Fe3 O4 @Au@Cu2-x S Heterostructures Designed for Tri-Modal Therapy: Photo- Magnetic Hyperthermia and 64 Cu Radio-Insertion.

Here, the synthesis and proof of exploitation of three-material inorganic heterostructures made of iron oxide-gold-copper sulfide (Fe3 O4 @Au@Cu2-x S) are reported. Starting with Fe3 O4 -Au dumbbell heterostructure as seeds, a third Cu2-x S domain is selectively grown on the Au domain. The as-synthesized trimers are transferred to water by a two-step ligand exchange procedure exploiting thiol-polyethylene glycol to coordinate Au and Cu2-x S surfaces and polycatechol-polyethylene glycol to bind the Fe3 O4 surface. The saline stable trimers possess multi-functional properties: the Fe3 O4 domain, of appropriate size and crystallinity, guarantees optimal heating losses in magnetic hyperthermia (MHT) under magnetic field conditions of clinical use. These trimers have indeed record values of specific adsorption rate among the inorganic-heterostructures so far reported. The presence of Au and Cu2-x S domains ensures a large adsorption which falls in the first near-infrared (NIR) biological window and is here exploited, under laser excitation at 808 nm, to produce photo-thermal heat alone or in combination with MHT obtained from the Fe3 O4 domain. Finally, an intercalation protocol with radioactive 64 Cu ions is developed on the Cu2-x S domain, reaching high radiochemical yield and specific activity making the Fe3 O4 @Au@Cu2-x S trimers suitable as carriers for 64 Cu in internal radiotherapy (iRT) and traceable by positron emission tomography (PET).

Scaling Up Magnetic Nanobead Synthesis with Improved Stability for Biomedical Applications.

The growing interest in multifunctional nano-objects based on polymers and magnetic nanoparticles for biomedical applications motivated us to develop a scale-up protocol to increase the yield of polymeric magnetic nanobeads while aiming at keeping the structural features at optimal conditions. The protocol was applied to two different types of magnetic ferrite nanoparticles: the Mn-ferrite selected for their properties as contrast agents in magnetic resonance imaging and iron oxide nanostar shaped nanoparticles chosen for their heat performance in magnetic hyperthermia. At the same time, some experiments on surface functionalization of nanobeads with amino modified polyethyelene glycol (PEG) molecules have provided further insight into the formation mechanism of magnetic nanobeads and the need to cross-link the polymer shell to improve the stability of the beads, making them more suitable for further manipulation and use. The present work summarizes the most important parameters required to be controlled for the upscaling of nanobead synthesis in a bench protocol and proposes an alternative cross-linking strategy based on prefunctionalization of the polymer prior to the nanobead formation as a key parameter to improve the nanobead structural stability in solutions at different pHs and during surface functionalization.

Forced- and Self-Rotation of Magnetic Nanorods Assembly at the Cell Membrane: A Biomagnetic Torsion Pendulum.

In order to provide insight into how anisotropic nano-objects interact with living cell membranes, and possibly self-assemble, magnetic nanorods with an average size of around 100 nm × 1 µm are designed by assembling iron oxide nanocubes within a polymeric matrix under a magnetic field. The nano-bio interface at the cell membrane under the influence of a rotating magnetic field is then explored. A complex structuration of the nanorods intertwined with the membranes is observed. Unexpectedly, after a magnetic rotating stimulation, the resulting macrorods are able to rotate freely for multiple rotations, revealing the creation of a biomagnetic torsion pendulum.

Subnanometer local temperature probing and remotely controlled drug release based on azo-functionalized iron oxide nanoparticles.

Local heating can be produced by iron oxide nanoparticles (IONPs) when exposed to an alternating magnetic field (AMF). To measure the temperature profile at the nanoparticle surface with a subnanometer resolution, here we present a molecular temperature probe based on the thermal decomposition of a thermo-sensitive molecule, namely, azobis[N-(2-carboxyethyl)-2-methylpropionamidine]. Fluoresceineamine (FA) was bound to the azo molecule at the IONP surface functionalized with poly(ethylene glycol) (PEG) spacers of different molecular weights. Significant local heating, with a temperature increase up to 45 °C, was found at distances below 0.5 nm from the surface of the nanoparticle, which decays exponentially with increasing distance. Furthermore, the temperature increase was found to scale linearly with the applied field at all distances. We implemented these findings in an AMF-triggered drug release system in which doxorubicin was covalently linked at different distances from the IONP surface bearing the same thermo-labile azo molecule. We demonstrated the AMF triggered distance-dependent release of the drug in a cytotoxicity assay on KB cancer cells.

CuFeS2 Nanoparticles Functionalized with a Thermoresponsive Polymer for Photothermia and Externally Controlled Drug Delivery.

CuFeS2 chalcopyrite nanoparticles (NPs) can generate heat under exposure to near-infrared laser irradiation. Here, we develop a protocol to decorate the surface of CuFeS2 NPs (13 nm) with a thermoresponsive (TR) polymer based on poly(ethylene glycol methacrylate) to combine heat-mediated drug delivery and photothermal heat damage. The resulting TR-CuFeS2 NPs feature a small hydrodynamic size (∼75 nm), along with high colloidal stability and a TR transition temperature of 41 °C in physiological conditions. Remarkably, TR-CuFeS2 NPs, when exposed to a laser beam (in the range of 0.5 and 1.5 W/cm2) at NP concentrations as low as 40-50 µg Cu/mL, exhibit a high heating performance with a rise in the solution temperature to hyperthermia therapeutic values (42-45 °C). Furthermore, TR-CuFeS2 NPs worked as nanocarriers, being able to load an appreciable amount of doxorubicin (90 µg DOXO/mg Cu), a chemotherapeutic agent whose release could then be triggered by exposing the NPs to a laser beam (through which a hyperthermia temperature above 42 °C could be reached). In an in vitro study performed on U87 human glioblastoma cells, bare TR-CuFeS2 NPs were proven to be nontoxic at a Cu concentration up to 40 µg/mL, while at the same low dose, the drug-loaded TR-CuFeS2-DOXO NPs displayed synergistic cytotoxic effects due to the combination of direct heat damage and DOXO chemotherapy, under photo-irradiation by a 808 nm laser (1.2 W/cm2). Finally, under a 808 nm laser, the TR-CuFeS2 NPs generated a tunable amount of reactive oxygen species depending on the applied power density and NP concentration.

Clickable Polymer Ligand-Functionalized Iron Oxide Nanocubes: A Promising Nanoplatform for 'Local Hot Spots' Magnetically Triggered Drug Release.

Exploiting the local heat on the surface of magnetic nanoparticles (MNPs) upon exposure to an alternating magnetic field (AMF) to cleave thermal labile bonds represents an interesting approach in the context of remotely triggered drug delivery. Here, taking advantages of a simple and scalable two-step ligand exchange reaction, we have prepared iron oxide nanocubes (IONCs) functionalized with a novel multifunctional polymer ligand having multiple catechol moieties, furfuryl pendants, and polyethylene glycol (PEG) side chains. Catechol groups ensure a strong binding of the polymer ligands to the IONCs surface, while the PEG chains provide good colloidal stability to the polymer-coated IONCs. More importantly, furfuryl pendants on the polymer enable to click the molecules of interest (either maleimide-fluorescein or maleimide-doxorubicin) via a thermal labile Diels-Alder adduct. The resulting IONCs functionalized with a fluorescein/doxorubicin-conjugated polymer ligand exhibit good colloidal stability in buffer saline and serum solution along with outstanding heating performance in aqueous solution or even in viscous media (81% glycerol/water) when exposed to the AMF of clinical use. The release of conjugated bioactive molecules such as fluorescein and doxorubicin could be boosted by applying AMF conditions of clinical use (16 kAm-1 and 110 kHz). It is remarkable that the magnetic hyperthermia-mediated release of the dye/drug falls in the concentration range 1.0-5.0 µM at an IONCs dose as low as 0.5 gFe/L and at no macroscopical temperature change. This local release effect makes this magnetic nanoplatform a potential tool for drug delivery with remote magnetic hyperthermia actuation and with a dose-independent action of MNPs.

Influence of Magnetic Scaffold Loading Patterns on Their Hyperthermic Potential Against Bone Tumors.

Magnetic scaffolds have been investigated as promising tools for the interstitial hyperthermia treatment of bone cancers, to control local recurrence by enhancing radio- and chemotherapy effectiveness. The potential of magnetic scaffolds motivates the development of production strategies enabling tunability of the resulting magnetic properties. Within this framework, deposition and drop-casting of magnetic nanoparticles on suitable scaffolds offer advantages such as ease of production and high loading, although these approaches are often associated with a non-uniform final spatial distribution of nanoparticles in the biomaterial. The implications and the influences of nanoparticle distribution on the final therapeutic application have not yet been investigated thoroughly. In this work, poly-caprolactone scaffolds are magnetized by loading them with synthetic magnetic nanoparticles through a drop-casting deposition and tuned to obtain different distributions of magnetic nanoparticles in the biomaterial. The physicochemical properties of the magnetic scaffolds are analyzed. The microstructure and the morphological alterations due to the reworked drop-casting process are evaluated and correlated to static magnetic measurements. THz tomography is used as an innovative investigation technique to derive the spatial distribution of nanoparticles. Finally, multiphysics simulations are used to investigate the influence on the loading patterns on the interstitial bone tumor hyperthermia treatment.

Highly Emitting Perovskite Nanocrystals with 2-Year Stability in Water through an Automated Polymer Encapsulation for Bioimaging.

Lead-based halide perovskite nanocrystals are highly luminescent materials, but their sensitivity to humid environments and their biotoxicity are still important challenges to solve. Here, we develop a stepwise approach to encapsulate representative CsPbBr3 nanocrystals into water-soluble polymer capsules. We show that our protocol can be extended to nanocrystals coated with different ligands, enabling an outstanding high photoluminescence quantum yield of ∼60% that is preserved over two years in capsules dispersed in water. We demonstrate that this on-bench strategy can be implemented on an automated platform with slight modifications, granting access to a faster and more reproducible fabrication process. Also, we reveal that the capsules can be exploited as photoluminescent probes for cell imaging at a dose as low as 0.3 µgPb/mL that is well below the toxicity threshold for Pb and Cs ions. Our approach contributes to expanding significantly the fields of applications of these luminescent materials including biology and biomedicine.

Co-loading of doxorubicin and iron oxide nanocubes in polycaprolactone fibers for combining Magneto-Thermal and chemotherapeutic effects on cancer cells.

Among the strategies to fight cancer, multi-therapeutic approaches are considered as a wise choice to put in place multiple weapons to suppress tumors. In this work, to combine chemotherapeutic effects to magnetic hyperthermia when using biocompatible scaffolds, we have established an electrospinning method to produce nanofibers of polycaprolactone loaded with magnetic nanoparticles as heat mediators to be selectively activated under alternating magnetic field and doxorubicin as a chemotherapeutic drug. Production of the fibers was investigated with iron oxide nanoparticles of peculiar cubic shape (at 15 and 23 nm in cube edges) as they provide benchmark heat performance under clinical magnetic hyperthermia conditions. With 23 nm nanocubes when included into the fibers, an arrangement in chains was obtained. This linear configuration of magnetic nanoparticles resemble that of the magnetosomes, produced by magnetotactic bacteria, and our magnetic fibers exhibited remarkable heating effects as the magnetosomes. Magnetic fiber scaffolds showed excellent biocompatibility on fibroblast cells when missing the chemotherapeutic agent and when not exposed to magnetic hyperthermia as shown by viability assays. On the contrary, the fibers containing both magnetic nanocubes and doxorubicin showed significant cytotoxic effects on cervical cancer cells following the exposure to magnetic hyperthermia. Notably, these tests were conducted at magnetic hyperthermia field conditions of clinical use. As here shown, on the doxorubicin sensitive cervical cancer cells, the combination of heat damage by magnetic hyperthermia with enhanced diffusion of doxorubicin at therapeutic temperature are responsible for a more effective oncotherapy.

Acidic pH-responsive nanogels as smart cargo systems for the simultaneous loading and release of short oligonucleotides and magnetic nanoparticles.

Smart materials able to sense environmental stimuli can be exploited as intelligent carrier systems. Acidic pH-responsive polymers, for instance, exhibit a variation in the ionization state upon lowering the pH, which leads to their swelling. The different permeability of these polymers as a function of the pH could be exploited for the incorporation and subsequent release of previously trapped payload molecules/nanoparticles. We provide here a proof of concept of a novel use of pH-responsive polymer nanostructures based on 2-vinylpyridine and divinylbenzene, having an overall size below 200 nm, as cargo system for magnetic nanoparticles, for oligonucleotide sequences, as well as for their simultaneous loading and controlled release mediated by the pH.

Polymer Coating and Lipid Phases Regulate Semiconductor Nanorods' Interaction with Neuronal Membranes: A Modeling Approach.

The interplay between nanoparticles (NPs) and cell membranes is extremely important with regard to using NPs in biology applications. With the aim of unraveling the dominating factors on the molecular scale, we have studied the interaction between polymer-coated semiconductor nanorods (NRs) made of cadmium selenium/cadmium sulfur and model lipid membranes. The zeta potential (ζ) of the NRs was tuned from having a negative value (-24 mV) to having a positive one (+11 mV) by changing the amine content in the polymer coating. Supported lipid bilayers (SLBs) and lipid monolayers (LMs) were used as model membranes. Lipid mixtures containing anionic or cationic lipids were employed in order to change the membrane ζ from -77 to +49 mV; lipids with saturated hydrophobic chains were used to create phase-separated gel domains. NR adsorption to the SLBs was monitored by quartz crystal microbalance with dissipation monitoring; interactions with LMs with the same lipid composition were measured by surface pressure-area isotherms. The results showed that the NRs only interact with the model membrane if the mutual Δζ is higher than 70 mV; at the air-water interface, positively charged NRs remove lipids from the anionic lipid mixtures, and the negative ones penetrate the space between the polar heads in the cationic mixtures. However, the presence of gel domains in the membrane inhibits this interaction. The results of the Derjaguin-Landau-Verwey-Overbeek model frame indicate that the interaction occurs not only due to electrostatic and van der Waals forces, but also due to steric and/or hydration forces.

Thermoresponsive Iron Oxide Nanocubes for an Effective Clinical Translation of Magnetic Hyperthermia and Heat-Mediated Chemotherapy.

The use of magnetic nanoparticles in oncothermia has been investigated for decades, but an effective combination of magnetic nanoparticles and localized chemotherapy under clinical magnetic hyperthermia (MH) conditions calls for novel platforms. In this study, we have engineered magnetic thermoresponsive iron oxide nanocubes (TR-cubes) to merge MH treatment with heat-mediated drug delivery, having in mind the clinical translation of the nanoplatform. We have chosen iron oxide based nanoparticles with a cubic shape because of their outstanding heat performance under MH clinical conditions, which makes them benchmark agents for MH. Accomplishing a surface-initiated polymerization of strongly interactive nanoparticles such as our iron oxide nanocubes, however, remains the main challenge to overcome. Here, we demonstrate that it is possible to accelerate the growth of a polymer shell on each nanocube by simple irradiation of a copper-mediated polymerization with a ultraviolet light (UV) light, which both speeds up the polymerization and prevents nanocube aggregation. Moreover, we demonstrate herein that these TR-cubes can carry chemotherapeutic doxorubicin (DOXO-loaded-TR-cubes) without compromising their thermoresponsiveness both in vitro and in vivo. In vivo efficacy studies showed complete tumor suppression and the highest survival rate for animals that had been treated with DOXO-loaded-TR-cubes, only when they were exposed to MH. The biodistribution of intravenously injected TR-cubes showed signs of renal clearance within 1 week and complete clearance after 5 months. This biomedical platform works under clinical MH conditions and at a low iron dosage, which will enable the translation of dual MH/heat-mediated chemotherapy, thus overcoming the clinical limitation of MH: i.e., being able to monitor tumor progression post-MH-treatment by magnetic resonance imaging (MRI).

Oil Core-PEG Shell Nanocarriers for In Vivo MRI Imaging.

Oil-in-water emulsions represent a promising carrier for in vivo imaging because of the possibility to convey poorly water-soluble species. To promote accumulation at the tumor site and prolong circulation time, reduction of carrier size and surface PEGylation plays a fundamental role. In this work a novel, simple method to design an oil-core/PEG-shell nanocarrier is reported. A PEG-shell is grown around a monodisperse oil-in-water nanoemulsion with a one-pot method, using the radical polymerization of poly(ethylene glycol)diacrylate. PEG polymerization is triggered by UV, obtaining a PEG-shell with tunable thickness. This core-shell nanosystem combines the eluding feature of the PEG with the ability to confine high payloads of lipophilic species. Indeed, the core is successfully loaded with a lipophilic contrast agent, namely super paramagnetic iron oxide nanocubes. Interestingly, it is demonstrated an in vitro and an in vivo MRI response of the nanocapsules. Additionally, when the nanosystem loaded with nanocubes is mixed with a fluorescent contrast agent, indo-cyanine green, a relevant in vitro photoacoustic effect is observed. Moreover, viability and cellular uptake studies show no significant cell cytotoxicity. These results, together with the choice of low cost materials and the scale up production, make this nanocarrier a potential platform for in vivo imaging.

Star poly(ε-caprolactone)-based electrospun fibers as biocompatible scaffold for doxorubicin with prolonged drug release activity.

In this work, a novel drug delivery system consisting of poly(ε-caprolactone) (PCL) electrospun fibers containing an ad-hoc-synthesized star polymer made up of a poly(amido-amine) (PAMAM) core and PCL branches (PAMAM-PCL) was developed. The latter system which was synthesized via the ring opening polymerization of ε-caprolactone, starting from a hydroxyl-terminated PAMAM dendrimer and characterized by means of 1H NMR, IR and DSC, was found to be compatible with both the polymer matrix and a hydrophilic chemotherapeutic drug, doxorubicin (DOXO), the model drug used in this work. The preparation of the dendritic PCL star product with an average arm length of 2000g/mol was characterized using IR and 1H NMR measurements. The prepared star polymer possessed a higher crystallinity and a lower melting temperature than that of the used linear PCL. Electrospun fibers were prepared starting from solutions containing the neat PCL as well as the PCL/PAMAM-PCL mixture. Electrospinning conditions were optimized in order to obtain defect free fibers, which was proven by the structural FE-SEM study. PAMAM moieties enhanced the hydrophilicity of the fibers, as proved by comparing the water absorption for the PCL/PAMAM-PCL fibers to that neat PCL fibers. The drug-loaded system PCL/PAMAM-PCL was prepared by directly introducing DOXO into the electrospinning solutions. The DOXO-loaded PCL/PAMAM-PCL showed a prolonged release of the drug with respect to the DOXO-loaded PCL fibers and elicited effective controlled toxicity over A431 epidermoid carcinoma, HeLa cervical cancer cells and drug resistant MCF-7 breast cancer cells. On the contrary, the drug-free PCL/PAMAM-PCL scaffold demonstrated no toxic effects on human dermal fibroblasts, suggesting the biocompatibility of the proposed system which can be used in cellular scaffold applications.

Multilayered Magnetic Nanobeads for the Delivery of Peptides Molecules Triggered by Intracellular Proteases.

In this work, the versatility of layer-by-layer technology was combined with the magnetic response of iron oxide nanobeads to prepare magnetic mesostructures with a degradable multilayer shell into which a dye quenched ovalbumin conjugate (DQ-OVA) was loaded. The system was specifically designed to prove the protease sensitivity of the hybrid mesoscale system and the easy detection of the ovalbumin released. The uptake of the nanostructures in the breast cancer cells was followed by the effective release of DQ-OVA upon activation via the intracellular proteases degradation of the polymer shells. Monitoring the fluorescence rising due to DQ-OVA digestion and the cellular dye distribution, together with the electron microscopy studying, enabled us to track the shell degradation and the endosomal uptake pathway that resulted in the release of the digested fragments of DQ ovalbumin in the cytosol.

Functionalization of strongly interacting magnetic nanocubes with (thermo)responsive coating and their application in hyperthermia and heat-triggered drug delivery.

Herein, we prepare nanohybrids by incorporating iron oxide nanocubes (cubic-IONPs) within a thermoresponsive polymer shell that can act as drug carriers for doxorubicin(doxo). The cubic-shaped nanoparticles employed are at the interface between superparamagnetic and ferromagnetic behavior and have an exceptionally high specific absorption rate (SAR), but their functionalization is extremely challenging compared to bare superparamagnetic iron oxide nanoparticles as they strongly interact with each other. By conducting the polymer grafting reaction using reversible addition-fragmentation chain transfer (RAFT) polymerization in a viscous solvent medium, we have here developed a facile approach to decorate the nanocubes with stimuli-responsive polymers. When the thermoresponsive shell is composed of poly(N-isopropylacrylamide-co-polyethylene glycolmethyl ether acrylate), nanohybrids have a phase transition temperature, the lower critical solution temperature (LCST), above 37 °C in physiological conditions. Doxo loaded nanohybrids exhibited a negligible drug release below 37 °C but showed a consistent release of their cargo on demand by exploiting the capability of the nanocubes to generate heat under an alternating magnetic field (AMF). Moreover, the drug free nanocarrier does not exhibit cytotoxicity even when administered at high concentration of nanocubes (1g/L of iron) and internalized at high extent (260 pg of iron per cell). We have also implemented the synthesis protocol to decorate the surface of nanocubes with poly(vinylpyridine) polymer and thus prepare pH-responsive shell coated nanocubes.

The One Year Fate of Iron Oxide Coated Gold Nanoparticles in Mice.

Safe implementation of nanotechnology and nanomedicine requires an in-depth understanding of the life cycle of nanoparticles in the body. Here, we investigate the long-term fate of gold/iron oxide heterostructures after intravenous injection in mice. We show these heterostructures degrade in vivo and that the magnetic and optical properties change during the degradation process. These particles eventually eliminate from the body. The comparison of two different coating shells for heterostructures, amphiphilic polymer or polyethylene glycol, reveals the long lasting impact of initial surface properties on the nanocrystal degradability and on the kinetics of elimination of magnetic iron and gold from liver and spleen. Modulation of nanoparticles reactivity to the biological environment by the choice of materials and surface functionalization may provide new directions in the design of multifunctional nanomedicines with predictable fate.

Nanoparticles for imaging, sensing, and therapeutic intervention.

Nanoparticles have the potential to contribute to new modalities in molecular imaging and sensing as well as in therapeutic interventions. In this Nano Focus article, we identify some of the current challenges and knowledge gaps that need to be confronted to accelerate the developments of various applications. Using specific examples, we journey from the characterization of these complex hybrid nanomaterials; continue with surface design and (bio)physicochemical properties, their fate in biological media and cells, and their potential for cancer treatment; and finally reflect on the role of animal models to predict their behavior in humans.

Heat-generating iron oxide nanocubes: subtle "destructurators" of the tumoral microenvironment.

Several studies propose nanoparticles for tumor treatment, yet little is known about the fate of nanoparticles and intimate interactions with the heterogeneous and ever-evolving tumor environment. The latter, rich in extracellular matrix, is responsible for poor penetration of therapeutics and represents a paramount issue in cancer therapy. Hence new strategies start aiming to modulate the neoplastic stroma. From this perspective, we assessed the efficacy of 19 nm PEG-coated iron oxide nanocubes with optimized magnetic properties to mediate mild tumor magnetic hyperthermia treatment. After injection of a low dose of nanocubes (700 µg of iron) into epidermoid carcinoma xenografts in mice, we monitored the effect of heating nanocubes on tumor environment. In comparison with the long-term fate after intravenous administration, we investigated spatiotemporal patterns of nanocube distribution, evaluated the evolution of cubes magnetic properties, and examined nanoparticle clearance and degradation processes. While inside tumors nanocubes retained their magnetic properties and heating capacity throughout the treatment due to a mainly interstitial extracellular location, the particles became inefficient heaters after cell internalization and transfer to spleen and liver. Our multiscale analysis reveals that collagen-rich tumor extracellular matrix confines the majority of nanocubes. However, nanocube-mediated hyperthermia has the potential to "destructure" this matrix and improve nanoparticle and drug penetration into neoplastic tissue. This study provides insight into dynamic interactions between nanoparticles and tumor components under physical stimulation and suggests that nanoparticle-mediated hyperthermia could be used to locally modify tumor stroma and thus improve drug penetration.

Immunocytochemistry, electron tomography, and energy dispersive X-ray spectroscopy (EDXS) on cryosections of human cancer cells doped with stimuli responsive polymeric nanogels loaded with iron oxide nanoparticles.

The cryosectioning technique is an alternative method for preparing biological material for Transmission Electron Microscopy (TEM). We have applied this technique to study the mechanism of cell internalization of stimuli-responsive polymeric nanogels exploited as cargo nanovectors. With respect to conventional TEM processing, cryosectioning technique better preserves the morphology of solvent-sensitive nanogels and enhances the visibility of membrane-bounded organelles inside the cell cytoplasm. In this chapter we describe the protocols we have established to perform Electron Microscopy (EM)-immunocytochemistry, Electron Tomography (ET), and Energy Dispersive X-ray Spectroscopy (EDXS) chemical analysis in Scanning TEM (STEM) on cryosections of HeLa cells treated with pH-responsive nanogels hosting short interference RNA (siRNAs) and iron oxide nanoparticles (IONPs).