You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction {#S1}
============

Preeclampsia is a special type of hypertensive disorders complicating pregnancy (HDCP) which is characterized by hypertension, proteinuria, hematological complications, and uteroplacental dysfunction ([@B26]). The fetal development is also significantly influenced by the pathogenesis of PE, such as amniotic fluid reduction, oxygenation defect, and growth restriction ([@B8]). During the pathogenesis of PE, the dysfunction in maternal vascular system and the abnormal activation of maternal-fetal immune system are proved to be the two main factors ([@B14]). The worldwide prevalence of PE in all pregnant women is from 3 to 8% and nearly 18% maternal deaths are caused by PE based on its high maternal morbidity and mortality ([@B4]). The primary prevention of PE is mainly performed through the treatment of aspirin, vitamin D, and low-dose calcium ([@B11]). The termination of pregnancy or fetus delivery is thought to be the only definitive treatment for PE ([@B27]). Although there has been great progress in the study of PE, the prediction and prevention of PE development is still a big challenge.

PE is proved to be an anti-angiogenic state. Soluble fms-like tyrosine kinase 1 (sFLT-1) is a shorter form of FLT-1 which lacks the intra-cytoplasmic and transmembrane domain and thus can circulates freely in maternal circulation ([@B23]). The ability of binding with vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) makes sFLT-1 to be the decoy receptor of these two factors in the maternal circulation ([@B21]). The sFLT1:PlGF ratio is proved to fluctuate during the normal pregnant. sFLT1 stays at a stable level till week 29--30 and then increases to a peak at week 40 ([@B20]). Meanwhile, The sFLT1:PlGF ratio decreases in week 9--12, stays at a low level from week 19--20 to 37--38, and then increases in week 39--40 ([@B20]). The tightly regulated sFLT1:PlGF ratio during pregnant is crucial for both placenta and fetal development. The elevated sFLT-1 level is observed in patients with PE ([@B15]). The pathogenesis of PE triggers the increasing of sFLT1 level in maternal circulation ([@B12]). The up-regulated sFLT-1 level in the maternal circulation decrease the levels of VEGF and PlGF and generates the endothelial dysfunction ([@B16]). It has been reported that sFLT-1 expression is up-regulated in the placenta and generated by villous and extravillous trophoblasts ([@B2]). Therefore, the overexpressed sFLT1 in placenta is considered to be a potential therapeutic target for PE. How to achieve efficient intervention of sFLT1 expression in the placenta is an urgent problem to be solved.

In recent, it has been proved that the systemically delivered sFLT1 siRNAs which are cholesterol conjugated and full chemical stabilized can accumulate in placentas, inhibit the expression of sFLT1, and alleviate proteinuria and hypertension ([@B25]). Chondroitin sulfate A (CSA) is a protein which is proved to exist on the surface of placental syncytiotrophoblasts and P-CSA-BP is effective for targeting lipid-polymer nanoparticle to the placenta ([@B31]). This research indicates that sFLT1-siRNA-based PE therapy is a hopeful therapeutic strategy. The discovery of P-CSA-BP makes it possible to develop a placenta-specific sFLT1-siRNA delivery method, which is thought to be a great progression in the improvement of sFLT1-siRNA-based PE therapeutic strategy.

In this research, we built up P-CSA-BP-conjugated nanoparticles loaded with sFLT1 siRNA to generate a new placenta-specific siRNA delivery method. We aimed to illustrate the effects of this method in placenta targeting and silencing of sFLT1 and analyze the toxic effect.

Materials and Methods {#S2}
=====================

Animals {#S2.SS1}
-------

Pregnant CD1 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were caged in pathogen-free animal room with 12 h light/12 h dark cycle. The gestation of mice was determined by checking vaginal plugs formation (E0.5 = vaginal plug day). At 14 and 15 days of gestation, the mice were treated by siRNA, NP~siRNA~, or T-NP~siRNA~ through intravenous injection in the tail vein at the siRNA dosage of 2 mg/kg. The accumulation of Cy5-labeled si*sFLT1* in mice placenta was observed through *ex vivo* images. Placenta tissue samples and blood samples were collected for further investigation. The procedures of animal experiments in this research were approved by the Shandong Provincial Hospital Affiliated to Shandong University.

Cell Culture {#S2.SS2}
------------

HTR-8/SVneo cell lines were obtained from the American Type Culture Collection (ATCC, VA, United States). RPMI-1640 medium (Gibco, New York, United States) with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Sigma, St. Louis, United States) was applied for the cell culture under the standard conditions.

Preparation and Characterization of Nanoparticles {#S2.SS3}
-------------------------------------------------

Lipid-polymer nanoparticles loaded with sFLT1 siRNAs (NP~si*sFLT1*~) were synthesized from carboxyl-polyethylene glycol-poly(D,L-lactide) (COOH-PEG~5K~-PLA~8K~), cationic lipid DOTAP (N-\[1-(2,3-dioleoyloxy)propyl\]-N,N,N-trimeth ylammonium chloride), and sFLT1 siRNAs using double emulsion method which was previously published in other research ([@B28]). Synthetic placental CSA-binding peptides (P-CSA-BP) were conjugated to NP~si*sFLT1*~ to generate the trophoblast-targeted nanoparticles (T-NP~si*sFLT1*~) based on the method from other study ([@B31]).

Size distribution and zeta potential of nanoparticles were measured through dynamic light scattering (DLS) in triplicate with the help of Zetasizer Nano ZS (Malvern, United Kingdom). The morphology of nanoparticles was visualized through transmission electron microscopy (TEM) (JEOL-2010 microscopy, Tokyo, Japan). The colloidal stability was investigated by incubating in phosphate buffered saline (PBS) with 10% fetal bovine serum (FBS) and analyzing the size of nanoparticles.

Gel Retardation Assay {#S2.SS4}
---------------------

The integrity of nanoparticles after 7 days' incubation in PBS with 10% BSA was investigated by gel retardation assay. Naked siRNA was used as a control. Samples were mixed with gel loading buffer containing 1% SDS and separated by 2% agarose gel electrophoresis. The samples in gel were observed by ethidium bromide staining.

Immunohistochemistry {#S2.SS5}
--------------------

Immunohistochemistry was employed to evaluate CSA expression in placenta tissues. After being deparaffinized and rehydrated, slides were treated by antigen retrieval buffer. Then the tissue sections were incubated with BSA for 1 h at room temperature. Then the sections were incubated with mouse monoclonal anti-chondroitin sulfate antibody (Abcam, Cambridge, United Kingdom) or control IgG (Abcam) at 4°C overnight. After being washed, the sections were incubated with HRP-donkey anti-mouse IgG (ThermoFisher, Waltham, United States). The sections were observed under a light microscope.

Immunofluorescence {#S2.SS6}
------------------

Cells were fixed by 4% paraformaldehyde for 30 min at room temperature and permeabilized by 0.05% Triton X-100 for 10 min. After being blocked by 5% BSA for 1 h at room temperature, cells were incubated with anti-chondroitin sulfate antibody (Abcam) at 4°C overnight. After being washed by PBS for three times, the cells were incubated with anti-mouse IgG FITC antibody (Abcam) for 1 h at room temperature. Cell nucleus were stained with DAPI. Cells were observed by confocal microscope.

Flow Cytometry {#S2.SS7}
--------------

5 × 10^4^ HTR-8/SVneo cells were planted in each well of a 24-well plate. After 24 h, cells were incubated with free Cy5 labeled siRNA (Cy5-siRNA), NP~Cy5--siRNA~, or T-NP~Cy5--siRNA~ (100 nM) for 4 h. After incubation, cells were collected and analyzed by FACSCalibur (BD Biosciences, Bedford, United States). Mean fluorescence intensity of Cy5 was calculated by FlowJo 7.6.1 software.

ELISA {#S2.SS8}
-----

fms-like tyrosine kinase 1 protein levels in cell medium or serum were evaluated by ELISA kit (R&D Systems, Minneapolis, United States) based on the manufacturer's instructions. Experiments were done in duplicate, and the protein levels were calculated using a standard curve. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were analyzed by colorimetric assay kits (Sigma, St. Louis, United States) based on the manufacturer's instructions.

Real-Time PCR {#S2.SS9}
-------------

Trizol reagent (Invitrogen, Waltham, MA, United States) was used for the extraction of total RNA. First Strand cDNA Synthesis Kit (Sigma, St. Louis, MO, United States) was employed to synthesis cDNA from relative RNA. Real-time PCR was executed by SYBR Green Real-Time PCR Master Mixes (ThermoFisher, Waltham, MA, United States) following manufacturer's instruction. β-actin and GAPDH were employed as internal control. All the primers were shown here:

1.  Human sFLT-1 F: GAAATGGTGAGTAAGGAAAGC

2.  Human sFLT-1 R: TACTGTCCCAGATTATGCGTTT

3.  Human β-actin F: CTGGTGCCTGGGGCG

4.  Human β-actin R: AGCCTCGCCTTTGCCG

5.  Mouse sFlt-1 F: CGACTCACTATAGGGAGACCC

6.  Mouse sFlt-1 R: TGGCCTGCTTGCATGATGTGCTGG

7.  Mouse GAPDH F: GTGGCAAAGTGGAGATTGTTGCC

8.  Mouse GAPDH R: GATGATGACCCGTTTGGCTCC

High-Performance Liquid Chromatography (HPLC) {#S2.SS10}
---------------------------------------------

High-performance liquid chromatography was employed to evaluate the amount of si*sFLT1* accumulated in different tissues. Tissues were harvested 1 day after injection. The performance of HPLC was based on the standard method ([@B31]). One day after injection, relative tissues were collected and homogenized in PBS. 10% perchloric acid was added into the mixture and vortexed for 3 min. After being centrifuged at 14,000 × *g* for 20 min, supernatants were collected and filtered (0.45 μm). Mobile phase was composed of 40 mM potassium phosphate dibasic and acetonitrile. Flow rate was 1.0 mL/min. Ultraviolet absorbance at 313 nm was detected.

Statistical Analysis {#S2.SS11}
--------------------

Statistical analysis was performed by GraphPad PRISM 6.0 software. Data were presented as mean ± SD. Student's *t*-test, one or two-way ANOVA analysis with a *post hoc* test were used to calculate the differences. Statistical analysis was significant when *P* value \< 0.05.

Results {#S3}
=======

The Preparation and Characterization of Trophoblast-Targeted sFLT1 siRNA-Encapsulated Nanoparticles {#S3.SS1}
---------------------------------------------------------------------------------------------------

As shown in [Figure 1A](#F1){ref-type="fig"}, NP~si*sFLT1*~ was constructed from COOH-PEG~5K~-PLA~8K~, DOTAP, and si*sFLT1* through double emulsion method and T-NP~si*sFLT1*~ was fabricated by conjugating P-CSA-BP to the surface of NP~si*sFLT1*~. DLS results shown that the resulted nanoparticles T-NP~si*sFLT1*~ had a relative larger particle size than NP~si*sFLT1*~ ([Figure 1B](#F1){ref-type="fig"}). The conjugation of P-CSA-BP enlarged the size of the nanoparticle. The morphology of these two nanoparticles was checked by TEM and the representative TEM images were shown in [Figure 1C](#F1){ref-type="fig"}. The P-CSA-BP could be observed on the surface of T-NP~si*sFLT1*~. Both of the nanoparticles had slightly positive zeta potentials ([Figure 1D](#F1){ref-type="fig"}). The incubation of siRNA-encapsulated nanoparticles in PBS demonstrated the colloidal stability of these two nanoparticles ([Figure 1E](#F1){ref-type="fig"}). Based on the results of agarose gel retardation assay, no significant siRNA release was observed, indicating that both nanoparticles were successfully protected the encapsulated siRNA ([Figure 1F](#F1){ref-type="fig"}). So, nanoparticles T-NP~si~*~*sFLT*1~* was successfully generated from NP~si~*~*sFLT*1~* and was proved to be stable.

![Preparation of trophoblast-targeted nanoparticles encapsulating sFLT1 siRNA. **(A)** Nanoparticles loaded with sFLT1 siRNA (denoted as NP~si*sFLT1*~) was constructed from COOH-PEG~5K~-PLA~8K~, cationic lipid DOTAP, and sisFLT1 using double emulsion method. Trophoblast-targeted nanoparticles (denoted as T-NP~si*sFLT1*~) was fabricated by conjugating peptides, which could specific bind with CSA on the trophoblasts, to the surface of NP~si*sFLT1*~. **(B)** Size distributions of NP~si*sFLT1*~ and T-NP~si*sFLT1*~ were examined by DLS. **(C)** Representative TEM images of NP~si*sFLT1*~ and T-NP~si*sFLT1*~. Scale bars = 100 nm. **(D)** Zeta potential of nanoparticles examined via DLS. **(E)** Effect of incubation in PBS on the particle size. **(F)** Gel retardation assay for examining the integrity of nanoparticles 7-day post construction.](fbioe-08-00064-g001){#F1}

T-NP~siRNA~ Specifically Target Trophoblast Cells *in vitro* {#S3.SS2}
------------------------------------------------------------

Chondroitin sulfate A is a protein which is proved to exist on the surface of placental syncytiotrophoblasts. Through the immunohistochemistry, we proved the expression of CSA in the tissue of mouse placenta ([Figure 2A](#F2){ref-type="fig"}). HTR-8/SVneo cells were human trophoblast cells and the expression of CSA was observed through immunofluorescence ([Figure 2B](#F2){ref-type="fig"}). To investigate the uptake of these two kinds of nanoparticles into HTR-8/SVneo cells, siRNA was labeled with Cy5 and cells were analyzed by flow cytometry after incubating with nanoparticles. Based on the result of flow cytometry, trophoblast cells incubated with T-NP~Cy5--siRNA~ had a significantly higher Cy5 signal intensity than those incubated with NP~Cy5--siRNA~ ([Figures 2C,D](#F2){ref-type="fig"}). Furthermore, the internalized NP~Cy5--siRNA~ and T-NP~Cy5--siRNA~ in HTR-8/SVneo cells were observed by confocal microscopy ([Figure 2E](#F2){ref-type="fig"}). These results indicated that T-NP~siRNA~ had a higher efficiency than NP~siRNA~ in targeting to trophoblast cells *in vitro.*

![T-NP~Cy5--siRNA~ specifically target trophoblast cells *in vitro*. **(A)** The expression of CSA in mouse placenta was examined by immunohistochemistry. Scale bar = 20 μm. **(B)** The expression of CSA on HTR-8/SVneo cells was examined by confocal microscopy. CSA was stained with IgG isotype control antibody or anti-CSA primary antibody and anti-mouse IgG (whole molecule) FITC antibody. Scale bar = 10 μm. **(C)** Flow cytometry analyses of HTR-8/SVneo cells after 4 h incubation with free Cy5 labeled siRNA (Cy5-siRNA), NP~Cy5--siRNA~ and T-NP~Cy5--siRNA~, the siRNA concentration was 100 nM. **(D)** Mean fluorescence intensity of Cy5 in HTR-8/SVneo cells after treated with Cy5-siRNA, NP~Cy5--siRNA~ and T-NP~Cy5--siRNA~. **(E)** Intracellular uptake of Cy5-siRNA, NP~Cy5--siRNA~ and T-NP~Cy5--siRNA~ were examined by confocal microscopy. Cell nuclei were stained with DAPI; the cytoskeleton was stained with Alexa Fluor 488 Phalloidin. Scale bar = 20 μm. Data was shown as mean ± SD. \*\*\**p* \< 0.001.](fbioe-08-00064-g002){#F2}

T-NP~sisFLT1~ Inhibit the Expression of sFLT1 *in vitro* {#S3.SS3}
--------------------------------------------------------

To demonstrate the function of these two sFLT1 siRNA-encapsulated nanoparticles in the inhibition of sFLT1 expression *in vitro*, HTR-8/SVneo cells were treated with free si~sFLT1~, NP~si*sFLT1*~ or T-NP~si*sFLT1*~ for 48 h. The mRNA levels of sFLT1 in cells were evaluated through RT-PCR and the results were shown in [Figure 3A](#F3){ref-type="fig"}. When compared with cells treated by NP~si*sFLT1*~, those cells treated by T-NP~si*sFLT1*~ shown a significant lower sFLT1 mRNA level and this phenomenon was enhanced by the increasing dosage. The protein level of sFLT1 was evaluated by ELISA and shown the same tendency with mRNA ([Figure 3B](#F3){ref-type="fig"}). The protein levels of sFLT1 in HTR-8/SVneo cells were not altered by the treatment of nanoparticles encapsulating nonsense siRNA (NP~siNC~ and T-NP~siNC~) ([Figure 3C](#F3){ref-type="fig"}). T-NP~si*sFLT1*~ had a higher efficiency than NP~si*sFLT1*~ in the inhibition of sFLT1 expression in trophoblast cells *in vitro.*

![Efficient silencing of sFLT1 by T-NP~si*sFLT1*~ *in vitro*. **(A)** Silencing of sFLT1 by different formulations in a dose-dependent manner. HTR-8/SVneo cells were treated with free si*sFLT1*, NP~si*sFLT1*~ or T-NP~si*sFLT1*~ for 48 h and sFLT1 mRNA levels were examined via RT-PCR. The final concentration of si*sFLT1* was 100 nM or 200 nM in present study. **(B)** sFLT1 protein levels produced by HTR-8/SVneo cells treated with different formulations for 48 h. sFLT1 protein levels were measured by ELISA (*n* = 3). **(C)** sFLT1 protein levels produced by HTR-8/SVneo cells after treated with T-NP encapsulating nonsense siRNA (T-NP~si~*~*NC*~*). Data was shown as mean ± SD. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001. ns. no significant difference.](fbioe-08-00064-g003){#F3}

T-NP~sisFLT1~ Selectively Accumulate in Placenta and Downregulate sFLT1 Expression in Mice {#S3.SS4}
------------------------------------------------------------------------------------------

The function of T-NP~si*sFLT1*~ was further checked in mouse model. Experimental design for testing accumulation ability of T-NP~Cy5--siRNA~ in pregnant CD1 mice was shown in [Figure 4A](#F4){ref-type="fig"}. To illustrate the accumulation of the nanoparticles, pregnant CD1 mice were intravenously injected with NP~Cy5--siRNA~ and T-NP~Cy5--siRNA~. Based on the *ex vivo* examination, the localization of T-NP~Cy5--siRNA~ in placenta was much higher than NP~Cy5--siRNA~ ([Figure 4B](#F4){ref-type="fig"}). HPLC was employed to quantitatively compare the accumulation of the nanoparticles in different organs. As shown in [Figure 4C](#F4){ref-type="fig"}, the amount of Cy5-siRNA in the placenta of mice treated by T-NP~Cy5--siRNA~ was significantly higher than in the placenta of mice treated by NP~Cy5--siRNA~. But the distribution of Cy5-siRNA shown no significant differences in other organs. So, P-CSA-BP on the surface of T-NP~si~*~*sFLT*1~* enhanced the nanoparticle's specific accumulation ability in mice placenta. Experimental design for testing silencing ability of T-NP~si*sFLT1*~ in pregnant CD1 mice was shown in [Figure 4D](#F4){ref-type="fig"}. We examined the mRNA levels of sFLT1 in the placenta of mice treated by different nanoparticles through RT-PCR. When compared with NP~si*sFLT1*~, the treatment of T-NP~si*sFLT1*~ significantly decreased the mRNA level of sFLT1 in the placenta of mice ([Figure 4E](#F4){ref-type="fig"}). Circulating sFLT1 levels in mice were evaluated by ELISA and shown the same tendency ([Figure 4F](#F4){ref-type="fig"}). So, P-CSA-BP on the surface of T-NPsisFLT1 also enhanced the nanoparticle's sFLT1 silencing ability in mice placenta. It was proved that T-NP~si*sFLT1*~ had a higher efficiency than NP~si*sFLT1*~ in accumulation in the placenta of mice and silencing sFLT1.

![T-NP~si*sFLT1*~ selectively accumulate in placenta and downregulate sFLT1 expression. **(A)** Experimental design for *in vivo* testing of T-NP~siRNA~ accumulation in pregnant CD1 mice. Pregnant CD1 mice treated with T-NP encapsulating Cy5-labeled si*sFLT1* at 14 days of gestation, and the injection dosage of Cy5-si*sFLT1* was 2 mg/kg. The biodistribution of T-NP~Cy5--siRNA~ was qualitatively and quantitively detected 1 day later. **(B)** *Ex vivo* images of placenta in the pregnant mice after Cy5-labled siRNA injection at 15 days of gestation. **(C)** Amount of Cy5 accumulated in indicated tissues 1 day after injection using HPLC. **(D)** Experimental design for *in vivo* silencing of sFLT1 via NP~si*sFLT1*~. Pregnant CD1 mice were intravenously injected with 2 mg/kg of si*sFLT1* on pregnancy days 14 and 15 days of gestation, and killed for examination on allowed to deliver pups on 19 days of gestation or allowed to delivery. Blood was collected on the indicated days. **(E)** sFLT1 mRNA expression in placenta on 19 days of gestation examined via RT-PCR (*n* = 8 biologically independent animals). **(F)** Circulating sFLT1 levels from mice injected with free si*sFLT1*, NP~si*sFLT1*~ or T-NP~si*sFLT1*~ measured by ELISA (*n* = 8 biologically independent animals). Data was shown as mean ± SD. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001.](fbioe-08-00064-g004){#F4}

Conjugating P-CSA-BP to the Surface of NP~sisFLT1~ Does Not Enhance the Toxic Effect {#S3.SS5}
------------------------------------------------------------------------------------

We further investigated whether conjugating P-CSA-BP to the surface of NP~si~*~*sFLT*1~* enhanced the nanoparticle's toxic effect in mice. The changes of ALT and AST were widely used for illustrating the severity of the toxic effect on liver. The levels of the maternal liver transaminases ALT and AST in mice was analyzed after treatment. The normal ALT and AST levels indicated that the treatment of NP~si*sFLT1*~ or T-NP~si*sFLT1*~ had no obvious adverse effects ([Figures 5A,B](#F5){ref-type="fig"}). We also checked the influence of these nanoparticles on the fetus. Both newborn pup number and weight were not influenced by the treatment of NP~si*sFLT1*~ or T-NP~si*sFLT1*~ ([Figures 5C,D](#F5){ref-type="fig"}). Both of these two nanoparticles had low maternal and fetal toxic effect. Thus, conjugating P-CSA-BP to the surface of NPsisFLT1 did not enhance the nanoparticle's toxic effect in mice.

![Toxic effect analysis of T-NP~si*sFLT1*~. Enzyme activities of ALT **(A)** and AST **(B)** at day 19 of gestation in mice injected with free si*sFLT1*, NP~si*sFLT1*~ or T-NP~si*sFLT1*~ (*n* = 8 biologically independent animals). Newborn pup number **(C)** and weights **(D)** from mice injected with free si*sFLT1*, NP~si*sFLT1*~ or T-NP~si*sFLT1*~ (*n* = 8 biologically independent animals). ns. no significant difference.](fbioe-08-00064-g005){#F5}

Discussion {#S4}
==========

Based on previous research, the pathogenesis of PE can be divided into two different stages ([@B22]). The first stage is asymptomatic, but abnormal placenta formation and accumulation of placenta released factors in maternal circulation can be observed. The symptomatic second stage is characterized by placental oxidative stress, hypertension and proteinuria, that eventually result in angiospasm in brain which caused eclampsia ([@B13]). Since the molecular mechanism during the pathogenesis of PE is not fully understand, PE still lacks reliable method for prediction and diagnosis and effective therapeutic strategy ([@B3]). Untreated PE is life threatening and will develops into eclampsia with several severe complications ([@B5]). Till now, the only effective therapy for PE is the early delivery of the pregnancy.

In recent years, the pathogenesis of PE is proved to have a strong correlation with the factors generated and released from placenta ([@B15]). Several different researches have demonstrated the abnormal increased level of anti-angiogenic molecule sFLT-1 in the circulation of women with PE ([@B10]; [@B18]). The accumulation of sFLT-1 has a strong correlation with the generation of hypertension through its function in antagonizing VEGF and PlGF ([@B9]). The sFLT-1/PlGF ratio is reported to be useful for the diagnosis and prognosis assessment of PE ([@B12]). sFLT-1 is also considered as a potential PE therapeutic target. [@B24], have proved that apheresis therapy is efficient in removing sFLT-1 in the circulation of women with PE and extends the pregnancies with no obvious adverse effects on both mother and fetus. [@B6], demonstrated that metformin inhibits the production of sFLT-1 in villous trophoblast and prevents the pathogenesis of PE. In both mouse and baboon PE model, systemically delivered modified sFLT1 siRNAs accumulate in placentas, inhibit sFLT1 expression, and alleviate hypertension and proteinuria ([@B25]). RNAi-based placental sFLT1 expression modulation is proved to be a novel therapeutic approach for PE. However, several limitations still exist in the development of this therapeutic strategy, such as siRNA degradation and difficulty in crossing cell membrane ([@B17]).

Nanoparticles can act as delivery system and have several benefits, including enhanced permeability, retention effect, and extravasation ability and are widely used in the therapy of cancer and other disease ([@B7]). The transfer ability of nanoparticles to different organs is critical for the therapy. It is reported that molecular shape, surface modification, and particle size determine whether the nanoparticle can cross the placenta barrier ([@B19]). Poly-amidoamine (PAMAM) dendrimers are shown to be excellent nanocarrier for siRNA delivery and siRNA-sFLT1-PAMAM complex is proved to be effective in inhibition sFLT1 expression and improving pregnancy outcomes in PE rat model ([@B30]). PEG-PLA nanoparticle generated by double-emulsion methods is a novel siRNA delivery system which is proved in both *in vivo* and *in vitro* cancer models ([@B29]). We aimed to use this PEG-PLA nanoparticular system for the delivery of sFLT1 siRNA into placenta and inhibiting the pathogenesis of PE.

Despite the placenta is unrestricted exposed to drugs in maternal circulation, only a small proportion of non-targeted nanoparticles are successfully transported into placenta ([@B31]). Thus, specific targeting the nanoparticles to placental tissues is critical for the development of nanoparticle-based therapeutic strategy for pregnancy-related disorders. CSA is a protein which exist on placental syncytiotrophoblasts. We confirmed the existence of CAS in both mice placenta tissues and human trophoblast HTR-8/SVneo cells. Synthetic placental CSA-binding peptide can specifically bind to trophoblasts in placenta but not to other cells expressing CSA in other tissues. It is reported that P-CSA-BP-conjugated nanoparticles is effective in targeting to the placenta and is a novel placenta-specific drug delivery option ([@B31]). Based on the PEG-PLA nanoparticular system, we synthesized P-CSA-BP-conjugated PEG-PLA nanoparticles encapsulating sFLT1 siRNA and analyzed its function both *in vivo* and *in vitro*. Conjugating P-CSA-BP enlarged the particle size and decreased the surface potential of PEG-PLA nanoparticles. Both NP~si*sFLT1*~ and T-NP~si*sFLT1*~ nanoparticles were proved to be stable in PBS and shown strong ability in the protection of encapsulated siRNA. HTR-8/SVneo cells were generated from isolated first trimester extravillous cytotrophoblasts through the infection with simian virus 40 large T antigen ([@B1]). In human trophoblast HTR-8/SVneo cells, the existence of P-CSA-BP on the surface of nanoparticles significantly enhanced its accumulation ability, which was shown by both flow cytometry analyses and immunofluorescence results. We also checked the efficiency of NP~si*sFLT1*~ and T-NP~si*sFLT1*~ nanoparticles in targeting to the placentas of pregnant CD1 mice. *Ex vivo* images and HPLC results demonstrated that nanoparticles with P-CSA-BP on the surface had a significantly stronger ability in accumulation in the placenta of mice. So, conjugating P-CSA-BP to the surface of PEG-PLA nanoparticles enhanced its ability in targeted delivery of drugs to the placenta.

In this research, we aimed to decrease the expression of sFLT1 in placenta through the trophoblast-targeted nanoparticles encapsulating sFLT1 siRNA. In HTR-8/SVneo cells, T-NP~si*sFLT1*~ nanoparticles had a higher efficiency in inhibition of sFLT1 expression than NP~si*sFLT1*~ nanoparticles, which was shown in both mRNA and protein levels. Also, after being injected into pregnant CD1 mice, T-NP~si*sFLT1*~ nanoparticles significantly decreased the sFLT1 mRNA level in placenta and sFLT1 protein level in serum. Trophoblast-targeted sFLT1 siRNA-encapsulated T-NP~si*sFLT1*~ nanoparticles had a strong ability in the inhibition of sFLT1 expression in placenta and decreasing the amount of sFLT1 in circulation.

Nanoparticles used as delivery system in the therapy of PE must be safe for both mother and fetus. It should have no adverse effects on tissues, do not cross the placenta into fetus, and show no apparent fetal toxicity. The injection of T-NP~si*sFLT1*~ nanoparticles did not influenced the enzyme activities of ALT and AST in pregnant mice. Meanwhile, the newborn pups number and weights were also not changed. These results illustrated that the utility of T-NP~si*sFLT1*~ nanoparticles as a sFLT1 siRNA placenta specific delivery system was safe in mice.

In order for this placenta targeted nanoparticles to be adopted and accepted clinically, further research and development are required. The maximum dosage of this nanoparticles should be tested. The nanoparticles should be optimized to improve the efficiency in encapsulating and releasing sFLT1 siRNA. More toxicity tests should be performed in different animal models to ensure the safety of this nanoparticles. Although we had demonstrated sFLT1 silencing function of T-NPsisFLT1 nanoparticles in mouse placenta, but its effects in the prevention or therapy against PE in mice was not investigated. So, further studies should be done to demonstrate the function of T-NPsisFLT1 nanoparticles in PE therapy.

In conclusion, the employment of T-NP~si*sFLT1*~ nanoparticles as a sFLT1 siRNA placenta specific delivery system significantly silenced sFLT1 in mice and was safe for both the mother and fetus. This nanoparticle might serve as a novel potential therapeutic strategy for PE.

Data Availability Statement {#S5}
===========================

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Ethics Statement {#S6}
================

The animal study was reviewed and approved by the Shandong Provincial Hospital Affiliated to Shandong University.

Author Contributions {#S7}
====================

LL, HY, and PC conceived the study. TX, QZ, DW, and YZ designed the experiments and analyzed the data. SW secured the funding and supervised the project.

Conflict of Interest {#conf1}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This study was supported by the Key Research & Development Project of Shandong Province (2019GSF108096), the National Natural Science Foundation of China (81801473, 81971409, 81741037, 81741038, and 81100447), the Shandong Medical and Health Technology Development Program of China (2016WS0412), the Chongqing Postdoctoral Research Special Funding Project (XmT2018077), the China Postdoctoral Science Foundation (2018M631067 and 2019T120814), the Open Project of NHC Key Laboratory of Fertility Control Technology (2018KF002), and the Jinan Science and Technology Program (201907011 and 201907012).

sFLT-1

:   fms-like tyrosine kinase 1

P-CSA -BP

:   Synthetic placental CSA binding peptide

PE

:   preeclampsia.

[^1]: Edited by: Larry D. Unsworth, University of Alberta, Canada

[^2]: Reviewed by: Augustine Rajakumar, Emory University, United States; Akihide Ohkuchi, Jichi Medical University, Japan

[^3]: This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology