Article www.acsnano.org Efficient Tumor Eradication at Ultralow Drug Concentration via Externally Controlled and Boosted Metallic Iron Magnetoplasmonic Nanocapsules Arnon Fluksman, Aritz Lafuente, Zhi Li, Jordi Sort, Silvia Lope-Piedrafita, Maria José Esplandiu, Josep Nogues, Alejandro G. Roca, Ofra Benny,* and Borja Sepulveda* See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: ACS Nano 2023, 17, 1946−1958 Read Online Downloaded via 83.41.163.172 on March 1, 2023 at 18:35:05 (UTC). ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: With the aim to locally enhance the efficacy of cancer nanotherapies, here we present metal iron based magnetoplasmonic drug-loaded nanocapsules (MAPSULES), merging powerful external magnetic concentration in the tumor and efficient photothermal actuation to locally boost the drug therapeutic action at ultralow drug concentrations. The MAPSULES are composed of paclitaxel-loaded polylactic-co-glycolic acid (PLGA) nanoparticles partially coated by a nanodome shape iron/silica semishell. The iron semishell has been designed to present a ferromagnetic vortex for incorporating a large quantity of ferromagnetic material while maintaining high colloidal stability. The large iron semishell provides very strong magnetic manipulation via magnetophoretic forces, enabling over 10-fold higher trapping efficiency in microfluidic channels than typical super- paramagnetic iron oxide nanoparticles. Moreover, the iron semishell exhibits highly damped plasmonic behavior, yielding intense broadband absorbance in the near-infrared biological windows and photothermal efficiency similar to the best plasmonic nanoheaters. The in vivo therapeutic assays in a mouse xenograft tumor model show a high amplification of the therapeutic effects by combining magnetic concentration and photothermal actuation in the tumor, leading to a complete eradication of the tumors at ultralow nanoparticle and drug concentration (equivalent to only 1 mg/kg PLGA nanoparticles containing 8 μg/kg of paclitaxel, i.e., 100−500-fold lower than the therapeutic window of the free and PLGA encapsulated drug and 13−3000-fold lower than current nanotherapies combining paclitaxel and light actuation). These results highlight the strength of this externally controlled and amplified therapeutic approach, which could be applied to locally boost a wide variety of drugs for different diseases. KEYWORDS: nanocapsules, photothermal therapy, magnetic manipulation, paclitaxel, breast cancer N anotherapies based on organic nanoparticles, e.g., liposomes, dendrimers, micelles, polymeric particles, conjugated or encapsulated with drugs have emerged as a valuable tool for overcoming the side effects of the clinical approval of only very few nanotherapies to date, which have not demonstrated the high-efficacy gains expected from the preclinical assays.6 Thus, strategies enhancing the delivery and efficacy of the therapeutic agents could reduce the time course of treatments while reducing treatment frequency chemotherapies by increasing both the circulation time and accumulation of the drug inside solid tumors by exploiting and dosages. their high drug loading capacity and the currently debated enhanced permeability and retention (EPR) in the tumor Received: June 16, 2022 vasculature.1,2 However, nanotherapies have not yet met these Accepted: November 28, 2022 expectations due to the final low nanoparticle concentration in Published: December 5, 2022 solid tumors.3,4 A recent analysis of nanotherapies in murine models showed that only an average 0.7% of the injected nanoparticles arrive at the tumor.5 These hurdles are behind © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsnano.2c05733 1946 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Magnetic nanostructures have been proposed to externally control and improve nanotherapy delivery.7−10 However, these nanostructures are mostly based on small superparamagnetic iron oxide nanoparticles, which limit the drug loading capacity and exhibit a weak magnetic moment that severely hampers their efficient external actuation with magnetic fields. The use of this type of particle stems from the difficulty to achieve colloidally stable ferromagnetic nanoparticles by chemical synthesis, as they tend to irreversibly aggregate by their intense magnetic dipolar interactions in the absence of a magnetic field.11 Interestingly, multifunctional dimeric nanoparticles combining plasmonic and magnetic moieties have also been proposed for simultaneous light and magnetic actuation.8,9 Nevertheless, these systems generally show a similar weak magnetic response and limited drug loading capabil- ities.10,12−14 Therefore, the development of drug vehicles with improved capacity of external control is required, as a way to achieve more efficient local release of the drug and to enhance the therapeutic activity.15 Here we present a biodegradable metal iron based magnetoplasmonic nanocapsule, which combines a large drug-loading capacity with the following unprecedented set Figure 1. MAPSULES structural properties. (A) Schematic of the of multiactuation and therapeutic capabilities: (i) highly drug-loaded ferromagnetic nanocapsules components and their efficient magnetic manipulation and concentration via functionalities. (B) SEM image of the self-assembled PLGA magnetophoretic forces keeping a high colloidal stability, (ii) nanoparticles capped with Fe (20 nm) and SiO2 (10 nm). (C) intense magnetic resonance imaging and strong optical TEM images of the ferromagnetic nanocapsules after dispersion in water for 3 h. (D) High-magnification TEM image of the semishell absorption to noninvasively visualize and track the nano- to show the thickness of the Fe and SiO2 layers, and (E) EDX capsules, and (iii) highly efficient and broadband optical mapping at the energies corresponding to Fe and Si atoms. heating in the two biological windows as adjuvant therapy. This set of features has enabled magnetic concentration of the nanocapsules at the tumor region and eradication of the strong ferromagnetic and highly damped plasmonic properties, tumors in mice at very low nanocapsule and drug while the SiO2 protects the Fe from a rapid oxidation and concentrations by exploiting the external photothermal provides negative charges to improve the colloidal stability. actuation. The MAPSULES are finally dispersed in water or buffer by mild ultrasonication. The obtained polydispersity index of the RESULTS AND DISCUSSION dispersed MAPSULES by dynamic light scattering was 0.2. Fabrication and Physicochemical Properties. The However, we consider that this value is overestimated due to active biodegradable magnetoplasmonic nanocapsules (MAP- the inherent anisotropy of the MAPSULES, which show SULES) are composed of acid terminated polylactic-co- different scattering cross sections depending on their glycolic-acid (PLGA) nanoparticles (diameter ca. 150 nm) orientation with respect to the polarization of the incident loaded with the anticancer drug paclitaxel, which are partially light, as will be discussed below. Once dispersed in water, the covered by metallic Fe (20 nm) and a SiO2 (10 nm) layers PLGA hydrolysis triggers the nanocapsule degradation and the (Figure 1A). paclitaxel release. According to the high-performance liquid The MAPSULES fabrication is based on a cost-effective and chromatography (HPLC) measurements, approximately 40% scalable combination of bottom-up and top-down processes, of the drug is released within the first 4 days (Figure S1F). involving nanoparticle synthesis, self-assembly, and physical This release profile correlates with the known burst kinetic vapor deposition.16,17 In this case, the fabrication process can effect in PLGA nanoparticles (NPs).18,19 The full degradation be divided into three steps (see Methods): (i) synthesis of the of the PLGA nanoparticle nearly takes one month, as can be paclitaxel-loaded PLGA nanoparticles by the emulsification- observed in Figure S1G. evaporation method, showing an average hydrodynamic The diameter of the nanocapsules and the thickness of the diameter of 150 nm (polydispersity index 0.07 ± 0.01), a Fe layer have been designed to simultaneously achieve high zeta potential of −30 mV given by the carboxylic groups at the saturation magnetization, vortex magnetic structure, and particle surface (Figure S1E), and paclitaxel loading efficiency intense broadband optical absorbance covering the whole of 38%, which corresponds to a drug payload of 0.8% (i.e., 0.8 near-infrared range. mg paclitaxel/100 mg PLGA × 100); (ii) electrostatic self- The MAPSULES show in-plane magnetization with a vortex assembly of the loaded PLGA nanoparticles on a positively magnetic configuration (Figure 2A) with a near zero charged solid support (e.g., silicon wafers or Kapton films) by remanence and a high susceptibility, i.e., M(H) slope.20 As a exploiting the negative charge of the PLGA particles, which consequence, the magnetic moment and the dipolar inter- generates a uniform monolayer of randomly distributed and actions between the nanocapsules are negligible in the absence well separated PLGA nanoparticles (Figure 1B), and (iii) of magnetic field, which is crucial to keep stable colloidal deposition of the Fe (20 nm) and SiO2 (10 nm) layers by dispersions. Importantly, the metallic iron layer offers a much electron beam evaporation to form the “nanodome” structure higher saturation magnetization (MS = 218 emu/g) than that (Figures 1C−E and S1A,B). The metallic Fe layer provides the of typical superparamagnetic iron oxide nanoparticles, such as 1947 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Figure 2. MAPSULES magnetic and optical properties. (A) In-plane and out-of-plane normalized hysteresis loops showing the preferential in-plane ferromagnetic vortex structure (inset image shows a schematic of the magnetic vortex configuration). (B) Magnetic trapping efficiency in a microfluidic channel at increasing flow rates (see experimental set up in Figure S2). (C) Nuclear magnetic resonance T2 contrast and r2 relaxivity. (D,E) Evolution of the nanoparticle absorbance during degradation in PBS at 37 °C at (D) pH 7 or (E) pH 4. (F) Demonstration of the efficient optical heating in the first and second biological windows, using 808 and 1064 nm lasers, respectively (incident light power 200 mW, beam diameter 5.2 mm − 0.95 W/cm2, MAPSULES concentration 9.75 μg/mL, and volume 600 μL). Fe3O4 (MS = 92 emu/g). The larger magnetization and the the large PLGA nanoparticle acting as nonmagnetic cargo in high colloidal stability of the metallic iron semishell enable the MAPSULES and the much higher viscous friction in the fabricating nanoparticles with more powerful magnetic liquid imposed by their much larger hydrodynamic diameter. features. As a comparison, a MAPSULE with a diameter of These results highlight their potential for efficient magnetic 150 nm exhibits a saturation magnetic moment ca. 300-fold trapping at the blood capillaries surrounding the tumor area. larger than that in superparamagnetic 16 nm edge magnetite The high magnetization of the metal iron layer also offers nanocubes. Over that size, the magnetite cubes become noninvasive and high-contrast imaging of the MAPSULES by ferromagnetic, and their colloidal stability is markedly nuclear magnetic resonance (NMR). The MAPSULES exhibit reduced.21 As the magnetophoretic forces are proportional to a very strong T2 signal in NMR showing a relaxivity r2 of 368 the magnetic moment, the high values that can be achieved s−1 mM−1 (Figure 2C), which is much higher than that of with the MAPSULES are the key for their efficient magnetic commercial contrast agents based on iron oxides (e.g., Feridex trapping and accumulation using moderate external magnetic and Resovist; r2 ≈ 120−189 s−1 mM−1).23 field gradients. To demonstrate the magnetic actuation Regarding the optical properties (Figure 2D), the capability via magnetophoretic forces, we compare the MAPSULES present an intense broadband absorbance that magnetic trapping efficacy of the MAPSULES and 14 nm slightly decreases toward shorter wavelengths due to the edge magnetite nanocubes by a spherical FeNdB magnet scattering reduction in this region. The undefined broad (diameter 18.8 mm, surface magnetic field 7.1 kOe, and field absorption band is a result of the highly damped plasmonic gradient according to Figure S2B) in a microfluidic channel behavior of the metallic iron, whose imaginary part of the mimicking the flow conditions in blood vessels (see Methods dielectric constant is much higher than that in the typical and Figure S2). The percentage of trapped nanoparticles by plasmonic metals, such as Ag and Au.24,25 the magnet is quantified by measuring the relative decrease of Interestingly, the absorbance variations can be used to the absorbance (ΔA/A) of the nanocapsule dispersions that analyze the degradation of the iron semishell during time. As flow through the microfluidic channel. As can be observed in can be observed in Figure 2D, the absorbance spectrum of the Figure 2B, even in a single fluidic pass, an extremely high nanocapsules shows a fast decrease in the whole visible range trapping efficiency (>50%) can be achieved for flow rates lower during the first 4 days (ca. 46% decrease) when the capsules than 1 mL/min, which are in the range of the blood flow in are kept at 37 °C and pH 7, which is accompanied by an mice (i.e., from 90 μL/min in the spleen to 1800 μL/min in increase of the absorbance in the ultraviolet region of the the liver).22 For higher speeds, the trapping percentage is spectrum. This optical evolution is consistent with the partial stabilized at ca. 20% per pass. Importantly, the trapping oxidation of the metallic iron layer, which initially shows efficiency of the MAPSULES is over 10-fold higher than that of plasmonic enhanced absorption in the near-infrared spectral the superparamagnetic magnetite nanocubes. It is worth region, into iron oxides and hydroxides showing increased highlighting that this large enhancement is achieved despite absorption in the blue and ultraviolet regions.26 In addition, 1948 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Figure 3. In vitro characterization. (A) Comparison of the viability of the unloaded and paclitaxel-loaded PLGA nanoparticles and the MAPSULES, keeping identical nanoparticles and drug concentration (10 μg/mL of PLGA nanoparticles and 0.08 μg/mL of paclitaxel). (B) Bright field images of the untreated (left) and MAPSULES-treated (right) MDA-MB-231 cells. The internalized MAPSULES are observed as black dots inside the cells. (C) Comparison of the viability under the different treatment conditions with the MAPSULES (P+), laser irradiation (L+), and magnetic concentration (M+). (D) Analysis of the photothermal effects in the cultured MDA-MB-231 cells treated with the MAPSULES and the MAPSULES after magnetic concentration compared to the untreated cells. * p < 0.05, *** p < 0.001 the absorbance of the supernatant in the ultraviolet region the MAPSULES and iron nanospheres despite the amount of followed a continuous increase that is related to the release of iron is 5-fold lower for the MAPSULES. Moreover, the partially water-soluble PLGA moieties and Fe3+ ions to the MAPSULES exhibit a flatter absorption spectrum, showing media (Figure S3). A negligible spectral contribution from the similar absorption in the first and second biological windows. surfactants and paclitaxel is expected in the supernatant Compared to iron nanodiscs, the absorption cross section is considering the fabrication process, which eliminates any substantially higher for the MAPSULES, and it is more remaining surfactants used in the synthesis of the PLGA isotropic, as the absorption variations for the different nanoparticles and the low solubility and small quantity of the orientations of the nanostructure with respect to the light paclitaxel compared to the PLGA (i.e., 0.8%). In addition, as polarization are much weaker. the nanoparticle internalization inside cells generally leads to These optical features are ideal for achieving high photo- nanoparticle trapping inside lysosomes with low internal pH thermal conversion efficiencies. Indeed, the photothermal (i.e., between 4 and 5), we have also analyzed the MAPSULES conversion efficiency in the first (wavelength 808 nm) and degradation at pH 4. As can observed in Figure 2E, the second biological windows (wavelength 1064 nm) are 63 and degradation at lower pH is substantially faster, with an almost 67%, respectively (Figure 2F), which is comparable with state- complete loss of the absorbance after 7 days. The rapid of-the-art nanoplasmonic heaters.27,28 The slightly increased degradation is due to the destabilization of the SiO2 layer at temperature variation at 1064 nm observed in Figure 2F is a low pH, thereby exposing the Fe layer, whose corrosion is also consequence of the water heating contribution at this enhanced by the higher solubility of the iron ions at low pH. wavelength (Figure S4E). This behavior is very relevant to promote a fast biodegradation In Vitro Analysis. To assess the biomedical potential of the of the MAPSULES but enables the preservation of the optical and magnetic properties during the necessary time (i.e., a few MAPSULES, we first analyzed their toxicity and therapeutic hours post injection) to externally control and enhance the activity in vitro by their exposure to cultures of human breast therapeutic effects, as it will be shown below. adenocarcinoma MDA-MB-231 cells under different treatment The highly damped plasmonic behavior of the metallic iron conditions (Figure 3). semishell induces a large reduction of the scattering cross We first compared the effects of the loaded and unloaded section of the nanocapsules while maintaining a high and PLGA nanoparticles and the MAPSULES. The tumor cells broadband absorption cross section (Figure S4A−C). This is were incubated for 48 h with equal PLGA and paclitaxel due to the higher penetration of the light electromagnetic field concentrations of 10 μg/mL of PLGA and 0.08 μg/mL of inside the iron film compared to the penetration in typical paclitaxel. In the case of the MAPSULES, this value plasmonic metals.24 The nanodome shape is also very relevant corresponds to a mass concentration of 28 μg/mL due to from the photothermal perspective, as it offers several benefits the mass contribution of the Fe and SiO2 layers. The unloaded with respect to other typical shapes of similar dimensions, such PLGA nanoparticles and the MAPSULES without drug did not as iron nanodiscs or nanospheres. The simulations in Figure reduce the viability of the cells compared to the untreated S4C show that the absorption cross section, which is the control cells (Figure 3A). In contrast, for both the paclitaxel- responsible for the optical heating, is approximately equal for loaded PLGA nanoparticles and the MAPSULES, the viability 1949 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Figure 4. In vivo biodistribution analysis. (A) Magnetic resonance T2-weighted images of hsd/foxn1 nude mice before and after tail vein injection of 2.5 mg/kg of unloaded MAPSULES (white dashed line points to the liver, the white line points to the the spleen, and the yellow line points to the kidneys). (B) Comparison of the organs’ accumulation at 2 h after injection with respect to the saline-treated mice. (C) Liver biodistribution over time. (D) Hematoxylin and eosin stained sections of the spleen, kidney, and liver of mice 72 h post intravenous injection of saline or unloaded MAPSULES. was reduced to 46 and 40%, respectively, due to the effect of °C (Figure 3D). Note that the light-induced amplification of the paclitaxel release (Figure 3A). the therapeutic effects could be substantially increased by These results confirmed the low toxicity of the MAPSULES, irradiating the samples inside an incubator at 37 °C. Moreover, the preservation of the paclitaxel release, and its therapeutic notice that the obtained temperature variation cannot be activity even in the MAPSULES morphology, indicating that compared to that of Figure 2F, as the experimental conditions the fabrication process does not degrade their properties. in terms of particle distribution, buffer volume, illuminated Moreover, the bright field images of the MAPSULES-treated area, and light path length in the sample are very different. MDA-MB-231 cells, taken after 4 h of incubation, confirm the In Vivo Toxicity and Biodistribution Analysis. Once the internalization of the MAPSULES, as shown by black dots low cytotoxicity and the therapeutic activity were demon- inside the cells (Figure 3B). strated in vitro, we analyzed in vivo the MAPSULES Next, we analyzed the effects of the magnetic concentration biodistribution and toxicity in Hsd:Athymic Nude-Foxn1nu and photothermal actuation of the MAPSULES on the viability mice by exploiting their intense T2 contrast in NMR. We of the cancer cells (Figure 3C,D). We compared the passive injected 2.5 mg/kg (i.e., 100 μg) of unloaded MAPSULES in administration of the MAPSULES with the administration mice (N = 2) via tail vein injection, and we monitored their under the effect of a magnetic field gradient generated by a distribution by T2-weighted imaging at different times (Figure spherical FeNdB magnet (diameter 12.7 mm, surface magnetic 4A). field 7.1 kOe) placed under the center of the well for 30 min The NMR images showed a very clear decrease in the T2 and/or the effect of the local photothermal actuation with a signal in the liver and spleen 2 h after the injection, while there near-infrared laser in the first biological window (wavelength was not a significant signal decrease in other organs, such as 808 nm, beam diameter 11 mm, power density 0.95 W/cm2) kidneys or muscle (Figure 4A,B). The T2 signal in the liver during 10 min. In all the cases, the MAPSULES concentration already increased at 24 h postinjection, and it was almost fully was equal to the previous experiment of Figure 3A. The recovered after 72 h (Figure 4C), indicating that the viability was analyzed 24 h after treatment using a MTT assay MAPSULES were cleared by the organism. To confirm the as well as with fluorescence areal scanning with calcein AM lack of toxicity, the spleen, kidneys, and liver were harvested 72 staining for viability distribution evaluation in each well. The h postinjection and stained by hematoxylin and eosin (H&E) results showed a complete lack of effect of the magnetic field for histological analysis to assess the MAPSULES effects in and light irradiation in the absence of nanoparticles (P-M-L+ these organs, compared to saline injection (Figure 4D). and P-M+L-) and a similar viability decrease to Figure 3A in Despite the initial accumulation of the MAPSULES in these the passively administrated MAPSULES (P+M-L-), and this organs shown by NMR, there was no tissue damage at 72 h. effect was homogeneous across the well, as shown by the Moreover, the mice did not suffer any weight loss or any fluorescence intensity map of Figure 3C. The viability was noticeable toxicity effects during the whole 3 week assay slightly reduced in the case of the light-treated samples (P+M- (Figure S7), thus indicating that the MAPSULES are safe. L+) with passive administration, in which the temperature These results suggest a secretion pathway through the liver, as increased up to 44 °C (Figure 3D). In the case of the expected for the size and surface charge of the MAPSULES magnetically actuated MAPSULES (P+M+L- and P+M+L+), and the passive delivery.29−32 It should be noted that in the viability reduction was higher, the therapeutic effect was contrast to other studies focused on nonbiodegradable Au more localized at the center of the well due to the magnetic particles,33 whose secretion rate is rather slow, a faster concentration, and the laser irradiation could increase this clearance of the MAPSULES due to their biodegradability is effect, as a consequence of the temperature increase up to 47 expected. To corroborate this hypothesis, we carried out mass 1950 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article spectrometry analysis of the liver and kidneys. As Figure S5A concentration (indicated by the black arrows in Figure 5C). shows, the iron levels in the liver increased right after the This result confirms that the magnetic strength of the injection but quickly decreased after 24 h, which correlates MAPSULES enables a highly efficient enrichment, not only with the data acquired by NMR and histopathology. On the next to the skin, where the magnetic field gradient is higher, other hand, Si increasingly accumulated in the liver for 72 h, but also inside the tumor. To quantify the amount of but no damage was observed in the organ. In contrast, neither MAPSULES in the tumor, we carried out mass spectrometry the iron nor the silicon levels raised in the kidneys, which analysis of the tumors harvested 24 h post injection. Figure confirms that the elimination route was not renal. S5B demonstrates that the magnetic concentration clearly In Vivo MAPSULES Treatment. Short-Term Effects. To increased the amount of iron and, consequently, the investigate the potential of the paclitaxel-loaded MAPSULES MAPSULES concentration inside the tumor. Considering the for magnetic and optically enhanced cancer chemotherapy, we mass of the tumors (ca. 200 mg) and the quantity of injected first compared the treatment response in Hsd:Athymic Nude- particles, it can be estimated that 0.9% of the MAPSULES Foxn1nu female MDA-MB-231 tumor bearing mice under six were trapped inside the tumor by passive delivery and 2.2% by different conditions: (i) PBS control (PBS), (ii) PBS and laser magnetic concentration. actuation (PBS+L), (iii) MAPSULES (MPs), (iv) MAPSULES Next, we analyzed the photothermal effects induced by the and magnetic concentration (MPs+M), (v) MAPSULES and near-infrared laser exposure for the different treatment laser actuation (MPs+L), and (vi) MAPSULES and magnetic conditions. As can be observed in Figure 5D,E, the group concentration plus laser actuation (MPs+M+L). The mice with passively delivered MAPSULES (MPs+L) exhibited only were placed on a 33 °C warming pillow and anesthetized for ca. 2 °C higher temperature increase induced by the laser 2.5 h during the treatment sequence of tail intravenous compared to the control (PBS+L). In contrast, for magneti- injection, magnetic concentration, and light actuation. A cally concentrated treatments, a significant 8 °C temperature volume of 150 μL (PBS or MAPSULES) was administered increase enhancement was achieved after 20 min of via tail vein injection. In the case of the MAPSULES treatment, illumination. This substantially higher temperature increase the total mass concentration was 2.8 mg/kg of MAPSULES, confirmed the capacity to magnetically amplify the MAP- which corresponds to 1 mg/kg of paclitaxel-loaded PLGA SULES concentration in the tumor. Note that the 2.4-fold nanoparticles and only 8 μg/kg of paclitaxel. These higher MAPSULES concentration in the tumor with magnetic concentrations are substantially lower than the typical free actuation determined by mass spectrometry was obtained 24 h and PLGA loaded with paclitaxel in mice, which are in the after the intravenous injection. However, a substantially higher 1.0−5.0 mg/kg range (Table 1).34−38 The magnetically MAPSULES concentration in the tumor is expected right after the magnetic concentration (i.e., 2 h post injection). Such a Table 1. Comparison of PLGA and Paclitaxel (PCX) concentration increase can be estimated from the temperature Concentrations of PLGA and Paclitaxel (PCX) in In Vivo rise in the tumor. As can be observed in Figure S5C, the Treatments photoinduced temperature rise is proportional to the particle concentration, at least up to 20 μg/mL of iron. In the case of PLGA [mg kg−1] PCX [mg kg−1] Reference the in vivo experiments, the temperature increase in the passive MAPSULES 0.8 8.0 × 10−3 This work delivery was only 2 °C higher than the control, whereas the PLGA-PEG NP 142.8 1.0 [34] magnetically actuated mice achieved 8 °C enhancement with Aptamer-PLGA 47.6 1.0 [35] respect to the control, thereby suggesting a 4-fold higher Ab-PLGA − 2.0 [36] concentration of the MAPSULES 2 h post injection. The MNP/T7-PLGA 14.8 4.0 [37] difference between the concentrations obtained by mass PLGA-PEG NC 833 5.0 [38] spectrometry (24 h post injection) and photothermal effects (2 h post injection) is probably due to the MAPSULES that actuated groups were exposed to a cylindrical FeNdB magnet were weakly attached at the periphery of the tumors, which (diameter 12 mm, height 10 mm, surface magnetic field 5 kOe, were cleared through the tumor blood vessels after removal of and field gradient according to Figure S2C) placed next to the the magnet. These results suggest that a longer magnetic tumor for 2 h right after the MAPSULES injection (Figure actuation time could be used to keep the 4-fold MAPSULES 5A). The laser actuated groups were irradiated for 20 min by concentration increase in the tumor, which could enhance even the 808 nm laser with a power density of 0.95 W/cm2 (beam more the therapeutic effect of the released paclitaxel. diameter 11 mm), and the temperature of the mice was The observed temperature increases in the control group monitored by a thermal camera (Figure 5A). (ca. 10 °C) are due to the non-negligible absorption of the 808 Interestingly, 2 h after the injection, the NMR images of the nm laser light by the skin, blood, and tumor tissues. tumor bearing mice exposed to the magnet revealed a clear Long-Term Therapeutic Effects. To evaluate the efficiency decrease in the T2 signal at the tumor site, demonstrating the and long-term effects of the MAPSULES under the different ability to magnetically accumulate the MAPSULES in the treatment conditions, the treated mice were monitored until tumor area (see the marked yellow circle in Figure 5B). reaching the exclusion criterion of over 1000 mm3 of tumor To determine if the MAPSULES could penetrate inside the volume or 21 days post injections as experiment end points. tumor mass, the tumors were harvested 2 h after the PBS or First, we examined the effect of magnetically driven MAPSULES injection with or without magnetic concentration, accumulation of the MAPSULES in tumor growth (Figure sectioned, and stained using Prussian blue/eosin to detect the 6A). The PBS injected group (PBS) reached the exclusion presence of iron. As shown in Figure 5C, while only few iron volume (>12-fold from initial size) at day 14 post injection. In signatures corresponding to the MAPSULES were observed in contrast, the mice with passively injected MAPSULES and the the tumor after the passive intravenous injection, high levels of MAPSULES with magnetic concentration (MPs and MPs+M) iron could be detected inside the tumor after the magnetic showed a substantial decrease in the tumor growth compared 1951 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Figure 5. In vivo short-term effects. (A) In vivo setup for the MAPSULES magnetic and light actuation. (B) NMR images of tumor bearing mice before and 2 h post MAPSULES injection and magnetic concentration (tumor circled in yellow). (C) Histological examination for iron detection using Prussian blue staining. MAPSULES accumulation regions are indicated by the black arrows in the zoom insets. (D) Representative thermal images of the laser-treated mice after intravenous injection of PBS or MAPSULES with and without magnetic concentration. (E) Plot of the average temperature increases as a function of time for each treatment condition. to the control, and the exclusion volume was reached at days power or irradiation time could have been reduced to achieve 17 and 19, respectively (Figure 6A). Although the magnetic similar enhanced therapeutic activity and complete elimination accumulation of the MAPSULES in the tumor was not of the tumor with the magnetically concentrated MAPSULES sufficient for the complete tumor eradication, the tumor under these experimental conditions. Such reduction could growth reduction is noteworthy taking into account the very also minimize the observed skin burn effect at the irradiated low concentration of the injected nanoparticles and drug regions in this experiment, although the mice completely compared to other therapies based on PLGA encapsulated recovered from this superficial side effect at the experimental paclitaxel (Table 1).34−38 end point (Figure S8). The observed reduction in the tumor Considering these promising therapeutic results, we next growth with laser irradiation in the absence of MAPSULES is evaluated the effect of laser radiation in the tumor area on the consistent with previous results of the effect of near-infrared MAPSULES therapeutic activity, using the same MAPSULES light heating on breast cancer tumor bearing mice, which concentration and laser irradiation conditions described above. showed that the locally induced optical hyperthermia could The analysis of the tumor growth over time (Figure 6B−D) showed an enhanced therapeutic effect for the passively induce immune cell activation and infiltration within the delivered MAPSULES (MPs+L) irradiated for 20 min tumor,39 being these effects highly dependent on the final compared to the irradiated control group (PBS+L) and the temperature. To gain insights into the effects of light nonirradiated groups at the 21 day end point, showing tumor irradiation without MAPSULES injection, we carried out an volumes of 550 and 900 mm3, respectively. Strikingly, in vivo experiment in mice irradiated with similar intensity but complete eradication of the tumors in the group with magnetic during shorter illumination times (see Figure S6). The results concentration and light actuation (MPs+M+L) was already clearly showed a negligible decrease of the tumor growth rate observed at day 14, which corresponded to the volume with respect to the control at shorter illumination times. These exclusion end point for the PBS control. This large results again suggest that an irradiation time of 15 min would amplification effect by light radiation suggests that the laser have probably been the ideal condition to maximize the 1952 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Figure 6. In vivo magnetic concentration and laser radiation effects. (A) Tumor growth curves in mice groups for 21 days or until reaching the volume of the exclusion criterion after magnet exposure treatments. (B) Tumor growth curves in mice groups after different laser radiation treatments for 21 days or until reaching the volume of the exclusion criterion after the different laser radiation treatments. (C) Tumor average weight for the different treatment conditions at the experiment end point given by the exclusion volume (PBS) or 21 days. (D) Representative images of the treated mice and harvested tumors at the laser radiation experiment end point. (E) Fluorescent images of stained sections for Ki67 (pink) (top row) and cleaved caspase-3 (red) (bottom row) and counterstained with DAPI for cell nucleus imaging (blue). (F) H&E staining of tumor sections harvested at the end point (scale bar 200 μm). photothermal effects of the MAPSULES with respect to the damage was observed in the other treated groups (Figure 6F). PBS+L condition. The enhanced apoptosis in the magnetically treated mice is To examine the cellular effects of the treatments, the tumors related to the enhanced paclitaxel concentration inside the were harvested, and representative tumors from each group tumors due to the magnetic MAPSULES enrichment. In were analyzed by H&E, Ki67, and cleaved capase-3 staining contrast, the observed necrosis can be associated with the (Figure 6E,F). The H&E staining revealed the presence of enhanced photothermal effect induced by the MAPSULES. To apoptotic bodies in the magnetically actuated groups, MPs+M evaluate the aggressiveness of the tumors, the sectioned tumors and MPs+M+L, while more severe effects with additional were stained for the detection of the proliferation marker Ki67 visible coagulative necrosis were observed in the light-treated (Figure 6E). Strong fluorescent signal was detected in all the groups, MPs+L and MPs+M+L. Conversely, no noticeable samples; however, light actuated groups, MPs+L and MPs+M 1953 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Table 2. Comparison of the MAPSULES Conditions and Other Multimodal Therapies Combining Paclitaxel and Light Actuation Optical settings Total PCX [mg kg−1] In vivo conditions Ref Type Conditions Tumor Injection Growth inhibition MAPSULES PTT (808 nm) 0.95 W cm−2 8.0 × 10−3 MDA-MB-231 iv Ablationc This work Poly(Ru/PTX)a PDT 671 nm, 0.2 W cm−2 15.0 4T1 iv 65% [41] Shape changeable NPsa 650 nm, 0.2 W cm−2 0.09 4T1 iv 85% [42] Liposomesa 450 nm, 0.2 W cm−2 24.0 PC3 it Ablationc [43] Chitosan@Au NPs PTT (808 nm) 1.2 W cm−2 0.7 MDA-MB-231 iv 97% [44] CuS NPsa 1 W cm−2 0.16 4T1 iv Ablationc [45] Gelatin-PtNPsb � 20.0 4T1 iv 83% [46] Polymeric micelleb 0.07 W cm−2 15.0 4T1 iv Ablation [47] a Several complete treatments (light + injection). bSeveral injections. cNot all the tumors from the group were ablated (iv: intravenous, it: intratumoral, PTT: photothermal therapy, PDT: photodynamic therapy). +L, exhibited a noticeable clear region where tissue was less expected range in the literature, showing an average of 0.6% for proliferative and cell density was reduced (Figure 6E, top row). the organic particles and 0.8% for the inorganic particles.3 In On the other hand, cleaved caspase-3 staining was performed contrast, the magnetic concentration provides a powerful to assess apoptotic cell death, showing elevated signal in the strategy to substantially increase the particle dose in the tumor MPs+M, MPs+L, and MPs+M+L groups (Figure 6E). These (2.2% at 24 h post injection). Notably, the magnetic actuation results are consistent with the H&E staining, confirming that was limited to only 2 h due to ethical considerations related to the magnetic concentration enables increased paclitaxel levels the mice wellbeing, despite the small size of the employed in the tumor, whereas the local photothermal actuation magnet (ca. 1 cm3). A longer magnetic actuation would drastically amplifies its effects. These findings confirm the possibly retain a larger fraction of trapped particles in the synergistic effect of chemo-photothermal combined therapy, tumor and, most probably, further enhance the therapeutic where the whole is larger than the sum of its parts.40 It is worth effects. The retention efficiency could be further improved by highlighting the achieved results using only one ultralow drug using more sophisticated quadrupolar magnetic actuators, dose by intravenous injection, which enabled the tumor which could enhance the concentration in the whole tumor ablation by combining the magnetic concentration and the volume. The combination of magnetic concentration and photothermally boosted chemotherapeutic effects, especially if enhanced photothermal effects endowed by the MAPSULES they are compared to other light and paclitaxel multimodal have enabled a complete eradication of the tumors using much therapies, which required multiple injections and/or a much lower drug and nanoparticle concentrations than the typical higher drug amount, i.e., between 13- and 3000-fold higher values used in the literature (between 100- and 500-fold lower than the MAPSULES treatment (Table 2).41−47 than the therapeutic window of the free or PLGA encapsulated drug and between 13- and 3000-fold lower than previously CONCLUSIONS described nanotherapies combining paclitaxel and light In conclusion, the presented MAPSULES constitute a cutting- actuation). Note that although the employed power density edge nanotherapeutic concept capable of merging externally of the 808 nm laser (ca. 1 W/cm2) is higher than the maximum controlled therapies with noninvasive imaging and drastically recommended exposure values for humans at this wavelength, enhanced therapeutic effects at very low drug concentrations. this power density is allowed for light irradiation in the second The high magnetic strength and colloidal stability of the metal biological window at 1064 nm. In this wavelength, the iron semishells in the MAPSULES are the key features for MAPSULES exhibit an even higher photothermal efficiency, enabling efficient external magnetic actuation via magneto- and penetration depth can be even higher for organs such as phoretic forces. The strong magnetism also provides an kidney, spleen, and liver.48,49 In regards to development outstanding T2 contrast in NMR to noninvasively visualize perspectives toward clinical utilization, it is relevant to the MAPSULES biodistribution and magnetic accumulation in emphasize that the PLGA nanocarriers have already been the tumor. On the other hand, the high optical absorbance approved by the FDA (e.g., Trelstar and Risperdal Consta). A with minimal scattering provided by the highly damped next important step is the scaling up of the MAPSULES plasmonic behavior of the Fe semishell enables highly efficient fabrication process. The self-assembly step takes less than 5 optical heating in the first and second NIR biological windows. min and could be easily automatized to coat much larger The therapeutic activity of the MAPSULES in vitro has surfaces and numbers of substrates. The fabrication process is demonstrated the capacity to preserve the activity of the currently limited by the Fe/SiO2 evaporation step, which takes paclitaxel during the MAPSULES fabrication process. The ca. 25 min per wafer in a research-oriented e-beam evaporator. MAPSULES have shown in vivo a total absence of side effects The duration of this process could be drastically reduced in and the expected biodistribution with accumulation in the liver industrial evaporators, which can load tens of larger wafers, and after passive intravenous injection. The magnetically actuated whose evaporation process can be continuously run without mice have shown a notable increase of the MAPSULES breaking the vacuum. Moreover, a combination of roll-to-roll retention in the tumor, as demonstrated by the NMR images, self-assembly and material deposition, which is typically used the mass spectrometry measurements, the enhanced photo- in the tape recording or packaging industries, could be used for thermal effects, and the histopathological analysis of the the cost-effective industrial production of the MAPSULES. harvested tumors. The obtained value of delivery efficiency Therefore, the promising therapeutic results of the MAP- inside the tumor by passive injection (0.9%) is within the SULES enable envisaging externally controlled therapeutic 1954 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article pathways with minimal side effects, which could be easily dispersion (total volume of 1 mL) was controllably flowed by a extended to other drugs and tumors and even to other diseases. syringe pump through the microfluidic channel at different flow rates. The experimental setup is shown in Figure S2. The optical absorbance spectra of the sample before and after the trapping experiments were METHODS measured to establish the concentration of nanoparticles in the Nanomaterial Preparation Section. Nanoprecipitation Syn- dispersion and the trapping efficiency. thesis Process of the Drug-Loaded PLGA Nanoparticles. First, a 5 Nuclear Magnetic Resonance. 1H-magnetic resonance imaging mL organic solution containing 100 mg of carboxylic terminated (MRI) studies were performed in a 70 kOe Bruker BioSpec 70/30 PLGA (ResomerR 502 H, Sigma), 2 mg of paclitaxel (Sigma), and USR (Bruker BioSpin GmbH, Ettlingen, Germany) system equipped 0.01% Tween80 (Sigma) in acetonitrile was added dropwise to a 10 with a mini-imaging gradient set (4000 Oe/m) using a linear volume mL aqueous solution containing 0.1% Solutol (Sigma) at room coil with a 72 mm inner diameter. The magnetic resonance data were temperature. Following 30 min of stirring at 550 rpm, the acetonitrile acquired and processed on a Linux computer using Paravision 5.1 was removed under reduced pressure using a rotary evaporator software (Bruker BioSpin GmbH, Ettlingen, Germany). For the (Heidolph, Germany), leading to the formation of the paclitaxel- relaxivity measurements of the MAPSULES, phantoms containing loaded PLGA nanoparticles. Paclitaxel-loaded PLGA nanoparticles various nanodome concentrations in 1% agar and PBS (1× ) mixture were used without any further purification. were prepared. The magnetic resonance images were obtained from Fabrication of the MAPSULES. The fabrication approach was two 2.5 mm slice thickness coronal sections with a field of view adapted from a previously developed method.17,50 Briefly, first, a 2 (FOV) of 90 × 60 mm2. Only transverse relaxation time (T2) mg/mL suspension of drug-loaded PLGA nanoparticles in Milli-Q measurements have been performed. A multislice multiecho sequence water was prepared and sonicated for 3 min. Meanwhile, the surfaces was used, with repetition time (TR) = 3 s, time of echo (TE) values of 4″ silicon wafers (Siegert Wafer GmbH) were incubated in between 10 and 600 ms in steps of 10 ms, and MTX = 128 × 128. poly(diallyldimethylammonium chloride) (PDDA) 2% solution Data were fitted to exponential curves to obtain the T2 relaxation (Sigma) for 3 min. Then, the wafers were rinsed with water and times. Transverse relaxivity values, r2, were obtained as the slope of blow dried with N2 gas, thereby yielding a monolayer of positively the linear regression of the relaxation rates (r), as the inverse of the charged PDDA on the surface. Next, the PLGA nanoparticle relaxation times (r2 = 1/T2) versus Fe concentration. dispersion was drop casted on the surface of the modified wafers A similar approach was used to image T2 contrast in nuclear and incubated for 3 min, followed by the same rinse and dry process magnetic resonance signal of the MAPSULES in vivo in mice. MRI with deionized water and N2 gas, to obtain a disordered and well experiments were performed at the Wohl Institute for Translational separated self-assembled monolayer of nanoparticles on the surface. Medicine at Hadassah Hebrew University Medical Center. Images Any excess of material outside the loaded PLGA nanoparticles was were acquired on a 7 T 240 mm bore, cryogen-free MR scanner based totally removed during this rising and drying process. on the proprietary dry magnet technology (MR Solutions, Guildford, Subsequently, electron beam evaporation (ATC-8E Orion, AJA UK) using a 38 mm inner diameter, 70 mm length mouse whole body International Inc.) was used to deposit 20 nm of Fe and 10 nm of volume coil. In detail, the unloaded MAPSULES nanocapsules were SiO2 on the Si wafer coated with the self-assembled PLGA particles. injected via the tail vein with a total volume of 150 μL with a total Then, MAPSULES were dispersed from the wafer by mild mass concentration of 2.5 mg/kg. For MRI acquisition, mice were ultrasonication in 2% poly(4-styrenesulfonic acid) solution and anesthetized with isoflurane vaporized with O2. Isoflurane is used at cleaned twice by using magnetic concentration with a FeNdB 3% for induction and at 1−2% for maintenance. The mice were cylindrical magnet (diameter 12 mm, thickness 10 mm) to remove the positioned on a heated bed, which allowed for continuous anesthesia supernatant, followed by redispersion in clean buffer. and breathing rate monitoring throughout the entire scan period. Morphological, Optical, Magnetic, and Colloidal Character- Coronal T1-weighted (T1W) spin−echo images were acquired for ization. The morphology and distribution of the nanodomes on the anatomical segmentation purposes (RT/ET = 1100/11 ms, FOV = 60 Si wafer were studied through scanning electron microscopy (SEM) mm, in-plane resolution = 230 μm, slice thickness = 1 mm). Coronal using a Quanta SEM 650 (Field Electron and Ion Company (FEI)) at and axial T2*W gradient echo images were acquired for MAPSULES 5 kV. Transmission electron microscopy, TEM, images were acquired detection (RT/ET = 300/10 ms, FOV = 60/35 mm, slice thickness = in an FEI Tecnai F20 at 200 kV. The samples for the TEM analysis 1 mm). The MRI tests were conducted at different times before and were prepared by placing a drop of the aqueous suspension of after (2, 24, 48, and 72 h) the MAPSULES injection. MAPSULES on a TEM grid and allowing it to air-dry for 2 h. Photothermal Heating Efficiency Measurement. A custom- The zeta potential and the hydrodynamic size of the PLGA made photothermal testing system was used to determine the particles and the MAPSULES were obtained through dynamic light photothermal conversion efficiency of nanocapsules in water as scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd.). previously described.16 The system consisted of (i) a NIR laser diode The mass concentration of Fe in the nanodomes dispersions was with the selected emission wavelength (808 nm, L808P500MM, determined by ICP-OES (Perking Elmer Optima 4300DV). Thorlabs, or 1064 nm, KA64FAMFA, Thorlabs) driven by a laser The visible-near-infrared (vis-NIR) spectroscopy studies of the diode controller (ITC4005, Thorlabs), (ii) an optical collimating and nanodome dispersions were carried out using a Lambda25 aligning system, (iii) an infrared camera (ETS320, FLIR) to monitor spectrometer (PerkinElmer) in the 400 to 1100 nm range, covering the temperature variations at the liquid surface, (iv) a power meter the visible light and near-infrared regions. (PM100D, Thorlabs), and (v) a computer with the data acquisition The magnetic characterization of the nanodomes was performed on software. The laser incident power upon the samples was set to 200 monolayers that were transferred from the Si wafer to a 10 mm mW for both lasers, and the temperature of the solution and the polydimethylsiloxane (PDMS) substrate to eliminate the magnetic power transmitted through the sample were continuously monitored signal from the bilayer that was deposited on the wafer surface. during the assays. The photothermal conversion efficiency η was Magnetization loops were acquired at room temperature using a calculated as follows:51 vibrating sample magnetometer (MicroSense, LOT QuantumDesign) with a maximum applied field of 20 kOe. The measurements were hS Tmax Q dis = Ax performed by applying the field either parallel or perpendicular to the I(1 10 ) (1) sample, i.e., in-plane or out-of-plane conditions. Magnetic Trapping in the Microfluidic Channel. The where h is the heat transfer coefficient, S is the laser irradiating magnetic trapping experiments were performed by setting a spherical surface, ΔTmax is the maximum temperature change optically induced, 19 mm diameter FeNdB permanent magnet (7.1 kOe at the surface Qdis is the heat dissipation due to the experimental setup, I is the laser and field gradient according to Figure S2C) attached to a narrow incident power (200 mW), and Ax is the absorbance at the irradiated microfluidic channel (3 mm wide, 0.8 mm height). A MAPSULES wavelength (x = 808 or 1064 nm). 1955 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article In order to get the value of η, hS for heat dissipating should be length × width2 calculated. During the cooling process, since there is no light tumor volume = 2 irradiating the nanoparticles, the dispersion will reach Tamb: When tumor volumes reached approximately 100 mm3, mice were dT randomly divided into groups and subjected to 150 μL tail Q diss = miCp , i = hS(T Tamb) i dt (2) intravenous injection of six different treatments: (i) PBS (PBS), (ii) PBS plus laser (PBS+L), (iii) MAPSULES (MPs), (iv) MAPSULES Here we can define the time constant τs and the dimensionless plus laser (MPs+L), (v) MAPSULES plus magnet (MPs+M), and (vi) parameter θ to simplify the integral as follows: MAPSULES plus magnet and laser (MPs+M+L). The magnetic actuated groups, MPs+M and MPs+M+L, were exposed to an N45 miCp , i T Tamb = i and = nickel-plated FeNdB cylindrical magnet (diameter 12 mm, thickness s hS Tmax Tamb 10 mm) placed next to the tumor for 2 h, while the mice were anesthetized on a warming pillow set to 33 °C. The laser actuated d 1 groups were radiated for 20 min by an 808 nm continuous wave laser = dt s (3) beam (0.95 W/cm2, beam diameter 11 mm) and the temperature was monitored using a thermal camera. The tumors were measured three Setting the boundary conditions for the cooling process, t = 0, θ = times a week for 21 days or until the exclusion criterion of 1000 mm3 1, the equation can be solved as eq 4, from which a linear relationship was reached (end points). At the end points, mice were sacrificed, can be deduced to obtain the τs value; consequently, hS can be imaged and the tumors were removed, photographed and weighted. calculated and introduced in eq 1 to calculate the photothermal efficiency. ASSOCIATED CONTENT 1t = es (4) * sı Supporting Information The total volume of the MAPSULES dispersion was 600 μL. The The Supporting Information is available free of charge at measured extinction values of the MAPSULES dispersion at 808 and https://pubs.acs.org/doi/10.1021/acsnano.2c05733. 1064 nm were 0.2192 and 0.1837 (Figure S4A), respectively. The MAPSULES characterization, trapping analysis, degra- constant time of the system was determined as 280 s, as shown in dation, photothermal efficiency calculation, mice weight Figure S4B. evolution, and tumor progression (PDF) Cell Culture. MDA-MB-231 breast adenocarcinoma tumor cells were purchased from ATCC (VA, USA) and maintained in DMEM, supplemented with 10% fetal calf serum (FCS) (Gipco, Brazil) media AUTHOR INFORMATION with 1% penicillin/streptomycin (Biological industries, Israel). For Corresponding Authors comparison of viability of the unloaded and paclitaxel-loaded PLGA nanoparticles and MAPSULES, 2 × 104 MDA-MB-231 cells were Ofra Benny − Institute for Drug Research (IDR), School of plated in a 96 well plate and incubated for 24 h. The PLGA and Pharmacy, Faculty of Medicine, The Hebrew University of MAPSULES treatments were introduced at identical particle Jerusalem, 9190501 Jerusalem, Israel; orcid.org/0000- concentrations with 10 μg/mL of PLGA nanoparticles and 0.08 μg/ 0002-2468-5978; Email: Ofra.Benny@mail.huji.ac.il mL of paclitaxel, and the plates were incubated for an additional 48 h Borja Sepulveda − Instituto de Microelectronica de Barcelona before the viability was evaluated using a MTT assay. The MTT (3- (IMB-CNM, CSIC), Campus UAB, 08193 Barcelona, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) was Spain; orcid.org/0000-0002-1562-7602; added to the cell cultures (final concentration 0.5 mg/mL) and Email: borja.sepulveda@csic.es incubated for another 4 h. Then, the supernatant was removed, and DMSO was added allowing the crystals to dissolve completely for Authors absorbance read at 570 nm by a SPECTRAFluor Plus plate reader Arnon Fluksman − Institute for Drug Research (IDR), School (Tecan, San Jose, CA, USA). Cells were also visualized using an inverted microscope (Nikon Eclipse Ti2, Japan) 4 h after incubation of Pharmacy, Faculty of Medicine, The Hebrew University of with the MAPSULES. In order to measure the effects of the magnetic Jerusalem, 9190501 Jerusalem, Israel concentration and photothermal actuation of the MAPSULES on the Aritz Lafuente − Catalan Institute of Nanoscience and cells viability, 5 × 104 cells were plated in a 35 mm Petri dish and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, incubated for 24 h before introducing magnets under the center of the 08193 Barcelona, Spain; Universitat Autònoma de well (12.7 mm spherical FeNdB magnet, 30 min) and/or laser Barcelona, Campus UAB, 08193 Barcelona, Spain; radiation (wavelength 808 nm, beam diameter 11 mm, power density orcid.org/0000-0002-1497-5744 0.95 W/cm2, 10 min). The temperature was monitored by an infrared Zhi Li − Catalan Institute of Nanoscience and Nanotechnology thermal camera (8320P, Infrared Cameras Inc. (ICI), Beaumont, TX, (ICN2), CSIC and BIST, Campus UAB, 08193 Barcelona, USA). The cells were incubated for 4 h following the treatments and then washed and incubated for another 24 h before the viability was Spain; orcid.org/0000-0003-2641-0297 evaluated. Cells were stained by calcein AM (2 μmol/L, 30 min), Jordi Sort − Universitat Autònoma de Barcelona, Campus washed with PBS, and the fluorescent signal was measured by aerial UAB, 08193 Barcelona, Spain; ICREA, 08010 Barcelona, scan using a plate reader (ex/em 488 nm/520 nm). The cell viability Spain; orcid.org/0000-0003-1213-3639 was also evaluated by MTT (as mentioned). Silvia Lope-Piedrafita − Universitat Autònoma de Barcelona, In Vivo Experiment. For the in vivo analysis of the therapeutic Campus UAB, 08193 Barcelona, Spain; orcid.org/0000- effect, 8-week-old Hsd:Athymic Nude-Foxn1nu female mice were 0002-8127-6425 purchased (Harlan, Rehovot, Israel), acclimated for a week, and Maria José Esplandiu − Catalan Institute of Nanoscience and stored in a specific pathogen-free (SPF) environment. All animal Nanotechnology (ICN2), CSIC and BIST, Campus UAB, procedures were carried out in accordance with the institutional and national guidelines, and protocols were approved by the ethics 08193 Barcelona, Spain committee of the Hebrew University Ein Kerem Medical School Josep Nogues − Catalan Institute of Nanoscience and IACUC (protocol MD-21-16572-5). Nude mice were inoculated Nanotechnology (ICN2), CSIC and BIST, Campus UAB, subcutaneously with 4 × 106 MDA-MB-231 cells, and the tumor 08193 Barcelona, Spain; ICREA, 08010 Barcelona, Spain; volumes were monitored and measured according to the equation: orcid.org/0000-0003-4616-1371 1956 https://doi.org/10.1021/acsnano.2c05733 ACS Nano 2023, 17, 1946−1958 ACS Nano www.acsnano.org Article Alejandro G. Roca − Catalan Institute of Nanoscience and (10) Urries, I.; Muñoz, C.; Gomez, L.; Marquina, C.; Sebastian, V.; Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Arruebo, M.; Santamaria, J. Magneto-Plasmonic Nanoparticles as 08193 Barcelona, Spain; orcid.org/0000-0001-6610- Theranostic Platforms for Magnetic Resonance Imaging, Drug 9197 Delivery and NIR Hyperthermia Applications. Nanoscale 2014, 6, 9230. Complete contact information is available at: (11) Gutiérrez, L.; de la Cueva, L.; Moros, M.; Mazarío, E.; de https://pubs.acs.org/10.1021/acsnano.2c05733 Bernardo, S.; de la Fuente, J. M.; Morales, M. P.; Salas, G. Aggregation Effects on the Magnetic Properties of Iron Oxide Author Contributions Colloids. Nanotechnology 2019, 30, 112001. A.F and A.L. contributed equally. (12) Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Drug/Dye- Loaded, Multifunctional Iron Oxide Nanoparticles for Combined Notes Targeted Cancer Therapy and Dual Optical/Magnetic Resonance The authors declare no competing financial interest. Imaging. Small 2009, 5, 1862−1868. (13) Kayal, S.; Ramanujan, R. V. Anti-Cancer Drug Loaded Iron- ACKNOWLEDGMENTS Gold Core-Shell Nanoparticles (Fe@Au) for Magnetic Drug Targeting. J. Nanosci. Nanotechnol. 2010, 10, 5527−5539. The authors would like to thank M. Saidian and Dr. V. (14) Gangopadhyay, P.; Gallet, S.; Franz, E.; Persoons, A.; Verbiest, Shelukhin from the Hebrew university center for nanoscience T. Novel Superparamagnetic Core(Shell) Nanoparticles for Magnetic and nanotechnology (HUCNN) and Prof. R. Abramovitch and Targeted Drug Delivery and Hyperthermia Treatment. IEEE Trans. N. Abudi from the Wohl institute for translational medicine for Magn. 2005, 41, 4194−4196. their technical support. This work was financially supported by (15) Farzin, A.; Etesami, S. A.; Quint, J.; Memic, A.; Tamayol, A. the European Research Council (ERC-StG) under the Magnetic Nanoparticles in Cancer Therapy and Diagnosis. Adv. European union’s horizon 2020 research and innovation Healthc. Mater. 2020, 9, 1901058. program, ERC proof of concept grant (ERC-2022-PoC1). (16) Li, Z.; Aranda-Ramos, A.; Güell-Grau, P.; Tajada, J. L.; Pou- We acknowledge financial support from grants No MAT2016- Macayo, L.; Lope Piedrafita, S.; Pi, F.; Roca, A. G.; Baró, M. D.; Sort, 77391-R, PID2019-106229RB-I00,PID2020-116844RB- J.; Nogués, C.; Nogués, J.; Sepúlveda, B. Magnetically Amplified C21,PGC2018-095032-B-100, PCIN2016-093 (M-ERA- Photothermal Therapies and Multimodal Imaging with Magneto- NET), RTI2018-095495-J-I00, and RYC2019-027449-I Plasmonic Nanodomes. Appl. Mater. Today 2018, 12, 430−440. (17) Li, Z.; Lopez-Ortega, A.; Aranda-Ramos, A.; Tajada, J. L.; Sort, funded by MCIN/AEI/10.13039/501100011033. We thank J.; Nogues, C.; Vavassori, P.; Nogues, J.; Sepulveda, B. Simultaneous the Ramon Areces foundation through grant CIVP19A5922. Local Heating/Thermometry Based on Plasmonic Magnetochromic The funding from Generalitat de Catalunya through the 2017- Nanoheaters. 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