Monitoring Lipopolysaccharide-induced Macrophage Polarization by Surface-Enhanced Raman Scattering

Monitoring Lipopolysaccharide-induced Macrophage Polarization by Surface-Enhanced Raman Scattering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Monitoring Lipopolysaccharide-induced Macrophage Polarization by Surface-Enhanced Raman Scattering Deniz Yilmaz, Mustafa Culha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4724386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2024 Read the published version in Microchimica Acta → Version 1 posted 11 You are reading this latest preprint version Abstract Macrophages are among the most important components of the innate immune system where the interaction of pathogens and their phagocytosis occur as the first barrier of immunity. When nanomaterials interact with the human body, they have to face macrophages as well. Thus, understanding of nanomaterials-macrophage interactions and underlying mechanisms is crucial. For this purpose, various methods are used. In this study, surface-enhanced Raman scattering (SERS) is proposed by studying lipopolysaccharide (LPS) induced macrophage polarization using gold nanoparticles (AuNPs) as an alternative to the current approaches. For this purpose, RAW 264.7 cells were polarized by LPS, and polarization mechanisms were characterized by nitrite release, reactive oxygen species (ROS) formation, and monitored using SERS. The spectral changes were interpreted based on the molecular pathways induced by LPS. Furthermore, polarized macrophages by LPS were exposed to the toxic AuNPs doses to monitor the enhanced phagocytosis and related spectral changes. It was observed that LPS induced macrophage polarization and enhanced AuNPs phagocytosis by activated macrophages elucidated clearly from SERS spectra in a label-free non-destructive manner. Gold nanoparticles SERS Macrophages Polarization Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Macrophages are one of the most important components of our immune system. Elie Metchnikoff won the Nobel prize in 1908 with the description of phagocytosis and stated that "the key of the immunity was to stimulate the phagocytes" [ 1 ]. After this discovery, the macrophages and their phagocytotic capacity have gained importance to understand their role in the defense mechanism. When the interaction of nanomaterials (NMs) with the immune system is considered, NMs can activate an innate immune response, complement system, and adaptive immune response [ 2 ]. For their effect on the immune system, their physicochemical properties such as size, distribution, aggregation, surface chemistry, composition, and crystallinity play an important role. In addition to their physicochemical properties, their altered properties as a result of their interaction in the biological matrix such as cell culture media or body fluids should be taken into consideration [ 3 ]. In a pioneering study, Shukla et al. investigated the interaction of 3 nm AuNPs with macrophages in detail in 2005 and found that small AuNPs used pinocytosis for internalization and localized mostly in lysosomes and perinuclear space. They also showed that AuNPs suppress the production of ROS, and do not cause any change in the proinflammatory cytokines production [ 4 ]. On the other hand, another report showed that AuNPs with three different sizes 3, 5, and 40 nm can cause a reduction of the macrophage number and increase the size of the cells and expression of proinflammatory genes IL-1, IL-6, and TNF-α as size-dependent manner [ 5 ]. Sumbayev et al. used 4 different sizes of AuNPs (5, 15, 20, and 35 nm) and showed that AuNPs down-regulate IL-1β in vitro and also in vivo in size-dependent manner [ 6 ]. To understand the interaction of NMs with the immune system, SERS is used as an alternative technique that can provide unique information from the molecules in the surrounding environment of SERS substrates. Although SERS is mostly used for the detection of inflammation markers [ 7 ], there are studies that investigate the interaction of NMs with macrophages [ 8 ], most of them include investigation of localization and distribution of designed nanoprobes inside the macrophages. There are also studies that include the design of the SERS nanoprobes for the detection of endogenous reactive oxygen species (ROS) or signaling molecules such as nitric oxide (NO) [ 9 ]. Moreover, activated macrophages were also investigated by Raman[ 10 ] and SERS for the detection of specific molecules such as intracellular adhesion molecule (ICAM-1) [ 11 ]. Cell culture media, where exogenous components were secreted, were also used for the tracking of macrophage activation by SERS [ 12 ]. However, the obtained intracellular SERS spectra were not examined to identify specific molecular pathways during the LPS-induced polarization of macrophages. In this report, LPS-induced polarization of macrophages was investigated by SERS. For this purpose, RAW 264.7 cells were polarized with LPS, and tracked by nitrite release from cells, formation of ROS, and SERS. Furthermore, the polarized cells are exposed to the high cytotoxic concentrations of AuNPs to monitor the phagocytosis. With the proposed study, it was found that during the polarization of macrophages and response to the toxic doses of AuNPs, significant changes can be detected in the intracellular SERS spectra, and obtained spectra can be used to interpret biomolecular changes during the mentioned pathways and responses. This study also shows that SERS can be used to interpret not only cell-material interactions but also cellular pathways along with intracellular biomolecular changes. 2. EXPERIMENTAL SECTION Synthesis and Characterization of Gold Nanoparticles (AuNPs) AuNPs were synthesized by citrate citrate-reducing method [ 13 ]. 0.1 mg/mL of gold (III) chloride trihydrate (HAuCl 4 .3H 2 O) (Sigma Aldrich) boiled, and then, 1% sodium citrate (Na 3 C 6 H 5 O 7 ) (Merck) was added into the solution quickly. The resulting mixture was kept boiling for 15 min and then cooled at room temperature. The synthesized AuNPs were characterized by using a UV/Vis spectrometer (Lambda 25, Perkin Elmer, USA) and Dynamic Light Scattering (DLS) (Nanozetasizer, Malvern). Cell Proliferation Assay For the investigation of the cytotoxic effect of AuNPs, cell proliferation was measured by WST-8 assay (Abcam). RAW 264.7 cells (American Type Culture Collection, ATCC) were seeded on each well of a 96-well plate with 15 000 cells·well − 1 density in triplicate in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated for 24h for attachment at 37⁰C under a 5% CO 2 humidified atmosphere. Then, cells were exposed to AuNPs at 2.5 nM, 5 nM, 6 nM, 7 nM, and 8 nM concentrations for 24 h [ 14 , 15 ]. Then, cells were washed and incubated with WST-8 containing medium for 2 h. Then, the supernatant was transferred to another 96-well plate and absorbance values were measured at 450 nm by a microplate reader (ELx800 Absorbance Reader, Biotek). As a positive control, 10% dimethyl sulfoxide (DMSO) was used. Macrophage Polarization and Nitrite Detection LPS-induced polarization of macrophages was examined with Griess reagent for nitrite detection. For this purpose, RAW 264.7 cells were seeded on a 96-well plate in triplicate at 10 6 cells·mL − 1 density. Cells were incubated for 24 h for attachment at 37⁰C under a 5% CO 2 humidified atmosphere. Then, cells were stimulated with 50, 100, 250, 500, and 1000 µg/mL of lipopolysaccharide (LPS) from E. coli (0111:B4, Sigma, USA) for 24 h with or without AuNPs. AuNPs were used with LPS to observe the polarization of macrophages, and also after LPS treatment to investigate AuNPs response of polarized macrophages. For the macrophage polarization, 2.5 nM AuNPs were added to the medium including increasing concentrations of LPS. For the response to AuNPs after polarization, cells were first activated with 1000 µg/mL LPS for 24 h. Then, old media was removed, and fresh media including different concentrations of AuNPs (2.5–8nM) were added into the cell culture and incubated for another 24 h. After the treatments with LPS and/or AuNPs, nitrite was detected from the supernatant using Griess reagent [ 16 ]. The supernatant was mixed with Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)] ethylenediamine dihydrochloride in 5% phosphoric acid) and incubated at room temperature for 10 min. After the incubation, the absorbance of the mixture was measured at 540 nm using a microplate reader. 100 µM N(G)-Nitro-L-arginine methyl ester (L-NAME) was used as a positive control. Cellular Reactive Oxygen Species (ROS) Assay For the determination of reactive oxygen species (ROS), RAW 264.7 cells were seeded in a 24-well plate in triplicate at 10 6 cells·mL − 1 density. After 24 h, cells were incubated with LPS and/or AuNPs for another 24 h. Then, cells were harvested, collected, washed with PBS, stained with 25 µM DCFDA, and incubated for 30 min at 37⁰C. After staining, cells were counted as 20,000 events and analyzed on a Guava easy-Cyte 5 benchtop flow cytometer. As a positive control, 1% DMSO was used. AuNPs Uptake The uptake of the AuNPs during and after the polarization of macrophages was examined using flow cytometry. Briefly, cells at 10 6 cells·mL − 1 density were seeded on each well of 24-well plates in triplicate. After 24 h, cells were exposed to LPS and/or AuNPs for another 24 h. After treatments, side scatter shift (SSC) of the cells was analyzed without further staining as 20,000 events on Guava easy-Cyte 5 (Merck Millipore, USA). SERS Measurements The SERS measurements were performed as described in our previous reports [ 17 , 18 ]. RAW 264.7 cells were seeded on approximately 1 cm 2 calcium fluoride (CaF 2 ) slides in 24-well plates at 10 6 cells·mL − 1 density. The cells were incubated and treated with LPS and/or AuNPs as mentioned above. After treatments, LPS and/or AuNPs containing media was removed, cells were washed with PBS, and measurements were obtained from the single living cells. A Renishaw inVia Reflex Raman spectrometer equipped with a high-speed encoded stage (Streamline) and a Leica DM2700 Dark Field upright microscope with an 830 nm laser source and 1200 line/mm grating was used for SERS measurements. A 150-mW laser power and 2 s exposure time with a spectral range of 470–1470 cm − 1 was used for spectral collection. Each single living cell was mapped with an average area of 10 µm x 10 µm with a 2 µm step size (2.5 µm laser spot size with a Leica long distance 20x objective, 0.40 NA) which resulted in a collection of a total of approximately 50 spectra per cell. The obtained spectra were averaged for each cell and a total of 30 cells were mapped. The 30 spectra from cells were averaged again and processed for background correction, smoothing, removal of cosmic spikes, and normalization using Wire 4.2 software. Tentative band assignments were given in Table S1 . Statistical Analysis For the emphasis of the variation of SERS spectra, principal component analysis (PCA) was applied to the obtained average SERS spectra from 30 cells for each treatment group. After PCA analysis, linear discriminant analysis (LDA) was applied for the obtained PC scores to observe the discrimination of the obtained spectra with different concentrations of LPS and/or AuNPs. Two-tailed student t-test was applied to the cellular assay data and intensity of the desired Raman shift. 3. RESULTS & DISCUSSION AuNPs Characterization and Cytotoxicity In this study, macrophage polarization along with response to the toxic doses of AuNPs was investigated by SERS. AuNPs were used for two different purposes. First, a low concentration of AuNPs (2.5 nM) without any cytotoxicity effect was used to enhance intracellular Raman scattering to observe molecular changes during LPS-induced macrophage polarization. On the other hand, slightly increasing concentrations of AuNPs (5–8 nM) were used to observe the response of non-polarized and polarized macrophages response to the toxic doses of AuNPs. In these cases, AuNPs not only enhanced the intracellular Raman scattering but also caused cytotoxicity and changed molecular dynamics. AuNPs were synthesized by citrate reducing method and characterized by UV/Vis and DLS, and the results are shown in Fig. 1 a, 1 b, and Table S2 . The SPR maximum of colloidal suspension of AuNPs is observed at 530 nm and shifted to 539 nm while hydrodynamic size increased from 52 nm to 72 nm and zeta potential of -26.8 mV increased to -18.5 mV in the cell culture media due to protein corona formation. After the characterization of synthesized AuNPs, their interaction with macrophages was investigated through cellular uptake, cell viability, ROS formation, and nitrite levels. The uptake of AuNPs by macrophages was investigated using flow cytometry in a dose-dependent manner. As shown in Fig. 1 c, starting from the lowest dose, a significant increase in the side scatter shift (SSC) was observed which resulted by the uptake of AuNPs. The cytotoxicity of AuNPs was investigated on RAW 264.7 based on cell viability, ROS formation, and nitrite secretion from macrophages, and results are shown in Fig. 1 d-f. As seen, the AuNPs caused a significant reduction on cell viability of macrophages with increasing concentrations except for the 2.5 nM AuNPs which is used as a control in SERS experiments due to the non-toxic behavior. Along with cytotoxicity, AuNPs also caused ROS formation and nitrite secretion from macrophages. However, ROS formation and nitrite secretion were found to be different from the cell viability assay. Although after 2.5 nM AuNPs, all concentrations caused a decrease in the cell viability, an increase in the ROS formation was observed starting with 6 nM, and an increase in the nitrite secretion was observed only after 7 nM. Moreover, 2.5 nM to 6 nM AuNPs caused also a significant reduction of nitrite secretion. This behavior can be explained by the inhibitory activity of AuNPs with nontoxic concentrations while induction of toxicity response with high concentrations. It is known that AuNPs could reduce nitrite levels by interfering with different important steps of inflammatory response such as inhibition of IL-1β-induced activation and block of MAPKs and Akt phosphorylation [ 6 ]. On the other hand, with high concentrations, cell death can be observed as a result of ROS formation and nitrite release [ 19 ]. To understand the AuNP response of non-polarized macrophages by SERS, macrophages were treated with increasing concentrations of AuNPs starting from nontoxic doses. As seen in Fig. 1 g, slight changes were observed with the increasing concentrations of AuNPs. The peak intensity changes originating from proteins (501, 618, 636, 1272–1352 cm − 1 ), amino acids (653, 1030, 755 cm − 1 ), and nucleic acids (678 cm − 1 ) were observed, which could be due to the death of the cells leading cellular disruption, protein degradation and denaturation, and DNA fragmentation [ 20 ]. However, dramatic changes due to cell death with high concentrations could not be observed as shown also in the canonical function plots and leave-one-out classification results in Figure S1 a and Table S3 . This was probably caused by the removal of dead cells during the elimination of cell culture media and washing steps before the SERS measurement. Investigation of Macrophage Polarization in the Presence of AuNPs For the investigation of the macrophage polarization by SERS, macrophages were polarized by LPS in the presence of AuNPs. The concentration of AuNPs was kept constant at a nontoxic concentration (2.5 nM) and the concentration of LPS increased gradually. When SSC shift after polarization of macrophages along with the AuNPs treatment was investigated (Fig. 2 a ) , a significant SSC shift was observed in both polarized cells without any treatment and polarized cells treated with AuNPs. The shift after polarization was observed due to the higher granulation rate of the cell after LPS uptake due to the increase in vacuoles within the activated cells [ 21 ]. Polarization of macrophages was tracked by investigation of ROS formation and nitrite secretion as shown in Fig. 2 b, c. As seen, with or without AuNP treatment, the presence of LPS caused ROS formation due to the macrophage polarization process. It is known that M1 macrophages produce nitrite and ROS when they are in contact with a pathogen. They use NADPH and NADPH oxidase through the pentose phosphate pathway for the production of ROS and when macrophages are stimulated by LPS, it promotes recognition by TLRs and interaction of LPS with receptors leads to the production of ROS and gene alterations [ 22 ]. When the LPS treatments are compared in the absence and presence of AuNPs, the presence of AuNPs causes a reduction in ROS production. This should be the result of the inhibitory effect of AuNPs as seen in nitrite formation results (Fig. 1 f). Thus, with the inhibition of polarization by AuNPs, lower ROS is achieved when compared to the LPS treatment in the absence of AuNPs. For the nitrite secretion, similar results with ROS formation were observed. The presence of LPS in the absence or presence of AuNPs caused significant nitrite release as expected due to being a marker of polarization into M1 type [ 23 ]. However, when LPS polarization was achieved with AuNPs treatment, a reduction in the nitrite secretion was observed. This is again due to the inhibitory effect of the AuNPs as explained above in the section of AuNPs cytotoxicity. Polarization mechanisms of macrophages into M1 type were tracked by SERS. To observe polarization, macrophages were treated with LPS and AuNPs simultaneously and the SERS spectra are shown in Fig. 2 d. As seen, when LPS and AuNPs were used together, the peak intensities attributed to from proteins (501, 618, 636, 653, 755, 838, 1030, 1180, 1218, 1272–1352 cm − 1 ) [ 24 ], and phospholipid (1130 cm − 1 ) [ 20 ] changed significantly which is caused by the M1 type polarization mechanisms induced by increasing concentrations of LPS treatment. When macrophages were treated with LPS, LPS internalized by cells with endocytosis as well as AuNPs [ 25 ]. Thus, when cells were exposed to LPS and AuNPs simultaneously, changes in the surrounding of AuNPs will be reflected in the intracellular SERS spectra. For the uptake of LPS and AuNPs, two scenarios can happen. It is known that AuNPs can interact with LPS [ 26 ] and in this case, they can be uptaken together and starting from the first interaction of LPS with membrane components, all changes in the surrounding of AuNPs will be reflected in the SERS spectra. Furthermore, LPS can be uptaken also by endocytosis pathways such as phagocytosis, micropinocytosis, and clathrin-mediated endocytosis. Also in this case, all the surroundings of AuNPs will be the same with LPS and LPS-caused changes will be reflected in the intracellular SERS spectra. For the LPS uptake, LPS first binds to a serum LPS binding protein (LBP), and LBP transfers the LPS monomer to the membrane-bound CD14, and then myeloid differentiation protein 2 (MD-2)/TLR4 complex initiating the LPS response [ 27 ]. After binding of LPS to MD-2, dimerization of MD-2/TLR4 complex is induced, and this induction initiates the myeloid differentiation factor (MyD88)-dependent and -independent pathways. With the activation of the MD-2/TLR4 complex, the Toll-interleukin-1 receptor (TIR) domain of TLR4 refolds and recruits 4 adaptor molecules TIRAP, MyD88, TRAM, and TRIF. With the recruitment of TIRAP and MyD88, MyD88 dependent pathway is activated through IκB kinase (IKK) which provides the activation of the nuclear factor (NF)-κB and the expression of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, IL-8, IL-12, IL-23 [ 28 ]. With the recruitment of TRAM and TRIF, the MyD88-independent pathway is activated, and this activation results in the activation of interferon regulatory factor (IRF3) which is used for the expression of type I IFNs including IFN-β [ 29 ]. During all these dynamics, all the events will be happening close to the AuNPs due to the interaction with LPS, and all these changes will be reflected in the intracellular spectra as peak intensity changes in the protein peaks at 501, 618, 636, 653, 755, 838, 1030, 1180, 1218, 1272–1352 cm − 1 due to the same microenvironment of AuNPs during internalization. As the second scenario, LPS and AuNPs can be uptaken separately, but even if uptake starts separately, the formed vesicles will be merged and continue to mature as early endosomes, late endosomes, and endolysosomes. During all these steps, many macromolecules such as proteins, cytoskeleton, and lipids play critical roles in organizing the development of the endocytosis process. After merging the vesicles that contain AuNPs and LPS, all the dynamics of the endocytosis will be reflected as well in the SERS spectra [ 17 , 18 ]. Thus, the variations in the intensities of the peaks attributed to the proteins and phospholipids shown above are kept responsible from the dynamics of the endocytosis pathway. To evaluate the variation of the observed intracellular spectra, a multivariate analysis was conducted. For this purpose, first principal component analysis was performed and then obtained principal scores were used to conduct linear discriminant analysis (LDA). Canonical function plots were extracted from LDA analysis results which provide separation of groups by showing the group. As shown in Figure S1 a and S1b , better separation was achieved with SERS spectra of polarized macrophages when compared with non-polarized macrophages treated with AuNPs in both cases. This is an expected result because more significant changes in the intracellular SERS spectra were also achieved after the polarization of macrophages. This can be caused due to the inclusion of the macrophage polarization process. In the case of non-polarized cells, only the amount of AuNPs was changed to observe the cytotoxic response to the AuNPs. However, in the case of polarization, all the mechanisms required for the polarization including the signaling pathway with endocytosis of LPS and AuNPs were reflected in the SERS spectra. Investigation of M1-Polarized Macrophages Response to the AuNPs After polarization of the macrophages, their ability to phagocyte AuNPs was also investigated by using high concentrations of AuNPs to cause cytotoxicity. When uptake of AuNPs by polarized macrophages was investigated as shown in Fig. 3 a, a significant SSC shift was observed in the presence of AuNPs after 2.5 nM when compared with polarized cells without any AuNPs treatment. This observation could be the result of an induced phagocytosis ability of activated macrophages. After polarization, macrophages can internalize more AuNPs than the non-activated macrophages as observed in the previous studies [ 21 , 30 ]. Thus, when the macrophages are activated, and then treated with AuNPs, this could cause a higher uptake rate of AuNPs. When ROS formation was evaluated after the AuNPs treatment, as shown in Fig. 3 b, it was found that up to 6 nM AuNPs, the formation of ROS was higher in the absence of AuNPs. After 6 nM, ROS formation increased with AuNPs treatment. These results can be associated with the cytotoxicity results of ANPs (Fig. 1 d-f). At the lower AuNPs concentrations, the ROS production of polarized macrophages was lower due to ROS formation caused by macrophage activation. On the other hand, after 6 nM concentration of AuNPs, serious cytotoxicity was observed, and this resulted in higher ROS production than the polarization process by LPS. After the investigation of ROS formation, nitrite secretion was also observed in polarized and non-polarized macrophages treated with AuNPs as shown in Fig. 3 c. When AuNPs were used after polarization, it was seen that even though there was a significant nitrite level increase with only LPS treatment, the presence of AuNPs significantly increased the nitrite secretion after polarization. These results are also correlated with the cytotoxicity of AuNPs. Without any polarization, AuNPs caused a dose-dependent nitrite secretion from macrophages (Fig. 1 f). After the polarization, the same pattern was observed however without any dose dependency. An incremental nitrite secretion was observed but it was significantly higher starting from 2.5 nM. The difference between non-polarized and polarized macrophages can be explained by the uptake rates of AuNPs. After 2.5 nM, the uptake of AuNPs was significantly higher in the polarized macrophages. Thus, due to the higher uptake rate, nitrite release becomes significantly higher even at lower concentrations. The response of the polarized macrophages against AuNPs was also examined by SERS. Figure 3 d shows the intracellular SERS spectra of polarized macrophages treated with 2.5 to 8 nM AuNPs. When the SERS spectra are compared, more dramatic changes were observed compared with non-polarized macrophages and simultaneous treatment of macrophages with LPS and AuNPs. When the cells were co-treated with LPS and AuNPs, LPS and AuNPs took the same route and found in the steps of macrophage polarization. On the other hand, when LPS was given first without AuNPs, macrophages were activated by LPS but then LPS including cell culture media was removed and different concentrations of AuNPs were added in the cell culture medium for the phagocytosis. This causes a response of polarized macrophages against AuNPs. On the SERS spectra, with the AuNPs concentration increase, dramatic intensity changes are observed with the peaks originating from not only proteins (618, 636, 653, 755, 838, 882, 1002, 1272, 1352 cm − 1 ) but also membrane structures including cholesterol (548 cm − 1 ), phospholipid (1130 cm − 1 ), nucleic acids (678 cm − 1 ). When AuNPs are added into the cell culture of polarized macrophages, they are phagocytosed. After the phagocytic receptors recognize a target particle and aggregate to initiate signaling pathways, the actin cytoskeleton is regulated for the membrane protrusions and formation of pseudopodia. As the first step of actin remodeling, the membrane-associated cortical cytoskeleton is disrupted, and then F-actin polymerization is initiated with actin filaments nucleation. Then, pseudopodia is extended, and a phagocytic cup is formed for the internalization of the target. Lastly, actin depolymerization occurs for the formation of phagosomes. During these steps, AuNPs were covered by a remodeled plasma membrane and actin is used for the remodeling of the membrane [ 31 ]. Interaction with the receptors and their mobility, membrane remodeling, and actin involvement during the phagosome formation could lead to the intensity changes of the peaks originating from lipid structures such as phospholipids (1130 cm − 1 ) and cholesterol (548 cm − 1 ). After the formation of pseudopods, phagocytic cup, and lastly phagosome forms, it matures and fuses with lysosome to form phagolysosomes. In the maturation process, several steps are involved. In the formation of early phagosomes, small GTPase Rab5 is used for the membrane fusion and recruited EEA1 to provide phagosome-early endosome fusion. After the fusion, maturation occurs and proteins of early phagocytosis are recycled back and proteins for the maturation are recruited to the site such as Rab7, Rab-interacting lysosomal protein (RILP). Then lumen is acidified, ILVs are formed, and phagosomes fuse with lysosome [ 32 ]. As seen, the maturation process of phagocytosis requires the recruitment of various proteins to the phagocytic site and then disassembling from the site and then again recruitment of new proteins for maturation. These types of alterations in the protein profile near or on the phagosomes are observed in the SERS spectra as the intensity changes of the peaks originating from proteins such as 618, 636, 653, 755, 838, 882, 1002, 1272, 1352 cm − 1 . When obtained spectral changes were examined by means of variation ( Figure S1 c and Table S3 ), it was found that the best separation between the control group and groups of AuNPs treatments was achieved by the response of polarized macrophages to the AuNPs. This is caused by more significant changes in the case of polarized macrophage response to the AuNPs. Furthermore, similar to the polarization results, good specificity, accuracy, and sensitivity rates were achieved in between 84.3–96.5%, 84.4–92.4%, and 50-84.8%respectively. Lower sensitivity results were achieved due to the heterogenous nature of cells and their unique responses to the external stimulus such as activation by LPS and interaction with AuNPs. 4. CONCLUSIONS In this study, LPS-induced macrophage polarization was examined by SERS. For this purpose, two main dynamics of macrophages were monitored. First, the activation mechanism of macrophages to the M1 type by LPS stimulation was tracked by SERS and it was found that polarization induction could result in significant changes in the obtained SERS spectra. Significant changes were interpreted based on the molecular pathways induced by LPS. Furthermore, when polarized macrophages were exposed to the high doses of AuNPs to trigger phagocytosis, more significant changes were observed. In conclusion, molecular dynamics of immune cells that respond to external stimuli such as LPS or AuNPs can be monitored through SERS spectral changes easily without requiring a label from single living cells. Declarations Conflicts of Interest The authors declare no conflict of interest. Competing Interests This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193). Funding This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193). Author Contribution Deniz Yılmaz: Conceptualization, Visualization, Methodology, Investigation, Formal analysis, Data curation, Writing - original draft. Mustafa Culha: Supervision, Conceptualization, Methodology, Investigation, Writing - review & editing. Acknowledgement This work has been supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193) and Yeditepe University. References Nathan C (2008) Metchnikoff’s Legacy in 2008. Nat Immunol 9:695–698. https://doi.org/10.1038/ni0708-695 Boraschi D, Duschl A (2014) Nanoparticles and the Immune System. Elsevier. https://doi.org/10.1016/C2012-0-06063-2 Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, Schlenk F, Fischer D, Kiouptsi K, Reinhardt C, Landfester K, Schild H, Maskos M, Knauer SK, Stauber RH (2013) Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8:772–781. https://doi.org/10.1038/nnano.2013.181 Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M (2005) Biocompatibility of Gold Nanoparticles and Their Endocytotic Fate Inside the Cellular Compartment: A Microscopic Overview, Langmuir 21 10644–10654. https://doi.org/10.1021/la0513712 Yen H, Hsu S, Tsai C (2009) Cytotoxicity and Immunological Response of Gold and Silver Nanoparticles of Different Sizes. Small 5:1553–1561. https://doi.org/10.1002/smll.200900126 Sumbayev VV, Yasinska IM, Garcia CP, Gilliland D, Lall GS, Gibbs BF, Bonsall DR, Varani L, Rossi F, Calzolai L (2013) Gold Nanopart Downregulate Interleukin-1β‐Induced Pro‐Inflammatory Responses Small 9:472–477. https://doi.org/10.1002/smll.201201528 Huang Q, Chen X, Fan M, Ruan S, Peng S, You R, Chen J, Lu Y (2023) SERS-based self-calibrating aptamer sensor for selective detection of IL-6. Sens Actuators B Chem 374:132828. https://doi.org/10.1016/j.snb.2022.132828 Heiner Z, Gühlke M, Živanović V, Madzharova F, Kneipp J (2017) Surface-enhanced hyper Raman hyperspectral imaging and probing in animal cells. Nanoscale 9:8024–8032. https://doi.org/10.1039/C7NR02762A Chen H-Y, Kouadio Fodjo E, Jiang L, Chang S, Li J-B, Zhan D-S, Gu H-X, Li D-W (2019) Simultaneous Detection of Intracellular Nitric Oxide and Peroxynitrite by a Surface-Enhanced Raman Scattering Nanosensor with Dual Reactivity. ACS Sens 4:3234–3239. https://doi.org/10.1021/acssensors.9b01740 Pavillon N, Hobro AJ, Akira S, Smith NI (2018) Noninvasive detection of macrophage activation with single-cell resolution through machine learning, Proceedings of the National Academy of Sciences 115 E2676–E2685. https://doi.org/10.1073/pnas.1711872115 Pissuwan D, Hattori Y (2017) Detection of Adhesion Molecules on Inflamed Macrophages at Early-Stage Using SERS Probe Gold Nanorods. Nanomicro Lett 9:8. https://doi.org/10.1007/s40820-016-0111-7 Shalabaeva V, Lovato L, La Rocca R, Messina GC, Dipalo M, Miele E, Perrone M, Gentile F, De Angelis F (2017) Time resolved and label free monitoring of extracellular metabolites by surface enhanced Raman spectroscopy. PLoS ONE 12:e0175581. https://doi.org/10.1371/journal.pone.0175581 Turkevich J, Stevenson PC, Hillier J (1951) A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss Faraday Soc 11:55. https://doi.org/10.1039/df9511100055 Kuku G, Altunbek M, Culha M (2017) Surface-Enhanced Raman Scattering for Label-Free Living Single Cell Analysis. Anal Chem 89:11160–11166. https://doi.org/10.1021/acs.analchem.7b03211 Sarıçam M, Ercan Ayra M, Culha M (2023) Systematic Investigation of Cellular Response to Hydroxyl Group Orientation Differences on Gold Glyconanoparticles. ACS Omega 8:42921–42935. https://doi.org/10.1021/acsomega.3c05920 Aydin A, Reis R, Sipahi H, Zeybekoðlu G, Çelik N, Kírmízíbekmez H, Kaklíkkaya N (2018) Hydroxytyrosol: The Phytochemical Responsible for Bioactivity of Traditionally used Olive Pits, Euroasian J Hepatogastroenterol 8 126–132. https://doi.org/10.5005/jp-journals-10018-1278 Yılmaz D, Culha M (2021) Investigation of the pathway dependent endocytosis of gold nanoparticles by surface-enhanced Raman scattering. Talanta 225:122071. https://doi.org/10.1016/j.talanta.2020.122071 Yılmaz D, Culha M (2022) Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering. Langmuir 38:6281–6294. https://doi.org/10.1021/acs.langmuir.1c03305 Villalpando-Rodriguez GE, Gibson SB (2021) Reactive Oxygen Species (ROS) Regulates Different Types of Cell Death by Acting as a Rheostat. Oxid Med Cell Longev 2021:1–17. https://doi.org/10.1155/2021/9912436 Kuku G, Saricam M, Akhatova F, Danilushkina A, Fakhrullin R, Culha M Surface-Enhanced Raman Scattering to Evaluate Nanomaterial Cytotoxicity on Living Cells. Anal Chem 88 (2016) 9813–9820. https://doi.org/10.1021/acs.analchem.6b02917 Kingston M, Pfau JC, Gilmer J, Brey R Selective inhibitory effects of 50-nm gold nanoparticles on mouse macrophage and spleen cells. J Immunotoxicol 13 (2016) 198–208. https://doi.org/10.3109/1547691X.2015.1035819 Ghesquière B, Wong BW, Kuchnio A, Carmeliet P (2014) Metabolism of stromal and immune cells in health and disease. Nature 511:167–176. https://doi.org/10.1038/nature13312 Paradkar P, Mishra L, Joshi J, Dandekar S (2017) In vitro macrophage activation: A technique for screening anti-inflammatory, immunomodulatory and anticancer activity of phytomolecules, https://nopr.niscpr.res.in/handle/123456789/40741 (accessed February 19, 2024) Talari ACS, Movasaghi Z, Rehman S, Rehman IU (2015) Raman Spectroscopy of Biological Tissues. Appl Spectrosc Rev 50:46–111. https://doi.org/10.1080/05704928.2014.923902 Tsai C-Y, Lu S-L, Hu C-W, Yeh C-S, Lee G-B, Lei H-Y (2012) Size-Dependent Attenuation of TLR9 Signaling by Gold Nanoparticles in Macrophages. J Immunol 188:68–76. https://doi.org/10.4049/jimmunol.1100344 Yang Y, Wang N, Zhu Y, Lu Y, Chen Q, Fan S, Huang Q, Chen X, Xia L, Wei Y, Zheng J, Liu X (2021) Gold nanoparticles synergize with bacterial lipopolysaccharide to enhance class A scavenger receptor dependent particle uptake in neutrophils and augment neutrophil extracellular traps formation. Ecotoxicol Environ Saf 211:111900. https://doi.org/10.1016/j.ecoenv.2021.111900 Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, Ikeda H (2003) Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 100:171–194. https://doi.org/10.1016/j.pharmthera.2003.08.003 Medzhitov R, Janeway CSJ (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173:89–97. https://doi.org/10.1034/j.1600-065X.2000.917309.x Moynagh PN (2005) TLR signalling and activation of IRFs: revisiting old friends from the NF-κB pathway. Trends Immunol 26:469–476. https://doi.org/10.1016/j.it.2005.06.009 Liu Z, Li W, Wang F, Sun C, Wang L, Wang J, Sun F (2012) Enhancement of lipopolysaccharide-induced nitric oxide and interleukin-6 production by PEGylated gold nanoparticles in RAW264.7 cells. Nanoscale 4:7135. https://doi.org/10.1039/c2nr31355c Rosales C, Uribe-Querol E (2017) Phagocytosis: A Fundamental Process in Immunity. Biomed Res Int 2017:1–18. https://doi.org/10.1155/2017/9042851 Harrison RE, Bucci C, Vieira OV, Schroer TA, Grinstein S (2003) Phagosomes Fuse with Late Endosomes and/or Lysosomes by Extension of Membrane Protrusions along Microtubules: Role of Rab7 and RILP. Mol Cell Biol 23:6494–6506. https://doi.org/10.1128/MCB.23.18.6494-6506.2003 Additional Declarations Competing interest reported. This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193). Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2024 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 30 Jul, 2024 Reviews received at journal 30 Jul, 2024 Reviews received at journal 27 Jul, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 18 Jul, 2024 Reviewers agreed at journal 18 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers invited by journal 17 Jul, 2024 Editor assigned by journal 15 Jul, 2024 Submission checks completed at journal 15 Jul, 2024 First submitted to journal 11 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4724386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333674147,"identity":"e0e651f7-f305-4f32-a58e-6450c50f666c","order_by":0,"name":"Deniz Yilmaz","email":"","orcid":"","institution":"Yeditepe University, Faculty of Engineering, Department of Genetics and Bioengineering","correspondingAuthor":false,"prefix":"","firstName":"Deniz","middleName":"","lastName":"Yilmaz","suffix":""},{"id":333674148,"identity":"7dfd1994-aadb-4022-867c-72a52d11d6c3","order_by":1,"name":"Mustafa Culha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHACxsMMDDZyINaBB8TqAWpJMwZrSSBBy+HEBhCLKC267WcMDhfUMKfPDzv8EGiLnZxuAwEtZmdyDA7POMaWu/F2mgFQS7Kx2QFCWm7wGBzmYePJ3Tg7AaTlQOI24rT8k0g3nJ3+gQQtvG0GCfLSOcTaciat4DBvX4LhBumcggMJBsT45fjhjY95vv2Xl5+dvvnDhwo7OYJa4MAArNKAWOUgIN9AiupRMApGwSgYUQAAIvlH4CRCOiQAAAAASUVORK5CYII=","orcid":"","institution":"Sabanci University Nanotechnology Research and Application Center (SUNUM)","correspondingAuthor":true,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Culha","suffix":""}],"badges":[],"createdAt":"2024-07-11 13:16:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4724386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4724386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-024-06635-3","type":"published","date":"2024-08-20T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61975783,"identity":"193db27e-da9a-4b9c-a9d3-b59955436fa1","added_by":"auto","created_at":"2024-08-07 18:32:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2130127,"visible":true,"origin":"","legend":"\u003cp\u003ea) UV/vis spectra of bare AuNPs and AuNPs in cell culture media (DMEM with 10% FBS). b) Hydrodynamic size measurements of bare AuNPs and AuNPs in cell culture media (DMEM with 10% FBS). c) WST-8 cell proliferation results of RAW 264.7 cells treated with different concentrations of AuNPs. 10% DMSO was used as a positive control (PC). d) SSC shift results when RAW 264.7 cells exposed to different concentrations of AuNPs. e) ROS assay results of RAW 264.7 cells treated with AuNPs. 1% DMSO was used as positive control (PC). f) Nitrite levels of RAW 264.7 cells treated with AuNPs. 100 µM L-NAME was used as positive control (PC). g) Average SERS spectra of RAW 264.7 cells treated with increasing concentrations of AuNPs. Statistically significant changes were calculated by two-paired Student’s t-test, and marked with stars, * for p≤0.05, ** for p≤0.01, and *** for p≤0.001. Colors for the p-values in the SERS spectra correspond to the associated concentration of AuNPs.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4724386/v1/0ab45ae4c760993a174e59d1.jpg"},{"id":61975784,"identity":"6d9fd5c2-5a6e-4d0e-befd-16afd5ca9887","added_by":"auto","created_at":"2024-08-07 18:32:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1796158,"visible":true,"origin":"","legend":"\u003cp\u003ea) SSC shift results when RAW 264.7 cells were exposed to different concentrations of LPS (0-1000 ug/mL) in the presence or absence of 2.5 nM AuNPs. b) ROS assay results of RAW 264.7 cells exposed to different concentrations of LPS in the presence or absence of 2.5 nM AuNPs. 1% DMSO was used as positive control (PC). c) Nitrite levels of RAW 264.7 cells exposed to different concentrations of LPS in the presence or absence of 2.5 nM AuNPs. 100 µM L-NAME was used as positive control (PC). g) Average SERS spectra of RAW 264.7 cells treated with different concentrations of LPS and 2.5 nM AuNPs. Statistically significant changes were calculated by two-paired Student’s t test, and marked with stars, * for p≤0.05, ** for p≤0.01, and *** for p≤0.001. Colors for the p-values in the SERS spectra correspond to the associated concentration of LPS.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4724386/v1/15d2a5ba034c685ecb773730.jpg"},{"id":61975786,"identity":"aeb4327b-6bc4-4eb2-a8c4-906553920daf","added_by":"auto","created_at":"2024-08-07 18:32:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1824968,"visible":true,"origin":"","legend":"\u003cp\u003ea) SSC shift results when polarized and non-polarized RAW 264.7 cells were exposed to different concentrations of AuNPs. b) ROS assay results of polarized and non-polarized RAW 264.7 cells exposed to different concentrations of AuNPs. 1% DMSO was used as a positive control (PC). c) Nitrite levels of polarized and non-polarized RAW 264.7 cells exposed to different concentrations of AuNPs. 100 µM L-NAME was used as a positive control (PC). g) Average SERS spectra of polarized (M1) and non-polarized (M0) RAW 264.7 cells treated with different concentrations of AuNPs. Statistically significant changes were calculated by two-paired Student’s t-test, and marked with stars, * for p≤0.05, ** for p≤0.01, and *** for p≤0.001. Colors for the p-values in the SERS spectra correspond to the associated concentration of AuNPs.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4724386/v1/86638e0438b4bc6741e95702.jpg"},{"id":63300321,"identity":"6dc3652c-1f12-48c1-81b4-61a167b725b1","added_by":"auto","created_at":"2024-08-26 16:13:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6100231,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4724386/v1/83db213a-b19d-41a2-bfb4-7a5f221c633a.pdf"},{"id":61975787,"identity":"b4454d8f-55e4-4453-9e14-cc1cd0ed2433","added_by":"auto","created_at":"2024-08-07 18:32:58","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":239788,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4724386/v1/7d72169731d9a5a95e95082b.docx"}],"financialInterests":"Competing interest reported. This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193).","formattedTitle":"Monitoring Lipopolysaccharide-induced Macrophage Polarization by Surface-Enhanced Raman Scattering","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eMacrophages are one of the most important components of our immune system. Elie Metchnikoff won the Nobel prize in 1908 with the description of phagocytosis and stated that \"the key of the immunity was to stimulate the phagocytes\" [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. After this discovery, the macrophages and their phagocytotic capacity have gained importance to understand their role in the defense mechanism.\u003c/p\u003e \u003cp\u003eWhen the interaction of nanomaterials (NMs) with the immune system is considered, NMs can activate an innate immune response, complement system, and adaptive immune response [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For their effect on the immune system, their physicochemical properties such as size, distribution, aggregation, surface chemistry, composition, and crystallinity play an important role. In addition to their physicochemical properties, their altered properties as a result of their interaction in the biological matrix such as cell culture media or body fluids should be taken into consideration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In a pioneering study, Shukla et al. investigated the interaction of 3 nm AuNPs with macrophages in detail in 2005 and found that small AuNPs used pinocytosis for internalization and localized mostly in lysosomes and perinuclear space. They also showed that AuNPs suppress the production of ROS, and do not cause any change in the proinflammatory cytokines production [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On the other hand, another report showed that AuNPs with three different sizes 3, 5, and 40 nm can cause a reduction of the macrophage number and increase the size of the cells and expression of proinflammatory genes IL-1, IL-6, and TNF-α as size-dependent manner [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Sumbayev et al. used 4 different sizes of AuNPs (5, 15, 20, and 35 nm) and showed that AuNPs down-regulate IL-1β \u003cem\u003ein vitro\u003c/em\u003e and also \u003cem\u003ein vivo\u003c/em\u003e in size-dependent manner [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo understand the interaction of NMs with the immune system, SERS is used as an alternative technique that can provide unique information from the molecules in the surrounding environment of SERS substrates. Although SERS is mostly used for the detection of inflammation markers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], there are studies that investigate the interaction of NMs with macrophages [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], most of them include investigation of localization and distribution of designed nanoprobes inside the macrophages. There are also studies that include the design of the SERS nanoprobes for the detection of endogenous reactive oxygen species (ROS) or signaling molecules such as nitric oxide (NO) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, activated macrophages were also investigated by Raman[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and SERS for the detection of specific molecules such as intracellular adhesion molecule (ICAM-1) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cell culture media, where exogenous components were secreted, were also used for the tracking of macrophage activation by SERS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the obtained intracellular SERS spectra were not examined to identify specific molecular pathways during the LPS-induced polarization of macrophages.\u003c/p\u003e \u003cp\u003eIn this report, LPS-induced polarization of macrophages was investigated by SERS. For this purpose, RAW 264.7 cells were polarized with LPS, and tracked by nitrite release from cells, formation of ROS, and SERS. Furthermore, the polarized cells are exposed to the high cytotoxic concentrations of AuNPs to monitor the phagocytosis. With the proposed study, it was found that during the polarization of macrophages and response to the toxic doses of AuNPs, significant changes can be detected in the intracellular SERS spectra, and obtained spectra can be used to interpret biomolecular changes during the mentioned pathways and responses. This study also shows that SERS can be used to interpret not only cell-material interactions but also cellular pathways along with intracellular biomolecular changes.\u003c/p\u003e"},{"header":"2. EXPERIMENTAL SECTION","content":"\u003cp\u003eSynthesis and Characterization of Gold Nanoparticles (AuNPs)\u003c/p\u003e \u003cp\u003eAuNPs were synthesized by citrate citrate-reducing method [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. 0.1 mg/mL of gold (III) chloride trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO) (Sigma Aldrich) boiled, and then, 1% sodium citrate (Na\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) (Merck) was added into the solution quickly. The resulting mixture was kept boiling for 15 min and then cooled at room temperature. The synthesized AuNPs were characterized by using a UV/Vis spectrometer (Lambda 25, Perkin Elmer, USA) and Dynamic Light Scattering (DLS) (Nanozetasizer, Malvern).\u003c/p\u003e \u003cp\u003eCell Proliferation Assay\u003c/p\u003e \u003cp\u003eFor the investigation of the cytotoxic effect of AuNPs, cell proliferation was measured by WST-8 assay (Abcam). RAW 264.7 cells (American Type Culture Collection, ATCC) were seeded on each well of a 96-well plate with 15 000 cells\u0026middot;well\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e density in triplicate in high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated for 24h for attachment at 37⁰C under a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. Then, cells were exposed to AuNPs at 2.5 nM, 5 nM, 6 nM, 7 nM, and 8 nM concentrations for 24 h [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Then, cells were washed and incubated with WST-8 containing medium for 2 h. Then, the supernatant was transferred to another 96-well plate and absorbance values were measured at 450 nm by a microplate reader (ELx800 Absorbance Reader, Biotek). As a positive control, 10% dimethyl sulfoxide (DMSO) was used.\u003c/p\u003e \u003cp\u003eMacrophage Polarization and Nitrite Detection\u003c/p\u003e \u003cp\u003eLPS-induced polarization of macrophages was examined with Griess reagent for nitrite detection. For this purpose, RAW 264.7 cells were seeded on a 96-well plate in triplicate at 10\u003csup\u003e6\u003c/sup\u003e cells\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e density. Cells were incubated for 24 h for attachment at 37⁰C under a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. Then, cells were stimulated with 50, 100, 250, 500, and 1000 \u0026micro;g/mL of lipopolysaccharide (LPS) from E. coli (0111:B4, Sigma, USA) for 24 h with or without AuNPs.\u003c/p\u003e \u003cp\u003eAuNPs were used with LPS to observe the polarization of macrophages, and also after LPS treatment to investigate AuNPs response of polarized macrophages. For the macrophage polarization, 2.5 nM AuNPs were added to the medium including increasing concentrations of LPS. For the response to AuNPs after polarization, cells were first activated with 1000 \u0026micro;g/mL LPS for 24 h. Then, old media was removed, and fresh media including different concentrations of AuNPs (2.5\u0026ndash;8nM) were added into the cell culture and incubated for another 24 h.\u003c/p\u003e \u003cp\u003eAfter the treatments with LPS and/or AuNPs, nitrite was detected from the supernatant using Griess reagent [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The supernatant was mixed with Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)] ethylenediamine dihydrochloride in 5% phosphoric acid) and incubated at room temperature for 10 min. After the incubation, the absorbance of the mixture was measured at 540 nm using a microplate reader. 100 \u0026micro;M N(G)-Nitro-L-arginine methyl ester (L-NAME) was used as a positive control.\u003c/p\u003e \u003cp\u003eCellular Reactive Oxygen Species (ROS) Assay\u003c/p\u003e \u003cp\u003eFor the determination of reactive oxygen species (ROS), RAW 264.7 cells were seeded in a 24-well plate in triplicate at 10\u003csup\u003e6\u003c/sup\u003e cells\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e density. After 24 h, cells were incubated with LPS and/or AuNPs for another 24 h. Then, cells were harvested, collected, washed with PBS, stained with 25 \u0026micro;M DCFDA, and incubated for 30 min at 37⁰C. After staining, cells were counted as 20,000 events and analyzed on a Guava easy-Cyte 5 benchtop flow cytometer. As a positive control, 1% DMSO was used.\u003c/p\u003e \u003cp\u003eAuNPs Uptake\u003c/p\u003e \u003cp\u003eThe uptake of the AuNPs during and after the polarization of macrophages was examined using flow cytometry. Briefly, cells at 10\u003csup\u003e6\u003c/sup\u003e cells\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e density were seeded on each well of 24-well plates in triplicate. After 24 h, cells were exposed to LPS and/or AuNPs for another 24 h. After treatments, side scatter shift (SSC) of the cells was analyzed without further staining as 20,000 events on Guava easy-Cyte 5 (Merck Millipore, USA).\u003c/p\u003e \u003cp\u003eSERS Measurements\u003c/p\u003e \u003cp\u003eThe SERS measurements were performed as described in our previous reports [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. RAW 264.7 cells were seeded on approximately 1 cm\u003csup\u003e2\u003c/sup\u003e calcium fluoride (CaF\u003csub\u003e2\u003c/sub\u003e) slides in 24-well plates at 10\u003csup\u003e6\u003c/sup\u003e cells\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e density. The cells were incubated and treated with LPS and/or AuNPs as mentioned above. After treatments, LPS and/or AuNPs containing media was removed, cells were washed with PBS, and measurements were obtained from the single living cells.\u003c/p\u003e \u003cp\u003eA Renishaw inVia Reflex Raman spectrometer equipped with a high-speed encoded stage (Streamline) and a Leica DM2700 Dark Field upright microscope with an 830 nm laser source and 1200 line/mm grating was used for SERS measurements. A 150-mW laser power and 2 s exposure time with a spectral range of 470\u0026ndash;1470 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used for spectral collection. Each single living cell was mapped with an average area of 10 \u0026micro;m x 10 \u0026micro;m with a 2 \u0026micro;m step size (2.5 \u0026micro;m laser spot size with a Leica long distance 20x objective, 0.40 NA) which resulted in a collection of a total of approximately 50 spectra per cell. The obtained spectra were averaged for each cell and a total of 30 cells were mapped. The 30 spectra from cells were averaged again and processed for background correction, smoothing, removal of cosmic spikes, and normalization using Wire 4.2 software. Tentative band assignments were given in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStatistical Analysis\u003c/p\u003e \u003cp\u003eFor the emphasis of the variation of SERS spectra, principal component analysis (PCA) was applied to the obtained average SERS spectra from 30 cells for each treatment group. After PCA analysis, linear discriminant analysis (LDA) was applied for the obtained PC scores to observe the discrimination of the obtained spectra with different concentrations of LPS and/or AuNPs. Two-tailed student t-test was applied to the cellular assay data and intensity of the desired Raman shift.\u003c/p\u003e"},{"header":"3. RESULTS \u0026 DISCUSSION","content":"\u003cp\u003eAuNPs Characterization and Cytotoxicity\u003c/p\u003e \u003cp\u003eIn this study, macrophage polarization along with response to the toxic doses of AuNPs was investigated by SERS. AuNPs were used for two different purposes. First, a low concentration of AuNPs (2.5 nM) without any cytotoxicity effect was used to enhance intracellular Raman scattering to observe molecular changes during LPS-induced macrophage polarization. On the other hand, slightly increasing concentrations of AuNPs (5\u0026ndash;8 nM) were used to observe the response of non-polarized and polarized macrophages response to the toxic doses of AuNPs. In these cases, AuNPs not only enhanced the intracellular Raman scattering but also caused cytotoxicity and changed molecular dynamics.\u003c/p\u003e \u003cp\u003eAuNPs were synthesized by citrate reducing method and characterized by UV/Vis and DLS, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, and \u003cb\u003eTable S2\u003c/b\u003e. The SPR maximum of colloidal suspension of AuNPs is observed at 530 nm and shifted to 539 nm while hydrodynamic size increased from 52 nm to 72 nm and zeta potential of -26.8 mV increased to -18.5 mV in the cell culture media due to protein corona formation.\u003c/p\u003e \u003cp\u003eAfter the characterization of synthesized AuNPs, their interaction with macrophages was investigated through cellular uptake, cell viability, ROS formation, and nitrite levels. The uptake of AuNPs by macrophages was investigated using flow cytometry in a dose-dependent manner. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, starting from the lowest dose, a significant increase in the side scatter shift (SSC) was observed which resulted by the uptake of AuNPs.\u003c/p\u003e \u003cp\u003eThe cytotoxicity of AuNPs was investigated on RAW 264.7 based on cell viability, ROS formation, and nitrite secretion from macrophages, and results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f. As seen, the AuNPs caused a significant reduction on cell viability of macrophages with increasing concentrations except for the 2.5 nM AuNPs which is used as a control in SERS experiments due to the non-toxic behavior. Along with cytotoxicity, AuNPs also caused ROS formation and nitrite secretion from macrophages. However, ROS formation and nitrite secretion were found to be different from the cell viability assay. Although after 2.5 nM AuNPs, all concentrations caused a decrease in the cell viability, an increase in the ROS formation was observed starting with 6 nM, and an increase in the nitrite secretion was observed only after 7 nM. Moreover, 2.5 nM to 6 nM AuNPs caused also a significant reduction of nitrite secretion. This behavior can be explained by the inhibitory activity of AuNPs with nontoxic concentrations while induction of toxicity response with high concentrations. It is known that AuNPs could reduce nitrite levels by interfering with different important steps of inflammatory response such as inhibition of IL-1β-induced activation and block of MAPKs and Akt phosphorylation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the other hand, with high concentrations, cell death can be observed as a result of ROS formation and nitrite release [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo understand the AuNP response of non-polarized macrophages by SERS, macrophages were treated with increasing concentrations of AuNPs starting from nontoxic doses. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, slight changes were observed with the increasing concentrations of AuNPs. The peak intensity changes originating from proteins (501, 618, 636, 1272\u0026ndash;1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), amino acids (653, 1030, 755 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and nucleic acids (678 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were observed, which could be due to the death of the cells leading cellular disruption, protein degradation and denaturation, and DNA fragmentation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, dramatic changes due to cell death with high concentrations could not be observed as shown also in the canonical function plots and leave-one-out classification results in \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e and \u003cb\u003eTable S3\u003c/b\u003e. This was probably caused by the removal of dead cells during the elimination of cell culture media and washing steps before the SERS measurement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInvestigation of Macrophage Polarization in the Presence of AuNPs\u003c/p\u003e \u003cp\u003eFor the investigation of the macrophage polarization by SERS, macrophages were polarized by LPS in the presence of AuNPs. The concentration of AuNPs was kept constant at a nontoxic concentration (2.5 nM) and the concentration of LPS increased gradually. When SSC shift after polarization of macrophages along with the AuNPs treatment was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, a significant SSC shift was observed in both polarized cells without any treatment and polarized cells treated with AuNPs. The shift after polarization was observed due to the higher granulation rate of the cell after LPS uptake due to the increase in vacuoles within the activated cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolarization of macrophages was tracked by investigation of ROS formation and nitrite secretion as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c. As seen, with or without AuNP treatment, the presence of LPS caused ROS formation due to the macrophage polarization process. It is known that M1 macrophages produce nitrite and ROS when they are in contact with a pathogen. They use NADPH and NADPH oxidase through the pentose phosphate pathway for the production of ROS and when macrophages are stimulated by LPS, it promotes recognition by TLRs and interaction of LPS with receptors leads to the production of ROS and gene alterations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen the LPS treatments are compared in the absence and presence of AuNPs, the presence of AuNPs causes a reduction in ROS production. This should be the result of the inhibitory effect of AuNPs as seen in nitrite formation results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Thus, with the inhibition of polarization by AuNPs, lower ROS is achieved when compared to the LPS treatment in the absence of AuNPs.\u003c/p\u003e \u003cp\u003eFor the nitrite secretion, similar results with ROS formation were observed. The presence of LPS in the absence or presence of AuNPs caused significant nitrite release as expected due to being a marker of polarization into M1 type [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, when LPS polarization was achieved with AuNPs treatment, a reduction in the nitrite secretion was observed. This is again due to the inhibitory effect of the AuNPs as explained above in the section of AuNPs cytotoxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePolarization mechanisms of macrophages into M1 type were tracked by SERS. To observe polarization, macrophages were treated with LPS and AuNPs simultaneously and the SERS spectra are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. As seen, when LPS and AuNPs were used together, the peak intensities attributed to from proteins (501, 618, 636, 653, 755, 838, 1030, 1180, 1218, 1272\u0026ndash;1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and phospholipid (1130 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] changed significantly which is caused by the M1 type polarization mechanisms induced by increasing concentrations of LPS treatment.\u003c/p\u003e \u003cp\u003eWhen macrophages were treated with LPS, LPS internalized by cells with endocytosis as well as AuNPs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, when cells were exposed to LPS and AuNPs simultaneously, changes in the surrounding of AuNPs will be reflected in the intracellular SERS spectra. For the uptake of LPS and AuNPs, two scenarios can happen. It is known that AuNPs can interact with LPS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and in this case, they can be uptaken together and starting from the first interaction of LPS with membrane components, all changes in the surrounding of AuNPs will be reflected in the SERS spectra. Furthermore, LPS can be uptaken also by endocytosis pathways such as phagocytosis, micropinocytosis, and clathrin-mediated endocytosis. Also in this case, all the surroundings of AuNPs will be the same with LPS and LPS-caused changes will be reflected in the intracellular SERS spectra.\u003c/p\u003e \u003cp\u003eFor the LPS uptake, LPS first binds to a serum LPS binding protein (LBP), and LBP transfers the LPS monomer to the membrane-bound CD14, and then myeloid differentiation protein 2 (MD-2)/TLR4 complex initiating the LPS response [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After binding of LPS to MD-2, dimerization of MD-2/TLR4 complex is induced, and this induction initiates the myeloid differentiation factor (MyD88)-dependent and -independent pathways. With the activation of the MD-2/TLR4 complex, the Toll-interleukin-1 receptor (TIR) domain of TLR4 refolds and recruits 4 adaptor molecules TIRAP, MyD88, TRAM, and TRIF. With the recruitment of TIRAP and MyD88, MyD88 dependent pathway is activated through IκB kinase (IKK) which provides the activation of the nuclear factor (NF)-κB and the expression of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, IL-8, IL-12, IL-23 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. With the recruitment of TRAM and TRIF, the MyD88-independent pathway is activated, and this activation results in the activation of interferon regulatory factor (IRF3) which is used for the expression of type I IFNs including IFN-β [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. During all these dynamics, all the events will be happening close to the AuNPs due to the interaction with LPS, and all these changes will be reflected in the intracellular spectra as peak intensity changes in the protein peaks at 501, 618, 636, 653, 755, 838, 1030, 1180, 1218, 1272\u0026ndash;1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the same microenvironment of AuNPs during internalization.\u003c/p\u003e \u003cp\u003eAs the second scenario, LPS and AuNPs can be uptaken separately, but even if uptake starts separately, the formed vesicles will be merged and continue to mature as early endosomes, late endosomes, and endolysosomes. During all these steps, many macromolecules such as proteins, cytoskeleton, and lipids play critical roles in organizing the development of the endocytosis process. After merging the vesicles that contain AuNPs and LPS, all the dynamics of the endocytosis will be reflected as well in the SERS spectra [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, the variations in the intensities of the peaks attributed to the proteins and phospholipids shown above are kept responsible from the dynamics of the endocytosis pathway.\u003c/p\u003e \u003cp\u003eTo evaluate the variation of the observed intracellular spectra, a multivariate analysis was conducted. For this purpose, first principal component analysis was performed and then obtained principal scores were used to conduct linear discriminant analysis (LDA). Canonical function plots were extracted from LDA analysis results which provide separation of groups by showing the group. As shown in \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e and \u003cb\u003eS1b\u003c/b\u003e, better separation was achieved with SERS spectra of polarized macrophages when compared with non-polarized macrophages treated with AuNPs in both cases. This is an expected result because more significant changes in the intracellular SERS spectra were also achieved after the polarization of macrophages. This can be caused due to the inclusion of the macrophage polarization process. In the case of non-polarized cells, only the amount of AuNPs was changed to observe the cytotoxic response to the AuNPs. However, in the case of polarization, all the mechanisms required for the polarization including the signaling pathway with endocytosis of LPS and AuNPs were reflected in the SERS spectra.\u003c/p\u003e \u003cp\u003eInvestigation of M1-Polarized Macrophages Response to the AuNPs\u003c/p\u003e \u003cp\u003eAfter polarization of the macrophages, their ability to phagocyte AuNPs was also investigated by using high concentrations of AuNPs to cause cytotoxicity. When uptake of AuNPs by polarized macrophages was investigated as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, a significant SSC shift was observed in the presence of AuNPs after 2.5 nM when compared with polarized cells without any AuNPs treatment. This observation could be the result of an induced phagocytosis ability of activated macrophages. After polarization, macrophages can internalize more AuNPs than the non-activated macrophages as observed in the previous studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, when the macrophages are activated, and then treated with AuNPs, this could cause a higher uptake rate of AuNPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen ROS formation was evaluated after the AuNPs treatment, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, it was found that up to 6 nM AuNPs, the formation of ROS was higher in the absence of AuNPs. After 6 nM, ROS formation increased with AuNPs treatment. These results can be associated with the cytotoxicity results of ANPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). At the lower AuNPs concentrations, the ROS production of polarized macrophages was lower due to ROS formation caused by macrophage activation. On the other hand, after 6 nM concentration of AuNPs, serious cytotoxicity was observed, and this resulted in higher ROS production than the polarization process by LPS.\u003c/p\u003e \u003cp\u003eAfter the investigation of ROS formation, nitrite secretion was also observed in polarized and non-polarized macrophages treated with AuNPs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec. When AuNPs were used after polarization, it was seen that even though there was a significant nitrite level increase with only LPS treatment, the presence of AuNPs significantly increased the nitrite secretion after polarization. These results are also correlated with the cytotoxicity of AuNPs. Without any polarization, AuNPs caused a dose-dependent nitrite secretion from macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). After the polarization, the same pattern was observed however without any dose dependency. An incremental nitrite secretion was observed but it was significantly higher starting from 2.5 nM. The difference between non-polarized and polarized macrophages can be explained by the uptake rates of AuNPs. After 2.5 nM, the uptake of AuNPs was significantly higher in the polarized macrophages. Thus, due to the higher uptake rate, nitrite release becomes significantly higher even at lower concentrations.\u003c/p\u003e \u003cp\u003eThe response of the polarized macrophages against AuNPs was also examined by SERS. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed shows the intracellular SERS spectra of polarized macrophages treated with 2.5 to 8 nM AuNPs. When the SERS spectra are compared, more dramatic changes were observed compared with non-polarized macrophages and simultaneous treatment of macrophages with LPS and AuNPs. When the cells were co-treated with LPS and AuNPs, LPS and AuNPs took the same route and found in the steps of macrophage polarization. On the other hand, when LPS was given first without AuNPs, macrophages were activated by LPS but then LPS including cell culture media was removed and different concentrations of AuNPs were added in the cell culture medium for the phagocytosis. This causes a response of polarized macrophages against AuNPs. On the SERS spectra, with the AuNPs concentration increase, dramatic intensity changes are observed with the peaks originating from not only proteins (618, 636, 653, 755, 838, 882, 1002, 1272, 1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) but also membrane structures including cholesterol (548 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), phospholipid (1130 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), nucleic acids (678 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eWhen AuNPs are added into the cell culture of polarized macrophages, they are phagocytosed. After the phagocytic receptors recognize a target particle and aggregate to initiate signaling pathways, the actin cytoskeleton is regulated for the membrane protrusions and formation of pseudopodia. As the first step of actin remodeling, the membrane-associated cortical cytoskeleton is disrupted, and then F-actin polymerization is initiated with actin filaments nucleation. Then, pseudopodia is extended, and a phagocytic cup is formed for the internalization of the target. Lastly, actin depolymerization occurs for the formation of phagosomes. During these steps, AuNPs were covered by a remodeled plasma membrane and actin is used for the remodeling of the membrane [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Interaction with the receptors and their mobility, membrane remodeling, and actin involvement during the phagosome formation could lead to the intensity changes of the peaks originating from lipid structures such as phospholipids (1130 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and cholesterol (548 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eAfter the formation of pseudopods, phagocytic cup, and lastly phagosome forms, it matures and fuses with lysosome to form phagolysosomes. In the maturation process, several steps are involved. In the formation of early phagosomes, small GTPase Rab5 is used for the membrane fusion and recruited EEA1 to provide phagosome-early endosome fusion. After the fusion, maturation occurs and proteins of early phagocytosis are recycled back and proteins for the maturation are recruited to the site such as Rab7, Rab-interacting lysosomal protein (RILP). Then lumen is acidified, ILVs are formed, and phagosomes fuse with lysosome [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. As seen, the maturation process of phagocytosis requires the recruitment of various proteins to the phagocytic site and then disassembling from the site and then again recruitment of new proteins for maturation. These types of alterations in the protein profile near or on the phagosomes are observed in the SERS spectra as the intensity changes of the peaks originating from proteins such as 618, 636, 653, 755, 838, 882, 1002, 1272, 1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen obtained spectral changes were examined by means of variation (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec\u003c/b\u003e and \u003cb\u003eTable S3\u003c/b\u003e), it was found that the best separation between the control group and groups of AuNPs treatments was achieved by the response of polarized macrophages to the AuNPs. This is caused by more significant changes in the case of polarized macrophage response to the AuNPs. Furthermore, similar to the polarization results, good specificity, accuracy, and sensitivity rates were achieved in between 84.3\u0026ndash;96.5%, 84.4\u0026ndash;92.4%, and 50-84.8%respectively. Lower sensitivity results were achieved due to the heterogenous nature of cells and their unique responses to the external stimulus such as activation by LPS and interaction with AuNPs.\u003c/p\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eIn this study, LPS-induced macrophage polarization was examined by SERS. For this purpose, two main dynamics of macrophages were monitored. First, the activation mechanism of macrophages to the M1 type by LPS stimulation was tracked by SERS and it was found that polarization induction could result in significant changes in the obtained SERS spectra. Significant changes were interpreted based on the molecular pathways induced by LPS. Furthermore, when polarized macrophages were exposed to the high doses of AuNPs to trigger phagocytosis, more significant changes were observed. In conclusion, molecular dynamics of immune cells that respond to external stimuli such as LPS or AuNPs can be monitored through SERS spectral changes easily without requiring a label from single living cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThis study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDeniz Yılmaz: Conceptualization, Visualization, Methodology, Investigation, Formal analysis, Data curation, Writing - original draft. Mustafa Culha: Supervision, Conceptualization, Methodology, Investigation, Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work has been supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 118Z193) and Yeditepe University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNathan C (2008) Metchnikoff\u0026rsquo;s Legacy in 2008. Nat Immunol 9:695\u0026ndash;698. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ni0708-695\u003c/span\u003e\u003cspan address=\"10.1038/ni0708-695\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoraschi D, Duschl A (2014) Nanoparticles and the Immune System. Elsevier. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/C2012-0-06063-2\u003c/span\u003e\u003cspan address=\"10.1016/C2012-0-06063-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, Schlenk F, Fischer D, Kiouptsi K, Reinhardt C, Landfester K, Schild H, Maskos M, Knauer SK, Stauber RH (2013) Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8:772\u0026ndash;781. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nnano.2013.181\u003c/span\u003e\u003cspan address=\"10.1038/nnano.2013.181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M (2005) Biocompatibility of Gold Nanoparticles and Their Endocytotic Fate Inside the Cellular Compartment: A Microscopic Overview, Langmuir 21 10644\u0026ndash;10654. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/la0513712\u003c/span\u003e\u003cspan address=\"10.1021/la0513712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYen H, Hsu S, Tsai C (2009) Cytotoxicity and Immunological Response of Gold and Silver Nanoparticles of Different Sizes. Small 5:1553\u0026ndash;1561. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/smll.200900126\u003c/span\u003e\u003cspan address=\"10.1002/smll.200900126\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSumbayev VV, Yasinska IM, Garcia CP, Gilliland D, Lall GS, Gibbs BF, Bonsall DR, Varani L, Rossi F, Calzolai L (2013) Gold Nanopart Downregulate Interleukin-1β‐Induced Pro‐Inflammatory Responses Small 9:472\u0026ndash;477. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/smll.201201528\u003c/span\u003e\u003cspan address=\"10.1002/smll.201201528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Q, Chen X, Fan M, Ruan S, Peng S, You R, Chen J, Lu Y (2023) SERS-based self-calibrating aptamer sensor for selective detection of IL-6. Sens Actuators B Chem 374:132828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.snb.2022.132828\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2022.132828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeiner Z, G\u0026uuml;hlke M, Živanović V, Madzharova F, Kneipp J (2017) Surface-enhanced hyper Raman hyperspectral imaging and probing in animal cells. Nanoscale 9:8024\u0026ndash;8032. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C7NR02762A\u003c/span\u003e\u003cspan address=\"10.1039/C7NR02762A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H-Y, Kouadio Fodjo E, Jiang L, Chang S, Li J-B, Zhan D-S, Gu H-X, Li D-W (2019) Simultaneous Detection of Intracellular Nitric Oxide and Peroxynitrite by a Surface-Enhanced Raman Scattering Nanosensor with Dual Reactivity. ACS Sens 4:3234\u0026ndash;3239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acssensors.9b01740\u003c/span\u003e\u003cspan address=\"10.1021/acssensors.9b01740\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavillon N, Hobro AJ, Akira S, Smith NI (2018) Noninvasive detection of macrophage activation with single-cell resolution through machine learning, Proceedings of the National Academy of Sciences 115 E2676\u0026ndash;E2685. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1711872115\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1711872115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePissuwan D, Hattori Y (2017) Detection of Adhesion Molecules on Inflamed Macrophages at Early-Stage Using SERS Probe Gold Nanorods. Nanomicro Lett 9:8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40820-016-0111-7\u003c/span\u003e\u003cspan address=\"10.1007/s40820-016-0111-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShalabaeva V, Lovato L, La Rocca R, Messina GC, Dipalo M, Miele E, Perrone M, Gentile F, De Angelis F (2017) Time resolved and label free monitoring of extracellular metabolites by surface enhanced Raman spectroscopy. PLoS ONE 12:e0175581. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0175581\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0175581\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurkevich J, Stevenson PC, Hillier J (1951) A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss Faraday Soc 11:55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/df9511100055\u003c/span\u003e\u003cspan address=\"10.1039/df9511100055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuku G, Altunbek M, Culha M (2017) Surface-Enhanced Raman Scattering for Label-Free Living Single Cell Analysis. Anal Chem 89:11160\u0026ndash;11166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.7b03211\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.7b03211\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarı\u0026ccedil;am M, Ercan Ayra M, Culha M (2023) Systematic Investigation of Cellular Response to Hydroxyl Group Orientation Differences on Gold Glyconanoparticles. ACS Omega 8:42921\u0026ndash;42935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsomega.3c05920\u003c/span\u003e\u003cspan address=\"10.1021/acsomega.3c05920\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAydin A, Reis R, Sipahi H, Zeybeko\u0026eth;lu G, \u0026Ccedil;elik N, K\u0026iacute;rm\u0026iacute;z\u0026iacute;bekmez H, Kakl\u0026iacute;kkaya N (2018) Hydroxytyrosol: The Phytochemical Responsible for Bioactivity of Traditionally used Olive Pits, Euroasian J Hepatogastroenterol 8 126\u0026ndash;132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5005/jp-journals-10018-1278\u003c/span\u003e\u003cspan address=\"10.5005/jp-journals-10018-1278\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYılmaz D, Culha M (2021) Investigation of the pathway dependent endocytosis of gold nanoparticles by surface-enhanced Raman scattering. Talanta 225:122071. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.talanta.2020.122071\u003c/span\u003e\u003cspan address=\"10.1016/j.talanta.2020.122071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYılmaz D, Culha M (2022) Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering. Langmuir 38:6281\u0026ndash;6294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.langmuir.1c03305\u003c/span\u003e\u003cspan address=\"10.1021/acs.langmuir.1c03305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillalpando-Rodriguez GE, Gibson SB (2021) Reactive Oxygen Species (ROS) Regulates Different Types of Cell Death by Acting as a Rheostat. Oxid Med Cell Longev 2021:1\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2021/9912436\u003c/span\u003e\u003cspan address=\"10.1155/2021/9912436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuku G, Saricam M, Akhatova F, Danilushkina A, Fakhrullin R, Culha M Surface-Enhanced Raman Scattering to Evaluate Nanomaterial Cytotoxicity on Living Cells. Anal Chem 88 (2016) 9813\u0026ndash;9820. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.6b02917\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.6b02917\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKingston M, Pfau JC, Gilmer J, Brey R Selective inhibitory effects of 50-nm gold nanoparticles on mouse macrophage and spleen cells. J Immunotoxicol 13 (2016) 198\u0026ndash;208. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/1547691X.2015.1035819\u003c/span\u003e\u003cspan address=\"10.3109/1547691X.2015.1035819\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhesqui\u0026egrave;re B, Wong BW, Kuchnio A, Carmeliet P (2014) Metabolism of stromal and immune cells in health and disease. Nature 511:167\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature13312\u003c/span\u003e\u003cspan address=\"10.1038/nature13312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParadkar P, Mishra L, Joshi J, Dandekar S (2017) In vitro macrophage activation: A technique for screening anti-inflammatory, immunomodulatory and anticancer activity of phytomolecules, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nopr.niscpr.res.in/handle/123456789/40741\u003c/span\u003e\u003cspan address=\"https://nopr.niscpr.res.in/handle/123456789/40741\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed February 19, 2024)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalari ACS, Movasaghi Z, Rehman S, Rehman IU (2015) Raman Spectroscopy of Biological Tissues. Appl Spectrosc Rev 50:46\u0026ndash;111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/05704928.2014.923902\u003c/span\u003e\u003cspan address=\"10.1080/05704928.2014.923902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai C-Y, Lu S-L, Hu C-W, Yeh C-S, Lee G-B, Lei H-Y (2012) Size-Dependent Attenuation of TLR9 Signaling by Gold Nanoparticles in Macrophages. J Immunol 188:68\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4049/jimmunol.1100344\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.1100344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Wang N, Zhu Y, Lu Y, Chen Q, Fan S, Huang Q, Chen X, Xia L, Wei Y, Zheng J, Liu X (2021) Gold nanoparticles synergize with bacterial lipopolysaccharide to enhance class A scavenger receptor dependent particle uptake in neutrophils and augment neutrophil extracellular traps formation. Ecotoxicol Environ Saf 211:111900. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2021.111900\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2021.111900\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, Ikeda H (2003) Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 100:171\u0026ndash;194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pharmthera.2003.08.003\u003c/span\u003e\u003cspan address=\"10.1016/j.pharmthera.2003.08.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedzhitov R, Janeway CSJ (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173:89\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1034/j.1600-065X.2000.917309.x\u003c/span\u003e\u003cspan address=\"10.1034/j.1600-065X.2000.917309.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoynagh PN (2005) TLR signalling and activation of IRFs: revisiting old friends from the NF-κB pathway. Trends Immunol 26:469\u0026ndash;476. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.it.2005.06.009\u003c/span\u003e\u003cspan address=\"10.1016/j.it.2005.06.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Li W, Wang F, Sun C, Wang L, Wang J, Sun F (2012) Enhancement of lipopolysaccharide-induced nitric oxide and interleukin-6 production by PEGylated gold nanoparticles in RAW264.7 cells. Nanoscale 4:7135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c2nr31355c\u003c/span\u003e\u003cspan address=\"10.1039/c2nr31355c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosales C, Uribe-Querol E (2017) Phagocytosis: A Fundamental Process in Immunity. Biomed Res Int 2017:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2017/9042851\u003c/span\u003e\u003cspan address=\"10.1155/2017/9042851\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison RE, Bucci C, Vieira OV, Schroer TA, Grinstein S (2003) Phagosomes Fuse with Late Endosomes and/or Lysosomes by Extension of Membrane Protrusions along Microtubules: Role of Rab7 and RILP. Mol Cell Biol 23:6494\u0026ndash;6506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.23.18.6494-6506.2003\u003c/span\u003e\u003cspan address=\"10.1128/MCB.23.18.6494-6506.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gold nanoparticles, SERS, Macrophages, Polarization","lastPublishedDoi":"10.21203/rs.3.rs-4724386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4724386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMacrophages are among the most important components of the innate immune system where the interaction of pathogens and their phagocytosis occur as the first barrier of immunity. When nanomaterials interact with the human body, they have to face macrophages as well. Thus, understanding of nanomaterials-macrophage interactions and underlying mechanisms is crucial. For this purpose, various methods are used. In this study, surface-enhanced Raman scattering (SERS) is proposed by studying lipopolysaccharide (LPS) induced macrophage polarization using gold nanoparticles (AuNPs) as an alternative to the current approaches. For this purpose, RAW 264.7 cells were polarized by LPS, and polarization mechanisms were characterized by nitrite release, reactive oxygen species (ROS) formation, and monitored using SERS. The spectral changes were interpreted based on the molecular pathways induced by LPS. Furthermore, polarized macrophages by LPS were exposed to the toxic AuNPs doses to monitor the enhanced phagocytosis and related spectral changes. It was observed that LPS induced macrophage polarization and enhanced AuNPs phagocytosis by activated macrophages elucidated clearly from SERS spectra in a label-free non-destructive manner.\u003c/p\u003e","manuscriptTitle":"Monitoring Lipopolysaccharide-induced Macrophage Polarization by Surface-Enhanced Raman Scattering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 18:32:53","doi":"10.21203/rs.3.rs-4724386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-30T10:18:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T09:19:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-27T15:22:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T14:36:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5049514340984776697792306185053918992","date":"2024-07-18T12:35:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240548563345671657479815310695420127741","date":"2024-07-18T10:34:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6018435572014994386010871108805538464","date":"2024-07-17T20:55:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T19:14:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-15T08:09:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-15T08:08:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2024-07-11T13:14:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4eb70356-54a4-4bea-a587-2d0d3667fa4f","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:04:02+00:00","versionOfRecord":{"articleIdentity":"rs-4724386","link":"https://doi.org/10.1007/s00604-024-06635-3","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2024-08-20 15:57:48","publishedOnDateReadable":"August 20th, 2024"},"versionCreatedAt":"2024-08-07 18:32:53","video":"","vorDoi":"10.1007/s00604-024-06635-3","vorDoiUrl":"https://doi.org/10.1007/s00604-024-06635-3","workflowStages":[]},"version":"v1","identity":"rs-4724386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4724386","identity":"rs-4724386","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}