Ionic liquid-coated gold core polymeric nanoparticles for selective neutrophil hitchhiking towards endometriosis treatment

Choline carboxylic acid-based ILs were prepared using a salt metathesis reaction as shown in scheme in Fig.  1a 47 , 48 . In brief, the carboxylic acid was transferred into a clean and dry round bottom flask followed by the careful drop-wise addition of the cation (choline bicarbonate), under uniform stirring. The reaction proceeds with the bicarbonate ion abstracting the acidic proton from the carboxyl group resulting in the immediate formation of CO 2  and H 2 O. The reaction mixture is allowed to stir for 24 h. at 300 rpm and 40 °C, yielding a viscous liquid i.e. IL. The IL was initially dried under reduced pressure (-40 mmHg) at 60 °C to remove excess water, followed by further drying in a vacuum oven at 60 °C and -40 mmHg for 48 h to eliminate residual moisture. The synthesized ILs were characterized by ¹H NMR spectroscopy (Fig.  S1 ), and their molecular structures are presented in Fig.  1b . Au-PLGA-IL NPs were prepared using a convenient three-step synthesis method as shown in Fig.  1c Fig. 1 Choline carboxylic acid-based ILs can be used to coat PLGA- AuNPs. a General synthesis scheme for the one-step salt metathesis procedure for the synthesis of ILs. b Molecular structures of choline carboxylic acid-based ILs used in this study. c Three-step modified Turkevich-nanoprecipitation method for the synthesis of Au-PLGA-IL NPs, prepared in Biorender. a General synthesis scheme for the one-step salt metathesis procedure for the synthesis of ILs. b Molecular structures of choline carboxylic acid-based ILs used in this study. c Three-step modified Turkevich-nanoprecipitation method for the synthesis of Au-PLGA-IL NPs, prepared in Biorender. The detailed synthesis protocol is provided in the Methods section 7.2.2. Firstly, citrate-stabilized AuNPs (AuNPs) were prepared using a modified Turkevich method by the reduction of gold precursor (HAuCl 4 •3H 2 O) using trisodium citrate (Na 3 C 6 H 5 O 7 ) as the reducing as well as the stabilizing agent 49 . Citrate-stabilized AuNPs are chosen due to their well-established biocompatibility, photostability, and extensive use in biomedical research 50 – 52 . The AuNPs were left to stabilize overnight without any disturbance at 25 °C. In the next step, PLGA-encapsulated AuNPs were prepared by using a modified solvent-evaporation method where PLGA (1 mg/mL in ACN i.e., organic phase) was added drop-wise to the aqueous phase containing AuNP solution under constant stirring (1200 rpm, 25 °C) for 3 h for the complete evaporation of the organic solvent (ACN). Since PLGA is hydrophobic, it rearranges itself into spherical micelles encapsulating AuNP and ultimately leading to the formation of Au-PLGA NPs referred to hereafter as bare NPs 35 , 36 , 53 . Finally, neat IL (section 7.2.2) is added to the bare NP solution leading to the formation of IL-coated Au-PLGA NPs. The hydrodynamic diameter (Dh) and polydispersity index (PDI) of NPs, as determined by dynamic light scattering (DLS), are summarized as follows. The citrate-stabilized gold nanoparticles (AuNPs) exhibited a Dh of 23.6 ± 0.6 nm with a PDI of 0.28. Upon encapsulation with PLGA, the resulting Au-PLGA NPs showed an increased Dh of 63.3 ± 1.6 nm and a higher PDI of 0.47. Further surface modification with ionic liquids led to significant changes in size and monodispersity. Specifically, the Au-PLGA-CA2HEP(1:1) NPs exhibited a Dh of 152.5 ± 4.3 nm with a PDI of 0.21, the Au-PLGA-CA3NE(1:2) NPs measured 173 ± 0.5 nm in Dh with a PDI of 0.18, and the Au-PLGA-CA2DEC(1:1) NPs showed a Dh of 139.5 ± 1.4 nm and a PDI of 0.17. These results, presented in Fig.  2a , demonstrate the systematic increase in particle size and improved uniformity following each modification step. Fig. 2 Physical characterization of Au-PLGA-IL NPs highlights that choline carboxylic acid-based ILs have successfully coated PLGA encapsulated AuNPs as evident by the increase in size and broader absorption band in the Vis-NIR spectrum, cryo-TEM images confirm the morphology. a Average hydrodynamic diameter distribution of respective Au-PLGA-IL NPs compared to bare NPs. b Average zeta potential of respective Au-PLGA-IL NPs compared to bare NPs. c Normalized Vis-NIR extinction spectra of different NP systems compared to bare NPs, the broader shift in the plasmonic band extending into the NIR region is due to the huge size increase of IL-coated NPs. d Cryo-TEM images of respective Au-PLGA-IL NPs. The reported error represents the standard deviation for triplicate measurements. One way ANOVA: F Zeta (3,8) = 200.02, p  = 7.3E-8. a Average hydrodynamic diameter distribution of respective Au-PLGA-IL NPs compared to bare NPs. b Average zeta potential of respective Au-PLGA-IL NPs compared to bare NPs. c Normalized Vis-NIR extinction spectra of different NP systems compared to bare NPs, the broader shift in the plasmonic band extending into the NIR region is due to the huge size increase of IL-coated NPs. d Cryo-TEM images of respective Au-PLGA-IL NPs. The reported error represents the standard deviation for triplicate measurements. One way ANOVA: F Zeta (3,8) = 200.02, p  = 7.3E-8. Zeta potential measurements were performed to evaluate the changes in surface charge induced by IL coating, with the results presented in Fig.  2b . The unmodified citrate-stabilized AuNPs exhibited a zeta potential of -41.3 ± 0.8 mV. Following PLGA encapsulation, the surface charge of the bare NPs became more negative at –51.7 ± 0.6 mV, indicating a change in surface coating. Upon functionalization with different ionic liquids, distinct changes in surface charge were observed due to variations in the IL cation/anion composition and their interaction with the PLGA polymer. Au-PLGA-CA2HEP(1:1),Au-PLGA-CA3NE(1:2), and Au-PLGA-CA2DEC(1:1) NPs exhibited comparable surface charges of -42.4  \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ±  0.6 mV and -40.6  \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ±  0.4 mV, and -42.8  \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ±  0.8 mV respectively. The small variations in the zeta potential of different IL-coated NPs possibly arise from the differing hydrophobicity, molar ratio of cation and anion of ILs, and the orientation and packing of IL moieties at the Au-PLGA NP interface 36 , 54 . The proof of surface modification of these ILs is further indicated by the shift in the absorbance maxima of Au-PLGA-IL NPs from the visible to the NIR region of the spectrum, as compared to bare NPs, as shown in Fig.  2c ., the broader absorption shift to higher wavelengths from the bare AuNP compared to their IL-coated counterparts is due to the considerable increase in NP size (aq.), which is consistent with the NP surface being modified with the ILs 55 – 57 . 1 H NMR spectroscopy was performed to confirm the chemical identity of PLGA polymer and IL on the surface of the AuNP. Figure  S2 provides the analyzed 1 H NMR spectra evidencing the successful coating of bare NPs with respective ILs. The morphology of the prepared NPs was analyzed by both cryo-transmission electron microscopy (cryo-TEM) and TEM. Figure  2d provides the respective cryo-TEM images of the prepared bare and Au-PLGA-IL NPs. The cryo-TEM images reveal a distinct core-shell geometry, with AuNPs encapsulated within the PLGA polymeric shell, followed by a fine layer of IL coating, thereby verifying the core-shell architecture of our Au-PLGA-IL NPs. All formulations displayed a quasi-spherical Au core with diameters below 20 nm, i.e., 15.2 ± 1.3 nm (measured by TEM, Fig.  S3 ), indicating that the PLGA and IL coatings did not alter the core morphology. Notably, the TEM-derived Au core sizes were substantially smaller than Dh measured by DLS (23.6 ± 0.6 nm, Fig.  2a ), highlighting the contribution of surface coating and hydration shells in solution. The photothermal properties of the synthesized NPs were evaluated by irradiating the solution of Au-PLGA-IL NPs by a continuous-wave (CW) NIR laser of 808 nm. The temperature changes in the NP solution were recorded using an IR-thermal imaging camera (PI400i, Optris, Portsmouth, NH). The 808 nm wavelength source was chosen due to its relevance in laser-based therapeutics as the NIR window (800–1200 nm). Also referred to as the biological window, irradiating with light in the NIR region will allow for penetration of light to potential endometrial tissue and efficient absorption of light by the Au-PLGA-IL NPs as there is minimal interference from other biological chromophores present in tissues such as water, blood, fat, or melanin in that region of the electromagnetic spectrum 58 – 61 . Although the λ max of prepared Au-PLGA-IL NPs falls within the visible region (500–600 nm) they can still effectively absorb the 808 nm NIR light due to the broad plasmonic band extending close to the NIR region by a collective surface plasmon resonance phenomenon 62 , 63 . The photothermal efficiency (PTE) of prepared Au-PLGA-NPs was calculated at normal human physiological temperature, i.e. 37 °C, to mimic an actual biological system. The samples were irradiated continuously using the 808 nm laser at a fixed power density of 1 W/cm 2 . We observed that the temperature rose rapidly within the first 5–10 min of irradiation and then attained a constant value at an average irradiation time of 30–35 min for the respective samples as shown in Fig.  3 . An irradiation time of 5 min and laser power density of 1 W/cm 2 were selected to perform In vitro photothermal studies as the temperature change observed (average of 5–7 °C) is likely sufficient to induce controlled cellular death 64 – 66 . Fig. 3 Au-PLGA-IL NPs showcase substantial temperature change during continuous irradiation with 808 nm NIR laser at 1 W/cm 2 . Heating/cooling curve for a ) Au-PLGA-CA2HEP(1:1) NPs; b ) Au-PLGA-CA3NE(1:2) NPs; c ) Au-PLGA-CA2DEC(1:1) NPs. Heating/cooling curve for a ) Au-PLGA-CA2HEP(1:1) NPs; b ) Au-PLGA-CA3NE(1:2) NPs; c ) Au-PLGA-CA2DEC(1:1) NPs. The maximum temperature changes obtained from the photothermal experiments were used to calculate the PTE of each material. The PTE values were determined to be 11.0 ± 0.3% for citrate-stabilized AuNPs and 10.43 ± 0.3% for Au-PLGA NPs (Fig.  S4 ). Upon ionic liquid modification, the PTE values increased to 33.6  ± 1.5% for Au-PLGA-CA2HEP(1:1), 12.3  \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ±  0.5% for Au-PLGA-CA3NE(1:2), and 15.3 ± 1.3% for Au-PLGA-CA2DEC(1:1), respectively (Fig.  S4 ). Detailed PTE calculation steps are provided in the Supporting Information (Section 1). These results indicate that IL functionalization significantly enhances the photothermal performance of Au-PLGA NPs. While unmodified AuNPs and Au-PLGA NPs exhibited comparable efficiencies, IL-coated systems displayed marked improvements, underscoring the role of IL shells in facilitating more efficient plasmonic energy dissipation and interfacial heat transfer 28 , 67 . Since our study focuses on an endometrial disease model, we selected the epithelial 12Z cell line to conduct our in vitro studies. The in vitro cytotoxicity of Au-PLGA-IL NPs towards the endometrial 12Z cell line was evaluated using a CellTiter-Glo® luminescent cell viability assay using a previously published protocol 34 . Fig.  4a provides the % cell viability of 12Z cells treated with six concentrations of bare and Au-PLGA-IL NPs (0–160 μg/mL relative to PLGA). Au-PLGA-IL NPs showed negligible cytotoxicity, with cell viabilities significantly greater than 80% for 12Z cells. This indicates their highly biocompatible nature towards 12Z cells, we further expect them to remain safe in healthy cells as well. Since the therapeutic response will arise from localized laser irradiation at endometrial sites in future applications, we expect off-target effects to be minimal. In addition, any NPs uptaken by healthy cells will likely undergo immune clearance, which will be further evaluated in planned in vivo studies 68 . Fig. 4 Au-PLGA-IL NPs are highly biocompatible towards epithelial 12Z cells and have shown impressive cellular uptake compared to the bare NPs, demonstrating the role of ILs in enhancing the cellular uptake as visible from TEM images of Au-PLGA-IL NP treated 12Z cells. a In vitro 12Z cell viability of adherent cells incubated with varying Au-PLGA-IL NPs concentrations for 24 h assessed by CellTiter-Glo® luminescent cell viability kit. Statistical analysis is performed by one-way ANOVA (NP concentration), followed by post-hoc tests b ICP-MS results quantifying Au content in 12 Z cells after 2 h and 6 h incubation periods respectively. c TEM micrographs of 12Z cells treated with Au-PLGA-IL NPs (6 h incubation) showing the cellular uptake through endocytosis into the cytoplasm. Error bars represent the standard deviation for triplicate measurements. ( n  = 3, mean ± SD, * p  < 0.05, ns-nonsignificant). a In vitro 12Z cell viability of adherent cells incubated with varying Au-PLGA-IL NPs concentrations for 24 h assessed by CellTiter-Glo® luminescent cell viability kit. Statistical analysis is performed by one-way ANOVA (NP concentration), followed by post-hoc tests b ICP-MS results quantifying Au content in 12 Z cells after 2 h and 6 h incubation periods respectively. c TEM micrographs of 12Z cells treated with Au-PLGA-IL NPs (6 h incubation) showing the cellular uptake through endocytosis into the cytoplasm. Error bars represent the standard deviation for triplicate measurements. ( n  = 3, mean ± SD, * p  < 0.05, ns-nonsignificant). Next, the cellular uptake efficiency of Au-PLGA-IL NPs in 12Z cells was quantified using inductively coupled plasma–mass spectrometry (ICP-MS). Briefly, 12Z cells were treated at a fixed concentration of 40 μg/mL, the highest NP concentration which showed no significant viability difference for all IL-coated NPs compared to bare NP. Figure  4b represents the % cellular uptake in 12Z cells for bare and respective Au-PLGA-IL NPs, for detailed ICP-MS measurements refer to Fig.  S5 and Table  S1 and S2 . From Fig.  4b , shows a significant increase in cellular uptake from 2 h to 6 h incubation period for the CA2HEP(1:1) and CA3NE(1:2) IL-coated NP respectively, whereas the uptake of bare NPs and CA2DEC(1:1) IL-coated NPs remains largely unchanged over the same incubation period, highlighting the role of surface coating in influencing the cellular uptake. Further TEM imaging was performed to evidence cellular internalization. Figure  4c shows the TEM images of Au-PLGA-NP treated 12Z cells. The presence of Au-PLGA-IL NPs in the cytoplasm of 12Z cells indicates that cellular uptake has occurred 69 – 71 . However, more inhibition studies using specific inhibitors must be performed to determine the particular cell entry pathway. The In vitro photothermal efficacy of Au-PLGA-IL NPs towards 12Z cells was evaluated using a series of experiments. Based on the photophysical characterization (“Photothermal efficiency of Au-PLGA-IL NPs”), 12Z cells were treated with Au-PLGA-IL NPs (40 μg/mL) followed by continuous exposure to an 808 nm NIR laser (1 W/cm 2 ) for 5 min at a starting temperature of 37 °C. Post-irradiation, cell viability was assessed using a mammalian Live/Dead Cell Staining Kit according to the vendor’s protocol using fluorescence-activated cell sorting (FACS) 72 . Detailed gating parameters can be found in Fig.  S6 . Figure  5a shows the distribution of live and dead 12Z endometrial cells following treatment, as quantified by FACS. In the untreated control group, 74.56 ± 1.47% of cells remained viable, while 25.53 ± 6.41% were non-viable. Treatment with Au-PLGA-CA2HEP(1:1) NPs significantly reduced cell viability to 30.0 ± 1.33%, with 76.87 ± 1.11% of cells identified as dead. Similarly, Au-PLGA-CA3NE(1:2) NPs resulted in 21.61 ± 2.43% live cells and 72.08 ± 1.09% dead cells. The most pronounced effect was observed with Au-PLGA-CA2DEC(1:1) NPs, which yielded 17.41 ± 0.35% live cells and 68.17 ± 1.14% dead cells. It is important to note that the selectivity of our NPs arises from neutrophil association rather than cell type, and the therapeutic efficacy arises from the localized irradiation, inducing cell death only at NP-accumulated sites. While healthy cells could be affected under direct irradiation, in our intended practical use of Au-PLGA-IL NPs, the laser would target endometrial regions with higher neutrophil recruitment and hence a higher amount of Au-PLGA-IL NPs. Since Au-PLGA-IL NPs are intrinsically biocompatible, we expect any off-target NPs to be cleared from the system via immune pathways 68 , 73 , 74 . Fig. 5 Au-PLGA-IL NPs exhibited controlled photothermal-induced apoptosis in 12Z epithelial cells, as confirmed by the higher percentage of apoptotic cells over necrotic cells and reduced % of live cells observed from the Live/Dead assay, while DNA damage assay revealed no significant genotoxicity, supporting a regulated cell death mechanism. a % Viability of 12Z cells post-treatment with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) quantified via FACS measurements. b % Apoptotic/Necrotic 12Z post-treatment with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) analyzed via FACS measurements. Live cell confocal images of 12Z cells treated with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) c Live/Dead assay. d Apoptosis/Necrosis assay. e Live cell confocal images obtained from DNA damage assay performed on 12Z cells with respective Au-PLGA-IL NPs under same photothermal experimental conditions. Represented error denotes the standard deviation from at least three independently prepared samples. a % Viability of 12Z cells post-treatment with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) quantified via FACS measurements. b % Apoptotic/Necrotic 12Z post-treatment with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) analyzed via FACS measurements. Live cell confocal images of 12Z cells treated with respective Au-PLGA-IL NPs (40 μg/mL) following NIR (808 nm) laser treatment (1 W/cm 2 for 5 min) c Live/Dead assay. d Apoptosis/Necrosis assay. e Live cell confocal images obtained from DNA damage assay performed on 12Z cells with respective Au-PLGA-IL NPs under same photothermal experimental conditions. Represented error denotes the standard deviation from at least three independently prepared samples. These results confirm the strong photothermal ablation potential of IL-coated NPs across all formulations. Figure  5c presents live cell confocal microscopy images of 12Z endometrial cells following photothermal treatment. Green fluorescence indicates live cells, while red fluorescence marks dead cells. A predominant red signal, especially in samples treated with Au-PLGA-IL NPs confirms extensive cell death after laser irradiation. This observation aligns with previous FACS data showing a high proportion of non-viable cells. In addition to establishing photothermal efficacy towards 12 Z cells, as consistent with the high percentage of dead cells compared to live cells, it is crucial to study the cellular death mechanism of 12Z cells for further controlled photothermal applications. The majority of cellular death occurs either via apoptosis i.e., controlled cell death, or necrosis i.e., uncontrolled cell death. Therefore, to identify the cellular death mechanism an apoptosis/necrosis assay was used according to the vendor’s protocol 66 , 75 . The experimental conditions were kept the same as for the live/dead assay. Following laser treatment, the respective samples were analyzed using FACS, as shown in Fig.  S7 . The distribution of live, apoptotic, and necrotic cell populations varied across formulations. For Au-PLGA-CA2HEP(1:1) nanoparticles, the percentage of live cells was 6.35 ± 3.35%, apoptotic cells were 74.13 ± 3.48%, and necrotic cells were 11.68 ± 0.48%. In the case of Au-PLGA-CA3NE(1:2) nanoparticles, 0.76 ± 0.47% of the cells remained live, 93.23 ± 1.31% were apoptotic, and 5.0 ± 0.88% were necrotic. For Au-PLGA-CA2DEC(1:1) nanoparticles, live cells constituted 0.5 ± 0.23%, apoptotic cells accounted for 97.70 ± 0.42%, and necrotic cells made up 1.14 ± 0.20%, as depicted in Fig.  5b . Further laser-treated cells were analyzed via live cell confocal microscopy as shown in Fig.  5d where the majority of cells showed a cyan fluorescent signal corresponding to apoptotic cells compared to a negligible magenta signal corresponding to necrotic cells. The difference in the apoptosis/necrosis values for Au-PLGA-CA3NE(1:2) NPs compared to Au-PLGA-CA2DEC(1:1) NPs can be attributed to the % higher uptake in case of Au-PLGA-CA3NE(1:2) NPs, which enables more effective intracellular localized heating during laser irradiation. Moreover, the structural differences between the anions of the two IL coatings i.e. 3-nonenoate and 2-decenoate, may influence cell membrane interactions, further contributing to different apoptosis/necrosis profiles for the two formulations. To assess the genotoxic potential of Au-PLGA-IL NPs post-photothermal treatment, a high-content DNA damage assay (HCS DNA Damage Kit, Thermo Fisher) was performed on 12Z epithelial cells using vendor’s protocol 76 . Fluorescence imaging with Hoechst, Dead Green™, and pH2AX markers enabled discrimination between viable, apoptotic, and DNA-damaged populations. Notably, cells exposed to Au-PLGA-IL NPs with laser irradiation showed minimal pH2AX foci formation, evident by the negligible change in the TRITC fluorescence signal, comparable to untreated controls, indicating negligible DNA double-strand breaks 76 . These results confirm that cell death observed under photothermal conditions primarily arises from controlled apoptosis rather than genotoxic stress, underscoring the biocompatibility and safety of the Au-PLGA-IL nanoplatform. To identify any potential inflammatory responses by Au-PLGA-IL NPs, we quantified IL-1β, IL-6, IL-10, and TNF-α cytokine profiles via enzyme‑linked immunosorbent assay (ELISA) in 12Z cell culture using the manufacturer’s protocol (Section 7.2.16) supernatants collected 24 h post-PTE under control, Au-PLGA-IL NP only, laser-only and Au-PLGA-IL NP + laser conditions. Calibration curves validated assay sensitivity within pg/mL -1 range (R 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\approx$$\end{document} ≈ 0.98-0.99: Fig.  S8 ). All cytokine concentrations were found to be below the detection threshold, consistent with the limited inflammatory competence of 12Z monocultures and loss of viable cells post-irradiation. ELISA confirms that the Au-PLGA-IL NP-mediated PTE does not provoke detectable cytokine release under the tested parameters. Thus, the in vitro photothermal results confirm that Au-PLGA-IL NPs have shown significant photothermal efficacy against 12Z cells via a controlled apoptotic cellular death pathway without inducing any genotoxic and inflammation features, providing a promising avenue for further in vivo trials. To assess the biosafety of our prepared Au-PLGA-IL NPs for potential further studies, a hemolysis assay was performed according to a previously published protocol with human adult female whole blood (anticoagulated with K 2 EDTA). Assessment of potential hemolytic activity towards red blood cells (RBCs) is necessary because destruction of RBCs can lead to severe side effects such as anemia, jaundice and several other pathological conditions 35 , 77 , 78 . Briefly, RBCs isolated from human adult female whole blood (sourced commercially from BioIVT, Westbury, NY) were treated with Au-PLGA-IL NPs (1:10 ratio of NPs to isolated RBCs, see methods section) in quadruplicate ( n  = 4) as displayed in Fig.  6a . Au-PLGA-IL NPs displayed negligible hemolysis confirming their high biocompatibility for intravenous delivery methods. Fig. 6 Au-PLGA-IL NPs showcase negligible hemolysis demonstrating their high Ex vivo biosafety and impressive affinity towards neutrophils indicating possible applications for targeted endometriosis treatment. IL coated Au-PLGA-IL NPs showed significantly higher neutrophil association compared to bare NPs based on Au content. a Hemolysis results do not show a significant increase compared to the control bare (Au-PLGA) nanoparticles. Triton-X (20% vol/vol) was used as a positive control ( n  = 4, mean ± SD, * p  < 0.0006). b % co-localization of bare and respective Au-PLGA-IL NPs with live neutrophil granulocytes obtained from FACS measurements ( n  = 4, mean ± SEM). c Live cell confocal images of Au-PLGA-IL NPs engaging on the surface of, or uptaken by, neutrophils (blue: Pacific Blue anti-human CD66b, green: FITC anti-human CD15, red: DiD NPs). d Quantification of gold uptake by neutrophils following treatment of adult human female whole blood (K2EDTA) with Au-PLGA-IL NPs ( n  = 3, mean ± SD, ** p  < 0.008). IL coated Au-PLGA-IL NPs showed significantly higher neutrophil association compared to bare NPs based on Au content. a Hemolysis results do not show a significant increase compared to the control bare (Au-PLGA) nanoparticles. Triton-X (20% vol/vol) was used as a positive control ( n  = 4, mean ± SD, * p  < 0.0006). b % co-localization of bare and respective Au-PLGA-IL NPs with live neutrophil granulocytes obtained from FACS measurements ( n  = 4, mean ± SEM). c Live cell confocal images of Au-PLGA-IL NPs engaging on the surface of, or uptaken by, neutrophils (blue: Pacific Blue anti-human CD66b, green: FITC anti-human CD15, red: DiD NPs). d Quantification of gold uptake by neutrophils following treatment of adult human female whole blood (K2EDTA) with Au-PLGA-IL NPs ( n  = 3, mean ± SD, ** p  < 0.008). To investigate the competitive co-localization of Au-PLGA-IL coated NPs with neutrophil granulocytes, DiD dye was incorporated into our NP system (for physical characterization see Fig.  S9 and Table  S3 ) to study cellular association via qualitative flow cytometric studies using commercially purchased human female whole blood (K 2 EDTA-anticoagulated) following a previously published protocol 36 , 38 . It is important to note that the flow cytometry experiment was conducted solely to evaluate NP association with live neutrophils and did not involve any photothermal treatment. Briefly, DiD loaded Au-PLGA -IL NPs (see methods section) at a concentration of 1 mg mL -1 (relative to PLGA) were mixed with whole blood in a 1:10 ratio (v/v) on ice. After 20 min incubation at 37 °C, respective whole blood components i.e., serum, white blood cells (WBCs), platelets, and red blood cells (RBCs) were mechanically isolated using centrifugation at 1000  g at 4 °C for 10 min 35 , 79 . To quantify the Au-PLGA-IL NPs association with neutrophils, WBC stock solutions were treated with FITC anti-human CD15 (SSEA-1) antibody (granulocyte marker) from Biolegend® (#323004) and Pacific Blue™ anti-human CD66b antibody (neutrophil marker) from STEMCELL™ technologies (#60086PB) using the vendor’s protocol 80 , 81 . The stained WBCs (treated with DiD loaded Au-PLGA -IL NPs) were analyzed via FACS. Detailed gating parameters for separated whole blood components can be found in Figs.  S10 , S11 , and S12 . Figure  6b illustrates the colocalization of IL-coated NPs with neutrophils, with the reported percentages indicating the proportion of neutrophils associated with nanoparticles. Bare nanoparticles exhibited an association rate of 36.91 ± 5.66% with neutrophils. In comparison, Au-PLGA-CA2HEP(1:1) nanoparticles showed a higher association of 61.35 ± 3.36%, followed by Au-PLGA-CA3NE(1:2) nanoparticles at 50.55 ± 6.35%, and Au-PLGA-CA2DEC(1:1) nanoparticles at 56.26 ± 4.07%. Figure  6c shows the corresponding live cell confocal images. It is critical to note that both the bare NPs and CA2DEC(1:1) IL-coated NPs undergo phagocytosis i.e. get internalized by the WBCs as part of their natural immune response, as indicated by the entire neutrophil fluorescing red from the internalized DiD dye. In contrast, CA2HEP(1:1) and CA3NE(1:2) IL-coated NPs bypass phagocytosis and attach themselves onto the neutrophil surface i.e., co-localization termed ‘hitchhiking’, which is shown via the red polka dot-like features on top of the neutrophils which come from the dye-loaded NPs attached to the outer membrane of the cell 82 , 83 . These results show an anion structure-dependent relationship exists between phagocytosis and co-localization when comparing the three IL-coated NPs. To further quantify the degree of NP-neutrophil interaction, ICP-MS was performed to determine Au content in isolated neutrophil fractions. Briefly, adult human female whole blood (K 2 EDTA anti-coagulated) was treated with Au-PLGA-IL NPs (40 μg mL⁻¹ relative to 1 mg mL⁻¹ PLGA), and neutrophils were isolated using a commercially available isolation kit (STEMCELL Technologies). ICP-MS analysis of neutrophil fractions (Fig.  S13 , Table  S4 ) showed that bare NP samples exhibited an average Au content of 8.50 ± 0.47%. In comparison, Au-PLGA-CA2HEP(1:1) showed a significantly higher uptake of 21.45 ± 1.58%, followed by Au-PLGA-CA3NE(1:2) at 20.82 ± 1.43% and Au-PLGA-CA2DEC(1:1) at 18.15 ± 0.61%. Although FACS and ICP-MS experiments were conducted on blood obtained from different donors, both datasets are complementary, the FACS data provides a qualitative assessment of the fraction of neutrophils with nanoparticles associated, while the ICP-MS data reflects Au content internalized or surface-bound to neutrophils. The increased Au content in ICP-MS data corelates well with the higher neutrophil association percentages observed from FACS analysis, validating the improved neutrophil targeting efficiency of Au-PLGA-IL NPs. IL coating thus imparts unique stealth and targeting properties to the nanoplatform. All Au-PLGA-IL NPs have shown significantly higher neutrophil targeting ability compared to bare (non-coated) NPs in whole blood. However, extensive in vivo studies still remain to be performed to assess biosafety and neutrophil targeting ability in live animals induced with endometriosis to gain a better understanding of the potential downstream biological differences between co-localization and phagocytosis, which is beyond the scope of the current project.

In this study, we present the synthesis of novel composite Au-PLGA-IL nanoparticles (NPs) using a modified Turkevich method combined with a nanoprecipitation method. Au-PLGA-IL NPs exhibit high in vitro biocompatibility towards 12Z endometrial cells and demonstrate excellent ex vivo safety in human female blood (K 2 EDTA), as evidenced by cytotoxicity and hemolysis experiments, confirming their high stability and bio-safety under physiological conditions. Additionally, we have tested the photothermal efficacy of Au-PLGA-IL NPs  in vitro, demonstrating their ability to induce cellular death via apoptosis in 12Z endometrial cells at a low laser power density (1 W/cm 2 , 808 nm) and a short irradiation time of 5 min, as confirmed by live/dead and apoptosis/necrosis assays. We have shown that the structural characteristics of IL coating on nanoparticles play the principal role in determining their biological interactions - specifically in modulating their competitive binding to neutrophils (WBCs) versus their uptake via phagocytosis. We performed ex vivo fluorescence activated cell sorting and live cell confocal imaging experiments using human female blood (K2EDTA), where CA2HEP (1:1) and CA3NE(1:2) IL-coated NPs showed distinct neutrophil co-localization whereas CA2DEC(1:1) IL coated NPs underwent phagocytosis when compared to bare (uncoated) NPs. These results highlight the high in vitro photothermal efficacy and neutrophil targeting abilities of Au-PLGA-IL NPs, with potential for future in vivo applications. Future studies will focus on testing the therapeutic potential of Au-PLGA-IL NPs in a suitable animal model.

Endometriosis is a chronic, inflammatory gynecological disorder that significantly impacts the lives of millions of women and people with uteri globally, with a prevalence of approximately 10% among reproductive-age women 1 – 4 . The disorder is characterized by the ectopic presence of endometrial-like tissue outside the uterine cavity, leading to the formation of lesions predominantly in the ovaries, fallopian tubes, and pelvic peritoneum 5 – 8 . However, in severe cases, these lesions can disseminate to distant sites, including the bladder, bowel, and even the lungs, through mechanisms that are not yet fully understood but may involve hematogenous or lymphatic spread 8 – 10 . The clinical manifestations of endometriosis are diverse and often debilitating. Patients typically experience severe pelvic pain, dysmenorrhea, and dyspareunia, which can significantly impair daily functioning and quality of life 11 , 12 . Furthermore, endometriosis is a leading cause of infertility, contributing to up to 50% of infertility cases in affected women, owing to the inflammatory milieu and anatomical distortion caused by adhesions and cysts 8 , 12 , 13 . The economic burden of endometriosis is also substantial, with direct and indirect costs rivaling those of chronic diseases like diabetes and rheumatoid arthritis, underscoring the urgent need for more effective management strategies 1 . Currently, treatment options for endometriosis are primarily limited to hormonal therapies and surgical interventions, each with inherent limitations 14 – 18 . Surgical excision of endometrial lesions is often pursued to provide symptomatic relief and improve fertility outcomes; however, the recurrence rate remains high, with approximately 40–50% of patients experiencing symptom return within five years 19 , 20 . Repeated surgeries are not only invasive but also increase the risk of complications and adhesions, which can further exacerbate pain and infertility 19 . Recent advancements in nanotechnology have introduced promising new approaches for the treatment of complex diseases like endometriosis 21 – 23 . Among these, gold nanoparticles (AuNPs) have garnered significant attention due to their unique optical properties, which enable them to efficiently convert light into heat, a phenomenon known as the plasmonic photothermal effect 21 , 24 , 25 . This property allows for the precise application of photothermal therapy, wherein targeted tissues are selectively ablated by localized heat generation, minimizing collateral damage to surrounding healthy tissue 26 , 27 . In this study, we propose an innovative therapeutic platform that integrates the plasmonic capabilities of AuNPs with biocompatible polymers and ionic liquids (ILs) to target and ablate endometrial lesions. ILs are composed of bulky, asymmetric organic cations and anions that exist in liquid form below 100 °C. ILs exhibit unique physicochemical properties, including low volatility and high thermal stability, making them ideal for biomedical applications. Our group has previously utilized choline carboxylic acid-based ILs to engineer the surface of polymeric and metallic nanoparticles for diverse biomedical applications, including drug delivery, bloodstain detection, antimicrobial therapies, and photothermal cancer treatment 28 – 33 . Building upon this work, we have additionally advanced the application of ILs to enable selective targeting of specific blood components, namely red blood cells (RBCs), white blood cells (WBCs), and platelets, demonstrating enhanced biodistribution, extended circulation time, and high biocompatibility 34 – 38 . Recently, Hamadani et al. published a report summarizing the mechanistic details regarding the membrane interaction of choline carboxylate IL-coated NPs with red blood cells: depending on the anion’s alkyl chain structure, each choline carboxylate IL self-assembled on PLGA NPs directed RBC hitchhiking via specific and distinct anion membrane transporters in situ, thereby giving the IL coatings their unique targeting properties 39 . The ILs used in this study: choline  trans -2-heptenoate (1:1) [CA2HEP(1:1)], choline   trans -3-nonenoate (1:2) [CA3NE(1:2)], and choline  trans -2-decenoate (1:1) [CA2DEC(1:1)] were selected for their distinct ability to interact selectively with white blood cell components based on our previous study, particularly neutrophil granulocytes 38 . WBCs are critical regulators of our immune system and perform crucial functions like antibody production, inflammatory response, tissue repair, infection control, and fighting against any foreign pathogens. Among them, neutrophils, the most abundant granulocytes, play a pivotal role as the first line of defense in innate immunity and are rapidly recruited to sites of inflammation, including endometrial lesions. Neutrophils regulate the growth and inflammation of endometrial tissue by recruiting signaling molecules like cytokines, releasing enzymes, and reactive oxygen species 40 , 41 . Leveraging these natural inflammatory functions and lesion-associated recruitment behavior of neutrophils, we designed IL-coated Au-PLGA nanoparticles capable of selectively co-localizing or “hitchhiking” with neutrophils. This strategy aims to harness neutrophil trafficking to enhance NP accumulation at diseased endometrial sites, where neutrophil recruitment is inherently elevated, thus facilitating effective photothermal therapy 42 . Importantly, during most of the menstrual cycle, except in the pre-menstrual/menstrual window (days 25–28), neutrophil recruitment to healthy tissue in the pelvic region is minimal 43 – 46 . Our new approach involves the use of IL-coated gold-core polymeric nanoparticles designed for selective neutrophil co-localization while providing a versatile platform for functionalization. We provide a non-surgical, minimally invasive treatment option for endometriosis that preserves fertility and addresses a critical unmet need in current clinical practice. By using ILs that show high affinity for neutrophils in whole blood, we can potentially deliver nanomaterials directly to endometrial sites. Following delivery, the gold cores can be irradiated with targeted light, resulting in localized hyperthermia and death of the tissue. This approach holds the potential to significantly reduce recurrence rates and improve patient outcomes compared to conventional therapies. Our research advances the understanding of nanoparticle-based therapies, offering a promising alternative for patients seeking effective management of endometriosis without the drawbacks associated with existing treatments. Here, we pioneer our composite gold-polymer-ionic liquid materials, detailing their synthesis, characterization, and In vitro and Ex vivo assessment for safety and photothermal efficacy.

Choline bicarbonate (80% in H 2 O), Deuterium oxide (99 atom % D), Tetrachloroauric (III) acid trihydrate (≥99.5%), Carboxylic-acid terminated Resomer® RG 504 H, Poly (D, L-lactide-co-glycolide) 50:50 (PLGA) (#71990), HPLC-grade Acetonitrile, Amicon Ultra-4 Centrifugal Filter Unit, Ultracel, 30 KDa, 4 mL, EMD Millipore UFC803096, BioXtra Triton™ X-100, Non-Ionic, Liquid, 12Z Human Endometrial Epithelial Cell Line, EmbryoMax® DMEM (1X), liquid (with 4,500 mg/L Glucose, 2.25 g/L Sodium Bicarb and L-Glut, without Sodium Pyruvate), L-Glutamine Solution - 200 mM, EmbryoMax® ES Cell Qualified Fetal Bovine Serum and TMS-AB2-C EmbryoMax® Penicillin-Streptomycin Solution, 100X were purchased from Sigma-Aldrich. 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine,4 Chlorobenzenesulfonate Salt solid (DiD) (# D7757), and Invitrogen™ HCS DNA Damage Kit, IL-1β (Invitrogen™ #KAC1211) ELISA kit, and IL-10 (Invitrogen™ #KHC0101) ELISA kit were purchased from ThermoFisher. Trypsin-EDTA (#SH30042.01) was obtained from Cytiva Life Sciences. 2-Heptenoic acid (≥97.0%) was obtained from 1PlusChem, 3-Nonenoic acid (≥97.0%) and trans-2-Decenoic Acid (≥95.0%) were purchased TCI Chemicals. Emsure® Ultrapure MilliQ water was obtained from a Millipore MilliQ purifier (#Milli-Q IQ 7000). Live/Dead Cell Staining Kit, Mammalian was purchased from ION BIOSCIENCES. Apoptosis and Necrosis Quantitation Kit Plus was purchased from Biotium. Adult human female whole blood (K2EDTA anticoagulated) was purchased from Bio-IVT. FITC anti-human CD15 (SSEA-1) Antibody was obtained from BioLegend®. Pacific Blue anti-human CD66b Antibody anti-CD66b, Clone G10F5 was purchased from STEMCELL Technologies™. IL-6 (BioGems #BGK0N0L5), and TNF-α (BioGems # BGK01375 ) ELISA kits were obtained from Biogems™. Choline carboxylic acid-based ILs were prepared using a one-step salt metathesis procedure. Choline bicarbonate (cation) was mixed with anions (2-heptanoic acid, 3-nonanoic acid, and 2-decanoic acid) in required molar ratios to yield CA2HEP(1:1), CA3NE(1:2) and CA2DEC(1:1) ILs respectively. Briefly, the amount needed of anion concerning the molar ratio was weighed out in a clean and dry 100 mL round bottom flask followed by the controlled drop-wise addition of choline bicarbonate at a constant stirring rate of 300 rpm to initiate the salt metathesis reaction. The immediate formation of effervescence confirms the initiation of the reaction. The reaction mixture was allowed to stir for 24 h (300 rpm, 40 °C) followed by rotary evaporation (15 mbar, 60 °C for 2 h) to remove excess water from the IL, and the IL was further dried in a vacuum oven (60 °C for 48 h) to remove any residual water content. A Karl Fischer coulometric titration was performed to calculate the water content in the dried IL. The IL was characterized using 1 H NMR spectroscopy (400 MHz Bruker Ascend) in D 2 O (99.96 atom % D) (Sigma-Aldrich) and by Karl Fischer Titration for water content (Metrohm Coulometer #899). (Fig.  S1 ). Au-PLGA-IL NP was synthesized using a three-step synthesis method. Step 1- Citrate-stabilized AuNP: In the first step, citrate-stabilized AuNPs were synthesized via a modified Turkevich method. Briefly, 10 ml of sterile MQ water was brought to boiling point in a clean and dry scintillation vial, 50 μL of HAuCl 4 .3H 2 O (100 mM) was added into the boiling solution under constant stirring at 1200 rpm followed by the addition of 400 μL of sodium citrate trihydrate solution (30 mg/mL), the solution changes color from light yellow to colorless to gray and ultimately wine red/ruby red indicating the reduction of Au +3 ion to citrate-stabilized AuNP. The AuNP solution was left overnight at room temperature to stabilize before further use. Step 2- PLGA encapsulation of citrate-stabilized AuNP (Bare NP): Bare NPs were synthesized using a modified version of the previously published solvent-evaporation method. Firstly, 1 mL of citrate-stabilized AuNP solution and 3 mL of sterile MQ water were mixed under vigorous stirring (1200 rpm at 25 °C) for 2 min for even distribution of AuNPs. Next, 1 mL of PLGA (1 mg/mL in ACN) solution was added drop-wise into the AuNP solution. The mixture was allowed to stir for 3 h at 25 °C for the complete evaporation of ACN (organic phase) yielding PLGA encapsulated AuNPs. Step 3a) IL-capping of Au-PLGA-IL NPs (for CA2HEP(1:1) and CA2DEC(1:1) ILs): After completion of step 2, 30 mg of neat IL was added into Bare NPs solution, the mixture was stirred at 800 rpm, 25 °C for 1 h and another 1 h at 900 rpm, 25 °C then filtered via 30 kDa MWCO centrifuge filtration at 25 °C to remove excess free IL yielding the final Au-PLGA-IL NPs. The concentrated Au-PLGA-IL NP pellet was diluted up to 1 mL (1 mg/mL) using a solvent of choice i.e MQ water, 0.9% USP-grade saline or 1x PBS pH 7.4/DPBS pH 7.2 depending on the experiment requirements. Step 3b) IL-capping of Au-PLGA-CA3NE(1:2) NPs: To fix the IL capping efficiency and stability issues, a special method was designed for synthesizing CA3NE(1:2) IL-coated NPs. Briefly, Bare NP solution (end product of step 2) was filtered via 30 kDa MWCO centrifuge filtration at 25 °C, the Bare NP concentrate was diluted up to 500 μL using sterile MQ water then a small drop (2.5 μL – 5 μL) of neat CA3NE(1:2) IL was added into the NP solution, the solution was gently inverted and allowed to sit undisturbed for 5 min at 25 °C yielding Au-PLGA-CA3NE(1:2) NPs, the NP solution was further diluted up to 1 mL using a solvent of choice (PBS, 0.9% saline and, MQ water). To perform FACS experiments (co-localization) a lipophilic carbocyanine far-red (Ex: 644 nm, Em: 663 nm) fluorescent dye DiD (DiIC18(5);1,1′-dioctadecyl-3,3,3′,3′tetramethylindodicarbocyanine, 4-chlorobenzene sulfonate salt) was incorporated into Au-PLGA-IL NPs using a three step-synthesis method. Step 1 and remain the same for respective Au-PLGA-IL NPs as mentioned in the previous section, slight modifications were made in step 2 as follows: To load DiD onto Bare NPs, 1 mg/mL DiD/ACN stock solution was prepared and mixed with a stock solution of 1 mg/mL PLGA/ACN at 1% by weight of the polymer. Next, 1 mL of citrate-stabilized AuNP solution and 3 mL of sterile MQ water were mixed under vigorous stirring (1200 rpm at 25 °C) for 2 min for even distribution of AuNPs. Next, 1 mL of PLGA-Did was added drop-wise into the AuNP solution. The mixture was allowed to stir for 3 h at 25 °C in the dark for the complete evaporation of ACN (organic phase) yielding DiD-loaded PLGA encapsulated AuNPs which were coated with respective IL counterparts as mentioned in Steps 3a and 3b under dark conditions to prevent degradation of DiD dye. The size (average hydrodynamic diameter) and surface charge were measured using a Zetasizer Pro, NanoZS (Malvern Instruments, UK). The DLS (size and surface charge) samples were prepared by mixing 50 uL of Au-PLGA-IL NP solution with 950 uL of MilliQ water. Disposable DLS cells were used for size measurements, and disposable zeta cells (DTS1070) were used for surface charge measurements. Both size and surface charge measurements were performed in triplicates to calculate the average value with each reading averaged from 15 internal runs. Errors represent the standard deviation from at least three independently prepared samples. The absorption properties of the prepared Au-PLGA-IL NPs were measured using a double reference Cary 5000 spectrophotometer (Cary 5000 UV-Vis-NIR, Agilent) in Aireka Cells® quartz glass cuvettes (Cuvette, 2 windows, Volume: 3.5 mL, Outer Dim(HxWxD): 45 × 12.5 × 12.5 mm, Path Length: 10 mm, Width: 10 mm, with teflon stopper, ES Quartz Glass: 190–2500 nm). Aliquots of 5 μL of 24 μg/mL of Au-PLGA-IL NPs solution were deposited on Formvar-coated copper grids. The excess liquid was wicked away, and the remaining thin film on the grid was allowed to dry for 24 h in a desiccator, then imaging was performed. The cells were fixed in a primary fixative agent (2% Paraformaldehyde+2.5% glutaraldehyde+2 mM CaCl 2 in 0.1 M sodium cacodylate buffer pH 7.4) for 2 h at room temperature. Subsequently, they were washed and post-fixed with 1% osmium tetroxide for 1 h. Afterward, the cells were washed again and dehydrated in a graded series of ethanol, starting from 30% and gradually increasing to 100% ethanol. Finally, the dehydrated cells were immersed in 100% epoxy and polymerized at 60°C for 1–2 days. The cells were sectioned using an ultramicrotome and were subsequently deposited on Formvar-coated copper grids. After sectioning, the grids were allowed to dry for 2 h in a desiccator. TEM imaging was performed using a JEOL 2100 TEM with an accelerating voltage of 200 kV to obtain clear images. The photothermal efficiency of Au-PLGA-IL NPs was measured using a continuous 808 nm NIR laser, and the temperature changes were recorded with an infrared thermal camera (PI400i, Optris, Portsmouth, NH) in sterile 1.5 mL Polypropylene Snap Cap Microcentrifuge Tubes (Vendor: Celltreat, Part Number: 229443), 1 mL of respective (40 μg/mL) were irradiated continuously at 1 W/cm 2 . (at 37 °C), the laser was switched off after 30–35 min and the sample was allowed to cool down to 37 °C. 1 mL of deionized water was irradiated at similar conditions for reference. The difference between the reference solution’s temperature change and Au-PLGA-IL NPs temperature change was used to calculate the photothermal efficiency, detailed calculations can be found in the supplementary information. 12Z cells were cultured in cell culture-treated 96-well plates (1.5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\,\times$$\end{document} ×  10 4 cell density per well) at 37 °C in a 5% humidified CO 2 incubator. The cell media was removed at ~80% confluency and replaced with respective Au-PLGA-IL NPs (0–160 μg/mL) dispersed in a fresh cell medium. The 96-well plate was further incubated for 24 h. The next day, the cell media was replaced with 100 uL of fresh media, and 100 µL of CellTiter-Glo(R) luminescent cell viability reagent was added to each well. The 96-well plate was incubated for another 15 min at 37 °C to stabilize the luminescent signal. Finally, the luminescence of each well was measured using a microplate reader (Biotek H1 Synergy Hybrid Multi-mode). The percentage cell viability was calculated against a negative control (0 μg/mL). Inductively coupled plasma mass spectrometry (ICP-MS) was employed to evaluate the cellular uptake of Au-PLGA-IL NPs in 12Z cells. 12Z cells were grown in 6-well polystyrene tissue culture-treated plates (1 × 105 cells/well) at 37 °C in a 5% humidified CO 2 incubator. At ~80% confluency the cell media was replaced and the cells were treated with respective Au-PLGA-IL NPs (40 μg/mL) dispersed in fresh cell medium followed by incubation for 2 h and 6 h. The cells were harvested using EDTA-Trypsin and collected via centrifugation (300 G for 10 min at 4 °C). The cells were further washed with pre-cooled PBS (3X, 500 µL) to remove any unbound NPs. The cells were digested using aqua-regia and Au content was quantified based on ICP-MS data, detailed information can be found in Fig.  S7 . 12Z cells were grown in 6-well polystyrene tissue culture-treated plates (1 × 105 cells/well) at 37 °C in a 5% humidified CO 2 incubator. At ~80% confluency the cell media was removed and treated with respective Au-PLGA-IL NPs (40 μg/mL) dispersed in fresh cell medium followed by incubation for 6 h. After 6 h the cell medium was removed and cells were washed three times (500 μL) with DPBS buffer to remove any free AuNPs. The respective NP-treated cell pellets were irradiated using the NIR laser (808 nm, 1 W/cm 2 for 5 min) in an incubator at 37 °C. After laser treatment, the respective pellets were treated with the Live/Dead Cell Staining Kit using the vendor’s protocol. The stained samples were analyzed using FACS to quantify the % cell viability and live cell confocal imaging to image % live/dead cells post-thermal treatment. Apoptosis and Necrosis Quantitation Kit Plus (Biotium) was used to evaluate the cellular death pattern of Au-PLGA-IL NPs treated 12Z cells after laser treatment. 12 Z cells were cultured in 6-well polystyrene tissue culture-treated plates (similar incubation and confluency conditions as mentioned in the cellular uptake section) and treated with respective Au-PLGA-IL NPs (40 μg/mL) dispersed in fresh cell medium followed by incubation for 6 h. After 6 h, the respective Au-PLGA-IL NP pellets were irradiated with (808 nm, 1 W/cm 2 for 5 min) in an incubator at 37 °C. After laser irradiation, the samples were then treated with the respective stains according to the vendor’s protocol i.e.CF®488 A Annexin V (in TE/0.1% BSA/0.1% NaN 3 ) for early apoptotic cells and Ethidium Homodimer III (EthD-III) in PBS for late apoptotic and necrotic cells. The respective samples were briefly incubated for 15 min in the dark at 25 °C before running FACS measurements on Attune NxT acoustic focusing cytometer (Model #AFC2) to quantify the % apoptosis and necrosis in each sample and, further live cell confocal microscopy was performed on each sample to distinguish between apoptotic and necrotic cells after laser treatment. All measurements were conducted with three independently prepared samples, a detailed protocol for staining can be found on the vendor’s website. Briefly, red blood cells were separated from commercially purchased human female (K2EDTA) whole blood by centrifugation at 1000  g and 4 °C. Next, the isolated red blood cells were washed with 0.9% saline. 1:100 RBC stocks were prepared in a 96-well plate; red blood cells and respective nanoparticle solution were added in 1:20 dilution followed by 1 h incubation at 37 °C in a rotary incubator. Next, samples were centrifuged at 4 °C at 500 g for 10 min. Following centrifugation, 100  μ L of supernatant from each sample was spectrophotometrically analyzed at 405 nm using a plate reader. (Biotek H1 Synergy Hybrid Multi-mode) to measure the absorbance of hemoglobin. Triton-X-100 and 0.9% saline were used as the positive and negative control, respectively. After subtracting the absorbance value of the negative control from each sample, the percentage of hemolysis was determined by normalizing it to the absorbance exhibited by the positive control Triton-X-100 (set at 100%). To investigate the competitive binding (co-localization) of Au-PLGA-IL coated NPs, DiD dye was incorporated into our NP system (physical characterization shown in Fig.  S3 ) to study co-localization via qualitative flow cytometric studies using commercially purchased human, female, k2EDTA anticoagulated (Bio-IVT, USA) blood following a previously published protocol 36 . Briefly, DiD loaded Au-PLGA-IL NPs (1 mg mL -1 , relative to PLGA) were mixed with 900 µL of whole blood in a 1:10 v/v ratio on ice. Initially, samples were mechanically mixed by inversion for 1 min, followed by 20 min incubation in a Roto-Therm rotational incubator (# H2020, SN: G10230243) and mixed at 37 °C at 50 RPM. Subsequently, all samples were centrifuged at 1000 x  g and 4 °C for 10 min to isolate respective whole blood components i.e., serum, white blood cells (WBCs), platelets, and red blood cells (RBCs). Following centrifugation each component was collected separately (except serum, which was discarded to get rid of unbound NPs) and washed three times using 0.9% surgical grade saline and centrifuged at 200 x  g , 4 °C for 5 min. After each wash, the supernatant was replaced with 0.9% saline pH 7.4 to restore the original volume (1 mL). Following the final wash, the isolated fractions were brought up to 1 mL with 0.9% saline to prepare final stock solutions for fluorescent-activated cell sorting (FACS). To study the specific binding affinity of our Au-PLGA-IL NPs towards neutrophils, WBC stock solutions were treated with FITC anti-human CD15 (SSEA-1) Antibody (granulocyte marker) and Pacific Blue™ anti-human CD66b Antibody (neutrophil marker) using vendor’s protocol 80 , 81 . The stained WBC (DiD loaded Au-PLGA-IL NPs) were analyzed via FACS using Attune NxT Flow Cytometer. FACS analysis was conducted at the flow rate of 12.5 µL min -1 (100,000 events) for four biological replicates. To further differentiate between phagocytosis and surface co-localization of respective candidates, live cell confocal microscopy was performed on DiD loaded Au-PLGA-IL NPs and bare NP treated human female k2EDTA anticoagulated (Bio-IVT, USA) blood. The whole blood components (RBCs, WBCs and platelets) were separated using the same method as mentioned in the above section. Briefly, washed and isolated human WBCs (10 5 cells) were transferred to a 5 mL centrifuge tube or 15 mL Falcon tube and resuspended to a final volume of 3 mL of cold staining buffer (1 x PBS + 0.5% BSA) containing anti-human direct fluorescent antibodies. Next an antibody cocktail consisting of 10 uL of FITC anti-human CD15 (SSEA-1) Antibody (granulocyte marker) and Pacific Blue™ anti-human CD66b Antibody (neutrophil marker) were added to the separated WBCs for neutrophil granulocyte staining using the vendor’s protocol followed by rotary-mixing of the cell-antibody solution (~5 min at 50 RPM) to ensure proper mixing of cells with antibody and then incubate at 4 °C for 2 h in the dark. The WBC-antibody solution was washed 3 times at 1000xg for 10 min with sterile 0.9% saline buffer at 4 °C to remove unbound antibody debris. After washing, the labeled cell pellets were transferred to a 6-well glass bottom plate containing 2 mL of 37 °C saline buffer. The lid was covered and immediately imaged using the Agilent Cytation C10 confocal plate reader (C10PHC2-SN). ( In vitro ) : 12 Z cells were cultured in a sterile Cellvis™ (P96-1.5H-N) 96-Well polystyrene tissue culture-treated multiple-well plate, black with #1.5 cover glass bottom surface (section 7.2.9) 12 Z cells were treated with respective Au-PLGA-IL NPs (40 μg/mL) dispersed in fresh cell medium, followed by incubation for 24 h. The control set (12Z cells only) were incubated in 70% ethanol for 20 min to ensure they were dead. Au-PLGA-IL NP treated samples (including bare NP) were irradiated with 808 nm laser for 5 min at 37 °C. After laser treatment, the respective samples, including the control, were treated with Invitrogen™ HCS DNA Damage Kit using vendor’s protocol. The treated samples were then analyzed by live cell confocal microscopy on an Agilent Cytation C10 confocal plate reader (C10PHC2-SN). All standards and samples were run in triplicates. 12 Z cells were cultured in 6-well polystyrene tissue culture-treated plates (section 7.2.9) and treated with respective Au-PLGA-IL NPs (40 μg/mL) dispersed in fresh cell medium followed by incubation for 24 h. The following experimental conditions were used: no NP / no laser, NP only, laser only, and NP + laser. At 24 h post-treatment, culture supernatants were harvested, clarified (300 g, 5 min), and stored at -80 °C until analysis. Cytokines IL-1β (Invitrogen KAC1211), IL-6 (BioGems BGK0N0L5), IL-10 (Invitrogen KHC0101), and TNF-α (BioGems BGK01375 ) were quantified by sandwich ELISA following the manufacturers’ instructions for cell-culture supernatants. Plates were read at 450 nm on a BioTek Cytation 5, and concentrations were computed from linear or 4-parameter logistic standard curves generated in each run. All standards and samples were run in triplicates. Cryo-TEM grids (lacey carbon, copper 300 mesh) were glow-discharged for 60 s at 20 mA using an EasiGlow system to render the carbon surface hydrophilic. 3 μL of each NP suspension (1 mg/ml) were applied to grids, blotted for 4.5 s with a blot force of –6, and plunge-frozen into liquid ethane using a Vitrobot Mark IV (4 °C, 100% relative humidity). Grids were stored in liquid nitrogen until imaging. Screening was carried out on a ThermoFisher Glacios microscope equipped with a Falcon direct electron detector. Images were acquired at multiple magnifications, beginning with grid square (GS) overview maps followed by hole-eucentric (HE) and data acquisition (DA) series. Representative images were selected from regions with suitable ice thickness and particle concentration. Statistical analysis was performed with Microsoft Excel 2021, and all data are presented as mean value ± standard error of the mean (SEM) obtained through at-least three independent experiments. A comparison between the two groups was done using two-tailed and one-tailed critical student t -tests. One-way ANOVA followed by post-hoc Tukey’s test was used to assess differences in zeta potential among formulations, with p  < 0.0125 considered statistically significant. Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

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