Structure and kinetics of lipid–nucleic acid complexes

The structure and function of lipid-based complexes (lipoplexes) have been widely investigated as cellular delivery vehicles for nucleic acids—DNA and siRNA. Transfection efficiency in applications such as gene therapy and gene silencing has been clearly linked to the local, nano-scale organization of the nucleic acid in the vehicle, as well as to the global properties (e.g. size) of the carriers. This review focuses on both the structure of DNA and siRNA complexes with cationic lipids, and the kinetics of structure evolution during complex formation.     

Design and enhanced gene silencing activity of spherical 2′-fluoroarabinose nucleic acids (FANA-SNAs)

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) – nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior – have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.

Optimizing FANA modified spherical nucleic acids (FANA-SNAs) for highly efficient delivery of nucleic acid therapeutics.     

Spiegelbildliche Nukleinsäuren als pharmakologische Wirkstoffe

Within the last decades we witnessed the discovery of a number of mechanisms that enable the use of nucleic acids for therapeutic purposes. Small RNA and DNA molecules can be used to specifically suppress the expression of individual genes. Aptamers provide an alternative to monoclonal antibodies. A prerequisite for the pharmacological use of nucleic acids is an enhanced stability towards the body's degrading enzymes. This can be achieved for instance by employing non‐natural mirror‐image nucleic acids. The article describes the basic principles of stereochemistry underlying this approach and shows how these translate into the discovery of mirror‐image aptamers. Furthermore, it explains why the stereospecificity of Watson‐Crick base pairing has precluded mirror‐image nucleic acids from gene silencing methods and introduces a new approach that may help to overcome this.     

A novel polyethyleneimine-decorated FeOOH nanoparticle for efficient siRNA delivery

Despite the promising prospect of small interfering RNA(siRNA) for the treatment of diverse diseases,it remains challenging to develop novel delive ry materials to desired tissues and cells.In this study,a novel iron oxyhydroxide(FeOOH) nanoparticle(NP) whose surface was modified with branched polyetherimide(PEI) was developed to deliver siRNA into the cancer cells.It was demonstrated that PEI-FeOOH(PFeOOH) efficiently complexed siRNA,mediated effective cellular uptake and endosomal escape,thereby triggering robust gene silencing in vitro.In addition,PFeOOH/siRNA formulation loading with anti-RRM2 siRNA effectively inhibited the growth of tumor tissues,and exhibited excellent safety profiles in vivo.Therefore,this study conceptually provided a FeOOH-based nucleic acid delivery vesicle which can potentially use to achieve diagnosis and therapy simultaneously.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

Cationic liposome-nucleic acid complexes: liquid crystal phases with applications in gene therapy

Cationic liposome (CL) carriers of nucleic acids are primarily studied because of their applications in gene delivery and gene silencing with CL-DNA and CL-siRNA (short-interfering RNA) complexes, respectively, and their implications to ongoing clinical gene therapy trials worldwide. A series of synchrotron-based small-angle-x-ray scattering studies, dating back to 1997, has revealed that CL-nucleic acid complexes spontaneously assemble into distinct novel liquid crystalline phases of matter. Significantly, transfection efficiency (TE; a measure of expression of an exogenous gene that is transferred into the cell by the lipid carrier) has been found to be dependent on the liquid crystalline structure of complexes, with lamellar complexes showing strong dependence on membrane charge density (σ(M)) and non-lamellar complexes exhibiting TE behavior independent ofσ(M). The review describes our current understanding of the structures of different liquid crystalline CL-nucleic acid complexes including the recently described gyroid cubic phase of CL-siRNA complexes used in gene silencing. It further makes apparent that the long-term goal of developing optimized liquid crystalline CL-nucleic acid complexes for successful medical applications requires a comprehensive understanding of the nature of the interactions of distinctly structured complexes with cell membranes and events leading to release of active nucleic acids within the cell cytoplasm.     

Injectable Biodegradable Chitosan‐Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide‐based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low‐transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan‐alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold‐mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex‐loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN‐γ secretion are seen with in vivo scaffold‐mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold‐mediated mRNA lipoplex delivery, while protein‐based immunization did not elicit IgG production until 2 weeks post‐injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.     

A Crosslinked Nucleic Acid Nanogel for Effective siRNA Delivery and Antitumor Therapy

Functional siRNAs are employed as cross‐linkers to direct the self‐assembly of DNA‐grafted polycaprolactone (DNA‐g‐PCL) brushes to form spherical and nanosized hydrogels via nucleic acid hybridization in which small interfering RNAs (siRNAs) are fully embedded and protected for systemic delivery. Owing to the existence of multivalent mutual crosslinking events inside, the crosslinked nanogels with tunable size exhibit not only good thermostability, but also remarkable physiological stability that can resist the enzymatic degradation. As a novel siRNA delivery system with spherical nucleic acid (SNA) architecture, the crosslinked nanogels can assist the delivery of siRNAs into different cells without any transfection agents and achieve the gene silencing effectively both in vitro and in vivo, through which a significant inhibition of tumor growth is realized in the anticancer treatment.     

Polymers for DNA delivery

Nucleic acid delivery has many applications in basic science, biotechnology, agriculture, and medicine. One of the main applications is DNA or RNA delivery for gene therapy purposes. Gene therapy, an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or expression inhibition of undesired genes. Following two decades of research, there are two major methods for delivery of genes. The first method, considered the dominant approach, utilizes viral vectors and is generally an efficient tool of transfection. Attempts, however, to resolve drawbacks related with viral vectors (e.g., high risk of mutagenicity, immunogenicity, low production yield, limited gene size, etc.), led to the development of an alternative method, which makes use of non-viral vectors. This review describes non-viral gene delivery vectors, termed "self-assembled" systems, and are based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids. It introduces the most important cationic polymers used for gene delivery. A transition from in vitro to in vivo gene delivery is also presented, with an emphasis on the obstacles to achieve successful transfection in vivo.     

Antisense and antigene inhibition of gene expression by cell-permeable oligonucleotide-oligospermine conjugates

Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.     

脂质纳米颗粒的制备及在基因治疗中的应用

基因治疗已经成为人类治疗疾病的一种重要手段.然而,为了将基因药物用于临床,需要更加复杂的递送系统.脂质纳米颗粒(LNPs)系统是目前领先的非病毒递送系统,在治疗诊断学方面取得了许多令人鼓舞的进展,其具有实现基因药物临床治疗应用的潜力.由于LNPs纳米尺寸的优势及类脂化合物的生物相容性和生物降解性,LNPs能够克服阻碍基...     

Rational design of smart supramolecular assemblies for gene delivery: chemical challenges in the creation of artificial viruses

Polymeric materials have been extensively developed as a delivery vehicle for nucleic acids over the past two decades. Many previous studies have demonstrated that synthetic delivery vehicles can be highly functionalized by chemical approaches to overcome biological barriers in nucleic acid delivery, similar to viruses. Based on our current knowledge, this tutorial review describes rational strategies in the design of polymeric materials to achieve construction of the versatile vehicles, that is "artificial viruses", for successful gene therapy, especially focusing on the chemical structures with the minimal adverse effects.     

Core–Shell Structured Chitosan-Polyethylenimine Nanoparticles for Gene Delivery: Improved Stability,Cellular Uptake,and Transfection Efficiency

The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan–polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.     

Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next‐generation delivery materials for RNA therapeutics.     

Reducible Dimeric Conjugates of Small Internally Segment Interfering RNA for Efficient Gene Silencing

The condensation of nucleic acids into compact nanoparticles with cationic carriers is a powerful tool for translocating exogenous nucleic acids into cells. To date, most efforts have been focused on the development of novel gene carriers for safe and efficient gene delivery. However, small interfering RNA (siRNA) is generally not strongly associated with cationic carriers due to its stiff structure and low spatial charge density. To overcome this limitation, this work introduces a well‐defined dimeric conjugate of small internally segment interfering RNA (sisiRNA) linked via a disulfide bond for enhanced cellular uptake and gene silencing. Dimeric sisiRNA is synthesized through oxidizing two monomeric sisiRNA molecules, each of which consists of a sense strand carrying a nick and an antisense strand modified with a thiol group at the 3′‐end. The nick in the sense strand enables the dimeric sisiRNA to be more effectively condensed into nanosized complexes due to the increased structural flexibility, which results in a higher gene silencing efficiency compared with the dimeric siRNA containing the intact sense strands. The results indicate that the discontinuity of the sense strands is a simple method of adding more flexibility to various siRNA‐based nanostructures for enhanced gene silencing.

     

Ultra‐pH‐Responsive and Tumor‐Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti‐Cancer Efficacy

RNA interference (RNAi) gene silencing technologies have shown significant potential for treating various diseases, including cancer. However, clinical success in cancer therapy remains elusive, mainly owing to suboptimal in vivo delivery of RNAi therapeutics such as small interference RNA (siRNA) to tumors. Herein, we developed a library of polymers that respond to a narrow pH change (ultra‐pH‐responsive), and demonstrated the utility of these materials in targeted and deep tumor‐penetrating nanoparticle (NP) for in vivo RNAi. The new NP platform is mainly composed of the following key components: i) internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration; ii) polyethylene glycol (PEG) chains to prolong blood circulation; and iii) sharp pH‐responsive hydrophobic polymer to improve endosome escape. Through systematic studies of structure–function relationship, the optimized RNAi NPs (<70 nm) showed efficient gene silencing and significant inhibition of tumor growth with negligible toxicities in vivo.     

Light-Induced Self-Escape of Spherical Nucleic Acid from Endo/Lysosome for Efficient Non-Cationic Gene Delivery

Developing non-cationic gene carriers and achieving efficient endo/lysosome escape of functional nucleic acids in cytosol are two major challenges faced by the field of gene delivery. Herein, we demonstrate the concept of self-escape spherical nucleic acid (SNA) to achieve light controlled non-cationic gene delivery with sufficient endo/lysosome escape capacity. In this system, Bcl-2 antisense oligonucleotides (OSAs) were conjugated onto the surface of aggregation-induced emission (AIE) photosensitizer (PS) nanoparticles to form core–shell SNA. Once the SNAs were taken up by tumor cells, and upon light irradiation, the accumulative ¹O₂ produced by the AIE PSs ruptured the lysosome structure to promote OSA escape. Prominent in vitro and in vivo results revealed that the AIE-based core–shell SNA could downregulate the anti-apoptosis protein (Bcl-2) and induce tumor cell apoptosis without any transfection reagent.     

Self-organization of nucleic acids in lipid constructs

Lipids and nucleic acids (NAs) can hierarchically self-organize into a variety of nanostructures of increasingly complex geometries such as the 1D lamellar, 2D hexagonal, and 3D bicontinuous cubic phases. The diversity and complexity of those lipid–NA assemblies are interesting from a fundamental perspective as well as being relevant to the performance in gene delivery and gene silencing applications. The finding that not only the chemical make of the lipid–NA constructs, but their actual supramolecular organization, affects their gene transfection and silencing efficiencies has inspired physicists, chemists, and engineers to this field of research. At the moment it remains an open question how exactly the different lipid–NA structures interact with cells and organelles in order to output an optimal response. This article reviews our current understanding of the structures of different lipid–NA complexes and the corresponding cellular interaction mechanisms. The recent advances in designing optimal lipid–based NA carriers will be introduced with an emphasis on the structure–function relations.