Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.     

Dendrimeric siRNA for Efficient Gene Silencing

Programmable molecular self‐assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second‐generation dendrimers of siRNA can be effectively complexed with a low‐molecular‐weight, cationic polymer (poly(β‐amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.     

Impact of lipid substitution on assembly and delivery of siRNA by cationic polymers

Characterization of a polymer library engineered to enhance their ability to protect and deliver their nucleotide cargo to the cells is reported. The ζ-potential continuously increased with higher polymer:siRNA weight ratio, and the ζ-potential of lipid-modified polymers:siRNA complexes were higher than PEI2 at all ratios. At polymer:siRNA ratio of 1:1, all lipid-substituted polymers showed complete protection against degradation. Lipid-modified polymers significantly increased the cellular uptake of siRNA complexes and down-regulation of GAPDH and P-gp (max. 66% and 67%, respectively). The results indicate that hydrophobic modification of low molecular PEI could render this otherwise ineffective polymer to a safe effective delivery system for intracellular siRNA delivery and protein silencing.     

Systemic Delivery of Bc12‐Targeting siRNA by DNA Nanoparticles Suppresses Cancer Cell Growth

Short interfering RNA (siRNA) is a promising molecular tool for cancer therapy, but its clinical success is limited by the lack of robust in vivo delivery systems. Rationally designed DNA nanoparticles (DNPs) have emerged as facile delivery vehicles because their physicochemical properties can be precisely controlled. Nonetheless, few studies have used DNPs to deliver siRNAs in vivo, and none has demonstrated therapeutic efficacy. Herein, we constructed a number of DNPs of rectangular and tubular shapes with varied dimensions using the modular DNA brick method for the systemic delivery of siRNA that targets anti‐apoptotic protein Bcl2. The siRNA delivered by the DNPs inhibited cell growth both in vitro and in vivo, which suppressed tumor growth in a xenograft model that specifically correlated with Bcl2 depletion. This study suggests that DNPs are effective tools for the systemic delivery of therapeutic siRNA and have great potential for further clinical translation.     

PAMAM Dendrimers for siRNA Delivery: Computational and Experimental Insights

Short double‐stranded RNAs, which are known as short interfering RNA (siRNA), can be used to specifically down‐regulate the expression of the targeted gene in a process known as RNA interference (RNAi). However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery. Polycationic branched macromolecules such as poly(amidoamine) (PAMAM) dendrimers show a strong binding affinity for RNA molecules and, hence, can provide an effective, reproducible, and relatively nontoxic method for transferring siRNAs into animal cells. Notwithstanding these perspectives, relatively few attempts have been made so far along these lines to study in detail the molecular mechanisms underlying the complexation process between PAMAMs and siRNAs. In this work we combine molecular simulation and experimental approaches to study the molecular requirements of the interaction of RNA‐based therapeutics and PAMAM dendrimers of different generations. The dendrimers and their siRNA complexes were structurally characterized, and the free energy of binding between each dendrimer and a model siRNA was quantified by using the well‐known MM/PBSA approach. DOSY NMR experiments confirmed the structural in silico prediction and yielded further information on both the complex structure and stoichiometry at low N/P ratio values. siRNA/PAMAM complex formation was monitored at different N/P ratios using gel retardation assays, and a simple model was proposed, which related the amount of siRNA complexed to the entropy variation upon complex formation obtained from the computer simulations.     

Potential Use of Dendrimer/α-Cyclodextrin Conjugate as a Novel Carrier for Small Interfering RNA (siRNA)

RNA interference (RNAi) is the mechanism of gene silencing-mediated messenger RNA degradation by small interference RNA (siRNA), which becomes a powerful tool for genetic analysis and novel gene therapy. However, one of the major obstacles for siRNA delivery is the difficulty to cross the biological membrane due to its hydrophilicity and high molecular weight. We evaluated the potential use of the starburst polyamidoamine dendrimer (generation 3) conjugate with α-cyclodextrin (α-CyD) having an average degree of substitution of 2.4 (α-CDE conjugate) as a siRNA carrier for RNAi. The ternary complex composed of pGL2 control vector (pDNA)/pGL2 siRNA/α-CDE conjugate showed higher pGL2 siRNA sequence-specific gene silencing effects without off-target effects than those of commercial transfection reagents such as Lipofectamine™2000 (LP), TransFast™ (TF) and Lipofectin™ (LF). These results suggest that α-CDE conjugate has the potential to be a novel carrier for siRNA.     

Unzipping and binding of small interfering RNA with single walled carbon nanotube: a platform for small interfering RNA delivery

In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference.     

Polymer siRNA conjugates synthesised by controlled radical polymerisation

Oligonucleotide therapeutics such as antisense RNA, micro RNA, mRNA and small interfering RNA have great potential to generate a novel therapeutic portfolio within the pharmaceutical market. The promising outlook of oligonucleotide therapeutics lies in their ability to knockdown genes responsible for disease progression. However, the efficient delivery of RNA medicines without causing toxicity remains a major challenge. With growing interest in siRNA therapeutics, a number of synthetic polymers have been developed to facilitate efficient in vitro and in vivo delivery. With the advent of controlled radical polymerisation (CRP) techniques – such as RAFT polymerisation and ATRP – new families of well-defined polymers with narrow molecular weight distribution and predictable molecular architecture potentially suitable to generate siRNA delivery devices are becoming available. In this review article we will describe and discuss how CRP can be utilised to generate siRNA delivery nanodevices.     

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI–PCL) (800 Da–40 kDa) and PEI–PCL–PEI (5–5–5 kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG–PCL) (5 kDa-4 kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100 nm. PEG–PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI–PCL–PEI and PEG–PCL show higher siRNA encapsulation efficiency than PEI–PCL/PEG–PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI–PCL–PEI/PEG–PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.     

植物多酚基元辅助递送siRNA的构效关系研究

表没食子儿茶素没食子酸酯(EGCG)在辅助低分子量阳离子聚合物递送siRNA的过程中表现优异,但其本身是一个结构较为复杂的植物多酚基元,可以进一步拆分为表没食子儿茶素(EGC)和没食子酸(GA)2种多酚基元.而这2种基元分子同样具有抑制癌细胞、杀伤肿瘤细胞以及通过氢键和疏水相互作用力来结合蛋白质或核酸的能力.本文拟探究各类植物多酚基元EGCG,EGC和GA结合核酸的能力和分别在辅助低分子量阳离子聚合物递送siRNA的过程中起到的作用.实验结果表明,EGC辅助ε-聚赖氨酸(PLL)递送siRNA的效率仅次于EGCG,而通过增加EGC的用量可以达到与EGCG接近的递送效果.     

Reducible Polyethylenimine Nanoparticles for Efficient siRNA Delivery in Corneal Neovascularization Therapy

The aim of this study is to establish the safe and effective ocular delivery system of therapeutic small interfering RNA (siRNA) in corneal neovascularization therapy. The major hurdle present in siRNA‐based corneal neovascularization (CNV) therapy is severe cytotoxicity caused by repetitive drug treatment. A reducible branched polyethylenimine (rBPEI)‐based nanoparticle (NP) system is utilized as a new siRNA carrier as a hope for CNV therapy. The thiolated BPEI is readily self‐crosslinked in mild conditions to make high molecular weight rBPEI thus allowing the creation of stable siRNA/rBPEI nanoparticles (siRNA‐rBPEI‐NPs). In the therapeutic region, the rBPEI polymeric matrix is effectively degraded into nontoxic LMW BPEI inside the reductive cytosol causing the rapid release of the encapsulated siRNA into the cytosol to carry out its function. The fluorescent‐labeled siRNA‐rBPEI‐NPs can release siRNA into the entire corneal region after subconjuctival injection into the eye of Sprague Dawley rats thus confirming the proof of concept of this system.

     

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d- fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru−bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d- fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).     

Identification of possible siRNA molecules for TDP43 mutants causing amyotrophic lateral sclerosis: In silico design and molecular dynamics study

The DNA binding protein, TDP43 is a major protein involved in amyotrophic lateral sclerosis and other neurological disorders such as frontotemporal dementia, Alzheimer disease, etc. In the present study, we have designed possible siRNAs for the glycine rich region of tardbp mutants causing ALS disorder based on a systematic theoretical approach including (i) identification of respective codons for all mutants (reported at the protein level) based on both minimum free energy and probabilistic approaches, (ii) rational design of siRNA, (iii) secondary structure analysis for the target accessibility of siRNA, (iii) determination of the ability of siRNA to interact with mRNA and the formation/stability of duplex via molecular dynamics study for a period of 15 ns and (iv) characterization of mRNA–siRNA duplex stability based on thermo-physical analysis. The stable GC-rich siRNA expressed strong binding affinity towards mRNA and forms stable duplex in A-form. The linear dependence between the thermo-physical parameters such as T_m, GC content and binding free energy revealed the ability of the identified siRNAs to interact with mRNA in comparable to that of the experimentally reported siRNAs. Hence, this present study proposes few siRNAs as the possible gene silencing agents in RNAi therapy based on the in silico approach.     

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor‐ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide‐modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.     

Poly(amidoamine)-based dendrimer/siRNA complexation studied by computer simulations: effects of pH and generation on dendrimer structure and siRNA binding

In this work we report, compare and discuss the results obtained from fully atomistic molecular dynamics simulations of generations 4, 5, and 6 of PAMAM-based dendrimers having NH(3) and triethanolamine as cores, forming complexes with a short interfering RNA (siRNA) at different pH values and at physiological ionic strength. By employing a detailed analysis we demonstrate how features such as molecular size, structural details, and protonation level of this category of dendrimers affect the dendrimer/siRNA complexation. Properties like the conformational flexibility of the dendrimer, the effective charge distribution of the assembly, and the level of intra- and intermolecular hydrogen bonding between the two molecular entities are all found to play a significant role in the mutual interactions between the nucleic acid and the hyperbranched molecules. All these features are of key importance in the multifaceted mechanism of dendrimer/gene complexation, and their understanding can provide valuable insight toward the design of more efficient nucleic acid nanocarriers.     

Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells

RNA interference (RNAi) is the process by which short-interfering RNA (siRNA) target a specific mRNA for degradation through interactions with an RNA-induced silencing complex (RISC). Here, a clear correlation between siRNA localization, cellular uptake, and RNAi activity was discovered by delivering siRNA into cells using siRNA-TAT(47-57) peptide, siRNA-TAT(47-57)-derived oligocarbamate conjugates, or nanoparticles. For successful RNAi, the localization of siRNA was distinctly perinuclear, suggesting that siRNA is targeted to these regions for interactions with RISC to induce RNAi. siRNA sequence variation and the presence of the target mRNA apparently did not change the subcellular localization pattern of siRNA. Intriguingly, siRNA conjugated to TAT(47-57) peptide or TAT(47-57)-derived oligocarbamate resulted in efficient RNAi activity and perinuclear localization of siRNA that was distinctly different from nonconjugated free TAT peptide nucleolar localization. These results suggest that interactions with RISC dictate siRNA localization even when siRNA is conjugated to TAT(47-57) peptide.     

Delivery of Survivin siRNA Using Cationic Diphenylalanine Vesicles

Small interfering RNA(siRNA) has been proved to be a powerful tool for silencing target gene in cells, raising the possibility that siRNA can be employed as a therapy for treating cancers and other genetic diseases. However, siRNA transfection has the limitation due to the difficulty in the delivery of siRNA to target cells and tissues. To explore an efficient biocompatible siRNA delivery system, cationic diphenylalanine vesicles(CDPVs) were constructed to transfer survivin siRNA to human ovarian cancer cells. The morphology of CDPVs was characterized by scanning electron microscopy(SEM) and the distribution of survivin siRNA was characterized by confocal laser scanning microscopy, which reveal that diphenylalanine and the survivin siRNA were successfully co-delivered. After co-incubation for 48 h, the CDPVs/siRNA exhibited enhanced tumor cell growth inhabitation and apoptosis inducted in human SK-OV-3 ovarian carcinoma cells. Overall, CDPVs is an efficient siRNA delivery system and has a promising prospect for cancer therapy.     

Engineered Hsp Protein Nanocages for siRNA Delivery

The efficient delivery of small interfering RNA (siRNA) to tumor cells still remains a great challenge. Of the various nanocarriers, protein nanocages have attracted extensive interest due to their unique structure and suitable characteristics derived from their proteinaceous nature. However, most reported protein nanocages that are developed are based on virus capsid proteins, which may raise safety concerns, including those related to gene mutation and carcinogenesis. The development of nonviral protein‐based systems for siRNA delivery is greatly needed. In this study, a novel siRNA delivery system based on heat shock protein (Hsp) nanocages is developed by a genetic engineering method. The delivery system could condense siRNA into stable complexes and protect the condensed siRNA from degradation. A cellular uptake analysis shows that siRNA is introduced into tumor cells mediated by Hsp‐R9 nanocages. Green fluorescent protein (GFP) expression in HeLa‐EGFP cells is significantly downregulated by Hsp‐R9/siRNA complexes. The results indicate that Hsp nanocages may be a good platform for siRNA delivery into tumor cells.     

Interaction of cholesterol-conjugated ionizable amino lipids with biomembranes: lipid polymorphism, structure-activity relationship, and implications for siRNA delivery

Delivery of siRNA is a major obstacle to the advancement of RNAi as a novel therapeutic modality. Lipid nanoparticles (LNP) consisting of ionizable amino lipids are being developed as an important delivery platform for siRNAs, and significant efforts are being made to understand the structure-activity relationship (SAR) of the lipids. This article uses a combination of small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) to evaluate the interaction between cholesterol-conjugated ionizable amino lipids and biomembranes, focusing on an important area of lipid SAR--the ability of lipids to destabilize membrane bilayer structures and facilitate endosomal escape. In this study, cholesterol-conjugated amino lipids were found to be effective in increasing the order of biomembranes and also highly effective in inducing phase changes in biological membranes in vitro (i.e., the lamellar to inverted hexagonal phase transition). The phase transition temperatures, determined using SAXS and DSC, serve as an indicator for ranking the potency of lipids to destabilize endosomal membranes. It was found that the bilayer disruption ability of amino lipids depends strongly on the amino lipid concentration in membranes. Amino lipids with systematic variations in headgroups, the extent of ionization, tail length, the degree of unsaturation, and tail asymmetry were evaluated for their bilayer disruption ability to establish SAR. Overall, it was found that the impact of these lipid structure changes on their bilayer disruption ability agrees well with the results from a conceptual molecular "shape" analysis. Implications of the findings from this study for siRNA delivery are discussed. The methods reported here can be used to support the SAR screening of cationic lipids for siRNA delivery, and the information revealed through the study of the interaction between cationic lipids and biomembranes will contribute significantly to the design of more efficient siRNA delivery vehicles.     

Development of an efficient transdermal delivery system of small interfering RNA using functional peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.