Magnetic field-induced plasmonic enhancement of near infrared fluorescence from a magnetoplasmonic nanoplatform for bioimaging applications

Magnetic field-induced plasmonic enhancement of near infrared fluorescence from a magnetoplasmonic nanoplatform for bioimaging applications | 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 Magnetic field-induced plasmonic enhancement of near infrared fluorescence from a magnetoplasmonic nanoplatform for bioimaging applications Siqi Gao, Jiantao Liu, Iuliia Golovynska, Zhenlong Huang, Yiqiang Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6912220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 17 You are reading this latest preprint version Abstract A phenomenon of plasmon-enhanced fluorescence (PEF) arises from interactions between fluorophores and metal nanostructures, leading to a substantial amplification of the fluorescence signal. Herein, we report a magnetic field (MF) induced on-demand PEF from the magnetoplasmonic nanoplatform and demonstrate its application in near infrared (NIR) bioimaging. The developed magnetoplasmonic nanoparticles (~ 50 nm diameter) feature a core-shell-satellite architecture comprising a Fe3O4 magnetic core, a mesoporous silica (mSiO2) shell housing IR775-silane NIR dye, and surface-anchored gold (Au) seeds (satellites). Application of an external MF causes the magnetophoretic movement and aggregation of the nanoparticles (NPs), resulting in a formation of localized plasmonic hotspots and, consequently, in a plasmonic enhancement of NIR fluorescence from IR775 dye molecules. Correspondingly, a substantial reduction of the fluorescence lifetime in the MF-treated area was observed, in addition to the enhanced fluorescence intensity. In vivo studies with NPs subcutaneously injected into mice revealed MF-activated amplification of NIR fluorescence. At 6 h post-injection, the injected region treated by MF exhibited 2.1-fold stronger NIR fluorescence signal than the MF-untreated one; the fluorescence enhancement correlated with the reduction of the emission lifetime (from 0.68 ns to 0.47 ns). At 96 h post-injection, the MF-treated region exhibited 6.8-fold more intense NIR fluorescence. Histological analysis showed absence of toxicity from the injected NPs, revealing their biocompatibility. Hence, a considerable potential of MF-induced PEF with the magnetoplasmonic nanoplatform for targeted NIR fluorescence bioimaging was demonstrated. This work also introduces MF-induced PEF as a powerful strategy for spatiotemporal control of optical signals, offering new opportunities for targeted imaging and sensing. magnetoplasmonic nanoparticles plasmon-enhanced fluorescence fluorescence lifetime imaging magnetic field-induced aggregation near infrared fluorescence bioimaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Plasmon-enhanced fluorescence (PEF) is a plasmonic phenomenon first reported in 1980 [1]. It originates from the plasmon resonance coupling between the frequencies emitted by the fluorophore and local surface plasmon resonance (LSPR) of the metal particles (NPs) that can result in a significant enhancement in the emission intensity [2, 3]. Use of PEF phenomenon can be advantageous for fluorescence bioimaging, particularly for near infrared (NIR) fluorescence imaging in vivo , as it would specifically benefit from an enhancement of fluorescence from imaging probes, allowing for deeper tissue imaging with a low background [4, 5, 6]. However, only a few studies reported PEF nanoformulations in in vivo imaging systems; PEF nanoformulations designed for in vivo fluorescence imaging generally lack high specificity/targeting ability and may reveal some toxicity in in vivo applications; At the same time, the PEF effect in nanoformulations renders to be unstable under the in vivo fluorescence imaging conditions [7, 8, 9]. Thus, to be employed in NIR imaging in vivo , PEF nanoformulations should possess good biocompatibility along with targeting ability for a specific application (e.g., tumor targeting in cancer theranostics). The PEF nanoformulations used in biosensing applications usually rely on the use of specific antibodies or aptamers as molecular recognition elements, which allow for specific binding to target analytes through the antibody-antigen or aptamer-nucleic acid interactions [10, 11]. However, when PEF nanoformulations come into contact with biological fluids in vivo , they encounter thousands of proteins, which reduces the detection sensitivity and specificity owing to inevitable non-specific adsorption [12], largely limiting an application of this approach for the in vivo NIR fluorescence imaging. On the other hand, an application of magnetic field (MF) targeting for localized bioimaging and therapy is widely reported, utilizing MF to magneticphoretically attract NPs loaded with imaging and/or therapeutic agents to targeted areas, where MF is the strongest. Unlike molecular targeting, magnetic targeting based on physical interactions is not limited by the specific receptor expression and maybe a more general active-targeting approach [13-17]. Recently, Y. Liu and colleagues proposed a multifunctional nanoplatform of upconversion/iron oxide (UCNP/Fe 3 O 4 ) NPs for magnetically targeted NIR-II imaging. The NIR-II imaging in vivo uncovers that UCNP/Fe 3 O 4 NPs tend to migrate toward the tumor under influence of MF from a magnet placed near the tumor, and exhibit intense tumor accumulation, about 6-fold higher than that without magnetic targeting [18]. D. Ni with colleagues reported magnetic NPs with 89 Zr radiolabeling and porphyrin molecules ( 89 Zr-MNP/TCPP) that exhibited a high tumor accumulation and significantly enhanced the fluorescence intensity under the presence of an external MF [19]. In addition, L. Chen with colleagues reported magnetoplasmonic nanocomposites of Au-shelled upconversion/iron oxide (MFNPs), which showed an ability to be magnetophoretically controlled and concentrated using the external MF. With the help of MF, the fluorescence intensity of tumor position was about 8-fold higher than that without MF targeting [20]. Hence, the application of an external MF is considered a simple but efficient method that can target some specific locations for in vivo fluorescence imaging. Very recently, we have reported Fe 3 O 4 @mSiO 2 @Au core@shell@satellites magnetoplasmonic NPs loaded with the chemotherapeutic drug doxorubicin for a magnetic field-induced and targeted combination of near-infrared photothermal therapy (NIR PTT) and chemotherapy [21]. When an external MF is applied to the dispersion of these NPs, it results in the magnetophoretic movement and aggregation of the NPs. The MF-induced aggregates reveal a notable absorption in NIR spectral range due to the plasmon resonance coupling between the Au satellites. As a result, an enhanced photothermal effect is observed in MF-treated NPs dispersion under 808 nm laser irradiation. The MF-induced, tumor targeted combination of NIR PTT with DOX chemotherapeutic action effectively kills cancer cells in vitro and restricts tumor growth in 4T1-tumor-bearing mice in vivo . Hence, our study revealed strong enhancement of NIR absorption of the magnetopasmonic NPs, which appeared only under the external magnetic field, resulting from the aggregation-induced plasmon resonance coupling. In this regard, it would be naturally to suggest that a presence of fluorophores within MF-induced aggregates may lead to an appearance of MF-induced PEF. Herein, we report a MF-induced PEF effect using Fe 3 O 4 @mSiO 2 @Au magnetoplasmonic nanoparticles surface-conjugated with a functionalized NIR fluorescent dye, IR775-silane (Scheme 1). The application of an external MF was shown to lead to the formation of Fe 3 O 4 @mSiO 2 @Au IR775 aggregates causing an increase in the IR775 fluorescence intensity with a simultaneous decrease in the fluorescence lifetime, which was attributed to the PEF effect resulted from the aggregation-induced increase in the amount of Au satellites in proximity to the IR775 fluorophores. In the in vivo studies, we achieved an efficient, magnetically induced PEF effect in the injected Fe 3 O 4 @mSiO 2 @Au-IR775 NPs, as the NIR fluorescence was significantly enhanced by the application of external MF. It also led to the prolonged accumulation of the NPs in the targeted region . 90 hours after ending of MF application, the fluorescence signal from the site of the injection of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs, which was treated by MF for 6 hours, was much higher than that from the injection site where MF was not applied. Moreover, NIR fluorescence lifetime imaging in vivo further confirmed the MF-induced PEF effect in the injected Fe 3 O 4 @mSiO 2 @Au-IR775 NPs: after the MF application, the NIR fluorescence lifetime significantly decreased in comparison with that before MF was applied, while the lifetime of the fluorescence from the control, MF-untreated site of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs injection remained the same. Taking into account the results of the histological studies revealing absence of noticeable toxicity from the injected NPs, this work provides a feasible but effective approach to induce PEF effect for in vivo NIR fluorescence imaging. At the same time, a possibility to induce and control PFE with the locally applied MF can provide interesting opportunities for multiple imaging and sensing applications. Results and Discussion The Fe 3 O 4 @mSiO 2 @Au-IR775 core@shell@satellites NPs were prepared by a layer-by-layer assembly approach using iron oxide, mesoporous silica, and gold as building blocks. Monodisperse Fe 3 O 4 NPs with an average diameter of 8 nm (Figure 1(a)) were first synthesized using a modified thermal decomposition protocol [22]. Then, a mSiO 2 shell (∼18 nm thick) was subsequently coated on the Fe 3 O 4 core using a reverse microemulsion approach [23]. A TEM performed after removal of the surfactant CTAB confirmed the formation of well-defined Fe 3 O 4 @mSiO 2 NPs with a 44 nm average diameter, single-core structure and a uniform mSiO 2 shell with ∼18 nm thickness (Figure 1(b)). Next, MPTES was used as a silane coupling reagent, which provided negatively charged sulfhydryl groups on the silica surface, allowing for attachment of the pre-synthesized Au NPs through formation of a strong Au-S bond. Finally, the functionalized NIR fluorescent dye IR775-silane was loaded into the mesoporous structure via a post-synthetic grafting method (Figure S1 and Experimental Section in Supporting Information); this process was facilitated by the abundant surface silanol groups (Si-OH) of mSiO 2 that serve as effective anchoring sites [24]. In such a way, Fe 3 O 4 @mSiO 2 @Au-IR775 NPs with core@shell@satellites structure were successfully obtained (Figure 1(c)). The element mapping images of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs (Figure 1(d)) prove that the prepared NPs have core of Fe (core), the mSiO 2 shell in the middle layer and the outer Au satellites. In order to further verify the structure of Fe 3 O 4 @mSiO 2 @Au-IR775 nanocomposites, energy dispersive spectroscopy (EDS) analysis was performed, quantitatively verifying the presence of Fe, Si, O, and Au elements (Figure S2 and Table S1); this result is in good agreement with TEM findings. In addition, X-ray photoelectron spectroscopy (XPS) was performed to further examine the chemical composition of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs. Figure 1(e) shows the XPS spectrum of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs over the scan range 0-1300 eV. As shown in Figure 1(f), after the Fe 3 O 4 core is coated with a mSiO 2 shell, the Fe 2p peak signal becomes very weak due to the shallow XPS detection depth limit of 10 nm [25], and the curve fitting analysis clearly reveals the characteristic peaks corresponding to Fe 2p 1/2 (724.0 eV) and Fe 2p 3/2 (711.4 eV). A clear Si 2p peak (103.0 eV) is observed, confirming a presence of Si element in the mSiO 2 shell (Figure 1(g)). The presence of Au 4f 7/2 (83.4 eV) and Au 4f 5/2 (87.0 eV) peaks further verified that the Fe 3 O 4 @mSiO 2 NPs were decorated with Au satellites (Figure 1(h)). Based on the TEM, EDX, and XPS results, we concluded that the Fe 3 O 4 @mSiO 2 @Au-IR775 NPs with the core@shell@satellites structure were successfully synthesized. Figure 2(a) shows the dynamic light scattering (DLS) results revealing that the hydrodynamic diameters of Fe 3 O 4 , Fe 3 O 4 @mSiO 2 and Fe 3 O 4 @mSiO 2 @Au-IR775 NPs are 7.5 nm, 43.8 nm and 50.7 nm, respectively. The progressive increase in nanoparticle size provides indirect evidence for the successful coating of Fe 3 O 4 core with the mSiO 2 shell and subsequent conjugation of Au seeds. The TEM and DLS results for pre-synthesized Au NPs are shown in Figure S3. Furthermore, the ζ potential of the Fe 3 O 4 @mSiO 2 NPs was found to change from -20.95 mV to -12.73 mV as a result of modification with MPTES; the following change of ζ potential from -12.73 mV to -24.90 mV is associated with the Au NPs and IR775-silane (Figure 2(b)). Overall, these characterization results on core-shell-satellites NPs are similar to those reported by us [21]. The optical properties of the NPs were investigated using optical absorption and fluorescence spectroscopies (Figure 2(c, d)). While Fe 3 O 4 and Fe 3 O 4 @mSiO 2 NPs exhibited no distinct absorption bands in 400-1000 nm spectral range, Fe 3 O 4 @mSiO 2 @Au-IR775 NPs displayed clear absorption bands peaked at ~520 nm and ~787 nm, which correspond to the localized surface plasmon resonance (LSPR) of Au satellites and the characteristic absorption of IR775-silane within the mSiO 2 shell, respectively. Notably, the IR775-silane absorption band in the Fe 3 O 4 @mSiO 2 @Au NPs exhibited a slight red-shift (787 nm) compared to free IR775-silane in methanol (775 nm), revealing change in polarity of the environment for the IR775-silane molecules [26, 27, 28]. A similar red-shift was observed in the fluorescence spectra, where the emission peak of IR775-silane shifted from 800 nm (in methanol) to 810 nm in the Fe 3 O 4 @mSiO 2 @Au-IR775 aqueous dispersion (Figure 2(d)). It is worth noting that the fluorescence intensity of IR775-silane in water was significantly quenched relatively to its methanol solution and the Fe 3 O 4 @mSiO 2 @Au NP-loaded dispersion. This attenuation likely arises from the propensity of IR775-silane to aggregate in aqueous media, leading to aggregation-caused quenching (Figure S4) [29]. This aggregation is confirmed by the appearance of the pronounced absorption band at ~710 nm; such a blue-shifted absorption band is knowingly associated with H-aggregates (e.g., dimers) of dye molecules [30]. As can be seen in Figures. S5 and S6, the fluorescence of IR775-silane in core-shell-satellites NPs reached saturation at loading concentration of 9 µg/mL, pointing towards aggregation of the dye fluorophores within the NPs at this and higher concentrations, which causes the fluorescence quenching [31, 32]. Figure 2(e) shows the magnetization characterization of Fe 3 O 4 @mSiO 2 @Au-IR775 at room temperature. One can see that the saturation magnetization (Ms) of Fe 3 O 4 @mSiO 2 @Au-IR775 is about 4.33 emu/g. Furthermore, Figure 2(f) shows the magnetization plot for near the zero magnetic field. Saturation remanence (Mrs) and coercivity (Hc) can be determined from the intersection of the hysteresis loop with two axes at 0.027 emu/g and 12.92 Oe, respectively. These two values indicate that a rather low residual magnetization is present when the external magnetic field is removed and that a low-intensity magnetic field is required to reduce the magnetization to zero. These results reveal that the Fe 3 O 4 in Fe 3 O 4 @mSiO 2 @Au-IR775 is in the superparamagnetic state [33, 34]. At the next stage, a behavior of NIR fluorescence from Fe 3 O 4 @mSiO 2 @Au-IR775 NPs under MF and without it was explored. While Figure 3(a) shows photographic images of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs water dispersion in a Petri dish without and with magnet application, Figure 3(b) presents corresponding NIR fluorescence images at different imaging camera exposure times (20, 50, and 100 ms). Notably, the Fe 3 O 4 @mSiO 2 @Au-IR775 at the corner of magnet (where MF is the strongest) produced the much stronger fluorescence compared to other areas under MF application, which can be associated with a plasmon coupling effect produced by the Au NPs becoming adjacent due to an MF-induced formation of Fe 3 O 4 @mSiO 2 @Au-IR775 aggregates/clusters [21, 35, 36]. As visualized in Figure 3(c), the total NIR fluorescence signal from the images of NPs under MF is clearly higher than that from NPs in absence of MF. It is worth noting that the NIR fluorescence from MF-gathered Fe 3 O 4 @mSiO 2 @Au-IR775 remains stable even 5 days after MF application (Figure S7). It should be noted that superparamagnetic NPs are well-established to aggregate under an external MF. Particularly, when exposed to MF, each nanoparticle acquires a magnetic dipole moment, leading to mutual attraction through magnetic dipole-dipole interactions. This phenomenon effectively drives the NPs together to form aggregates, a process commonly referred to as “magnetic chaining” [37]. Figure S8 shows TEM image illustrating the formation of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs aggregates. In turn, the magnetically-activated formation of the Fe 3 O 4 @mSiO 2 @Au aggregates leads to a shortening of distance between the satellite Au NPs, which, in turn, can cause their surface plasmon resonance coupling [21]. As illustrated in Figures S9-S13, the finite-difference time domain (FDTD) simulations reveal that the MF-induced formation of “dimer” and “trimer” structures of Fe 3 O 4 @mSiO 2 @Au NPs leads to an appearance of intense “hot spots” in electric field strength when the interparticle distance between adjacent Au NPs reduces. When the distance between adjacent Au NPs in “dimer” and “trimer” structures of Fe 3 O 4 @mSiO 2 @Au NPs is 1 nm, the electric field enhancement factors (|E/E 0 | 2 ) are 11.11 and 9.20. It is known that the excitation rate of fluorophores in PEF phenomenon tis directly proportional to the localized electric field intensity: the enhanced field strength correlates with increased photon absorption probability, consequently boosting fluorophore excitation rate. On the other hand, when fluorophores are close to metal NPs, the metal NPs can modify the local density of optical states (LDOS) around the fluorophores, increasing their radiative decay rate [38]. To verify an existence of PEF effect in the MF-induced aggregates of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs, we explored changes in fluorescence lifetime of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs aqueous dispersion with applied MF and without it using fluorescence lifetime imaging microscopy (FLIM) [39]. Figure 3(d, e) show the average fluorescence lifetime images and histograms for Fe 3 O 4 @mSiO 2 @Au-IR775 aqueous dispersion without and with MF application. The average fluorescence lifetime (τ m ) for Fe 3 O 4 @mSiO 2 @Au-IR775 NPs acquired from different locations of the Petri dish with NPs dispersion in the absence of MF was measured to be 0.68 ns, 0.66 ns, 0.69 ns for locations L1, L2, L3, respectively. In contrast, τ m was found to be significantly shorter (0.56 ns, 0.58 ns, 0.49 ns, and 0.46 ns) when an external MF was applied, with a clear correlation between areas with shorter lifetimes in FLIM images and higher fluorescence signal in the fluorescence images (Figure 3(d, e)). The obtained results suggest that a plasmon coupling effect produced by the Au NPs becoming adjacent due to an MF-induced formation of Fe 3 O 4 @mSiO 2 @Au aggregates/clusters [21], resulting in a powerful PEF effect and leading to a significant shortening of the fluorescence lifetime and the amplification of fluorescence intensity [40, 41]. Interestingly, the FLIM of some areas, where Fe 3 O 4 @mSiO 2 @Au aggregates were formed under an external applied MF (“e.g., “corners” C3 and C4 in Figure 3(d, e)), did not show a notable reduction of the average fluorescence lifetime (approximately 0.65 ns and 0.63 ns, respectively), while the fluorescence signal at these locations is noticeably increased compared to that at the locations L1, L2, L3, where MF was not applied. It means that PEF effect in the areas C3 and C4 was not as pronounced and the observed fluorescence enhancement was mainly associated with the increased concentration of IR775. Correspondingly, it is naturally to suggest that the magnitude of the PEF effect is proportional to the MF strength at the selected locations and correlates with the fluorescence lifetime change. At the same time, we hypothesize that there is a MF strength threshold range, above which PEF effect for Fe 3 O 4 @mSiO 2 @Au-IR775 NPs becomes substantial. For fluorescent molecules (IR775-silane) far away from metal nanoparticles (Au satellites), the fluorescence quantum yield Q 0 and lifetime τ 0 can be expressed as follows [42, 43]: Where Г is radiation decay rate and K nr is nonradiation decay rate. When IR775 fluorophores are in the vicinity of Au satellites, their radiation decay increases with the generation of an extra radiative rate (Γ m ) caused the Au NPs. In this case, the quantum yield Q m and lifetime τ m can be expressed as [44, 45] where Г m is plasmon-enhanced radiation decay rate. As seen in Equations (3) and (4), the quantum yield Q m of IR775 increases with an increase in the value of Γ + Γ m (at constant K nr ), while the fluorescence lifetime decreases. Thus, when the distance between the Au NPs and the IR775 fluorophore is within an appropriate range, the excitation and emission efficiency of IR775 are greatly enhanced, resulting in a significant enhancement of the fluorescence intensity [41]. It is worth also noting that the photostability of IR775-fluorophores should also increase in this case [12, 46, 47]. At the next stage of the study, a possibility to obtain a MF-induced PEF effect with Fe 3 O 4 @mSiO 2 @Au-IR775 NPs in vivo was assessed in small animals (mice). First, 200 μL of aqueous dispersion of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs were twice subcutaneously (s.c.) injected in two different places on the back of one mouse. Next, a small round magnet NdFeB magnet (5 mm diameter, 1 mm thick) was fixed at one of the locations for 6 hours and then removed. NIR fluorescence imaging was performed at certain intervals after injection (0, 6, 24, 36, 48, 72, and 96 h), as illustrated in Figure 4(a, b). Figure 4(b, c) shows that a significant enhancement of the fluorescence intensity was observed in the injection region after 6 hours of MF application, while only very weak enhancement was seen in the location where the magnet was not applied (probably after excessive water was drained off the injection site). Along with this, the fluorescence area decreased, suggesting gathering of NPs by the applied magnet. The quantification of the fluorescence signal in the images is shown in Figure 4(c), revealing about 2.1-fold more intense fluorescence after 6 h of MF application in comparison with that in absence of the external MF. Furthermore, as seen in Figure 4(c), the fluorescence signal from the injected and MF-treated Fe 3 O 4 @mSiO 2 @Au-IR775 NPs exhibited a prolonged retention in the injection site: 96 h post-injection (and 90 h after the magnet was removed) NIR fluorescence from MF-treated subcutaneously injected NPs was ~6.8-fold more intense than that from the subcutaneously injected and MF-untreated NPs. This is apparently associated with the MF-induced formation of Fe 3 O 4 @mSiO 2 @Au-IR775 aggregates, which cannot be cleared from the body as fast as the non-aggregated NPs. After acquisition of the in vivo imaging 96 hours post-injection of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs, the mouse was sacrificed and its organs (skin, liver, kidneys, lungs, heart, spleen) were resected and imaged immediately. As demonstrated in Figure 4(d), the images of the mouse skin samples harvested from two injection sites (with and without applied MF) also confirm an enhanced retaining of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs at the site where MF was applied. Figure 4(e) shows the ex vivo images of the resected organs of mouse injected with Fe 3 O 4 @mSiO 2 @Au-IR775 NPs, revealing that the fluorescence signal is seen from liver and undistinguishable from kidney, spleen, lungs, and heart. It is known that subcutaneously injected NPs are drained into the lymphatic system, reach lymph nodes and, subsequently, enter the bloodstream. While smaller NPs (typically less than 10 nm) can be filtered by kidneys and excreted in urine, larger NPs are captured by liver, processed and excreted into bile, followed by excretion from the body with feces [48, 49]. An enhanced fluorescence signal from the liver is clearly associated with the hepatobiliary elimination pathway and it can be mainly associated with the non-aggregated NPs, while the NPs in MF-induced aggregates mainly retain at the injection site. To confirm that the observed increase in the fluorescence intensity 6 h post-injection is associated with MF-induced PFE effect, FLIM of the injection sites was performed in vivo in another s.c. injected mouse at 0 h and 6 h after injection, accessing the changes in fluorescence lifetimes for Fe 3 O 4 @mSiO 2 @Au-IR775 NPs before and after MF application (Figure 5(a, b)). No noteworthy difference between fluorescence lifetimes at two injection sites (IS 1 and IS 2) was found before MF application (the average fluorescence lifetimes at IS 1 and IS 2 were found to be 0.68 ns and 0.71 ns, respectively). Similarly, no notable difference in the fluorescence intensity was observed. Six hours after injection, the fluorescence lifetime of the Fe 3 O 4 @mSiO 2 @Au-IR775 NPs at IS 2 (where the MF was not applied did not change much (from 0.71 ns to 0.64 ns). In contrast the FLIM image of IS 1 reveal two clearly distinct areas. While the average fluorescence lifetime in area 1 (A1) was found to be 0.47 ns (changing from 0.68 ns at 0 h time point, apparently as a result of MF application), the average fluorescence lifetime in area 2 (A2) was ~0.64 ns, which is almost the same as in IS 1 at 0 h time point and in IS 2, where MF was not applied. Moreover, fluorescence intensity in A1 is also drastically higher than in A2. We believe that the obtained data clearly prove the PEF effect occurrence in A1, while it is not revealed at A2. One can suggest that the difference between A1 and A2 is in MF strength; this difference is similar to that shown in Figure 3(d, e) (C1 and C2 vs C3 and C4). Overall. the obtained FLIM images allow us to solidify our hypothesis that not only shortening of the fluorescence lifetime correlates with the MF strength, but there is a MF strength threshold range, below which PEF effect for Fe 3 O 4 @mSiO 2 @Au-IR775 NPs is insignificant. It should be also noted that the lifetime changes correlate with the changes in fluorescence intensity, similarly as in the batch experiment (Figure 3) the shorted lifetime, the more intense fluorescence is. Hence, the FLIM imaging results presented in Figure 5 confirmed that a significant localized PEF effect was induced in vivo by the application of external MF to the s.c. injected Fe 3 O 4 @mSiO 2 @Au-IR775 NPs. Finally, to assess potential toxicity of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs in in vivo applications, the s.c. injected mice were sacrificed at different time points (4 and 21 days) after injection, their vital organs (lungs, liver, spleen, kidneys, and heart) were collected and histological analysis was performed. Figure 6 shows representative images of histopathological analysis of lungs, liver, spleen, kidneys, and heart sections stained with H&E at different treatment groups. The results of the histological analysis revealed the injected dosage of the Fe 3 O 4 @mSiO 2 @Au-IR775 NPs (200 µL, 1 mg/mL) did not cause any detectable toxicity in the s.c. injected mice, suggesting good biocompatibility safety of the Fe 3 O 4 @mSiO 2 @Au-IR775 nanoformulation in in vivo applications. Conclusions This study reports a phenomenon of magnetic field-induced plasmon-enhanced fluorescence using Fe 3 O 4 @mSiO 2 @Au-IR775 core@shell@satellite magnetoplasmonic nanoparticles and its application for NIR fluorescence bioimaging. The synthesized NPs demonstrate magnetophoretic ability and also form aggregates under external MF. The MF-induced aggregation may lead to plasmon coupling between Au satellites of different NPs, enhancing local electric field strength and consequently amplifying the fluorescence of IR775 fluorophores in the shell of Fe 3 O 4 @mSiO 2 @Au-IR775 NPs. This localized enhancement of fluorescence intensity correlates with localized shortening of the fluorescence lifetime, confirming PEF effect induced by MF application to NPs. The application of external MF to the NPs subcutaneously injected into mice lead to the significant enhancements of NIR fluorescence intensity (~2.1-fold after 6 h of MF application) in comparison with that from the injected NPs in absence of MF. Besides, fluorescence from the subcutaneously injected and MF-treated NPs at 90 hours after MF application (and 96 h post injection) was ~6.8-fold more intense than that from the subcutaneously injected and MF-untreated NPs, suggesting at MF-induced NPs aggregates cannot be cleared from the body as fast as the NPs non-treated with MF. Importantly, the FLIM imaging in vivo revealed that fluorescence lifetime from the part of injected and MF-treated NPs was significantly shortened (from ~0.68 ns to ~0.47 ns) after MF application, along with a significant increase in fluorescence intensity. At the same time, the average lifetime from another part of MF-treated NPs injection site was found to be the same as in the MF-untreated injection site 6 h after injection (0.64 ns). The obtained FLIM imaging results allowed us to suggest that the fluorescence lifetime shortening correlates with the MF strength and there is a threshold for MF strength, below which PEF effect for Fe 3 O 4 @mSiO 2 @Au-IR775 NPs is insignificant. Histological studies of the main mouse organs showed no detectable toxicity for treated mice, suggesting good biocompatibility of the Fe 3 O 4 @mSiO 2 @Au-IR775 nanoformulation. Thus, it was demonstrated that the MF-induced PEF effect in a magnetoplasmonic nanoplatform can be employed for targeted NIR fluorescence bioimaging. A possibility to control PFE by the external MF with variable strength may be of interest for other imaging and sensing applications. Declarations Funding This work has been supported by the National Natural Science Foundation of China (grants W2431056, 62361136586, 62475163) and Shenzhen Science and Technology Program (JCYJ20220818100202005, JCYJ20170818090620324). Author information Siqi Gao and Jiantao Liu contributed equally to this work. Authors and Affiliations Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China Siqi Gao, Jiantao Liu, Iuliia Golovynska, Zhenlong Huang, Yiqiang Wang, Hao Xie, Rana Zaki Abdul Bari, Hao Xu, Junle Qu, Tymish Y. Ohulchanskyy Contributions Siqi Gao: Investigation, Software, Formal analysis, Data curation, Writing-original draft. Jiantao Liu: Investigation, Writing-original draft. Iuliia Golovynska: Data curation, Formal analysis. Zhenlong Huang: Investigation. Yiqiang Wang: Investigation. Hao Xie: Investigation. Rana Zaki Abdul Bari: Investigation. Hao Xu: Investigation. Junle Qu: Funding acquisition, Supervision. Tymish Y. Ohulchanskyy: Conceptualization, Methodology, Supervision, Funding acquisition, Writing-review & editing. Corresponding author Correspondence to Tymish Y. Ohulchanskyy Ethics declarations Ethical approval All animal studies were performed with the requirement of the Animal Ethical and Welfare Committee of Shenzhen University (Approval No. SZUHSC-01) Competing interests The authors declare that they have no competing interests. References Glass AM, et al. Interaction of metal particles with adsorbed dye molecules: absorption and luminescence. Opt Lett. 1980; 5(9): 368. Zhang YJ, et al. Plasmonic core-shell nanomaterials and their applications in spectroscopies. Adv Mater. 2021; 33(50): e2005900. Miller MM, Lazarides AA. Sensitivity of metal nanoparticle surface plasmon resonance to the dielectric environment. J Phys Chem B. 2005; 109(46): 21556-21565. Hong G, et al. 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Adv Funct Mater. 2016; 26(22): 3837-3858. Itoh T, Yamamoto YS, Ozaki Y. Plasmon-enhanced spectroscopy of absorption and spontaneous emissions explained using cavity quantum optics. Chem Soc Rev. 2017; 46(13): 3904-3921. Prakash S, et al. Microscopic perspective of synergy between localized surface plasmon resonance and disruption of dye aggregates in metal nanoparticle-enhanced fluorescence. ACS Appl Nano Mater. 2023; 6(19): 17539-17547. Ray K, Lakowiczb JR. Metal-enhanced fluorescence lifetime imaging and spectroscopy on a modified SERS substrate. J Phys Chem C Nanomater Interfaces. 2013; 117(30): 15790-15797. Goldys EM, et al. Fluorescence amplification by electrochemically deposited silver nanowires with fractal architecture. J Am Chem Soc. 2007; 129(40): 12117-12122. Aslan K, et al. Annealed silver-island films for applications in metal-enhanced fluorescence: interpretation in terms of radiating plasmons. J Fluoresc. 2005; 15(5): 643-654. Lakowicz JR. Radiative decay engineering: biophysical and biomedical applications. Anal Biochem. 2001; 298(1): 1-24. Tobias AK, Jones M. Metal-enhanced fluorescence from quantum dot-coupled gold nanoparticles. J Phys Chem C. 2019; 123(2): 1389-1397. Gan W, et al. Atomically thin boron nitride as an ideal spacer for metal-enhanced fluorescence. ACS Nano. 2019; 13(10): 12184-12191. Gao Y, et al. More symmetrical "hot spots" ensure stronger plasmon-enhanced fluorescence: from Au nanorods to nanostars. Anal Chem. 2021; 93(4): 2480-2489. Yan Y, et al. High-throughput single-particle analysis of metal-enhanced fluorescence in free solution using Ag@SiO 2 core-shell nanoparticles. ACS Sens. 2017; 2(9): 1369-1376. Kennedy J, et al. In vivo studies investigating biodistribution of nanoparticle-encapsulated rhodamine B delivered via dissolving microneedles. J Control Release. 2017; 265: 57-65. Huang X, et al. Effect of injection routes on the biodistribution, clearance, and tumor uptake of carbon dots. ACS Nano. 2013; 7(7): 5684-93. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx GraphicalAbstract.tif Scheme1.tif.jpg Scheme 1. Schematic illustration of (a) synthesis of magnetoplasmonic Fe 3 O 4 @mSiO 2 @Au-IR775 nanoparticles, (b) plasmon-enhanced NIR fluorescence induced in situ by magnetic field locally applied to magnetoplasmonic NPs, and (c) depictions of the nanoparticles used in (a). Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 29 Jun, 2025 Reviews received at journal 29 Jun, 2025 Reviews received at journal 26 Jun, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 22 Jun, 2025 Reviews received at journal 21 Jun, 2025 Reviewers agreed at journal 21 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers invited by journal 18 Jun, 2025 Editor assigned by journal 18 Jun, 2025 Submission checks completed at journal 18 Jun, 2025 First submitted to journal 17 Jun, 2025 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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Ohulchanskyy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYHACxgcQOoFI9TwMDMwGJGthkyBNi71E8rHKH38OM/Cz5xgw/NxBjC0SaWm3eXgOM0j2vDFg7D1DlJYcs9sMEocZDG7kGDAzthGppfCHwWEGe5K0MPAkAG2RIFrLmWfJ0jwH0nkkzjwrONhLjBb29uSDH3/8sZbjb0/e+OAnMVoYBBIgtoGIA8RoYGDgJ1LdKBgFo2AUjGAAAA0aLzgkM2SuAAAAAElFTkSuQmCC","orcid":"","institution":"Shenzhen University","correspondingAuthor":true,"prefix":"","firstName":"Tymish","middleName":"Y.","lastName":"Ohulchanskyy","suffix":""}],"badges":[],"createdAt":"2025-06-17 08:39:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6912220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6912220/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03691-6","type":"published","date":"2025-09-29T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85390490,"identity":"3100a1c9-7713-4965-bec9-067e44bfa5cc","added_by":"auto","created_at":"2025-06-25 10:24:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1036369,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003eNPs (a), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e NPs (b), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs (c). (d) Elemental mappings showing the spatial distribution of Fe (green), Si (magenta), O (cyan), and Au (red) in the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. (e) XPS spectra of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, and (f) Fe 2p, (g) Si 2p and (h) Au 4f peaks from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 XPS spectra shown in (e). \u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/504d7b2ae122f259b3779be7.jpg"},{"id":85390499,"identity":"f3adf205-ada3-4426-ac7e-f3580d17718b","added_by":"auto","created_at":"2025-06-25 10:24:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":737302,"visible":true,"origin":"","legend":"\u003cp\u003e(a) DLS data for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. (b) ζ potentials for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e, MPTES-modified Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2 \u003c/sub\u003eand Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. (c) Absorbance of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e, Au, IR775-silane in water, IR775-silane in methanol, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. (d) Spectral of fluorescence intensity of IR775-silane in water, IR775-silane in methanol, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs in water. (e) Hysteresis loop of the magnetophoretic response of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, and (f) the curve of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs with the external magnetic field is near zero, wherein the two markers present the values of saturation remanence (M\u003csub\u003ers\u003c/sub\u003e) and coercivity (H\u003csub\u003ec\u003c/sub\u003e), respectively.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/0020ee25e29be7d0651c54f1.jpg"},{"id":85390492,"identity":"4d0889c2-3d2f-4082-a03c-0c26b75a2b2b","added_by":"auto","created_at":"2025-06-25 10:24:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":646705,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photographs of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aqueous dispersion in Petri dish without (upper row) and with (down row) applied MF, and (b) the corresponding NIR fluorescence images obtained by imaging camera at different exposure times (1: 20 ms, 2: 50 ms, and 3: 100 ms) and (c) fluorescence intensities for different camera exposure times using 808 nm laser irradiation. (d) Average fluorescence lifetime (τ\u003csub\u003em\u003c/sub\u003e) images and (e) lifetime distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aqueous dispersions without and with applied MF. Scale bar is 1 mm.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/d582a5850026ac2f22b8ada8.jpg"},{"id":85391642,"identity":"2fca077d-0b11-4c3e-8e46-008d058ff0bf","added_by":"auto","created_at":"2025-06-25 10:32:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":800972,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the \u003cem\u003ein vivo\u003c/em\u003e MF-induced PEF effect revealed by NIR fluorescence imaging. (b) NIR fluorescence images of the mouse at different time points after subcutaneous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs in different regions on the back of the mouse, with and without applied MF. (c) Fluorescence intensity versus time with and without applied MF regions in mouse. (d, e) \u003cem\u003eEx vivo\u003c/em\u003e NIR fluorescence imaging of main organs (heart, liver, spleen, lungs, kidneys, and skin) collected from mouse sacrificed 96 h after subcutaneous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/6e89308a2d2d0e19ca705b21.jpg"},{"id":85392469,"identity":"d525a561-2168-4126-b1d0-60b2db2c084f","added_by":"auto","created_at":"2025-06-25 10:40:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":567636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e NIR fluorescence lifetime imaging of mice acquired at 0 h (a) and 6 h (b) post subcutaneous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, comparing regions with and without MF application, and corresponding fluorescence intensity profile before and after applied MF of Figure (a, b). Scale bar is 1 mm.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/37bc609ca2297a0e3edb9912.jpg"},{"id":85390501,"identity":"313e09ee-101b-42c8-896f-00a4443a00ab","added_by":"auto","created_at":"2025-06-25 10:24:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2006229,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of histopathological analysis show the lungs, liver, spleen, kidneys, and heart sections stained with H\u0026amp;E at different treatment groups (control (healthy mouse without injection), mouse 4 days after s.c. injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs and mouse 21 days after injection). Graphs show the histopathology scoring of lungs, liver, spleen, kidneys, and heart tissue in different groups. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/3b3ad898730f5c41b06ca987.jpg"},{"id":92883997,"identity":"7bf10df3-5237-4ab4-9600-ff8c01489f20","added_by":"auto","created_at":"2025-10-06 16:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6415458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/766ed1a3-47ef-4f4c-939a-4a223b3f95ca.pdf"},{"id":85390496,"identity":"5dd47d72-c4c6-4be3-b951-ee01c4dcc701","added_by":"auto","created_at":"2025-06-25 10:24:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2239014,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/0794297332b3a315ae0c1576.docx"},{"id":85390493,"identity":"60050c00-0e4d-4653-90eb-2d5aca9edea0","added_by":"auto","created_at":"2025-06-25 10:24:05","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3012876,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/4c9b839be3986e9f89333e58.tif"},{"id":85391648,"identity":"924fff27-c6b1-4138-a0d3-437cfdf752b3","added_by":"auto","created_at":"2025-06-25 10:32:05","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":750670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSchematic illustration of (a) synthesis of magnetoplasmonic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 nanoparticles, (b) plasmon-enhanced NIR fluorescence induced \u003cem\u003ein situ\u003c/em\u003e by magnetic field locally applied to magnetoplasmonic NPs, and (c) depictions of the nanoparticles used in (a).\u003c/p\u003e","description":"","filename":"Scheme1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6912220/v1/59fef020ca52b26f5f3ee82b.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Magnetic field-induced plasmonic enhancement of near infrared fluorescence from a magnetoplasmonic nanoplatform for bioimaging applications","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlasmon-enhanced fluorescence (PEF) is a plasmonic phenomenon first reported in 1980 [1]. It originates from\u0026nbsp;the plasmon resonance coupling between the frequencies emitted by the fluorophore and local surface plasmon resonance (LSPR) of the metal particles (NPs) that can result in a significant enhancement in the emission intensity [2, 3]. Use of PEF phenomenon can be advantageous for fluorescence bioimaging, particularly for near infrared (NIR)\u003cem\u003e\u0026nbsp;\u003c/em\u003efluorescence imaging \u003cem\u003ein vivo\u003c/em\u003e, as it would specifically benefit from an enhancement of fluorescence from imaging probes, allowing for deeper tissue imaging with a low background [4, 5, 6]. However, only a few studies reported PEF\u0026nbsp;nanoformulations in \u003cem\u003ein vivo\u003c/em\u003e imaging systems; PEF nanoformulations designed for \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging generally lack high specificity/targeting ability and may reveal some toxicity in \u003cem\u003ein vivo\u003c/em\u003e applications; At the same time, the PEF effect in nanoformulations renders to be unstable under the \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging conditions [7, 8, 9]. Thus, to be employed in NIR imaging \u003cem\u003ein vivo\u003c/em\u003e, PEF\u0026nbsp;nanoformulations should possess good biocompatibility along with targeting ability for a specific application (e.g., tumor targeting in cancer theranostics).\u003c/p\u003e\n\u003cp\u003eThe PEF nanoformulations used in biosensing applications usually rely on the use of specific antibodies or aptamers as molecular recognition elements, which allow for specific binding to target analytes through the antibody-antigen or aptamer-nucleic acid interactions [10, 11]. However, when PEF\u0026nbsp;nanoformulations come into contact with biological fluids \u003cem\u003ein vivo\u003c/em\u003e, they encounter thousands of proteins, which reduces the detection sensitivity and specificity owing to inevitable non-specific adsorption [12], largely limiting an application of this approach for the \u003cem\u003ein vivo\u003c/em\u003e NIR fluorescence imaging. On the other hand, an application of magnetic field (MF) targeting for localized bioimaging and therapy is widely reported, utilizing MF to magneticphoretically attract NPs loaded with imaging and/or therapeutic agents to targeted areas, where MF is the strongest. Unlike molecular targeting, magnetic targeting based on physical interactions is not limited by the specific receptor expression and maybe a more general active-targeting approach [13-17]. Recently, Y. Liu\u0026nbsp;and colleagues proposed a multifunctional nanoplatform of upconversion/iron oxide (UCNP/Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) NPs for magnetically targeted NIR-II imaging. The NIR-II imaging\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e uncovers that UCNP/Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eNPs tend to migrate toward the tumor under influence of MF from a magnet placed near the tumor, and exhibit intense tumor accumulation, about 6-fold higher than that without magnetic targeting [18]. D. Ni with colleagues reported magnetic NPs with \u003csup\u003e89\u003c/sup\u003eZr radiolabeling and porphyrin molecules (\u003csup\u003e89\u003c/sup\u003eZr-MNP/TCPP) that exhibited a high tumor accumulation and significantly enhanced the fluorescence intensity under the presence of an external MF [19]. In addition, L. Chen with colleagues reported magnetoplasmonic nanocomposites of Au-shelled upconversion/iron oxide (MFNPs), which showed an ability to be magnetophoretically controlled and concentrated using the external MF. With the help of MF, the fluorescence intensity of tumor position was about 8-fold higher than that without MF targeting [20]. Hence, the application of an external MF is considered a simple but efficient method that can target some specific locations for \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVery recently, we have reported Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au core@shell@satellites magnetoplasmonic NPs loaded with the chemotherapeutic drug doxorubicin for a magnetic field-induced and targeted combination of near-infrared photothermal therapy (NIR PTT) and chemotherapy [21]. When an external MF is applied to the dispersion of these NPs, it results in the magnetophoretic movement and aggregation of the NPs. The MF-induced aggregates reveal a notable absorption in NIR spectral range due to the plasmon resonance coupling between the Au satellites. As a result, an enhanced photothermal effect is observed in MF-treated NPs dispersion under 808 nm laser irradiation. The MF-induced, tumor targeted combination of NIR PTT with DOX chemotherapeutic action effectively kills cancer cells \u003cem\u003ein vitro\u003c/em\u003e and restricts tumor growth in 4T1-tumor-bearing mice \u003cem\u003ein vivo\u003c/em\u003e. Hence, our study revealed strong enhancement of NIR absorption of the magnetopasmonic NPs, which appeared only under the external magnetic field, resulting from the aggregation-induced plasmon resonance coupling. In this regard, it would be naturally to suggest that a presence of fluorophores within MF-induced aggregates may lead to an appearance of MF-induced PEF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHerein, we report a MF-induced PEF effect using Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au magnetoplasmonic nanoparticles surface-conjugated with a functionalized NIR fluorescent dye, IR775-silane (Scheme 1). The application of an external MF was shown to lead to the formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au IR775 aggregates causing an increase in the IR775 fluorescence intensity with a simultaneous decrease in the fluorescence lifetime, which was attributed to the PEF effect resulted from the aggregation-induced increase in the amount of Au satellites in proximity to the IR775 fluorophores. In the \u003cem\u003ein vivo\u003c/em\u003e studies, we achieved an efficient, magnetically induced PEF effect in the injected Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, as the NIR fluorescence was significantly enhanced by the application of external MF. It also led to the prolonged accumulation of the NPs in the targeted region\u003cem\u003e.\u003c/em\u003e 90 hours after ending of MF application, the fluorescence signal from the site of the injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, which was treated by MF for 6 hours, was much higher than that from the injection site where MF was not applied. Moreover, NIR fluorescence lifetime imaging \u003cem\u003ein vivo\u003c/em\u003e further confirmed the MF-induced PEF effect in the injected Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs: after the MF application, the NIR fluorescence lifetime significantly decreased in comparison with that before MF was applied, while the lifetime of the fluorescence from the control, MF-untreated site of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs injection remained the same. Taking into account the results of the histological studies revealing absence of noticeable toxicity from the injected NPs, this work provides a feasible but effective approach to induce PEF effect for \u003cem\u003ein vivo\u003c/em\u003e NIR fluorescence imaging. At the same time, a possibility to induce and control PFE with the locally applied MF can provide interesting opportunities for multiple imaging and sensing applications.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 core@shell@satellites NPs were prepared by a layer-by-layer assembly approach using iron oxide, mesoporous silica, and gold as building blocks. Monodisperse Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs with an average diameter of 8 nm (Figure 1(a)) were first synthesized using a modified thermal decomposition protocol [22]. Then, a mSiO\u003csub\u003e2\u003c/sub\u003e shell (\u0026sim;18 nm thick) was subsequently coated on the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ecore using a reverse microemulsion approach [23].\u0026nbsp;A TEM performed\u0026nbsp;after removal of the surfactant CTAB confirmed the formation of well-defined Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eNPs with a 44 nm average diameter, single-core structure and a uniform mSiO\u003csub\u003e2\u003c/sub\u003e shell with \u0026sim;18 nm thickness (Figure 1(b)). Next, MPTES was used as a silane coupling reagent, which provided negatively charged sulfhydryl groups on the silica surface, allowing for attachment of the pre-synthesized Au NPs through formation of a strong Au-S bond. Finally, the functionalized NIR fluorescent dye IR775-silane was loaded into the mesoporous structure via a post-synthetic grafting method (Figure S1 and Experimental Section in Supporting Information); this process was facilitated by the abundant surface silanol groups (Si-OH) of mSiO\u003csub\u003e2\u003c/sub\u003e that serve as effective anchoring sites [24]. In such a way, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs with core@shell@satellites structure were successfully obtained (Figure 1(c)). The element mapping images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs (Figure 1(d)) prove that the prepared NPs have core of Fe (core), the mSiO\u003csub\u003e2\u003c/sub\u003e shell in the middle layer and the outer Au satellites. In order to further verify the structure of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 nanocomposites, energy dispersive spectroscopy (EDS) analysis was performed, quantitatively verifying the presence of Fe, Si, O, and Au elements (Figure S2 and Table S1); this result is in good agreement with TEM findings. In addition, X-ray photoelectron spectroscopy (XPS) was performed to further examine the chemical composition of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. Figure 1(e) shows the XPS spectrum of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs over the scan range 0-1300 eV. As shown in Figure 1(f), after the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e core is coated with a mSiO\u003csub\u003e2\u003c/sub\u003e shell, the Fe 2p peak signal becomes very weak due to the shallow XPS detection depth limit of 10 nm [25], and the curve fitting analysis clearly reveals the characteristic peaks corresponding to Fe 2p\u003csub\u003e1/2\u003c/sub\u003e (724.0 eV) and Fe 2p\u003csub\u003e3/2\u003c/sub\u003e (711.4 eV). A clear Si 2p peak (103.0 eV) is observed, confirming a presence of Si element in the mSiO\u003csub\u003e2\u003c/sub\u003e shell (Figure 1(g)). The presence of Au 4f\u003csub\u003e7/2\u003c/sub\u003e (83.4 eV) and Au 4f\u003csub\u003e5/2\u003c/sub\u003e (87.0 eV) peaks further verified that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e NPs were decorated with Au satellites (Figure 1(h)). Based on the TEM, EDX, and XPS results, we concluded that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs with the core@shell@satellites structure were successfully synthesized.\u003c/p\u003e\n\u003cp\u003eFigure 2(a) shows the dynamic light scattering (DLS) results revealing that the hydrodynamic diameters of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs are 7.5 nm, 43.8 nm and 50.7 nm, respectively. The progressive increase in nanoparticle size provides indirect evidence for the successful coating of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ecore with the mSiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eshell and subsequent\u0026nbsp;conjugation of Au seeds. The TEM and DLS results for pre-synthesized Au NPs are shown in Figure S3. Furthermore, the \u0026zeta; potential of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e NPs was found to change from -20.95 mV to -12.73 mV as a result of modification with MPTES; the following change of \u0026zeta; potential from -12.73 mV to -24.90 mV is associated with the Au NPs and IR775-silane (Figure 2(b)). Overall, these characterization results on core-shell-satellites NPs are similar to those reported by us [21]. The optical properties of the NPs were investigated using optical absorption and fluorescence spectroscopies (Figure 2(c, d)). While Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e NPs exhibited no distinct absorption bands in 400-1000 nm spectral range, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs displayed clear absorption bands peaked at ~520 nm and ~787 nm, which correspond to the localized surface plasmon resonance (LSPR) of Au satellites and the characteristic absorption of IR775-silane within the mSiO\u003csub\u003e2\u003c/sub\u003e shell, respectively. Notably, the IR775-silane absorption band in the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au NPs exhibited a slight red-shift (787 nm) compared to free IR775-silane in methanol (775 nm), revealing change in polarity of the environment for the IR775-silane molecules [26, 27, 28]. A similar red-shift was observed in the fluorescence spectra, where the emission peak of IR775-silane shifted from 800 nm (in methanol) to 810 nm in the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aqueous dispersion (Figure 2(d)). It is worth noting that the fluorescence intensity of IR775-silane in water was significantly quenched relatively to its methanol solution and the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au NP-loaded dispersion. This attenuation likely arises from the propensity of IR775-silane to aggregate in aqueous media, leading to aggregation-caused quenching (Figure S4) [29]. This aggregation is confirmed by the appearance of the pronounced absorption band at ~710 nm; such a blue-shifted absorption band is knowingly associated with H-aggregates (e.g., dimers) of dye molecules [30]. As can be seen in Figures. S5 and S6, the fluorescence of IR775-silane in core-shell-satellites NPs reached saturation at loading concentration of 9 \u0026micro;g/mL, pointing towards aggregation of the dye fluorophores within the NPs at this and higher concentrations, which causes the fluorescence quenching [31, 32].\u003c/p\u003e\n\u003cp\u003eFigure 2(e) shows the magnetization characterization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 at room temperature. One can see that the saturation magnetization (Ms) of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 is about 4.33 emu/g. Furthermore, Figure 2(f) shows the magnetization plot for near the zero magnetic field. Saturation remanence (Mrs) and coercivity (Hc) can be determined from the intersection of the hysteresis loop with two axes at 0.027 emu/g and 12.92 Oe, respectively. These two values indicate that a rather low residual magnetization is present when the external magnetic field is removed and that a low-intensity magnetic field is required to reduce the magnetization to zero. These results reveal that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ein Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 is in the superparamagnetic state [33, 34].\u003c/p\u003e\n\u003cp\u003eAt the next stage, a behavior of NIR fluorescence from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs under MF and without it was explored. While Figure 3(a) shows photographic images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs water dispersion in a Petri dish without and with magnet application, Figure 3(b) presents corresponding NIR fluorescence images at different imaging camera exposure times (20, 50, and 100 ms). Notably, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 at the corner of magnet (where MF is the strongest) produced the much stronger fluorescence compared to other areas under MF application, which can be associated with a plasmon coupling effect produced by the Au NPs becoming adjacent due to an MF-induced formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aggregates/clusters [21, 35, 36]. As visualized in Figure 3(c), the total NIR fluorescence signal from the images of NPs under MF is clearly higher than that from NPs in absence of MF. It is worth noting that the NIR fluorescence from MF-gathered Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 remains stable even 5 days after MF application (Figure S7). It should be noted\u0026nbsp;that superparamagnetic NPs are well-established to aggregate under an external MF. Particularly, when exposed to MF, each\u0026nbsp;nanoparticle\u0026nbsp;acquires a magnetic dipole moment, leading to mutual attraction through magnetic dipole-dipole interactions. This phenomenon effectively drives the NPs together to form aggregates, a process commonly referred to as \u0026ldquo;magnetic chaining\u0026rdquo; [37]. Figure S8 shows TEM image illustrating the formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs aggregates. In turn, the magnetically-activated formation of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au aggregates leads to a shortening of distance between the satellite Au NPs, which, in turn, can cause their surface plasmon resonance coupling [21]. As illustrated in Figures S9-S13, the finite-difference time domain (FDTD) simulations reveal that the MF-induced formation of \u0026ldquo;dimer\u0026rdquo; and \u0026ldquo;trimer\u0026rdquo; structures of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au NPs leads to an appearance of intense\u0026nbsp;\u0026ldquo;hot spots\u0026rdquo; in electric field strength when the interparticle distance between adjacent Au NPs reduces. When the distance between adjacent Au NPs in \u0026ldquo;dimer\u0026rdquo; and \u0026ldquo;trimer\u0026rdquo; structures of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au NPs is 1 nm, the electric field enhancement factors (|E/E\u003csub\u003e0\u003c/sub\u003e|\u003csup\u003e2\u003c/sup\u003e) are 11.11 and 9.20. It is known that the excitation rate of fluorophores in PEF phenomenon tis directly proportional to the localized electric field intensity: the enhanced field strength correlates with increased photon absorption probability, consequently boosting fluorophore excitation rate. On the other hand, when fluorophores are close to metal NPs, the metal NPs can modify the\u0026nbsp;local density of optical states (LDOS) around the fluorophores, increasing their radiative decay rate [38].\u003c/p\u003e\n\u003cp\u003eTo verify an existence of PEF effect in the MF-induced aggregates of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, we explored changes in fluorescence lifetime of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs aqueous dispersion with applied MF and without it using fluorescence lifetime imaging microscopy (FLIM) [39]. Figure 3(d, e) show the average fluorescence lifetime images and histograms for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aqueous dispersion without and with MF application. The average fluorescence lifetime (\u0026tau;\u003csub\u003em\u003c/sub\u003e) for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs acquired from different locations of the Petri dish with NPs dispersion in the absence of MF was measured to be 0.68 ns, 0.66 ns, 0.69 ns for locations L1, L2, L3, respectively. In contrast, \u0026tau;\u003csub\u003em\u003c/sub\u003e was found to be significantly shorter (0.56 ns, 0.58 ns, 0.49 ns, and 0.46 ns) when an external MF was applied, with a clear correlation between areas with shorter lifetimes in FLIM images and higher fluorescence signal in the fluorescence images (Figure 3(d, e)). The obtained results suggest that a plasmon coupling effect produced by the Au NPs becoming adjacent due to an MF-induced formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au aggregates/clusters [21], resulting in a powerful PEF effect and leading to a significant shortening of the fluorescence lifetime and the amplification of fluorescence intensity [40, 41]. Interestingly, the\u0026nbsp;FLIM of some areas, where\u0026nbsp;Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au aggregates were formed under an external applied MF (\u0026ldquo;e.g., \u0026ldquo;corners\u0026rdquo; C3 and C4 in Figure 3(d, e)), did not show a notable reduction of the average\u0026nbsp;fluorescence lifetime (approximately 0.65 ns and 0.63 ns, respectively), while the fluorescence signal at these locations is noticeably increased compared to that at the locations L1, L2, L3, where MF was not applied. It means that PEF effect in the areas C3 and C4 was not as pronounced and the observed fluorescence enhancement was mainly associated with the increased concentration of IR775. Correspondingly, it is naturally to suggest that the magnitude of the PEF effect is proportional to the MF strength at the selected locations and correlates with the fluorescence lifetime change.\u003c/p\u003e\n\u003cp\u003eAt the same time, we hypothesize that there is a MF strength threshold range, above which PEF effect for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs becomes substantial. For fluorescent molecules (IR775-silane) far away from metal nanoparticles (Au satellites), the fluorescence quantum yield \u003cem\u003eQ\u003csub\u003e0\u003c/sub\u003e\u003c/em\u003e and lifetime \u0026tau;\u003csub\u003e0\u003c/sub\u003e can be expressed as follows [42, 43]:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAdUAAABoCAYAAACnmmroAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAApESURBVHhe7d1LSNTdH8fxr39alhtbCIGQWoLUwpQWkY9lF80KRUy6YYIiaKsIiijoghtv1CYNIehCpLgx1C4YlSIuDDXciWYheEFyU+77P9/j7zeO84w2Osf86bxfcJg55zdoT8/i0/fcJur3vwQAAITtf84rAAAIE6EKAIAlhCoAAJYQqgAAWEKoAgBgCaEKAIAlhCoAAJYQqqt06dIliYqKCtr0GQAg8hCqq/T06VOpra2VoqIi0fsz3DYwMCDbt293PgUAiCSEqmW7du2S5ORkpwcAiCSEqmVbt26VkpISpwcAiCSEKgAAlhCqAABYQqgCAGAJoQoAgCWeC9WmpiZJSkoy5z31aMrdu3edJwAAeJtnvqR8bm5O8vLy5OfPn/LixQtzNGVkZESys7OloqJCrl696nzSG/SCh2fPnpn3MTEx0tnZKSkpKaYPAIhMnqlUL1++bF4/fPhgAlXpqwbqmzdvTOh6iV7+4F748OPHDwIVADwkMTHReTdP+9XV1U5PTLb09PQ4PXs8Eart7e3S0dEhNTU15pxnoG/fvsmvX7+cHgAAS9PlwydPnji9+UD9+vWr05tXX18vxcXFZsnRpnUPVa1AHzx4ICdPngxa7Q0NDcnOnTtl27ZtzggAAMFpBfry5Us5ePCgMyIyOjoqWVlZTm+Bjp87d87p2bHuoaoVqFaiZ86ccUYWaOBOTEzIiRMnglaw/rjgHgAi29jYmDQ0NMjZs2edkT+rqqpaNC0crnUP1enpadmyZYvs2LHDGVnw6dMn+fLli2RmZjojS/Nf4wxs+gwAsLn19fUFrUiXc+DAAbOXxxZPrKnOzs6aitTf4OCgme++ceNGSJuAVlKpBvuM1xoAYGXGx8clPj7e6YVOp4FtWfdQ1cDU9VRdV9UjNOr169dy7NgxE4buURoNWW1LWUmlGuwzXmsAgI3HE5Xqw4cPzb8udu/ebaq0O3fumDOgdXV15nl3d7dZe9Wm7wEACBQXF2fWVVcq8PhNODwRqroJ6d69e+YLvrVK03nxnJwc56nIq1evzO5fPbf68eNHZxQAgAW6Qendu3dOb4FeIqTj169f/8+mpN7eXt++HQ3XcAPWE6Gq51T37Nljpnx1ute96EGng+/fvy/R0dESGxtrxmxZq93C+mc/evSo72dptT01NSUJCQmmr8+8dpEFAGwW5eXl/zl7+vbtW9/S2rVr15zReY2Njb4xXVstKysz71dr3UNVA+bQoUPy/ft3uXnzpln/1KpU7/3t6uqSK1eumJ3BuktYW7Bdwquhv6e2tlaKiop8f9natFrW371aWnW3trbKkSNHzFkp9x8J+fn55oKL9+/f//F4EABgdfRSh1u3boV0W5JWsJWVlU5vvh8uz9z9uxytWN1NSrqxyb3GMFxaRerlEv4bmTQAm5ubpaSkxBlZOa1MNax1XVj/3JOTk1JaWuo8BQCsNZ3GXW5Xr14Scf78+UWXRNiwIUJ1rQQL1T/RqWp16tQp8xqMfqalpUXS09MlNTWVe4EBIEJ4Yk11s9HNVDMzM5KRkUGgAkAEIVQt06lfXVPVhfHh4WFnFAAQCQjVEPjvFD59+rRpbj9wp7BupkpLSzPnaXWT0nIXVgAANhdCNQS65qpLz9ra2tpMc/uB67F6h+T+/fvNeqqu2RYUFJjqFQCw+RGqFml46s5h9yCxbmbKy8uT3Nxc3xWMAIDNK2J3/+q0rR55UTExMdLZ2RnSpqKldv/quE4LK/fn6ZcEuGNKj9msZKcxAGBjiegjNetFvzBAz8PqRiY9v6rrru4tS4WFheYVALDxEKrrwK129Xao/v5+3xWMy519BQB4H2uqAABYQqiuAz12o9wvZnfvNQYAbGxM/wIAYAmVKgAAlhCqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqiuwODgoCQlJZlXAAACEaoham9vl3379sns7KwzAgDAYoRqiE6dOiWTk5MSHx/vjAAAsBihCgCAJYQqAACWEKoAAFhCqAIAYAmhCgCAJYQqAACWEKoh0gsf9u7dK58/fzbnVfXcKgAA/gjVEKWkpMiPHz/k9+/fpum5VQCANyUmJprXsbExiYqK8jVXRUWF9PT0OD17PBGqc3NzUlZWtug//NKlS85TAABCpxny5MkT817D0y2GsrKyJDs724zX19dLcXGxNDU1mb4tUf/+ot/O+3Xz+PFj+eeff+TRo0dy+PBhqkAAwKpoiGqenD171hlZoJWpBuno6KgzMh/ANmPQE5VqSUmJbN26VX79+iWHDh1yRv9Mq1n/6ta/UekCQGTRqd6Ghoaggepyp4VdVVVVUl1d7fTC55k11f7+fvMNMBquoXr69KmvrA9s+gwAEDn6+vrMFO9Sent75datW05v3oEDB+TDhw9OL3yeWVN9/vy5ZGZmOiOhCbVSDfbcKw0AYMf4+PiSX3qiVaw6ePCgefXnPx0cLk+Eqk77asBolarrqy49xrLcd5eGWqkGe+6VBgBYe8ePH5dr1645vbXjmelfPf954cIFycnJMf3u7m4Tttr0PQAAy4mLi/NVpP50HdWtRnWzUuCO38B11nB4YvdvMFevXpWLFy9KbGysNDY2yu3bt50nAAAEp7OebqxpwCYkJJj3/vxjz92kpFWsG67hTAd7plINFB0dbQLVlrXYKaxrwUePHvX9nLq6OpmamjL/E7Wvz/QzAIC/o7y83FeJ6vqqBmhg86dFmzstrGGqdyaEw7OhumPHDpmenjZN34dL11hra2ulqKho0V/uwMCAbN++3fnUyugacGtrqxw5ckRevnxpqmsN0fz8fOno6JD379+vaDczACA8eqmD7vAN5bYkvQiisrLS6c33w+XZUM3IyJCRkRHT9P1a2bVrlyQnJzu9lXM3Wemf8fXr19LV1SU1NTW+tWEAwN+lFade8rAcvSRCw9f/TOvbt2/D3szk2TXVtaDTs0NDQyGfYXUvzV/uhif9TEtLi6Snp0tqaqq5IxgAEJk8W6luFB8/fpSZmRlTqRKoABDZCNUw6KYkXVPVKYPh4WFnFAAQqQjVAP67hE+fPm2a2w/cJaybqNLS0sw5Wt2ktNxFFQCAzY9QDeB/S1NbW5tpbj9wLVbvi9y/f79ZT9X12oKCAlO9AgAiE6G6Shqezc3NvvuKdTNTXl6e5Obmmh3LAIDIEzG7f3Xq9tmzZ+Z9TEyMdHZ2/nFj0VK7f3Vcp4WV+7MmJiZ8Y0rPw4a6yxgAsDlE1JGav03PreplELqJqbS01Ky5ujcsFRYWmlcAwOZBqK4ht9LVG6H0+2LdaxeXO/cKANi4WFMFAMASQnUN6ZEbpeutyr3LGACwOTH9CwCAJVSqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqgCAGAJoQoAgCWEKgAAlhCqAABYQqgCAGCFyP8Blfs58P1XK2YAAAAASUVORK5CYII=\" height=\"104\" width=\"469\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere Г is radiation decay rate and K\u003csub\u003enr\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/sub\u003eis nonradiation decay rate. When IR775 fluorophores are in the vicinity of Au satellites, their radiation decay increases with the generation of an extra radiative rate (\u0026Gamma;\u003csub\u003em\u003c/sub\u003e) caused the Au NPs. In this case, the quantum yield \u003cem\u003eQ\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and lifetime \u0026tau;\u003csub\u003em\u003c/sub\u003e can be expressed as [44, 45]\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"452\" height=\"103\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere Г\u003csub\u003em\u003c/sub\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003eis plasmon-enhanced radiation decay rate. As seen in Equations (3) and (4), the quantum yield Q\u003csub\u003em\u003c/sub\u003e of IR775 increases with an increase in the value of \u0026Gamma; + \u0026Gamma;\u003csub\u003em\u0026nbsp;\u003c/sub\u003e(at constant\u0026nbsp;K\u003csub\u003enr\u003c/sub\u003e), while the fluorescence lifetime decreases.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThus, when the distance between the Au NPs and the IR775 fluorophore is within an appropriate range, the excitation and emission efficiency of\u0026nbsp;IR775\u0026nbsp;are greatly enhanced, resulting in a significant enhancement of the fluorescence intensity [41].\u0026nbsp;It is worth also noting that the photostability of IR775-fluorophores should also increase in this case\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[12, 46, 47].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the next stage of the study,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ea possibility to obtain a MF-induced PEF effect with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs \u003cem\u003ein vivo\u003c/em\u003e was assessed in small animals (mice). First, 200 \u0026mu;L of aqueous dispersion of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs were twice subcutaneously (s.c.) injected in two different places on the back of one mouse. Next, a small round magnet NdFeB magnet (5 mm diameter, 1 mm thick) was fixed at one of the locations for 6 hours and then removed. NIR fluorescence imaging was performed at certain intervals after injection (0, 6, 24, 36, 48, 72, and 96 h), as illustrated in Figure 4(a, b). Figure 4(b, c) shows that a significant enhancement of the fluorescence intensity was observed in the injection region after 6 hours of MF application, while only very weak enhancement was seen in the location where the magnet was not applied (probably after excessive water was drained off the injection site). Along with this, the fluorescence area decreased, suggesting gathering of NPs by the applied magnet. The quantification of the fluorescence signal in the images is shown in Figure 4(c), revealing about 2.1-fold more intense fluorescence after 6 h of MF application in comparison with that in absence of the external MF. Furthermore, as seen in Figure 4(c), the fluorescence signal from the injected and MF-treated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs exhibited a prolonged retention in the injection site: 96 h post-injection (and 90 h after the magnet was removed) NIR fluorescence from MF-treated subcutaneously injected NPs was ~6.8-fold more intense than that from the subcutaneously injected and MF-untreated NPs. This is apparently associated with the MF-induced formation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 aggregates, which cannot be cleared from the body as fast as the non-aggregated NPs. After acquisition of the \u003cem\u003ein vivo\u003c/em\u003e imaging 96 hours post-injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, the mouse was sacrificed and its organs (skin, liver, kidneys, lungs, heart, spleen) were resected and imaged immediately. As demonstrated in Figure 4(d), the images of the mouse skin samples harvested from two injection sites (with and without applied MF) also confirm an enhanced retaining of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs at the site where MF was applied. Figure 4(e) shows the \u003cem\u003eex vivo\u003c/em\u003e images of the resected organs of mouse injected with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs, revealing that the fluorescence signal is seen from liver and undistinguishable from kidney, spleen, lungs, and heart. It is known that subcutaneously injected NPs are drained into the lymphatic system, reach lymph nodes and, subsequently, enter the bloodstream. While smaller NPs (typically less than 10 nm) can be filtered by kidneys and excreted in urine, larger NPs are captured by liver, processed and excreted into bile, followed by excretion from the body with feces [48, 49]. An enhanced fluorescence signal from the liver is clearly associated with the hepatobiliary elimination pathway and it can be mainly associated with the non-aggregated NPs, while the NPs in MF-induced aggregates mainly retain at the injection site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm that the observed increase in the fluorescence intensity 6 h post-injection is associated with MF-induced PFE effect, FLIM of the injection sites was performed \u003cem\u003ein vivo\u003c/em\u003e in another s.c. injected mouse at 0 h and 6 h after injection, accessing the changes in fluorescence lifetimes for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs\u0026nbsp;before and after MF application (Figure 5(a, b)). No noteworthy difference between fluorescence lifetimes at two injection sites (IS 1 and IS 2) was found before MF application (the average fluorescence lifetimes at IS 1 and IS 2 were found to be\u0026nbsp;0.68 ns and 0.71 ns, respectively). Similarly, no notable difference in the fluorescence intensity was observed. Six hours after injection, the fluorescence lifetime of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs at IS 2 (where the MF was not applied did not change much (from 0.71 ns to 0.64 ns). In contrast the FLIM image of IS 1 reveal two clearly distinct areas. While the average fluorescence lifetime in area 1 (A1) was found to be 0.47 ns (changing from 0.68 ns at 0 h time point, apparently as a result of MF application), the average fluorescence lifetime in area 2 (A2) was ~0.64 ns, which is almost the same as in IS 1 at 0 h time point and in IS 2, where MF was not applied. Moreover, fluorescence intensity in A1 is also drastically higher than in A2. We believe that the obtained data clearly prove the PEF effect occurrence in A1, while it is not revealed at A2. One can suggest that the difference between A1 and A2 is in MF strength; this difference is similar to that shown in Figure 3(d, e) (C1 and C2 vs C3 and C4). Overall. the obtained FLIM images allow us to solidify our hypothesis that not only shortening of the fluorescence lifetime correlates with the MF strength, but\u0026nbsp;there is a MF strength threshold range, below which PEF effect for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs is insignificant. It should be also noted that the lifetime changes correlate with the changes in fluorescence intensity, similarly as in the batch experiment (Figure 3) the shorted lifetime, the more intense fluorescence is. Hence, the FLIM imaging results presented in Figure 5 confirmed that a significant localized PEF effect was induced \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eby the application of external MF to the s.c. injected Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs.\u003c/p\u003e\n\u003cp\u003eFinally, to assess potential toxicity of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs in \u003cem\u003ein vivo\u003c/em\u003e applications, the s.c. injected mice were sacrificed at different time points (4 and 21 days) after injection, their vital organs (lungs, liver, spleen, kidneys, and heart) were collected and histological analysis was performed. Figure 6 shows representative images of histopathological analysis of lungs, liver, spleen, kidneys, and heart sections stained with H\u0026amp;E at different treatment groups. The results of the histological analysis revealed the injected dosage of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs (200 \u0026micro;L, 1 mg/mL) did not cause any detectable toxicity in the s.c. injected mice, suggesting good biocompatibility safety of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 nanoformulation in \u003cem\u003ein vivo\u003c/em\u003e applications. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study reports a phenomenon of magnetic field-induced plasmon-enhanced fluorescence using Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 core@shell@satellite magnetoplasmonic nanoparticles and its application for NIR fluorescence bioimaging. The synthesized NPs demonstrate magnetophoretic ability and also form aggregates under external MF. The MF-induced aggregation may lead to plasmon coupling between Au satellites of different NPs, enhancing local electric field strength and consequently amplifying the fluorescence of IR775 fluorophores in the shell of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs. This localized enhancement of fluorescence intensity correlates with localized shortening of the fluorescence lifetime, confirming PEF effect induced by MF application to NPs. The application of external MF to the NPs subcutaneously injected into mice lead to the significant enhancements of NIR fluorescence intensity (~2.1-fold after 6 h of MF application) in comparison with that from the injected NPs in absence of MF. Besides, fluorescence from the subcutaneously injected and MF-treated NPs at 90 hours after MF application (and 96 h post injection) was ~6.8-fold more intense than that from the subcutaneously injected and MF-untreated NPs, suggesting at MF-induced NPs aggregates cannot be cleared from the body as fast as the NPs non-treated with MF. Importantly, the FLIM imaging \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003erevealed that fluorescence lifetime from the part of injected and MF-treated NPs was significantly shortened (from ~0.68 ns to ~0.47 ns) after MF application, along with a significant increase in fluorescence intensity. At the same time, the average lifetime from another part of MF-treated NPs injection site was found to be the same as in the MF-untreated injection site 6 h after injection (0.64 ns). The obtained FLIM imaging results allowed us to suggest that the fluorescence lifetime shortening correlates with the MF strength and there is a threshold for MF strength, below which PEF effect for Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 NPs is insignificant. Histological studies of the main mouse organs showed no detectable toxicity for treated mice, suggesting good biocompatibility of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e@Au-IR775 nanoformulation. Thus, it was demonstrated that the MF-induced PEF effect in a magnetoplasmonic nanoplatform can be employed for targeted NIR fluorescence bioimaging. A possibility to control PFE by the external MF with variable strength may be of interest for other imaging and sensing applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been supported by the National Natural Science Foundation of China (grants W2431056, 62361136586, 62475163) and Shenzhen Science and Technology Program (JCYJ20220818100202005, JCYJ20170818090620324).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiqi Gao and Jiantao Liu contributed equally to this work.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eKey Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China\u003c/p\u003e\n\u003cp\u003eSiqi Gao, Jiantao Liu, Iuliia Golovynska, Zhenlong Huang, Yiqiang Wang, Hao Xie, Rana Zaki Abdul Bari, Hao Xu, Junle Qu, Tymish Y. Ohulchanskyy\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eSiqi Gao: Investigation, Software, Formal analysis, Data curation, Writing-original draft. Jiantao Liu: Investigation, Writing-original draft. Iuliia Golovynska: Data curation, Formal analysis. Zhenlong Huang: Investigation. Yiqiang Wang: Investigation. Hao Xie: Investigation. Rana Zaki Abdul Bari: Investigation. Hao Xu: Investigation. Junle Qu: Funding acquisition, Supervision. Tymish Y. Ohulchanskyy: Conceptualization, Methodology, Supervision, Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Tymish Y. Ohulchanskyy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eAll animal studies were performed with the requirement of the Animal Ethical and Welfare Committee of Shenzhen University (Approval No. SZUHSC-01)\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGlass AM, et al. Interaction of metal particles with adsorbed dye molecules: absorption and luminescence. Opt Lett. 1980; 5(9): 368.\u003c/li\u003e\n \u003cli\u003eZhang YJ, et al. Plasmonic core-shell nanomaterials and their applications in spectroscopies. Adv Mater. 2021; 33(50): e2005900.\u003c/li\u003e\n \u003cli\u003eMiller MM, Lazarides AA. Sensitivity of metal nanoparticle surface plasmon resonance to the dielectric environment. 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ACS Nano. 2013; 7(7): 5684-93.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"magnetoplasmonic nanoparticles, plasmon-enhanced fluorescence, fluorescence lifetime imaging, magnetic field-induced aggregation, near infrared fluorescence bioimaging","lastPublishedDoi":"10.21203/rs.3.rs-6912220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6912220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"A phenomenon of plasmon-enhanced fluorescence (PEF) arises from interactions between fluorophores and metal nanostructures, leading to a substantial amplification of the fluorescence signal. Herein, we report a magnetic field (MF) induced on-demand PEF from the magnetoplasmonic nanoplatform and demonstrate its application in near infrared (NIR) bioimaging. The developed magnetoplasmonic nanoparticles (~ 50 nm diameter) feature a core-shell-satellite architecture comprising a Fe3O4 magnetic core, a mesoporous silica (mSiO2) shell housing IR775-silane NIR dye, and surface-anchored gold (Au) seeds (satellites). Application of an external MF causes the magnetophoretic movement and aggregation of the nanoparticles (NPs), resulting in a formation of localized plasmonic hotspots and, consequently, in a plasmonic enhancement of NIR fluorescence from IR775 dye molecules. Correspondingly, a substantial reduction of the fluorescence lifetime in the MF-treated area was observed, in addition to the enhanced fluorescence intensity. In vivo studies with NPs subcutaneously injected into mice revealed MF-activated amplification of NIR fluorescence. At 6 h post-injection, the injected region treated by MF exhibited 2.1-fold stronger NIR fluorescence signal than the MF-untreated one; the fluorescence enhancement correlated with the reduction of the emission lifetime (from 0.68 ns to 0.47 ns). At 96 h post-injection, the MF-treated region exhibited 6.8-fold more intense NIR fluorescence. Histological analysis showed absence of toxicity from the injected NPs, revealing their biocompatibility. Hence, a considerable potential of MF-induced PEF with the magnetoplasmonic nanoplatform for targeted NIR fluorescence bioimaging was demonstrated. This work also introduces MF-induced PEF as a powerful strategy for spatiotemporal control of optical signals, offering new opportunities for targeted imaging and sensing.","manuscriptTitle":"Magnetic field-induced plasmonic enhancement of near infrared fluorescence from a magnetoplasmonic nanoplatform for bioimaging applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 10:23:53","doi":"10.21203/rs.3.rs-6912220/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-30T03:41:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-29T14:36:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T11:56:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T14:51:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315416333632469270087615942824734306108","date":"2025-06-23T02:18:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-22T01:19:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47906219724966402367306795479182896271","date":"2025-06-21T10:10:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101944109692214233617787507353752287372","date":"2025-06-21T03:48:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46691406142251358904291282603484549706","date":"2025-06-20T14:52:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308920346140757073699452479922616448935","date":"2025-06-20T00:45:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256642430313634656583565310268181781019","date":"2025-06-19T04:11:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42216577409763764970428755053646316155","date":"2025-06-19T04:09:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257367357663556409005043231229527860120","date":"2025-06-19T04:01:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-19T03:46:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T05:53:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-18T05:36:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-06-17T08:35:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fcda3a19-5acd-4f3b-9ae3-3db7ec149097","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:06:36+00:00","versionOfRecord":{"articleIdentity":"rs-6912220","link":"https://doi.org/10.1186/s12951-025-03691-6","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-09-29 15:57:42","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-06-25 10:23:53","video":"","vorDoi":"10.1186/s12951-025-03691-6","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03691-6","workflowStages":[]},"version":"v1","identity":"rs-6912220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6912220","identity":"rs-6912220","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}