How can membranes be disrupted

Journal of the American Chemical Society , 12 , Brash, Lei Wang, Hong Chen. ACS Nano , 14 11 , Kladko, Maxim A. Zakharzhevskii, Vladimir V. The Journal of Physical Chemistry Letters , 11 21 , Jonas, Michael A. ACS Materials Letters , 2 11 , Richardson, Frank Caruso. ACS Nano , 14 10 , Belling, Liv K. Kawakami, Jack Takahashi, Isaura M. Frost, Yao Gong, Thomas D. Young, Joshua A. Jackman, Steven J. Dissanayaka, John C.

Vaughan, Nitish Kunte, G. Mills, Guillaume M. Laurent, L. Adriana Avila. Juliano, Jean-Philippe Pellois. ACS Chemical Biology , 15 9 , Langmuir , 36 33 , Chemical Reviews , 11 , ACS Nano , 14 5 , Thompson, Tom W. Chemoenzymatic Semisynthesis of Proteins. Chemical Reviews , 6 , Bioconjugate Chemistry , 31 3 , ACS Nano , 14 2 , Schubert, Anchao Feng, San H. ACS Macro Letters , 9 2 , Dixit, Renate Starr, Morgan L. Dundon, Pranee I. Ballas, Hideaki Tsutsui, Stephen J.

Forman, Christine E. Brown, Masaru P. Nano Letters , 20 2 , Analytical Chemistry , 92 2 , Chemical Reviews , 24 , Kondow-McConaghy, Dakota J. Brock, Kristin Graham, Elizabeth C. Hager, Andrea L. ACS Chemical Biology , 14 12 , Nano Letters , 19 10 , Nanostructured Materials for Intracellular Cargo Delivery. Accounts of Chemical Research , 52 9 , Langmuir , 35 24 , Bioconjugate Chemistry , 30 4 , Smith, Laura I.

Selby, Angus P. Johnston, Georgina K. Bioconjugate Chemistry , 30 2 , Journal of the American Chemical Society , 50 , Metagenomic assembly deciphered the type-dependent effects of surfactants on the fates of antibiotics resistance genes during sludge fermentation and the underlying mechanisms.

Science of The Total Environment , , Voelcker , Roey Elnathan. Cellular nanotechnologies: Orchestrating cellular processes by engineering silicon nanowires architectures.

Pijpers , Heiner Friedrich , Loai K. Abdelmohsen , David S. Williams , Jan C. Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy.

Recent electroporation-based systems for intracellular molecule delivery. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed.

Disease results from failure of either the preventive or resealing mechanisms. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilisation through immune cells secretion of DNA networks, as we demonstrate by trapping E.

Proteolipid membranes represent the main means through which biological cells sustain chemical heterogeneity at the micro- and nanoscale, enabling most of their astounding responses. Several biomolecular agents, from small molecules to large protein complexes, have evolved the ability to sculpt lipid membranes changing their morphology, chemical composition, and physical properties.

The action of membrane-destabilizing biological agents is central to several biosensing, diagnostic, therapeutic, and synthetic-biological platforms. In view of this applicative potential, efforts have been devoted to engineering natural membrane-sculpting entities 15 , 17 , and even mimic their responses with purely synthetic analogues.

DNA nanotechnology, with its yet unparalleled ability to construct nanoscale motifs of near-arbitrary shape and responsiveness, offers an ideal toolkit for mimicking the complex responses of membrane-active biological machinery 18 , Simple amphiphilic DNA nanostructures have been utilized to engineer membrane adhesion and the formation of artificial tissues in both synthetic membranes 20 , 21 , 22 , 23 and cells Trans-membrane DNA constructs can mimic biological pores 25 , 26 and simple enzymes 27 , while membrane-adhering DNA-origami have been shown to establish local membrane curvature 28 , Here we demonstrate the design of responsive, DNA-based particles capable of permeabilizing and disrupting model lipid membranes when triggered by a molecular cue.

The particles self-assemble in a one-pot reaction from cholesterol-modified and plain synthetic oligonucleotides, designed to form three distinct motifs. This protective layer suppresses particle—membrane interactions while, combined with a tailored thermal annealing protocol, also enables precise control over particle size.

The corona can be removed via toehold-mediated strand displacement 30 , exposing the amphiphilic core. Unprotected particles readily aggregate with each other and deposit onto nearby membranes, leading to their enhanced permeability and rupture. The membrane-disrupting DNA particles mimic the action of biological toxins and could form the basis of smart therapeutic platforms, where target cells or pathogens are locally damaged if a molecular trigger is present.

To explore this analogy we demonstrate that aggregating DNA particles can capture and immobilize motile Escherichia coli E. Figure 1 a shows the three DNA motifs from which the core—shell particles are formed. We have recently introduced these amphiphilic nanostructures and explored their unique tendency to self-assemble into macroscopic single crystals with programmable structure and stimuli-responsiveness 35 , 36 , In the present contribution, however, C-stars are not exploited for their prowess to crystallize although we still observe crystals , but rather for the exquisite control one can afford on their self-assembly and the ability of C-star aggregates to disrupt lipid membranes.

As with previous designs, here each C-star assembles from four different oligonucleotides, making up the central junction, and four identical cholesterol-modified strands The former mediate the interactions with the corona, while the latter are included as potential anchoring points for molecular cargoes.

Core motifs C-stars assemble from four different strands forming the central junction blue and four identical cholesterol-functionalized oligonucleotides orange. Inner corona motifs green and outer corona motifs red each self-assemble from six different oligonucleotides. All DNA strands are mixed in stoichiometric ratios. At this stage, individual corona motifs assemble but remain detached from the particles. All six arms of the inner corona motifs end with ssDNA overhangs.

The sequences of all involved oligonucleotides are reported in Supplementary Table 1 , while Figs. The self-assembly pathway of protected DNA particles of prescribed size is sketched in Fig.

All ssDNA components of all motifs are included at once in stoichiometric ratios, namely, such that the molar ratio of assembled core, inner corona, and outer corona motifs is , and each motif can potentially connect to others leaving no overhangs unbound.

We shall see below that this is, in fact, unlikely to happen. The molar composition of the samples is reported in Supplementary Table 2. Aggregates of these dimensions would display negligible Brownian motion and be unsuitable as membrane-disrupting agents. The result is a dense hydrophilic brush that stabilizes the now core—shell DNA particles against further coalescence.

The two-step self-assembly protocol we introduce enables precise control over particles size, as demonstrated with differential dynamic microscopy DDM 38 , 39 in Fig. The particle size dependency on t g is well described by a standard diffusion-reaction growth model, and discussed in Supplementary Discussion 1 Particle size can be prescribed by tuning t g.

Dashed lines are fits to a diffusion-reaction growth model see Supplementary Discussion 1. Top Bright-field snapshots from the videos used in the DDM analysis, showing visibly larger aggregates for increasing t g. Contrast has been enhanced to enable visualization. A limited increase in size is observed, demonstrating particle stability against coalescence. Supplementary Fig. Selected micrographs represent data obtained in a single experiment.

Scale bars nm. In d and e core motifs are labeled with fluorescein cyan and outer corona motifs with Alexa Fluor red. For both d and e image acquisition was performed twice independently. Once the protective corona is in place, the particles remain stable at room temperature for extended periods of time.

Indeed, Fig. This slight post-assembly growth could be a consequence of a small degree of further particle coalescence or, for larger sizes, equilibration of the barometric particle distribution.

These effects bear no impact on the trends observed in Fig. Notably, particle samples can be stored long-term at room temperature, and retain their colloidal stability for at least 14 days post-assembly Supplementary Fig. Particles prepared at various t g have been imaged with TEM, and found to possess a roughly spherical morphology Fig.

In these conditions, fluorescent labeling of the core and outer-corona motifs enables a direct verification of the sought core—shell architecture with confocal microscopy, as demonstrated in Fig.

These can be removed via centrifugation and washing Fig. If a very slow annealing is performed, similar to the one we developed for C-star crystallization 36 , also the core-motifs used here are found to self-assemble into macroscopic single crystals, which however also feature a clear corona Fig.

With the motif architectures discussed to this point, the protective corona surrounding the particles can only be selectively displaced upon temperature increase.

However, a simple design update, summarized in Fig. Supplementary Figs. The confocal micrographs show a polyhedral core—shell particle before left and 1 min after trigger addition right. Core motifs are labeled with fluorescein cyan and outer corona motifs with Alexa Fluor red. Note the increase in the background fluorescence from free corona motifs after the corona displacement. The increase in R H observed upon trigger addition follows from the formation of larger aggregates, while the increase in the fluorescence trace is caused by their progressive sedimentation at the bottom of the cell, where the signal is recorded.

Red triangles indicate a control fluorescent trace measured in the absence of trigger. The constant and low value confirms the absence of spontaneous sedimentation. The displacement of the corona exposes the amphiphilic C-star cores, resuming particle coalescence and aggregate growth, as demonstrated in Fig.

Here, upon trigger addition, DDM detects a clear increase in mean hydrodynamic size. Aggregation is visible in both bright field and fluorescence microscopy, as is the formation of branched gel-like particle aggregates at later times Fig. These dense structures sink to the bottom of the experimental chamber as they grow, allowing us to track particle aggregation by monitoring fluorescence intensity on that plane.

If the protective corona is displaced from small diffusive particles in the presence of lipid-bilayer membranes, the exposed hydrophobic moieties of the C-star cores produce an attractive interaction with membranes, as sketched in Fig. Confocal micrographs in Fig. Upon addition of the trigger strand and displacement of the hydrophilic corona, unprotected particles adhere to each other and to the GUVs owing to the hydrophobic nature of cholesterol molecules.

Legends as in panel d Top: confocal micrographs of membrane rupture induced by unprotected particles. Core C-stars are shown in cyan, the lipid membrane in red. Bottom: unprotected particles significantly increase the spontaneous leakage rate compared to control samples of unperturbed GUVs and those exposed to stabilized DNA particles. Data shown in panels b , c , and d represent three and two independent experiments, respectively.

The mass concentration of GUVs was 3. Particle accumulation on the membranes is found to have a significant destabilizing effect on the GUVs, often leading to their bursting or collapse. A typical GUV disruption event, following triggered membrane—particle interaction, is shown in Fig. Here, as particles accumulate on the membrane surface and aggregates grow larger, the initial bilayer architecture is compromised and the lipids start mixing with the amphiphilic DNA aggregates.

This process initially leads to shrinkage of the GUV, likely due to embedding of the membrane material in the DNA aggregates, and ultimately to its complete collapse. In other cases, as shown in Supplementary Fig.

Polydispersity in membrane tension could be a discriminating factor between more fragile and resilient vesicles. In Fig. In the case of unprotected particles, the observed trend is readily explained: after the initial transient in which massive particle accumulation on the membranes takes place, the formation of large non-diffusive aggregates depletes the number of diffusive particles capable of targeting still intact GUVs.

Therefore, vesicles that happen to remain intact at this stage are no longer at risk. This hypothesis is confirmed by Fig. Here the asymptotic fraction of surviving vesicles decreases monotonically with increasing C-star concentration, consistent with the picture that more GUVs can be disrupted if more particles are present at early times, before the process is arrested by large-scale particle aggregation.

Consistent with this picture, Supplementary Fig. Interestingly, the trend is well fitted by the Hill equation, typically used to describe dose-dependent toxicity in live organisms The rapid saturation of the fraction of surviving GUVs in the sample with protected particles is more puzzling Fig.

The observed behavior could indicate a degree of heterogeneity in particle shielding. Specifically, a small fraction of the particles could be less well protected than others and able to disrupt the vesicles at early stages.

The fluorescein emission recorded in GUVs exposed to protected particles decreases at a rate very similar to that observed in the absence of any DNA, probably driven by a slow natural leakage rate and photobleaching.

In turn, corona displacement leads to a substantially higher rate of fluorescence decrease, ascribed to enhanced membrane leakage Fig. The same trends are observed in bulk fluorimetry experiments performed on Large Unilamellar Vesicles and exploiting the self-quenching properties of concentrated calcein solutions Supplementary Fig. Similar to what we observed for the ability of unprotected particles to destabilize GUVs, also permeabilization is enhanced if particle concentration increases, as summarized in Fig.

To test this potential functionality we performed experiments, summarized in Fig. To quantify the ability of the DNA-particle-network to inhibit bacteria motility we collected bright-field microscopy videos and extracted the pixel-intensity standard deviation over 7 consecutive frames. Colormaps in Fig. Once activated by the addition of the trigger strand, particles assemble into a sticky DNA network.

Swimming E. Core C-stars fluorescein are shown in cyan, E. See Supplementary Fig. Bottom: epifluorescence micrographs in the DNA cyan and E. An increasing signal indicates bacterial growth, which occurs in the presence of DNA particles but is absent for the control DNA-only sample, suggesting that E. Data shown in panels b — e was acquired in two independent experiments. Here, triggered and non-triggered samples display a comparable and steady fluorescence increase, confirming growth and highlighting that the clear difference in the motility parameter between the two samples Figs 5 c, d, and S16 is ascribable to bacteria immobilization, rather than a difference in growth rate.

In fact, E. This trend is confirmed by turbidity measurements shown in Supplementary Fig. Detailed inspection of the microscopy images, exemplified in Supplementary Fig. The latter point indicates a greater robustness of the cell-wall against destabilization by the amphiphilic DNA network compared to bare lipid membranes, likely as a consequence of presence of the protective lipopolysaccharide layer In summary, we present the rational design of a multi-component system of amphiphilic and unmodified DNA nanostructures that self-assemble in one-pot reactions to form core—shell particles with programmable size.

The stabilizing hydrophilic corona of the particles, instrumental in controlling their size via a two-step thermal annealing protocol, can be isothermally displaced by a DNA trigger. Corona removal leads to particle aggregation and, if lipid vesicles are present, to their disruption and enhanced permeability.

Vesicle disruption occurs following the interaction of the bilayer with the amphiphilic core of the unprotected DNA particles, and the formation of large DNA-lipid aggregates on the vesicles. The responsive membrane-active DNA particles mimic the action of molecular and nanoscale biological agents evolved to damage cell membranes, and could find application as targeted cell-killing nanodevices with therapeutic value.

At late aggregation stages, the DNA particles form extended networks that envelop cell-sized vesicles, and are able to arrest the motion of swimming E. The latter behavior is reminiscent of the ability of innate-immune cells to immobilize pathogens in sticky DNA webs 31 , 32 , 33 , 34 , and could form the basis of antimicrobial technologies.

Here, the removal of the corona could lead to an apoptosis-like response, with the permeabilization of the synthetic-cell membrane and release of its content, a highly sought-after functionality for conditional drug release. Finally, the approach introduced here for the production of size-controlled, stable, core—shell particles could be easily adapted to create smart drug delivery systems All sequences are shown in Supplementary Table 1.

Cholesterol modified and fluorescently labeled oligonucleotides were purified by the supplier using high-performance liquid chromatography HPLC , while the non-functionalized strands were purified using standard desalting.

Samples for assessing the self-assembly and properties of individual nanostructures were prepared by replacing the cholesterol-modified strands with oligonucleotides of identical sequence, but lacking the cholesterol moiety.

The concentration of each oligonucleotide are reported in Supplementary Table 2. For the preparation of macroscopic spherical aggregates Fig. Samples for the preparation of large polyhedral single-crystal aggregates Fig. Protected particles of controlled size Fig.

For experiments aimed at monitoring particle-size dependence on t g and their long-term stability Fig. Note that changing oligonucleotide concentrations with respect to the values reported in SI Table 2 may lead to reduced particle stability.