Noninvasive Detection and Thermal Ablation Therapy of Endometriosis Using Silica-Coated Gold Nanorods

AuNR@Si(F)-PEG NPs were prepared as previously described. Briefly, AuNRs were prepared via a seed-mediated growth method using cetyltrimethylammonium bromide (CTAB) as a shape-directing and stabilizing agent. [ 42 , 43 ] The as-prepared AuNRs showed an average particle size of (52.75 ± 1.30 nm × 20.71 ± 1.40 nm) with an extinction peak at 770 nm ( Figure 1A , C ). To enhance the stability and for conjugation with FITC, the synthesized AuNRs were subjected to silica shell coating followed by functionalization with FITC-APTES to yield AuNR@Si(F) NPs. To improve biocompatibility and blood circulation, the NPs were further conjugated with m-PEG-acetic acid (mPEG-AA) using an EDC coupling reaction. The extinction spectra for the AuNR@Si(F)-PEG NPs showed characteristic maximal peaks of AuNR and FITC at 780 and 490 nm, respectively. Excitation of NPs at 490 nm showed an emission peak at 520 nm, which corresponds to the FITC emission with a quantum yield of 0.13% ( Figure 1B ). The AuNR@Si(F)-PEG NPs showed uniform size distribution with an average particle size of (55.81± 1.30 nm × 28.33± 1.80 nm) with a silica shell thickness of 5.2 ± 1.8 nm ( Figure 1D ). As shown in Figure 1B inset, the FITC conjugated AuNRs showed bright green fluorescence under UV light. Owing to the strong NIR absorption peak at 780 nm, the synthesized AuNR@Si(F)-PEG NPs can act as an exogenous contrasting agent for PAI while simultaneously acting as a fluorescence imaging agent due to the inclusion of FITC. A representative phantom image for the synthesized AuNRs with varying concentrations of Au is shown in Figure 1E . The intense signals indicate the PA contrasts from AuNRs with a linearity in the signal intensity with [Au] concentration ( Figure 1F ). Dynamic light scattering (DLS) and zeta potential measurements were performed in every synthetic step to confirm the surface modifications ( Figure S1A – B , Supporting Information ). CTAB-AuNRs showed an average size of 55.84 ± 5.3 nm with a zeta potential value of +38.1 mv due to the presence of CTAB. Silica modification reduced the zeta potential from +38.1 mv to +10.1 mv with a slight increase in the hydrodynamic size of AuNR@Si with an average particle size of 60.98 ± 2.8 nm. The decrease in zeta potential value compared to the CTAB-AuNR indicated the formation of a silica shell on the surface of AuNR. Conjugation of FITC-APTES with AuNR@Si further increased the zeta potential from +10.1 mv to +18.4 mv with an increase in the hydrodynamic size of AuNR@Si(F) NPs from 60.98 ± 2.8 nm to 85.19 ± 3.8, due to presence of free amino groups. Finally, the conjugation of mPEG-acetic acid with free amino groups on AuNR@Si(F) using EDC coupling reaction increased the hydrodynamic size of AuNR@Si(F)-PEG NPs to 93.40 ± 3.3 nm with an overall zeta potential value of −11.0 mv. As shown in Figure S1A , the DLS studies showed an acceptable PDI value ranging between 0.1 to 0.2, indicating a highly monodisperse particles in every surface modification. The presence of various functional groups and surface modifications on AuNR was further confirmed by Fourier transform infrared spectroscopy (FT-IR) analysis ( Figure S2 , Supporting Information ). CTAB-AuNR showed two major bands at ~ 2933 and ~ 28545 cm −1 due to the asymmetrical and symmetrical vibrations of CH 2 units from CTAB molecules, respectively. As the CTAB-AuNRs is functionalized with TEOS for silica modification, these peaks showed a reduced intensity due to the replacement of CTAB with silica. IR spectrum showed two characteristic peaks, 1098 cm −1 and 810 cm −1 , corresponding to the Si-O-Si bonds. The peak at 937 cm −1 indicated the presence of Si-OH bonds in the AuNR@Si NPs. As shown in Figure S2 , the conjugation of FITC with APTES was confirmed by the IR peaks at 1635 cm −1 corresponding to the C=N stretching vibration of imines. The peaks at 1104, 920, and 840 cm −1 confirm the IR stretching bands for Si-O-Si bonds in FITC-APTES. Conjugation of this FITC-APTES with AuNR@Si showed the characteristic peaks for imine (1635 cm −1 ) and Si-O-Si (1098 cm −1 , 917 cm −1 , and 837 cm −1 ), respectively. IR spectrum of PEGylated AuNR@Si(F)-PEG NPs showed characteristic peaks at 2885 cm −1 corresponding to symmetric and antisymmetric C-H vibrations in PEG. The IR band at 1640 cm −1 corresponds to the -NH-CO- bond from APTES and PEG acetic acid. The Si-O-Si peaks were characterized by the peak positions at 1103, 919, and 839 cm −1 in the PEG conjugated AuNR@Si(F)-PEG NPs. The additional peaks in the IR spectra at 1155 and 1046 cm −1 confirm the -C-O- stretching vibrations in the PEG backbone. The stability of AuNR@Si(F)-PEG NPs was evaluated using optical absorption spectroscopy and DLS measurements in various dispersing media, including water, simulated body fluid (SBF), fetal bovine serum (FBS), and phosphate buffered saline (PBS) (pH 7.4). Results showed no significant changes in the absorption or emission spectra over time, indicating stability in different environments for AuNR@Si(F)-PEG NPs ( Figure S3A – D ; Figure S4A – D , Supporting Information ). DLS analysis confirmed consistent nanoparticle size across all media, with a PDI value below 0.2 for up to 15 days ( Figure S5A , Supporting Information ). To assess whether FITC was leaching, the nanoparticles were centrifuged (8000 rpm, 10 minutes), and the fluorescence of the filtrate was monitored. This process was repeated over several days. No detectable fluorescence was observed, confirming the absence of FITC release in any dispersing media used ( Figure S5B , Supporting Information ). Additionally, fluorescence quantum yield calculations revealed no substantial variations during the stability study, further supporting the integrity of the nanoparticles ( Figure S5C , Supporting Information ). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was conducted for the AuNR@Si(F)-PEG NPs at concentrations from 1 to 75 μg/mL to assess their cytotoxicity in 12Z human endometrial epithelial cells. As depicted in Figure S6 , Supporting Information , the nanoparticles are nontoxic to the cells at concentrations up to 75 μg/ml. These results suggested that the biocompatible nanoparticles have no significant impact on cell viability with 12Z cells in vitro. As shown in Figure 2A – B , the accumulation and cellular uptake of NPs was examined in a dose-dependent manner. The NPs were accumulated in the cytoplasm of the cell, indicating AuNR@Si(F)-PEG can be used as a cell tracking and imaging agent. On quantification using Image J software, the NP uptake was 96.2% for 20 μg/ml [Au] content, whereas a 93.8 % uptake was observed for [Au] concentration of 50 μg/ml. Gold nanorods (AuNRs) exhibit strong light absorption within the biological transparency window, with a peak absorbance at 780 nm ( Figure 1A ). Their ability to convert laser energy into heat makes them promising candidates for photothermal therapy. To assess their photothermal efficiency, a 0.5 mg/mL AuNR solution was exposed to an 808 nm laser (2 W/cm 2 ) in multiple 10-minute cycles, allowing cooling between exposures. The cooling rate constant was calculated ( Figure S7A – B , Supporting Information ). A FLIR thermal imaging camera was used to monitor the temperature within the NPs during the laser irradiation studies ( Figure S7C – D , Supporting Information ). The AuNR@Si(F)-PEG NPs demonstrated a progressive increase in maximum temperature, reaching 49.5 °C. As a control, CTAB-AuNRs were tested to evaluate the impact of silica coating on laser-induced heating. CTAB-AuNR showed a maximum rise in temperature reaching 43.7 °C. The calculated photothermal conversion efficiencies (η) for AuNR@Si(F)-PEG NPs and CTAB-AuNRs were 52.1% and 48.4%, respectively. Notably, DLS measurements revealed no significant size changes in AuNR@Si(F)-PEG NPs compared to CTAB-AuNRs after repeated heating cycles ( Figure S8A , Supporting Information ). TEM imaging further confirmed the stability of the silica-coated NPs, whereas CTAB-AuNRs exhibited morphological deformations ( Figure S8B – C , Supporting Information ). The absorbance and fluorescence measurements further supported the stability of AuNR@Si(F)-PEG NPs after the laser studies without any change in the peak positions ( Figure S8D – E , Supporting Information ). Thus, during the photothermal studies, the FITC dye retained its emission properties due to the presence of silica shell and PEG on the surface of AuNRs. These findings highlight the enhanced stability and reusability of AuNR@Si(F)-PEG NPs, making them suitable for photothermal-based biomedical applications. AuNRs are known for their high photothermal conversion efficiency upon NIR light irradiation, which can be exploited for tumor ablation. Motivated by the promising stability and remarkable light-to-heat conversion capabilities of AuNR@Si(F)-PEG NPs, we conducted photothermal toxicity experiments on 12Z cells in vitro using an 808 nm laser at 2.0 W/cm 2 for 1 minute using various concentrations of Au. We observed 30% cell viability after treatment with 20 μg/ml of AuNR and laser exposure. Similarly, for a concentration of 10 μg/ml of AuNR, photothermal studies showed a cell viability of 50%. This indicates PTT can effectively induce cell death in 12Z cells. Conversely, non-radiation experiments demonstrated no statistically significant reduction in cell viability ( Figure 2C and Figure S6 , Supporting Information ). These in vitro findings suggest that AuNR@Si(F)-PEG NPs can be used as photothermal ablation agents for endometriosis. The induced early apoptosis in the 12Z cells was further validated using live and dead cell staining studies. As shown in Figure 2D , when combined with nanoparticle and laser treatments, we saw distinctive apoptotic cell death as indicated by propidium iodide staining (red color) under fluorescence microscopy. Using Image J software, we quantified the cell death and results showed a 47.8% cell death under laser irradiation using AuNR@Si(F)-PEG NPs. In contrast, the cells treated with NPs without laser treatment and cells treated with laser alone showed significant live cells, as confirmed by the calcein-AM staining (green color). The induction of endometriosis mouse model was conducted through a meticulous procedure involving the excision and transplantation of uterine horns from hyperestrogenic donor Balb/c mice into recipient Balb/c mice, as reported previously ( Figure 3A ). [ 44 , 45 ] We examined the presence of endometriosis lesions by microscopic observations of endometriotic cysts and histological analyses. Utilizing IVIS Spectrum in vivo imaging, we further confirmed the endometriosis development as shown in Figure 3B – C . Endometriosis is an angiogenesis-associated disease characterized by a high neovascularization surrounding endometriotic lesions. [ 46 , 47 ] Because of the leaky blood vessels and poor lymphatic drainage in the lesions, AuNRs can passively accumulate due to the enhanced permeability and retention (EPR) effect. [ 11 ] Additionally, PEGylation improves the adhesion of the AuNRs to the lesions. EM lesions have larger openings compared to normal tissues, facilitating the easier passage of PEGylated nanoparticles into the lesions. The structural abnormalities in these lesions, in contrast to normal tissues, play a significant role in allowing PEGylated NPs to adsorb onto the lesions. The presence of AuNR@Si(F)-PEG NPs in the lesion was further confirmed by excising the lesion from the mice after the injection of NPs, and analyzed under fluorescence microscope. As shown in Figure 3D a bright green fluorescence was observed at the lesion site corresponding to FITC from the NPs. Further validation of endometriosis tissue using estrogen and progesterone receptors is necessary for differentiating the pathology of endometriosis from other similar tissue types [ 48 , 49 ] . We used immunostaining to identify estrogen receptor 1 (ESR1) and progesterone receptors (PGR). The presence of these receptors showed characteristic features of stroma and glands, which appeared in brown after staining studies ( Figure S9A – B , Supporting Information ). After four weeks of lesion establishment in the recipient mice, we conducted noninvasive whole-body PAI. Subsequently, we excised the lesions guided by fluorescence. The NPs were intravenously injected into mice bearing endometrial lesions, and the PA signals from the AuNR were monitored over time. As shown in Figure 4A , a clear and distinct PA signal (green) for AuNR was detected and was colocalized with total hemoglobin (HbT) (yellow) in the lesions. This observation suggests an anatomical localization of AuNR@Si(F)-PEG accumulation within tissues, concurrently exhibiting heightened blood flow surrounding the ectopic endometriotic lesions. The PAI showed peak NP uptake at 2 hrs as indicated by higher signal intensity, and they also showed a sustained signal lasting for 24 hrs. After 48 hrs, there were no PA signals in the lesions, indicating clearance of NPs, which is critical for nano-based therapies ( Figure S10A , Supporting Information ). [ 50 ] A statistically significant rise in particle signals within the lesion was observed for 2 h, compared to the pre-injection and other post-injection time points ( Figure 4B and Figure S10B , Supporting Information ). The nanoparticle’s presence did not influence the total hemoglobin saturation ( Figure 4C ). PA images were collected 2 hrs post-injection, the mice were euthanized, and the ectopic endometriosis lesion and other major organs (heart, liver, lung, spleen, kidney, uterus) were collected. The biodistribution of AuNR@Si(F)-PEG NPs in the organs was investigated under IVIS fluorescence imaging. As shown in Figure 5A , a strong fluorescent signal was observed in the liver and the lesion. Since the liver acts as a biological filtration for reticuloendothelial system (RES) uptake, a large accumulation of NPs occurs during circulation. [ 51 – 53 ] Quantitative analysis showed a significant increase in the FITC signal from the lesions compared to the other organs or the uterus ( Figure 5B ). To quantify the Au content, we have performed inductively coupled plasma optical emission spectroscopy (ICP-OES) for the major organs and lesions collected. As shown in Figure 5C , the major uptake of AuNR was observed in liver with an uptake of 28.2 μg [Au]/g of liver. The uptake in endometriosis lesion was observed to be 12.8 μg [Au]/g of lesion. Spleen and kidney showed an uptake of 2.4 μg [Au]/g of the organ weight. There was no detectable Au content in the other organs, such as lungs, heart, and uterus. A histological examination of ectopic endometriosis lesions was further performed to validate the results obtained from PA and fluorescence imaging. Endometriosis tissue reveals the presence of endometrial epithelial glands and stroma, and E-cadherin is strongly expressed in endometrial surface epithelium. From the tissue section, we detected E-cadherin-positive gland-like structures encircled by stromal cells, exhibiting similar structures and staining intensity to the eutopic endometrium, thus confirming the identity of endometriosis-like tissue ( Figure 5C ). Additionally, gold enhancement staining was performed on tissue sections from the endometriosis-like lesions, revealing enlarged gold labels in the lesions after the nanoparticle injection ( Figure S11 , Supporting Information ). Further hematoxylin and eosin (H&E) staining from the lesion tissue was done, and bright-field microcopy images were taken. Subsequent histological examination of the endometriotic tissues revealed distinct endometrial glands (black arrows) and stroma (green arrows), accompanied by noticeable vascular structures, including small arteries, as illustrated in Figure S12 ( Supporting Information ). [ 54 ] This meticulously conducted study through imaging and histopathology highlights the establishment of the endometriosis model and systemic distribution of functional nanoparticles within the ectopic endometriosis lesions, offering potential diagnostic and therapeutic avenues. The therapeutic effect of AuNR@Si(F)-PEG NPs as photothermal therapy (PTT) agents was evaluated using laser irradiation in vivo after the intravenous injection of NPs. For this study, we have prepared a stitch model from the mice, as shown in Figure 6A from the previous reports. [ 44 ] The mice weight used for in vivo experiments was ≈24–25 gm, and we did the post-care medications after surgery by injecting Meloxicam (0.25 mg/kg of mice). After validating the lesion site in the stitch model ( Figure S13 , Supporting Information ), AuNR@Si(F)-PEG NPs (50 μg/mL) were administrated into the mice via IV. injection, and after one hour of injection the lesion was irradiated using an 808 nm laser for 2 minutes. Control mice were used for the study in which the same dose of NPs was administrated but without laser application. After laser irradiation, the mice underwent PAI, and the signal intensity was monitored in real-time. A strong PA signal intensity was observed for both non-treated and laser-treated mice after the first dose of NPs ( Figure 6B ). Subsequently, during the second and third doses of NPs + laser irradiation, there was a decrease in PA signal intensity as compared to the non-treated mice. After the fourth dose + laser irradiation, there was a decrease in the PA signal intensity by 6 times as compared to the non-treated mice ( Figure 6B – C ). At the same time, the control mice without laser irradiation showed prominent PA signals at the lesion during the entire treatment period without laser irradiation ( Figure 6B ). To validate this further, we have performed biodistribution studies for the lesions after the treatment. As shown in Figure 6D , a strong fluorescence signal from FITC was observed in the lesion for the non-treatment mice, whereas the PTT-treated mice showed no fluorescence. This experiment correlates the targeting ability of NPs via the EPR effect at the lesion site. Moreover, PA imaging, which noninvasively determines the particle signals, can be used as a guidance for target validation and for treatment monitoring. A noticeable decrease in the lesion size (4 times) was observed for the mice after laser irradiation compared to the non-treated mice ( Figure 6D ), and there was no change in the body weight of the mice after the PTT, indicating the mice remained healthy throughout treatment ( Figure S14 , Supporting Information ). The photothermal effect of AuNR resulted in apoptosis at the lesion during the PTT and resulted in lesser uptake during the additional dosage of NPs. The H&E staining results show that the loss of graft and stroma glands in the PTT-treated mice as compared to the non-treated mice was detected, which indicates the designed NPs can be used as therapeutic agents for endometriosis ( Figure S15A – B , Supporting Information ). The cleaved Caspase-3 expression from the tissues further validated the early apoptosis at the lesion after the PTT with NPs, as compared to the mice injected with only NPs (no laser) and those with only the laser treatment (no NPs) ( Figure S16 , Supporting Information ).

In conclusion, our study demonstrates the fabrication of a nanoparticle system, AuNR@Si(F)-PEG NPs, for the specific targeting and photothermal therapy for endometriosis. We have incorporated FITC onto the surface of silica-coated AuNRs to enable multi-scale (micro to macro) identification of the lesions by fluorescent imaging and PAI. Owing to the high PA signal intensity from AuNRs, we can easily target the lesion site in vivo under PA image guidance. The studies showed a signal enhancement in the lesion for 3 h and sustained signals lasting 24 hours. Histopathological studies showed the characteristic features of the endometriosis lesions, and gold enhancement assay showed the particle uptake at the lesions from the mice. Comprehensive in vitro and in vivo studies showed that our designed PEGylated NPs can target and be preferentially taken up by the lesions via the EPR effect. Furthermore, the therapeutic effect of NPs was studied using laser-assisted photothermal heating and monitored the efficacy via PAI signals at different administration and irradiation times in vivo. The reduced lesion size and PA signal intensity from NPs during the treatment procedures indicated that NPs can be used as a theranostic agent for endometriosis treatment. Our multifunctional NPs, designed with multimodal detection capabilities and therapeutic efficacy, will help to diagnose, treat, and understand endometriosis.

Endometriosis is a complex disease without clinically approved noninvasive diagnostic biomarkers. [ 8 , 55 ] Clinical trials ( NCT03376451 ) for validating specific biomarkers for diagnosing and monitoring the recurrence of endometriosis are underway, but results are pending. Current noninvasive imaging techniques struggle to detect all lesion types and sizes, highlighted by a recent review for their poor methodological quality and inability to match the accuracy of surgical evaluation. [ 56 ] However, using targeted contrast agents shows promise for improving detection through imaging techniques like PAI. [ 19 , 24 ] To have nanomaterials better suited for therapeutic and diagnostic purposes in endometriosis, there needs to be an effort to achieve the highest sensitivity and specificity and additional work to ensure they are safe for use in living systems. Here, we have designed a AuNR-based theranostic system for detecting, imaging, and treatment of endometriosis lesions both in vitro and in vivo. Our studies utilized mouse models with established endometriosis lesions and defined vasculature as confirmed by the H & E staining studies. AuNRs can target the lesions due to the EPR effect, and PEGylation allows prolonged systemic circulation of NPs with preferential accumulation in the angiogenic endometriosis lesion. [ 11 ] The attachment of FITC to the surface of AuNRs allowed for detecting lesions ex vivo via fluorescence imaging. The endometriosis lesions were further detected using the PAI technique, and a prominent, sustained PA signal was observed in the lesion for 24 hours, followed by complete clearance of the particles after 48 hours. Thus, the present study highlighted the development of a new multimodal imaging nanoparticle for the accurate detection of endometriosis lesions. AuNRs possess excellent photothermal properties and have been used for tumor ablation studies. [ 57 ] Using AuNRs for photothermal therapy offers a promising approach to treating endometriosis by utilizing their unique plasmonic absorption to generate localized heat, leading to the destruction of the targeted tissue. This method leverages our ability to functionalize the nanorods for specific targeting and provides a minimally invasive treatment option. Furthermore, the nanoagents can offer adaptable utility in theranostic, to assess their delivery and therapeutic efficacy in endometriosis treatment via noninvasive imaging. [ 58 ] In AuNR-assisted PTT, temperatures typically reach between 41°C to 47°C, inducing apoptosis (programmed cell death), or higher temperatures (above 50°C) causing necrosis (uncontrolled cell death). The heat denatures cellular proteins, disrupts cell membranes, and damages the tumor vasculature, leading to the death of cells and, in the case of cancer, the reduction of the tumor mass. We have studied the photothermal ablation effect of AuNRs in both in vitro and in vivo. The measured cleaved caspase-3 activity showed an early apoptosis effect in the lesions after the PTT treatment. Indirectly, lesion size was assessed in real-time using the PA signal intensity of AuNRs at the lesion site. Studies showed that an injection of 50 μg/ml of AuNRs reduced lesion size (4 times) after the laser treatment. Guo et al showed that TYL peptide conjugated hollow gold nanostructures with an administration dose of 2.5mg/Kg can reduce the lesion volume by 92.7% upon PTT. [ 59 ] Moses et al showed the use of silicon naphthalocyanine as a NIR imaging and PTT based therapeutic for endometriosis with an administration dose of 3 mg/kg, can suppress the lesion volume dramatically. [ 48 ] The present study highlight the importance of a non-invasive pathway for endometriosis lesion detection and simultaneous therapeutical applications with a lower dose of NPs, such as 0.05 mg/kg of mice with a 77% reduction in the lesion volume. In summary, our theranostic nanoparticle system can be used for the simultaneous detection and treatment of endometriosis lesions. In this study, we have used two mice models: a systemic model and a stitch model. The stitch model was beneficial for the initial efficacy testing of the nanoparticle agent because it identified the exact location of the lesions. On the other hand, the systemic model allowed the further validation of the targeting potential of our designed nanoparticles in a more realistic scenario. AuNRs and PAI-based tools hold significant promise for enhancing laparoscopic procedures and advancing targeted therapeutic strategies. In future work, the application of AuNRs as adjuvants in laparoscopic surgery could improve visualization and enable real-time monitoring of surgical sites, thus enhancing precision and outcomes. Additionally, modifications in these tools for active targeting could facilitate the selective delivery of therapeutic agents to tumor sites, minimizing side effects and improving treatment efficacy. Furthermore, integrating these technologies into existing laparoscopic frameworks could pave the way for innovative minimally invasive procedures, leading to better patient recovery and overall surgical success. Continued research in this field may result in the development of multifunctional platforms that combine imaging and therapy, transforming the landscape of minimally invasive surgery.

Hexadecyltrimethylammonium bromide (CTAB), gold (III) chloride (HAuCl4), silver nitrate (AgNO3), sodium borohydride (NaBH4), L-ascorbic acid (AA), tetraethyl orthosilicate (TEOS), 3-amino- propyltriethoxysilane (APTES), sodium hydroxide (NaOH), Methanol (MeOH), Fluorescein isothiocyanate (FITC), 3-(4,5-dimethylthiazolyl-2)-25-diphenyl tetrazolium bromide (MTT), were purchased from Sigma-Aldrich (St. Louis, MO, USA). M-PEG-acetic acid was purchased from Biopharma PEG (Watertown, MA, USA). Gold nanorods (AuNRs) were synthesized by the seed-mediated growth method. [ 42 ] A seed solution was prepared by mixing 0.25 mL of 0.01 M HAuCl4 with 7.5 mL of 0.1 M CTAB solution. After stirring for 2–3 minutes, 0.6 mL of an aqueous 0.01 M ice-cold NaBH4 solution was added, and the reaction was kept stirred for 3 h at 30 °C. A growth solution was prepared by mixing 4.75 mL of 0.10 M CTAB, 0.2 mL of 0.01 M HAuCl4, and 0.030 mL of 0.01 M AgNO3 solutions in the order, one by one. To this well-stirred solution, 0.032 mL of 0.10 M AA was added and mixed well. To this colorless solution, 0.01 mL of premade Au seed solution was added, and mixed gently. The solution was left undisturbed for another 4 h. Following this period, the pink-colored solution was centrifuged (8000 rpm, 10 minutes, 3 times), redispersed in distilled water, and characterized by spectroscopy and transmission electron microscopy studies. As prepared AuNRs (2.9 nM) were dispersed in 5 mL of water and mixed with 20% of TEOS solution in MeOH and 0.1 mL of 0.1 N NaOH solution. The solution was stirred at 40 °C for 1 h. Silica-coated AuNR was collected by centrifugation (8000 rpm, 10 minutes, 3 times) and redispersed in 5 mL of distilled water. [ 60 ] The FITC-APTES conjugate was synthesized by combining FITC (10 mg) with 44 μL of APTES (with a molar ratio of FITC: APTES = 1:10) in 0.75 mL of ethanol, kept in darkness for 2 days. Subsequently, 50 μL of the resulting FITC-APTES solution was introduced into the AuNR@Si NP suspension in ethanol and stirred for 3 h. The resulting particle suspension, AuNR@Si-FITC nanoparticles, underwent three ethanol washes to eliminate unreacted components and were finally dispersed in 3 mL of aqueous solution. As prepared AuNR@Si-FITC NPs (2.4 nM, Optical density 1), were diluted with water. To this, m-PEG-acetic acid (25 mg) and EDC-HCl (50 mg), was added and the reaction was stirred for 12 h at room temperature. The product obtained was centrifuged (8000 rpm, 10 minutes, two times), washed with water, and redispersed in 3 mL of water for further characterization. Transmission electron microscopy (TEM) imaging was performed using a 2200FS transmission electron microscope manufactured by JEOL in Japan. Specimens for TEM were prepared by applying a small quantity of nanoparticle suspension in water onto Cu grids coated with carbon. UV–visible absorption and fluorescent spectrum of the nanoparticles were recorded using a microplate reader (SpectraMax; Molecular Devices). FTIR spectrum for the samples was recorded using a PerkinElmer Spectrum 2 FTIR instrument. DLS and Zeta potential measurements were performed on Zeta Sizer Nano, Malvrn Instruments. Briefly, 12Z cells were seeded in 6 well plates and allowed to incubate at 37 °C with 5% CO 2 overnight. The following day, the cells were treated with particles at the concentrations such as 20 μg/ml; 50 μg/ml. The cells were again incubated overnight, and the next day they were counterstained with calcein AM and DAPI to allow for the visualization of the cell membrane and nucleus respectively. The cells were then imaged on a Keyence fluorescence microscope. All animal experiments performed were approved by the Institutional Animal Care and Use Committee (IACUC ID: PROTO202200363) at Michigan State University, while animal care and wellbeing throughout the study was monitored by the Center for Animal Resources (CAR) at Michigan State University. A total of 6, 8-week-old female adult Balb/c mice were used for the studies. 17-B-estradiol (100 μL, 1 μg/mL in sesame oil) was administrated from day 0 and continued till 3 days. Afterward, the donor mice were sacrificed, and the uterine horns were removed under sterile conditions. The uterine horn was opened longitudinally with scissors and cut into small fragments of about 1 mm3 in a petri dish with a scalpel. Tissue fragments were placed into 500 μL PBS and tissue fragments were injected into the peritoneal cavity of the recipient mouse. The abdominal incision was closed with sutures for the peritoneum and wound clips for the skin. The mice were left for three weeks to develop endometriosis-like tissues and used for the studies. One of the mice was sacrificed for histopathological studies. The remaining mice were then divided into two groups control group and treatment group. The control group mice were injected with PBS and the treatment group mice were injected with AuNR@Si(F)-PEG NPs. For the in vivo photothermal therapy studies, we used a stitch model mouse. After the estradiol administration, the mice were sacrificed and the uterus horn of the donor mice was opened longitudinally, sliced, and kept in 500 μL PBS. These tissue fragment then sutured into the muscle near the uterus in the recipient mice by making an incision. The incision was closed using sutures and wound clips for skin. The mice were left 3 weeks to allow for the growth of endometriosis type lesions. Photoacoustic imaging was conducted with the Invision 512-echo preclinical multispectral optoacoustic tomographic imaging (MSOT) system from iThera Medical (Munich, Germany). The transducer operated at a central frequency of 5 MHz. MSOT utilizes pulsed laser lights spanning wavelengths from 680 to 980 nm to visualize images and detects ultrasound waves emitted by photo-absorbing molecules like oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and imaging agents within tissue. Before imaging, mice were anesthetized with isoflurane, their abdominal area was shaved and cleared of hair using a commercial hair removal cream, and warm ultrasound gel was applied. Mice were then secured in a holder with a thin polyethylene membrane to ensure acoustic coupling and submerged in warm water during imaging. Isoflurane and oxygen were supplied through a breathing mask throughout the process. Imaging was conducted by moving the stage over the abdominal area, with 10 averages per illumination wavelength (680, 700, 730, 760, 800, and 850 nm) and a scanning step size of 0.3 mm, completing in less than 20 minutes per mouse. Acquired images were reconstructed using a back-projection algorithm, and linear spectral unmixing was performed using ViewMSOT software to create component images for each absorber (gold, Hb, HbO 2 , HbT) from the composites. In post-processing, all layers of multispectral images were auto-scaled using an imaging threshold tool to eliminate low-signal visualization. Quantitative analysis of fluorescence intensity was carried out using the IVIS Spectrum in vivo imaging system in conjunction with Living Image software from PerkinElmer (Waltham, MA). Fluorescence measurements were derived from normalized radiant efficiency units obtained through manual selection of regions of interest after adaptive fluorescence background subtraction. For fluorescence imaging of tissue sections, frozen samples embedded in OCT were sectioned at 10 μm thickness and mounted on slides. These slides were then cover slipped using DAPI mounting media from Vector Laboratories (Burlingame, CA, Cat. #H-1800) to facilitate imaging. Immunostaining procedures involved fixation of frozen tissue sections in 4% paraformaldehyde, followed by treatment with 0.3% hydrogen peroxide in methanol and washing with Triton X-100 solution. Blocking was carried out using 10% normal goat serum (NGS) in pH 7.5 PBS, followed by overnight incubation with primary antibodies diluted in 10% NGS in PBS at 4°C. The primary antibody used was anti-E-cadherin (dilution 1:1000; CS-3195, Cell Signaling Technology, Danvers, MA). Subsequently, species-specific fluorescently tagged secondary antibodies (Donkey anti-Rabbit IgG Alexa Fluor 594, Invitrogen, Carlsbad, CA, Cat. # A-21207) were applied before coverslipping with DAPI mounting media for imaging. Imaging of the immunostained samples was conducted using a Leica DFC9000 GTC THUNDER microscope coupled with a camera that can capture images at a rate of 90 fps full frame via the Camera Link interface. 6 μm tissue slides were washed with PBS (2x for 5 minutes) to remove OCT. Slides were dried by hand to avoid damaging the tissue and circled by a PAP Pen to allow for low-volume staining. 10% normal goat serum solution was applied to the slides for 2 hours at room temperature as a blocking solution. Slides were then drained without washing. HRP-conjugated primary antibodies were applied to cover the tissue at a 1:300 dilution in PBS, and the slides were incubated overnight at 40C. The next day, the slides were washed three times in PBS to ensure that any unbound HRP conjugated antibody was removed completely. Then, prepared 3,3’-diaminobenzidine (DAB) was applied to the slides for 5 minutes. The slides were washed with tap water (2x for 5 minutes) and counterstained with nuclear fast red. Slides were washed twice in tap water, dried by hand, and then allowed to dry on the bench top. Then, the slides were mounted, cover slipped and imaged on a Keyence optical microscope. H&E staining was performed in the following manner. First, the OCT was removed from the tissue by water washing. Then, the tissue was stained for 30 seconds in hematoxylin solution. The tissue was then washed twice in water, blued for 1 minute and 30 seconds, and washed one additional time in water. Then the tissue was stained for 30 seconds in an eosin solution followed by three washes in ethanol. Coverslips were then affixed to the slides using poly-mount xylene for optimal imaging. Brightfield imaging of H&E-stained slides was performed using a Keyence optical microscope. Microscopic visualization enhancement using the Gold-Enhance LM kit (Nanoprobes, Inc, Yaphank, NY, Cat. #2112–28) was conducted on frozen tissue samples embedded in OCT, sliced at 10 μM, and then mounted on slides. The protocol followed the manufacturer’s instructions. The 12 Z cells in DMEM media were seeded at 5000 cells/well in 96 well plates. NP treatment was performed by diluting particles to the desired concentration in media. The following day, the plates were washed and redispersed the cells in media. For laser studies, each well was then subjected to an 808 nm laser, for 2 minutes (2 mW/cm2), and left overnight. Following this period, a standard MTT assay was performed, and the cell viability was measured using optical spectroscopy. For these studies, we prepared suture model mice. Afterward, AuNR@Si(F)-PEG NPs (50 μg/mL) were administered to the mice via IV injection. The lesions were then subjected to laser irradiation for 2 minutes using an 808 nm laser (2 mW/cm2). The in vivo PTT study performed in such a way the NPs were injected via IV on day 1 and day 3. The laser irradiation was performed on every alternative day from day 1 to day 5. The tumor size and AuNR signal intensity were monitored using a PAI system. The treatment was continued every other day, and images taken each day were recorded. To determine the photothermal conversion efficiency (η) for CTAB-AuNRs, and AuNR@Si(F)-PEGNPs results from Figure S7A – B were analyzed. The cooling rate constant (𝜃) was calculated from Figure S7B as shown in equation 1 . (1) θ = ( T − T surr ) / ( T m a x − T surr ) Where T (°C) = temperature at any time point within the cooling cycle, Tsurr (°C) = temperature of the solvent, Tmax (°C) = maximum temperature reached within the cooling cycle. Tsurr was determined using a vial of water under the same conditions. Once θ was calculated for every given temperature within the cooling cycle, a τ (s) time constant is determined using the inverse relationship between the time and −ln(θ) of the same cycle. τ is found to be the slope of the linear correlation, which is seen in eq 2 . t (s) = any given time during the cooling period. (2) t = τ l n ( θ ) After deducing the value of τ for each of the particles, the value for hs (J/s°C) can be calculated. hs is represented by the heat transfer coefficient (h) and total surface area of the solution (s) and is calculated using eq 3 . m (g) = mass of the solution, C (J/g °C) = specific heat capacity of the solution. (3) h s = m C τ Finally, the photothermal conversion efficiency can be calculated using eq 4 . Qsurr (J/s) was determined from a vial of water exposed to the same conditions. I(W) = laser power, A808 = absorbance of the particle solution at 808 nm. (4) η = h ( T m a x − T s u r r ) − Q surr / I ( 1 − 10 − A 808 ) Statistical analysis was conducted using GraphPad, with all data presented as mean ± SD. Cell-based experiments were conducted in a sterile environment with a minimum sample size of n = 6. Whereas in vivo experiments performed for n = 3. Significance was assessed via the student’s unpaired t-test, considering P-values below 0.05 as statistically significant.

Endometriosis (EM), a prevalent gynecological disorder affecting millions of women worldwide, is characterized by the presence of endometrial-like tissue outside the uterine cavity, leading to chronic pelvic pain, infertility, and substantially impaired quality of life .[ 1 – 3 ] Despite affecting an estimated 10% of reproductive-age women, EM remains severely underdiagnosed, often taking 7 to 10 years from symptom onset to confirmation. [ 4 , 5 ] Traditional imaging modalities such as ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT) scans have been utilized for diagnosis since the 1970s but their effectiveness remains limited for EM lesions. [ 6 – 8 ] However, while CT scans are effective for identifying thoracic endometriosis, they often fail to image pelvic lesions, which are more common. [ 9 ] MRI provides improved detection of deep-infiltrating and peritoneal lesions but requires precisely timed scans during the menstrual cycle and the use of peristaltic inhibitors. Moreover, smaller superficial endometriosis lesions and are unable to be readily visualized by MRI. Ultrasound often fails to provide sufficient information due to the lack of acoustic impedance diversity between EM lesions and nearby tissues. [ 10 ] Due to the limited utility of imaging modalities for diagnosis, invasive laparoscopic surgery and biopsy remain the gold standard despite related risks such as bowel injuries and bleeding. Reliance on invasive procedures contributes to a significant delay in diagnosing EM. Thus, there is a critical need for specific, sensitive, and non-invasive diagnostics for EM, which will ultimately lead to early treatment and prevention of disease progression. Recently, advancements in biomedical imaging techniques have opened new avenues for accurate diagnosis of endometriosis and a better understanding of endometriosis pathology. [ 11 ] Studies using targeted nanoparticles, such as those functionalized with RGD peptides or folic acid, have shown enhanced localization of magnetic nanoparticles to EM lesions for MRI detection. [ 12 , 13 ] Among emerging techniques, photoacoustic imaging (PAI) stands out due to its exceptional ability to combine optical contrast with ultrasound resolution—allowing real-time, high-resolution imaging deep within tissues. [ 14 , 15 ] PAI utilizes the photoacoustic effect based on the ‘light-in/sound-out’ approach, where nanosecond pulsed lasers are absorbed by tissues, inducing rapid thermoelastic expansion, and generating ultrasonic waves that are detected and reconstructed into high-resolution images. [ 16 ] PAI can exploit endogenous contrasts such as hemoglobin and lipids, enabling non-invasive visualization of vascular networks and tissue composition with exquisite detail. Researchers have explored PAI for visualizing EM lesions, taking advantage of their unique bleeding patterns and optical absorption characteristics during the menstrual cycle. Notably, PAI demonstrated remarkable contrast imaging of EM tissues implanted subcutaneously in a nude mouse model, achieving high sensitivity and specificity of imaging. [ 17 ] However, the use of endogenous blood contrast at the wavelength of 532 nm (label-free imaging) is limited in tomographic imaging applications to detect deep-seated lesions because of the restricted tissue penetration ability of visible light (<2 mm). Moreover, certain EM lesions lack inherent colorization and share thermoelastic properties with normal tissues, further diminishing the effectiveness of endogenous contrast agents for PAI. [ 18 , 19 ] Exogenous contrast agents especially nanoparticles (NPs) can overcome these limitations by tailoring absorption properties, improving photothermal conversion efficiency, and enabling high-contrast, target-specific imaging. [ 20 , 21 ] Exogenous nanoparticle contrast agents are able to label and visualize lesions with a high signal-to-noise ratio in vivo, which allows for clear distinction between EM lesions and surrounding normal tissues. [ 22 , 23 ] Recent advancements in PAI technology have facilitated the development of targeted contrast agents for molecular imaging, allowing specific interrogation of key biomarkers associated with EM pathogenesis. [ 19 , 24 ] Furthermore, in vivo imaging by contrast agents holds promise for delineating molecular signatures and elucidating the underlying mechanisms driving lesion development and disease progression. Notably, NPs offer theranostic potential, serving both as imaging agents and tools for photothermal therapy at the disease site. [ 25 – 27 ] Nanoparticles (NPs), such as gold [ 28 , 29 ] and silica-based materials [ 30 , 31 ] , convert absorbed light in to heat under NIR laser exposure leading to localize tissue ablation. This precise heating allows targeted lesion destruction while sparing surrounding healthy tissue, making it a promising method for minimally invasive treatment. [ 32 , 33 ] Gold nanoparticles (AuNPs) are promising exogenous PAI contrast agents due to their tunable optical properties, high heat conductivity, and biocompatibility. [ 34 , 35 ] While anisotropic AuNPs like nanorods and nanoplates show strong NIR absorption, they are prone to shape distortion under repeated laser exposure, causing signal loss. [ 36 – 39 ] This results in PA signal decay, making anisotropic gold nanostructures unsuitable for continuous high-contrast imaging. Thus, it is crucial to develop gold nanoparticles with strong, angle-independent optical absorption and high photostability for effective PAI. To enhance stability and maintain imaging performance, AuNPs can be coated with silica shells, which also allow further surface functionalization. [ 36 , 40 , 41 ] In this work, we highlight the advantages of PAI in detecting EM lesions by the intravenous injection of contrast agents in murine models of endometriosis. We utilized PEGylated, FITC-tagged, silica-coated gold nanorods (AuNR@Si(F)-PEG), known for their robust PA response, photothermal properties, and biocompatibility. AuNRs are known for their excellent PA properties, light-to-heat conversion ability under UV–vis–NIR laser irradiation, and biocompatibility. The silica shell improves colloidal and photostability in biological media and allows labeling with fluorescein isothiocyanate (FITC) for ex vivo detection. Taking advantage of the enhanced permeability and retention (EPR) effect, these nanoparticles accumulate in EM lesions. PAI and fluorescence imaging confirmed strong nanoparticle signals two hours post-injection, sustained over 24 hours. Additionally, the photothermal ablation effect of AuNRs, which induces considerable cell death in the EM lesions, thereby shrinking the lesion sizes, was shown by histological analysis in vitro and in vivo. These findings support a novel approach for non-invasive EM diagnosis and therapy using multifunctional nanoparticles potentially accelerating clinical translation of image-guided, targeted treatment strategies.

Supporting Information is available from the Wiley Online Library or from the author.