Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Biomarkers of nanomaterials hazard from multi-layer data.

There is an urgent need to apply effective, data-driven approaches to reliably predict engineered nanomaterial (ENM) toxicity. Here we introduce a predictive computational framework based on the molecular and phenotypic effects of a large panel of ENMs across multiple in vitro and in vivo models. Our methodology allows for the grouping of ENMs based on multi-omics approaches combined with robust toxicity tests. Importantly, we identify mRNA-based toxicity markers and extensively replicate them in multiple independent datasets. We find that models based on combinations of omics-derived features and material intrinsic properties display significantly improved predictive accuracy as compared to physicochemical properties alone.

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides.

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles.

Oligonucleotides (ONs) comprise a rapidly growing class of therapeutics. In recent years, the list of FDA-approved ON therapies has rapidly expanded. ONs are small (15-30 bp) nucleotide-based therapeutics which are capable of targeting DNA and RNA as well as other biomolecules. ONs can be subdivided into several classes based on their chemical modifications and on the mechanisms of their target interactions. Historically, the largest hindrance to the widespread usage of ON therapeutics has been their inability to effectively internalize into cells and escape from endosomes to reach their molecular targets in the cytosol or nucleus. While cell uptake has been improved, "endosomal escape" remains a significant problem. There are a range of approaches to overcome this, and in this review, we focus on three: altering the chemical structure of the ONs, formulating synthetic, lipid-based nanoparticles to encapsulate the ONs, or biologically loading the ONs into extracellular vesicles. This review provides a background to the design and mode of action of existing FDA-approved ONs. It presents the most common ON classifications and chemical modifications from a fundamental scientific perspective and provides a roadmap of the cellular uptake pathways by which ONs are trafficked. Finally, this review delves into each of the above-mentioned approaches to ON delivery, highlighting the scientific principles behind each and covering recent advances.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics.

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers' RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Profiling of Sub-Lethal in Vitro Effects of Multi-Walled Carbon Nanotubes Reveals Changes in Chemokines and Chemokine Receptors.

Engineered nanomaterials are potentially very useful for a variety of applications, but studies are needed to ascertain whether these materials pose a risk to human health. Here, we studied three benchmark nanomaterials (Ag nanoparticles, TiO2 nanoparticles, and multi-walled carbon nanotubes, MWCNTs) procured from the nanomaterial repository at the Joint Research Centre of the European Commission. Having established a sub-lethal concentration of these materials using two human cell lines representative of the immune system and the lungs, respectively, we performed RNA sequencing of the macrophage-like cell line after exposure for 6, 12, and 24 h. Downstream analysis of the transcriptomics data revealed significant effects on chemokine signaling pathways. CCR2 was identified as the most significantly upregulated gene in MWCNT-exposed cells. Using multiplex assays to evaluate cytokine and chemokine secretion, we could show significant effects of MWCNTs on several chemokines, including CCL2, a ligand of CCR2. The results demonstrate the importance of evaluating sub-lethal concentrations of nanomaterials in relevant target cells.

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery.

RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.

Multiparametric Profiling of Engineered Nanomaterials: Unmasking the Surface Coating Effect.

Despite considerable efforts, the properties that drive the cytotoxicity of engineered nanomaterials (ENMs) remain poorly understood. Here, the authors inverstigate a panel of 31 ENMs with different core chemistries and a variety of surface modifications using conventional in vitro assays coupled with omics-based approaches. Cytotoxicity screening and multiplex-based cytokine profiling reveals a good concordance between primary human monocyte-derived macrophages and the human monocyte-like cell line THP-1. Proteomics analysis following a low-dose exposure of cells suggests a nonspecific stress response to ENMs, while microarray-based profiling reveals significant changes in gene expression as a function of both surface modification and core chemistry. Pathway analysis highlights that the ENMs with cationic surfaces that are shown to elicit cytotoxicity downregulated DNA replication and cell cycle responses, while inflammatory responses are upregulated. These findings are validated using cell-based assays. Notably, certain small, PEGylated ENMs are found to be noncytotoxic yet they induce transcriptional responses reminiscent of viruses. In sum, using a multiparametric approach, it is shown that surface chemistry is a key determinant of cellular responses to ENMs. The data also reveal that cytotoxicity, determined by conventional in vitro assays, does not necessarily correlate with transcriptional effects of ENMs.

Sequential delivery of synergistic drugs by silica nanocarriers for enhanced tumour treatment.

Herein hybrid silica nanoparticles have been engineered to direct the sequential delivery of multiple chemotherapeutic drugs in response to external stimuli such as variations in pH. The nanocarriers consist of conventional MCM-41-type nanoparticles, which have been functionalised with an organic ligand (or stalk) grafted onto the external surface. The stalk is designed to "recognise" a complementary molecule, which serves as a "cap" to block the pores of the nanoparticles. First, camptothecin is introduced into the pores by diffusion prior to capping the pore apertures via molecular recognition. The cap, which is a derivative of 5-fluorouracil, serves as a second cytotoxic drug for synergistic chemotherapy. In vitro tests revealed that negligible release of the drugs occurred at pH 7.4, thus avoiding toxic side effects in the blood stream. In contrast, the stalk/cap complex is destabilised within the endolysosomal compartment (pH 5.5) of cancer cells, where release of the drugs was demonstrated. Furthermore, this environmentally responsive system exhibited a synergistic effect of the two drugs, where the pH-triggered release of the cytotoxic cap followed by diffusion-controlled release of the drug cargo within the pores led to essentially complete elimination of breast cancer cells.

Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity.

Aggregation and intracellular deposition of the protein α-synuclein is an underlying characteristic of Parkinson's disease. α-Synuclein assemblies also undergo cell-cell spreading, facilitating propagation of their cellular pathology. Understanding how cellular interactions and uptake of extracellular α-synuclein assemblies depend on their physical attributes is therefore important. We prepared fragmented fluorescently labeled α-synuclein amyloid fibrils of different average lengths (∼80 nm to >1 µm) and compared their interactions with SH-SY5Y cells. We report that fibrils of all lengths, but not monomers, bind avidly to the cell surface. Their uptake is inversely dependent on their average size, occurs via a heparan sulfate dependent endocytic route, and appears to have a size cutoff of ∼400 nm. The uptake of α-synuclein fibrils, but not monomers, correlates with their cytotoxicity as measured by reduction in metabolic activity, strongly suggesting an intracellular basis for α-synuclein fibril toxicity, likely involving endolysosomes.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

Cationic gold nanoparticles elicit mitochondrial dysfunction: a multi-omics study.

Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer's disease peptide Aß(1-42).

Proteoglycans (PGs) have been found in Alzheimer's disease amyloid-ß (Aß) plaques and their glycosaminoglycan chains reportedly influence Aß aggregation, neurotoxicity and intracellular accumulation in cell and animal models, but their exact pathophysiological role(s) remain unclear. We have studied the cellular uptake of fluorescently labelled Aß(1-42) and Aß(1-40) peptides in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached heparan and chondroitin sulfate chains. After 24 h of incubation, CHO-K1 accumulates more Aß(1-42) and Aß(1-40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aß uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aß(1-42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aß endocytosis. Neither Aß(1-42) nor Aß(1-40) could stimulate uptake of a 10 kDa dextran (a general endocytosis marker) suggesting that Aß-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aß(1-42)-positive vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aß(1-42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aß(1-42) is aggregated in both cell types. We also report that CHO-pgsA-745 cells perform less endocytosis than CHO-K1 and, albeit this does not explain their difference in Aß internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aß uptake that may be relevant for our understanding of their role in AD pathology.

Macrophage activation status determines the internalization of mesoporous silica particles of different sizes: Exploring the role of different pattern recognition receptors.

Mesoporous silica-based particles are promising candidates for biomedical applications. Here, we address the importance of macrophage activation status for internalization of AMS6 (approx. 200 nm in diameter) versus AMS8 (approx. 2 µm) mesoporous silica particles and the role of different phagocytosis receptors for particle uptake. To this end, FITC-conjugated silica particles were used. AMS8 were found to be non-cytotoxic both for M-CSF-stimulated (anti-inflammatory) and GM-CSF-stimulated (pro-inflammatory) macrophages, whereas AMS6 exhibited cytotoxicity towards M-CSF-stimulated, but not GM-CSF-stimulated macrophages; this toxicity was, however, mitigated in the presence of serum. AMS8 triggered the secretion of pro-inflammatory cytokines in M-CSF-activated cells. Class A scavenger receptor (SR-A) expression was noted in both M-CSF and GM-CSF-stimulated macrophages, although the expression was higher in the former case, and gene silencing of SR-A resulted in a decreased uptake of AMS6 in the absence of serum. GM-CSF-stimulated macrophages expressed higher levels of the mannose receptor CD206 compared to M-CSF-stimulated cells, and uptake of AMS6, but not AMS8, was reduced following the downregulation of CD206 in GM-CSF-stimulated cells; particle uptake was also suppressed by mannan, a competitive ligand. These studies demonstrate that macrophage activation status is an important determinant of particle uptake and provide evidence for a role of different macrophage receptors for cell uptake of silica particles.

Cytotoxic and Proinflammatory Effects of Metal-Based Nanoparticles on THP-1 Monocytes Characterized by Combined Proteomics Approaches.

Thorough characterization of toxic effects of nanoparticles (NP) is desirable due to the increasing risk of potential environmental contamination by NP. In the current study, we combined three recently developed proteomics approaches to assess the effects of Au, CuO, and CdTe NP on the innate immune system. The human monocyte cell line THP-1 was employed as a model. The anticancer drugs camptothecin and doxorubicin were used as positive controls for cell death, and lipopolysaccharide was chosen as a positive control for proinflammatory activation. Despite equivalent overall toxicity effect (50 ± 10% dead cells), the three NP induced distinctly different proteomics signatures, with the strongest effect being induced by CdTe NP, followed by CuO and gold NP. The CdTe toxicity mechanism involves down-regulation of topoisomerases. The effect of CuO NP is most reminiscent of oxidative stress and involves up-regulation of proteins involved in heat response. The gold NP induced up-regulation of the inflammatory mediator, NF-κB, and its inhibitor TIPE2 was identified as a direct target of gold NP. Furthermore, gold NP triggered activation of NF-κB as evidenced by phosphorylation of the p65 subunit. Overall, the combined proteomics approach described here can be used to characterize the effects of NP on immune cells.

Imidazopyridine-fused [1,3]-diazepinones part 2: Structure-activity relationships and antiproliferative activity against melanoma cells.

We recently described a pyrido-imidazodiazepinone derivative which could be a promising hit compound for the development of new drugs acting against melanoma cells. In this study, a series of 28 novel pyrido-imidazodiazepinones were synthesized and screened for their in vitro cytotoxic activities against the melanoma MDA-MB-435 cell line. Among the derivatives, seven of them showed 50% growth inhibitory activity at 1 µM concentration, and high selectivity against the melanoma cell line MDA-MB-435.

A 3D co-culture microtissue model of the human placenta for nanotoxicity assessment.

There is increasing evidence that certain nanoparticles (NPs) can overcome the placental barrier, raising concerns on potential adverse effects on the growing fetus. But even in the absence of placental transfer, NPs may pose a risk to proper fetal development if they interfere with the viability and functionality of the placental tissue. The effects of NPs on the human placenta are not well studied or understood, and predictive in vitro placenta models to achieve mechanistic insights on NP-placenta interactions are essentially lacking. Using the scaffold-free hanging drop technology, we developed a well-organized and highly reproducible 3D co-culture microtissue (MT) model consisting of a core of placental fibroblasts surrounded by a trophoblast cell layer, which resembles the structure of the in vivo placental tissue. We could show that secretion levels of human chorionic gonadotropin (hCG) were significantly higher in 3D than in 2D cell cultures, which indicates an enhanced differentiation of trophoblasts grown on 3D MTs. NP toxicity assessment revealed that cadmium telluride (CdTe) and copper oxide (CuO) NPs but not titanium dioxide (TiO2) NPs decreased MT viability and reduced the release of hCG. NP acute toxicity was significantly reduced in 3D co-culture MTs compared to 2D monocultures. Taken together, 3D placental MTs provide a new and promising model for the fast generation of tissue-relevant acute NP toxicity data, which are indispensable for the safe development of NPs for industrial, commercial and medical applications.

Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance.