Targeting modulation of the choroid plexus blood-CSF barrier and CSF hypersecretion via lipid nanoparticle-mediated co-delivery siRNA and resveratrol

doi:10.21203/rs.3.rs-4869812/v1

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Targeting modulation of the choroid plexus blood-CSF barrier and CSF hypersecretion via lipid nanoparticle-mediated co-delivery siRNA and resveratrol

https://doi.org/10.21203/rs.3.rs-4869812/v1

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Hydrocephalus is one of the most common neurological disorders, but pharmacotherapy options are currently lacking due to the complex pathogenesis. The blood-CSF barrier (B-CSFB), consisting of choroid plexus (ChP) epithelial cells, is a crucial gate for the entry of peripheral immune cells and its dysfunction emerges as a new contributor to hydrocephalus pathology. Meanwhile, SPAK-mediated CSF hypersecretion in ChP epithelial cells plays an important role in the pathogenesis of hydrocephalus. Here, we fabricated a transferrin receptor-targeted nano-drug (siR/RSV@TNP) that can intelligently navigate to the blood-CSF barrier and prepared for combined delivery of resveratrol (RSV) and SPAK siRNA (siSPAK)for synergetic hydrocephalus therapy. As expected, siR/RSV@TNP fulfilled its function of knocking down SPAK expression, relieving inflammation and oxidative stress, retrieving blood-CSF barrier integrity, and ultimately preventing ventriculomegaly and hydrocephalus. The current study demonstrates proof of concept that targeting the choroid plexus blood-CSF barrier and cerebrospinal fluid hypersecretion offers a promising and innovative approach for alleviating hydrocephalus.

Health sciences/Diseases/Neurological disorders/Hydrocephalus

Biological sciences/Biotechnology/Biomaterials/Drug delivery

Hydrocephalus

Targeted delivery

the blood-CSF barrier

CSF hypersecretion

gene delivery

Anti-inflammatory

tight junction

Hydrocephalus is one of the most common neurosurgical disorders, imposing a significant financial burden on the global healthcare system¹. Annually, an estimated 400,000 new cases of hydrocephalus are diagnosed worldwide². Elevated intracranial pressure (ICP) in hydrocephalus can lead to visual loss, developmental delay, brain damage, and even death³. Currently, there is no reliable pharmaceutical treatment for hydrocephalus. Surgical cerebrospinal fluid (CSF) shunting, which drains CSF from the brain to another part of the body via a catheter, remains the primary treatment⁴. However, approximately 40–50% of patients experience shunt failure due to obstruction or infection. And 60% of surgically treated patients endure lifelong neurological symptoms, such as motor skills impairment, cognitive deficits, or epilepsy. Thus, non-invasive therapies based on targeted nano-delivery systems represent a significant advancement for hydrocephalus patients across all age groups.

In recent years, increasing attention has been given to the relationship between the structural and functional integrity of the blood-cerebrospinal fluid barrier (BCSFB) and hydrocephalus. The BCSFB, comprised of choroid plexus epithelial cells, serves as a crucial gatekeeper at the brain–immune system interface and is an underestimated gate for entry of immune cells during inflammatory disorders^5–7. Previous research has indicated that increased immune cell infiltration into the choroid plexus is associated with a loss of BCSFB integrity^7–9. For example, Kahle et al. have identified that the recruitment of peripheral monocytes to the choroid plexus (ChP), triggered by intraventricular hemorrhage or infection, significantly contributes to hydrocephalus pathogenesis¹⁰. In our prior investigations, we observed disrupted BCSFB integrity, evidenced by ZO-1 staining, alongside heightened macrophage accumulation in the choroid plexus during hydrocephalus¹¹. Similarly, Zhang et al. demonstrated that BCSFB dysfunction exacerbates hydrocephalus, attributing mitochondrial reactive oxygen species (ROS) to the destruction of tight junctions in choroid plexus epithelial cells¹². Collectively, the evidence supports that dysfunction of BCSFB serves as a great contributor to hydrocephalus pathology, and addressing the comprised BCSFB represents a potential novel therapeutic strategy for hydrocephalus.

In addition, the ChP secretes over 500 ml/day CSF into the brain’s ventricular spaces, which relies on the vectorial ion transport across the apical and basolateral membranes of the ChP epithelial cells, coupled with water transport—either co-transported or osmotically driven^13–16. Notably, SPAK-NKCC1, a multi-ion transporter complex, prominently expressed on the apical surface of ChP cells, accounts for roughly half of CSF production¹³. Furthermore, substantial research has established the close relationship between the expression and functional status of the SPAK-NKCC1 complex and CSF secretion^10,15. Robert et al. have identified SPAK as a pivotal regulator of a multi-ion transporter complex, including NKCC1, ATP1A1, KCNJ13, and CLIC6, all implicated in CSF hypersecretion in hydrocephalus¹⁰. Therefore, targeting SPAK represents a promising approach to mitigate CSF hypersecretion. Considering all the pathological alterations in ChP epithelial cells in hydrocephalus, it is plausible to conclude that the diseased BCSFB-targeted and CSF hypersecretion-targeted modulation strategy can be a novel and promising remedy for hydrocephalus treatment.

To verify our hypothesis, we designed a multifunctional lipid nanoparticle by encapsulating SPAK siRNA (siSPAK) and resveratrol within a T7 peptide-decorated, glutathione (GSH)-responsive lipopolyplex (siR/RSV@TNP). In view of the increased expression of SPAK and the subsequent overproduction of hydrocephalus, we employed siSPAK to lower the expression of pathogenic genes and consequently decrease the excessive production of CSF. Nonetheless, the transition of siRNA from the laboratory to clinical use has been hindered by concerns regarding its effective delivery, primarily due to its instability in circulation and vulnerability to degradation by RNases and phosphatases when entrapped in lysosomes. To address the obstacles associated with gene delivery, we utilized a GSH-responsive lipopolyplex featuring disulfide-bonded cationic complexes. This complexes effectively condensed siRNA and responded to intracellular redox environments to facilitate siRNA release. Additionally, a lipid layer was incorporated to conceal the positive charge and extend circulation time. Moreover, T7 peptide, which can specifically bind to TfR was further modified to enhance distribution within ChP epithelial cells¹⁷. Resveratrol, a natural polyphenol known for its anti-inflammatory and antioxidant properties, has been harnessed in our system to modulate disrupted BCSFB integrity.¹⁸. Resveratrol could significantly inhibit the inflammatory response through the TLR4/NF-κB pathway and reduce the inflammatory cytokines¹⁸. Besides, it can alter the antioxidant activity by reducing the production of ROS and increasing antioxidant enzymes; and its therapeutic effects have been demonstrated in many clinical trials¹⁹. Recent studies have highlighted how inflammatory cytokines and NF-κB pathways can compromise the integrity of the BCSFB barriery^20–23, making resveratrol particularly suitable for modulating BCSFB integrity and ChP inflammation in hydrocephalus.

As anticipated, siR/RSV@TNP not only preserved BCSFB integrity and attenuated macrophage infiltration in the ChP but also suppressed CSF hypersecretion without significant toxicity. To our knowledge, this study represents the first attempt to regulate both BCSFB integrity and CSF hypersecretion using a self-regulated, multifunctional nano-modulator in hydrocephalus treatment.

2.1. Materials

Low-weight molecular polyethylenimine (PEI) was purchased from RHAWN. Cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained from AVT (shanghai) Pharmaceutical Tech Co., Ltd. (Shanghai, China). 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), was purchased from Bide Pharmatech Co., Ltd. (Shanghai, China).1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)] (DSPE-PEG-MAL) were obtained from Ponsure Biological Co., Ltd. (Shanghai, China). Sphingomyelin was purchased from Jiuding Chemical Co., Ltd. (Shanghai, China). T7 peptide (Cys-His-Ala-Ile-Tyr-Pro-Arg-His) was synthesized by Taopu Biotech (Nanjing, China). The SPAK siRNA1 (sense:CCAUGUUGGAUAUCAUCAATT, antisense:UUGAUGAUAUCCAACAUGGTT),SPAKsiRNA2(sense:GGAACAGGUGAGAGGCUAUTT,antisense:AUAGCCUCUCACCUGUUCCTT),SPAK siRNA3 (sense:CAGCGCCUUAC CACAAAUATT, antisense:UAUUUGUGG UAAGGCGCUGTT) and SPAK siRNA 4 (sense: GAACUUAAUGACAUACGAUTT, antisense: AUCGUAUGUCAUUAAGUUCTT) were designed and purchased from Genepharma company (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Solarbio (L8880, Beijing, China). Kaolin was purchased from Sigma-Aldrich (K7375, America). Resveratrol was purchased from MedChemExpress (HY-16561, USA).

2.2. Synthesis of sPEI and DSPE-PEG-T7

The synthesis procedure was executed in accordance with the documented protocol, albeit with certain modifications. The pH of a 500 mg aqueous solution of PEI800 was adjusted to 7.2 using 0.5 mol/L HCl through incremental additions. Following the evaporation of water, the resulting yellow solid was dissolved in 30 ml of methanol in a round-bottom flask and purged with nitrogen three times. A calculated excess amount (5-fold molar excess to PEI800) of propylene sulfide was added, and the reaction was carried out at 60 ℃ for 24 hours. The mixture was evaporated to dryness under reduced pressure, dissolved in methanol, and precipitated in cold diethyl ether twice. The resulting precipitate was dissolved in DMSO and stirred at room temperature for 48 hours. The product was then purified by dialysis against water (MWCO 3500) and lyophilized. The chemical structure was confirmed using 1H NMR (in D2O, 400 MHz): δ 3.14–2.66 (m, NCH2CH2N, NCH2CHMeS), 1.25–1.05 (m, CH3).

To synthesize DSPE-PEG-T7, DSPE-PEG2000-MAL was dissolved in DMF at a concentration of 10mg/mL and subsequently a 1.5-fold molar excess of T7 peptide was introduced. The resulting solution was stirred at 40 ℃ under a nitrogen atmosphere for 24 hours. Following the completion of the reaction, the mixture underwent dialysis against deionized water for a duration of 24 hours. The ultimate product was acquired through freeze-drying and subjected to time-of-flight mass spectrometry for structural confirmation.

2.3. Preparation and characterization of polyplexes with different weight ratios

In order to evaluate the encapsulation efficiency of siRNA and the polyplexes' responsiveness to glutathione (GSH), a gel electrophoresis assay was executed. The siRNA and sPEI were dissolved separately in DEPC water to produce stock solutions with concentrations of 1 mg/mL and 20 µM, correspondingly. Following a predetermined array of weight ratios (w/w = 0, 0.3, 0.5, 1, 2, 3, 4), different volumes of the sPEI solution were combined with 2 uL of siRNA and incubated at room temperature for 10 minutes. Subsequently, the nucleic acid loading buffer was introduced and samples containing various weight ratios were loaded and subjected to electrophoresis at 100 V for 20 minutes. To assess the response to glutathione (GSH), the complexes were treated with a 10 mmol/L GSH solution at 37°C and visualized using TEM imaging.

2.4. SPAK Knockdown Efficiency Assessments of Polyplexes

To assess the efficiency of siR/RSV@TNP in knockdown SPAK and its downstream ion channels. The primary ChP epithelial cells were seeded in the 24-well and 6-well plates. These cells were treated with polyplexes containing 50nmol SPAK siRNA for 48 h, the branched polyethyleneimine (PEI, 25 kDa) was used as a positive control. RT-qPCR was performed as described previously²⁴. The expression of SPAK an NKCC1 protein was detected using the Western Blotting analysis, employing specific antibodies for SPAK (ABclonal, A2275) and NKCC1 (Proteintech, 13884-1-AP).

2.5. Preparation and characterization of siR/RSV@TNP

According to a previous report, T7-modified resveratrol-loaded lipopolyplex was prepared by reverse evaporation method^25,26. Various lipids and resveratrol were dissolved either in chloroform, ethanol, or methanol-ethanol (v/v = 1:1) with a concentration of 4 mg/mL. The organic phase was obtained by mixing cholesterol, sphingomyelin, DOPC, DOPS, DOPE, DSPE-PEG2000, DSPE-PEG2000-RAP, and resveratrol were added drop by drop to the water phase. The mixture was transferred to ultrasonication for 5 min in an ice bath and the organic phase was removed using a rotary evaporator to get lipopolyplex solution. The siSPAK was mixed with sPEI for 30 min. Then the mixture was added to the lipopolyplex solution and incubated for 4 h at room temperature. The Dynamic light scattering (DLS) was performed to assess the size and zeta potential of siR/RSV@TNP and the TEM was utilized to observe the morphology of siR/RSV@TNP. To further determine DLC (%) and EE (%) of pioglitazone, HPLC analysis was carried out. The release process of resveratrol was investigated via the dialysis method. Briefly, siR/RSV@TNP solution (1 mL, containing 1 mg resveratrol) was sealed in a dialysis bag (MWCO = 2000 Da) and dialyzed against 10 mL of PBS containing 1% tween 80 at different pH (pH = 7.4 or 5.0). At predetermined time intervals, the dialysate was withdrawn for HPLC analysis.

2.6. Experimental cells and animals

We cultured primary ChP epithelial cells using a previously described method²⁷. Initially, the ChP was isolated from the brain and placed into freshly prepared sterile dissection media, comprising 1×DMEM and 1× penicillin-streptomycin. The tissue was finely minced in a 1.5 mL sterile EP tube, digested with pancreatin, and then repeatedly aspirated using a 200-µL pipette. Following digestion, the tissue was centrifuged and resuspended in a specialized ChP epithelial cell medium, containing 10% fetal bovine serum (FBS), 10,000 units/mL penicillin-streptomycin, and 10 ng/mL human epidermal growth factor (EGF) in DMEM. The ChP epithelial cells were cultured for 14 days with media changes every 2–3 days before being utilized for experiments. RAW264.7 cells were obtained from the National Collection of Authenticated Cell Cultures (Beijing, China). The cells were cultured at 37°C in DMEM supplemented with 1% penicillin-streptomycin and 10% FBS in a 5% CO₂ atmosphere.

Male Kunming mice aged 7‒8 weeks and weighing 25‒30g were purchased from the Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, China). The mice were maintained in a temperature-regulated environment on a 12-hour light/dark cycle and were provided with a standard rat diet and unrestricted access to water. All animal experiments were carried out in accordance with guidelines evaluated and approved by West China Hospital of Sichuan University.

2.7. Cell uptake and lysosomal escape assay

For the cellular uptake assay, ChP epithelial cells were seeded into 12-well plates to achieve a specific density. FITC-siR@NP and FITC-siR@TNP were then added to the cells. After 1-hour and 4-hour incubation periods, the cells were washed with PBS, and the cellular uptake efficacy was assessed using flow cytometry. Confocal laser scanning microscopy (CLSM) was employed to visualize the cellular uptake. Briefly, the cells were incubated with FITC-siR@NP and FITC-siR@TNP for 1 hour and 4 hours, followed by DAPI staining for 5 minutes to label the nuclei. The cells were then washed and fixed with 4% PFA before observation. For the lysosomal escape assay, choroid plexus epithelial cells were plated onto glass slides. FITC and Lysotracker Red DND99 were used to label siRNA and lysosomes, respectively, for tracking purposes.

2.8. Cytotoxicity assay

To assess cell viability, a CCK 8 assay was conducted. Specifically, choroid plexus epithelial cells were seeded in 96-well plates and subjected to different treatments. After the treatment period, 10 µL of CCK-8 reagent is added to each well containing 100 µL of culture medium. The absorbance of each well is measured at 450 nm using a microplate reader, with the absorbance values correlating directly to cell viability.

2.9. Alleviating intracellular ROS

Primary ChP epithelial cells were seeded in 12-well plates and incubated with various formulations for 48 hours, followed by a 2-hour incubation with H₂O₂(200µmol/L). After removing the medium, cells were treated with serum-free medium containing 10 µmol/L DCFH-DA (S0033S, Beyotime) for 30 minutes at 37°C. Subsequently, cells were collected and washed three times with PBS. Fluorescent intensity was measured using a flow cytometer (Beckman, USA).

2.10. Kaolin-induced hydrocephalus models

The hydrocephalus mice model was induced via kaolin injection into the right lateral ventricle^28,29. After the successful induction of anesthesia, the mice were carefully positioned in a stereotaxic frame. A cranial burr hole was drilled at coordinates 0.3 mm posterior and 1 mm lateral to bregma, ensuring precision. The surgical site was covered with sterile gauze to prevent contamination. A micro-syringe needle was inserted into the lateral ventricle (depth:3.7 mm) and a 10 µL sterile suspension of 3% kaolin (ultrasonic emulsification about 15 minutes) in artificial CSF in saline was injected slowly into the lateral ventricle. The needle was kept in place for an additional 10 minutes to prevent backflow. Finally, the incision site was sutured to ensure proper wound healing.

2.11. Western Blot

Primary ChP epithelial cells and mouse choroid plexus tissues were collected after different treatments. The lysates were then centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA assay. Equal amounts of protein were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were transferred onto PVDF membranes, which were subsequently blocked with 5% non-fat milk in TBST for 1 hour at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies: pNK-κB (CST, 3033), NF-κB (CST,8242), TNF-α (Abcam, ab307164), p-SPAK (Sigma, 07-2273), pmTOR (Abcam, ab131538), pPI3K (Abcam, ab191606), p-AKT (Abcam, ab38449), CCL2 (Abcam, ab7202), IL-1β (Abcam, ab283818), p-NKCC1 (Sigma, ABS1004). After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system. Band intensities were quantified using ImageJ software and normalized to the expression of β-Actin as a loading control.

2.12. Quantitative Real-time PCR

Total RNA from the cells and ChP samples was extracted using TRIzol reagent from Thermo Fisher Scientific (Waltham, MA, USA). cDNA synthesis was conducted with a reverse transcription kit from Vazyme (Nanjing, China). Subsequently, RT-qPCR was performed using the qPCR SYBR Green Master Mix (Servicebio, China) according to the manufacturer’s protocol. The primer sets for the targeted genes are listed in Supporting Information Table S1. Gene expression levels were quantified and normalized to GAPDH or ACTIN.

2.13. In vivo imaging in hydrocephalus mice

DiD-encapsulated lipid nanoparticles, with or without T7 modification, were administered to facilitate further tracking of biodistribution. Both the siR/DiD@NP and siR/DiD@TNP lipid nanoparticles were intravenously injected into Kunming mice. The mice were euthanized 2 hours after injection to retrieve major organs for ex vivo imaging, followed by quantification of fluorescence intensity using the Lumina III Imaging System from PerkinElmer (Waltham, USA). The brains were fixed in 4% paraformaldehyde for 24 hours, and then underwent a structured dehydration process before frozen sectioning. DAPI staining was applied to visualize nuclei on the sections, which were promptly examined under a fluorescence microscope, specifically utilizing the Leica DMi8 (Weztlar, Germany). To further determine the co-localization of different lipid nanoparticles with choroid plexus, immunofluorescence staining was performed on choroid plexus endothelial cells, and images were captured by an inverted fluorescence microscope.

2.14. Cranial MRI scans

The mice were imaged 72 hours after Kaolin injection using a Bruker BioSpec 7T MRI (Bruker, Germany) under isoflurane anesthesia. During imaging, breathing, and heart rates were monitored to ensure appropriate anesthesia depth. Axial T2-weighted images were obtained, and the lateral ventricle volumes were measured using 3D-Slicer software (NIH, MD, USA).

2.15. Immunofluorescence staining

Immunofluorescence staining was performed on brain tissue and cell samples to visualize specific proteins. Brain tissues were fixed in 4% paraformaldehyde, cryoprotected in sucrose, and sectioned at 20 µm thickness. Sections were permeabilized with 0.3% Triton X-100 and blocked with 5% bovine serum albumin (BSA). Primary antibodies were incubated overnight at 4°C, followed by washing and incubation with fluorescently labeled secondary antibodies for 1 hour at room temperature. The following antibodies were used: pNK-κB (CST, 3033), TNF-α (Abcam, ab307164), p-SPAK (Sigma, 07-2273), CCL2(Abcam, ab7202), p-NKCC1 (Sigma, ABS1004), ZO-1(Proteintech, 21773-1-AP), IBA-1(Proteintech;10904-1-AP), SPAK (ABclonal, A2275). DAPI was used to stain nuclei. For cell staining, primary choroid plexus epithelial cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Primary antibodies were applied overnight at 4°C, followed by secondary antibody incubation and DAPI staining. Samples were mounted with an antifade medium and imaged using a fluorescence microscope.

2.16. Statistical analyses

The results are presented as the mean ± SD, with error bars depicted in each graph. Statistical comparisons between two groups were conducted using Student's t-test, while comparisons among three or more groups were analyzed using one-way ANOVA followed by Dunnett's test. A P-value of less than 0.05 was considered statistically significant for all analyses.

3.1. Construction and characterization of siR/RSV@TNP

The GSH-responsive polymers designed for delivering SPAK siRNA were prepared following established procedures by connecting low molecular weight polyethyleneimine PEI600 with a disulfide bond. The structure of the polymer (named as sPEI) was confirmed by ¹H NMR to verify the successful synthesis of sPEI (Supporting Fig. S1). Due to the limited gene loading capacity of low molecular weight PEI, we examined whether the complexes could enhance gene loading capacity following the introduction of disulfide bond linkage. The gene loading capacity of sPEI was assessed by agarose gel electrophoresis. As demonstrated in Fig. 1A, LMW PEI600 was unable to fully encapsulate siRNA at a weight ratio of 4, whereas sPEI effectively encapsulated siRNA entirely at a weight ratio of 1, similar to the traditional gene transfection vector polyethyleneimine 25K (PEI25K). What’s more, the MTT results proved that sPEI showed lower toxicity compared to PEI25K (Supporting Fig. S2), indicating its improved biosafety for gene delivery. Considering the instability of siRNAs in physiological environments, where they are prone to degradation leading to a loss of their gene silencing efficacy, we conducted tests to confirm the ability of sPEI to shield siRNAs from degradation. As demonstrated in Fig. 1B, the naked siRNA experienced considerable degradation after 6 hours when exposed to serum. In contrast, sPEI, similar to PEI25K, effectively shielded the siRNA from degradation. Notably, even after 24 hours incubation with serum, the siRNA retained its intact structure. Since disulfide bond structures can break in response to intracellular reduction leading to disruption of the nanostructure and facilitate the release of siRNA for gene silence, we examined the reduction responsiveness of the lipid nanoparticles. In Fig. 1C, after incubated with GSH, the spherical structure appeared predominantly disrupted and numerous dispersed fragments were generated. This observation indicates the responsiveness of the polyplexes (siR@sPEI) to GSH. Overall, these results underscore the potential of sPEI for gene delivery. Besides, to optimize the pharmacological effects of siRNA, the sequence of SPAK siRNA was screened using real-time PCR and Western Blot. Among the candidates, SPAK siRNA4 demonstrated the highest inhibitory efficiency, achieving approximately 80% inhibition in primary ChP epithelial cells (Supporting Fig.S3). Consequently, SPAK siRNA4 was chosen for subsequent experiments.

To fulfill the need for systematic administration and further improve the treatment effectiveness, a resveratrol-encapsulated, T7-functionalized (confirmed in Supporting Fig. S4) lipid layer was utilized to shield the positive charge of siR@sPEI polyplexes and to exert combined therapeutical effects. As shown in Supporting Fig. S5, the zeta potential of siR/RSV@TNP decreased from + 29.7 ± 5.6 to ‒18.9 ± 2.5 mV, suggesting the successful coating of the lipid layer. As shown in Fig. 1E, siR/RSV@TNP with a size of 68.7 ± 29.9 nm was obtained, and transmission electron microscopy (TEM) images demonstrated a typical lipid-vesicle structure (Fig. 1D). Additionally, the size of siR/RSV@TNP showed minimal change within 24 h in PBS and 10% serum (Fig. 1E), indicating its excellent stability. The DLC and EE of RSV in siR/RSV@TNP were 4.35% and 96.16%, respectively (Supporting Fig.S6). We also investigated the release characteristics of RSV and demonstrated that over 80% of the drug could be released from the nano-formulation (siR/RSV@TNP) within 24 hours (Supporting Fig.S7).

Furthermore, it is crucial for siRNA to escape from the endo/lysosome to induce a pharmacological response. Hence, the lysosomal escape capability of lipid nanoparticles was examined. The findings revealed that following a 1-hour incubation of FITC-siR@TNP with ChP epithelial cells, a significant overlap was observed between red and green fluorescence, indicating the presence of FITC-siR@RP within the endosome/lysosome. However, as the incubation time increased, the red fluorescence became distinct from the green fluorescence (Fig. 1F and G). This suggested that our lipid nanoparticles facilitated the successful escape of siRNA from the endosome/lysosome, thereby preventing the degradation of siRNA in the lysosome.

3.2. In vivo and vitro targeting efficacy of siR/RSV@TNP to blood-CSF barrier

Given the substantial presence of transferrin receptors on the ChP^30–33, our study aimed to investigate the potential enhancement of lipid nanoparticle targeting for the ChP through T7 modification, thereby improving gene delivery efficiency. The in vitro targeting ability of lipid nanoparticles was assessed using primary ChP epithelial cells. Analysis using flow cytometry revealed that the T7-modified Cy5-siR@TNP group significantly increased cellular endocytosis efficiency compared to both free Cy5-siR and Cy5-siR@NP groups (Fig. 1H). This finding was further supported by confocal imaging results (Supporting Fig.S8). Subsequently, we explored the in vivo targeting efficacy using an IVIS Lumina III Imaging System. The results in Fig. 1I and 1J demonstrated stronger fluorescent signals in the brain with DiD-loaded siR/DiD@TNP compared to unmodified siR/DiD@NP, with no significant difference observed in other major organs (Supporting Fig. S9 and S10), indicating the benefit of T7 modification for specific targeting of the choroid plexus. Further validation through confocal imaging analysis of brain distribution confirmed that siR/Did@TNP accumulated in the choroid plexus at a significantly higher level than unmodified siR/Did@NP (Supporting Fig. S11). These results provide evidence that T7 modification can facilitate preferential targeting of the choroid plexus for drug delivery.

3.3. Functional evaluation of lipid nanoparticles in knocking down the expression of SPAK and its downstream ion channels NKCC1 and KCNJ13 in ChP Epithelial Cells

Firstly, in vitro cytotoxicity of lipid nanoparticles in primary ChP epithelial cells and RAW264.7 cells were performed. The lipid nanoparticles exhibited low toxicity in primary ChP epithelial cells within the concentration range of 0.25-7 µg/mL but showed significant cytotoxicity at concentrations above 3 µg/mL in RAW264.7 cells (Fig. 1K and Supporting Fig. S12). Hence, a concentration of 3 µg/mL was chosen as the optimal concentration for subsequent cell experiments.

We then investigated the effect of siR/RSV@TNP on SPAK mRNA expression in primary ChP epithelial cells (Fig. 2A). As expected, the lipid nanoparticles effectively silenced the SPAK gene in primary ChP epithelial cells, with downregulation efficiencies of 39% in the siR@NP group and 54% in the siR/RSV@TNP group (Fig. 2B). In contrast, the group treated with pure SPAK siRNA exhibited low efficiency, likely due to the negative charge of free siRNA, which impedes cellular entry without a transfection reagent. These results were further corroborated by Western blot analyses (Fig. 2C) and immunofluorescence (Fig. 2D).

SPAK has also been identified as a key regulator of a multi-ion transporter complex, including NKCC1, ATP1A1, KCNJ13, and CLIC6¹⁰. We then examined whether SPAK knockdown could downregulate its downstream ion transporters, such as NKCC1 and KCNJ13. The results demonstrated that RSV/siSPAK@TNP significantly reduced NKCC1 protein expression (Fig. 2E), with downregulation efficiencies of 25% for NKCC1 mRNA (Fig. 2F) and 32% for KCNJ13 mRNA (Fig. 2G). These findings indicate that SPAK siRNA-loaded lipid nanoparticles facilitate siRNA transfection and suggest their potential role in treating hydrocephalus.

We further explored whether the lipid nanoparticle could inhibit pSPAK and pNKCC1 expression in disease conditions. As depicted in Fig. 2H, 2I, and Supporting Fig. S13, siR/RSV@TNP significantly reduced pSPAK and pNKCC1 expression. These results demonstrate that SPAK knockdown via siRNA can inhibit LPS-induced pSPAK and pNKCC1 expression, thereby restraining CSF hypersecretion.

3.4. siR/RSV@TNP protects the BCSFB integrity and inhibits macrophage recruitment in hydrocephalus in vitro

Previous studies have established a link between hydrocephalus and the loss of BCSFB integrity, as well as increased macrophage infiltration into the ChP^10,12. To determine whether siR/RSV@TNP could preserve ChP blood-CSF barrier integrity and limit macrophage infiltration, we employed lipopolysaccharide (LPS) to induce BCSFB disruption, as previous reports have shown that LPS can impair BCSFB and increase its permeability^34–36. Tight junction (TJ) scaffold proteins in ChP epithelial cells are crucial for BCSFB integrity, and their loss leads to barrier impairment^9,37. Therefore, we evaluated the expression of tight junction protein ZO-1. As shown in Fig. 3A and 3B, ZO-1 expression was significantly reduced following LPS incubation. Treatment with various formulations upregulated ZO-1 expression to varying extents, with the most pronounced upregulation observed in the siR/RSV@TNP group. We further investigated whether siR/RSV@TNP could inhibit macrophage recruitment. Using a 2D Transwell co-culture system (Fig. 3C), we cultured RAW264.7 cells in the upper chamber and primary ChP epithelial cells in the lower chamber. After treatment with LPS and different formations, siR/RSV@TNP significantly inhibited macrophage migration from the upper to the lower chamber (Fig. 3D, E). Our previous study underscored the crucial role of the CCL2 signaling molecule in recruiting monocytes to the ChP in hydrocephalus¹¹. We then examined the effect of siR/RSV@TNP on CCL2 expression in LPS-treated ChP epithelial cells. Results from immunofluorescence (Fig. 3F), Western blot (Fig. 3G), and RT-qPCR (Fig. 3H) demonstrated that siR/RSV@TNP significantly inhibited CCL2 signaling. Hence, these findings validated that resveratrol-loaded groups effectively restrained macrophage infiltration by inhibiting CCL2 signaling and protecting the BCSFB barrier integrity (Fig. 3I).

3.5. siR/RSV@TNP protects the BCSFB integrity by alleviating the ROS and inflammation in the ChP epithelial cells

Prior studies have demonstrated that reactive oxygen species (ROS) and increased activation of the NF-κB pathway led to the disruption of the BCSFB^9,38. Zhang et al found that mitochondrial dysfunction in ChP epithelial cells contributes to BCSFB impairment by increasing ROS release in hydrocephalus¹². Additionally, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, produced in peripheral tissues and the choroid plexus, can negatively impact BCSFB tight junctions through the NF-κB and MMP9 pathways ^{20–22,39,40}.

In this regard, we investigated whether the siR/RSV@TNP lipid nanoparticle could inhibit oxidative stress in primary ChP epithelial cells. The results showed that siR/RSV@TNP significantly decreased the fluorescence intensity of DCFH-DA compared to H₂O₂-induced positive control cells, indicating its ROS-eliminating properties (Fig. 4A, B). What’s more, we examined the anti-inflammatory activity of the nanoparticle by measuring the expression of pro-inflammatory factors at the mRNA and protein levels in ChP epithelial cells. We first assessed NF-κB signaling after treatment with siR/RSV@TNP. The results demonstrated that siR/RSV@TNP significantly inhibited LPS-induced upregulation of pNF-κB, as shown by Western blot analysis (Fig. 4C) and immunofluorescence (Fig. 4F). Besides, as shown in Fig. 4C-G, TNF-α and IL-1β expression significantly increased in LPS-treated epithelial cells compared to the control group, while siR/RSV@TNP noticeably reduced TNF-α and IL-1β production.

In addition, Kahle et al. revealed that a macrophage-derived cytokine storm can act on ChP epithelial cells, activating inflammation and leading to CSF hypersecretion¹⁰. They also found that these interactions activate the PI3K-AkT-mTOR signaling pathway. We investigated whether the lipid nanoparticle could mediate the crosstalk between macrophages and ChP epithelial cells. By treating ChP epithelial cells with the conditional medium of inflammatory macrophages (MCM), we observed that siR/RSV@TNP significantly inhibited cell crosstalk, reduced inflammatory cytokines, and restrained the PI3K-AkT-mTOR signaling pathway in ChP epithelial cells (Fig. 4H-J). Overall, these findings demonstrated that siR/RSV@TNP can alleviate ROS and inflammation in ChP epithelial cells (Fig. 4K).

3.6. siR/RSV@TNP inhibited the occurrence and progression of hydrocephalus

Based on these results, we established a Kaolin-induced mouse hydrocephalus model to investigate the therapeutic effect of siR/RSV@TNP in vivo. Mice received tail injections of various lipid nanoparticles every 24 hours, starting 24 hours before injecting Kaolin into the lateral ventricles and continuing until 48 hours post-injection. The mice were randomly divided into five groups and treated with PBS, RSV@NP, siR/RSV@NP, and siR/RSV@TNP. The therapeutic effect was assessed using 7T cranial magnetic resonance imaging (MRI) 72 hours after the Kaolin injection. As expected, the siR/RSV@TNP group showed a significant reduction in lateral ventricle volume compared to the PBS group (Fig. 5A and B). Additionally, hematoxylin-eosin (HE) staining of major organs showed no substantial damage, suggesting the safety of our system (Fig.S14). Compared with RSV@NP and siR/RSV@NP groups, siR/RSV@TNP exhibited the most significant therapeutic effect, highlighting the importance of targeted combination therapy.

What’s more, RT-qPCR results demonstrated that siR/RSV@TNP significantly decreased the mRNA expression level of SPAK (Fig. 5C). Immunofluorescence showed siR/RSV@TNP significantly decreased the expression of SPAK in vivo (Fig. 5D). The immunofluorescence photographs of pSPAK and pNKCC1, two major markers of CSF hypersecretion, showed a marked reduction in ChP epithelial cells after siR/RSV@TNP injection (Fig. 5E, Supporting Fig. S15). Given that inflammatory factors are also responsible for BCSFB integrity, we measured proinflammatory cytokine levels in the ChP. The level of IL-6 and CCL2 levels significantly decreased across the different treatment groups (Fig. 5F and 5G). These findings demonstrated that siR/RSV@TNP effectively mitigates hydrocephalus, downregulated SPAK, and alleviated ChP inflammation.

3.7. Influence of siR/RSV@TNP on the BCSFB integrity and ChP inflammation based on the ChP transcriptome and Associated Differentially Expressed Genes

To further investigate the impact of siR/RSV@TNP on gene expression in the choroid plexus (ChP) of the mouse brain, we performed RNA-sequencing on ChP tissues from hydrocephalus and siR/RSV@TNP administrated hydrocephalus mouse. We performed statistical analysis on genes that were statistically expressed by more than |log2foldchange|>0.6 and presented them in the form of heatmap and volcano plots (Fig. 6A, B). Compared to the untreated hydrocephalus group, a total of 3896 genes were significantly altered in expression after being administrated with siR/RSV@TNP, where 1409 genes were downregulated and 2487 genes were upregulated. Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were enriched in cytokine production, leukocyte migration, myeloid leukocyte activation, and regulation of innate immune response (Fig. 6C), while the upregulated genes were associated with cell junctions and cell-substrate adhesion (Fig. 6D). KEGG pathway enrichment analysis indicated that the downregulated differentially expressed genes (DEGs) were involved in several inflammation-related pathways, including the chemokine signaling pathway, NF-κB signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway (Fig. 6E). These pathways are known to contribute to the onset and progression of hydrocephalus¹⁰. In contrast, the upregulated DEGs were linked to focal adhesion and tight junctions (Fig. 6F).

Notably, many upregulated DEGs related to tight junctions, such as cldn2, cldn3, ocln, cldn9, and ctnnb1 were identified (Fig. 6G). Tight junctions, formed by occludin, claudins, and the associated cytosolic protein zonula occludens-1, are critical for maintaining B-CSFB integrity⁴¹. The upregulation of these tight junction-related genes suggests that siR/RSV@TNP helps protect and repair B-CSFB integrity in hydrocephalus.

We also observed the downregulation of specific DEGs, including Trem2, CCLs, ILs, and TLRs, which are directly involved in ChP inflammation and macrophage recruitment and activation (Fig. 6H). Both CCL2 and CCR2, key molecules in recruiting peripheral monocytes to the ChP⁸, were significantly downregulated. To verify the above findings, we labeled the BCSFB with zonula occludens-1 protein (ZO-1) to observe changes in tight junctions after treatment. Figure 6I shows a marked loss of ZO-1 in the hydrocephalus group, while siR/RSV@TNP treatment reversed this destruction. Additionally, we used IBA-1 staining to assess ChP macrophage infiltration (Fig. 6J). The results showed there was a substantial increase in macrophage infiltration in hydrocephalus mice, which significantly decreased after treatment, especially in the siR/RSV@TNP group. Overall, these results demonstrate that siR/RSV@TNP can alleviate hydrocephalus by restoring BCSFB integrity and inhibiting ChP inflammation (Scheme 1).

Hydrocephalus is the most common neurosurgical disorder worldwide. Most patients are treated with VP shunt or endoscopic third ventriculostomy (ETV), both of which have significant long-term morbidity and high failure rates ^42–46. Currently, drug treatments for hydrocephalus are unavailable, largely due to the poorly understood cellular and molecular mechanisms and the limited efficacy of drug exposure in the brain.

SPAK is highly expressed and predominantly localized to the apical membrane of ChP epithelial cells⁴⁷, and increased pSPAK is closely linked to CSF hypersecretion and hydrocephalus¹⁰. Although inhibitors like STOCK1S-50699 and Closantel, targeting the ATP-binding site of SPAK, have been proposed, their application is limited due to poor in vivo pharmacokinetics and retinal toxicity^48,49. Zhang et al. designed a novel SPAK inhibitor, ZT-1a, which effectively inhibits NKCC1 and decreases SPAK-dependent phosphorylation, however, this drug has not yet been clinically approved⁵⁰. Small interfering RNAs (siRNAs) offer a promising therapeutic strategy due to their high specificity and low toxicity compared to other targeted drugs^51,52. However, siRNAs cannot easily penetrate cell membranes due to their anionic properties and are quickly degraded by serum nucleases, resulting in a short half-life⁵². To address these challenges, we designed GSH-responsive polymers to deliver SPAK siRNA to the ChP. We utilized sPEI with a proton sponge effect to help siRNA escape from lysosomes and be released into the cytosol. Results confirmed the lysosome escape ability and GSH-sensitivity of our system. After treatment with siR/RSV@TNP, the expressions of SPAK and abnormally upregulated pSPAK-pNKCC1 were reversed both in vitro and in vivo. Additionally, siR/RSV@TNP significantly protected the integrity of the BCSFB by inhibiting reactive oxygen species (ROS) and inflammation in ChP epithelial cells. In vivo ChP transcriptomic analysis revealed that the downregulated genes were enriched in leukocyte migration and myeloid leukocyte activation, while the upregulated genes were associated with cell junctions and cell-substrate adhesion. Immunofluorescence demonstrated increased expression of ZO-1 and reduced macrophages in ChP. Previous research has indicated that the recruitment of peripheral macrophages to the choroid plexus coincides with disruptions in its BCSFB integrity^8,9. Kahle et al. have highlighted the pivotal role of choroid plexus macrophages in hydrocephalus pathogenesis¹⁰. Prolonged inflammation mediated by macrophages in the choroid plexus can lead to scarring in the ependyma and arachnoid, as well as obstruction of brain parenchymal glymphatics and meningeal lymphatics, thereby impairing CSF resorption⁵³. Our present study demonstrated that modulating BCSFB integrity can be a promising therapeutic target to treat hydrocephalus. Furthermore, to enhance the specific distribution of our nano-drug in the ChP epithelial cells, we incorporated a T7 peptide into our nano-system. Transferrin, an 80 kDa serum glycoprotein, facilitates iron transport into proliferating cells via the transferrin receptor (TfR), which is abundantly expressed in ChP epithelial cells^30,31,54,55. Our results showed that T7-conjugated siR/RSV@NP accumulates more effectively in the ChP and brain, exhibiting superior therapeutic efficacy compared to non-targeted lipid nanoparticles. Hence, this study underscores TfR as a promising target for efficient drug delivery to the BCSFB, offering the potential for treating hydrocephalus.

In summary, we have successfully designed and developed a novel multi-functional nano-modulator termed siR/RSV@TNP, tailored for efficient delivery of siSPAK and resveratrol to ChP epithelial cells. T7 peptide was conjugated to the surface of NPs for targeted delivery to the blood-cerebrospinal fluid barrier. Upon entering the ChP epithelial cells, SPAK siRNA could be released in the intracellular redox environment and therefore inhibited SPAK-dependent CSF hypersecretion. Meanwhile, resveratrol acted to restore BCSFB integrity by suppressing inflammation and oxidative stress. Collectively, our results demonstrated that siR/RSV@TNP effectively attenuated ventriculomegaly and hydrocephalus, proving the efficacy of this strategy in modulating B-CSFB function and controlling CSF hypersecretion for hydrocephalus therapy. We anticipate that this nano-drug, synthesized in our study, represents a promising therapeutic approach for hydrocephalus with significant potential for future clinical applications. The versatility of siR/RSV@TNP, designed as a non-invasive therapeutic option, offers a promising modality for the treatment of hydrocephalus, allowing for targeted delivery of therapeutic agents and effectively addressing the underlying pathophysiological mechanisms of the condition. By minimizing side effects typically associated with conventional invasive treatments, siR/RSV@TNP has the potential to enhance patient compliance and improve overall outcomes in hydrocephalus management. Furthermore, its adaptability may enable the integration of additional therapeutic agents in the future, paving the way for more comprehensive treatment strategies tailored to the specific needs of hydrocephalus patients.

Acknowledgments

We are grateful to the National Natural Science Foundation of China (82201501), the Natural Science Foundation of Sichuan Province (2023NSFC1581), and 1·3·5 projects for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (No. 2020HXFH013), and Postdoctoral Research Fund of West China Hospital, Sichuan University (No.2024HXBH136), and Sichuan Science and Technology Program (22ZDYF2619, 2022NSFC1380) for financial support.

Author Contributions

Huile Gao and Qiguang Wang designed the research. Qiguang Wang, Xue Xia, and Huan Zhang carried out the experiments and performed data analysis. Lei Zhu performed RNA-seq analysis. Qiguang Wang and Yue Li performed tissue pathological staining and analysis. Huan Zhang and Jian Cheng performed MRI studies. Qiguang Wang, Xuhui Hui, Yulong Shi, and Yuzhao Tang constructed rat PHH models and analyses. Qiguang Wang and Xue Xia wrote the manuscript. Qiguang Wang, Xue Xia, and Huile Gao revised the manuscript. All authors discussed the results and contributed to the final manuscript.

Conflict of interest

All the authors declare they have no competing interests.

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Scheme 1 is available in the Supplementary Files section.

There is NO Competing Interest.

Supplentarymaterials.docx
Supplentary materials
Scheme1.png
Scheme 1. The multi-functional nano-modulator (siR/RSV @TNP) preferably navigates to the choroid plexus epithelial cells. It can significantly restore blood-cerebrospinal fluid barrier (B-CSFB) integrity and inhibit CSF hypersecretion by co-delivery of resveratrol (RSV) and SPAK siRNA. It can effectively attenuate ventriculomegaly and represent a promising therapeutic approach for hydrocephalus.

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Targeting modulation of the choroid plexus blood-CSF barrier and CSF hypersecretion via lipid nanoparticle-mediated co-delivery siRNA and resveratrol

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Synthesis of sPEI and DSPE-PEG-T7

2.3. Preparation and characterization of polyplexes with different weight ratios

2.4. SPAK Knockdown Efficiency Assessments of Polyplexes

2.5. Preparation and characterization of siR/RSV@TNP

2.6. Experimental cells and animals

2.7. Cell uptake and lysosomal escape assay

2.8. Cytotoxicity assay

2.9. Alleviating intracellular ROS

2.10. Kaolin-induced hydrocephalus models

2.11. Western Blot

2.12. Quantitative Real-time PCR

2.13. In vivo imaging in hydrocephalus mice

2.14. Cranial MRI scans

2.15. Immunofluorescence staining

2.16. Statistical analyses

3. Results

3.1. Construction and characterization of siR/RSV@TNP

3.2. In vivo and vitro targeting efficacy of siR/RSV@TNP to blood-CSF barrier

3.4. siR/RSV@TNP protects the BCSFB integrity and inhibits macrophage recruitment in hydrocephalus in vitro

3.6. siR/RSV@TNP inhibited the occurrence and progression of hydrocephalus

4. Discussion and Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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