Eliahu Heldman

Multifunctional RNA Nanoparticles

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.

Cationic vesicles from novel bolaamphiphilic compounds

Effective targeted drug delivery by cationic liposomes is difficult to achieve because of their rapid clearance from the blood circulation. Bolaamphiphiles that form monolayer membrane may provide vesicles with improved stability, as shown for archaeosomes. We investigated a series of bolaamphiphiles with acetylcholine head groups and systematic structural changes in their hydrophobic domain for their ability to form stable nanovesicles. Bolaamphiphiles with two aliphatic chains separated by a short amide midsection produced spherical nanovesicles ranging in diameter from 80 to 120 nm. These vesicles lost their encapsulated material within 24 hours of incubation in phosphate-buffered saline (PBS). Similar bolaamphiphiles with a longer midsection produced a mixture of fibers and more stable nanovesicles. Bolaamphiphiles with ester amide midsection produced only spherical nanovesicles that were stable during incubation in PBS for several days. Vesicles made from bolaamphiphiles with acetylcholine head groups conjugated to the aliphatic chain via the amine were less stable than vesicles made from bolaamphiphiles with head groups conjugated to the aliphatic chain via the acetyl group. Vesicles that were stable in vitro showed good stability in the blood circulation after intravenous administration to mice. These results help in elucidating the bolaamphiphile structures needed to form stable cationic vesicles for targeted drug delivery.

Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles.

Bolaamphiphilic cationic vesicles with acetylcholine (ACh) surface groups were investigated for their ability to deliver a model protein-bovine serum albumin conjugated to fluorescein isothiocyanate (BSA-FITC) across biological barriers in vitro and in vivo. BSA-FITC-loaded vesicles were internalized into cells in culture, including brain endothelial b.End3 cells, at 37 °C, but not at 4 °C, indicating an active uptake process. To examine if BSA-FITC-loaded vesicles were stable enough for in vivo delivery, we tested vesicle stability in whole serum. The half-life of cationic BSA-FITC-loaded vesicles with ACh surface groups that are hydrolyzed by choline esterase (ChE) was about 2 h, whereas the half-life of vesicles with similar surface groups, but which are not hydrolyzed by choline esterase (ChE), was over 5 h. Pyridostigmine, a choline esterase inhibitor that does not penetrate the blood-brain barrier (BBB), increased the stability of the ChE-sensitive vesicles to 6 h but did not affect the stability of vesicles with ACh surface groups that are not hydrolyzed by ChE. Following intravenous administration to pyridostigmine-pretreated mice, BSA-FITC encapsulated in ChE-sensitive vesicles was distributed into various tissues with marked accumulation in the brain, whereas non-encapsulated (free) BSA-FITC was detected only in peripheral tissues, but not in the brain. These results show that cationic bolaamphiphilic vesicles with ACh head groups are capable of delivering proteins across biological barriers, such as the cell membrane and the blood-brain barrier (BBB). Brain ChE activity destabilizes the vesicles and releases the encapsulated protein, enabling its accumulation in the brain.

Inhibition of specific adenylyl cyclase isoforms by lithium and carbamazepine, but not valproate, may be related to their antidepressant effect.

OBJECTIVES Lithium, valproate, and carbamazepine decrease stimulated brain cyclic-AMP (cAMP) levels. Adenylyl cyclase (AC), of which there are nine membrane-bound isoforms (AC1-AC9), catalyzes the formation of cAMP. We have recently demonstrated preferential inhibition of AC5 by lithium. We now sought to determine whether carbamazepine and valproate also preferentially inhibit specific AC isoforms or decrease cAMP levels via different mechanisms. METHODS COS7 cells were transfected with one of AC1-AC9, with or without D1-dopamine receptors. Carbamazepines and valproates effect on forskolin- or D1 agonist-stimulated ACs was studied. The effect of Mg(2+) on lithiums inhibition was studied in membrane-enriched fraction from COS7 cells co-expressing AC5 and D1 receptors. AC5 knockout mice were tested for a behavioral phenotype similar to that of lithium treatment. RESULTS Carbamazepine preferentially inhibited forskolin-stimulated AC5 and AC1 and all D1 agonist-stimulated ACs, with AC5 and AC7 being the most sensitive. When compared to 1 or 3 mM Mg(2+), 10 mM Mg(2+) reduced lithium-induced AC5 inhibition by 70%. In silico modeling suggests that among AC isoforms carbamazepine preferentially affects AC1 and AC5 by interacting with the catechol-estrogen site. Valproate did not affect any forskolin- or D1 receptor-stimulated AC. AC5 knockout mice responded similarly to antidepressant- or lithium-treated wild-types in the forced-swim test but not in the amphetamine-induced hyperactivity mania model. CONCLUSIONS Lithium and carbamazepine preferentially inhibit AC5, albeit via different mechanisms. Lithium competes with Mg(2+), which is essential for AC activity; carbamazepine competes for ACs catechol-estrogen site. Antidepressant-like behavior of AC5 knockout mice in the forced-swim test supports the notion that AC5 inhibition is involved in the antidepressant effect of lithium and carbamazepine. The effect of lithium and carbamazepine to lower cAMP formation in AC5-rich dopaminergic brain regions suggests that D1-dopamine receptors in these regions are involved in the antidepressant effect of mood stabilizers.

Delivery of analgesic peptides to the brain by nano-sized bolaamphiphilic vesicles made of monolayer membranes

Inefficient drug delivery to the brain is a major obstacle for pharmacological management of brain diseases. We investigated the ability of bolavesicles - monolayer membrane vesicles self-assembled from synthetic bolaamphiphiles that contain two hydrophilic head groups at each end of a hydrophobic alkyl chain - to permeate the blood-brain barrier and to deliver the encapsulated materials into the brain. Cationic vesicles with encapsulated kyotorphin and leu-enkephalin (analgesic peptides) were prepared from the bolalipids GLH-19 and GLH-20 and studied for their analgesic effects in vivo in experimental mice. The objectives were to determine: (a) whether bolavesicles can efficiently encapsulate analgesic peptides, (b) whether bolavesicles can deliver these peptides to the brain in quantities sufficient for substantial analgesic effect, and to identify the bolavesicle formulation/s that provides the highest analgetic efficiency. The results indicate that the investigated bolavesicles can deliver analgesic peptides across the blood-brain barrier and release them in the brain in quantities sufficient to elicit efficient and prolonged analgesic activity. The analgesic effect is enhanced by using bolavesicles made from a mixture the bolas GLH-19 (that contains non-hydrolyzable acetylcholine head group) and GLH-20 (that contains hydrolysable acetylcholine head group) and by incorporating chitosan pendants into the formulation. The release of the encapsulated materials (the analgesic peptides kyotorphin and leu-enkephalin) appears to be dependent on the choline esterase (ChE) activity in the brain vs. other organs and tissues. Pretreatment of experimental animals with pyridostigmine (the BBB-impermeable ChE inhibitor) enhances the analgesic effects of the studied formulations. The developed formulations and the approach for their controlled decapsulation can serve as a useful modality for brain delivery of therapeutically-active compounds.

Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable surface groups

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 esters 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.

Steric environment around acetylcholine head groups of bolaamphiphilic nanovesicles influences the release rate of encapsulated compounds.

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.

Specific inhibition of adenylyl-cyclase isoform 5 by mood stabilizers may be related to their mechanism of action

Lithium, valproate and carbamazepine decrease brain cAMP. Adenylyl-cyclase (AC), which synthesizes cAMP has nine membrane-bound isoforms (AC1-AC9). In this study we used COS7 cells transfected with cDNA of each of the isoforms to study the effect of a therapeutic concentration of each of lithium, carbamazepine and valproate on ACs activity. AC5 was the most inhibitable isoform by lithium and carbamazepine either when stimulated by forskolin or by a D1 agonist. Ten mM Mg2+ reduced lithium-induced AC5 inhibition by 70% and in silico analysis suggested that carbamazepine preferentially affects AC1 and AC5 by interacting with two amino-acids at the catechol-estrogen binding site region. Valproate did not inhibit any AC isoform suggesting it decreases cAMP levels via a different mechanism. AC5 knockout mice behaved in the forced-swim-test similarly to antidepressant- or lithium-treated wildtypes implying that AC5 inhibition may be involved in the antidepressant effect of lithium and carbamazepine. Specific AC5 inhibitors may be mood-stabilizers or antidepressants.