Assessing the influence of small structural modifications in simple DNA-based nanostructures on their role as drug nanocarriers.

DNA nanotechnology leverages Watson-Crick-Franklin base-pairing interactions to build complex DNA-based nanostructures (DNS). Due to DNA specific self-assembly properties, DNS can be designed with a total control of their architecture, which has been demonstrated to have an impact on the overall DNS features. Indeed, structural properties such as the shape, size and flexibility of DNS can influence their biostability as well as their ability to internalise into cells. We present here two series of simple DNS with small and precise variations related to their length or flexibility and study the influence that these structural changes have on their overall properties as drug nanocarriers. Results indicate that shorter and more flexible DNS present higher stability towards nuclease degradation. These structural changes also have a certain effect on their cell internalisation ability and drug release rate. Consequently, drug-loaded DNS cytotoxicity varies according to the design, with lower cell viability values obtained in the DNS exhibiting faster drug release and larger cell interaction rates. In summary, small changes in the structure of simple DNS can have an influence on their overall capabilities as drug nanocarriers. The effects reported here could guide the design of simple DNS for future therapeutic uses.

A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aß42 Aggregation.

The aggregation of the amyloid ß (Aß) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aß deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aß assemblies. Because these intracellular Aß aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aß is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aß (Aß42). Intracellularly, Aß42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aß, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aß42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aß42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aß aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aß.

DNA-Liposome Hybrid Carriers for Triggered Cargo Release.

The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA-liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics.

One-Step Generation of Multisomes from Lipid-Stabilized Double Emulsions.

Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.

DNA-Based Nanocarriers to Enhance the Optoacoustic Contrast of Tumors In Vivo.

Optoacoustic tomography (OT) enables non-invasive deep tissue imaging of optical contrast at high spatio-temporal resolution. The applications of OT in cancer imaging often rely on the use of molecular imaging contrast agents based on near-infrared (NIR) dyes to enhance contrast at the tumor site. While these agents afford excellent biocompatibility and minimal toxicity, they present limited optoacoustic signal generation capability and rapid renal clearance, which can impede their tumor imaging efficacy. In this work, a synthetic strategy to overcome these limitations utilizing biodegradable DNA-based nanocarrier (DNA-NC) platforms is introduced. DNA-NCs enable the incorporation of NIR dyes (in this case, IRDye 800CW) at precise positions to enable fluorescence quenching and maximize optoacoustic signal generation. Furthermore, these DNA-NCs show a prolonged blood circulation compared to the native fluorophores, facilitating tumor accumulation by the enhanced permeability and retention (EPR) effect. In vivo imaging of tumor xenografts in mice following intravenous administration of DNA-NCs reveals enhanced OT signals at 24 h when compared to free fluorophores, indicating promise for this method to enhance the optoacoustic signal generation capability and tumor uptake of clinically relevant NIR dyes.

Continuous Flow Reactors from Microfluidic Compartmentalization of Enzymes within Inorganic Microparticles.

Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.

Complexity in Lipid Membrane Composition Induces Resilience to Aß42 Aggregation.

The molecular origins of Alzheimer's disease are associated with the aggregation of the amyloid-ß peptide (Aß). This process is controlled by a complex cellular homeostasis system, which involves a variety of components, including proteins, metabolites, and lipids. It has been shown in particular that certain components of lipid membranes can speed up Aß aggregation. This observation prompts the question of whether there are protective cellular mechanisms to counterbalance this effect. Here, to address this issue, we investigate the role of the composition of lipid membranes in modulating the aggregation process of Aß. By adopting a chemical kinetics approach, we first identify a panel of lipids that affect the aggregation of the 42-residue form of Aß (Aß42), ranging from enhancement to inhibition. We then show that these effects tend to average out in mixtures of these lipids, as such mixtures buffer extreme aggregation behaviors as the number of components increases. These results indicate that a degree of quality control on protein aggregation can be achieved through a mechanism by which an increase in the molecular complexity of lipid membranes balances opposite effects and creates resilience to aggregation.

Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly.

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials, advanced hybrid carriers can be derived. Here, we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy, and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development toward the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarriers.

Modulating the Mechanical Performance of Macroscale Fibers through Shear-Induced Alignment and Assembly of Protein Nanofibrils.

Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.

An active DNA-based nanoprobe for photoacoustic pH imaging.

We report an active DNA construct capable of probing pH through a photoacoustic (PA) ratiometric analysis approach. Our nanoprobe enables different PA readout in tissue mimicking phantoms in the range between pH 6.8 to 7.8 at physiologically relevant sodium concentrations. Thus, it represents a promising platform to probe pH values relevant to the tumor microenvironment using PA.

A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

Distance dependent photoacoustics revealed through DNA nanostructures.

Molecular rulers that rely on the Förster resonance energy transfer (FRET) mechanism are widely used to investigate dynamic molecular processes that occur on the nanometer scale. However, the capabilities of these fluorescence molecular rulers are fundamentally limited to shallow imaging depths by light scattering in biological samples. Photoacoustic tomography (PAT) has recently emerged as a high resolution modality for in vivo imaging, coupling optical excitation with ultrasound detection. In this paper, we report the capability of PAT to probe distance-dependent FRET at centimeter depths. Using DNA nanotechnology we created several nanostructures with precisely positioned fluorophore-quencher pairs over a range of nanoscale separation distances. PAT of the DNA nanostructures showed distance-dependent photoacoustic signal enhancement and demonstrated the ability of PAT to reveal the FRET process deep within tissue mimicking phantoms. Further, we experimentally validated these DNA nanostructures as a novel and biocompatible strategy to augment the intrinsic photoacoustic signal generation capabilities of small molecule fluorescent dyes.