Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing.

Utilizing small interfering RNA (siRNA) for gene therapy has been widely studied based on its gene silencing capability. However, unmodified naked siRNA delivery to target cells is challenging because of short half-lives in blood serum and difficulty in uptake due to its negatively charged surface. To bypass these biological barriers in siRNA delivery, we investigated the use of cationic bolaamphiphile (bola) vesicles, GLH-19, GLH-20, and the mixture of two, GLH-19/GLH-20. GLH-19 has an acetylcholine head group while GLH-20 has an acetylcholine ester head group which can be hydrolyzed by choline esterase (ChE) after it penetrates the brain blood barrier (BBB). Using in-silico, in-vitro, and in-vivo studies, we characterized the delivery of siRNA/bola complexes in terms of stability, protection, transfection, and gene silencing. From our in-vitro experiments and atomic level in silico studies, we found that the GLH-19 vesicles have not only higher stability but also better protection and higher transfection efficiency than the GLH-20 vesicles. On the other hand, both of the GLH-19/siRNA and GLH-20/siRNA complexes showed similar efficiency of eGFP silencing in MDA-MB-231 cells. We also investigated the in-vivo distribution of siRNA/GLH-19 and siRNA/GLH-19/GLH-20 complexes in the liver, tumor, and brain. We found that the siRNA/GLH-19 complexes were delivered into tumors while no major accumulation was observed in the liver. We also found that siRNA/GLH-19/GLH-20 complexes were efficiently delivered into the brain. Therefore, our in-silico, in-vitro, and in-vivo studies showed that both siRNA/GLH-19 and siRNA/GLH-20 complexes can be used for siRNA delivery with different characteristics. When the two bolas are mixed together, GLH-19 provides enhanced stability, protection, and transfection, while GLH-20 provides efficient BBB penetration and siRNA release due to the hydrolyzation of GLH-20 head group by ChE.

In this study we have investigated a new class of cationic lipids – “bolaamphiphiles” or “bolas” – for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5μg/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.

The present study focused on synthesizing bolaamphiphiles from the readily available and inexpensive castor oil, a vegetable oil, which contains about 90% of ricinoleic acid. Two classes of symmetric and asymmetric bolaamphiphiles with acetylcholine head groups were synthesized and characterized by spectroscopic analysis. These novel bolaamphiphilic compounds self-assemble in aqueous media to form stable cationic spherical nano-sized vesicles that are potential drug delivery systems.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

OBJECTIVE: To demonstrate delivery of therapeutic amounts of tenofovir into the brain by novel nano-vesicles (V-Smart TM ) prepared from synthetic bolaamphiphiles for treatment of neuro-HIV.BACKGROUND: Many AIDS patients, on Highly Active Anti-Retroviral Treatment (HAART), have non-detectable HIV in the blood and, yet, measurable amounts in the CNS because of poor BBB penetrability of some HAART drugs, particularly tenofovir. Persistent HIV reservoirs in the CNS are considered responsible for neurological impairment in AIDS patients.We have synthesized novel bolaamphiphiles that form stable monolayer-membrane vesicles, characterized by high encapsulation efficiency, penetrability through the BBB and efficient release of the encapsulated material within the brain. Here we describe optimized V-Smart TM vesicles capable of delivering tenofovir into the brain to make HAART treatment more effective.DESIGN/METHODS Nano-vesicles were formed from various formulations that contain our novel bolaamphiphiles and tenofovir. Tenofovir encapsulation was determined following the removal of non-encapsulated tenofovir by size-exclusion chromatography. The vesicle formulation was optimized to achieve high tenofovir encapsulation and good BBB permeability. Optimized vesicles were administered to mice and at various times after vesicle administration, brain tenofovir levels were determined by LC/MS.RESULTS: Optimal vesicle formulations produced 20-40% tenofovir encapsulation. Incorporation of new bolaamphiphiles with chitosan head groups to the formulation increased significantly the penetration of the vesicles to the brain. A single injection (iv) of tenofovir-loaded vesicles (at a dose 10-fold below toxic levels) resulted in brain tenofovir concentration of ~6 ug/gm tissue, much higher than the therapeutic levels of ~ 100ng/gm, which is a typical blood level in HAART patients.CONCLUSIONS: V-Smart TM vesicles, optimized for brain delivery of tenofovir, successfully delivered therapeutic amounts of the drug into the brain after a single iv administration. The results indicate the feasibility of using the V-SmartTM vesicles for lowering viral load in the CNS of HIV patients.Study Supported by: The Campbell Foundation Disclosure: Dr. Heldman has received personal compensation for activities with Lauren Sciences. Dr. Heldman has received research support from Lauren Sciences. Dr. Linder has received personal compensation for activities with Lauren Sciences. Dr. Linder has received research support from Lauren Sciences. Dr. Grinberg has received personal compensation for activities with Lauren Sciences. Dr. Grinberg has received research support from Lauren Sciences. Dr. Popov has received personal compensation for activities with Lauren Sciences. Dr. Popov has received research support from Lauren Sciences. Dr. Hollander has received personal compensation for activities with Lauren Sciences. Dr. Hollander has received research support from Lauren Sciences.

Supported biomimetic membranes hold potential for applications such as biosensors and water purification by filtration. The current paper reports on the preparation of a supported bolaamphiphile membrane on two polymeric nanofiltration membranes: NF-270 made of polyamide with carboxylic surface charges and NTR-7450 made of sulfonated polyethersulfone with sulfonic surface charges. The results, obtained using a proprietary microfluidic device, confirmed that the supported membrane coverage on the polymeric membranes was governed by the double-layer interactions, in agreement with previous studies on silica, mica, and gold. Remarkably, the formation of the biomimetic membrane was more favorable on the sulfonated polyethersulfone than on the polyamide surface, although both surfaces exhibit a similar surface charge density. It is suggested that the higher dissociation constant of sulfonic groups was the main reason for the higher coverage of NTR-7450. Finally, spinach aquaporins, which are trans-membrane proteins that facilitate the water transport, were incorporated into a supported membrane on NTR-7450. The incorporated aquaporin resulted in enhanced pressure-driven water transport through the membrane, however, for still unclear reasons, the transport was not selective. Despite this failure, the results provide new insights into formation of biomimetic membrane on water permeable polymeric substrates, as a generic approach towards biomimetic water filters.

Most conventional cancer therapeutics gain limited access to many types of tumors while having considerable adverse effects, resulting in low therapeutic efficacy and high toxicity. Therefore, research has now focused on the development of novel drug delivery systems (DDS) with the goal of maintaining high therapeutic drug levels at malignant cells and as low as possible drug levels in other cells. The introduction of nanotechnology has addressed some of these problems and opened up new avenues for improved cancer therapy. The design of nanoparticles for DDS takes into consideration issues such as targeting, controlled drug release and enhanced penetration via biological barriers. In this review we describe the design principles of targeted DDS for cancer therapy and the types of nanoparticles that are under development. Emphasis is put on lipid-based nanoparticles, particularly bolaamphiphilic vesicles that have tremendous potential in delivering therapeutic and diagnostic agents to specific cells following systemic administration.

OBJECTIVE: To demonstrate delivery of GDNF to brain regions affected in Parkinson's (PD) by novel nano-vesicles (V-SmartTM) targeted to dopaminergic neurons for treatment of PD. BACKGROUND: Neurotrophins like GDNF have good potential as therapeutic agents in PD. However, neurotrophins do not penetrate the BBB and a targeted delivery system is needed to derive their therapeutic potential. Here we describe optimized V-SmartTM vesicles (based on our nano-vesicles shown to cross the BBB and deliver proteins and siRNA) capable of delivering GDNF to the striatum and the substantia nigra -- brain regions affected in PD. DESIGN/METHODS: Nano-vesicles were formed from synthetic bolaamphiphiles containing: acetylcholine head groups that enable release from the vesicles upon exposure to AChE; targeting ligand that enables targeting to dopaminergic neurons; and chitosan moieties that enable penetration through the BBB. Carboxyfluorescein-loaded vesicles were added to cultured cells, then studied by FACS. GDNF-loaded vesicles were administered intravenously to mice, which were then perfused with PBS at various times after vesicle administration, brains removed, homogenized or sectioned for histology, and brain GDNF determined quantitatively by ELISA and localized by histofluorescence. RESULTS: Vesicles decorated with targeting ligand bind to cultured cells that express receptor significantly more than naked vesicles do, whereas no difference is found between naked and decorated vesicles in cultured cells that do not express receptor, suggesting that in vivo specific targeting to dopaminergic cells would occur. GDNF, encapsulated in vesicles decorated with targeting ligand, was administered intravenously to mice and found in significant amounts in the brain with preferential localization in the striatum and substantia nigra -- brain regions rich in dopaminergic cells. CONCLUSIONS: V-SmartTM vesicles, optimized for targeted delivery of GDNF, successfully delivered GDNF into areas of the brain affected by Parkinson's and, thus, show great potential for PD therapy. Efficacy studies are currently being conducted in PD animal models. Study Supported by: The Michael J. Fox Foundation

Two bolaamphiphilic compounds with identical acetylcholine (ACh) head groups, but with different lengths of an alkyl chain pendant adjacent to the head group, as well as differences between their hydrophobic skeleton, were investigated for their ability to self-assemble into vesicles that release their encapsulated content upon hydrolysis of their head groups by acetylcholinesterase (AChE). One of these bolaamphiphiles, synthesized from vernolic acid, has an alkyl chain pendant of five methylene groups, while the other, synthesized from oleic acid, has an alkyl chain pendant of eight methylene groups. Both bolaamphiphiles formed stable spherical vesicles with a diameter of about 130 nm. The ACh head groups of both bolaamphiphiles were hydrolyzed by AChE, but the hydrolysis rate was significantly faster for the bolaamphiphile with the shorter aliphatic chain pendant. Likewise, upon exposure to AChE, vesicles made from the bolaamphiphile with the shorter alkyl chain pendant released their encapsulated content faster than vesicles made from the bolaamphiphile with the longer alkyl chain pendant. Our results suggest that the steric environment around the ACh head group of bolaamphiphiles is a major factor affecting the hydrolysis rate of the head groups by AChE. Attaching an alkyl chain to the bolaamphiphile near the ACh head group allows self-assembled vesicles to form with a controlled release rate of the encapsulated materials, whereas shorter alkyl chains enable a faster head group hydrolysis, and consequently faster release, than longer alkyl chains. This principle may be implemented in the design of bolaamphiphiles for the formation of vesicles for drug delivery with desired controlled release rates.

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

However, non-modified naked siRNAs have short half-lives in blood serum and encounter difficulties in crossing biological membranes due to their negative charge. These obstacles can be overcome by using siRNAs complexed with bolaamphiphiles, consisting of two positively charged head groups that flank an internal hydrophobic chain. Bolaamphiphiles have relatively low toxicities, long persistence in the blood stream, and most importantly, in aqueous conditions can form poly-cationic micelles thus, becoming amenable to association with siRNAs. Herein, two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations are compared for their abilities to complex with siRNAs and deliver them into cells inducing gene silencing. Our explicit solvent molecular dynamics (MD) simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies are supported by various in vitro and in cell culture experimental techniques as well as by some in vivo studies. Results demonstrate that depending on the application, the extent of siRNA chemical protection, delivery efficiency, and further intracellular release can be varied by simply changing the type of bolaamphiphile used.

Bolaamphiphiles - amphiphilic molecules consisting of two hydrophilic headgroups linked by a hydrophobic chain - form highly stable vesicles consisting of a monolayer membrane that can be used as vehicles to deliver drugs across biological membranes, particularly the blood-brain barrier (BBB). We prepared new vesicles comprising bolaamphiphiles (bolavesicles) that encapsulate iron oxide nanoparticles (IONPs) and investigated their suitability for targeted drug delivery. Bolavesicles displaying different headgroups were studied, and the effect of IONP encapsulation upon membrane interactions and cell uptake were examined. Experiments revealed more pronounced membrane interactions of the bolavesicles assembled with IONPs. Furthermore, enhanced internalization and stability of the IONP-bolavesicles were observed in b.End3 brain microvessel endothelial cells - an in vitro model of the blood-brain barrier. Our findings indicate that embedded IONPs modulate bolavesicles' physicochemical properties, endow higher vesicle stability, and enhance their membrane permeability and cellular uptake. IONP-bolavesicles thus constitute a promising drug delivery platform, potentially targeted to the desired location using external magnetic field. (C) 2013 Elsevier B.V. All rights reserved.

Supported biomimetic membranes (SBMs) on solid substrates have been commonly prepared from vesicle-forming double-tail lipids, such as zwitterionic phospholipids, using the method of vesicle fusion. Here we report on the preparation of SBMs on silica surfaces via a similar process of "micelle fusion" from a cationic single-tail bolaamphiphile GLH-20 that forms spherical and elongated thread-like micelles in solution. We demonstrate that, in contrast to zwitterionic phospholipids, GLH-20 self-assembles into a stable contiguous SBM at both low and high ionic strengths. The cationic charge of GLH-20 promotes the formation of a stable SBM through enhanced double-layer interactions with the negatively charged silica surface. It is also shown that spinach aquaporin PM-28 was successfully incorporated within bolaamphiphile SBM in a manner similar to SBMs prepared by vesicle/proteoliposome fusion; thereby the inherent curvature of the micelle surface does not inhibit protein reconstitution. The results suggest that SBMs based on charged bolaamphiphiles might be an attractive platform for applications such as water purification and biosensors, where the stability and low defect rate of SBMs in diverse conditions are crucial for achieving desired performance.

Stable nano-sized vesicles with a monolayer encapsulating membrane were prepared from novel bolaamphiphiles with choline ester head groups. The head groups were covalently bound to the alkyl chain of the bolaamphiphiles either via the nitrogen atom of the choline moiety, or via the choline ester's methyl group. Both types of bolaamphiphiles competed with acetylthiocholine for binding to acetylcholine esterase (AChE), yet, only the choline ester head groups bound to the alkyl chain via the nitrogen atom of the choline moiety were hydrolyzed by the enzyme. Likewise, only vesicles composed of bolaamphiphiles with head groups that were hydrolyzed by AChE released their encapsulated material upon exposure to the enzyme. Injection of carboxyfluorescein (CF)-loaded vesicles with cleavable choline ester head groups into mice resulted in the accumulation of CF in tissues that express high AChE activity, including the brain. By comparison, when vesicles with choline ester head groups that are not hydrolyzed by AChE were injected into mice, there was no accumulation of CF in tissues that highly express the enzyme. These results imply that bolaamphiphilic vesicles with surface groups that are substrates to enzymes which are highly expressed in target organs may potentially be used as a drug delivery system with controlled site-directed drug release.

Antigen-presenting cells (APCs) act as vehicles that transfer HIV to their target CD4(+) cells through an intercellular junction, termed the virologic synapse. The molecules that are involved in this process remain largely unidentified. In this study, we used photoaffinity labeling and a proteomic approach to identify new proteins that facilitate HIV-1 transfer. We identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between APCs and CD4(+) target cells. Monoclonal antibodies against the β-subunit of ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4(+) target cells. Likewise, the specific inhibitors of ATPase, citreoviridin and IF1, completely blocked APC-mediated transfer of HIV-1 at the APC-target cell interaction step. Confocal fluorescent microscopy showed localization of extracellular ATP synthase at junctions between APC and CD4(+) target cells. We conclude that ectopic ATP synthase could be an accessible molecular target for inhibiting HIV-1 proliferation in vivo.