Introduction

Macrophages are present in all parts of the body and play an essential role in defending against foreign pathogens and maintaining homeostasis. Macrophages are differentially activated along a spectrum ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages, depending on their microenvironmental signals. This dynamic change in macrophage function is defined as macrophage polarization1. M1 macrophages are responsible for pro-inflammatory responses by producing key factors, such as interleukin (IL)-6, IL-12, and tumor necrosis factor α. When activated by IL-4 or IL-13, macrophages are instead polarized to the M2 phenotype. M2 macrophages contribute to developing an anti-inflammatory milieu that promotes immunosuppression and allows the resolution of inflammation, followed by tissue repair. These cells express high levels of soluble IL-1 receptor (sIL-1R) as a decoy receptor, an IL-1R antagonist with anti-inflammatory properties, and a mannose receptor (CD206) for immune homeostasis. Many diseases (i.e., such as diabetes, atherosclerosis, rheumatoid arthritis, obesity, and cancers) are associated with chronic inflammation2. Rebalancing the biased macrophage polarity may provide a novel approach to treating inflammation-related diseases3.

The macrophage polarization may be modulated by delivering nucleotides, such as small RNAs, including microRNAs (miRNAs), into macrophages. However, modulating the macrophage polarity by nucleotide delivery is a critical challenge in treating inflammatory diseases because the delivery efficiency to macrophages remains low. Macrophages possess unique characteristics that make them particularly difficult to transfect. In particular, macrophages are equipped with pattern recognition receptors that detect foreign nucleic acids, potentially triggering inflammatory responses or degradation of the delivered material4. miRNAs such as miR-10a, differentially expressed in distinctly polarized macrophages, are vital post-transcriptional mediators that control inflammatory responses5,6. Several groups, including us, demonstrated that small RNAs delivery to macrophages can rebalance the biased macrophage polarization in vivo and in vitro7,8,9. However, improving nucleotide delivery efficiency to macrophages remains demanding.

Nanoparticles (NPs) are widely used as nucleotide delivery carriers because they can penetrate cell membranes without interfering with biological barriers10. Among NPs, quantum dots (QDs) have emerged as indispensable tools in biomedical research, especially for simultaneous detection and quantitative, multiplexed, and long-term fluorescence imaging11. QDs, with their nanoscale size and surface modifiability, could potentially overcome macrophage nucleotide delivery challenges by enhancing cellular uptake and stability of nucleotide cargo, thus improving delivery efficiency12. In addition to their delivery capabilities, QDs have enabled the tracking of biomolecules, such as proteins and nucleic acids within cells, providing insights into cellular processes13. These advantages make QDs ideal agents for intracellular tracking and drug delivery systems in vitro and in vivo14,15,16. Although the therapeutic application of QDs is often a concern because of their potential toxicity at high concentrations, recent advances in QDs surface engineering and bioconjugation strategies have shown promising results in mitigating these concerns and expanding their therapeutic potential17. Owing to their stable chemical properties and large surface area, QDs are potential carriers for loading small RNAs, peptides, and proteins18. QDs can potentially promote the efficient delivery of small RNAs into target cells and track the distribution of small RNAs in vitro and in vivo19. However, the application of QDs as small RNAs carriers for regulating macrophage polarization has not yet been elucidated. In this study, we used carboxyl-QDs coated with polyethyleneimine (PEI) and conjugated to miR-10a (QDP/miR-10a) to regulate macrophage polarization. We demonstrate that QDP/miR-10a efficiently promotes macrophages M1-to-M2 conversion without causing cytotoxicity in vitro and in vivo. QDP/miR-10a, a driver of M1-to-M2 conversion in macrophage polarity, restored impaired phenotypes in the endometrium of a mouse model of Asherman’s syndrome (AS) characterized by sterile uterine inflammation with fibrosis. For the first time, we suggest that QDP is a biocompatible nanocarrier with dual modes for delivering and tracking small RNA to modulate macrophage polarization for treating various inflammatory diseases, including AS.

Results

Carboxyl-QDs modified with polyethyleneimine (QDP) is an efficient delivery carrier of small-size nucleotides to macrophages

In this study, we used three distinct types of QDs, each functionalized with a different surface charge. ZnS-coated CdSe QDs (CdSe/ZnS) were conjugated with amine groups (amine-QDs, A-QDs), carboxyl groups (carboxyl-QDs, C-QDs), and polyethylene glycol (PEG-QDs, P-QDs). These A-QDs, C-QDs, and P-QDs were 15 nm in size with the expected charges (Fig. S1, Supporting Information). When three types of QDs were treated to RAW 264.7 cells, a mouse macrophage cell line, at various concentrations up to 40 nM, P-QDs (94.79–98.75%) and C-QDs (95.04–98.14%), but not A-QDs (<10.72%), were effectively internalized (Fig. S2A–C, Supporting Information). None of the three QDs caused cytotoxic effects even at a concentration of 40 nM, the highest concentration (Fig. S2D–F, Supporting Information). Since carboxyl groups electrostatically bind to PEI, QDP was used to deliver nucleotides to RAW 264.7 cells (Fig. 1A). Dynamic light scattering (DLS) and scanning electron microscopy analyses showed that QDP and QDP/fluorescein amidite-labeled small RNA (FAM-small RNA) become positively charged and larger (79.0 and 115.5 nm, respectively) than C-QDs (Fig. 1B–D). The gel retardation assay showed that QDPs were efficiently conjugated with FAM-small RNA from 5 nM, the lowest concentration tested (Fig. 1E). To optimize the small RNAs delivery efficiency, we extended the QDP/FAM-small RNA concentration range up to 120 nM. QDP/FAM-small RNA did not affect the cell viability of RAW 264.7 cells, even at 120 nM (Fig. 1F), indicating that increasing the C-QDs concentration improved the small RNA delivery efficiency while maintaining cell viability.

Fig. 1: Characterization of QDs complex for small RNAs delivery in the RAW 264.7 cells.
figure 1

A A schematic diagram shows that carboxyl-QDs (C-QDs) were initially modified with polyethyleneimine (PEI), and the C-QDs/PEI (QDP) was conjugated with small RNAs to synthesize the QDs. Size distribution and morphology of assessed QDP/small RNAs using dynamic light scattering (B) and scanning electron microscopy (C). n = 3 per group. Scale bar: 500 nm. The schematic diagram was created with BioRender.com. D Surface charge changes in the zeta-potential of various QD complexes. E Agarose gel electrophoresis retardation assay of QDP/small RNA. Ten pmol FAM-small RNA were used in this experiment. The white arrow indicates complete retardation of the nucleotide at 5 nM. F In vitro cytotoxicity of QDP/FAM-small RNA as assessed using the CCK-8 assay in RAW 264.7 cells at 24 h after treatment. n = 3 per group. Each bar represents the mean ± SD of three independent experiments. Statistical analyses were performed using a t-test. G The fluorescence-activated cell sorting (FACS) histograms represent the delivery efficiency of the QDP/FAM-small RNA at 80 and 120 nM at 24 h after transfection. H The graph shows the average delivery efficiency of FAM-small RNA at various concentrations of C-QDs. Each bar represents the mean ± SD of three independent experiments. I FACS histograms and J transfection efficiency comparison of QDP and commercial reagent (CR, Lipofectamine 3000) in RAW264.7 cells, 24 h post-transfection. n = 3 per group. K Fluorescence images show uptake efficiency with boxed area magnified. Nucleus (Blue), QDP (Red), FAM-small RNA (Green). The scale bar represents 75 μm. L Western blotting verified the silencing efficiency of the QDP/small RNA for GAPDH at 24 h after transfection in the RAW 264.7 cell. α-Tubulin was used as a loading control. The expression of the GAPDH protein was normalized to α-Tubulin. Each bar represents the mean ± SD of three independent experiments. Statistical analyses were performed using a t-test. *p < 0.05.

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Macrophages are known to have low transfection efficiency. Fluorescence-activated cell sorting (FACS) analyses showed that the percentage of cells positive for FAM-small RNA 24 h after transfection with commercial transfection reagent (CR) was approximately 90% in 293 T cells but less than 50% in RAW 264.7 cells (Fig. S3A, B, Supporting Information). Accordingly, the silencing efficiency of small interfering RNA (siRNA) for GAPDH at the protein level was much lower in RAW 264.7 cells compared to 293 T cells (Fig. S3C, Supporting Information). When QDP was used, FAM-small RNA were successfully delivered to RAW 264.7 cells, with higher efficiencies at 80 and 120 nM (Fig. 1G, H). When QDP/FAM-small RNA at 80 nM were delivered to RAW 264.7 cells, they had two times higher delivery efficiency than CR in RAW 264.7 cells (Fig. 1I–K). To evaluate the silencing efficiency of QDP-delivered small RNA, siGAPDH targeting GAPDH, a housekeeping gene that is stably expressed in most cells, was assessed. The silencing efficiency of QDP/siGAPDH was also significantly higher than that of the CR (Fig. 1L).

QDP/small RNA are internalized via clathrin and caveolin-mediated endocytosis into macrophages

Exposure to an ice-cold medium at 4 °C is a standard method for inducing nonspecific inhibition of cell endocytosis20. When RAW 264.7 cells were exposed to an ice-cold medium at 4 °C, the uptake efficiency of C-QDs and QDP/FAM-small RNA significantly decreased (Fig. 2A, B), suggesting that the uptake of C-QDs and QDP/FAM-small RNA depends on endocytosis. RAW 264.7 cells were treated with several pharmacological endocytosis inhibitors to identify the specific pathways involved in C-QDs and QDP/FAM-small RNA uptake. Whereas Cytochalasin D (an inhibitor of both phagocytosis and macropinocytosis) and amiloride (a macropinocytosis inhibitor) did not interrupt their internalization, CPZ and Dynasore, inhibitors for clathrin- and clathrin/caveolin-mediated endocytosis, effectively prevented it. Interestingly, MβCD (a caveolin-mediated endocytosis inhibitor) significantly decreased the uptake efficiency of QDP/FAM-small RNA but not C-QDs (Fig. 2C, D).

Fig. 2: Cellular uptake mechanisms of QDP/small RNA.
figure 2

A, B FACS histograms represent the uptake efficiency of C-QDs and QDP/FAM-small RNA at 37 °C and 4 °C incubation. The graph shows the average uptake efficiency of C-QDs at both temperatures. C, D FACS histograms represent the uptake efficiency of C-QDs and QDP/FAM small RNA in the presence of various endocytosis inhibitors. The graph shows the average uptake efficiency of C-QDs in the presence of various inhibitors. Each bar represents the mean ± SD of three independent experiments. Statistical analyses were performed using a t-test. *p < 0.05, **p < 0.01. E Confocal images represent immunofluorescence staining for EEA1 (early endosome marker) and LAMP-1 (late endosome marker) in QDP/FAM-small RNA-treated cells. White arrowheads indicate the co-localization of the QDP and endosomes. Yellow arrowheads indicate the QDP escaped from the endosomes. Green arrowheads indicate free FAM-small RNA released into the cytoplasm. Nucleus (Blue), QD (Red), FAM-small RNA (Green), Endosome (Magenta). The scale bar represents 10 μm.

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To track the intracellular distribution of QDP/FAM-small RNA after internalization, immunofluorescence staining was performed for early endosome antigen 1 (EEA1) and lysosome-associated membrane protein (LAMP1), which are early and late endosome markers, respectively (Fig. 2E). QDP/FAM- small RNA were observed in early endosomes within 5 min, escaped mostly at 10 min and entirely at 30 min, and were observed in late endosomes (LAMP1) at 2 and 4 h. The signals of FAM-small RNA free from QDP escaped from the late endosomes and were released into the cytoplasm at 4 h. These results suggest that QDP/small RNAs are internalized via clathrin- and/or caveolin-mediated endocytosis and that the delivered small RNA efficiently escape from the endosome in macrophages.

QDP/miR-10a efficiently promotes M1-to-M2 conversion by suppressing NF-kB, MAPK, and AKT pathways in macrophages

We then examined whether QDP/miR-10a contributes to the plasticity of macrophage polarization in vitro (Fig. 3A). Whereas LPS significantly increased the mRNA levels of M1 markers such as inducible nitric oxide synthase (iNos) and suppressor of cytokine signaling 3 (Socs3) in macrophages, QDP/miR-10a pretreatment effectively repressed them. Furthermore, QDP/miR-10a significantly increased the mRNA levels of M2 markers such as arginase 1 (Arg1) and mannose receptor C-type 1 (Mrc1) in macrophages exposed to LPS, suggesting that QDP-delivered miR-10a effectively promoted M1-to-M2 conversion.

Fig. 3: Effect of miR-10a delivered by QDP in modulating macrophage polarity by suppressing LPS-induced NF-κB, MAPK, and AKT signaling pathways.
figure 3

A The relative mRNA levels of M1 (iNos, Socs3) and M2 (Arg1, Mrc1) markers were analyzed by real-time RT-PCR in the RAW264.7 cells. Statistical analyses were performed using a t-test *p < 0.05 vs. LPS, **p < 0.01 vs. LPS, #p < 0.05 vs. Naïve. B Western blotting analyses for the effects of QDP/miR-10a for the suppression of NF-κB, MAPK, and AKT signaling pathways that were induced activation by LPS in the RAW264.7 cells. α-Tubulin was used as a loading control.

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To investigate how QDP/miR-10a modulates macrophage polarity, LPS-induced major signaling pathways were examined in macrophages (Fig. 3B). QDP/miR-10a pretreatment effectively reduced LPS-induced phosphorylation of NF-κB, the extracellular signal-regulated kinase (ERK1/2), and p38. Furthermore, LPS-induced AKT phosphorylation was also significantly attenuated by QDP/miR-10a. Accordingly, Western blotting for the M1 markers cyclooxygenase-2 (COX2) and iNOS showed that QDP/miR-10a significantly reduced the expression levels of these inflammatory mediators in M1 macrophages stimulated with LPS.

Intravenous delivery of QDP/miR-10a recovers balanced macrophage polarity in the impaired uterus in mice without systemic toxicity

We then examined the safety and therapeutic potential of intravenous (i.v.) delivery of QDP/miR-10a in mice with AS (Fig. 4A). As shown in our previous studies, AS was experimentally induced in the uterus by physical insults to mimic the clinical phenotypes of patients with AS7. Intravenous delivery of QDP/miR-10a did not cause systemic toxicity (Fig. 4B), as evaluated by blood chemistry tests. Furthermore, when immune cell profiles were examined after QDP/miR-10a delivery, all hematological parameters were comparable between the PBS and QDP/miR-10a groups (Fig. 4C). QDP/miR-10a treatment effectively decreased the M1/M2 macrophage ratio by increasing the M2 macrophages population 24, 48, and 72 h after QDP/miR-10a treatment (Figs. 4D and S4, Supporting Information).

Fig. 4: QDP/miR10-a induces polarization of M2 macrophages without general toxicity after injection of QDP/miR10-a in vivo.
figure 4

A A schematic diagram illustrating the experimental procedures to examine the polarization of M2 macrophages after injection of QDP/miR-10a in mice with Asherman’s syndrome (AS). The schematic diagram was created with BioRender.com. B Biochemical assays of serum 24 h after QDP/miR10-a treatment. C Cell counts of various immune cells in whole blood 24 h after QDP/miR10-a treatment. D Bar graph of FACS data showing the population of M0, M1, and M2 macrophages at different time points. Macrophages were labeled with M0 (F4/80+CD80−CD206−), M1 (F4/80+CD80+), and M2 (F4/80+CD206+) markers for FACS at 24, 48, and 72 h after QDP/miR10-a treatment.

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QDP/miR-10a targeting imbalanced macrophage polarity improves impaired uterine phenotypes of mice with AS

We then examined whether QDP/miR-10a-induced rebalancing of biased macrophage polarity alleviated the impaired uterine phenotypes in mice with AS (Fig. 5A). Real-time RT-PCR revealed that QDP/miR-10a not only significantly decreased the mRNA levels of M1 macrophages markers, such as iNos and Socs3, but also increased those of M2 markers, such as Arg1 and Mrc1, in the uteri of mice with AS (Fig. 5B). Furthermore, QDP/miR-10a decreased the mRNA expression of fibrosis markers, such as collagen type I alpha 1 chain (Col1a1) and collagen type III alpha 1 chain (Col3a1), and increased the mRNA expression of major angiogenic factors, including hypoxia-inducible factor-1α (Hif-1α) and angiopoietin 1 (Ang1) (Fig. 5C, D). Immunofluorescence staining for COL1A1 reinforced the effective reduction of fibrosis by QDP/miR-10a (Fig. 5E). Furthermore, co-immunofluorescence staining of KI-67 (a cell cycle marker) and CD31 (an endothelial marker) showed that QDP/miR-10a promoted angiogenesis in the impaired uterus for effective tissue regeneration in AS uterine horns (Fig. 5E, F).

Fig. 5: QDP/miR-10a promotes M2 macrophage polarization to enhance uterine regeneration in Asherman’s syndrome mice.
figure 5

A A schematic diagram illustrating the experimental procedures to examine the therapeutic effects of QDP/miR10-a for uterine regeneration in mice with AS. Uterine tissues were collected 14 days after intravenous injection. n = 4 or 5 mice in each group. The schematic diagram was created with BioRender.com. B Relative mRNA levels of M1 (iNos, Socs3) and M2 (Arg1, Mrc1) markers were analyzed by real-time RT-PCR in the uterus of mice with AS after injection of QDP/miR10-a. C, D Relative mRNA levels of fibrosis-related genes (Col1a1, Col13a1) and angiogenesis-related genes (Hif-1α, Ang1) were analyzed by real-time RT-PCR in the uterus of mice with AS after injection of QDP/miR10-a. E Histological analyses of uterine tissue from AS mice and AS mice treated with QDP/miR-10a, using hematoxylin and eosin (HE) staining. Immunofluorescence staining for COL1A1 in uterine sections of mice with AS after injection of QDP/miR-10a. Green and red colors indicate COL1A1 and nucleus, respectively. Co-immunofluorescence staining for CD31 and KI-67 in mice with AS after injection QDP/miR10-a. Blue, green, and red colors indicate CD31, KI-67, and nucleus, respectively. Yellow arrows indicate KI-67 positive nuclei (cyan color) in endothelial cells. Scale bar, 100 μm. F Graphs depicting the percentage of KI-67 positive cells/CD31 positive cells. Statistical analyses were performed using a t-test. *p < 0.01.

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Discussion

QDs offer potentially invaluable benefits such as drug delivery and in vivo biomedical imaging. However, QDs can harm human health and the environment under certain conditions. Several studies have reported on the potential toxicity of QDs. ZnS and CdS QDs have low to moderate toxicity, as shown by an increase in catalase activity and lipid peroxidation levels in zebrafish21. Human umbilical vein endothelial cells proliferate slowly upon exposure to CdTe/ZnS QDs22. Graphene QDs significantly decrease mouse oocyte maturation rates23, although they switched macrophage polarization from M1 to M2, attenuating inflammatory diseases, including inflammatory bowel disease (IBD)24. They suggested that the potential toxicity of QDs and their derivatives should be considered before their application in biological systems. Therefore, it is critical to mention that the application of QDP/small RNAs in this study effectively modulated macrophage polarization without cytotoxicity in vitro (Fig. 1F) and in vivo (Fig. 4). Furthermore, intravenous delivery of QDP/miR-10a stably regulated macrophage polarity even 72 h after treatment without systemic cytotoxicity, suggesting the biocompatibility of our QDP/miR-10a in alleviating inflammatory phenotypes in the impaired uterus with AS in mice. Our previous study demonstrated that improving the uterine microenvironment using human perivascular stem cells (hPVSCs) restored implantation rates and mitigated intrauterine growth restriction in mice with AS, emphasizing the potential of fibrosis reduction to enhance fertility25. Similarly, QDP/miR-10a may support reproductive recovery by restoring uterine function. Future long-term evaluations will focus on monitoring sustained fibrosis reduction, successful embryo implantation, and the number of live pups born to comprehensively assess the restoration of reproductive function. The size, charge, concentration, outer coating bioactivity (capping materials and functional groups), and biochemical stability determine QDs toxicity. The smallest QDs exhibited the highest in vitro cytotoxicity. A low negative surface charge is characteristic of most toxic QDs, whereas positively charged QDs are less cytotoxic26. In this study, the size of the C-QDs increased by approximately 79 nm after coating with PEI, and the surface charge became positive in the QDP. These changes render our QDP less toxic and more suitable for conjugating with small RNAs. Previously, Wang et al. and Lin et al. reported that exposure of C-QDs to RAW 264.7 cells for 24 or 48 h resulted in cytotoxicity, even at low concentrations such as 1.25 and 2.5 nM, suggesting the potential toxicity of C-QDs27,28. However, we did not find cytotoxicity even when RAW 264.7 cells were treated with C-QDs and QDP at 80 nM for 4 h (Fig. 1). Furthermore, when QDP/miR-10a was intravenously given to mice with AS daily for a week at a high concentration of 800 nM, the immune cell profiles and blood biochemical parameters were comparable to those of the control mice (Fig. 4). This discrepancy highlights the importance of the exposure time to QDs and QDs modifiers. In this study, RAW264.7 cells were treated with C-QDs, QDP, and QDP/small RNAs for 4 h, and in vivo delivery of QDP/miR-10a had 24 h intervals. This underscores the importance of both the concentration and exposure time when evaluating the safety and biocompatibility of QDP for therapeutic applications.

NPs are internalized through different uptake pathways depending on the target cell and particle type29. Understanding these pathways is crucial to the design of nanoparticle-based RNA delivery systems, as targeting specific endocytosis routes can enhance delivery efficiency and reduce off-target effects. Previous studies have shown that QDs are internalized via clathrin- and caveolin-mediated endocytosis, with pathways influenced by physicochemical properties such as size, charge, and surface modifications30. We hypothesize that the uptake of our QDs via these pathways is driven by their surface properties. Although the uptake mechanisms of QDs have been described in various cell types20, they need to be further studied in macrophages. We observed that the negatively charged C-QDs, approximately 15 nm in size, were mainly internalized through clathrin-mediated endocytosis (Fig. 2C). Positively charged QDP/FAM-small RNA with increased sizes utilized both clathrin- and caveolin-mediated endocytosis (Fig. 2). Clathrin- and caveolae-dependent endocytosis are common routes for NP internalization. Consistent with the results of this study, other studies have reported that clathrin-dependent endocytosis shifts to caveolin-mediated endocytosis as the size increases31. In addition to the size, the charge of NPs significantly influences their internalization pathways32. While positively charged NPs are predominantly internalized via caveolin-mediated endocytosis, negatively charged NPs exhibit greater internalization via clathrin-mediated endocytosis29. We designed a size-controlled liposome to target macrophages based on the idea that macrophages have a unique biological activity to engulf large particles, called phagocytosis, which others do not possess. Accordingly, phagocytosis inhibitors effectively inhibited the internalization of our macrophages-targeting liposomes7. Hence, the uptake pathways of QDs and their modifiers are contingent on their size and surface charge.

QDP/miR-10a was efficiently internalized into macrophages, inhibiting M1 features and promoting M2 in vivo and in vitro (Figs. 3–5). Previous studies reported that miR-10a inhibits the pro-inflammatory NF-κB pathway and promotes M2 macrophage polarization33. We also demonstrated that macrophage-specific targeting of miR-10a with size-controlled liposomes promoted M2 macrophage polarization, which resolved inflammatory responses in the uteri of mice with AS7. In addition to miR-10a, other miRNAs play essential roles in modulating macrophage polarity. miR‑182 and miR‑146a in macrophages could directly inhibit the expression of TLR4, leading to NF‑κB inactivation to allow M2 polarization34. Our result showed that QDP/miR-10a inhibits NF-κB, ERK1/2, p38, and AKT signaling pathways in LPS-treated RAW 264.7 cells. It suggests that QDP can be used as a biocompatible carrier of small RNAs such as miR‑182 and miR‑146a to convert M1-biased macrophages to alleviate phenotypes of various inflammatory diseases, such as IBD and rheumatoid arthritis (RA) in vivo. In a mouse model of IBD, graphene QDs inhibited Th1/Th17 polarization and promoted M2 macrophage polarization, mitigating intestinal inflammation24. Polymers and liposomes encapsulating anti-inflammatory factors such as roburic acid and triptolide were used to reprogram M1-biased macrophages to reduce inflammation and support wound healing with reduced toxicity in rats with RA35,36. However, QDs-based therapeutic approaches targeting the dysregulated macrophage polarity have not yet been applied to these inflammatory diseases. Considering that QDP can be easily conjugated with various types of small RNAs, its therapeutic potential as a small RNAs carrier can be expanded to alleviate the pathological phenotypes in a broad range of inflammatory diseases. Collectively, our results suggest that QDP provides a higher transfection efficiency of small RNAs to macrophages, and it is a biocompatible small RNAs nanocarrier with dual modes to modulate macrophage polarization for treating inflammatory diseases such as AS in vivo.

In conclusion, our study demonstrates that QDP is an effective and biocompatible nanocarrier for delivering small RNA and modulating macrophage polarization. QDP/miR-10a shows potential as a safe therapeutic complex for treating inflammatory diseases, including AS, without causing adverse immune responses or systemic toxicity (Fig. 6). These findings suggest the broad applicability of QDP in treating various inflammatory conditions. Collectively, our results indicate that QDP provides a higher transfection efficiency of small RNA to macrophages, and it is a biocompatible small RNA nanocarrier with dual modes to modulate macrophage polarization for treating inflammatory diseases such as AS in vivo. Further studies are needed to evaluate the long-term safety and efficacy of this approach, as well as to explore its translational potential in clinical settings.

Fig. 6: A schematic illustration showing how QDP/miR-10a treatment in a mouse model of Asherman’s syndrome successfully shifts M1 macrophages to an M2 phenotype, leading to the alleviation of uterine fibrosis and inflammation, and promoting uterine tissue regeneration.
figure 6

The schematic illustration was created with BioRender.com.

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Methods

Materials

The branched PEI (MW 25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Functionalized QDs with amine (Amine-QDs; Ocean nanotech, San Diego, CA, USA), carboxyl (Carboxyl-QDs; Invitrogen, Carlsbad, CA, USA), and polyethylene glycol (PEG-QDs; Ocean nanotech) were purchased and used in experiments with DMEM high-glucose media (Hyclone, Logan, UT, USA).

Preparation and characterization of QDP

To prepare the PEI-modified QDs, carboxyl-QDs (5–120 nM) and 1 mL of PEI (2 mg/mL) were mixed in a tube. The carboxyl-QDs/PEI (QDP) solution was incubated on a shaker for 3 h at room temperature and then purified by centrifugation at 13,000 rpm for 10 min. After centrifugation, the supernatant containing the free PEI was gently removed to avoid disturbing the QDP pellet. The pellet was then washed with 1 mL of deionized water and centrifuged three times, followed by washing. After the last washing, the pellet of QDP was dispersed in 100 µL of deionized water. To fabricate the QDP complex, 10 pmol of small RNAs (siRNA and miRNA) were added to various concentrations of QDP for 15 min. Variously modified QDs were suspended in 600 µL deionized water, and the size distribution and surface charge of variously modified QDs were measured by DLS (Malvern ZetaSizer Nano ZS, Malvern, UK). An agarose gel electrophoresis retardation assay of the QDP and QDP/small RNAs was performed on a 0.5% agarose gel. The QD complex samples (20 µL/lane) were run for 30 min.

Cell culture

Human embryonic kidney cells (HEK293T) and murine macrophages (RAW 264.7) were used for all experiments. Cells were maintained in DMEM media containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 1% penicillin, and streptomycin (1% P/S; Hyclone) in a 5% CO2 atmosphere at 37 °C incubator.

Treatment of QDs complex on RAW 264.7 cells

One day before treatment, the cells were seeded at a density of 1 × 105 cells/well in 24-well plates in DMEM culture media. The following day, the DMEM was removed, and the cells were washed three times with PBS. Various concentrations of QDs complexes were prepared in 1 mL of serum-free DMEM (SF-DMEM), and then cells were treated with the QDs complexes for 4 h. After 4 h of treatment, the uptake efficiency of the QDs complexes was determined using fluorescence microscopy (Carl Zeiss, Oberkochen, Germany) and/or measured by FACS.

Cell transfection

RAW 264.7 and 293 T cells were transfected with small RNAs. Small RNAs were transfected into cells using Lipofectamine 3000 (Invitrogen) for 4 h. One day before transfection, cells were seeded at a density of 2 × 105 cells/well in 12-well plates in DMEM.

Cell viability

Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Donginbiotech, Seoul, Korea). Cells were seeded at a density of 2.5 × 105 cells/well to evaluate cytotoxicity in 12-well plates. The QDs were incubated with cells for 4 h at various concentrations. After 24 h, the cells were washed with DPBS and then incubated for 3 h at 37 °C in serum-free media containing 10% CCK-8 solution. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

RNA preparation, reverse transcription-PCR (RT-PCR), and real-time RT-PCR

Total RNA was extracted from the RAW 264.7 cells and uterine tissues using Trizol Reagent (Ambion, Carlsbad, CA, USA) according to the manufacturer’s protocols. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA) with random primers and oligo dT for cDNA synthesis. The synthesized cDNA was used for PCR with specific gene primers at appropriate cycles and annealing temperatures. The PCR products were subjected to electrophoresis. Real-time RT-PCR was performed to quantify expression levels using the fluorescence of SYBR Green Dye (Bio-Rad, Waltham, USA). To compare transcript levels between samples, a standard curve of cycle thresholds from several serial dilutions of a cDNA sample was established and then used to calculate the relative abundance of each gene. The values were then normalized to the relative amounts of rPL7 cDNA. All PCR reactions were performed in duplicate.

Western blotting

RAW 264.7 and 293 T cells were lysed in a lysis buffer containing PRO-PREP (iNtRON, Seongnam, Korea) solution and 1× phosphatase inhibitor (Roche Applied Science, Indianapolis, IN, USA). The protein samples (10 µg/lane) were then separated by 8–15% SDS-PAGE, transferred onto a nitrocellulose membrane (Bio-Rad, Waltham, MA, USA), and blocked with 5% skim milk (Bio-Rad) in TBS (Bio-Rad) containing 0.1% Tween 20 (Sigma-Aldrich). After blocking, membranes were subjected to Western blotting with appropriate primary antibody overnight at 4 °C. The following day, the membranes were incubated with a secondary antibody in 5% skim milk for 1 h at room temperature. The signals were developed using an ECL Western blotting substrate kit (Bio-Rad) and detected using ChemiDoc XRS+ (Bio-Rad) system with Image Lab software.

Fluorescence-activated cell sorting (FACS)

Cells were washed with PBS and incubated in Accutase (Millipore, Burlington, MA, USA) for 5 min at 37 °C. Following the incubation, the cells were detached and centrifuged at 6000 rpm for 3 min at 4 °C. The supernatant was removed, and the cell pellet was resuspended in 0.1% BSA in 1× DPBS by three rounds. FACS was performed using the Beckman Coulter Flow Cytometer and CyExpert software (Beckman Coulter, Brea, CA, USA). Data shown represent the mean fluorescence signals from 10,000 cells.

Experimentally induced murine model of AS

All mice were housed following the institutional guidelines for laboratory animals (Animal Care Facility of CHA University). This study was approved by the Institutional Animal Care and Use Committee (IACUC, Approval Number: IACUC230161). As previously reported7, 8-week-old female ICR mice (Orient Bio, Gapyeong, Gyeonggi, Korea) were used to create a mouse model of AS. A vertical incision was made in the abdominal wall following intraperitoneal injection of 240 mg/kg avertin to expose the uterus. At the utero-tubal junction, a small incision was made in each uterine horn, and each horn was traumatized in a standardized manner using a 27 G needle (Korea vaccine, Ansan, Korea) entering through the lumen, rotated, and withdrawn 10 times. All mice were euthanized using cervical dislocation, and tissues were collected immediately after euthanasia.

Isolation of uterine cells

Uteri were dissected, minced into small pieces, and incubated in HBSS (GIBCO, Basel, Switzerland) containing dispase (2.4 U/mL) and pancreatin (25 mg/mL) for 1 h at 4 °C followed by 1 h at room temperature. Tissues were incubated at 37 °C for 10 min, and then the supernatant (the epithelial cell-rich fraction) was collected. The remaining stromal cell-rich pellet was then digested with collagenase (0.5 mg/mL) after filtration through a 70 μm nylon mesh. The cells were incubated with fluorochrome-conjugated antibodies for 30 min at room temperature. FACS was used to examine the cells after rinsing with washing buffer.

Immunofluorescence staining

Uterine tissues were fixed in 4% paraformaldehyde for histological analysis. The fixed tissues were washed, dehydrated, and embedded in paraffin (Leica Biosystems, Wetzlar, Germany). Paraffin-embedded tissues were sectioned at a thickness of 5 μm using a microtome. Uterine sections were deparaffinized and rehydrated. Sections were subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0). Nonspecific staining was blocked using protein block serum (Dako, Carpinteria, CA, USA) for 1 h. Sections were incubated with primary antibodies at 4 °C overnight. The following day, the sections were incubated with the appropriate secondary antibodies for 1 h at room temperature. The sections were counterstained with DAPI (1:1000, Thermo, Rockford, IL, USA) or Topro-3-iodide (Life Technologies, Carlsbad, CA, USA) and mounted. Images were captured under a microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss).

Inhibition of endocytosis

To investigate endocytosis-mediated internalization pathways of C-QDs and QDP/FAM-small RNA, RAW 264.7 cells were placed in an ice-cold medium (0–4 °C) and incubated at 4 °C for 4 h. To inhibit the endocytosis pathway, five different pharmacological inhibitors were used: 20 µM CytoD (an inhibitor of phagocytosis; Sigma-Aldrich), 50 µM Amiloride (an inhibitor of macropinocytosis; Sigma-Aldrich), 10 µM CPZ (an inhibitor for clathrin-coated vesicles; Sigma-Aldrich), 100 mM MβCD (an inhibitor for the formation of caveolin-coated vesicles; Sigma-Aldrich), and 50 µM Dynasore (an inhibitor for the scission of clathrin- and caveolin-coated vesicles; Sigma-Aldrich). RAW 264.7 cells were pretreated with various pharmacological inhibitors to block specific endocytosis pathways. The cells were pretreated with the inhibitors for 1 h and then incubated with carboxyl-QDs and QDP/FAM-small RNA for 4 h in the presence of inhibitors. After treatment, the uptake efficiencies of carboxyl-QDs and QDP were measured using FACS.

Evaluation of immune cell profiles and general toxicity after intravenous delivery of QDP/miR-10a

Whole blood was withdrawn 24 h after the intravenous delivery of saline or QDP/miR-10a into mice. As previously described7, immune cell profiles and general toxicity were immediately evaluated in these blood samples.

Statistical analysis

All values represent the means ± SD. The analysis was used for the Student’s t test for statistical evaluation. A p value of less than 0.05 was considered statistically significant. GraphPad Prism version 8 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses.