Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging.

Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2 weeks for cells labeled by endocytosis.

The impact of species and cell type on the nanosafety profile of iron oxide nanoparticles in neural cells.

BACKGROUND: While nanotechnology is advancing rapidly, nanosafety tends to lag behind since general mechanistic insights into cell-nanoparticle (NP) interactions remain rare. To tackle this issue, standardization of nanosafety assessment is imperative. In this regard, we believe that the cell type selection should not be overlooked since the applicability of cell lines could be questioned given their altered phenotype. Hence, we evaluated the impact of the cell type on in vitro nanosafety evaluations in a human and murine neuroblastoma cell line, neural progenitor cell line and in neural stem cells. Acute toxicity was evaluated for gold, silver and iron oxide (IO)NPs, and the latter were additionally subjected to a multiparametric analysis to assess sublethal effects. RESULTS: The stem cells and murine neuroblastoma cell line respectively showed most and least acute cytotoxicity. Using high content imaging, we observed cell type- and species-specific responses to the IONPs on the level of reactive oxygen species production, calcium homeostasis, mitochondrial integrity and cell morphology, indicating that cellular homeostasis is impaired in distinct ways. CONCLUSIONS: Our data reveal cell type-specific toxicity profiles and demonstrate that a single cell line or toxicity end point will not provide sufficient information on in vitro nanosafety. We propose to identify a set of standard cell lines for screening purposes and to select cell types for detailed nanosafety studies based on the intended application and/or expected exposure.

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro-in vivo gap.

The number of newly engineered nanomaterials is vastly increasing along with their applications. Despite the fact that there is a lot of interest and effort is being put into the development of nano-based biomedical applications, the level of translational clinical output remains limited due to uncertainty in the toxicological profiles of the nanoparticles (NPs). As NPs used in biomedicines are likely to directly interact with cells and biomolecules, it is imperative to rule out any adverse effect before they can be safely applied. The initial screening for nanotoxicity is preferably performed in vitro, but extrapolation to the in vivo outcome remains very challenging. In addition, generated in vitro and in vivo data are often conflicting, which consolidates the in vitro-in vivo gap and impedes the formulation of unambiguous conclusions on NP toxicity. Consequently, more consistent and relevant in vitro and in vivo data need to be acquired in order to bridge this gap. This is in turn in conflict with the efforts to reduce the number of animals used for in vivo toxicity testing. Therefore the need for more reliable in vitro models with a higher predictive power, mimicking the in vivo environment more closely, becomes more prominent. In this review we will discuss the current paradigm and routine methods for nanotoxicity evaluation, and give an overview of adjustments that can be made to the cultivation systems in order to optimise current in vitro models. We will also describe various novel model systems and highlight future prospects.

Cationic Amphiphilic Drugs Boost the Lysosomal Escape of Small Nucleic Acid Therapeutics in a Nanocarrier-Dependent Manner.

Small nucleic acid (NA) therapeutics, such as small interfering RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome the multiple extra- and intracellular barriers upon in vivo administration. Interaction with target cells typically triggers endocytosis and sequesters the NPs in endosomes, thus hampering the pharmacological activity of the encapsulated siRNAs that occurs in the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal escape is largely inefficient. As a result, the bulk of the endocytosed NA drug is rapidly trafficked toward the degradative lysosomes that are considered as a dead end for siRNA nanomedicines. In contrast to this paradigm, we recently reported that cationic amphiphilic drugs (CADs) could strongly promote functional siRNA delivery from the endolysosomal compartment via transient induction of lysosomal membrane permeabilization. However, many questions still remain regarding the broader applicability of such a CAD adjuvant effect on NA delivery. Here, we report a drug repurposing screen (National Institutes of Health Clinical Collection) that allowed identification of 56 CAD adjuvants. We furthermore demonstrate that the CAD adjuvant effect is dependent on the type of nanocarrier, with NPs that generate an appropriate pool of decomplexed siRNA in the endolysosomal compartment being most susceptible to CAD-promoted gene silencing. Finally, the CAD adjuvant effect was verified on human ovarian cancer cells and for antisense oligonucleotides. In conclusion, this study strongly expands our current knowledge on how CADs increase the cytosolic release of small NAs, providing relevant insights to more rationally combine CAD adjuvants with NA-loaded NPs for future therapeutic applications.

Repurposing cationic amphiphilic drugs as adjuvants to induce lysosomal siRNA escape in nanogel transfected cells.

Cytosolic delivery remains a major bottleneck for siRNA therapeutics. To facilitate delivery, siRNAs are often enclosed in nanoparticles (NPs). However, upon endocytosis such NPs are mainly trafficked towards lysosomes. To avoid degradation, cytosolic release of siRNA should occur prior to fusion of endosomes with lysosomes, but current endosomal escape strategies remain inefficient. In contrast to this paradigm, we aim to exploit lysosomal accumulation by treating NP-transfected cells with low molecular weight drugs that release the siRNA from the lysosomes into the cytosol. We show that FDA-approved cationic amphiphilic drugs (CADs) significantly improved gene silencing by siRNA-loaded nanogels in cancer cells through simple sequential incubation. CADs induced lysosomal phospholipidosis, leading to transient lysosomal membrane permeabilization and improved siRNA release without cytotoxicity. Of note, the lysosomes could be applied as an intracellular depot for triggered siRNA release by multiple CAD treatments.

Choose your cell model wisely: The in vitro nanoneurotoxicity of differentially coated iron oxide nanoparticles for neural cell labeling.

Currently, there is a large interest in the labeling of neural stem cells (NSCs) with iron oxide nanoparticles (IONPs) to allow MRI-guided detection after transplantation in regenerative medicine. For such biomedical applications, excluding nanotoxicity is key. Nanosafety is primarily evaluated in vitro where an immortalized or cancer cell line of murine origin is often applied, which is not necessarily an ideal cell model. Previous work revealed clear neurotoxic effects of PMA-coated IONPs in distinct cell types that could potentially be applied for nanosafety studies regarding neural cell labeling. Here, we aimed to assess if DMSA-coated IONPs could be regarded as a safer alternative for this purpose and how the cell model impacted our nanosafety optimization study. Hereto, we evaluated cytotoxicity, ROS production, calcium levels, mitochondrial homeostasis and cell morphology in six related neural cell types, namely neural stem cells, an immortalized cell line and a cancer cell line from human and murine origin. The cell lines mostly showed similar responses to both IONPs, which were frequently more pronounced for the PMA-IONPs. Of note, ROS and calcium levels showed opposite trends in the human and murine NSCs, indicating the importance of the species. Indeed, the human cell models were overall more sensitive than their murine counterpart. Despite the clear cell type-specific nanotoxicity profiles, our multiparametric approach revealed that the DMSA-IONPs outperformed the PMA-IONPs in terms of biocompatibility in each cell type. However, major cell type-dependent variations in the observed effects additionally warrant the use of relevant human cell models. STATEMENT OF SIGNIFICANCE: Inorganic nanoparticle (NP) optimization is chiefly performed in vitro. For the optimization of iron oxide (IO)NPs for neural stem cell labeling in the context of regenerative medicine human or rodent neural stem cells, immortalized or cancer cell lines are applied. However, the use of certain cell models can be questioned as they phenotypically differ from the target cell. The impact of the neural cell model on nanosafety remains relatively unexplored. Here we evaluated cell homeostasis upon exposure to PMA- and DMSA-coated IONPs. Of note, the DMSA-IONPs outperformed the PMA-IONPs in each cell type. However, distinct cell type-specific effects were witnessed, indicating that nanosafety should be evaluated in a human cell model that represents the target cell as closely as possible.