RESUMEN
N-acylethanolamines (NAE, also called ethanolamides) are significant lipid signaling molecules with anti-inflammatory, pain-relieving, cell-protective, and anticancer properties. Here, we present the use of a hitherto unreported group of Δ3-NAE and also some Δ4- and Δ5-NAE, in in vitro and in vivo assays to gain a better understanding of their structure-bioactivity relationships. We have developed an efficient synthetic method to rapidly produce novel unlabeled and 13 C-labeled Δ3-NAE (NAE-18:5n-3, NAE-18:4n-6) and Δ4-NAE (NAE-22:5n-6). The new NAE with shorter carbon backbone structures confers greater neuroprotection than their longer carbon backbone counterparts, including anandamide (Δ5-NAE-20:4n-6) in a focal ischemia mouse model of stroke. This study highlights structure-dependent protective effects of new NAE following focal ischemia, in which some of the new NAE, administered intranasally, lead to significantly reduced infarct volume and improved recovery of limb use. The relative affinity of the new NAE toward cannabinoid receptors was assessed against anandamide, NAE-22:6n-3 and NAE-20:5n-3, which are known cannabinoid receptor ligands with high-binding constants. Among the newly synthesized NAE, Δ4-NAE-22:5n-6 shows the greatest relative affinity to cannabinoid receptors hCB1 and hCB2 , and inhibition of cyclic adenosine monophosphate activity through hCB2 compared to anandamide.
Asunto(s)
Neuroprotección , Accidente Cerebrovascular , Animales , Etanolaminas , Ratones , Receptores de CannabinoidesRESUMEN
Lyotropic phases of amphiphiles are a prototypical example of self-assemblies. Their structure is generally determined by amphiphile shape and their phase transitions are primarily governed by composition. In this paper, we demonstrate a new paradigm for membrane shape control where the electrostatic coupling of charged membranes to short DNA (sDNA), with tunable temperature-dependent end-to-end stacking interactions, enables switching between the inverted gyroid cubic structure (QIIG) and the inverted hexagonal phase (HIIC). We investigated the structural shape transitions induced in the QIIG phase upon complexation with a series of sDNAs (5, 11, 24, and 48 bp) with three types of end structure ("sticky" adenine (A)-thymine (T) (dAdT) overhangs, no overhang (blunt), and "nonsticky" dTdT overhangs) using synchrotron small-angle X-ray scattering. Very short 5 bp sDNA with dAdT overhangs and blunt ends induce coexistence of the QIIG and the HIIC phase, with the fraction of QIIG increasing with temperature. Phase coexistence for blunt 5 bp sDNA is observed from 27 °C to about 65 °C, where the HIIC phase disappears and the temperature dependence of the lattice spacing of the QIIG phase indicates that the sDNA duplexes melt into single strands. The only other sDNA for which melting is observed is 5 bp sDNA with dTdT overhangs, which forms the QIIG phase throughout the studied range of temperature (27 °C to 85.2 °C). The longer 11 bp sDNA forms coexisting QIIG and HIIC phases (with the fraction of QIIG again increasing with temperature) only for "nonsticky" dTdT overhangs, while dAdT overhangs and blunt ends exclusively template the HIIC phase. For 24 and 48 bp sDNAs the HIIC phase replaces the QIIG phase at all investigated temperatures, independent of sDNA end structure. Our work demonstrates how the combined effects of sDNA length and end structure (which determine the temperature-dependent stacking length) tune the phase behavior of the complexes. These findings are consistent with the hypothesis that sDNAs and sDNA stacks with lengths comparable to or larger than the cubic unit cell length disfavor the highly curved channels present in the QIIG phase, thus driving the QIIG-to-HIIC phase transition. As the temperature is increased, the breaking of stacks due to thermal fluctuations restores increasing percentages of the QIIG phase.
RESUMEN
Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(ß') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(ßF), L(ßL), and L(ßI)) constituting the L(ß') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.
Asunto(s)
ADN/química , Lípidos/química , Liposomas/química , Animales , Cationes/química , Bovinos , Lípidos/síntesis química , Modelos Moleculares , Estructura MolecularRESUMEN
Cationic liposome-DNA (CL-DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL-DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL-DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL-DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL-DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL-DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL-DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome.
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ADN/química , Endosomas/metabolismo , Liposomas/química , Polietilenglicoles/química , Transfección/métodos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Terapia Genética , Liposomas/efectos adversos , Ratones , Modelos Químicos , Polietilenglicoles/efectos adversosRESUMEN
Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.
Asunto(s)
Disulfuros/química , Técnicas de Transferencia de Gen , Lípidos/química , Animales , Cationes , Citoplasma/metabolismo , ADN/química , Etidio/farmacología , Fibroblastos/citología , Vectores Genéticos , Luz , Espectroscopía de Resonancia Magnética/métodos , Ratones , Microscopía/métodos , Dispersión de Radiación , Dispersión del Ángulo PequeñoRESUMEN
The self-assembly of a custom-synthesized pentavalent cationic lipid (MVL5) and glycerol monooleate (GMO) with small interfering RNA (siRNA) results in the formation of a double-gyroid bicontinuous inverted cubic phase with colocalized lipid/siRNA domains as shown by synchrotron X-ray scattering and fluorescence microscopy. The high charge density (due to MVL5) and positive Gaussian modulus of the GMO-containing membranes confer optimal electrostatic and elastic properties for endosomal escape, enabling efficient siRNA delivery and effective, specific gene silencing.