Nanoceria as a non-steroidal anti-inflammatory drug for endometriosis theranostics

The levels of phospho-STAT3 (pSTAT3) were significantly higher in epithelial cells of the eutopic endometrium from women with endometriosis compared to controls [ 23 ]. Using a non-human primate model [ 43 ], we observed aberrant overexpression of pSTAT3 in the eutopic endometrium from the same baboons over time, after endometriosis induction [ 23 ]. To delve deeper into the dysregulation of pSTAT3 following endometriosis, we surgically induced endometriosis in mice [ 44 ]. pSTAT3 levels ( p < 0.001) were significantly higher in the eutopic endometrium of mice with endometriosis compared to the sham group ( Fig. 1 ). This result suggests that abnormal activation of STAT3 may play a crucial role in the pathogenesis of endometriosis. To identify STAT3 targets in the uterus, we performed transcriptomic analysis in uterine-specific Stat3 knock-out ( Pgr cre /+ Stat3 f/f ; Stat3 d/d ) mice [ 25 ] at gestational day (GD) 3.5. Differential expression analysis revealed that a total of 1,382 genes (931 increased, 451 decreased) were significantly dysregulated ( p <0.05) due to the Stat3 loss ( Table S1 , Supporting Information ). The canonical pathways linked to the dysregulated genes analyzed by the Ingenuity Pathway Analysis (IPA) ( Table S2 , Supporting Information ). Our pathway analysis revealed that the up-regulated pathways included interferon, macrophage, natural killer cell, and T and B cell signaling ( Fig. 2A ). Given that STAT3 plays a pivotal role in immunosuppression [ 23 , 25 ], we investigated the inflammatory and immune-related pathways. Further, RT-qPCR validated the different differentially expressed genes from the transcriptomic data coding for interferon signaling ( Stat1, Ifi35, Ifit3, Irf9, Psmb8, Tpi1, Jak2, and Adamts4 ), role of macrophages ( Adamts4, Ccl5, Ccnd1, Il15, Il18, Mmp3, Tlr1, Tnfsf13b, Traf1, Wnt5b, Stat3, Tlr3, Tlr4, Fcgr1a, Camk2d, and Nfatc1 ), B cell receptor signaling ( Camk2d, Nfatc1, Mapk8, Ptprc, Vav1, Ptpn6, Rac2, Lyn, and Fcgr2b ), T cell receptor signaling ( Nfatc1, Mapk8, Ptprc, Vav1, Lck, and Lcp2 ), and natural killer (NK) cell signaling ( Vav1, Lck, Lcp2, Ptpn6, and Rac2 ) ( Fig. 2B ). Overall, these data suggest that endometriosis induces aberrant activation of STAT3 that results in inflammatory condition and immune dysfunction. The endometrial immune cell population, e . g ., macrophages, regulatory T cells, and natural killer, exhibited an aberrant activity in endometriosis versus healthy control [ 12 , 13 ]. Therefore, we first assessed uterine macrophages in control and Stat3 d/d mice by immunostaining F4/80 and CD11b, the two most common cell surface markers for macrophages/monocytes in mice [ 45 , 46 ]. Although both F4/80 and CD11b positive cells have been observed in the control Stat3 f/f uterus, their expressions were significantly higher in Stat3 d/d mice compared to control mice ( p <0.01 and p <0.05, respectively; Fig. 3A ). We additionally examined the presence of markers indicative of M1 (iNOS) and M2 (ARG1) macrophages in the uteri of both Stat3 d/d and Stat3 f/f mice ( Fig. 3A ). Our results showed that the loss of Stat3 led to significantly increased levels of iNOS expression and a decrease in ARG1 expression compared to the control group ( p <0.0001) ( Fig. 3A ). T cells are also crucial for host immune functions in maintaining health and preventing disease [ 47 ]. It has been reported that mature T cells expressed either CD8 or CD4 glycoprotein on their surface [ 48 ]. Similar to macrophages, we observed that both T cell surface glycoproteins (CD4, CD8α) were highly expressed in Stat3 d/d uteri compared to their control counterpart ( p <0.05; Fig. 3B ). In contrast, the uterine expression of natural killer cell receptor (NKR) is downregulated in the Stat3 d/d mice compared to Stat3 f/f ( p <0.05; Fig. 3C ). These results suggest that endometriosis induces overexpression of pSTAT3 that causes inflammatory condition, and STAT3 targeting may activate innate immunity to inhibit the development of endometriosis. Tofacitinib, a widely used JAK inhibitor, has been proposed as a therapeutic choice for endometriosis due to its ability to block JAK/STAT activation [ 10 ]. We determined the effect of tofacitinib on the progression of endometriosis and implantation. Endometriosis was induced in 8-week-old female mice with dual fluorescence reports ( Pgr cre /+ Rosa26 mTmG /+ ) [ 44 ]. Following intraperitoneal (IP) injection of either vehicle or tofacitinib (10 mg/Kg body weight for four weeks), we evaluated the treatment efficacy on the development of endometriosis ( Fig. 4A ). We observed a significant reduction in the number of ectopic lesions in tofacitinib-treated mice compared to the vehicle group ( p <0.001, Fig. 4B ). Since STAT3 plays a critical role in implantation [ 25 ], we examined the effect of tofacitinib on early pregnancy in mice. Eight-week-old female mice were treated with vehicle or same dose of tofacitinib for four weeks and then the effect of tofacitinib was assessed at GD5.5 implantation stage ( Fig. 4C ). While the vehicle group mice had normal implantation, tofacitinib-treated female mice exhibited complete implantation failure ( Fig. 4D ). To elucidate the molecular basis of the implantation failure in tofacitinib-treated mice, we conducted the histological analysis of the uteri and assessed the expression of molecular markers related to implantation and decidualization ( e . g ., COX2, E-cadherin, and PGR) at GD5.5 [ 49 ]. In vehicle-treated mice, the implantation sites exhibited typical luminal closure ( Fig. 4E a and b ). We also observed abundant expression of COX2 and PGR in decidual cells but not E-cadherin in implantation sites ( Fig. 4E c – e ). In contrast, the endometrium from mice treated with tofacitinib displayed a fully open luminal epithelial layer ( Fig. 4E f and g ). Implantation failure of tofacitinib-treated mice was confirmed with absent COX2 expression as well as strong expression of PGR and E-cadherin in luminal epithelial cells ( Fig. 4E h – i ). These results suggest that tofacitinib results in implantation failure due to its systemic inhibitory effect on JAK/STAT signaling in the uterus. Nanoceria has been developed as a powerful non-steroidal anti-inflammatory drug (NSAID) due to its unique antioxidant and enzymatic properties [ 28 , 50 ]. We synthesized nanoceria by growing cerium oxide nanoclusters on albumin substrate via the biomineralization process. Albumin protein, used as a conventional substrate for nanosynthesis, has been explored to enhance the biocompatibility, blood circulation time, and targeting efficacy of NPs [ 28 , 51 ]. Since albumin is known to accumulate and retain at sites of inflammation [ 52 ], albumin-nanoceria can target the inflammatory ectopic lesions but not eutopic endometrium. The albumin-nanoceria were chemically post-conjugated to a near-infrared (NIR) fluorescent dye, indocyanine green (ICG) ( Fig. S5 ), to further allow for in vivo and ex vivo visualization of particle localization. The resulting albumin-nanoceria particles (nanoceria) were shown to contain small, highly crystalline cerium-oxide nanoparticles CeO 2 core ≈ 5 nm, together with protein shell ~ 25 nm by transmission electron microscope (TEM) ( Fig. 5A ) and shown to have a hydrodynamic diameter of 28.65 nm (PDI: 0.471) by dynamic light scattering (DLS) ( Fig. 5B ). The particles were shown to be stable in biological conditions over a week ( Fig. S6 ). The particles have a mixed surface oxygen valence containing both Ce 3+ and Ce 4+ at a ratio of 5.4: 4.6, as confirmed by X-ray photoelectron spectroscopy (XPS) ( Fig. 5C ), allowing them to scavenge reactive oxygen as demonstrated by DCF-DA assay ( p <0.001, Fig. 5D ). Resulting enzymatic activity was validated by superoxide dismutase (SOD) and catalase assays ( Fig. 5E ) demonstrating the characteristic multi-enzyme mimetic behavior of the nanoceria and their efficacy as an anti-inflammatory agent. In spite of their therapeutic effects in biomedical applications, nanoceria-based therapeutics to the clinic have been impaired due to potential cytotoxicity arising from the use of cerium oxide [ 53 , 54 ]. Off-target effects and cell damage by cerium oxide are major concerns. Fortunately, the use of an albumin substrate has been shown to mitigate the adverse effects of nanoceria on biological systems [ 28 , 55 ]. In our in vitro experiments, we observed that even at concentrations as high as 200 μg Ce/mL, our nanoceria does not induce cell death or inhibit proliferation ( Fig. S1A , Supporting Information ). Additionally, to further confirm the biocompatibility of our particles used in this study, we performed a blood chemistry experiment to validate normal organ function and levels of metabolites in mice treated with nanoceria. The panels included liver function, kidney function, and metabolic function. Notably, our results indicate no adverse effects of the particles on mice as there are no statistically significant differences in the blood markers detected in our controls (PBS and tofacitinib) and nanoceria groups ( Fig. S1B , Supporting Information ). These findings suggest that our nanoceria (albumin-nanoceria), unlike traditional nanoceria, may offer a safe and effective therapeutic option. Following the synthesis and characterization of the particles, their anti-inflammatory therapeutic efficacy needed to be evaluated in vitro. It is known that macrophages are abundant in endometriosis and that the pro-inflammatory M1 subtype dominates [ 12 ]. Modulation of macrophage phenotype away from M1 toward M2 would enhance treatment efficacy and reduce disease severity. Nanoceria with a mixed surface valence state (Ce 3+ /Ce 4+ ) have the ability to polarize macrophages [ 56 , 57 ]. To evaluate our nanoceria’s anti-inflammatory and immunomodulatory effects, we performed flow cytometry and cell sorting (FACS) to differentiate between macrophage subtypes by detecting surface markers. The FACS study allows visualization of entire populations of cells rapidly and, in our case, will enable us to compare macrophage cells treated with our nanoceria, STAT3 inhibitor (5,15-DPP) and tofacitinib, or inflammatory cytokines (LPS and IFN-γ). We identified pro-inflammatory M1-like cells using CD80 expression as a characteristic surface marker. We found that nanoceria and the inhibitors (5,15-DPP and tofacitinib) can treat pro-inflammatory states induced by LPS and IFN-γ and return J774 cells to a non-inflammatory state ( Fig. 6A ). The results of nanoceria treatment reducing the pro-inflammatory response in LPS/IFN-γ-treated J774 cells suggest that nanoceria inhibits inflammatory signaling via STAT-3 inactivation [ 33 , 34 ]. Notably, our nanoceria showed lower CD80 signal than either of the inhibitors. This indicates our nanodrug is an effective anti-inflammatory capable of treating the pro-inflammatory macrophages associated with endometriosis. To further validate immunomodulatory effects of nanoceria, immunofluorescence imaging was performed using CD80 and ARG1 as pro-inflammatory M1 and anti-inflammatory M2 markers, respectively. This experiment not only confirmed the surface marker expression observed with FACS but also allowed visualization of cell morphology after treatment. Our imaging data showed low expression of both CD80 and ARG1 in our undifferentiated control, high CD80 and low ARG1 in the pro-inflammatory LPS/IFN-γ control, and low CD80 and high ARG1 in our anti-inflammatory IL4/IL13 control, validating our ability to differentiate between phenotypes. When cells were then treated with our nanoceria, we saw that they had high ARG1 expression compared to 5,15 DPP-treated cells [ 58 ] and showed similar ARG1 expression to tofacitinib-treated cells, indicating the strong anti-inflammatory performance of our nanoceria as compared with traditional therapeutics. None of the three treatment conditions showed elevated expression of CD80 ( Fig. 6B ). Additionally, cells treated with nanoceria exhibited a notably rounded morphology, further confirming successful anti-inflammatory treatment. Given that our nanoceria are formulated by coating with an albumin protein shell they can be successfully internalized by phagocytic cells and well-accumulated at sites of angiogenic, vascular leakage of endometriosis [ 59 ]. Ectopic lesions appear disorganized, permeable, and have reduced lymphatic drainage, while endometrium within the uterus is histologically organized. In fact, the preferential accumulation of nanoparticles in ectopic lesions, compared to the uterus, has been reported [ 60 , 61 ]. We made ICG-conjugated nanoceria (albumin-nanoceria-ICG) to allow for their fluorescence and photoacoustic detection in the tissues. To evaluate targeting efficiency of nanoparticles in ectopic lesions, ICG-conjugated nanoceria was administered to mice with endometriosis via tail-vain injection, and in vivo photoacoustic imaging was performed at 3-hour post-injection. Strong photoacoustic contrasts from nanoceria (green) were detected from the ectopic lesions (arrowhead) and endogenous signals from oxyhemoglobin (red; an indicator of the blood flow) were overlaid in the ectopic lesions ( Fig. 7Aa ). The in vivo targeting of ectopic lesions and eutopic uterus was also further validated with fluorescence imaging of the GFP-positive endometrial cells from genetically engineered Pgr cre /+ Rosa26 mTmG /+ mouse by using fluorescence microscopy ( Fig. 7Ab ). Next, we investigated the biodistribution of the nanoparticles. After 24 hours of IP injection of nanoparticles in endometriosis mice, ectopic lesions, eutopic uterus, liver, spleen, and heart were collected and examined ICG signal from the tissues using optical imaging systems (IVIS and PEARL NIR imager). ICG signals from nanodrugs were strongly detected on the ectopic lesion and liver, while they scarcely were present in the eutopic uterine tissues ( Fig. 7B ). Liver accumulation is frequently seen in systemic administrations of all types of nanoparticles for many use cases [ 62 ]. When fluorescence intensities from the tissues were calculated, it was revealed 3-fold higher target accumulation in ectopic lesions compared to nonspecific uptake in the uterus ( p < 0.05; Fig. 7C ). The specific targeting was further validated through tissue sectioning and fluorescence microscopy imaging of the uterus and ectopic lesions, showing strong ICG signal in the ectopic lesions but not the uterus ( Fig. 7D ). The ectopic lesion-specific signal indicates the successful enrichment of the nanodrugs from systemic circulation. Therefore, our results demonstrated that the nanoceria exhibit strong therapeutic targeting in ectopic lesions without off-target effects in the uterus. Next, we evaluated the effect of nanoceria on endometriosis. Endometriosis was induced in female mice by autologous uterine tissue transplantation followed by four-week treatment (120 μg/kg body weight) with either vehicle or nanoceria ( Fig. 8A ). The number of ectopic lesions in nanoceria-treated mice was significantly smaller than in vehicle-treated mice ( p <0.01; Fig. 8B ). We then assessed the effect of nanoceria on early pregnancy. Female mice were injected with a vehicle or nanoceria (120 μg/kg body weight) for four weeks. After mating with fertile males, we examined implantation sites at GD 5.5 ( Fig. 8C ). Nanoceria did not affect the number of implantation sites ( Fig. 8D ). To further characterize the effects of nanoceria in early pregnancy, we examined histological and molecular analysis at implantation sites from mice treated with vehicle or nanoceria. Implantation sites from mice treated with both vehicle and nanoceria presented typical luminal epithelial closure and well differentiated decidual cells ( Fig. 8E ). In addition, both groups represent an identical strong expression of COX2 and PGR but lack of E-cadherin at implantation sites ( Fig. 8E ). Taken together, these results demonstrated that, unlike tofacitinib, nanoceria does not cause detrimental effects on early pregnancy including implantation and decidualization while effectively reducing development of endometriosis. Stat3 depletion affects the uterine immunological and inflammatory microenvironment, while endometriosis causes STAT3 activation. Given nanoceria’s anti-inflammatory and immunomodulatory properties, we determined whether its therapeutic actions relate to STAT3 signaling-targeted immune mobilization. The ectopic lesions developed in our mouse model of endometriosis exhibited distinct endometrial glands and stromal layers as confirmed by immunohistochemical analysis of vimentin and E-cadherin ( Fig. S2 , Supporting Information ). Nanoceria-treated mice showed lower levels of F4/80 and CD11b in ectopic lesions compared to mice treated with the vehicle ( p < 0.01; Fig. 9A ). Interestingly, nanoceria decreased the population of pro-inflammatory M1 macrophages (iNOS expression) while increasing the levels of the anti-inflammatory macrophages (ARG1 expression) ( p < 0.01 and p < 0.05, respectively; Fig. 9A ). Treatment with nanoceria also significantly reduced the expression of T cell surface glycoproteins (CD4 and CD8α) in ectopic lesions compared to the control group ( p < 0.001 and p < 0.01, respectively; Fig. 9B ). There were no changes in NKR expression in mice treated with nanoceria or vehicle ( Fig. 9C ). Notably, the immune cell population alterations in the ectopic lesions of mice treated with nanoceria are entirely opposite to those observed in the eutopic uteri of Stat3 d/d animals. To ascertain the direct relationship between STAT activation and the observed effects of nanoceria, we analyzed the expression of pSTAT3 in endometriosis mice treated with nanoceria compared to those treated with the vehicle. We observed that nanoceria-treated mice showed significantly lower epithelial pSTAT3 expression compared to vehicle group ( Fig. 10 ). These findings suggest that targeting STAT3 signaling with NP-based nanodrugs could be a promising future treatment for endometriosis.

Cerium nitrate hexahydrate (99.999% purity; catal. #202991), hydrogen peroxide (catal. #216763), bovine serum albumin (BSA, catal. #05470), Ultra Centrifugal Filters (catal. #UFC810008), 5,15 DPP (catal. # D4071), tofacitinib (catal. #PZ0017), 3-(4,5-Dimethylthiazolyl-2)-25-Diphenyl Tetrazolium Bromide (MTT) (catal. #102227), and estradiol (catal. #E8875) were purchased from Sigma-Aldrich Chemicals (Atlanta, GA, USA). Sodium Hydroxide (catal. #S318-500-500G), MINI Dialysis Devices, 10K MWCO (catal. #69570), paraformaldehyde, PRILLS (catal. #19202), eBioscience Flow Cytometry Staining Buffer (catal. #00-4222-57), NucBlue Live Cell Stain ReadyProbes Reagent (DAPI) (catal. # R37605 ), Dimethyl-Sulfoxide (catal. #022914.K2), MMLV Reverse Transcriptase (catal. #28025013), Triton X-100 (catal. #BP151-500), and SYBR Green Master Mix (catal. #A25742) were purchased from Thermo Fischer Scientific. ICG-NHS ester (catal. #POS1604) was purchased from DiagnoCine LLC (Hackensack, NJ). Superoxide dismutase (SOD; catal. #706002) and catalase assay kits (catal. #707002) were purchased from Cayman chemicals (Ann Arbor, MI). IL-4 (catal. #78045), IL-13 (catal. #78030), IFNγ (catal. #78020) were purchased from STEM CELLS Technologies (Cambridge, MA). APC anti-CD80 (catal. #104714), PE anti-CD206 (catal. #141706), and Zombie Green fixable viability dye (catal. #423112) were purchased from BioLegend (San Diego, California). RNeasy total RNA Isolation Kit (catal. #74106) was purchased from Qiagen, (Valencia, CA). DAPI mounting solution (catal. #H-1800), 10% normal goat serum (catal. #S-1000), anti-mouse secondary antibody (catal. #BA-9200), and anti-rabbit secondary antibody (catal. #BA-1000 were purchased from Vector Laboratories (Burlingame, CA). Horseradish peroxidase (catal. #43-4324) was purchased from Invitrogen (Waltham, MA). Primary antibodies including anti-pSTAT3 (catal. #ab76315), anti-iNOS (catal. #ab15323), and anti-NKR-P1C (catal. #ab289542) were purchased from Abcam (Cambridge, United Kingdom). Primary antibodies including anti-F4/80 (catal. #70076), anti-CD11c (catal. #97585), Anti-Arg-1 (catal. #93668), anti-CD4 (catal. #25229), and anti-CD8α (catal. #98941) were purchased from Cell Signaling Technologies (Danvers, MA). We followed the approved protocol from the University of Missouri Animal Care and Use Committee for the animal study. Experimental mice were bred and maintained in a specially designated animal care facility at the University of Missouri, ensuring adherence to ethical standards. Mice were kept under a 12-hour light-dark cycle with controlled environmental factors such as humidity and temperature. Housing conditions included cages with a maximum of five mice per cage, allowing free access to ad libitum food and water. Stat3 conditional-knockout mice were generated by breeding Pgr cre /+ with Stat3 f/f mice using Cre-LoxP approach [ 39 , 40 ]. Breeding pairs typically comprised one male and one female mouse, occasionally adjusted to optimize breeding outcomes, such as pairing one male with two females. Albumin-nanoceria were synthesized via biomineralization. Briefly, 80 mg of bovine serum albumin (BSA) was dissolved in 4 mL by stirring at 40°C, and 0.5 mL of 1 mM cerium (III) nitrate was added into the BSA solution. After stirring for 15 minutes, the pH was adjusted to 8.5 by adding 2 M NaOH. Then, 600 uL of 30% hydrogen peroxide (H 2 O 2 ) was added as a catalyst. This solution was stirred continuously for 2 hours at 40°C. The resulting particles were then washed by centrifugal filtration through a 100 kDa membrane at 3240 rpm. The supernatant was collected and underwent dialysis in a 10 kDa membrane overnight at 4°C. The final product was collected and stored at 4°C. Albumin-ICG particles were synthesized using the desolvation method. Briefly, 80 mg of BSA was dissolved in 2 mL water alongside 3 mg ICG-NHS ester. After the BSA was completely dissolved, 6 mL of ethanol was added to the solution dropwise. After adding ethanol, the particles were stirred for 30 minutes at room temperature. The resulting particles were then washed by centrifugal filtration through a 100 kDa membrane at 3240 rpm. The supernatant was collected and underwent dialysis in a 10 kDa membrane overnight at 4°C. The final product was collected and stored at 4°C. A 0.5 mg/mL ICG-NHS ester solution was prepared in water. This solution was combined with the prepared, albumin-nanoceria for a final ratio of 1:100 ICG to albumin (mg:mg). This solution was rotated on a shaker overnight. Then, the resulting solution underwent dialysis in a 10 kDa membrane overnight at 4°C. Then, the particles were concentrated using a 100 kDa membrane centrifugal filter and adjusted to desired concentration. The final product was collected and stored at 4°C. Stable chemical conjugation of ICG to the albumin-nanoceria was confirmed by measuring the percentage of ICG remaining in the particle after repeated centrifugal filtration. Particle stability was evaluated over time by diluting particle stock 1:10 in simulated body fluid (SBF) with hydrodynamic diameter readings taken by DLS over a week. Dynamic light scattering (DLS, Zeta Sizer Nano, Malvern Instruments) was used to evaluate the hydrodynamic diameter and colloidal stability of albumin-nanoceria. Crystallinity and size of cerium-oxide nanoclusters were evaluated by using a 2200FS high-resolution transmission electron microscope (HRTEM, JEOL). Samples for X-ray photoelectron spectroscopy (XPS) was prepared by lyophilization, and investigated using an AXIS SUPRA+ system (KratosAnalytical Ltd.). Albumin concentration in albumin-nanoceria was evaluated via bicinchoninic acid (BCA, Thermo Scientific), while ICG quantification was evaluated relative to a standard curve by absorbance comparison at 390 nm. Cerium concentration was determined by an inductively coupled plasma-optical emission spectroscopy (ICP-OES, Varian 710-ES Axial ICP-OES) Initially, J774 cells were seeded in 24-well plates at 50,000 cells per well and incubated overnight at 37°C and 5% humidity. The following day, the cells (except the untreated, which received no treatment, and anti-inflammatory, which received IL4/IL13 treatment controls) were treated with LPS and IFN-gamma (10 ng/mL) and incubated overnight. The following day, cells were either not treated, treated with albumin-nanoceria (150 μgCe/mL), treated with 5,15 DPP (STAT3 inhibitor) (1 nmol), or treated with tofacitinib (JAK7 inhibitor) (1 mmol). All groups were incubated overnight under the conditions described above. The next day, the cells were detached, stained with APC-CD80, PE-CD206, and Zombie green fixable viability dye, washed, fixed with 1% paraformaldehyde for half an hour, and washed again. The fixed cells were resuspended in flow buffer. The fixed cells were run on an Accuri c6 flow cytometer and the data was analyzed using FCS Express analysis software. Cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Briefly, J774 cells were seeded in a 96-well plate at 50k cells/well. They were incubated overnight at 37°C and 5% humidity, and the following day were treated with albumin-nanoceria at 0, 5, 10, 50, 100, or 200 μg Ce/mL. After one more overnight incubation, the supernatant is discarded and replaced with MTT solution and incubated for 4 hours. Finally, the solution is removed, and the resulting formazan crystals are dissolved using DMSO. Absorbance was collected using a SoftMax Pro plate reader (Molecular Devices, CA) at 570 nm. Background used for calculations was collected at 610 nm from the same plate. Initially, J774 cells were seeded in an 8-well slide at 50k cells/well and incubated overnight at 37°C and 5% humidity. The following day, the cells were treated as described above in the FACS section then incubated overnight. The following day, the cells were washed, fixed with 4% paraformaldehyde for half an hour, washed again, incubated with 10% normal goat serum for half an hour, and finally incubated with primary antibodies (anti-CD80 and anti-Arg-1; 1:300) overnight at 4°C. The following day, the cells were washed again, and incubated with secondary antibodies (1:100) for two hours at room temperature to allow for fluorescence imaging. Finally, the cells were washed, stained with DAPI, and imaged using a Leica Thunder Microscope. Stat3 f/f or Stat3 d/d female mice were bred with fertile C57BL/6 male mice. The presence of a vaginal plug in the morning marked the gestational day 0.5 (GD 0.5). Euthanasia was performed via cervical dislocation under anesthesia, and uterine samples were collected at GD 3.5 and 5.5. Upon collection, uterine tissues were promptly preserved by freezing at −80°C for RNA/protein extraction or fixed in 4% paraformaldehyde for histological examination. Whole uterine samples obtained at GD 5.5 were photographed and weighed before fixation in 4% (vol/vol) paraformaldehyde. Subsequently, they underwent processing through a graded alcohol series for paraffin embedding. Implantation sites were identified based on their visible characteristics and then counted, with confirmation through histological analysis. Uterine tissue samples underwent total RNA extraction employing the RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA, Cat. #74106) following established protocol [ 41 ]. Assessment of RNA purity and preliminary concentration estimation was conducted using NanoDrop. Subsequently, all RNA samples were subjected to analysis utilizing a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE) to verify concentration and purity, ensuring an RNA Integrity Number (RIN) exceeding 8.0 and a concentration within the range of 100–200 ng/μl prior to microarray hybridization. RNA pooling from more than three mice per genotype at GD 3.5 preceded microarray analysis, employing GeneChip ® Mouse Genome 430 2.0 Arrays (Affymetrix), following established procedures. Array data underwent quantile normalization using Bioconductor. Differentially expressed genes in the uteri of Stat3 f/f and Stat3 d/d mice at GD 3.5 were identified through a two-sample comparison, with significant fold changes exceeding 1.5. Classification of aberrantly expressed genes was performed using QIAGEN Ingenuity Pathway Analysis (IPA) to analyze canonical pathways. As mentioned, the extraction of total RNA from uterine tissues was performed using the RNeasy Mini Kit. cDNA was synthesized from 3 μg of total RNA using MMLV Reverse Transcriptase (Thermo Fisher Scientific, Cat. #28025013) and random hexamers. Subsequently, the cDNA was quantified through real-time PCR SYBR green analysis employing an Applied Biosystems StepOnePlus system according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). For the PCR reaction, PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Cat. #A25742) along with validated primers ( Table S3 , Supporting Information ) were utilized. The amplification protocol comprised an initial denaturation step at 95 °C for ten minutes, followed by forty cycles of amplification with denaturation at 95 °C for fifteen seconds, annealing at 60 °C for one minute, and extension at 72 °C for one minute. Additionally, a melt curve analysis was conducted with cycles of denaturation at 95 °C for fifteen seconds, annealing at 60 °C for one minute, and further denaturation at 95 °C for fifteen seconds. Finally, the expression levels were normalized to ribosomal protein L7 (Rpl7) transcripts. We followed previously published protocols for surgically inducing endometriosis [ 42 ]. Eight-week-old wild-type mice were administered subcutaneous injections of 100 μL of 1 μg/mL estradiol (E2; Sigma Aldrich, Cat. #E8875) dissolved in sesame oil for three consecutive days. Six hours following the final E2 injection, under anesthesia, a small midline abdominal incision was made. One uterine horn was removed and longitudinally opened with scissors, then cut into small fragments using a scalpel and placed in a Petri dish. These tissue fragments were then injected into the peritoneal cavity of the same mice. Finally, the abdominal incision was closed using sutures for the peritoneum and wound clips for the skin. Fluorescence-guided dissection and brightfield imaging were conducted using a Nikon fluorescent dissection microscope along with NIS-Elements imaging software (Nikon Instruments, Melville, NY). Tissue samples embedded in OCT were frozen and cryosectioned at a thickness of 10 μM, followed by placement on slides for fluorescence imaging. Subsequently, a DAPI mounting solution (Vector Laboratories, Burlingame, CA, Cat. #H-1800) was applied, and coverslips were added. For immunostaining, the tissue sectioned was treated with 0.3% hydrogen peroxide in methanol and washed in 1/40 Triton X-100 (Fisher Scientific, Cat. #BP151-500). Afterward, the sections were blocked with 10% normal goat serum (NGS; Vector Laboratories, Cat. #S-1000) in pH 7.5 PBS and incubated with primary antibodies diluted in 10% NGS in PBS overnight at 4 °C. Specific dilutions for primary antibodies including anti-pSTAT3 (1:1000 dilution; #ab76315; Abcam), anti-F4/80 (1:800 dilution; #70076; Cell Signaling), anti-CD11c (1:250 dilution; #97585; Cell Signaling), anti-iNOS (1:100 dilution; ab15323; Abcam), Anti-ARG 1 (1:2000 dilution; #93668; Cell Signaling), anti-CD4 (1:100 dilution; #25229; Cell Signaling), anti-CD8α (1:500 dilution; #98941; Cell Signaling), and anti-NKR-P1C (1:200 dilution; ab289542; Abcam) were employed. Following incubation, an appropriate species-specific fluorescently tagged secondary antibody [anti-mouse (1:500 dilution; #BA-9200; Vector Laboratories) or anti-rabbit (1:500 dilution; #BA-1000; Vector Laboratories) conjugated to horseradish peroxidase (1:1000 dilution; #43-4324; Invitrogen) was applied before mounting and cover slipping with DAPI mounting media for imaging. Imaging procedures were conducted using PhenoImager HT (Akoya Biosciences Inc.), with whole slide contextual viewing facilitated by Phenochart 2.0.0 (Akoya Bioscience). Animal imaging study was approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University. In vivo fluorescence images were obtained using an IVIS imaging system (PerkinElmer, Waltham, MA) and a Pearl Trilogy NIRF imaging system (LI-COR Biosciences, Lincoln, NE) at various time points (1, 3, 6, 24 h) after intraperitoneal injection of PBS, ICG-conjugated nanoceria into endometriosis mice. The mice were kept under anesthesia with 2% isoflurane, and imaging was performed with white-lighted filter and 800 nm filter. The regions of interest (ROI) were drawn and calculated by Image J software. For biodistribution, 24 h after injection with ICG-conjugated nanoceria, the mice were sacrificed and the liver, spleen, heart, uterus, and ectopic lesions were taken. Organs were placed on a black paper background to reduce noise and measured using a Pearl Trilogy NIRF imaging system. Photoacoustic imaging was performed using inVision 512-echo preclinical multispectral optoacoustic tomographic (MSOT) imaging system (iThera Medical, Germany). Endometriosis was induced in 8-week-old female mice with dual fluorescence reports ( Pgr cre /+ Rosa26 mTmG /+ ). ICG-conjugated nanoceria (albumin–nanoceria–ICG; 480 μg/kg body weight) were injected into the endometriosis mice via tail-vein injection. After 3 hours of injection, the mice were placed in the animal holder, had ultrasound coupling gels applied, and were wrapped in a thin polyethylene membrane. Anesthesia (2% isoflurane and oxygen) was then supplied through a breathing mask. Imaging was performed in 0.2 mm steps, and all acquisition was performed using 10 averages per wavelength, with the wavelengths chosen as (680, 700, 730,760, 800, and 850 nm). Image analysis was performed by using ViewMSOT software. Statistical analyses were conducted utilizing Prism9 software from GraphPad (San Diego, CA, USA). To assess the distinction between two variables, the Student’s t -test was employed. For datasets involving more than two variables, one-way ANOVA was utilized, with Tukey’s post hoc test applied for multiple comparisons. Statistical significance was defined as p < 0.05. Mean values along with standard error of the mean (SEM) or standard deviations (SD) were employed to present numerical data (mean ± SEM/SD). The results section and accompanying figure legends explicitly state P-values obtained from statistical tests.

Nanoceria exhibits remarkable therapeutic potential as a non-steroidal anti-inflammatory drug capable of reducing the size of the ectopic lesion. At the molecular level, this nanodrug effectively counteracts aberrant immune cell function. This mechanism potentially involves the inhibition of abnormal STAT3 activation. Further, the exceptional ability of our nanodrug to efficiently accumulate within ectopic lesions underscores its capacity to mitigate off-target effects associated with fertility. Our results warrant an additional assessment of this nanoparticle to improve both its therapeutic and diagnostic effectiveness, potentially positioning it as a theranostic agent.

Our research revealed that STAT3 activation contributes to changes in endometrial immune cell populations (macrophages, T-cells, B-cells, and NK cells), which leads to immune suppression and inflammation linked to endometriosis. Nanoceria, an immune-modulatory and anti-inflammatory nanodrug, can safely counteract these adverse effects, prevent endometriosis, and restore fertility. We previously reported that total STAT3 protein levels remain unchanged during endometriosis [ 23 ]. However, pSTAT3, a marker of STAT3 activation, significantly increased in the eutopic endometrium of women with endometriosis [ 23 ] and correlated with disease progression in female baboons over time [ 11 ]. In our mouse model of endometriosis, we also observed significantly higher pSTAT3 levels in the eutopic endometrium of endometriosis mice compared to sham mice. Transcriptomic analysis of Stat3 d/d mice shows that STAT3 loss directly affects uterine inflammation and immune status. Altogether, targeting STAT3 could feasibly activate innate immunity, which holds promising therapeutic potential against endometriosis. Despite its anti-inflammatory potential, nanoceria remains largely underutilized in treating endometriosis. In fact, the only previous use of nanoceria for endometriosis relied on intraperitoneal (IP) injection of nanoceria to reduce oxidative stress [ 38 ]. However, other nanoparticle (NP)-based therapeutic strategies have been employed in recent years [ 63 ], demonstrating that nanomedicine is a viable option for the treatment of endometriosis. Drug delivery by using polymeric NPs is a classical approach in endometriosis nanomedicine. A pilot study discovered that methotrexate containing lipid NPs reduced dyspareunia, pelvic pain, and dyschezia in deep-infiltrating endometriosis patients [ 64 ]. In another study, the anti-endometriosis effects of albumin-glucose oxidase NPs were reported in a mouse model [ 59 ]. However, these studies do not test NPs’ inhibitory effects on endometriosis progression or their impact on subsequent implantations and fertility. PLGA nanoparticles loaded with various drugs, including doxycycline, have been shown to effectively inhibit angiogenesis and cellular proliferation while increasing apoptosis of endometrial cells [ 63 ]. However, the nano-drug delivery approach still fails to overcome off-target side effects associated with traditional therapeutics. In contrast, laser ablation of ectopic lesions using photosensitizing NPs capable of absorbing laser light and emitting heat can destroy abnormal tissue. Laser ablation has been shown to effectively reduce the size of ectopic lesions [ 65 ], however, it has some major drawbacks. First, laser ablation must either be performed during a surgical procedure or on lesions very close to the skin as the laser’s depth of penetration in tissue is limited [ 66 ]. Second, laser ablation enhances inflammation in the region surrounding the ablated tissue [ 67 ], which may increase the risk of further lesions developing. Finally, magnetic hyperthermia has been employed in the treatment of endometriosis [ 60 ]. If properly targeted, hyperthermia can cause targeted apoptosis in regions of interest, but care must be taken not to allow excess accumulation in healthy tissues near the target sites. The liver, a major organ of mononuclear phagocytes, is likely to take up a large portion of the superparamagnetic iron-oxide nanoparticles [ 68 ] and may be damaged by off-target hyperthermia. Nanoceria efficiently inhibits inflammation by scavenging free radicals and regulating inflammatory cytokines such as NF-kB, IL-6, and IL-8 [ 69 ]. Our nanoparticle formulation reduces the size and severity of ectopic lesions but does not impair decidualization and implantation for pregnancy. In nanoceria-treated mice at GD5.5, characteristic COX2 expression is observed around the embryo in endometrial decidual cells, accompanied by PGR expression supporting and maintaining decidual tissue, and lost epithelial E-cadherin expression [ 28 , 49 ]. Endometriosis, characterized by immune system dysfunction, leads to elevated levels of macrophages, T cells, and B cells in the peritoneal fluid of affected women due to immune dysregulation, which in turn enables the survival of endometrial fragments during retrograde menstruation [ 12 , 70 ]. This phenomenon is echoed in the uterine cells of Stat3 d/d mice compared to those of the control counterparts. Control of immune cell homeostasis by nanoceria in mice with endometriosis offers insights into how this novel nanomedicine could potentially prevent the condition by targeting STAT3 signaling and mitigating its aberrant activation. It is important to note that while selective JAK/STAT3 inhibitors like tofacitinib can protect against endometriosis, they cannot restore fertility. This limitation may be due to their systemic impact on overall STAT3 signaling, which is crucial for normal growth and development [ 71 ]. Our targeting mechanism relies on particle accumulation in angiogenic inflamed regions, facilitated by leaky vasculature where extravasation of proteins and small molecules occurs [ 72 ]. Albumin, comprising 50–60% of blood plasma proteins, is particularly prevalent in the expanded interstitial space characteristic of inflammation [ 73 ]. Our albumin-nanoceria showed promoted preferential accumulation in ectopic lesions by exploiting so-called ‘enhanced permeability and retention (EPR)’ effect, which is prevalent in solid cancer tumors and associated with endometriosis [ 74 ]. In addition to the passive targeting, exploiting active targeting through incorporating targeting ligands, such as angiogenic markers like vascular endothelial growth factor (VEGF), into the particles could further enhance and ensure specific nanoparticle targeting to ectopic lesions [ 75 , 76 ]. VEGF is highly overexpressed in ectopic tissue and was suggested as a promising marker for visualization of ectopic lesions [ 77 ]. In this study, we used ICG to validate the targeting of ectopic lesions via fluorescence and photoacoustic imaging. As a NIR fluorescent dye active within the biological transparency window, ICG holds great potential for in vivo imaging at deeper tissue penetrations [ 78 ]. Our nanoceria conjugated with ICG (albumin-nanoceria-ICG) can thus achieve diagnostic capabilities in real-time without affecting its anti-inflammatory properties, both in fluorescence and photoacoustic imaging after accumulating on the lesion [ 28 , 79 , 80 ]. Particularly, photoacoustic imaging, based on the principle of ‘light-in/sound-out’, combines the sensitivity and contrast of optical imaging with the depth and resolution of ultrasound [ 81 ]. Therefore, the systemically administered nanoceria, which provides sensitive and durable photoacoustic tomographic image signals, can quantitatively differentiate the location and volume of deep-seated ectopic lesions in the peritoneal cavity [ 82 ]. As a new class of theranostic agent, this could help to track the treatment efficacy of the anti-inflammatory nanoceria via noninvasive imaging.

Endometriosis—endometrial-like tissue growth outside the uterus—is a widespread gynecological disease that affects roughly 1 in 10 reproductive-age women (200 million) worldwide [ 1 , 2 ]. Endometriosis is a major cause of infertility and pelvic pain [ 3 , 4 ], and its prevalence increases to 50–60% in women with chronic pelvic pain and infertility [ 5 ]. Endometriosis is associated with reduced health-related quality of life, diminished mental well-being, adverse effects on intimate relationships, and reduced social activity. Although current treatments, including over-the-counter analgesics, hormonal therapy, and surgical excision of lesions offer temporary relief, they are linked to various side effects and significant recurrence rates (21.5% after two years and 40–50% at five years) [ 6 – 8 ]. This chronic estrogen-dependent inflammatory condition is characterized by an upsurge in the elevated production of pro-inflammatory cytokines [ 9 ], leading to immune suppression [ 10 , 11 ]. The immune system, a complex and multifaceted player, is deeply involved in the pathophysiology of endometriosis [ 12 ]. Furthermore, endometriosis disrupts the distribution of immune cell populations [ 13 ], increasing numbers of peritoneal macrophages, regulatory T cells, and B cells [ 14 , 15 ]. Pro-inflammatory M1 macrophages are linked to the proliferation of endometriosis and are implicated in adverse health effects such as pain and infertility [ 16 – 18 ]. These innate immune cells secrete pro-inflammatory cytokines like IL-6 and recruit additional immune cells, perpetuating an inflammatory response [ 19 ] associated with pain and excessive menstrual blood loss. M1 macrophages are also associated with implantation failure and infertility, explaining these aspects of the disease [ 16 ]. Conversely, anti-inflammatory M2 macrophages are associated with better outcomes in endometriosis patients [ 17 ]. Therefore, employing anti-inflammatory and immunomodulatory agents as well as agents inducing a hypoestrogenic state to reduce M1 cells and increase M2 cells, holds promise for endometriosis therapy. Recent research has investigated the efficacy of such treatments, including synthetic steroids, progestin, GnRH agonists, and hydroxychloroquine for managing endometriosis [ 20 , 21 ]. Systemic effects, however, are often associated with the use of such therapies [ 22 ] which may have adverse consequences on implantation and fertility. Therefore, focusing on endometriosis-specific molecular pathways and facilitating hypoestrogenic and immunomodulatory effects may offer an alternative therapeutic approach. Signal transducer and activator of transcription-3 (STAT3) protein plays important roles in the development and function of various tissues throughout the body. STAT3 is particularly essential for embryo attachment, implantation, and decidualization in the uterus [ 23 , 24 ]. However, phosphorylation activates STAT3, which contributes to estrogen dominance and facilitates the development and progression of endometriosis [ 23 ]. Triggering by inflammation, altered cytokines such as IL-6, IL-11, and epidermal growth factor can further phosphorylate STAT3 (pSTAT3) [ 10 , 25 ]. Our previous study found higher pSTAT3 levels in the eutopic endometrium of women with endometriosis and showed that induction of endometriosis results activation of STAT3 in non-human primate [ 23 ]. In a mouse model, the effects of treating endometriosis with 10 mg/kg of Tofacitinib—a JAK/STAT3 activation inhibitor and immunomodulator—have also been explored [ 10 ]. However, targeted therapies for inflammation would be superior to traditional therapies as they could allow us to avoid off-target effects, such as infertility [ 24 ], by preventing the drug from acting in the healthy uterine tissue resulting in reduced disease burden and restored fertility. Nanomedicine holds promise for treating diseases where conventional therapies face significant challenges [ 26 ]. Nanoparticles (NPs) have been investigated for various conditions, including cancer, heavy metal poisoning, infections, and traumatic injuries. Nonetheless, managing inflammatory conditions is one area where NPs hold tremendous potential to transform treatment [ 27 ]. Albumin-nanoceria, specifically, has shown the ability to modulate macrophages away from an M1 and towards an M2 phenotype in a mouse model of rheumatoid arthritis [ 28 ]. This is attributed to nanoceria’s unique anti-inflammatory properties. Due to surface oxygen valence switching between Ce 3+ and Ce 4+ , nanoceria can act as both a superoxide dismutase (SOD) and a catalase allowing for continuous scavenging of reactive oxygen species in their microenvironment with the ratio of Ce 3+ and Ce 4+ determining the overall catalytic behavior of the particles [ 29 – 31 ]. This multienzymatic activity contributes to its anti-inflammatory effect [ 32 ]. There is ample evidence that nanoceria can be used as a STAT3 inhibitor and reduce the downstream expression of STAT3 in sepsis [ 33 , 34 ]. These findings suggest that nanoceria has the capacity to influence multiple inflammation pathways. Furthermore, the nanoceria can be tailored to a theranostic nanomedicine through albumin-indocyanine green (ICG) conjugates, which enables noninvasive visualization of therapeutic delivery and accumulation at targeted tissues. ICG is an FDA-approved, near-infrared (NIR) fluorescent dyes that can be used in dual-mode of imaging: fluorescence (optical) and photoacoustic (optoacoustic) [ 28 , 35 , 36 ]. Current diagnostic methods for endometriosis are imperfect, invasive, and lengthy; to receive a confirmed diagnosis of endometriosis, patients still must undergo laparoscopic surgery and histology [ 37 ]. A targeted, theranostic nanoparticle could offer non-invasive imaging and diagnosis, but nanoceria remains underutilized in endometriosis [ 38 ]. Here, we investigated the role of STAT3 in the uterine inflammatory and immune status using uterine-specific Stat3 knock-out ( Pgr cre /+ Stat3 f/f ; Stat3 d/d ) mice. Our transcriptomic analysis identified dysregulation of inflammatory and immune-related pathways in Stat3 d/d mice. Then, we examined the effect of tofacitinib and nanoceria on endometriosis and implantation using an endometriosis mouse model. Although tofacitinib exhibited a reduction in endometriosis, it caused implantation failure due to its systemic effects. In contrast, nanoceria did not impair implantation but suppressed the development of ectopic lesions. We observed that nanoceria possesses unique enzymatic and anti-inflammatory properties, making it a promising candidate for endometriosis treatment. Its prolonged anti-inflammatory therapeutic effects offer continuous management of ectopic lesions, potentially reducing the need for extended use of conventional medications.