Controlling the Reversible Assembly of Liposomes through a Multistimuli Responsive Anchored DNA.

We present a novel approach to reversibly control the assembly of liposomes through an anchored multistimuli responsive DNA oligonucleotide decorated with an azobenzene moiety (AZO-ON1). We show that liposomes assembly can be simultaneously controlled by three external stimuli: light, Mg(2+), and temperature. (i) Light alters the interaction of AZO-ON1 with liposomes, which influences DNA coating and consequently liposomes assembly. (ii) Mg(2+) induces the assembly, hence variation in its concentration enables for reversibility. (iii) Double-stranded AZO-ON1 is more efficient than single-stranded AZO-ON1 in triggering the assembly of liposomes and temperature has been used for controllable assembly through DNA thermal denaturation. Our multiresponsive AZO-ON1 represents a unique example in which multiple stimuli can be simultaneously applied to regulate the reversible assembly of liposomes.

Blockable Zn10 L15 Ion Channels through Subcomponent Self-Assembly.

Metal-organic anion channels based on Zn10 L15 pentagonal prisms have been prepared by subcomponent self-assembly. The insertion of these prisms into lipid membranes was investigated by ion-current and fluorescence measurements. The channels were found to mediate the transport of Cl- anions through planar lipid bilayers and into vesicles. Tosylate anions were observed to bind and plug the central channels of the prisms in the solid state and in solution. In membranes, dodecyl sulfate blocked chloride transport through the central channel. Our Zn10 L15 prism thus inserts into lipid bilayers to turn on anion transport, which can then be turned off through addition of the blocker dodecyl sulfate.

DNA-Tile Structures Induce Ionic Currents through Lipid Membranes.

Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

Voltage-dependent properties of DNA origami nanopores.

We show DNA origami nanopores that respond to high voltages by a change in conformation on glass nanocapillaries. Our DNA origami nanopores are voltage sensitive as two distinct states are found as a function of the applied voltage. We suggest that the origin of these states is a mechanical distortion of the DNA origami. A simple model predicts the voltage dependence of the structural change. We show that our responsive DNA origami nanopores can be used to lower the frequency of DNA translocation by 1 order of magnitude.

Self-assembly modulation in ionic PAMAM derivatives.

The self-assembly behaviour both in the bulk and water of a series of amphiphilic dendrimers constituted by second generation PAMAM ionically functionalized with different amounts of myristic acid is shown here. The number of acids in the dendrimer determines the liquid-crystal properties and the structural parameters of their supramolecular organization. Most of them present mesomorphism, organizing in a smectic A mesophase, with a layer spacing decreasing when increasing the number of acids. All these dendrimers form well-defined nanoobjects in water. Micelles and broken lamellae have been found for compounds with low acid contents. In contrast, dendrimers with higher fatty acid contents self-assemble forming nanospheres with a lamellar nanostructure. All compounds are able to trap the hydrophobic molecule 9,10-diphenylanthracene independent of the acid contents. Interestingly, the trapped hydrophobic molecule dominates the self-assembly trend of the dendrimers with low acid contents and thus different nanoobjects are found after the encapsulation.

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.

Lipid-coated nanocapillaries for DNA sensing.

We report a simple and efficient way to accomplish the chemical modification of glass nanopores by means of lipid self-assembly. Lipid coating improves the success rate of these glass nanopores as biosensors to detect λ-DNA.

Heparin-Coated Dendronized Hyperbranched Polymers for Antimalarial Targeted Delivery.

The rampant evolution of resistance in Plasmodium to all existing antimalarial drugs calls for the development of improved therapeutic compounds and of adequate targeted delivery strategies for them. Loading antimalarials in nanocarriers specifically targeted to the parasite will contribute to the administration of lower overall doses, with reduced side effects for the patient, and of higher local amounts to parasitized cells for an increased lethality toward the pathogen. Here, we report the development of dendronized hyperbranched polymers (DHPs), with capacity for antimalarial loading, that are coated with heparin for their specific targeting to red blood cells parasitized by Plasmodium falciparum. The resulting DHP-heparin complexes exhibit the intrinsic antimalarial activity of heparin, with an IC50 of ca. 400 nM, added to its specific targeting to P. falciparum-infected (vs noninfected) erythrocytes. DHP-heparin nanocarriers represent a potentially interesting contribution to the limited family of structures described so far for the loading and targeted delivery of current and future antimalarial compounds.

Dendron-functionalised hyperbranched bis-MPA polyesters as efficient non-viral vectors for gene therapy in different cell lines.

Gene therapy has become a relevant tool in the biomedical field to treat or even prevent some diseases. The effective delivery of genetic material into the cell remains a crucial step to succeed in this purpose. In the search for efficient non-viral vectors, a series of amino-terminated dendronized hyperbranched polymers (DHPs) of different generations based either on bis-MPA or bis-GMPA have been designed. All of them have demonstrated an accurate ability to complex two types of genetic materials, a plasmid DNA and a siGFP, yielding dendriplexes. Moreover, some of them have proved to be able to deliver the genetic material inside the cells, resulting in the effective accomplishment of the desired genetic modification and improving the activity of some commercial transfection reagents. Different cell lines, including cancer and mesenchymal stem cells, have been studied here to evaluate the ability of DHPs as vectors for transfection. Treatments based on mesenchymal stem cells are gaining importance due to their pluripotency. Thus, it is of special relevance to introduce a genetic modification into a mesenchymal cell line as it allows it to act over a wide spectrum of tissues after inducing cellular differentiation.

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.

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.

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.

Micellar Nanocarriers from Dendritic Macromolecules Containing Fluorescent Coumarin Moieties.

The design of efficient drug-delivery vehicles remains a big challenge in materials science. Herein, we describe a novel class of amphiphilic hybrid dendrimers that consist of a poly(amidoamine) (PAMAM) dendritic core functionalized with bisMPA dendrons bearing cholesterol and coumarin moieties. Their self-assembly behavior both in bulk and in water was investigated. All dendrimers exhibited smectic A or hexagonal columnar liquid crystal organizations, depending on the generation of the dendrimer. In water, these dendrimers self-assembled to form stable spherical micelles that could encapsulate Nile Red, a hydrophobic model compound. The cell viability in vitro of the micelles was studied in HeLa cell line, and proved to be non-toxic up to 72 h of incubation. Therefore, these spherical micelles allow the encapsulation of hydrophobic molecules, and at the same time provided fluorescent traceability due to the presence of coumarin units in their chemical structure, demonstrating the potential of these dendrimers as nanocarriers for drug-delivery applications.

Janus Dendrimers to Assess the Anti-HCV Activity of Molecules in Cell-Assays.

The use of nanocarriers has been revealed as a valid strategy to facilitate drug bioavailability, and this allows for expanding the drug libraries for the treatment of certain diseases such as viral diseases. In the case of Hepatitis C, the compounds iopanoic acid and 3,3',5-triiodothyroacetic acid (or tiratricol) were identified in a primary screening as bioactive allosteric inhibitors of viral NS3 protease, but they did not exhibit accurate activity inhibiting viral replication in cell-based assays. In this work, dendritic nanocarriers are proposed due to their unique properties as drug delivery systems to rescue the bioactivity of these two drugs. Specifically, four different amphiphilic Janus dendrimers synthesized by combining 2,2'-bis(hydroxymethyl)propionic acid (bis-MPA) and 2,2'-bis(glyciloxy)propionic acid (bis-GMPA) functionalized with either hydrophilic or lipophilic moieties at their periphery were used to entrap iopanoic acid and tiratricol. Interestingly, differences were found in the loading efficiencies depending on the dendrimer design, which also led to morphological changes of the resulting dendrimer aggregates. The most remarkable results consist of the increased water solubility of the bioactive compounds within the dendrimers and the improved antiviral activity of some of the dendrimer/drug aggregates, considerably improving antiviral activity in comparison to the free drugs. Moreover, imaging studies have been developed in order to elucidate the mechanism of cellular internalization.

Promising nanomaterials in the fight against malaria.

For more than one hundred years, several treatments against malaria have been proposed but they have systematically failed, mainly due to the occurrence of drug resistance in part resulting from the exposure of the parasite to low drug doses. Several factors are behind this problem, including (i) the formidable barrier imposed by the Plasmodium life cycle with intracellular localization of parasites in hepatocytes and red blood cells, (ii) the adverse fluidic conditions encountered in the blood circulation that affect the interaction of molecular components with target cells, and (iii) the unfavorable physicochemical characteristics of most antimalarial drugs, which have an amphiphilic character and can be widely distributed into body tissues after administration and rapidly metabolized in the liver. To surpass these drawbacks, rather than focusing all efforts on discovering new drugs whose efficacy is quickly decreased by the parasite's evolution of resistance, the development of effective drug delivery carriers is a promising strategy. Nanomaterials have been investigated for their capacity to effectively deliver antimalarial drugs at local doses sufficiently high to kill the parasites and avoid drug resistance evolution, while maintaining a low overall dose to prevent undesirable toxic side effects. In recent years, several nanostructured systems such as liposomes, polymeric nanoparticles or dendrimers have been shown to be capable of improving the efficacy of antimalarial therapies. In this respect, nanomaterials are a promising drug delivery vehicle and can be used in therapeutic strategies designed to fight the parasite both in humans and in the mosquito vector of the disease. The chemical analyses of these nanomaterials are essential for the proposal and development of effective anti-malaria therapies. This review is intended to analyze the application of nanomaterials to improve the drug efficacy on different stages of the malaria parasites in both the human and mosquito hosts.

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.

Proton conductive ionic liquid crystalline poly(ethyleneimine) polymers functionalized with oxadiazole.

Two novel series of ionic liquid crystal polymers that display proton conductive properties are presented here. These materials are based on linear (l-PEI) or branched (b-PEI) poly(ethyleneimine) functionalized with unsymmetrical oxadiazole carboxylic acids derived from 1,3,4-oxadiazole (1,3,4-OXA m ) or 1,2,4-oxadiazole (1,2,4-OXA m ). The subscript "m" indicates the length of the spacer between the rigid moiety and the carboxyl group, namely m = 4 and 10. The occurrence of proton transfer from the carboxylic acid to the amine groups was confirmed by FTIR and NMR measurements. The liquid crystalline properties were investigated by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffraction (XRD). All ionic complexes displayed enantiotropic smectic A mesophases and in the case of the l-PEI derivatives a nematic phase was also observed at high temperatures. All investigated derivatives presented good proton conductivity values as determined by electrochemical impedance spectroscopy (EIS). Therefore, these ionic LC hyperbranched polymers represent an effective approach for the preparation of proton-transporting polymeric materials with potential applications in electrochemical devices.

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.

Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug - we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.