Maleimide-acetylcholine headed bolaamphiphilic vesicles made from ricinoleic acid: Prospective active targeted drug delivery systems.

Based on ricinoleic acid, two asymmetric bolaamphiphiles with unsymmetrical hydrophobic skeletons and two different hydrophilic head groups were designed and synthesized. The first bola compound had acetylcholine (ACh) and maleimide (MAL) head groups while the second was derived from the first bolaamphiphile by thiol-ene conjugation of its maleimide moiety with l-glutathione and possessed ACh and l-glutathione-MAL head groups. Both synthetic bolaamphiphiles were characterized by common spectroscopic methods. The asymmetric bola compound with ACh and MAL head groups was investigated for its ability to self-aggregate into nanoparticles and showed to form in aqueous media nano-sized vesicles that were stable, positively charged and had symmetrical monolayer membrane with antiparallel packing. These vesicles prepared with or without membrane stabilizers such as cholesterol (CHOL) and cholesteryl hemisuccinate (CHEMS) were able to encapsulate carboxyfluorescein (CF), a water soluble and self-quenching marker and particularly those without additives were more CF encapsulating. The synthesis of bolaamphiphile with ACh-l-glutathione-MAL head groups gives evidence that the bola with ACh and MAL head groups can be utilized as a precursor of a plethora of asymmetric bolas.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

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.

Progress in lipid-based nanoparticles for cancer therapy.

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.

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.

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.

In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery.

Specific small interfering RNAs (siRNAs) designed to silence different oncogenic pathways can be used for cancer therapy. 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.Molecular Therapy-Nucleic Acids (2013) 2, e80; doi:10.1038/mtna.2013.5; published online 19 March 2013.

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.

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 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.

Interfacial and self-assembly properties of bolaamphiphilic compounds derived from a multifunctional oil.

The self-assembly characteristics in aqueous solutions of cationic bolaamphiphiles with systematic changes in their chemical structure is described with respect to their interfacial properties within water and at the air/water interface. Six cationic bolaamphiphiles were synthesized from multifunctional vernonia oil with the following variations: (a) two different alkyl chain lengths connecting the head groups, (b) polar ester or hydrogen bonding amide groups within the hydrophobic domain, and (c) an acetylcholine cationic head group with different conjugation sites to the alkyl chain. Surface tension measurements were used for determining critical aggregation concentration (CAC) values and air/water interfacial parameters such as 'effectiveness', surface excess concentration and area occupied by one molecule in the air/water interface. Fluorescent studies with pyrene were used to characterize CAC properties within the aqueous volume and transmission electron microscopy (TEM) for determining the aggregate structure's size, homogeneity and morphology. A bolaamphiphile molecular structure vs. interfacial property relationship was derived from this data which could be used to determine the molecular structure properties needed to generate interfacial forces to form either spherical vesicles or fibrous networks. The effects of the aliphatic chain length, head group orientation and functional groups within the hydrophobic domain on CAC, surface tension properties and self-aggregate morphology are described. Most bolaamphiphiles studied had CAC values in the 10-190 µM range, while two out of the six were found to assemble into MLM spherical vesicles with diameters ranging up to 120 nm suitable for drug delivery applications. Others formed a gelatinous network of fibers or multi-lamellar vesicles.

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.

Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes.

Novel cationic amphiphilic compounds were prepared from vernonia oil, a natural epoxidized triglyceride, and studied with respect to vesicle formation, encapsulation of biomaterials such as DNA, and their physical stability and transport through isolated plant cuticle membranes. The amphiphiles studied were a single-headed compound III (a quaternary ammonium head group with two alkyl chains) and a triple-headed compound IV, which is essentially three molecules of compound III bound together through a glycerol moiety. Vesicles of the two amphiphiles, prepared by sonication in water and solutions of uranyl acetate or the herbicide 2,4-D (2,4-dichloropenoxy acetic acid), were examined by TEM, SEM, AFM, and confocal laser systems and had a spherical shape which encapsulated the solutes with diameters between 40 and 110 nm. Vesicles from amphiphile IV could be made large enough to encapsulate a condensed 5.2kb DNA plasmid (pJD328). Vesicles of amphiphile IV were also shown to pass intact across isolated plant cuticle membranes and the rate of delivery of encapsulated radio-labeled 2,4-D through isolated plant cuticle membranes obtained with these vesicles was clearly greater in comparison to liposomes prepared from dipalmitopyl phosphatidylcholine (DPPC) and the control, nonencapsulated 2,4-D. Vesicles from amphiphiles III and IV were found to be more stable than those of liposomes from DPPC. The data indicate the potential of vesicles prepared from the novel amphiphile IV to be a relatively efficient nano-scale delivery system to transport DNA and other bioactive agents through plant biological barriers. This scientific approach may open the way for further development of efficient in vivo plant transformation systems.

Pentacyclic triterpenoids from Embelia schimperi.

Five oleanane-type pentacyclic triterpenoids were isolated by chromatographic separation of a chloroform extract of the stem bark of Embelia schimperi. Three of these compounds have a methyleneoxy bridge. Two compounds, embelinone and schimperinone, are reported here for the first time from a natural source (they have been synthesized previously during chemical transformations). Their structures were determined by spectroscopic techniques, among which 2-D NMR was useful for complete characterization. Three of the triterpenoids exhibited mild antibacterial properties against the gram-positive bacterial strain Rhodococcus sp.