Method Article

A Mouse Model of Endometriosis Using Ex Vivo Cy5.5 Dye-Doped Silica Nanoparticle Labeling for Lesion Visualization

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DOI:

10.3791/72070

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August 4th, 2026

 ,  ,  ,  , 
1Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, 2Department of Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University

* These authors contributed equally

In This Article

Summary

This study demonstrates the induction of endometriosis using ex vivo labeling with Cy5.5 dye-doped silica nanoparticles, enabling fluorescent visualization of endometriotic lesions without genetic manipulation.

Abstract

Endometriosis is a common gynecological disorder characterized by the growth of endometrial-like tissue outside the uterine cavity, leading to chronic inflammation, pelvic pain, and infertility. Reliable mouse models are essential for investigating the molecular, hormonal, immune, and environmental mechanisms underlying disease development and progression. Conventional methods for identifying endometriotic lesions often rely on genetically engineered fluorescent reporter mice, which are time-consuming and costly to generate. Here, we present a simple, non-genetic approach to inducing and visualizing endometriosis in mice by ex vivo labeling donor uterine tissue fragments with Cy5.5-doped silica nanoparticles prior to transplantation. The nanoparticles exhibit favorable physicochemical properties, high fluorescence stability, and minimal cytotoxicity, enabling efficient labeling of uterine tissue without compromising experimental procedures. Following intraperitoneal implantation into recipient mice, Cy5.5-labeled endometriotic lesions can be readily distinguished from surrounding host tissues using fluorescence imaging. This protocol provides a reproducible and versatile method for lesion visualization and tracking, facilitating studies of endometriosis pathogenesis and the preclinical evaluation of diagnostic and therapeutic strategies without the need for genetic manipulation.

Introduction

Endometriosis is a common gynecological disease in which cells lining the endometrium grow outside the uterus. The resulting endometriotic lesions lead to a chronic inflammatory condition1, and affected women commonly experience pelvic pain and infertility2. Dysfunction in endometrial stromal and epithelial cells enhances the survival and development of endometriotic lesions3,4,5,6. This common disorder afflicts approximately 11% of women in the US alone. It is a major cause of infertility and pelvic pain7, and its prevalence increases to 50%–60% in women with chronic pelvic pain and infertility8,9,10,11,12,13. The estimated yearly cost to diagnose and treat endometriosis exceeds $22 billion in the United States14,15. Current treatments include surgical removal of the lesions or induction of a hypoestrogenic state. However, recurrence after surgery is common, and existing therapies are often ineffective9,16. Diagnosis is typically established only through surgical visualization and histological verification17,18. Moreover, endometriosis is frequently undiagnosed or misdiagnosed, with a diagnostic delay of 9–11 years19,20. This delay imposes a significant emotional and financial burden on both patients and the healthcare system20,21. Therefore, there is an urgent need to develop noninvasive methods for both the diagnosis and treatment of endometriosis.

In vitro models have advanced endometriosis research. However, they remain limited in their ability to fully replicate the complexity of disease. A major challenge lies in the multifactorial nature of endometriosis, which involves intricate interactions among hormones, inflammation, immune cells, nerves, and diverse cell types within a dynamic three-dimensional environment. In addition, in vitro systems do not capture the natural fluctuations of ovarian steroid hormones, limiting the ability to study disease effects on the reproductive tract21,22,23. Thus, a model of induced endometriosis using intact mice provides a versatile platform to investigate the individual and interactive roles of the immune system24, hormones25,26, and environmental factors27,28 in endometriosis development and its impacts on the reproductive tract29. Additionally, the use of transgenic mouse models, in which specific genes are selectively deleted or overexpressed in the endometrium, enables investigators to elucidate the roles of distinct molecular pathways in the development and progression of endometriosis. However, accurately distinguishing endometriotic lesions from surrounding host tissues remains a significant technical challenge.

Dye-doped silica nanoparticles are versatile and robust probes for cellular tracking and bioimaging. They are simple to synthesize, highly dispersive, photostable, biocompatible, and efficient in cell labeling30,31. The silica backbone stabilizes hydrophobic fluorophores within the matrix, making them efficient, high-quantum-yield (QY) fluorescent molecular reporters. Over the years, such fluorescent silica nanoparticles have been widely adopted as bright, durable ex vivo labels for cells and tissue structures prior to transplantation and subsequent imaging in vivo32,33,34,35. Here, we describe a detailed procedure for the induction of endometriosis in mice with ex vivo labeling using Cy5.5 dye-doped silica nanoparticles, as used in a previous publication35, enabling their application as stable imaging markers for lesion tracking.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Missouri (Protocol No.65323) and were performed in accordance with institutional guidelines and applicable national regulations for the care and use of laboratory animals.

1. Preparation of Cy5.5-APTES conjugate

  1. Dissolve 1 mg of Cyanine 5.5-NHS ester in 100 µL of dimethyl sulfoxide (DMSO) in a clear 11.1 mL glass threaded vial. Immediately cover the vial with aluminum foil to protect the solution from light.
    NOTE: Cy 5.5-APTES conjugate can be stored at 4 °C at room temperature for one week.
  2. Add 1 µL of (3-aminopropyl)triethoxysilane (APTES) to the dye solution.
  3. Add 0.5 µL of triethylamine (TEA) and mix immediately to facilitate the reaction.
  4. Stir the reaction mixture at 300 rpm at 30 °C in the dark for 24 h to allow covalent coupling between Cy5.5 and APTES.
  5. Seal the vial with its cap and continue protecting it from light by wrapping it with aluminum foil.

2. Synthesis of Cy5.5 dye-doped silica nanoparticles

  1. Add 1 µL of concentrated ammonia solution (28%) to a new clear glass threaded vial.
  2. Add 8.5 mL of absolute ethanol (molecular biology grade).
  3. Add 350 µL of tetraethyl orthosilicate (TEOS).
  4. Add 15 µL of the prepared Cy5.5-APTES solution to the silica synthesis reaction mixture.
  5. Stir the reaction mixture at 500 rpm at room temperature in the dark for 24 h to enable covalent incorporation of the dye into the silica matrix.

3. Purification and dispersion

  1. Transfer the entire reaction mixture into a 15 mL centrifuge tube.
  2. Prepare a balance tube containing an equal volume of distilled water for centrifugation.
  3. Centrifuge both tubes at 12,074 × g for 10 min at 4 °C. Confirm that a visible pellet has formed at the bottom of the tube after centrifugation.
  4. Wash the pellet with 10 mL of 95% ethanol by centrifuging at 12,074 × g for 10 min at 4 °C. Repeat this wash 3x, resuspending the pellet by sonication for approximately 3–5 min between washes, or until fully dispersed.
  5. Wash the pellet with 5 mL of distilled water and centrifuge at 12,074 × g for 10 min at 4 °C. Repeat this wash 2x, resuspending the pellet by sonication for approximately 3–5 min between washes, or until fully dispersed.
  6. Resuspend the pellet in 5 mL of distilled water by sonication until completely dispersed (no solid fragments observed to confirm dispersion). Confirm that the particle suspension appears visibly blue after complete dispersion.
  7. Transfer the suspension to a glass vial, wrap the vial with aluminum foil to protect it from light, and store it at room temperature.
  8. Assess particle dispersion and fluorescence using an in vivo imaging system (IVIS). Use an excitation wavelength of 680 nm and an emission wavelength of 710 nm for Cy5.5.

4. Endometriosis induction using ex vivo labeling with Cy5.5 dye-doped silica nanoparticles

  1. Inject an 8-week-old female donor mouse (strain: C57Bl6J X129Sv) subcutaneously with E2 (100 µL of 1 µg/mL in sesame oil) once daily in the morning for three consecutive days for hormonal synchronization.
  2. Euthanize the donor mouse by cervical dislocation 6 h after the final E2 injection, and remove the uterus.
  3. Mince the uterine tissue into approximately 1 mm3 fragments in a Petri dish using a scalpel30,36.
  4. Incubate the uterine tissue fragments (45 mg) with 20% (v/v) Cy5.5 dye-doped silica nanoparticles in a 24-well plate containing complete RPMI-1640 medium (10% FBS) for 3 h at 37 °C in 5% CO2.
  5. Anesthetize the recipient mouse (strain: C57Bl6J X129Sv) with 3% isoflurane in oxygen and make a 0.5 cm midline abdominal incision.
    NOTE: Place the mouse in an induction chamber with 3% isoflurane until anesthesia is achieved. Shave the abdomen and transfer the mouse to the surgical platform. Position the anesthetic nose cone before beginning surgery.
  6. Wash the ex vivo-labeled uterine tissue fragments with room-temperature culture medium by centrifugation at 380 × g for 2 min. Repeat the wash 3x.
  7.  Under anesthesia, make a 1 cm midline abdominal incision in the recipient mice. Suspend 45 mg of Cy5.5-labeled tissue fragments in 500 µL of PBS and inject the suspension into the peritoneal cavity using a 20-gauge needle.
  8. Close the peritoneum with absorbable suture and close the skin with wound clips. Place the mouse in a warm environment and monitor it until recovery from anesthesia.
  9. Euthanize the mouse by cervical dislocation at the selected experimental endpoint and collect the endometriotic lesions.

Results

Dye-doped silica nanoparticles can be used as imaging probes for cell-labeling applications. Cy5.5 dye-doped silica nanoparticles (Cy5.5-SiNPs) were synthesized in two steps using a silica sol-gel process and functionalized with the fluorescent cyanine dye Cy5.5 using silane chemistry (Figure 1). Transmission electron microscopy (TEM) revealed uniform spherical nanoparticles with diameters of 156.67 ± 15.23 nm (Figure 1A). Dynamic light scattering (DLS) demonstrated that the synthesized nanoparticles (SiNPs and Cy5.5-SiNPs) were colloidally stable, with hydrodynamic diameters (Hd) of 130.67 ± 3.20 nm (PDI = 0.152 ± 0.009) and 185.9 ± 5.44 nm (PDI = 0.14 ± 0.009), respectively (Figure 1B). The zeta potentials of bare SiNPs and Cy5.5-SiNPs were −32.39 ± 3.55 mV and −50.53 ± 2.47 mV, respectively (Figure 1C). Photoluminescence spectroscopy (excitation wavelength = 680 nm; emission wavelength = 710 nm) confirmed the successful incorporation of Cy5.5 into the silica nanoparticles, and the Cy5.5-SiNPs exhibited characteristic fluorescence (Figure 1D). Successful dye conjugation was further confirmed by the photoluminescence spectrum. In addition, the quantum yield (QY) of the Cy5.5-SiNPs (φ = 0.31) was comparable to that of free Cy5.5 dye (φ = 0.23)36.

To evaluate nanoparticle biocompatibility, 12Z endometrial epithelial cells were incubated overnight with Cy5.5-SiNPs in DMEM at 37 °C under 5% CO₂. Cell viability was assessed using an MTT assay after treatment with nanoparticle concentrations of 0, 0.5, 1, 2.5, 5, and 10 µg/mL (Figure 1E). No cytotoxicity was observed at concentrations up to 10 µg/mL. Cells incubated with 5 µg/mL Cy5.5-SiNPs exhibited efficient nanoparticle uptake, with fluorescence microscopy demonstrating particle localization in the perinuclear region (Figure 1F). In contrast, no Cy5.5 fluorescence was detected in untreated control cells. The prepared Cy5.5-SiNPs also demonstrated aqueous stability, with hydrodynamic diameters ranging from 170–190 nm in deionized water and maintaining stable fluorescence intensity for seven days, as determined by DLS and fluorescence imaging, respectively (Figure 1G, H).

To establish the suitability of Cy5.5 dye-doped silica nanoparticles for ex vivo tissue labeling, their fluorescence properties were validated before endometriosis induction. IVIS imaging demonstrated robust fluorescence emission from the nanoparticle suspensions, whereas no detectable fluorescence signal was observed in the negative control containing water alone, confirming the specificity and sensitivity of Cy5.5 fluorescence detection (Figure 2).

The concentration and incubation time for ex vivo labeling of uterine tissue fragments were subsequently optimized. Fluorescence was detected in uterine tissue fragments incubated with 20% (v/v) Cy5.5 dye-doped silica nanoparticles for 3 h at 37 °C under 5% CO235. IVIS imaging confirmed strong and consistent fluorescence signals from the labeled tissue fragments. These results demonstrate that Cy5.5 dye-doped silica nanoparticles efficiently label uterine tissue fragments without genetic modification (Figure 3).

The ability of Cy5.5 dye-doped silica nanoparticle-labeled uterine tissue fragments to induce endometriosis and facilitate lesion identification was then evaluated35. Labeled tissue fragments were injected into the peritoneal cavity of recipient mice, and disease progression was monitored for one month. At the experimental endpoint, endometriotic lesions were readily identified within the peritoneal cavity. Cy5.5-positive lesions were clearly distinguished from the surrounding host tissues by IVIS imaging and fluorescence microscopy, enabling rapid and unambiguous lesion identification. Cy5.5 fluorescence within the lesions was further confirmed by fluorescence microscopy.

These results demonstrate that ex vivo labeling with Cy5.5 dye-doped silica nanoparticles supports the successful induction of endometriosis and enables the reliable visualization of endometriotic lesions in vivo (Figure 4).

Silica nanoparticles (SiNPs) characterization; diagrams: size, zeta potential, fluorescence, cell assays.
Figure 1: Physical characterization of Cy5.5 dye-doped silica nanoparticles (Cy5.5-SiNPs). (A) Representative transmission electron microscopy (TEM) image of Cy5.5-SiNPs. Particle diameter = 156.67 ± 15.23 nm. Scale bar = 100 nm. (B) Dynamic light scattering (DLS) measurements of the hydrodynamic diameters of SiNPs (130.67 ± 3.20 nm, PDI = 0.152 ± 0.009) and Cy5.5-SiNPs (185.9 ± 5.44 nm, PDI = 0.14 ± 0.009). (C) Zeta potential measurements of SiNPs (−32.39 ± 3.55 mV) and Cy5.5-SiNPs (−50.53 ± 2.47 mV), demonstrating the decrease in zeta potential following Cy5.5 conjugation. (D) Fluorescence profile of Cy5.5-SiNPs (excitation wavelength = 680 nm; emission wavelength = 710 nm). Inset: Bright-field and fluorescence images of Cy5.5-SiNPs dispersed in water. (E) MTT cell viability assay of 12Z cells treated with Cy5.5-SiNPs at concentrations of 0, 0.5, 1, 2.5, 5, and 10 µg/mL. (F) Cellular uptake of Cy5.5-SiNPs by 12Z cells. Cells were treated with PBS (control) or Cy5.5-SiNPs. Blue = DAPI; red = Cy5.5-SiNPs. Scale bar = 200 µm. (G) Size stability of Cy5.5-SiNPs stored in water for 7 days, demonstrating no significant change in hydrodynamic diameter. (H) Fluorescence stability of Cy5.5-SiNPs stored in water for 7 days, demonstrating no significant change in fluorescence intensity. Inset: Representative fluorescence images acquired on days 1, 3, and 6. NS = not significant. Values are presented as mean ± SD. Please click here to view a larger version of this figure.

Dye-doped silica nanoparticles in solution and corresponding IVIS fluorescence spectrum chart.
Figure 2: Confirmation of Cy5.5 dye-doped silica nanoparticles. (A) Representative image of Cy5.5 dye-doped silica nanoparticles. (B) Detection of Cy5.5 fluorescence from Cy5.5 dye-doped silica nanoparticles using IVIS imaging. Please click here to view a larger version of this figure.

Non-treatment vs. CY5.5 IVIS spectrum; fluorescence imaging comparison in culture dishes.
Figure 3: Ex vivo labeling of uterine tissue fragments with Cy5.5 dye-doped silica nanoparticles. The left panel shows untreated control uterine tissue fragments, and the right panel shows uterine tissue fragments labeled with Cy5.5 dye-doped silica nanoparticles. Cy5.5 fluorescence was detected using IVIS imaging. Please click here to view a larger version of this figure.

CY5.5 fluorescence imaging, IVIS spectrum, microscope, endometriotic lesion, H&E histology.
Figure 4: Detection of endometriotic lesions labeled with Cy5.5 dye-doped silica nanoparticles. (A) IVIS image of the peritoneal cavity. (B) Fluorescence microscopy of Cy5.5-labeled endometriotic lesions enables clear identification within the peritoneal cavity. (C) Representative fluorescence microscopy images of endometriotic lesions. Yellow fluorescence indicates the Cy5.5 signal, and nuclei were counterstained with DAPI. (D) Representative image of an endometriotic lesion. The arrow indicates Cy5.5 dye-doped silica nanoparticles. Please click here to view a larger version of this figure.

Discussion

The present study introduces a Cy5.5 dye-doped silica nanoparticle-based approach as a practical and efficient method for visualizing endometriotic lesions in mouse models35. This strategy offers several advantages, including its simplicity, flexibility, and non-genetic nature, eliminating the need to generate transgenic reporter animals. Compared with conventional visualization approaches, such as fluorescent reporter mouse models, this method reduces technical complexity while maintaining robust labeling efficiency, thereby broadening its accessibility for endometriosis research. In addition, silica nanoparticles provide a stable platform for incorporating fluorescent dyes and for their efficient retention within tissue, thereby supporting reproducible lesion identification.

Distinguishing endometriotic lesions from surrounding host tissue is essential for studying disease initiation and progression in mouse models. Rosa26mTmG reporter mice ubiquitously express a membrane-targeted tandem dimer Tomato fluorescent protein (mT), which is converted to membrane-targeted green fluorescent protein (mG) following Cre-mediated excision in specific target tissues37. Although numerous transgenic mouse models are available to investigate gene function in endometriosis, generating fluorescent reporter animals is labor-intensive and time-consuming. In contrast, ex vivo labeling with Cy5.5 dye-doped silica nanoparticles provides a rapid, flexible, and non-genetic alternative for lesion visualization while remaining compatible with a wide range of existing mouse models.

Successful implementation of this protocol depends on careful control of several critical experimental parameters. During nanoparticle synthesis, catalyst concentration, solvent composition, TEOS addition rate, mixing conditions, and washing efficiency directly influence particle size, fluorescence intensity, and labeling reproducibility. The Stöber method provides a simple, scalable, and reproducible approach for synthesizing fluorescent silica nanoparticles with precise control over particle size and stable dye incorporation38,39,40,41. Compared with reverse microemulsion methods, the Stöber approach minimizes surfactant-associated purification challenges while preserving Cy5.5 fluorescence under mild reaction conditions39,40,41,42. Increasing the catalyst concentration generally increases particle size40, whereas a faster TEOS addition rate promotes nucleation, resulting in smaller particles41. Thorough washing is essential to remove unreacted reagents and improve nanoparticle stability during storage.

Several additional factors are important for achieving reproducible ex vivo tissue labeling and lesion establishment. Uniform uterine tissue fragment size, homogeneous nanoparticle dispersion, and consistent incubation conditions improve labeling efficiency and reduce experimental variability. Processing unlabeled control tissues in parallel is also essential for distinguishing true Cy5.5 fluorescence from tissue autofluorescence and nonspecific background signals. During lesion induction, consistent hormonal priming of donor mice, gentle implantation of labeled tissue fragments, and appropriate postoperative care minimize tissue trauma and nonspecific inflammation, thereby improving the reproducibility of lesion formation.

Despite these advantages, several limitations should be considered. The persistence and stability of the fluorescence signal in vivo may vary after implantation due to tissue remodeling, cellular proliferation, and immune-mediated clearance. As an exogenous fluorescent label, the Cy5.5 signal is expected to decrease gradually as labeled cells divide, potentially limiting the feasibility of long-term longitudinal studies. In addition, variability in tissue preparation and nanoparticle uptake may influence labeling efficiency between experiments, highlighting the importance of standardized experimental procedures.

Although the present study focuses on ex vivo lesion detection, Cy5.5 is a far-red/near-infrared (NIR) fluorescent dye that minimizes tissue autofluorescence and enables deeper penetration of photons. These optical properties make Cy5.5 well-suited for in vivo imaging applications. Accordingly, the high-quantum-yield Cy5.5 dye-doped silica nanoparticle labeling strategy provides a robust platform for noninvasive longitudinal lesion tracking using NIR fluorescence and photoacoustic imaging43,44.

Future studies should focus on improving fluorescence durability and further optimizing labeling consistency across experimental conditions. Systematic evaluation of signal retention will help define the optimal imaging window for longitudinal studies, while continued refinement of nanoparticle design and labeling conditions may enhance sensitivity and extend the applicability of this approach to other disease models. Overall, this nanoparticle-based strategy provides a rapid, reproducible, and versatile alternative to genetic labeling approaches for visualizing endometriotic lesions and should facilitate mechanistic studies and the preclinical evaluation of diagnostic and therapeutic strategies.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) (R01HD108895 to T.K. and J.W.J.).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
(3-Aminopropyl)triethoxysilane (APTES)Sigma-Aldrich440140Purity ≥99%
17β-EstradiolMilliporeSigmaE8875Purity ≥98%
Ammonium hydroxideFisher ScientificA669S-50028% ammonium hydroxide solution
BD Autoclip Wound Closing SystemBD427631Surgical wound clips
Clear glass threaded vialFisherbrandFS60910A-311.1 mL clear glass vial
Cyanine 5.5 NHS esterSigma-AldrichGEPA15502Near-infrared fluorescent dye
Ethanol, absoluteFisher ScientificBP2818Molecular biology grade
Fetal bovine serum (FBS)Gibco16000044Cell culture supplement
High-speed centrifugeBeckman Coulter Life SciencesAvanti J-EHigh-speed centrifuge
In vivo imaging systemPerkinElmerIVIS SpectrumFluorescence imaging system
IsofluraneBaxterNDC1001-9-360-60Inhalation anesthetic
Magnetic stir barFisherbrand14-513-648 mm
Magnetic stir plateChelmsfordSP88854200Variable-speed magnetic stirrer
Refrigerated centrifugeEppendorf5427 REquipped with FA-45-48-11 rotor
RPMI-1640 mediumGibco11835-030Cell culture medium
Sesame oilMilliporeSigmaS3547Vehicle for estradiol preparation
Tetraethyl orthosilicate (TEOS)Sigma-Aldrich86578Purity ≥99%
Triethylamine (TEA)Sigma-AldrichT0886Purity ≥99%
Ultrasonic bathFisherbrandCPX1800HFor nanoparticle dispersion by sonication

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Endometriosis LesionsEx Vivo LabelingSilica NanoparticlesCy5 5 LabelingFluorescence ImagingUterine TissueIntraperitoneal ImplantationNon Genetic Approach

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