This article is licensed under CC-BY 4.0 www.acsami.org Research Article Modular Drug-Loaded Nanocapsules with Metal Dome Layers as a Platform for Obtaining Synergistic Therapeutic Biological Activities Arnon Fluksman, Aritz Lafuente, Ron Braunstein, Eliana Steinberg, Nethanel Friedman, Zhanna Yekhin, Alejandro G. Roca, Josep Nogues, Ronen Hazan, Borja Sepulveda,* and Ofra Benny* Cite This: ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Multifunctional drug-loaded polymer−metal nanocapsules have attracted increasing attention in drug delivery due to their multifunc- tional potential endowed by drug activity and response to physicochemical stimuli. Current chemical synthesis methods of polymer/metal capsules require specific optimization of the different components to produce particles with precise properties, being particularly complex for Janus structures combining polymers and ferromagnetic and highly reactive metals. With the aim to generate tunable synergistic nanotherapeutic actuation with enhanced drug effects, here we demonstrate a versatile hybrid chemical/physical fabrication strategy to incorporate different functional metals with tailored magnetic, optical, or chemical properties on solid drug-loaded polymer nanoparticles. As archetypical examples, we present poly(lactic-co-glycolic acid) (PLGA) nanoparticles (diameters 100−150 nm) loaded with paclitaxel, indocyanine green, or erythromycin that are half-capped by either Fe, Au, or Cu layers, respectively, with application in three biomedical models. The Fe coating on paclitaxel-loaded nanocapsules permitted efficient magnetic enhancement of the cancer spheroid assembly, with 40% reduction of the cross-section area after 24 h, as well as a higher paclitaxel effect. In addition, the Fe- PLGA nanocapsules enabled external contactless manipulation of multicellular cancer spheroids with a speed of 150 μm/s. The Au- coated and indocyanine green-loaded nanocapsules demonstrated theranostic potential and enhanced anticancer activity in vitro and in vivo due to noninvasive fluorescence imaging with long penetration near-infrared (NIR) light and simultaneous photothermal− photodynamic actuation, showing a 3.5-fold reduction in the tumor volume growth with only 5 min of NIR illumination. Finally, the Cu-coated erythromycin-loaded nanocapsules exhibited enhanced antibacterial activity with a 2.5-fold reduction in the MIC50 concentration with respect to the free or encapsulated drug. Altogether, this technology can extend a nearly unlimited combination of metals, polymers, and drugs, thus enabling the integration of magnetic, optical, and electrochemical properties in drug-loaded nanoparticles to externally control and improve a wide range of biomedical applications. KEYWORDS: drug delivery, nanocapsules, multifunctional nanocapsules, Janus metal−polymer nanocapsules 1. INTRODUCTION fabrication methods are based on the coencapsulation of the The advances in nanotechnology have generated a flourishing polymer and the metal NP-containing solutions. The obtained field of nanoparticulate systems for therapeutic and diagnostic products using these methods are typically isotropic, in the form of nanocapsules with metallic cores that are shelled by a applications.1 A wide diversity of materials has been used to polymeric layer or, alternatively, polymeric nanocapsules formulate nanoparticles (NPs), including polymers, lipids, conjugated with metallic NPs on their surface, resulting in metals, carbon, and inorganic nonmetallic solids.2 NPs are used “patchy” nanocapsules or core−satellite nanocapsules.10−13 in drug delivery to stabilize the active compounds, enhance the Metals such as iron, gold, palladium, silver, copper, and circulation time of the encapsulated drugs, and improve the titanium were shown to be promising candidates for the selectivity and tissue targeting. The improved pharmacological functionalization of drug carriers by exploiting the biological, properties eventually lead to an enhanced therapeutic index while minimizing the off-target effects.3,4 In recent years, multifunctional platforms for drug delivery Received: May 19, 2023 have been developed by combining one or more materials with Accepted: September 29, 2023 different properties, thus providing new possibilities usually Published: October 20, 2023 not available when used separately.5−9 In this regard, drug- loaded nanocapsules (NCs) comprising solid polymers with different metals have been extensively studied. Common © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsami.3c07188 50330 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article Scheme 1. Schematic Representation of the Metal-Coated Janus Nanocapsule Fabricationa a The drug-loaded nanocapsules were prepared by the emulsification−evaporation technique, followed by their electrostatic self-assembly on positively charged silicon wafers to enable the deposition of the metal half-coating. The metal-coated nanocapsules were finally dispersed in water by bath sonication. biochemical, or physical properties of the metals and and silica templates were fabricated with metals for different polymers.11,14−22 However, these core−shell systems have purposes, such as cell targeting and biosensing,28 ultrasound substantial drawbacks, such as limited drug loading, metal and magnet propulsion,29 drug delivery, photothermal masking by the polymeric shell which often limits their therapy,30 opto-magnetic nanoheating/thermometry,31 and physicochemical response, and unpredictable release kinetics theranostic applications.32 Nevertheless, the application of from polymer matrices.11,14,17 Another significant drawback in drug-loaded biodegradable polymer nanoparticles as templates chemical synthesis methods that combine inorganic phases for the development of active multiactuated nanocapsules is (metals or metal oxides) and organic phases (polymers) is the currently in its early stages of exploration. Interestingly, a need to optimize each formulation element separately, which recent work has demonstrated their potential in magnetically can be a time-consuming and complex process. Furthermore, if amplified chemo/photothermal cancer therapy,33 indicating a changes to the inorganic component are required, it often promising direction for future advancements in this area. demands an entirely new fabrication process, adding further However, there is still a need to demonstrate the capacity to complexity and potential inefficiencies to the overall extend this modular nanotherapeutic concept to other drugs procedure.12 In the case of anisotropic Janus metal/polymer and metals to achieve synergistic therapeutic effects in other nanoparticles, the chemical synthesis processes are even more biomedical applications. complex. The most common methods to chemically synthesize Here, we present a tunable and robust strategy to fabricate Janus particles include: (i) interfacial or emulsion polymer- multiple combinations of metal coating on drug-loaded ization,23 although they are mainly restricted to Janus polymer nanocapsules as templates to achieve synergistic enhanced structures; (ii) seed-mediated growth,24 which involves the therapeutic effects. This fabrication strategy involves the self- controlled growth of metal nanoparticles on preformed assembly of drug-loaded nanocapsules in a solid support and polymer particles or vice versa; and (iii) pickering emulsion25 the deposition of the different metal layers by physical vapor using metal nanoparticles as emulsifiers to stabilize emulsions deposition. Therefore, once the drug-loaded polymer nano- of two immiscible liquids, leading to the formation of polymer capsule is optimized and self-assembled, any metal layer or forms around the metal nanoparticles. These processes are multilayer can be deposited without the need for any further even more complex when they involve the incorporation of a optimization process. To demonstrate the wide versatility and third component, as the drug, inside the polymer particle. applicability of this polymer/metal nanostructure, we show the As an alternative, Janus metal/polymer nanostructures can control of both the active encapsulated substance and the be fabricated by adapting physical vapor deposition techniques deposited metal. This enables the integration layers with typically used in the semiconductor, optic, aerospace, and solar customized magnetic, optical, and chemical properties to industries for metal depositions,26,27 to deposit nanodome- enhance the drug effects. As proof of concept, we selected a shaped metal coatings on the polymer nanoparticles, i.e., biodegradable and biocompatible copolymer, poly(lactic-co- polymer nanoparticles half-coated by metal layers. Such glycolic acid) (PLGA),34 loaded with three different drugs and nanoparticles have shown some promising biomedical coated with three different metals to synergistically boost their applications.5 For example, polystyrene, poly(acrylic acid), therapeutic effects. We first show ferromagnetic iron-capped 50331 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article carriers (Fe-PLGA NCs) loaded with the drug paclitaxel, Detaching the nanocapsules from the silicon wafer surface is which enable the enhancment of assembly and external, another critical step for obtaining concentrated dispersions of the contactless control of multicellular spheroids by using metal−polymer hybrid NP solution. A sonication bath was used as magnetic forces as well as increasing the efficacy of the loaded intense mechanical vibration to properly detach the NPs. In our high- power ultrasonic bath (Sonorex Digiplus, Bandelin, Berlin, Germany), drug in the cancer spheroids. In our second demonstration, we 5 min at 100% sonication power was adequate for removing the NPs show plasmonic gold-coated carriers (Au-PLGA NCs) loaded from the wafers to a 0.2% PSS solution. with indocyanine green (ICG) for theranostic application, 2.3. Morphology and Composition Characterization Using enabling noninvasive imaging and simultaneous photother- Scanning Electron Microscopy (SEM) and Transmission mal−photodynamic therapies with near-infrared (NIR) light. Electron Microscopy (TEM)/Energy-Dispersive X-ray Spectros- Finally, we demonstrate antibacterial copper-coated carriers copy (EDS). The morphology of the nanocapsules and their surface distribution on the wafer were examined using the environmental (Cu-PLGA NCs) loaded with the antibiotic erythromycin, scanning electron microscope (SEM) Quanta 200 (FEI Company, showing a synergistic antibacterial effect resulting from the The Netherlands) and the transmission electron microscope (TEM) release of copper cations. Altogether, our examples illustrate Tecnai G2 F20 equipped with an energy-dispersive X-ray spectros- the capacity to easily integrate any active metal layer in drug- copy detector (EDS) (EDAX, TSL, AMETEK, Hillsboro, OR) for loaded polymer nanoparticles without perturbing the activity surface elemental identification. The chemical analysis was performed and release of the drugs. This integration serves to significantly in the frame mode, representing the sum of the elemental amplify the drug effects, thereby showcasing its potential across composition of the imaged nanocapsules and the silicon substrate. a broad spectrum of biomedical applications. In the case of Cu-PLGA samples, to avoid undesired signal from the equipment, electron energy loss spectroscopy (EELS) was performed for elemental mapping. 2. MATERIALS AND METHODS 2.4. Particle Size, Charge, and Encapsulation Efficiency (EE %) Determinations. Particles were characterized according to their 2.1. Materials. Acid-terminated poly(D,L-lactic-co-glycolic acid) size, size distribution, and ζ potential. Dynamic light scattering (DLS) (PLGA) (RG502H 7000−17 000 Da, viscosity: 0.16−0.24 dL/g, 0.1% was performed at a scattering angle of 90° and at a temperature of 25 (w/v) in chloroform), poly(allylamine hydrochloride) (PAH), °C using a Zetasizer Nano ZSP (ZEN5600, Malvern Instruments, poly(diallyldimethylammonium chloride) (PDDA), (3-aminopropyl)- U.K.) to determine the mean diameter, polydispersity index (PDI), trimethoxysilane (APTMS), poly(sodium 4-styrenesulfonate) (PSS), and electric charge of the nanocapsules. The nanocapsule morphology agarose, poly(vinyl alcohol) (PVA), coumarin-6, rhodamine 6G, and size were examined and imaged using a transmission electron Dulbecco’s phosphate-buffered solution (PBS), 2′,7′-dichlorofluor- microscope (TEM) (JEM 1400Plus, JEOL, Japan, with a charge- escein diacetate (DCFH-DA), and Dulbecco’s modified Eagle’s coupled camera, Gatan Orius SC600). Samples were negatively medium (DMEM) were purchased from Sigma-Aldrich (MO). stained with a 2% aqueous solution of uranyl acetate (1:1) and placed Tween 80 was obtained from Fisher BioReagent (NJ). Solutol on a glow-discharged carbon-coated copper TEM grid (Ted Pella, (Kolliphor HS 15) and erythromycin were purchased from Glentham Inc., Redding, CA). The mean size was calculated from at least 20 (U.K.), and indocyanine green (ICG) was obtained from Chem- nanocapsules from two fields and presented as the mean ± standard Impex (IL). Thiazolyl blue tetrazolium bromide (MTT) was deviation using ImageJ software. The encapsulation efficiency (EE%) purchased from Alfa Aesar (Lancashire, U.K.). was determined for each encapsulant thrice by centrifugation (10,000 2.2. Preparation of Metal-Deposited Polymeric NPs. The rpm for 10 min) of 1 mL of the nanocapsule solution and metal-coated nanocapsule synthesis process is described in Scheme 1. resuspension in 1 mL of acetonitrile. The concentrations were Drug-loaded PLGA NPs were prepared using the emulsification− measured by high-performance liquid chromatography (HPLC, evaporation technique, as previously reported.35 Briefly, in the case of Shimadzu, OR) using a C18 column for: (i) erythromycin fluorescent nanocapsules, PLGA (100 mg), Tween 80 (0.01%), and (acetonitrile to 0.025 M ammonium dihydrogen phosphate in coumarin-6 or rhodamine 6G (25 μg) were dissolved in 5 mL of deionized water (60:40) at pH 7 and a detection wavelength of 205 acetonitrile. The other loaded nanocapsules were prepared similarly nm)37 and (ii) paclitaxel (acetonitrile to DDW with 0.1% trifluor- by substituting the coumarin with 0.2 mg/mL of paclitaxel or ICG in acetic acid (80:20) and a detection wavelength of 227 nm).38 The the organic phase. To generate the solid nanocapsules, the organic ICG concentration was evaluated using a SPECTRAFluor Plus plate solution was gradually poured into 10 mL of an aqueous solution reader (Tecan, San Jose, CA) in 780/820 nm Ex/Em. The containing solutol (0.1%) while stirring for 15 min. Acetonitrile was encapsulation efficiency was calculated using the equation given evaporated using an evaporator (Basis Hei-Vap Value, Heidolph below (eq 1) instruments, Germany) and the nanocapsules were collected by centrifugation and redispersed in double distilled water (DDW). In amount of encapsulated drug the particular case of erythromycin encapsulation (1 mg/mL), EE% = × 100 total drug added (1) acetonitrile was replaced by ethyl acetate (3 mL), the amount of PLGA was increased to 120 mg, the solutol was replaced by 2% PVA, 2.5. In Vitro Drug Release Profile. The loaded nanocapsules and the emulsion was generated by a 10 min probe sonication (Sonic were removed from the wafers and counted, concentrated to 0.5 mL ruptor 400, OMNI International, GA) after the stirring step.36 in deionized water by centrifugation, and then loaded into a dialysis To self-assemble the loaded nanocapsules, silicon wafers (100 mm tube (Pur-A-Lyzer Maxi Dialysis Kit MW cutoff 3.5 kDa, Sigma- boron doped p-type Si wafer, University Wafers, MA) were cleaned Aldrich) that was immersed in dissolution media (8 mL, 1% Tween and activated by oxygen plasma for 2 min at 100% power (Femto- 80 in PBS) under constant stirring at 900 rpm. At fixed time intervals, QLS, Diener Electronics, Germany). Then, the wafers were 0.5 mL of the buffer solution was withdrawn and replaced with fresh immediately incubated with a 2% positive polyelectrolyte polymer buffer, and the amount of released fluorophore (coumarin-6) was PDDA in water for 5 min, rinsed with DDW, and dried under N2. The evaluated using a plate reader in 440/528 nm Ex/Em. positively charged surface was incubated with the nanocapsule 2.6. Cell Lines. MDA-MB-231 breast adenocarcinoma tumor cells solution for 5 min and then rinsed and dried again to yield a self- were purchased from ATCC (VA) and maintained in DMEM assembled monolayer of well-separated nanocapsules on the Si supplemented with 10% fetal calf serum (FCS) (Gipco, Brazil) in a surface. medium with 1% penicillin/streptomycin (Biological Industries, The different metal films were deposited on the coated wafers using Israel). a high vacuum thin film electron beam deposition system (TFDS- 2.7. Magnet-Assisted Spheroid Formation. The magnetic 462B, VST Ltd., Israel) inside a glovebox. The substrate holder attraction level of the Fe-PLGA nanocapsules was evaluated by using temperature was set to −5 °C without rotation. their capability to induce the aggregation of multicellular spheroids. 50332 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article For cell labeling, MDA-MB-231 cells were first plated at 7 × 103 cells/ stained with hematoxylin and eosin (H&E). Sections were blocked by well and incubated for 24 h; then, Fe-PLGA nanocapsules at a 3% goat serum for 2 h, then stained with cleaved caspase-3 (1:500; concentration of 1.4 × 109 NCs/mL (equivalent to 5 μg/mL of iron) cat. no. 9664, Cell Signaling Technology, Inc.) overnight at 4 °C, were added to the medium and incubated for 4 h to allow them to followed by 2 h of incubation with Cy5 antibody (Alexa Fluor 647, interact with the cancer cells. The spheroids were prepared as ab150083, abcam) and 20 min of counterstaining with 6-diamidino-2- previously described, but with mild modifications.39 A preheated 2% phenylindole (Dapi), and then coverslipped and imaged by a agarose solution plated in 96-well plates at 50 μL/well and plate was fluorescence microscope. incubated for 10 min for gelation. Cells were counted after 2.9. Reactive Oxygen Species (ROS) Production. Gold-capped trypsinization and seeded at 4 × 104 cells/mL on top of the PLGA nanocapsules loaded with ICG were examined for their ability agarose-coated wells. A nickel-plated cylindrical neodymium magnet to generate reactive oxygen species, using 2′,7′-dichlorodihydrofluor- (magnetization N45, supermagnente, Gottmadingen, Germany), with escein diacetate (DCFH-DA) staining41 and its effect on cell viability. a magnetic field and field gradient according to Figure S6b, was fixed Cells were plated at 4 × 104 cells/well 24 h prior to treatment for 1 h under the desired wells, followed by overnight incubation for spheroid with 3 × 109 nanocapsules/mL (i.e., 12 μg/mL) of ICG-loaded or maturation. The spheroids’ brightfield and fluorescence images were unloaded Au-PLGA nanocapsules. Then, the wells were radiated using taken with an ECLIPSE Ti2 inverted microscope (NIKON, Tokyo, a laser diode (λ = 808 nm at 1 W/cm2) for 0, 2, 5, or 10 min, and the Japan) to visualize the interaction of rhodamine 6G-loaded Fe-PLGA medium was replaced by 100 μL of medium containing 10 μM NCs with the spheroid and the manipulation of the spheroid inside DCFH-DA. The plates were incubated for 1 h and washed three the well using a magnet near the well wall. The viability of the times, 100 μL of fresh medium was added, and fluorescence was read spheroids was measured after 5 days of incubation with empty or immediately using ex/em 485/530. The results were normalized by paclitaxel-loaded Fe-PLGA nanocapsules by the WST-8 colorimetric the fluorescence readout from untreated cells, thus being presented as assay (Sigma-Aldrich). The WST-8 reagent was added at a 1:10 ratio the reactive oxygen species increase factor. The cell viability was to the cell medium, and the plates were incubated for 3 h, after which evaluated by the MTT assay 72 h post laser radiation. Cells were absorbance was measured using a SPECTRAFluor Plus plate reader at incubated with 0.5 mg/mL of MTT reagent for 4 h. Then, the 450 nm. supernatant was removed and dimethyl sulfoxide (DMSO) was 2.8. Photothermal Effects of Hybrid NPs. For the in vitro added. The absorbance was read at 570 nm by a SPECTRAFluor Plus photothermal effect, Au-PLGA NPs were removed from a wafer and plate reader. concentrated by centrifugation to 37 μg/mL of Au. The photothermal 2.10. Antibacterial Effect of Cu-PLGA NPs. Copper-capped conversion efficiency at 808 nm was measured for: (i) commercial PLGA NPs loaded with the antibiotic erythromycin were evaluated gold nanoshells (AuNS) and (ii) ICG-loaded and (iii) unloaded Au- for their effect on Gram-positive Staphylococcus aureus (S. aureus PLGA NPs. The following equation (eq 2) was used40 C127). Bacteria (6 × 105 CFU/mL in lysogeny broth) were incubated with erythromycin (0.02−12 μg/mL) in 96-well plates at 37 °C. The hS Tmax Q dis minimum inhibitory concentration (MIC) was determined by = A808 I(1 10 ) (2) measuring the optical density (OD), at 600 nm, for each well every 20 min over a period of 20 h using the Eon microplate where h, S, and Qdis represent the heat transfer coefficient, area of the spectrophotometer (Gen5 v2.07, BioTek Instruments, VT).42 To illuminated spot, and heat dissipated from the light absorbed by the compare between treatments, the lowest concentration of the polystyrene cell, respectively. In addition, A808 is the absorbance value antibiotic to inhibit 50% of the isolates (MIC50) was measured. of the nanocapsule dispersion at 808 nm, ΔTmax represents the Uncoated erythromycin-loaded PLGA NPs were tested for their temperature increase after 20 min of radiation, and I is the laser power ability to inhibit bacterial growth using an erythromycin concentration (267 mW). range from 0.02 to 12 μg/mL, which was determined by HPLC. The visualization of ICG-loaded Au-PLGA NCs was performed by Erythromycin-loaded Cu-PLGA NCs at concentrations with an an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). For the erythromycin concentration from 9.6 × 10−3 to 0.6 μg/mL were analysis of the in vivo therapeutic effects, 8-week-old Hsd:Athymic studied using the same method to evaluate the synergistic effect of Nude-Foxn1nu female mice were purchased (Harlan, Rehovot, copper in the delivery system by subtracting the background Israel), stored in a specific pathogen-free (SPF) unit, and acclimated absorbance from the NCs. To examine the morphological changes, for a week. All animal procedures were conducted according to the TEM images of the bacteria were acquired after 2 h of treatment. The institutional and national guidelines, and protocols were approved by colony-forming units (CFU/mL) of each treatment were measured the Hebrew University Ein Kerem Medical School IACUC (protocol after 20 h of treatment by decimally diluting the sample 8 times, and MD-21-16572-5). Mice were injected subcutaneously (S.C.) with 5 × the colonies were counted after 24 h of incubation in LB-agar plates 106 MDA-MB-231 cells/mouse in 100 μL PBS. Tumors were spotted with 10 μL from each dilution at 37 °C. monitored until an approximate size of ∼120 mm3 was reached; then, 2.11. Statistical Analysis. All data were analyzed with GraphPad the mice were divided into four groups. The loaded Au-PLGA NCs Prism 8.0.1 software. Results are presented as mean ± standard error. were injected intratumorally at a concentration of 1.82 × 1011 NCs/ One-way analysis of variance (ANOVA) was applied in the statistical mL with a volume of 50 μL (i.e., 4 mg/kg of Au) into two of the difference comparation. In the cases in which the data did not follow a groups, whereas the other two received only PBS intratumorally, and normal distribution, specifically in the context of the Au-PLGA in vivo the mice were imaged by IVIS. Immediately after injection, the two experiment, statistical analysis was performed using a Kruskal−Wallis groups were irradiated by a laser diode (B1-808-1500-15A, test to assess differences among multiple groups. Subsequently, a Sheaumann, MA, driven by a laser diode controller ITC4005, Dunn’s post hoc test was conducted to compare pairs of groups to Thorlabs, Germany) with emission wavelength λ = 808 nm and light evaluate the therapeutic effect of the treatment. Statistically significant intensity of 1 W/cm2 for 5 min. The thermal images were acquired by differences were determined as *p < 0.05, **p < 0.01, and ***p < an infrared thermal camera (8320P, Infrared Cameras Inc. (ICI), 0.001. Beaumont, TX). The tumors were monitored, and their volumes were measured for 14 days post laser irradiation using the following equation (eq 3) 3. RESULTS AND DISCUSSION 3.1. Drug-Loaded Nanocapsule Fabrication and width2 × length Metal Coating. The drug-loaded PLGA nanocapsules were tumor volume = 2 (3) fabricated using an established method of emulsification− Each tumor volume was normalized to its initial volume (n = 5). Five evaporation according to the details described in Section 2.35 days post laser irradiation, the selected mice from the laser-treated Highly uniformed nanocapsules with diameter ca. 100 nm were groups were euthanized and the tumors were removed, sectioned, and obtained, with a PDI lower than 0.1 (Table 1) and negative 50333 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article Table 1. Physicochemical Characteristics of NPs before and after Metal Depositions mean size by DLS mean size by TEM ± SD ζ potential encapsulation efficiency ± SD nanocapsule layers (nm) PDI (nm) (mV) encapsulant (%) PLGA 108.1 0.07 106 ± 11 −30 ± 3 Fe-PLGA Fe�20 nm 138.8 0.16 124 ± 12 −19 ± 2 paclitaxel 50 ± 3 SiO2�10 nm Au-PLGA Ti�2 nm 131.2 0.31 117 ± 11 −34 ± 1 indocyanine 55 ± 3 Au�20 nm green Cu-PLGA Cu�20 nm 191.7 0.31 149 ± 32 −36 ± 1 erythromycin 36 ± 3 SiO2�10 nm Figure 1. (A) SEM image of metal-coated nanocapsules on the wafer. TEM images and element mapping of representative metal-coated nanocapsules corresponding to (B) Fe (red) and Si (green) in the Fe-PLGA nanocapsules, (C) Au (yellow) in the Au-PLGA nanocapsules, and (D) Cu (orange) and Si (green) in the Cu-PLGA nanocapsules. (E) Release profile of the dye loaded into uncapped and metal-capped PLGA NPs over time. charge (ζ potential −30.2 mV). Prior to metal deposition, we Ti layer was first deposited. Note that the direct deposition developed a robust protocol for the electrostatic self-assembly approach is a straightforward method to control the thickness of the PLGA nanocapsules on silicon surfaces to enable metal of the metal layers, which allows fine-tuning their functional evaporation on only half of their surface. The silicon wafers properties. For example, by changing the thickness of the shell were first activated by plasma, resulting in the formation of layers, it is possible to control the magnetization reversal in the silanol groups (−Si−OH)43 and then coated with a monolayer Fe coating or to tune the wavelength of the plasmonic of the positively charged polyelectrolyte PDDA, which resonance of Au and the photothermal response.45 It is also facilitated the electrostatic attachment of the negatively important to emphasize that the growth of these anisotropic charged PLGA nanocapsules to the surface. When examining multilayers on the polymer particles (e.g., SiO2 on Fe or Cu as the nanocapsule distribution and morphology on the wafer protection from oxidation) to optimize their performance surface by SEM, it was found that PDDA was the most would be extremely complex by using chemical synthesis adequate linker, providing dense and well-separated nano- methods. Notably, although in our case we have used the capsules on the surface in comparison with the other tested positive molecules (PAH and APTMS) (Figure S1). additional layers for structural purposes, the same concept To form the metal (iron, gold, or copper) semishell that could be used to introduce new functionalities in the facilitate efficient release of the loaded drug, we used highly structures. For example, combining Fe and Au layers can directional electron beam evaporation on the self-assembled provide dual magnetic resonance and X-ray contrast for arrays of the loaded PLGA nanocapsules. Since iron (Fe) and noninvasive imaging, Fe and platinum (Pt) could merge copper (Cu) tend to oxidize over time, an additional 10 nm magnetic imaging with antineoplastic capabilities, and Cu and silica (SiO2) layer was deposited over the 20 nm metal layer as silver (Ag) could merge the antibacterial capabilities and a protective shield.44 To guarantee the firm attachment of the enable efficient photothermal actuation to boost the 20 nm Au layer to the PLGA nanocapsules and wafer, a 2 nm therapeutic effects even further.46 50334 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 2. Fe-PLGA nanocapsules for the magnetic manipulation of spheroids. (A) Picture showing the magnetic concentration of dispersed nanocapsules after 90 s. (B) Schematic illustration of the magnetically assisted spheroid formation. (C) Absorbance at 590 nm of MDA-MB-231 cells incubated with Fe-PLGA nanocapsules for 5 h and the eosin/Prussian blue staining of cells before and after incubation (scale 50 μm). (D) Fluorescence and brightfield (BF) images of spheroids containing rhodamine 6G-loaded Fe-PLGA nanocapsules after 48 h (scale 200 μm). (E) Brightfield microscopy images (scale 500 μm) and cross-section area measurements of the spheroids, formed with empty Fe-PLGA nanocapsules with and without magnetic concentration after 24 and 48 h. (F) Spheroid magnetic manipulation inside the well showing the positions before (blue) and after (orange) using an external magnetic field gradient. (G) Viability of the spheroids formed without nanocapsules, with empty Fe- PLGA nanocapsules and paclitaxel-loaded Fe-PLGA nanocapsules with (+) or without (−) magnetic manipulation. The SEM images of Figure 1A show the expected array of extension of the drug release (Figure 1E), thereby preserving self-assembled nanocapsules coated with the metal layer on the water hydrolysis of the polymer chains that triggers the their upper half, whereas the TEM images with EDS/EELS polymer degradation.50 In the case of the ICG-loaded mapping analysis verify the presence and thickness of the nanocapsules, a similar release pattern is expected, considering different deposited layer, i.e, Fe/SiO2 for the Fe-PLGA that the formulation of the polymer nanocapsules loaded is nanocapsules (Figure 1B), Ti/Au for the Au-PLGA nano- identical to that in the paclitaxel or coumarin cases, which is capsules (Figure 1C), and Cu/SiO2 for the Cu-PLGA confirmed with the observed therapeutic activity of the drug. nanocapsules (Figure 1D). Both the self-assembly of the 3.2. Physicochemical Characterization. The main loaded PLGA nanocapsules and the final dispersion of the physicochemical parameters of the uncoated and coated metal-capped nanocapsules were optimized to maximize the nanocapsules are summarized in Table 1. The DLS measure- final concentration and to minimize the generation of particle ments showed slightly larger nanoparticle diameters with aggregates, yielding a ca. 85% recovery from the wafer (Figures respect to the TEM measurements (Figure S4), as expected S2 and S3D,E). from their larger hydrodynamic diameter. In the case of the To compare the drug release profiles and kinetics of the Cu-coated nanocapsules, the difference is larger due to the PLGA nanocapsules with and without metal deposition, release presence of small aggregates in the self-assembled monolayer, studies were performed by detecting the druglike fluorophore resulting from the different formulations of the PLGA molecule coumarin-6. Interestingly, no significant differences nanocapsules. In all of the examined cases, a stable colloidal were observed in the release profile and kinetics between the dispersion was obtained with negative ζ potential values metal-coated and uncoated nanocapsules (∼41% over 168 h), ranging from ∼−18 to −36 mV. The high polydispersity index being similar to other reports on the release kinetics of (PDI) values observed in Au- and Cu-coated nanocapsules are coumarin-6 from PLGA NPs (33−45% over 168 h).47−49 A due to their high optical anisotropy, which generates different similar release pattern of the paclitaxel drug was also observed scattering intensities depending on the particle orientation in our previous work for Fe-coated and uncoated polymer with respect to the polarization of the incident light, thus nanoparticles.33 These results indicate that neither the metal overestimating the actual polydispersity observed in the SEM layer nor the deposition process reduced the percentage and images. In the case of Fe-coated nanoparticles, the PDI is lower 50335 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article as a result of the highly damped plasmonic behavior of the nanocapsules (Figure S4). After an initial increase of the nanostructured Fe,51,52 which reduces the optical anisotropy. absorbance signal in the first 2 h, no major difference occurred Therefore, for optically anisotropic structures, SEM/TEM or between 3 and 5 h. As a result, we selected 4 h as the other nanoparticle analysis techniques should be used to optimized uptake time. The confocal fluorescence images of accurately determine the PDI.53,54 Finally, the nanocapsules cells incubated with the Fe-PLGA nanocapsules loaded with exhibited a loading efficiency over 50% for paclitaxel and ICG, rhodamine 6G for 4 h showed a large quantity of internalized whereas it was slightly lower (36%) for erythromycin. The nanocapsules inside the cells, although a significant fraction of typical optical absorbance values of the dispersed Fe-, Au-, and particles was still at the cell membrane (Figure S7). In contrast, Cu-coated nanocapsules are gathered in Figure S5, showing the after 24 h of incubation, the majority of the nanocapsules was expended broadband absorption for the Fe-PLGA NCs and already internalized (Figure S7). Prussian blue staining to more defined plasmonic bands for the Au- and Cu-coated visualize the presence of iron showed an intense signal in the particles, although the Cu-PLGA NC absorbance was treated cells after 4 h of incubation compared to the significantly lower. nontreated cells (Figure 2C; right panel). Next, we analyze the synergistic effects that can be achieved To evaluate both the cellular uptake and the distribution of for the different metal-PLGA-drug formulations. the nanocapsules in the spheroids, Fe-PLGA nanocapsules 3.3. Fe-PLGA Nanocapsules Loaded with Paclitaxel loaded with rhodamine 6G showed a qualitatively high uptake for Magnetically Enhanced and Manipulated Cell of the nanocapsules with a very homogeneous distribution in Spheroids with Boosted Therapeutic Effect. The Fe- the spheroid after 24 h of assembly (Figure 2D). coated nanocapsules offer a very efficient external magnetic To compare the spheroid formation rate with and without manipulation, which is granted by their large volume, V, and magnetic actuation, the cross-section area of the formed the strong ferromagnetism of the metal Fe layer, whose spheroid was calculated from images taken at 24 and 48 h, saturation magnetization, MS, is 3-fold higher than that of the showing a significant decrease (40%) in the spheroid area for typical iron oxides used for biomedical applications. This the magnetically actuated cells, thus indicating the formation of confers them a total moment, m = V × MS, ca. 250 times larger more condensed and stable spheroids (Figure 2E).55 Such than a Fe3O4 nanoparticle of 20 nm. The diameter and efficient magnetic actuation is not only important for thickness of the Fe layer have been designed to exhibit a improving spheroidal formation but can also be useful for magnetic vortex (Figure S6a),33,45 thereby showing near zero externally manipulating the whole spheroid in an aqueous remanence and enabling high colloidal stability, despite the medium, as can be observed in Figure 2F and the video in the large content of ferromagnetic material. This is due to the Supporting Information. These images showed the capability negligible magnetic dipole−dipole interactions between the to magnetically displace the spheroids inside the well at a coated nanocapsules in the absence of an external magnetic speed of 150 μm/s, despite the low nanocapsule concentration. field. However, in the presence of a magnetic field gradient, the To validate the synergistic effect of an active compound large induced magnetic moment in the Fe semishell allows the loaded in the nanocapsules, we used paclitaxel-loaded Fe- generation of magnetophoretic forces that are 2 orders of PLGA NCs to evaluate the anticancer therapeutic effect in magnitude larger than those in superparamagnetic iron oxide breast cancer carcinoma spheroids. Whereas spheroids treated nanoparticles.33 Here we exploit the high drug-loading capacity with empty Fe-PLGA nanocapsules or PBS were still vital after and the magnetic strength to demonstrate the ability to (i) 5 days, paclitaxel-loaded Fe-PLGA NPs decreased the viability magnetically manipulate cells and improve the efficiency in the to 60 and 48% in the unmanipulated and magnetically formation of three-dimensional (3D) cell cancer spheroids and manipulated spheroids, respectively (Figure 2G), thus high- (ii) enhance the effects of encapsulated compounds. While lighting the magnetically enhanced therapeutic effect. This is some previous studies have demonstrated magnetically probably due to the higher drug concentration in the induced multicellular spheroids using NPs that drive cell magnetically actuated spheroids resulting from their higher aggregation,55 in our case, the Fe-PLGA nanocapsules enable a cell (and nanocapsule) density. simultaneous combination of magnetic actuation and drug This magnetically controlled spheroid model may be release in the same system, thus accelerating cell assembly at relevant for the screening of drug candidates in more low particle concentrations. physiologically relevant cancer models.56 In addition, there is The high magnetic strength of the Fe-PLGA nanocapsules is a major limitation regarding the penetration of drug carriers reflected in Figure 2A, in which the response to an external into tissues and 3D cultures, such as spheroids. Therefore, magnetic force was demonstrated by placing a magnet close to locating the drug in the center of the cell mass, as shown in our a concentrated solution of Fe-PLGA nanocapsules, resulting in study, may potentially facilitate the assessment of the drug complete attraction and accumulation of all of the nano- responses in spheroids or other 3D cultures.57 This could be capsules to the magnet in 90 s. utilized in personalized medicine for screening anticancer To enable cell actuation with magnetic forces for improving drugs on spheroids obtained from patient’s biopsies.57 the assembly of the spheroids, the nanocapsules should be The magnetic nanocapsules could also be applied for tissue efficiency-internalized or adhered at the cell surface before engineering, as spheroids could be magnetically assembled in placing the magnet to trigger the cell concentration (as complex shapes and scaffolds.58 Moreover, the encapsulation illustrated in Figure 2B). MDA-MB-231 cancer cells were of growth factors inside Fe-PLGA nanocapsules could improve analyzed for their uptake capacity during 5 h of incubation the assembly of the spheroids and support long-term culture, with the Fe-PLGA nanocapsules at a concentration of 1.4 × as previously shown with PLGA microspheres loaded with 109 nanocapsules/mL (i.e., an Fe concentration of 5 μg/mL). transforming growth factor-β1 (TGF-β1) in human mesen- After intensive washes with PBS, the nanocapsules’ uptake was chymal stem cell spheroids.59 The concentration of the Fe- assessed by measuring the absorbance at 590 nm (Figure 2C; PLGA nanocapsules used for magnet-assisted spheroid left panel), which corresponds to the absorbance peak of the formation (5 μg/mL) is in line with concentrations used 50336 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 3. Application of Au-PLGA nanocapsules as dual photodynamic and photothermal therapy. (A) Visible−NIR spectrum of Au-PLGA nanocapsules and nanoshells. The vertical line indicates the wavelength used in the experiments, λ = 808 nm. (B) ROS production (bars) and viability (lines) of untreated and ICG-loaded Au-PLGA NC-treated MDA-MB-231 cells vs irradiation time (808 nm, 0.95 W/cm2). (C) Fluorescence images of ICG-loaded Au-PLGA NCs in solution compared to PBS and after the intratumoral injection. (D) Photo-induced temperature increase by ICG-loaded Au-PLGA nanocapsules in vivo in MDA-MB-231 tumor-bearing mice (808 nm, 1 W/cm2), and (E) representative thermal camera images. (F) Photographs of the mouse treated with Au-PLGA nanocapsules and laser at different time points during the experiment. (G) Evolution of the tumor volumes post radiation. (H) H&E and caspase-3 staining of the mice sacrificed on day 5 treated with laser and PBS or ICG-loaded Au-PLGA NCs (blue represents Dapi and red represents cleaved caspase-3; scale: 200 μm and insert scale: 50 μm). with the commercially available NanoShuttle-PL superpar- first biological window (peak at 870 nm) (Figure 3A) to amagnetic iron oxide nanoparticles (SPIONs),55 but without ensure high light penetration in the tissues. The incorporation the drug-loading capability. It is also worth mentioning that the of ICG in the capsules induces a slight blue shift of the metallic iron semishell in the nanocapsules can also provide a resonance (Figure 3A), as a consequence of the ICG high photothermal conversion efficiency and a high contrast in absorption band centered at approximately 780 nm. magnetic resonance imaging, 33 thereby expanding the Next, we analyzed the photothermal conversion efficiency at repertoire of functionalities of these nanocapsules for a typical wavelength used in the first biological window (i.e., therapeutic applications. 808 nm) for unloaded and ICG-loaded Au-PLGA nano- 3.4. Synergistic Photodynamic/Photothermal Ther- capsules, compared to commercial Au nanoshells over silica apy and Noninvasive In Vivo Imaging Using Indoc- nanoparticles that have been widely used in photothermal yanine Green (ICG)-Loaded Au-PLGA Nanocapsules for therapies.62 As Table 2 shows, the photothermal efficiencies Tumor Eradication. Gold NPs have been widely utilized to generate photothermal therapies with near-infrared (NIR) Table 2. Photothermal Conversion Efficiency Comparison radiation to locally induce cancer cell death.60 Conversely, the sample Abs at 808 nm ΔT (°C) η small molecule ICG has been used as a photodynamic agent AuNS 1.09 10.5 0.50 due to its capability to generate reactive oxygen species (ROS) Au-PLGA NCs 0.91 14.8 0.76 upon NIR irradiation.61 Therefore, simultaneous photothermal ICG-Au-PLGA NCs 0.82 14.5 0.79 and photodynamic local actuation could enhance the nano- therapy performance by combining (i) the oxidative stress produced by the photodynamic agent and (ii) the hyper- present remarkably high 76 and 79% values for the unloaded thermia irreversible cell damage. To demonstrate the capacity and ICG-loaded Au-PLGA nanocapsules, respectively (Figure to merge both effects, we fabricated Au-PLGA nanocapsules S8). Therefore, the incorporation of ICG does not affect the loaded with ICG following the same fabrication strategy. The photothermal efficiency. In contrast, standard gold nanoshells Au thickness and size of the PLGA particles were selected to with 20 nm Au and a resonance at 800 nm showed an exhibit strong and broadband plasmonic resonance within the efficiency of only 50%. Such photothermal efficiency enhance- 50337 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 4. Synergistic antibacterial activity of Cu-PLGA nanocapsules. (A) Antibacterial activity of free erythromycin, PLGA-erythromycin, and Cu- PLGA-erythromycin NCs against S. aureus as a function of the erythromycin concentration, evaluated by the optical density method after 20 h of incubation. (B) Determination of the colony-forming units (CFU per mL) after 20 h treatment of free erythromycin (0.5 μg/mL) compared to loaded PLGA nanocapsules, unloaded Cu-PLGA nanocapsules, and loaded Cu-PLGA nanocapsules at a concentration of 2.5 × 1010 nanocapsules/ mL. TEM images of (C) untreated and (D−F) erythromycin-loaded Cu-PLGA nanocapsule-treated bacteria (magnification 10k and 40k). ment is due to the nanodome shape of the Au coating in the growth was exhibited with the nanocapsule- and laser-treated nanocapsules, which enables a drastic reduction of the group (3.5-fold of the initial tumor volume), compared to the scattering cross section compared to complete nanoshells.45 groups of nanocapsules without laser, only laser, and PBS To explore the synergistic photothermal and photodynamic control (6.5, 9.3, and 9.1, respectively; Figure 3G). The effects, ICG-loaded Au-PLGA nanocapsules (1.5 × 1010 NCs/ sectioned tumors, stained by H&E and cleaved caspase-3 to mL, equivalent to 60 μg/mL of Au) were added to 4 × 104 evaluate necrosis and apoptosis, displayed a necrotic core and cell/mL MDA-MB-231 cells and evaluated for ROS higher levels of apoptotic fluorescence signals for the laser- and production after laser irradiation (808 nm, 0.95 W/cm2). nanocapsule-treated mice, compared with the laser-treated Compared to untreated cells, the cells treated with ICG-loaded mice injected with PBS, as a consequence of the combination Au-PLGA nanocapsules produced a significantly higher ROS of the higher photothermal effect and the oxidative stress concentration as the exposure time increased, being about 2- caused by ICG61 (Figure 3H). Our observations supported the fold higher after 10 min of irradiation. These ROS levels notion that the combination of photodynamic and photo- directly correlated with the drastic decrease in cell viability thermal therapies using ICG and gold-containing formulations compared to nontreated cells after 5 min of irradiation (Figure can lead to efficient anticancer effects in vivo.63−65 In contrast 3B). to these technologies in which ICG is either conjugated to gold The high fluorescence of the ICG-loaded nanocapsules was nanoclusters,63 acting as a coating layer on the gold cluster demonstrated by the IVIS device (Figure 3C; left panel), thus cores,64 or coencapsulated with gold nanorods in a polymer showing that there is no significant fluorescence quenching by matrix,65 our approach enables more versatile combinations the Au semishell. Importantly, the ICG-loaded nanocapsules and adjustments of the gold layer thickness and potentially can be used for noninvasive visualization in vivo with very supports the coencapsulation of ICG with additional drugs strong contrast (Figure 3C; right panel). inside the NCs. To study the efficacy of the photothermal−photodynamic In addition to the photothermal effects, the Au semishell nanotherapy in vivo, MDA-MB-231 tumor-bearing mice were also provides an intense X-ray contrast,45 which can be useful treated with intratumoral injection (50 μL) of either PBS or for noninvasive deep tissue imaging by computed tomography. ICG-loaded Au-PLGA nanocapsules (1.82 × 1011 NCs/mL It is also possible to integrate Fe and Au layers that can equivalent to 4 mg of Au per kg of mouse). The mice were complement the optical treatment with magnetic guidance to imaged by IVIS to visualize the presence of ICG inside the enhance the therapeutic effects locally. The Fe/Au combina- tumor (Figure 3C), showing a homogeneous distribution. tion could also enable simultaneous opto-magnetic heating Next, a 5 min laser exposure was monitored via thermal thermometry in vivo.31 imaging of the tumor site, which increased the local 3.5. Synergistic Antibacterial Treatment with Cu- temperature by 6.9 °C for the PBS-treated group and 16.6 PLGA Nanocapsules Loaded with Erythromycin. Copper, °C for the nanocapsule-treated group (Figure 3D,E). The among other metals, has been widely studied for its healing from the radiation was monitored for 2 weeks post antibacterial activity due to its ability to inhibit the growth injection, demonstrating almost complete healing of the of a wide spectrum of microorganisms.66 The antibacterial radiated area (Figure 3F). A substantial reduction in tumor mechanism of the action of copper is based on “contact killing” 50338 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article and was linked to the capacity of the metal to release copper (10 μg disks) dipped in silver NP solution increased the zone ions, which causes membrane damage and eventually DNA of inhibition by 68% compared to undipped disks,73 and the degradation.67 A combination of a commercial antibiotic drug azithromycin (15 μg disks) zone of inhibition was increased by with this metal may potentially enhance the antimicrobial 48 and 11% when the disks were impregnated with silver NP activity. Therefore, erythromycin was loaded in Cu-PLGA or zinc NP solution, respectively.74 nanocapsules to evaluate the antibacterial activity against the Furthermore, the combination of Cu and Fe layers could Gram-positive bacteria, S. aureus. To determine the minimum also offer additional functionalities to locally enhance the inhibitory concentration, MIC50 S. aureus cultures were first therapeutic effects by magnetic manipulation and to boost treated with free erythromycin at concentrations of 0.02−12 even further the therapeutic effects by photothermal actuation. μg/mL. The MIC50 value was about 0.5 μg/mL (Figure 4A). The antibacterial activity of the uncoated erythromycin-loaded 4. CONCLUSIONS PLGA nanocapsules was tested on S. aureus to compare the Transitioning from single-function nanocarriers to multifunc- activities of the free and loaded drugs, showing a similar tional ones, which integrate multiple components in a single inhibition profile after 20 h of incubation (Figure 4A), with a system, has been recognized as an important modality for MIC50 erythromycin value of 0.5 μg/mL. Erythromycin therapy and theranostics.12 This is in line with the growing concentrations were determined by HPLC (Figure S9). To acknowledgment that a single mechanism of action is typically evaluate the synergistic bactericidal effect, MIC50 was also insufficient to treat complex diseases featuring multiple and measured in erythromycin-loaded Cu-PLGA nanocapsules, parallel biological processes.75 In this study, we established a showing a remarkable 2.5-fold lower MIC50 concentration robust and versatile platform for manufacturing multifunctional value (i.e., 0.2 μg/mL, equal to 2.5 × 1010 NC/mL) (Figure nanocapsules using physical metal deposition on self- 4A), thus indicating the powerful concomitant effect of the Cu assembled drug-loaded PLGA nanocapsules. Here, we have cap. After 24 h of incubation at 37 °C, copper ion release from demonstrated the multifunctionality and synergistic therapeu- Cu-PLGA NCs was observed, with approximately 80% of the tic effects using three metal−drug−polymer combinations, initial copper content released to the medium (Figure S10). providing in each of them a proof of concept in a specific In addition, the antibacterial effects of copper and the relevant application. In the case of iron−PLGA nanocapsules, antibiotic drug were evaluated by determining the colony- we demonstrated the capacity to magnetically improve the forming units (CFU/mL) after 20 h of different treatments formation of denser 3D cultures (40% reduction in their cross- using the MIC50 concentration. As can be observed in Figure section area after 24 h), thus opening the path to a better 4B, the free erythromycin at 0.5 μg/mL resulted in 4 × 107 understanding of the drug efficiency and resistance in an CFU/mL, i.e., significantly lower than untreated bacteria (1.6 improved representation of a tumor microenvironment.76 In × 109 CFU/mL). To highlight the effect of Cu, we also the case of gold−PLGA nanocapsules, we showed a dual analyzed the CFU for the unloaded Cu-PLGA NCs and anticancer photodynamic−photothermal therapy in combina- erythromycin-loaded Cu-PLGA nanocapsules in comparison to tion with noninvasive fluorescence imaging with high- uncapped erythromycin-loaded PLGA nanocapsules at the penetration NIR light, for enhanced therapeutic effect, showing same particle concentration of 2.5 × 1010 NC/mL, achieving a a 3.5-fold reduction of the tumor volume growth with only 5 remarkable reduction up to 2.6 × 107 and 1.42 × 108 CFU/mL min of NIR illumination. With copper−PLGA nanocapsules, compared to 1.2 × 107 CFU/mL, respectively. These results we exploited the innate antibacterial properties of copper to demonstrate the synergistic effects of the Cu layer and the improve the antibacterial activity and reduce the dosage of an antibiotic drug. encapsulated antibiotic drug (a 2.5-fold increase in the MIC50 To clarify how the erythromycin-loaded Cu-PLGA nano- value), which is a relevant step in the fight against antibiotic capsules affect the bacteria, we used TEM to examine the resistance. morphological changes that occur in S. aureus after 2 h of A central advantage of this approach is the very wide range treatment. Untreated bacteria displayed a normal undamaged of metal−drug combinations that can be envisaged to enhance structure (Figure 4C). In contrast, erythromycin-loaded Cu- the drug effects and the simple and very accurate control over PLGA nanocapsules interacted with the outer layer of S. the thickness and the number of active materials in the aureus, resulting in an amorphous shape, with blurry and structures, without the need of any further fabrication disturbed membranes (Figure 4D−F, nanocapsules marked in optimization. Therefore, this modular metal−polymer nano- red). The bright areas in the center also indicate a decrease in capsule concept provides a high level of versatility that can be cytoplasmatic material due to the membrane damage.68,69 exploited to explore new biomedical applications. In addition to Cu, decorations with other metals that are known for their bactericidal activities such as silver, zinc, titanium, nickel, and palladium could be used to enhance the ■ * ASSOCIATED CONTENT sı Supporting Information antibacterial effects.70 These types of metal-coated nano- The Supporting Information is available free of charge at capsules containing antibiotic, antifungal, or antiviral drugs https://pubs.acs.org/doi/10.1021/acsami.3c07188. circumvent the necessity of using conjugating, stabilizing, and reducing agents during the fabrication of metallic nanoparticles Spheroid (MOV) loaded with antimicrobial drugs.71 Moreover, since one of the Investigation of NCs and silicon surface association greatest risks to human health is the increasing antibiotic regarding thin layer formation (Figure S1) and NC resistance, the reactive metals can be an interesting alternative removal (Figure S2); evaluations of NC size, morphol- to eradicate resistant bacteria, allowing the administration of ogy, and metal composition using DLS and SEM/EDS lower initial doses.72 Our results are in line with other studies (Figure S3); TEM images of the three representative on metal−antibiotic combination effects on S. aureus, metal-coated nanocapsules (Figure S4); UV/vis absorb- specifically with the macrolide family, where erythromycin ance spectra of the metal-coated nanoparticles (Figure 50339 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article S5); magnetic properties of the Fe-PLGA NCs and critical inputs of A.G.R., J.N., and R.H., with B.S. and O.B. magnetic fields used for the formation of the cell leading as principal investigators, providing conceptualization spheroids (Figure S6); confocal and SEM images of the and supervision. The manuscript was written through the Fe-PLGA nanocapsules loaded with rhodamine 6G to contributions of all authors. All of the photos/artwork were demonstrate the internalization of the nanocapsules taken/created by the authors of this manuscript. All authors (Figure S7); measurements of Au-PLGA NC photo- have given approval to the final version of the manuscript. thermal conversion efficiency (Figure S8); HPLC Funding measurement for the detection of erythromycin (Figure This work was financially supported by the European Research S9); and determination of the release of copper ion from Council (ERC-StG) under the European Union’s Horizon Cu-PLGA NCs (Figure S10) (PDF) 2020 Research and Innovation Program (Grant Agreement No. 0305260), the ERC proof of concept grant (ERC-2022- ■ AUTHOR INFORMATION Corresponding Authors PoC1) (Grant Agreement No. 756762), and the M-ERA.NET transnational grant. We acknowledge funding from Generalitat de Catalunya through the 2017-SGR-292 and 2021-SGR-0065 Borja Sepulveda − Instituto de Microelectronica de Barcelona projects. We acknowledge the financial support from the (IMB-CNM, CSIC), 08193 Bellaterra, Barcelona, Spain; Spanish Ministerio de Ciencia, Innovación y Universidades Email: borja.sepulveda@csic.es (MICINN) through the MAT2016-77391-R, PDC2022- Ofra Benny − Institute for Drug Research (IDR), School of 133036-I00, PID2019- 106229RB-I00, PCIN2016-093 (M- Pharmacy, Faculty of Medicine, The Hebrew University of ERA-NET), RTI2018-095495-J-I00, and RYC2019-027449-I Jerusalem, 9112102 Jerusalem, Israel; orcid.org/0000- funded by MCIN/AEI/10.13039/501100011033. We thank 0002-2468-5978; Email: Ofra.Benny@mail.huji.ac.il the Ramon Areces foundation through grant CIVP19A5922. ICN2 is funded by the CERCA programme/Generalitat de Authors Catalunya. The ICN2 has been supported by the Severo Arnon Fluksman − Institute for Drug Research (IDR), School Ochoa Centres of Excellence programme [SEV-2017-0706] of Pharmacy, Faculty of Medicine, The Hebrew University of and is currently supported by the Severo Ochoa Centres of Jerusalem, 9112102 Jerusalem, Israel; orcid.org/0000- Excellence programme, Grant CEX2021-001214-S, both 0003-1057-6209 funded by MCIN/AEI/10.13039.501100011033). Aritz Lafuente − Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, Notes Barcelona, Spain; Universitat Autònoma de Barcelona, The authors declare no competing financial interest. 08193 Cerdanyola del Vallès, Barcelona, Spain; orcid.org/0000-0002-1497-5744 Ron Braunstein − Institute of Biomedical and Oral Research ■ ACKNOWLEDGMENTS The authors would like to thank Dr. E Berenshtein, V. Belzer, (IBOR), Faculty of Dental Medicine, The Hebrew University M. Saidian, and Dr. V. Shelukhin for their technical support. of Jerusalem, 9112102 Jerusalem, Israel ToC, abstract, graphics, and Scheme 1 are created with Eliana Steinberg − Institute for Drug Research (IDR), School BioRender.com. of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 9112102 Jerusalem, Israel Nethanel Friedman − Institute for Drug Research (IDR), ■ ABBREVIATIONS 3D- three-dimensional School of Pharmacy, Faculty of Medicine, The Hebrew Au- gold University of Jerusalem, 9112102 Jerusalem, Israel CFU- colony-forming units Zhanna Yekhin − Department of Bone Marrow Cu- copper Transplantation and Cancer Immunotherapy, Hadassah DDW- double distilled water Medical Center, The Faculty of Medicine, The Hebrew DLS- dynamic light scattering University of Jerusalem, 9112102 Jerusalem, Israel EDS- energy-dispersive X-ray spectroscopy Alejandro G. Roca − Catalan Institute of Nanoscience and EE- encapsulation efficiency Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, Ery- erythromycin Barcelona, Spain Fe- iron Josep Nogues − Catalan Institute of Nanoscience and H&E- hematoxylin and eosin Nanotechnology (ICN2), CSIC and BIST, 08193 Bellaterra, HPLC- high-performance liquid chromatography Barcelona, Spain; ICREA, 08010 Barcelona, Spain; I.T.- intratumoral orcid.org/0000-0003-4616-1371 ICG- indocyanine green Ronen Hazan − Institute of Biomedical and Oral Research IVIS- in vivo imaging system (IBOR), Faculty of Dental Medicine, The Hebrew University MAPSULES [MPs]- magneto-plasmonic nanocapsules of Jerusalem, 9112102 Jerusalem, Israel MDA-MB-231- epithelial breast cancer cell line Complete contact information is available at: MIC- minimum inhibitory concentration https://pubs.acs.org/10.1021/acsami.3c07188 NIR- near-infrared NPs/NCs- nanoparticles/nanocapsules Author Contributions NTA- nanoparticle tracking analysis A.F., B.S., and O.B. designed the study and wrote the PDDA- poly(diallyldimethylammonium chloride) manuscript. A.F. and A.L. performed majority of the PDI- polydispersity index experiments, characterization, and validation with help from PLGA- poly(lactic-co-glycolic acid) R.B., E.S., N.F., and Z.Y. The manuscript was revised through ROS- reactive oxygen species 50340 https://doi.org/10.1021/acsami.3c07188 ACS Appl. Mater. Interfaces 2023, 15, 50330−50343 ACS Applied Materials & Interfaces www.acsami.org Research Article S.C.- subcutaneous of Recombinant IFN γ by Targeting the Cell Surface. Biomater. Sci. SEM- scanning electron microscopy 2014, 2, 1080−1089. SiO2- silicon oxide (17) Perlman, O.; Weitz, I. S.; Sivan, S. S.; Abu-khalla, H.; TEM- transmission electron microscopy Benguigui, M.; Shaked, Y.; Azhari, H. Copper Oxide Loaded PLGA Ti- titanium Nanospheres : Towards a Multifunctional Nanoscale Platform for Ultrasound-Based Imaging and Therapy. Nanotechnology 2018, 29, ■ REFERENCES (1) Baetke, S. C.; Lammers, T.; Kiessling, F. Applications of No. 185102, DOI: 10.1088/1361-6528/aab00c. 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