Frame-guided Assembly Research Articles

DNA-based nanostructures for RNA delivery.

RNA-based therapeutics have emerged as a promising approach for the treatment of various diseases, including cancer, genetic disorders, and infectious diseases. However, the delivery of RNA molecules into target cells has been a major challenge due to their susceptibility to degradation and inefficient cellular uptake. To overcome these hurdles, DNA-based nano technology offers an unprecedented opportunity as a potential delivery platform for RNA therapeutics. Due to its excellent characteristics such as programmability and biocompatibility, these DNA-based nanostructures, composed of DNA molecules assembled into precise and programmable structures, have garnered significant attention as ideal building materials for protecting and delivering RNA payloads to the desired cellular destinations. In this review, we highlight the current progress in the design and application of three DNA-based nanostructures: DNA origami, lipid-nanoparticle (LNP) technology related to frame guided assembly (FGA), and DNA hydrogel for the delivery of RNA molecules. Their biomedical applications are briefly discussed and the challenges and future perspectives in this field are also highlighted.

A facile method to prepare non-cationic mRNA lipid-nanoparticles based on frame guided assembly strategy

In recent years, lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) has become a promising tool in vaccine development and protein replacement therapy. However, the cationic or ionizable lipid components in the current LNPs also bring the concerns on toxicity and immunogenicity. In this study, we have developed a novel cationic lipid-independent strategy to prepare mRNA-LNPs through frame-guided assembly (FGA). A general mRNA compacting method has been demonstrated to fold long mRNA strands into compact mRNA particles, which was subsequently served as the amphiphilic frames to guide the assembly of phospholipid through hydrophobic interaction. We have also demonstrated high mRNA encapsulation efficiency, good stability and intracellular delivery capability of our mRNA-LNPs, which is expected as a promising tool in mRNA therapeutics and vaccines.

Preparing liposomes through frame guided assembly with high-loading functional nucleic acids.

Recently, a frame guided assembly (FGA) has been demonstrated as a robust tool to prepare liposomes with customized morphologies. However, the potential application of FGA liposomes in delivering nucleic acid drugs is still limited by the low payload. In this study, by systemically investigating the correlation between the leading hydrophobic group (LHG) density and the initial detergent concentration, it has been demonstrated that frames with a low LHG density, which may facilitate the increase of the load of the nucleic acid drug, could be guided to form liposomes at low initial detergent concentrations. By capitalizing on this phenomenon, FGA liposomes with a high loading of ASO/siRNA have been successfully prepared and applied to treat pathogenic genes.

Self-assembly of DNA-organic hybrid amphiphiles by frame-guided assembly strategies

It has been reported that DNA-Organic hybrid amphiphiles in aqueous solution could self-assemble into various morphologies including micelles, fibers and vesicles, where the hydrophobic parts intend to aggregate to avoid touching with water and the hydrophilic DNA are exposed at the shell. The regulation of their self-assembling morphologies and sizes usually needs to change the molecular inner structure, or assembling conditions and methods. Hence, it is a huge challenging to obtain aggregate with single variable of morphology or size. Inspired by the diversity of cell membrane morphologies in living organisms, a novel Frame-Guided Assembly (FGA) strategy is firstly proposed. The amphiphiles are guided to assemble along the leading hydrophobic groups (LHGs) anchored on the frame and finally form morphologies with certain shape and uniform size in accordance with the predesigned frame. It has been successfully applied to direct self-assembly behaviors of amphiphiles, including DNA and Dendron hybrids, DNA and Polymer hybrids and small molecules lipids. This strategy breaks the limits of traditional assembly method, and makes it possible to prepare aggregates with programmed geometry and size, and lays foundation for the application in the life science and potentially solves the issues of nano assemblies and cellular uptake mechanism.

Frame-Guided Assembly of Amphiphiles

ConspectusAmphiphiles tend to self-assemble into various structures and morphologies in aqueous environments (e.g., micelles, tubes, fibers, vesicles, and lamellae). These assemblies and their properties have made significant impact in traditional chemical industries, e.g., increasing solubility, decreasing surface tension, facilitating foaming, etc. It is well-known that the molecular structure and its environment play a critical role in the assembly process, and many theories, including critical packing factor, thermodynamic models, etc., have been proposed to explain and predict the assembly morphology. It has been recognized that the morphology of the amphiphilic assembly plays important roles in determining the functions, such as curvature-dependent biophysical (e.g., liposome fusion and fission) and biochemical (e.g., lipid metabolism and membrane protein trafficking) processes, size-related EPR (enhanced permeability and retention) effects, etc. Meanwhile, various nanomaterials have promised great potential in directing the arrangement of molecules, thus generating unique functions. Therefore, control over the amphiphilic morphology is of great interest to scientists, especially in nanoscale with the assistance of functional nanomaterials. However, how to precisely manipulate the sizes and shapes of the assemblies is challenged by the entropic nature of the hydrophobic interaction. Inspired by the "cytoskeleton-membrane protein-lipid bilayer" principle of the cell membrane, a strategy termed "frame-guided assembly (FGA)" has been proposed and developed to direct the arrangement of amphiphiles. The FGA strategy welcomes various nanomaterials with precisely controlled properties to serve as scaffolds. By introducing scattered hydrophobic molecules, which are defined as either leading hydrophobic groups (LHGs) or nucleation seeds onto a selected scaffold, a discontinuous hydrophobic trace along the scaffold can be outlined, which will further guide the amphiphiles in the system to grow and form customized two- or three-dimensional (2D/3D) membrane geometries.Topologically, the supporting frame can be classified as three types including inner-frame, outer-frame, and planar-frame. Each type of FGA assembly possesses particular advantages: (1) The inner-frame, similar to endoskeletons of many cellular structures, steadily supports the membrane from the inside and exposes the full surface area outside. (2) The outer-frame, on the other hand, molds and constrains the membrane-wrapped vesicles to regulate their size and shape. It also allows postengineering of the frame to precisely decorate and dynamically manipulate the membrane. (3) The planar-frame mediates the growth of the 2D membrane that profits from the scanning-probe microscopic characterization and benefits the investigation of membrane proteins.In this Account, we introduce the recent progress of frame-guided assembly strategy in the preparation of customized amphiphile assemblies, evaluate their achievements and limitations, and discuss prospective developments and applications. The basic principle of FGA is discussed, and the morphology controllability is summarized in the inner-, outer-, and planar-frame categories. As a versatile strategy, FGA is able to guide different types of amphiphiles by designing specific LHGs for given molecular structures. The mechanism of FGA is then discussed systematically, including the driving force of the assembly, density and distribution of the LHGs, amphiphile concentration, and the kinetic process. Furthermore, the applications of FGA have been developed for liposome engineering, membrane protein incorporation, and drug delivery, which suggest the huge potential of FGA in fabricating novel and functional complexes.

Delivery and controllable release of anti-sense DNA based on frame-guided assembly strategy

Using nanoparticles to deliver antisense oligonucleotides is a fabulous way which can solve the difficulty of penetrating cell membrane on account of high molecular weight and high negative charges. Herein, we have reported a frame-guided assembly strategy to prepare drug delivery system for anti-sense DNA. The in vitro experiments demonstrated that the anti-sense oligonucleotides vesicles perform good cytotoxicity to the cancer cells by downregulation of the expression of anti-apoptotic Bcl-2 gene, finally resulting in the apoptosis. It is feasible that taking advantage of the programmable assembly process and good stability of the frame-guided assembly (FGA) systems, this strategy can deliver a wide variety of drugs.

Frame-guided assembly from a theoretical perspective.

The molecular self-assembly of various structures such as micelles and vesicles has been the subject of comprehensive studies. Recently, a new approach to design these structures, the frame-guided assembly, has been developed to progress toward fabrics of predefined shape and size, following an initially provided frame of guiding elements. Here, we study the frame-guided assembly in a two-dimensional membrane via computer simulations based on a single-bead coarse grained surfactant model in continuous space. In agreement with the experiment, the assembly process already starts for surfactant concentrations below the critical micelle concentration. Furthermore, upon increasing temperature, the formation process gets more delocalized. Additionally, the assembly process of the resulting membrane plane is modeled by a lattice gas model. It displays a similar phenomenology but additionally allows for the derivation of analytical mean-field predictions. In this way, a fundamental understanding of frame-guided assembly can be gained.

Frame-guided assembly of DNA nanohydrogels via clamped hybridization chain reactions

Centering on gold nanoparticles, we developed a new DNA nanohydrogel construction strategy by combining frame-guided assembly principle with clamped hybridization chain reaction, and achieved size control by regulating gelation time and concentration. On-demand cargo loading and releasing were demonstrated by employing fluorescence labelled ssDNA sequences as models.

Construction of Liposomes Mimicking Cell Membrane Structure through Frame‐Guided Assembly

AbstractThe shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame‐Guided Assembly (FGA) strategy, we successfully prepared hetero‐liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton–membrane protein–lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications.

Construction of Liposomes Mimicking Cell Membrane Structure through Frame-Guided Assembly.

The shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame-Guided Assembly (FGA) strategy, we successfully prepared hetero-liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton-membrane protein-lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications.

PH-responsive Frame-Guided Assembly with hydrophobicity controllable peptide as leading hydrophobic groups

Frame-Guided Assembly (FGA) strategy has been recently reported to prepare vesicles with customized shapes and sizes. However, the effects of the interaction between leading hydrophobic groups (LHGs) and amphiphiles on the thermodynamic and kinetic control of the FGA process haven't been fully understood. In this work, we employed the pH low-insertion peptide (pHLIP) as the LHGs because its interaction with lipids could be finely tuned by pH and investigated the mechanism of FGA in detail. Our study demonstrated the peptide frames could successfully guide the assembly of lipids to form hetero-liposomes below the pH transition point owing to the strong peptide and lipids interaction. The pH-dependent kinetic controlled FGA process was proved and factors affecting the FGA process were also investigated systematically. We believe this pH-responsive FGA strategy improved our knowledge on the mechanism of the FGA and provide inspiration in understanding the sophisticated assembly behavior in life.

Redox triggered disassembly of frame-guided assemblies

The recently reported Frame Guided Assemblies (FGA) strategy has been successfully applied to prepare assemblies with designed shapes and sizes, which exhibit good stability, providing a potential platform for drug delivery. Herein to realize controllable drug release, this work has introduced the cleavable disulfide bond into the frame, which plays important roles in both guiding and supporting the formed assemblies. It has been demonstrated that upon stimuli, the FGA vesicles could be deformed to release the loaded molecules under physiological condition, demonstrating the importance of leading hydrophobic groups. Furthermore, the disassembly of 2D frame-guided assembly system has also been realized through the same method. These responsive FGA systems may provide new nano-vehicles for intracellular drug delivery which might benefit the design of responsive nanomaterials and controllable drug delivery systems.

The investigation of the stability of DNA-b-PPO vesicles formed through frame guided assembly

Frame-guided assembly is a novel assembly method which can control the shape and size of vesicles accurately. It has been reported that the DNA- b -PPO vesicles could be stable over two weeks. In this communication we have introduced restriction sites of EcoR1 in the interior DNA scaffold to the formed vesicles in order to further study their stability. Dynamic light scattering (DLS), transmission electron microscope (TEM) and fluorescence spectrum were used to characterize the digestion process. The final results revealed that the vesicles were still stable even after two weeks cultivated with EcoR1, which suggests potential in future biological applications.

A Modularly Designable Vesicle for Sequentially Multiple Loading.

The vesicle is one of the most intriguing platforms for drug delivery, which is believed to improve drug efficacy. In the past few decades, a great deal of materials have been explored to make vesicles, including lipids, block copolymers, dendrons, erythrocyte membranes, and even DNA. Other than shape and size control, most efforts are focused on achieving certain functions, for example, an abundance of stimuli-responsive features are introduced to vesicles, which can be applied to controllable release, such as pH, redox, light, radiation, enzyme etc. Besides, crosslinking or pegylation is used to increase vesicles' stability and elongate circulation time. By incorporating affinity ligands, vesicles can further accumulate to diseased cells or tissues to achieve targeting properties. Recently, multidrug delivery is believed to show a synergy effect in cancer therapy and has become a new direction in this field. However, coloading hydrophilic-hydrophobic small molecules, oligonucleotides, and peptides in the same size- and shape-controlled vesicle through a stepwise manner with high efficiency is still challenging. Herein, a modularly designable vesicle is reported for sequential multiple loading based on frame-guided assembly, which is believed to be an outstanding platform for drug delivery in the future.

An Addressable 2D Heterogeneous Nanoreactor to Study the Enzyme-Catalyzed Reaction at the Interface.

Membrane plays significant role in cellular enzymatic reactions. To better understand its function on membrane integral or bound enzymes, DNA origami and frame-guided assembly strategy are combined to construct a given-size, addressable enzyme-containing nanomembrane as a heterogeneous reactor to explore the enzymatic catalyst reaction on the membrane. The enzymes in the membrane are located precisely. This new kind of membrane can enrich hydrophobic substrate molecules in aqueous solution to the embedded enzymes. Otherwise, this nanomembrane shows the capability of substrate selectivity, which plays important role in the highly efficient and specific properties of enzymes in vivo.

Cuboid Vesicles Formed by Frame‐Guided Assembly on DNA Origami Scaffolds

AbstractWe describe the use of a frame‐guided assembly (FGA) strategy to construct cuboid and dumbbell‐shaped hetero‐vesicles on DNA origami nanostructure scaffolds. These are achieved by varying the design of the DNA origami scaffolds that direct the distribution of the leading hydrophobic groups (LHG). By careful selection of LHGs, different types of amphiphiles (both polymer and small‐molecule surfactants) were guided to form hetero‐vesicles, demonstrating the versatility of the FGA strategy and its potential to construct asymmetric and dynamic hetero‐vesicle assemblies with complex DNA nano‐scaffolds.

Cuboid Vesicles Formed by Frame-Guided Assembly on DNA Origami Scaffolds.

We describe the use of a frame-guided assembly (FGA) strategy to construct cuboid and dumbbell-shaped hetero-vesicles on DNA origami nanostructure scaffolds. These are achieved by varying the design of the DNA origami scaffolds that direct the distribution of the leading hydrophobic groups (LHG). By careful selection of LHGs, different types of amphiphiles (both polymer and small-molecule surfactants) were guided to form hetero-vesicles, demonstrating the versatility of the FGA strategy and its potential to construct asymmetric and dynamic hetero-vesicle assemblies with complex DNA nano-scaffolds.

Precisely Controlled 2D Free-Floating Nanosheets of Amphiphilic Molecules through Frame-Guided Assembly.

2D assembly of amphiphilic molecules in aqueous solution is a challenging and intriguing topic as it is normally thermodynamically unfavorable. However, through frame-guided assembly strategy and using DNA origami as the frame, monodispersed and shape-defined nanosheets are prepared. As leading hydrophobic groups (LHGs) are anchored on the frames, amphiphilic molecules in aqueous solution are guided to assemble in the hydrophobic region. By adjusting the distribution of the LHGs, the size and shape of the assemblies can be controlled precisely.

Frame-Guided Assembly of Amphiphiles.

Frame-guided assembly, a recently discovered strategy for amphiphilic assembly, is discussed as a strategy for constructing vesicle assemblies with programmed geometries and dimensions under identical surrounding conditions. The strategy is inspired by the cytoskeletal-membrane protein-lipid bilayer structure and shows great potential in the understanding and controlling of the amphiphilic assembly process. Both the principles and basic requirements are discussed, along with recently reported examples. The prospects and potential investigations of frame-guided assembly are also proposed.

Using Small Molecules to Prepare Vesicles with Designable Shapes and Sizes via Frame‐Guided Assembly Strategy

Following the principle of frame-guided assembly (FGA), a small amphiphilic molecule, sodium dodecyl sulfate, is shown to form vesicles with cholesterol as the leading hydrophobic group. These findings not only demonstrate the generality of the FGA but also provide a clue to understand the formation mechanism of the cell membrane, and even the origin of life.