Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon Interaction with anionic cellular lipids, resulting in DNA release At the early stages of interaction, We found a universal behavior of lipoplex/anionic lipid (AL) Mixtures the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs This finding is in excellent agreement with previous Suggestions on the mechanism of DNA unbinding from lipoplexes by ALs Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs Furthermore. we investigated the effect of the membrane charge density and a general correlation resulted the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release Lastly, the fort-nation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases Remarkably. also phase transition rates and [DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes These results highlight the phase properties of the carrier lipid/cellular lipid Mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carrier

Tailoring lipoplex composition to the lipid composition of plasma membrane: a Trojan horse for cell entry?

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raftmediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells.Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing

The molecular and crystal structure of an allylpalladium(II) triazenido complex: di--(1,3-di-p-tolyltriazenido)di(1-3--allyl)dipalladium(II)

The structure of [(1-3--C3H5)Pd(II)(p-CH3C6H4NNNC6H4CH3-p)]2 was detd. by single-crystal x-ray anal. The compd. has space group P21/c, with a 8.510(2), b 40.652(9), c 9.762(2) Å, 103.61(2)°, and d.(calcd.) = 1.50. R = 0.041, Rw = 0.060, based on 3978 independent reflections. The 2 -allylpalladium residues are bridged by 2 1,3-di-p-tolyltriazenido groups, gaining an approx. square planar coordination around each heavy atom. The 2 allyl units are stereochem. equiv., with the central C atoms pointing outwards. The rigid triazenido groups force the 2 Pd atoms into close contact (2.86 Å). The arom. rings are somewhat rotated with respect to the bonded N-N-N planes, but some -conjugation over the whole ligand is still retained

Allylic palladium(II) triazenido complexes: synthesis and spectroscopic studies

Reaction of [Pd(1-3-η-allyl)Cl]2 with lithium triazenide (triazenide = p-XC6H4NN-NC6H4X-p; X = Cl, H, CH3) affords dimeric complexes of the type [Pd(1-3-η-allyl)(triazenide)]2. In the solid state the triazenido ligands are bridging two palladium atoms with their terminal nitrogen atoms, as shown by a preliminary X-ray determination of the complex with X = CH3. The allyl groups are stereochemically equivalent. 1H NMR spectra demonstrate the presence of two conformers in solution. The major component has the same configuration found in the solid. The other conformer has stereochemically non equivalent allyl groups. The concentration ratio of the two conformers is independent of the temperature, suggesting the absence of intramolecular processes and of palladium- triazenido bond breaking. This point is discussed also by comparing the (1-3-η-allyl)(triazenide)palladium (II) dimers with the closely related(1-3-η-allyl)(acetate)palladium(II) complexes. © 1975

ON THE ROLE OF HYDRONIUM IONS IN THE PROTONATED MICELLAR AGGREGATES OF BILE SALTS

Abstract In previous work, structural units observed in bile salt crystals and fibres have been successfully used to represent bile salt micellar aggregates in aqueous solutions and electromotive force measurements have shown that protonated micellar species are present below some critical values of pH. This paper deals with the crystal structures of 3 alpha,12 alpha-dihydroxy-5 beta-cholanoylglycine (HGDC), 3 alpha,12 alpha-dihydroxy-5 beta-cholanoyltaurine (HTDC) and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyltaurine (HTUDC), which were solved to obtain models of protonated micellar aggregates. The models are compared with those found in crystals and fibres of sodium and rubidium salts of HGDC and HTDC (NaGDC, NaTDC, RbGDC, RbTDC) in order to verify whether the acid structures match with the salt structures. The HGDC packing resembles that of a NaTDC crystal and is stabilized mainly by hydrogen bonds as well as by dipole-dipole interactions between acetone molecules and carboxylic groups. Three different 3(1) helices are identified. One of these can be easily transformed into the 7/1 helix which satisfactorily describes the NaGDC, NaTDC, RbGDC and RbTDC micellar aggregates. The HTDC and HTUDC crystal structures are practically the same. Strong hydrogen bonds between H3O+ (hydronium ion) and three oxygen atoms of the anions show O ... O distances within the range 2.4-2.6 Angstrom, owing to additional ion-ion and ion-dipole interactions. Very probably, H3O+ replaces Na+ in the micellar aggregates without remarkably changing their structure because the H3O+... O and Na+... O distances are very close. Inspection of previous electromotive force data indicates that the glycodeoxycholate and taurodeoxycholate micellar aggregates' proton affinities increase as their sizes increase and that those of the bigger aggregates seem to converge, even though the proton affinity of COO- is greater than that of SO3-. These findings strongly suggest that micellization induces H3O+ binding. HTDC and HTUDC form micellar aggregates which increase their apparent hydrodynamic radius by adding HCl

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm2/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm2/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm(2)/s). In the cytosol, the lipoplexes’ motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes