Demystifying the long noncoding RNA landscape of small EVs derived from human mesenchymal stromal cells.

INTRODUCTION: The regenerative capacity of mesenchymal stromal cells or medicinal signaling cells (MSCs) is largely mediated by their secreted small extracellular vesicles (sEVs), and the therapeutic efficacy of sEVs can be enhanced by licensing approaches (e.g., cytokines, hypoxia, chemicals, and genetic modification). Noncoding RNAs within MSC-derived sEVs (MSC-sEVs) have been demonstrated to be responsible for tissue regeneration. However, unlike miRNA fingerprints, which have been explored, the landscape of long noncoding RNAs (lncRNAs) in MSC-sEVs remains to be described. OBJECTIVES: To characterize lncRNA signatures in sEVs of human adipose-derived MSCs with or without inflammatory cytokine licensing and depict MSC-sEV-specific and MSC-enriched lncRNA repertoires. METHODS: sEVs were isolated from MSCs with or without TNF-α and IFN-γ (20 ng/mL) stimulation. High-throughput lncRNA sequencing and an in silico approach were employed to analyze the profile of lncRNAs in sEVs and predict lncRNA-protein interactomes. RESULTS: sEVs derived from human MSCs and fibroblasts carried a unique landscape of lncRNAs distinct from the lncRNAs inside these cells. Compared with fibroblast-derived sEVs (F-sEVs), 194 MSC-sEV-specific and 8 upregulated lncRNAs in MSC-sEVs were considered "medicinal signaling lncRNAs"; inflammatory cytokines upregulated 27 lncRNAs in MSC-sEVs, which were considered "licensing-responsive lncRNAs". Based on lncRNA-protein interactome prediction and enrichment analysis, we found that the proteins interacting with medicinal signaling lncRNAs or licensing-responsive lncRNAs have a tight interaction network involved in chromatin remodeling, SWI/SNF superfamily type complexes, and histone binding. CONCLUSION: In summary, our study depicts the landscape of lncRNAs in MSC-sEVs and predicts their potential functions via the lncRNA-protein interactome. Elucidation of the lncRNA landscape of MSC-sEVs will facilitate defining the therapeutic potency of MSC-sEVs and the development of sEV-based therapeutics.

Intranuclear Delivery of DNA Nanostructures via Cellular Mechanotransduction.

DNA nanostructures are attractive gene carriers for nanomedicine applications, yet their delivery to the nucleus remains inefficient. We present the application of extracellular mechanical stimuli to activate cellular mechanotransduction for boosting the intranuclear delivery of DNA nanostructures. Treating mammalian cells with polythymidine-rich spherical nucleic acids (poly(T) SNAs) under gentle compression by a single coverslip leads to up to ∼50% nuclear accumulation without severe endosomal entrapment, cytotoxicity, or long-term membrane damage; no chemical modification or transfection reagent is needed. Gentle compression activates Rho-ROCK mechanotransduction and causes nuclear translocation of YAP. Joint compression and treatment with poly(T) oligonucleotides upregulate genes linked to myosin, actin filament, and nuclear import. In turn, Rho-ROCK, myosin, and importin mediate the nuclear entry of poly(T) SNAs. Treatment of endothelioma cells with poly(T) SNAs bearing antisense oligonucleotides under compression inhibits an intranuclear oncogene. Our data should inspire the marriage of DNA nanotechnology and cellular biomechanics for intranuclear applications.

Mammalian Cells Exocytose Alkylated Gold Nanoparticles via Extracellular Vesicles.

Understanding the exocytosis of nanoparticles (NPs) from cells is valuable because it informs design rules of NPs that support desirable cellular retention for nanomedicine applications, but investigations into the mechanism for the exocytosis of NPs remain scarce. We elucidate the mechanism for the exocytosis of dodecyl-terminated, polyethylene glycol-coated gold NPs (termed "dodecyl-PEG-AuNP"). The Au core enables ultrastructural differentiation of the exocytosed NPs from the nearby extracellular vesicles (EVs). The PEG shell prevents interparticle agglomeration or aggregation that disfavors exocytosis. The minute amounts of alkyl chains on the PEG shell not only promote cellular uptake but also improve exocytosis by up to 4-fold higher probability and upregulate exocytosis- and vesicle-related genes. After entering Kera-308 keratinocytes and trafficking to multivesicular bodies and lysosomes, these NPs exit the cell predominantly via unconventional exocytosis, accompanied by enhanced secretion of sub-100 nm, CD81-enriched exosomes. The pathway for NP exocytosis and subpopulation of EVs that are secreted alongside the exocytosed NPs depends on dodecyl loading. This work provides insights into dissecting the mechanism of NP exocytosis and its relationship with EV secretion.

A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells.

Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multivesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for "always-on" dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.

Alkyl-Terminated Gold Nanoparticles as a Self-Therapeutic Treatment for Psoriasis.

We present a self-therapeutic nanoparticle for topical delivery to epidermal keratinocytes to prevent and treat psoriasis. Devoid of known chemical or biological antipsoriatic drugs, this sub-15 nm nanoparticle contains a 3 nm gold core and a shell of 1000 Da polyethylene glycol strands modified with 30% octadecyl chains. When it is applied to imiquimod-induced psoriasis mice without an excipient, the nanoparticle can cross the stratum corneum and preferentially enter keratinocytes. Applying the nanoparticles concurrently with imiquimod prevents psoriasis and downregulates genes that are enriched in the downstream of the interleukin-17 signaling pathway and linked to epidermis hyperproliferation and inflammation. Applying the nanoparticles after psoriasis is established treats the psoriatic skin as effectively as standard steroid and vitamin D analog-based therapy but without hair loss and skin wrinkling. The nanoparticles do not accumulate in major organs or induce long-term toxicity. Our nanoparticle offers a simple, safe, and effective alternative for treating psoriasis.

Sub-10 nm Substrate Roughness Promotes the Cellular Uptake of Nanoparticles by Upregulating Endocytosis-Related Genes.

Nanosubstrate engineering is an established approach for modulating cellular responses, but it remains infrequently exploited to facilitate the intracellular delivery of nanoparticles (NPs). We report nanoscale roughness of the extracellular environment as a critical parameter for regulating the cellular uptake of NPs. After seeding cells atop a substrate that contains randomly immobilized gold NPs (termed AuNP-S) with sub-10 nm surface roughness, we demonstrate that such cells internalize up to ∼100-fold more poly(ethylene glycol)-coated AuNPs (Au@PEG NPs) than those cells seeded on a conventional flat culture plate. Our result is generalizable to 4 different cell types and Au@PEG NPs modified with 13 different hydrocarbyl functional groups. Conditioning cells to AuNP-S not only leads to upregulation of clathrin- and integrin-related genes, but also supports elevated uptake of Au@PEG NPs via clathrin-mediated endocytosis. Our data suggest a simple and robust method for boosting the intracellular delivery of nanomedicines by nanosubstrate engineering.

Effect of Surface Modification with Hydrocarbyl Groups on the Exocytosis of Nanoparticles.

Designing nanoparticles (NPs) with desirable cell type-specific exocytosis properties, say promoting their exocytosis from scavenging cell types (e.g., macrophages and endothelial cells) or suppressing their exocytosis from target disease cell types (e.g., cancer cells), improves the application of nanomedicines. However, the design parameters available for tuning the exocytosis of NPs remain scarce in the "nano-cell" literature. Here, we demonstrate that surface modification of NPs with hydrocarbyl functional groups, commonly found in biomolecules and NP-based drug carriers, is a critical parameter for tuning the exocytosis of NPs from RAW264.7 macrophages, C166 endothelial cells, and HeLa epithelial cancer cells. To exclude the effect of hydrophobicity, we prepare a collection of hydrophilic NPs that bear a gold NP (AuNP) core, a dense polyethylene glycol (PEG) shell, and different types of hydrocarbyl groups (X) that are attached to the distal end of the PEG strands (termed "Au@PEG-X NPs"). For all three cell types tested, modification of NPs with straight-chain dodecane leads to a >10-fold increase in the level of cellular uptake, drastically higher than those of all other types of X tested. However, the probability of exocytosis of NPs significantly depends on the types of cell and X. Notably, NPs modified with cyclododecanes are most likely to be exocytosed by RAW264.7 and C166 cells (but not HeLa cells), accompanied by the release of intralumenal vesicles to the extracellular milieu. These data suggest a reductionist approach for rationally assembling bionanomaterials for nanomedicine applications by using hydrocarbyl functional groups as building blocks.

Intrapulmonary Cellular-Level Distribution of Inhaled Nanoparticles with Defined Functional Groups and Its Correlations with Protein Corona and Inflammatory Response.

Concerns over the health risks associated with airborne exposure to ultrafine particles [PM0.1, or nanoparticles (NPs)] call for a comprehensive understanding in the interactions of inhaled NPs along their respiratory journey. We prepare a collection of polyethylene glycol-coated gold nanoparticles that bear defined functional groups commonly identified in atmospheric particulates (Au@PEG-X NPs, where X = OCH3, COOH, NH2, OH, or C12H25). Regardless of the functional group, these ∼50 nm NPs remain colloidally stable following aerosolization and incubation in bronchoalveolar lavage fluid (BALF), without pronouncedly crossing the air-blood barrier. The type of BALF proteins adhered onto the NPs is similar, but the composition of protein corona depends on functional group. By subjecting Balb/c mice to inhalation of Au@PEG-X NPs for 6 h, we demonstrate that the intrapulmonary distribution of NPs among the various types of cells (both found in BALF and isolated from the lavaged lung) and the acute inflammatory responses induced by inhalation are sensitive to the functional group of NPs and postinhalation period (0, 24, or 48 h). By evaluating the pairwise correlations between the three variables of "lung-nano" interactions (protein corona, intrapulmonary cellular-level distribution, and inflammatory response), we reveal strong statistical correlations between the (1) fractions of albumin or carbonyl reductase bound to NPs, (2) associations of inhaled NPs to neutrophils in BALF or macrophages in the lavaged lung, and (3) level of total protein in BALF. Our results provide insights into the effect of functional group on lung-nano interactions and health risks associated with inhalation of PM0.1.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Nano-Cell Interactions of Non-Cationic Bionanomaterials.

Advances in nanotechnology have empowered the design of bionanomaterials by assembling different types of natural biomolecules (e.g., nucleic acids, proteins, and lipids) as building blocks into nanoparticles (NPs) of 1-100 nm in diameter. Such bionanomaterials form the basis of useful nanomedicine applications, such as targeted delivery, gene regulation, molecular diagnostics, and immunomodulation. To achieve optimal performance in these applications, it is imperative that the NPs be delivered effectively to the organs, tissues, and cells of interest. A rational approach to facilitating the delivery of NPs is to develop a detailed and comprehensive understanding in their fundamental interactions with the biological system (or nano-bio interactions). Rigorous nano-bio research can provide mechanistic insights for circumventing the bottlenecks associated with inefficient and nonspecific delivery of NPs, catalyzing the clinical translation of nanomedicines. Cationic liposomes and lipid NPs are conventional carriers of therapeutic cargoes into cells due to their high ability to penetrate the cell membrane, a barrier comprised by an anionic phospholipid bilayer. Yet, cationic NPs tend to cause cytotoxicity and immune responses that may hamper their clinical translation. Contrary to cationic NPs, non-cationic NPs (be they near-neutral or anionic in surface charge) generally exhibit higher biocompatibility but enter mammalian cells in much less pronounced amounts. Intriguingly, some types of non-cationic NPs exhibit high biocompatibility and cellular uptake properties, all attractive features for intracellular delivery. In this Account, we present our studies of the interactions of non-cationic bionanomaterials with cells (or nano-cell interactions). To start with, we introduce the use of near-neutral poly(ethylene glycol)-coated NPs for probing the roles of two rarely explored physicochemical parameters on cellular uptake, namely, extracellular compression and alkylation. We next present the nano-cell interactions of two representative types of anionic bionanomaterials that effectively enter mammalian cells and have found widespread applications in the past decade, including DNA-coated NPs and polydopamine (PDA)-coated NPs. In our cell-based studies, we dissect the route of intracellular trafficking, pathway proteins that dictate cellular uptake, and trafficking of NPs. We further touch on our recent quantitative analysis of the cellular-level distribution of NPs in various organs and tissues of diseased animal models. Our results offer important design rules of NPs for achieving effective intracellular delivery and may even guide us to explore nanomedicine applications that we did not conceive before, such as using DNA-coated NPs for targeting atherosclerotic plaques and PDA-coated plasmonic nanoworms for photothermal killing of cancer cells. We conclude with our perspectives in elucidating nano-bio interactions via a reductionist approach, calling for closer attention to the role of functional groups and more refined studies on the organelle-level distribution of NPs and the genetic basis of in vivo distribution of NPs.

Morphological Diversity, Protein Adsorption, and Cellular Uptake of Polydopamine-Coated Gold Nanoparticles.

Polydopamine (PDA)-coated nanoparticles are adhesive bionanomaterials widely utilized in intracellular applications, yet how their adhesiveness affects their colloidal stability and their interactions with serum proteins and mammalian cells remain unclear. In this work, we systematically investigate the combined effects of dopamine (DA) concentration and polymerization time (both reaction parameters spanning 2 orders of magnitude) on the morphological diversity of PDA-coated nanoparticles by coating PDA onto gold nanoparticle cores. Independent of the DA concentration, Au@PDA NPs remain largely aggregated upon several hours of limited polymerization; interestingly, extended polymerization for 2 days or longer yield randomly aggregated NPs, nearly monodisperse NPs, or worm-like NP chains in the ascending order of DA concentration. Upon exposure to serum proteins, the specific type of proteins adsorbed to the Au@PDA NPs strongly depends upon the DA concentration. As DA concentration increases, less albumin and more hemoglobin subunits adhere. Moreover, cellular uptake is a strong function of polymerization time. Serum-stabilized Au@PDA NPs prepared by limited polymerization enter Neuro-2a and HeLa cancer cells more abundantly than those prepared by extended polymerization. Our data underscore the importance of DA concentration and polymerization time for tuning the morphology and degree of intracellular delivery of PDA-coated nanostructures.

Promoting intracellular delivery of sub-25 nm nanoparticles via defined levels of compression.

Many investigations into the interactions between nanoparticles and mammalian cells entail the use of culture systems that do not account for the effect of extracellular mechanical cues, such as compression. In this work, we present an experimental set-up to systematically investigate the combined effects of nanoparticle size and compressive stress on the cellular uptake and intracellular localization of poly(ethylene glycol)-coated gold nanoparticles (Au-PEG NPs). Specifically, we employ an automated micromechanical system to apply defined levels of compressive strain to an agarose gel, which transmits defined amounts of unconfined, uniaxial compressive stress to a monolayer of C2C12 mouse myoblasts seeded underneath the gel without compromising cell viability. Notably, uptake of Au-PEG NPs smaller than 25 nm by compressed myoblasts is up to 5-fold higher than that by uncompressed cells. The optimal compressive stress for maximizing the cellular uptake of sub-25 nm NPs monotonically increases with NP size. With and without compression, the Au-PEG NPs enter C2C12 cells via energy-dependent uptake; they also enter compressed cells via clathrin-mediated endocytosis as the major pathway. Upon cellular entry, the Au-PEG NPs more readily reside in the late endosomes or lysosomes of compressed cells than uncompressed cells. Results from our experimental set-up yield mechanistic insights into the delivery of NPs to cell types under extracellular compression.

Toward Understanding in Vivo Sequestration of Nanoparticles at the Molecular Level.

A longstanding and widely accepted bottleneck in the targeted delivery of intravenously injected nanoparticles lies in their clearance by macrophages in the liver and spleen. In this Perspective, we call for deeper understanding of the critical role of endothelial cells in the sequestration of nanoparticles in vivo. In this issue of ACS Nano, Campbell et al. used a combination of real-time imaging and genome-editing methods to demonstrate that stabilin-2 is an important receptor for removing anionic liposomes from blood circulation in a zebrafish model. Such mechanistic insights at the molecular level will provide a more holistic picture of the in vivo sequestration of administered nanoparticles beyond the cellular level and pose valuable design considerations for redistributing nanoparticles in vivo.

Effect of Alkylation on the Cellular Uptake of Polyethylene Glycol-Coated Gold Nanoparticles.

Alkyl groups (CnH2n+1) are prevalent in engineered bionanomaterials used for many intracellular applications, yet how alkyl groups dictate the interactions between nanoparticles and mammalian cells remains incomprehensively investigated. In this work, we report the effect of alkylation on the cellular uptake of densely polyethylene glycol-coated nanoparticles, which are characterized by their limited entry into mammalian cells. Specifically, we prepare densely PEGylated gold nanoparticles that bear alkyl chains of varying carbon chain lengths (n) and loading densities (termed "alkyl-PEG-AuNPs"), followed by investigating their uptake by Kera-308 keratinocytes. Strikingly, provided a modest alkyl mass percentage of 0.2% (2 orders of magnitude lower than that of conventional lipid-based NPs) in their PEG shells, dodecyl-PEG-AuNPs (n = 12) and octadecyl-PEG-AuNPs (n = 18) can enter Kera-308 cells 30-fold more than methoxy-PEG-AuNPs (no alkyl groups) and hexyl-PEG-AuNPs (n = 6) after 24 h of incubation. Such strong dependence on n is valid for all serum concentrations considered (even under serum-free conditions), although enhanced serum levels can trigger the agglomeration of alkyl-PEG-AuNPs (without permanent aggregation of the AuNP cores) and can attenuate their cellular uptake. Additionally, alkyl-PEG-AuNPs can rapidly enter Kera-308 cells via the filipodia-mediated pathway, engaging the tips of membrane protrusions and accumulating within interdigital folds. Most alkyl-PEG-AuNPs adopt the "endo-lysosomal" route of trafficking, but ∼15% of them accumulate in the cytosol. Regardless of intracellular location, alkyl-PEG-AuNPs predominantly appear as individual entities after 24 h of incubation. Our work offers insights into the incorporation of alkyl groups for designing bionanomaterials for cellular uptake and cytosolic accumulation with intracellular stability.