Staying on track - Keeping things running in a high-end scientific imaging core facility.

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.

Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA.

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.

Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

An inhibitor-mediated beta-cell dedifferentiation model reveals distinct roles for FoxO1 in glucagon repression and insulin maturation.

OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.

Cross-modality imaging of bisphosphonate-treated murine jawbones.

In this proof-of-principle study, we established and implemented a cross-modality imaging (CMI) pipeline to characterize and compare bisphosphonate (BIS)-treated jawbones of Sprague-Dawley rats after tooth extraction after physical therapies (photobiomodulation and extracorporeal shockwave therapy (PBMT and ESWT)). We showcase the feasibility of such a CMI approach and its compatibility across imaging modalities to probe the same region of interest (ROI) of the same jawbone. Jawbones were imaged in toto in 3D using micro-Computed Tomography to identify ROIs for subsequent sequential 2D analysis using well-established technologies such as Atomic Force Microscopy and Scanning Electron Microscopy, and recent imaging approaches in biomedical settings, such as micro-X-Ray Fluorescence Spectroscopy. By combining these four modalities, multiscale information on the morphology, topography, mechanical stiffness (Young's modulus), and calcium, zinc and phosphorus concentrations of the bone was collected. Based on the CMI pipeline, we characterized and compared the jawbones of a previously published clinically relevant rat model of BIS-related osteonecrosis of the jawbone (BRONJ) before and after treatment with BISs, PBMT and ESWT. While we did not find that physical therapies altered the mechanical and elemental jawbone parameters with significance (probably due to the small sample size of only up to 5 samples per group), both ESWT and PBMT reduced pore thicknesses and bone-to-enamel distances significantly compared to the controls. Although focused on BIS-treated jawbones, the established CMI platform can be beneficial in the study of bone-related diseases in general (such as osteoarthritis or -porosis) to acquire complementary hallmarks and better characterize disease status and alleviation potentials.

Effect of hydroxypropyl-ß-cyclodextrin in fluid and semi-solid submicron emulsions on physiological skin parameters during regular in vivo application.

OBJECTIVE: The aim of the present study was to evaluate the effect of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in cosmetic submicron emulsions and submicron emulsion gels on physiological skin parameters during regular application in a clinical set-up. METHODS: Formulation morphology was investigated using cryo-transmission electron microscopy. Stability of the employed formulations was determined by photon correlation spectroscopy, measurement of pH and rheological properties. Effect on physiological skin parameters was evaluated during regular application over four weeks in a parallel group study (n = 15, healthy forearm skin) with a Corneometer, Sebumeter, skin-pH-Meter, Aquaflux and an Epsilon sensor. Confocal Raman spectroscopy was employed to monitor urea and NMF levels. RESULTS: Both submicron emulsions and gels showed satisfying storage stability irrespective of cyclodextrin incorporation. No statistically significant effects on skin barrier function and any of the observed parameters were obtained, indicating good skin tolerability of all tested formulations. CONCLUSION: Results suggest good skin tolerability of the developed cosmetic submicron emulsions and gels with HP-ß-CD.

OBJECTIF: Le but de la présente étude était d'évaluer l'effet de l'hydroxypropyl-ß-cyclodextrine (HP-ß-CD) dans les émulsions cosmétiques submicroniques et les gels d'émulsion submicronique sur les paramètres physiologiques de la peau lors d'une application régulière dans une configuration clinique. MÉTHODES: La morphologie de la formulation a été étudiée en utilisant la microscopie électronique à transmission cryo. La stabilité des formulations employées a été déterminée par spectroscopie de corrélation de photons, mesure du pH et des propriétés rhéologiques. L'effet sur les paramètres physiologiques de la peau a été évalué lors d'une application régulière pendant quatre semaines dans une étude de groupe parallèle (n = 15, peau d'avant-bras saine) avec un cornéomètre, un sébumètre, un pH-mètre cutané, un Aquaflux et un capteur Epsilon. La spectroscopie Raman confocale a été utilisée pour surveiller les niveaux d'urée et de NMF. RÉSULTATS: Les émulsions et les gels submicroniques ont montré une stabilité de stockage satisfaisante indépendamment de l'incorporation de cyclodextrine. Aucun effet statistiquement significatif sur la fonction de barrière cutanée et aucun des paramètres observés n'a été obtenu, indiquant une bonne tolérance cutanée de toutes les formulations testées. CONCLUSION: Les résultats suggèrent une bonne tolérance cutanée des émulsions et des gels cosmétiques submicroniques développés avec HP-ß-CD.

Extracellular Vesicles in Human Skin: Cross-Talk from Senescent Fibroblasts to Keratinocytes by miRNAs.

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.

Protein A- and Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant.

Detection of disease-related biomarkers in plasma provides a possibility for early clinical diagnosis. However, highly abundant proteins in plasma, such as human immunoglobulin (hIgG) are a main impediment to biomarker discovery and analysis. Therefore, rapid and easy depletion of hIgG in the plasma is beneficial for biomarker discovery. In this work, citrate-capped gold nanoparticles (GNPs) were synthesized and conjugated with cysteine-tagged recombinant Protein A (rProtA) and Protein G (ProtG), respectively. The resultant protein-GNP bioconjugates were thoroughly characterized by surface plasmon resonance spectroscopy, hydrodynamic light scattering (DLS), electrophoretic light scattering (ELS) and rotary metal shadowing transmission electron microscopy (TEM) measurements. In order to quantitatively control the amount of the rProt A and ProtG on the GNP surface, binding studies and isotherm measurements have been performed. rProtA-GNP conjugate exhibited better binding capacities towards hIgG. Its surface coverage with rProtA molecules was determined by protein quantification after hydrolysis of the rProtA-GNP conjugate, GNP removal and subsequent amino acid assay by HPLC with fluorescence detection. Binding isotherms acquired with hIgG revealed their maximal capacity for depletion experiments. Depletion efficiency of around 90% could be achieved in a standard solution. With optimized amount of rProtA-GNP and ProtG-GNP, respectively, hIgG could be efficiently extracted from real samples (human plasma and hIgG-spiked cell culture supernatant). A benchmarking study with ProteinA-modified magnetic particles (Dynabeads) was performed as well. The results document that these rProtA-GNP and ProtG-GNP affinity nanoparticles could be a promising alternative to magnetic bead based immunoaffinity trapping and constitutes a flexible platform for both depletion of hIgG from human plasma and antibody affinity capture from cell culture supernatants in process control of biopharmaceuticals by simple solution handling (via pipetting) and centrifugation steps.

DRC2/CCDC65 is a central hub for assembly of the nexin-dynein regulatory complex and other regulators of ciliary and flagellar motility.

The nexin-dynein regulatory complex (N-DRC) plays a central role in the regulation of ciliary and flagellar motility. In most species, the N-DRC contains at least 11 subunits, but the specific function of each subunit is unknown. Mutations in three subunits (DRC1, DRC2/CCDC65, DRC4/GAS8) have been linked to defects in ciliary motility in humans and lead to a ciliopathy known as primary ciliary dyskinesia (PCD). Here we characterize the biochemical, structural, and motility phenotypes of two mutations in the DRC2 gene of Chlamydomonas Using high-resolution proteomic and structural approaches, we find that the C-terminal region of DRC2 is critical for the coassembly of DRC2 and DRC1 to form the base plate of N-DRC and its attachment to the outer doublet microtubule. Loss of DRC2 in drc2 mutants disrupts the assembly of several other N-DRC subunits and also destabilizes the assembly of several closely associated structures such as the inner dynein arms, the radial spokes, and the calmodulin- and spoke-associated complex. Our study provides new insights into the range of ciliary defects that can lead to PCD.

Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance.

Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic, and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction.

Different Potential of Extracellular Vesicles to Support Thrombin Generation: Contributions of Phosphatidylserine, Tissue Factor, and Cellular Origin.

Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

Antagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life Cycles.

Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, nonlinear pH changes. The time scales are tunable by variation of the enzyme concentrations and additional buffer substances. The resulting material system operates in full autonomy after injection of the chemical fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.

Photonic Devices Out of Equilibrium: Transient Memory, Signal Propagation, and Sensing.

Soft photonic materials are important for sensors, displays, or energy management and have become switchable under static equilibrium conditions by integration of responsive polymer features. The next step is to equip such materials with the ability for autonomously dynamic and self-regulating behavior, which would advance their functionality and application possibilities to new levels. Here, this study shows the system integration of a nonlinear, biocatalytic pH-feedback system with a pH-responsive block copolymer photonic gel, and demonstrates autonomous transient memories, remotely controlled signal propagation, and sensing. This study utilizes an enzymatic switch to program the lifetime of the reflective state of a photonic gel, and induces propagation of pH-waves extinguishable by illumination with UV-light. The described combination of nonlinear chemistry and responsive photonic gels opens pathways toward out-of-equilibrium photonic devices with active and autonomous behavior useful for sensing, computation, and communication.

3D DNA Origami Cuboids as Monodisperse Patchy Nanoparticles for Switchable Hierarchical Self-Assembly.

The rational design of anisotropic interaction patterns is a key step for programming colloid and nanoparticle self-assembly and emergent functions. Herein, we demonstrate a concept for harnessing the capabilities of 3D DNA origami for extensive supracolloidal self-assembly and showcase its use for making truly monodisperse, patchy, divalent nanocuboids that can self-assemble into supracolloidal fibrils via programmable DNA hybridization. A change in the number of connector duplexes at the patches reveals that multivalency and cooperativity play crucial roles to enhance superstructure formation. We further show thermal and chemical switching of the superstructures as the first steps toward reconfigurable self-assemblies. This concept lays the groundwork for merging monodisperse 3D DNA origami, featuring programmable patchiness and interactions, with nanoparticle self-assembly.

Simple Platform Method for the Synthesis of Densely Functionalized Microgels by Modification of Active Ester Latex Particles.

This study reports a simple and versatile synthesis route for the preparation of highly uniform and densely functionalized aqueous microgels by modification of latex particles composed of an active ester monomer (pentafluorophenyl acrylate; PFPA). The hydrophobic nature of the PFPA allows synthesizing very uniform latex particles via emulsion polymerization, whose size can be controlled by the surfactant concentration, while the degree of crosslinking is a function of the added crosslinker. The high reactivity of the PFPA groups toward nucleophilic substitution delivers a platform method to synthesize functional microgels by reaction with functional amines. This study demonstrates this process for the dense functionalization of the entire particle with an amine carrying a pH-responsive unit. This study further describes the influence of the crosslinking degree on the ability for swelling of the resulting microgels in aqueous dispersion.

Polyacid microgels with adaptive hydrophobic pockets and ampholytic character: synthesis, solution properties and insights into internal nanostructure by cryogenic-TEM.

Microgels with internal and reconfigurable complex nanostructure are emerging as possible adaptive particles, yet they remain challenging to design synthetically. Here, we report the synthesis of highly charged poly(methacrylic acid) (PMAA) microgels incorporating permanent (poly(methyl methacrylate) (PMMA)) and switchable hydrophobic pockets (poly(N,N'-diethylaminoethyl methacrylate) (PDEAEMA)) via emulsion polymerization. We demonstrate detailed tuning of the size, crosslinking density and tailored incorporation of functional comonomers into the polyacid microgels. Analysis via cryo-TEM and pyrene probe measurements reveal switchable hydrophobic pockets inside the microgels as a function of pH. The particles show a rich diversity of internal phase-segregation, that adapts to the surrounding conditions. Large amounts of hydrophobic pockets even lead to hydrophobic bridging between particles. The study shows ways towards tailored polyelectrolyte microgels with narrow dispersity, high charge density, as well as tailored and reconfigurable hydrophobic compartments and interactions.

Biocatalytic Feedback-Driven Temporal Programming of Self-Regulating Peptide Hydrogels.

Switchable self-assemblies respond to external stimuli with a transition between near-equilibrium states. Although being a key to present-day advanced materials, these systems respond rather passively, and do not display autonomous dynamics. For autonomous behavior, approaches must be found to orchestrate the time domain of self-assemblies, which would lead to new generations of dynamic and self-regulating materials. Herein, we demonstrate catalytic control of the time domain of pH-responsive peptide hydrogelators in a closed system. We program transient acidic pH states by combining a fast acidic activator with the slow, enzymatic, feedback-driven generation of a base (dormant deactivator). This transient state can be programmed over orders of magnitude in time. It is coupled to dipeptides to create autonomously self-regulating, dynamic gels with programmed lifetimes, which are used for fluidic guidance, burst release, and self-erasing rapid prototyping.

Generic concept to program the time domain of self-assemblies with a self-regulation mechanism.

Nature regulates complex structures in space and time via feedback loops, kinetically controlled transformations, and under energy dissipation to allow non-equilibrium processes. Although man-made static self-assemblies realize excellent control over hierarchical structures via molecular programming, managing their temporal destiny by self-regulation is a largely unsolved challenge. Herein, we introduce a generic concept to control the time domain by programming the lifetimes of switchable self-assemblies in closed systems. We conceive dormant deactivators that, in combination with fast promoters, enable a unique kinetic balance to establish an autonomously self-regulating, transient pH-state, whose duration can be programmed over orders of magnitude-from minutes to days. Coupling this non-equilibrium state to pH-switchable self-assemblies allows predicting their assembly/disassembly fate in time, similar to a precise self-destruction mechanism. We demonstrate a platform approach by programming self-assembly lifetimes of block copolymers, nanoparticles, and peptides, enabling dynamic materials with a self-regulation functionality.