Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids.

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3ß-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

Forgotten fatty acids-Surface properties supply conclusive evidence for including carotenoic acids.

The term "fatty acids" is conceptually well defined with regard to fats, whose extent of saturation or unsaturation is precisely indicated in the content description of foodstuff. In contrast, the term "fatty acid" gives no hint of being associated with "soap" (Na and K salts of fatty acids). Fatty acids in edible fats or in cleaning soaps have one thing in common: they are colorless. The prevalence of colorless fats and unadulterated white soaps has ensured that fatty acids are not associated with color. However, colored conjugated polyunsaturated fatty acids do exist, occurring abundantly in nature or manufactured at large scale. We endeavor to extricate conjugated polyenoic acids from oblivion by Based on the presented results (alkalicarotenoates have similar surface properties to alkalicarboxylates, carotenoic acids react like carboxylic acids to lipids), we argue for inclusion of conjugated carotenoic acids in fatty acid inventories and organic chemistry textbooks. Carotenoic acids and -salts have outstanding qualities by combining visibility and traceability with biological activity.

Surface properties of polyene glycol phospholipid monolayers.

We studied the surface properties of monolayers composed of polyunsaturated conjugated ethylene glycol phospholipids (carotenoid lipids), compared the data with monolayers of dipalmitoylphosphatidylcholine (DPPC) to which carotenoids were added and evaluated the impact of the unsaturated glycol lipids on monolayers with the glycerolipid DPPC. The carotenoid based glycol lipids formed monolayers at the air/water interface. Using the Langmuir method we obtained series of pressure-area (π-A) isotherms and determined the limiting area A per molecule of three glycol lipids, C30:9-C0A=42.6±1.4Å2, C30:9-C2A=76.1±2.5Ǻ2 and C30:9-C12A=354.0±12.0Å2 and their mixtures with DPPC at various mole fraction X. C30:9-C0 and C30:9-C2 did not affect significantly the shape of the isotherm, but caused their slight shift toward a lower and larger molecular area, respectively. C30:9-C12 at mole fractions X>0.02 affected the shape of isotherm. The compressibility modulus Cs-1 of monolayers depended on the surface pressure. Cs-1 value was substantially higher for DPPC monolayers in comparison with those of pure glycol lipids. At low surface pressure π=5-10mN/m and low mole fractions X<0.02 the glycol lipids formed complexes with DPPC; at higher surface pressure the separation of pure components took place. The dipole potential of the monolayers composed of cationic glycol lipids C30:9-C2 and C30:9-C12 was higher in comparison with those of zwitterionic DPPC and C30:9-C0. This may be connected with various contributions of dipole moments of the molecules and their orientation in the monolayer.

Polyene-based cationic lipids as visually traceable siRNA transfer reagents.

Cationic lipids are promising non-viral vectors for the cellular delivery of nucleic acids. Important considerations for the development of new delivery vectors are enhanced uptake efficiency, low toxicity and traceability. Traceable gene transfer systems however typically require the inclusion of a labeled excipient, and highly sensitive imaging instrumentation to detect the presence of the label. Recently, we reported the synthesis and characterization of colored, polyene cationic phospholipidoids composed of a rigid, polyenoic acid of predetermined dimension (C20:5 and C30:9) paired with flexible saturated alkyl chains of varying lengths (12:0, 14:0, 16:0, 18:0, 20:0 carbons). Herein, the potential of these cationic phospholipids as siRNA carriers was evaluated through standard liposomal formulations in combination with a neutral helper lipid DOPE. The polyene-based lipids were compared with a standard cationic lipid for siRNA-delivery into luciferase expressing HR5-CL11 cells. Within the series of lipids screened, knockdown results indicated that polyene cationic phospholipids paired with longer saturated alkyl chains are more effective as gene transfer agents, and perform comparably with the commercial lipid EPC. Furthermore, the chromophore associated with the polyene chain allowed tracking of the siRNA delivery using direct observation. The polyene lipoplexes were tracked on both a macroscopic and microscopic level either as a single-component or as a multi-component lipoplex formulation. When combined with a reference EPC, effective knockdown and tracking abilities were combined in a single preparation.

Cationic polyene phospholipids as DNA carriers for ocular gene therapy.

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.

Novel cationic polyene glycol phospholipids as DNA transfer reagents--lack of a structure-activity relationship due to uncontrolled self-assembling processes.

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains.

Synthesis, self-assembling and gene delivery potential of a novel highly unsaturated, conjugated cationic phospholipid.

The synthesis and self-assembling properties of a model compound in a new class of cationic phospholipids with a highly unsaturated conjugated fatty acid are described. In addition, the potential of this new lipid as a nucleic acid carrier was evaluated through lipoplex formulations employing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as helper lipid with and without the polycationic peptide protamine, together with a plasmid DNA (pDNA). Lipoplexes composed of this novel unsaturated lipid exhibited pDNA binding and protection from DNase I degradation when formulated with protamine. The new cationic lipid revealed transfection efficiency comparable to the commercial reference 1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (EPC) in Chinese hamster ovary-K1 (CHO-K1) cells and performed equally to the standard reference Lipofectamine 2000 when the formulation included protamine.

Hydrophilic carotenoids: facile syntheses of carotenoid oxime hydrochlorides as long-chain, highly unsaturated cationic (bola)amphiphiles.

Stable cationic carotenoid aggregates - predominantly of the J-type - develop when the hydrochlorides of carotenoid aldoximes and ketoximes are exposed to water. The oxime hydrochlorides are obtained by simple syntheses from commercially available food color carotenoids. Bluish-purple, unstable transient compounds were observed during hydrochlorination performed at liquid nitrogen temperature.