Samantha E. Wilner

An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.

Self-Sorting and Coassembly of Fluorinated, Hydrogenated, and Hybrid Janus Dendrimers into Dendrimersomes.

The modular synthesis of a library containing seven self-assembling amphiphilic Janus dendrimers is reported. Three of these molecules contain environmentally friendly chiral-racemic fluorinated dendrons in their hydrophobic part (RF), one contains achiral hydrogenated dendrons (RH), while one denoted hybrid Janus dendrimer, contains a combination of chiral-racemic fluorinated and achiral hydrogenated dendrons (RHF) in its hydrophobic part. Two Janus dendrimers contain either chiral-racemic fluorinated dendrons and a green fluorescent dye conjugated to its hydrophilic part (RF-NBD) or achiral hydrogenated and a red fluorescent dye in its hydrophilic part (RH-RhB). These RF, RH, and RHF Janus dendrimers self-assembled into unilamellar or onion-like soft vesicular dendrimersomes (DSs), with similar thicknesses to biological membranes by simple injection from ethanol solution into water or buffer. Since RF and RH dendrons are not miscible, RF-NBD and RH-RhB were employed to investigate by fluorescence microscopy the self-sorting and coassembly of RF and RH as well as of phospholipids into hybrid DSs mediated by the hybrid hydrogenated-fluorinated RHF Janus dendrimer. The hybrid RHF Janus dendrimer coassembled with both RF and RH. Three-component hybrid DSs containing RH, RF, and RHF were formed when the proportion of RHF was higher than 40%. With low concentration of RHF and in its absence, RH and RF self-sorted into individual RH or RF DSs. Phospholipids were also coassembled with hybrid RHF Janus dendrimers. The simple synthesis and self-assembly of DSs and hybrid DSs, their similar thickness with biological membranes and their imaging by fluorescence and (19)F-MRI make them important tools for synthetic biology.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission

Significance Janus particles (JPs) are structures with two distinct faces. This study reports the discovery of an unprecedented class of JPs denoted Janus dendrimersomes (JDSs) coassembled from amphiphilic Janus dendrimers (JDs) with fluorinated, hydrogenated, and hybrid fluorinated–hydrogenated hydrophobic chains. JDSs consist of fluorinated and hydrogenated vesicles connected in a dumbbell-like shape. They appear as one structure in a broader fission-like pathway, which is revealed by varying the ratios of the three JD components. This pathway implies that non–protein-mediated fission and fusion is a likely mechanism by which similar structures form in synthetic and biological systems. These results highlight the potential importance of non–protein-mediated fusion and fission for biology and medicine. A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated–fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving “undruggable” targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205+ DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR.

Synthesis and Characterization of Aptamer-Targeted SNALPs for the Delivery of siRNA

Aptamers selected against cell surface receptors represent a unique set of ligands that can be used to target nanoparticles and other therapeutics to specific cell types. Here, we describe a method for using aptamers to deliver stable nucleic acid lipid particles (SNALPs) encapsulating small interfering RNA (siRNA) to cells in vitro. Using this method, we have demonstrated the ability of aptamer-conjugated SNALPs to achieve target-specific delivery and siRNA-mediated knockdown of a gene of interest. We also describe methods to characterize SNALP size, siRNA encapsulation efficiency, and aptamer conjugation efficiency.

Controlling lipid micelle stability using oligonucleotide headgroups

Lipid-based micelles provide an attractive option for therapeutic and diagnostic applications because of their small size (<20 nm) and ability to self-assemble and improve the solubility of both hydrophobic drugs and dyes. Their use, however, has been challenged by the fact that these particles are inherently unstable in serum becaue of interactions with protein components, which drives the micelle equilibrium to the monomeric state. We have engineered serum stabilized micelles using short quadruplex forming oligonucleotide extensions as the lipid headgroup. Quadruplex formation on the surface of the particles, confirmed by (1)H NMR, results in slight distortion of the otherwise spherical micelles and renders them resistant to disassembly by serum proteins for >24 h. Using antisense oligonucleotides we demonstrated that disruption of the quadruplex leads to micelle destabilization and cargo release. The ability to use oligonucleotide interactions to control lipid particle stability represents a new approach in the design of programmed nanoscale devices.

Dendrimersomes exhibit lamellar-to-sponge phase transitions

Lamellar to nonlamellar membrane shape transitions play essential roles in key cellular processes, such as membrane fusion and fission, and occur in response to external stimuli, including drug treatment and heat. A subset of these transitions can be modeled by means of thermally inducible amphiphile assemblies. We previously reported on mixtures of hydrogenated, fluorinated, and hybrid Janus dendrimers (JDs) that self-assemble into complex dendrimersomes (DMSs), including dumbbells, and serve as promising models for understanding the complexity of biological membranes. Here we show, by means of a variety of complementary techniques, that DMSs formed by single JDs or by mixtures of JDs undergo a thermally induced lamellar-to-sponge transition. Consistent with the formation of a three-dimensional bilayer network, we show that DMSs become more permeable to water-soluble fluorophores after transitioning to the sponge phase. These DMSs may be useful not only in modeling isotropic membrane rearrangements of biological systems but also in drug delivery since nonlamellar delivery vehicles can promote endosomal disruption and cargo release.

Erratum: An RNA alternative to human transferrin: A new tool for targeting human cells (Molecular Therapy - Nucleic Acids (2013) 2 e79 Doi:10.1038/mtna. 2013.6)

1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA; 2Universidade do Estado de Santa Catarina, Centro de Ciências Agroveterinárias, Lages, Santa Catarina, Brazil; 3Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas, USA; 4STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany. Matthew Levy 1301 Morris Park Avenue, Price Center, Room 519, Bronx, NY, 10461, USA. Correspondence: E-mail: matthew.levy@einstein.yu.edu Received 11 November 2011; revised 2 March 2012; accepted 26 March 2012

Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural Phenolic Acids to Discover Monodisperse Unilamellar Dendrimersomes

Natural, including plant, and synthetic phenolic acids are employed as building blocks for the synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest examples of programmed synthetic macromolecules. Amphiphilic Janus dendrimers are synthesized from a diversity of building blocks including natural phenolic acids. They self-assemble in water or buffer into vesicular dendrimersomes employed as biological membrane mimics, hybrid and synthetic cells. These dendrimersomes are predominantly uni- or multilamellar vesicles with size and polydispersity that is predicted by their primary structure. However, in numerous cases, unilamellar dendrimersomes completely free of multilamellar assemblies are desirable. Here, we report the synthesis and structural analysis of a library containing 13 amphiphilic Janus dendrimers containing linear and branched alkyl chains on their hydrophobic part. They were prepared by an optimized iterative modular synthesis starting from natural phenolic acids. Monodisperse dendrimersomes were prepared by injection and giant polydisperse by hydration. Both were structurally characterized to select the molecular design principles that provide unilamellar dendrimersomes in higher yields and shorter reaction times than under previously used reaction conditions. These dendrimersomes are expected to provide important tools for synthetic cell biology, encapsulation, and delivery.