Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.
ABSTRACT: 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 5?g/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.
Project description: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.
Project description: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.
Project description:Obesity is a major risk factor for diabetes, heart disease and other health problems. Adipose tissue plays a central role in the development of obesity and obesity-associated diseases. Gene therapy targeting adipose tissue may provide a promising strategy for obesity treatment. However, nucleic acid delivery to adipose tissue or even cultured adipocytes is challenging due to low delivery efficacy and high toxicity of the current cationic lipid based delivery systems, or monoamphiphiles. Herein, we report using dendritic peptide bolaamphiphiles (bolas) to deliver siRNA to primary adipocytes and hepatocytes. The bola consists of two l-Lysine dendrons connected to a fluorocarbon core through disulfide linkages. The Lysine dendrons are functionalized with l-histidine and l-tryptophan to promote endosomal escape and cellular uptake. The bola exhibited over 70% knockdown of GAPDH gene in both primary adipocytes and hepatocytes. Importantly, different from Lipofectamine that significantly reduced genes involved in lipolysis, lipogenesis, fatty acid oxidation and ketogenesis, the bolas had little to no effect on these genes. These results demonstrate the bola as a promising new vector for clinical and experimental applications for delivery of siRNA to metabolic organs.
Project description:Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.
Project description:The application of small interfering RNA (siRNA) for cancer treatment is a promising strategy currently being explored in early phase clinical trials. However, efficient systemic delivery limits clinical implementation. We developed and tested a novel delivery system comprised of (i) an internalizing streptavidin-conjugated monoclonal antibody (mAb-SA) directed against CD22 and (ii) a biotinylated diblock copolymer containing both a positively charged siRNA condensing block and a pH-responsive block to facilitate endosome release. The modular design of the carrier facilitates the exchange of different targeting moieties and siRNAs to permit its usage in a variety of tumor types. The polymer was synthesized using the reversible addition fragmentation chain transfer (RAFT) technique and formed micelles capable of binding siRNA and mAb-SA. A hemolysis assay confirmed the predicted membrane destabilizing activity of the polymer under acidic conditions typical of the endosomal compartment. Enhanced siRNA uptake was demonstrated in DoHH2 lymphoma and transduced HeLa-R cells expressing CD22 but not in CD22 negative HeLa-R cells. Gene knockdown was significantly improved with CD22-targeted vs. nontargeted polymeric micelles. Treatment of DoHH2 cells with CD22-targeted polymeric micelles containing 15 nmol/l siRNA produced 70% reduction of gene expression. This CD22-targeted polymer carrier may be useful for siRNA delivery to lymphoma cells.
Project description:In this study, we report a new dipeptide functionalization strategy for developing new dendritic bolaamphiphile vectors for efficient siRNA transfection. A focused library of dipeptides was constructed using four amino acids: l-arginine, l-histidine, l-lysine, and l-tryptophan. The dipeptides were coupled to two dendritic bolaamphiphile scaffolds that we developed previously, allowing us to quickly access a focused library of discrete vectors with multivalent dendritic dipeptide functionalities. The resulting discrete bolaamphiphiles were screened for siRNA delivery in vitro in HEK-293 and HeLa cells. Bolaamphiphiles functionalized with dipeptides containing Lys or Arg and either His or Trp were the most effective for in vitro siRNA delivery. Necessary cationic charge to ensure efficient siRNA binding are provided by Arg and Lys residues, whereas endosomal escape is provided through pH responsive buffering of His or membrane interactions of Trp. The most effective vectors (F10 HR/RH) exhibited greater than 75% gene silencing in multiple cell lines and exhibited serum stability.
Project description:Nanoparticles show great potential for drug delivery. However, suitable nanostructures capable of loading a range of drugs together with the co-delivery of siRNAs, which avoid the problem of cation-associated cytotoxicity, are lacking. Herein, we report an small interfering RNA (siRNA)-based vesicle (siRNAsome), which consists of a hydrophilic siRNA shell, a thermal- and intracellular-reduction-sensitive hydrophobic median layer, and an empty aqueous interior that meets this need. The siRNAsome can serve as a versatile nanostructure to load drug agents with divergent chemical properties, therapeutic proteins as well as co-delivering immobilized siRNAs without transfection agents. Importantly, the inherent thermal/reduction-responsiveness enables controlled drug loading and release. When siRNAsomes are loaded with the hydrophilic drug doxorubicin hydrochloride and anti-P-glycoprotein siRNA, synergistic therapeutic activity is achieved in multidrug resistant cancer cells and a tumor model.
Project description:The studies of bolaamphiphile-based nanoparticles as delivery vectors are still rudimentary and under development. In this study, several asymmetric bolaamphiphiles containing lysine and another moiety with special functions, such as pH-sensitive or cell-targeting property, were designed and synthesized. The potentials of these bolaamphiphile-based nanoparticles as versatile vectors for both nucleic acids and chemical drugs were studied. With the presence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), these amphiphiles could be prepared into bolasomes, which showed good DNA binding ability and could condense plasmid DNA into nanoparticles with appropriate size and surface potential. Lys-His, which has a pH-sensitive histidine on one head, exhibited higher transfection efficiency than the symmetric counterpart and comparable efficiency to commercially available transfection reagent. Mechanism studies confirmed that the bolaplexes formed from Lys-His might induce the highest cellular uptake and the best endosomal escape ability. On the other hand, these bolaamphiphiles also exhibited good drug loading ability. The self-assembly vesicles could efficiently encapsulate the hydrophobic anti-cancer drug doxorubicin (DOX) in aqueous solution with high drug loading content and encapsulation efficiency. Confocal laser scanning microscopy (CLSM) experiment and cell viability assay exhibited a controlled release of the drug with the assistance of bolasomes. It was shown that such bolaamphiphiles have great potential as nano-vectors for both drug and gene or their co-delivery.
Project description: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.
Project description:The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.