for 5–10 minutes.

5. The construction method according to claim 2, characterized in that, The mass ratio of TLR4 siRNA to the four lipids in S3 is 1:5 to 1:10, and the concentration of TLR4 siRNA in citrate buffer is 75 to 100 μg / mL.

6. The construction method according to claim 2, characterized in that, The S4 hydration time is 30-40 minutes, and the ultrasonic time is 10-30 minutes.

7. The construction method according to claim 2, characterized in that, The molar ratio of placental chondroitin sulfate A-binding peptide CSA-BP to DSPE-PEG2000-NHS in liposomes in S5 is 1:1 to 1:5, and the vortexing time is 60 to 90 min.

8. The use of the placenta-targeting liposomal TLR4 siRNA delivery system of claim 1 in the preparation of a medicament for the prevention and / or treatment of maternal pregnancy complications and the prevention of neurodevelopmental disorders in offspring.

9. The application according to claim 8, characterized in that, The drug specifically targets placental tissue.

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