Three-Dimensional Imaging of Lipid Gene-Carriers: Membrane Charge Density Controls Universal Transfection Behavior in Lamellar Cationic Liposome-DNA Complexes

Abstract

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L(alpha)(C) and inverted hexagonal H(II)(C) nanostructures, and mouse fibroblast cells. Confocal images of L(alpha)(C) complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L(alpha)(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L(alpha)(C) complexes climbs exponentially over approximately four decades with increasing sigma(M) below an optimal charge density (sigma(M)(*)), and saturates for at a value rivaling the high transfection efficiency of H(II)(C) complexes. In contrast, the transfection efficiency of H(II)(C) complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L(alpha)(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L(alpha)(C) complexes and H(II)(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

Alteration of epitope recognition pattern in Ag85B and ESAT‐6 has a profound influence on vaccine‐induced protection against Mycobacterium tuberculosis

“…The target cells for adenovirus delivered in liposomes, on the other hand, may be quite different. It has recently been demonstrated that cationic liposomes are taken up primarily by endocytosis within the APC compartment [34,35] and we have shown that delivery of antigen by DDA liposomes both targets to, and accelerates antigen loading of, professional APC (Korsholm et al, submitted). We therefore hypothesize that liposome delivery of adenovirus redirects the virus particles to MHC class II-rich professional APC in which expressed protein is degraded in endocytic vesicles and presented to CD4 cells.…”

Effect of Headgroup on DNA−Cationic Surfactant Interactions

“…44,45 Another important factor for the transfection efficiency is the structure of the formed complexes, especially if the composition is not optimized. The efficiency of the transfection is not only dependent on the entrance of the complex into the cell but also the escape of the complexes from the endosomal membrane.…”

“…44,45 Inverted hexagonal structures are very efficient in releasing the DNA into the cell since these structures are much more prone to fusion. 45 With this in mind, one can rationalize that the presence of hydrophilic groups in the headgroups of the lipids can induce changes in terms of lipid packing that can lead to substantial differences in the self-assembly structures that are formed, when compared with similar lipids without those groups. Also they can, if sufficiently long, provide stability in serum and avoid the aggregation of the CL-DNA complexes due to steric repulsion.…”

Como estudar interações entre nanopartículas e sistemas biológicos

“…Sendo assim, a simples mistura de DNA e lipossomas catiônicos na solução leva a reorganização de suas estruturas formando agregados em múltiplas camadas podendo estar organizadas no estado líquido-cristalinas a depender, entre outras coisas, da relação entre as cargas positivas e negativas dos eletrólitos, das características estruturais dos lipossomas (como raio de curvatura), do tamanho do fragmento de nucleotídeo, pH, etc. 28 Lin et al 29 desenvolveram um estudo comparativo em que foram preparados dois complexos entre DNA e lipossomas com estruturas distintas: lamelar (L c α ), utilizando os lipídios DOTAP/DOPC e hexagonal invertida (H II c ) e os lipídios DOTAP/DOPE. Essas estruturas, ao serem colocadas em contato com células de fibroblastos de rato, apresentaram perfis de transfecção distintos.…”