Challenges for optical nanothermometry in biological environments.

Temperature monitoring is useful in medical diagnosis, and essential during hyperthermia treatments to avoid undesired cytotoxic effects. Aiming to control heating doses, different temperature monitoring strategies have been developed, largely based on luminescent materials, a.k.a. nanothermometers. However, for such nanothermometers to work, both excitation and emission light beams must travel through tissue, making its optical properties a relevant aspect to be considered during the measurements. In complex tissues, heterogeneity, and real-time alterations as a result of therapeutic treatment may have an effect on light-tissue interaction, hindering accuracy in the thermal reading. In this Tutorial Review we discuss various methods in which nanothermometers can be used for temperature sensing within heterogeneous environments. We discuss recent developments in optical (nano)thermometry, focusing on the incorporation of luminescent nanoparticles into complex in vitro and in vivo models. Methods formulated to avoid thermal misreading are also discussed, considering their respective advantages and drawbacks.

Size-Dependent Transport and Cytotoxicity of Mitomycin-Gold Nanoparticle Conjugates in 2D and 3D Mammalian Cell Models.

This work aims at learning how the size of gold nanocarriers influences the transport of DNA-alkylating antitumoral drugs. For this purpose, we devised conjugates of mercaptoethylmitomycin C (MEMC), a DNA alkylating agent, with gold nanoparticles of different sizes (2, 5, and 14 nm), and studied how size affects drug cytotoxicity, tumor penetrability, cellular uptake, and intracellular localization using two-dimensional (2D) and three-dimensional (3D) cell models. We show that only small, 2 nm, nanoparticles can transport MEMC efficiently to the cell nucleus, whereas MEMC cell uptake is much lower when delivered by these small nanoparticles than with the larger ones. 3D cellular models showed that smaller nanoparticles can transport MEMC toward deeper areas of tumor spheroids as compared to larger nanoparticles. We discuss the insights of this work toward the efficient delivery of DNA-targeting drugs.

Cellular Uptake of Gold Nanoparticles Triggered by Host-Guest Interactions.

We describe an approach to regulate the cellular uptake of small gold nanoparticles using supramolecular chemistry. The strategy relies on the functionalization of AuNPs with negatively charged pyranines, which largely hamper their penetration in cells. Cellular uptake can be activated in situ through the addition of cationic covalent cages that specifically recognize the fluorescent pyranine dyes and counterbalance the negative charges. The high selectivity and reversibility of the host-guest recognition activates cellular uptake, even in protein-rich biological media, as well as its regulation by rational addition of either cage or pyranine.

Current Challenges toward In Vitro Cellular Validation of Inorganic Nanoparticles.

An impressive development has been achieved toward the production of well-defined "smart" inorganic nanoparticles, in which the physicochemical properties can be controlled and predicted to a high degree of accuracy. Nanoparticle design is indeed highly advanced, multimodal and multitargeting being the norm, yet we do not fully understand the obstacles that nanoparticles face when used in vivo. Increased cooperation between chemists and biochemists, immunologists and physicists, has allowed us to think outside the box, and we are slowly starting to understand the interactions that nanoparticles undergo under more realistic situations. Importantly, such an understanding involves awareness about the limitations when assessing the influence of such inorganic nanoparticles on biological entities and vice versa, as well as the development of new validation strategies.

Plasmonic Surfaces for Cell Growth and Retrieval Triggered by Near-Infrared Light.

Methods for efficient detachment of cells avoiding damage are required in tissue engineering and regenerative medicine. We introduce a bottom-up approach to build plasmonic substrates using micellar block copolymer nanolithography to generate a 2D array of Au seeds, followed by chemical growth leading to anisotropic nanoparticles. The resulting plasmonic substrates show a broad plasmon band covering a wide part of the visible and near-infrared (NIR) spectral ranges. Both human and murine cells were successfully grown on the substrates. A simple functionalization step of the plasmonic substrates with the cyclic arginylglycylaspartic acid (c-RGD) peptide allowed us to tune the morphology of integrin-rich human umbilical vein endothelial cells (HUVEC). Subsequent irradiation with a NIR laser led to highly efficient detachment of the cells with cell viability confirmed using the MTT assay. We thus propose the use of such plasmonic substrates for cell growth and controlled detachment using remote near-IR irradiation, as a general method for cell culture in biomedical applications.

Glycans as biofunctional ligands for gold nanorods: stability and targeting in protein-rich media.

Poly(ethylene glycol) (PEG) has become the gold standard for stabilization of plasmonic nanoparticles (NPs) in biofluids, because it prevents aggregation while minimizing unspecific interactions with proteins. Application of Au NPs in biological environments requires the use of ligands that can target selected receptors, even in the presence of protein-rich media. We demonstrate here the stabilizing effect of low-molecular-weight glycans on both spherical and rod-like plasmonic NPs under physiological conditions, as bench-marked against the well-established PEG ligands. Glycan-coated NPs are resistant to adsorption of proteins from serum-containing media and avoid phagocytosis by macrophage-like cells, but retain selectivity toward carbohydrate-binding proteins in protein-rich biological media. These results open the way toward the design of efficient therapeutic/diagnostic glycan-decorated plasmonic nanotools for specific biological applications.

An iron oxide nanocarrier for dsRNA to target lymph nodes and strongly activate cells of the immune system.

The success of nanoparticle-based therapies will depend in part on accurate delivery to target receptors and organs. There is, therefore, considerable potential in nanoparticles which achieve delivery of the right drug(s) using the right route of administration to the right location at the right time, monitoring the process by non-invasive molecular imaging. A challenge is harnessing immunotherapy via activation of Toll-like receptors (TLRs) for the development of vaccines against major infectious diseases and cancer. In immunotherapy, delivery of the vaccine components to lymph nodes (LNs) is essential for effective stimulation of the immune response. Although some promising advances have been made, delivering therapeutics to LNs remains challenging. It is here shown that iron-oxide nanoparticles can be engineered to combine in a single and small (<50 nm) nanocarrier complementary multimodal imaging features with the immunostimulatory activity of polyinosinic-polycytidylic acid (poly (I:C)). Whilst the fluorescence properties of the nanocarrier show effective delivery to endosomes and TLR3 in antigen presenting cells, MRI/SPECT imaging reveals effective delivery to LNs. Importantly, in vitro and in vivo studies show that, using this nanocarrier, the immunostimulatory activity of poly (I:C) is greatly enhanced. These nanocarriers have considerable potential for cancer diagnosis and the development of new targeted and programmable immunotherapies.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on bilayer properties, biodistribution, and immune responses.

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses.

3D Bioprinted Tumor-Stroma Models of Triple-Negative Breast Cancer Stem Cells for Preclinical Targeted Therapy Evaluation.

Breast cancer stem cells (CSCs) play a pivotal role in therapy resistance and tumor relapse, emphasizing the need for reliable in vitro models that recapitulate the complexity of the CSC tumor microenvironment to accelerate drug discovery. We present a bioprinted breast CSC tumor-stroma model incorporating triple-negative breast CSCs (TNB-CSCs) and stromal cells (human breast fibroblasts), within a breast-derived decellularized extracellular matrix bioink. Comparison of molecular signatures in this model with different clinical subtypes of bioprinted tumor-stroma models unveils a unique molecular profile for artificial CSC tumor models. We additionally demonstrate that the model can recapitulate the invasive potential of TNB-CSC. Surface-enhanced Raman scattering imaging allowed us to monitor the invasive potential of tumor cells in deep z-axis planes, thereby overcoming the depth-imaging limitations of confocal fluorescence microscopy. As a proof-of-concept application, we conducted high-throughput drug testing analysis to assess the efficacy of CSC-targeted therapy in combination with conventional chemotherapeutic compounds. The results highlight the usefulness of tumor-stroma models as a promising drug-screening platform, providing insights into therapeutic efficacy against CSC populations resistant to conventional therapies.

A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

3D bioprinted breast tumor-stroma models for pre-clinical drug testing.

The use of three-dimensional (3D) bioprinting has been proposed for the reproducible production of 3D disease models that can be used for high-throughput drug testing and personalized medicine. However, most such models insufficiently reproduce the features and environment of real tumors. We report the development of bioprinted in vitro 3D tumor models for breast cancer, which physically and biochemically mimic important aspects of the native tumor microenvironment, designed to study therapeutic efficacy. By combining a mix of breast decellularized extracellular matrix and methacrylated hyaluronic acid with tumor-derived cells and non-cancerous stromal cells of biological relevance to breast cancer, we show that biological signaling pathways involved in tumor progression can be replicated in a carefully designed tumor-stroma environment. Finally, we demonstrate proof-of-concept application of these models as a reproducible platform for investigating therapeutic responses to commonly used chemotherapeutic agents.

Remodeling arteries: studying the mechanical properties of 3D-bioprinted hybrid photoresponsive materials.

3D-printed cell models are currently in the spotlight of medical research. Whilst significant advances have been made, there are still aspects that require attention to achieve more realistic models which faithfully represent the in vivo environment. In this work we describe the production of an artery model with cyclic expansive properties, capable of mimicking the different physical forces and stress factors that cells experience in physiological conditions. The artery wall components are reproduced using 3D printing of thermoresponsive polymers with inorganic nanoparticles (NPs) representing the outer tunica adventitia, smooth muscle cells embedded in extracellular matrix representing the tunica media, and finally a monolayer of endothelial cells as the tunica intima. Cyclic expansion can be induced thanks to the inclusion of photo-responsive plasmonic NPs embedded within the thermoresponsive ink composition, resulting in changes in the thermoresponsive polymer hydration state and hence volume, in a stimulated on-off manner. By changing the thermoresponsive polymer composition, the transition temperature and pulsatility can be efficiently tuned. We show the direct effect of cyclic expansion and contraction on the overlying cell layers by analyzing transcriptional changes in mechanoresponsive mesenchymal genes associated with such microenvironmental physical cues. The technique described herein involving stimuli-responsive 3D printed tissue constructs, also described as four- dimensional (4D) printing, offers a novel approach for the production of dynamic biomodels.

Hybrid core-shell nanoparticles for cell-specific magnetic separation and photothermal heating.

Hyperthermia, as the process of heating a malignant site above 42 °C to trigger cell death, has emerged as an effective and selective cancer therapy strategy. Various modalities of hyperthermia have been proposed, among which magnetic and photothermal hyperthermia are known to benefit from the use of nanomaterials. In this context, we introduce herein a hybrid colloidal nanostructure comprising plasmonic gold nanorods (AuNRs) covered by a silica shell, onto which iron oxide nanoparticles (IONPs) are subsequently grown. The resulting hybrid nanostructures are responsive to both external magnetic fields and near-infrared irradiation. As a result, they can be applied for the targeted magnetic separation of selected cell populations - upon targeting by antibody functionalization - as well as for photothermal heating. Through this combined functionality, the therapeutic effect of photothermal heating can be enhanced. We demonstrate both the fabrication of the hybrid system and its application for targeted photothermal hyperthermia of human glioblastoma cells.

Combination of Live Cell Surface-Enhanced Raman Scattering Imaging with Chemometrics to Study Intracellular Nanoparticle Dynamics.

Surface-enhanced Raman scattering (SERS)-encoded nanoparticles are used for bioimaging, on account of their well-defined Raman spectra and biocompatibility, which allow long incubation times with high signal stability and no cytotoxicity. However, reliable analysis of SERS bioimaging requires quantification of the amount of encoded nanoparticles that have been taken up by cells and the effect of subsequent dilution due to cellular division (mitosis). Although methods such as elemental analysis and flow cytometry can be used to quantify nanoparticle uptake, these are both end-point measurements in which a cell population is screened rather than looking at individual cells. In contrast, SERS imaging can be applied at multiple timepoints to the same individual cells without damaging the biological sample. We present the application of both supervised and unsupervised multivariate analyses, to quantify the intracellular amount of SERS tags in individual MCF7 living cells, toward the characterization of cellular uptake in vitro. The obtained results from both methodologies were validated by standard elemental analysis techniques.

SERS and Fluorescence-Active Multimodal Tessellated Scaffolds for Three-Dimensional Bioimaging.

With the ever-increasing use of 3D cell models toward studying bio-nano interactions and offering alternatives to traditional 2D in vitro and in vivo experiments, methods to image biological tissue in real time and with high spatial resolution have become a must. A suitable technique therefore is surface-enhanced Raman scattering (SERS)-based microscopy, which additionally features reduced photocytotoxicity and improved light penetration. However, optimization of imaging and postprocessing parameters is still required. Herein we present a method to monitor cell proliferation over time in 3D, using multifunctional 3D-printed scaffolds composed of biologically inert poly(lactic-co-glycolic acid) (PLGA) as the base material, in which fluorescent labels and SERS-active gold nanoparticles (AuNPs) can be embedded. The combination of imaging techniques allows optimization of SERS imaging parameters for cell monitoring. The scaffolds provide anchoring points for cell adhesion, so that cell growth can be observed in a suspended 3D matrix, with multiple reference points for confocal fluorescence and SERS imaging. By prelabeling cells with SERS-encoded AuNPs and fluorophores, cell proliferation and migration can be simultaneously monitored through confocal Raman and fluorescence microscopy. These scaffolds provide a simple method to follow cell dynamics in 4D, with minimal disturbance to the tissue model.

Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3ß-[N-(N',N'-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses.

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3ß-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

Shielded Silver Nanorods for Bioapplications.

Silver is arguably the best plasmonic material in terms of optical performance. However, wide application of Ag and Ag-containing nanoparticles is usually hindered by two major drawbacks, namely, chemical degradation and cytotoxicity. We report herein a synthetic method for highly monodisperse polymer-coated Ag nanorods, which are thereby protected against external stimuli (oxidation, light, heat) and are noncytotoxic to various cell lines. The monodispersity of Ag nanorods endows them with narrow plasmon bands, which are tunable into the near-infrared biological transparency window, thus facilitating application in bioanalytical and therapeutic techniques. We demonstrate intracellular surface-enhanced Raman scattering (SERS) imaging using Ag nanorods encoded with five different Raman reporter molecules. Encoded Ag nanorods display long-term stability in terms of size, shape, optical response, and SERS signal. Our results help eliminate concerns of instability and cytotoxicity in the application of Ag-containing nanoparticles with enhanced optical response, toward the development of bioapplications.

Live-Cell Surface-Enhanced Raman Spectroscopy Imaging of Intracellular pH: From Two Dimensions to Three Dimensions.

Visualization of intracellular pH (i-pH) using surface-enhanced Raman spectroscopy (SERS) plays an important role toward understanding of cellular processes including their interactions with nanoparticles. However, conventional two-dimensional SERS imaging often fails to take into consideration changes occurring in the whole-cell volume. We therefore aimed at obtaining a comprehensive i-pH profile of living cells by means of three-dimensional (3D) SERS imaging, thereby visualizing dynamic i-pH distribution changes in a single cell. We devised here a biocompatible and highly stable SERS pH probe, comprising plasmonic gold nanostars functionalized with a pH-sensitive Raman reporter tag-4-mercaptobenzoic acid-and protected by a cationic biocompatible polymer, poly-l-arginine hydrochloride (PA). The positively charged PA coating plays a double role in enhancing cell uptake and providing chemical and colloidal stability in cellular environments. The SERS-active pH probe allowed visualization of local changes in i-pH, such as acidification during nanoparticle (NP) endocytosis. We provide evidence of i-pH changes during NP endocytosis via high-resolution 3D SERS imaging, thereby opening new avenues toward the application of SERS to intracellular studies.

Reversible Control of Protein Corona Formation on Gold Nanoparticles Using Host-Guest Interactions.

When nanoparticles (NPs) are exposed to biological media, proteins are adsorbed, forming a so-called protein corona (PC). This cloud of protein aggregates hampers the targeting and transport capabilities of the NPs, thereby compromising their biomedical applications. Therefore, there is a high interest in the development of technologies that allow control over PC formation, as this would provide a handle to manipulate NPs in biological fluids. We present a strategy that enables the reversible disruption of the PC using external stimuli, thereby allowing a precise regulation of NP cellular uptake. The approach, demonstrated for gold nanoparticles (AuNPs), is based on a biorthogonal, supramolecular host-guest interactions between an anionic dye bound to the AuNP surface and a positively charged macromolecular cage. This supramolecular complex effectively behaves as a zwitterionic NP ligand, which is able not only to prevent PC formation but also to disrupt a previously formed hard corona. With this supramolecular stimulus, the cellular internalization of AuNPs can be enhanced by up to 30-fold in some cases, and even NP cellular uptake in phagocytic cells can be regulated. Additionally, we demonstrate that the conditional cell uptake of purposely designed gold nanorods can be used to selectively enhance photothermal cell death.