Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D₃⁵⁰⁺) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.     

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of K_Chol = 5.94 (± 0.8) × 10⁴ M^–1, K_DDAB = 8.33 (± 0.90) × 10⁵ M^–1, K_DOTAP = 1.05 (± 0.30) × 10⁵ M^–1 and K_DOPE = 2.75 (± 0.50) × 10⁴ M^–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.     

Intranasal Immunization with DOTAP Cationic Liposomes Combined with DC-Cholesterol Induces Potent Antigen-Specific Mucosal and Systemic Immune Responses in Mice

Despite the progress made by modern medicine, infectious diseases remain one of the most important threats to human health. Vaccination against pathogens is one of the primary methods used to prevent and treat infectious diseases that cause illness and death. Vaccines administered by the mucosal route are potentially a promising strategy to combat infectious diseases since mucosal surfaces are a major route of entry for most pathogens. However, this route of vaccination is not widely used in the clinic due to the lack of a safe and effective mucosal adjuvant. Therefore, the development of safe and effective mucosal adjuvants is key to preventing infectious diseases by enabling the use of mucosal vaccines in the clinic. In this study, we show that intranasal administration of a cationic liposome composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N'',N''-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposome) has a potent mucosal adjuvant effect in mice. Intranasal vaccination with ovalbumin (OVA) in combination with DOTAP/DC-chol liposomes induced the production of OVA-specific IgA in nasal tissues and increased serum IgG1 levels, suggesting that the cationic DOTAP/DC-chol liposome leads to the induction of a Th2 immune response. Additionally, nasal-associated lymphoid tissue and splenocytes from mice treated with OVA plus DOTAP/DC-chol liposome showed high levels of IL–4 expression. DOTAP/DC-chol liposomes also enhanced OVA uptake by CD11c⁺ dendritic cells in nasal-associated lymphoid tissue. These data demonstrate that DOTAP/DC-chol liposomes elicit immune responses via an antigen-specific Th2 reaction. These results suggest that cationic liposomes merit further development as a mucosal adjuvant for vaccination against infectious diseases.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.     

Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study

The possibility of cationic (di-oleoyltrimethylammonium propane, DOTAP)/(l-α-dioleoylphosphatidyl-ethanolamine, DOPE) liposomes to act as carriers of boronated compounds such as 1,2-dicarba-closo-dodecaboran(12)-1-ylmethyl](β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside and 1,2-di-(β-d-gluco-pyranosyl-ox)methyl-1,2-dicarba-closo-dodeca-borane(12) has been investigated by Electron Spin Resonance (ESR) of n-doxyl stearic acids (n-DSA) and Quasi-Elastic Light Scattering (QELS). Both these carboranes have potential use in Boron Neutron Capture Therapy (BNCT), which is a targeted therapy for the treatment of radiation resistant tumors. They were shown to give aggregation both in plain water and in saline solution. Carborane aggregates were, however, disrupted when DOTAP/DOPE liposome solutions were used as dispersing agents. The computer analysis of the ESR spectra from carborane-loaded liposomes allowed to establish an increase of the order degree in the liposome bilayer with increasing carborane concentration, together with a decreased mobility. The same discontinuities of both correlation time and order parameter with respect to temperature variations were observed in carborane-containing and carborane-free liposomes. This suggested that a homogeneous dispersion of nitroxides and carboranes occurred in the liposome bilayer. The ESR line shape analysis proved that no dramatic changes were induced in the liposome environment by carborane insertion. QELS data showed that the overall liposome structure was preserved, with a slight decrease in the mean hydrodynamic radius and increase in polydispersity caused by the guest molecules.     

NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.     

Binding oligonucleotides to Escherichia coli and Bacillus stearothermophilus 5 S RNA.

Binding complementary tri- and tetranucleotides to Escherichia coli A19 and Bacillus stearothermophilus 799 5 S RNAs permitted identification of single-stranded regions in these RNAs. Sequences around positions 10, 30, 60, 70, 85 and 95 are in a single-stranded conformation in both 5 S RNAs. It is concluded that the overall structure of bacterial 5 S RNA has been conserved during evolution. Two types of structural conservation have been observed at specific sites of the 5 S RNA: firstly, nucleotide sequence and single strandedness and secondly, single strandedness only. The oligonucleotide binding data for E. coli 5 S RNA are in general agreement with a previous study (Lewis and Doty, 1970) and do not support fully any proposed structural model.     

Mechanism of Membrane Permeation Induced by Synthetic β-Hairpin Peptides

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ∼50% of the liposomes that contain small (R_h <1.5 nm) markers, only ∼15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 β-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.     

Oxidative stress can affect the gene silencing effect of DOTAP liposome in an in vitro translation system

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.     

Physico-chemical aspects of the interaction between DNA and oppositely charged mixed liposomes

The mechanism of complex formation between DNA and oppositely charged dioctadecyldimethylammonium bromide/dioleoyl phosphatidylethanolamine (DODAB/DOPE) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/DOPE mixed liposomes, as well as the physico-chemical properties of DNA-mixed liposome complexes, were examined. Fluorescence microscopy showed that the interaction between DNA and oppositely charged mixed liposomes started at very low liposome concentrations and induced a discrete coil-globule transition in individual DNA molecules. The DNA size distribution was bimodal in a wide range of liposome concentrations. The critical concentration of the cationic lipid needed for the complete compaction of single DNA molecules depended on the composition of the charged mixed DODAB/DOPE and DOTAP/DOPE liposomes. Cryogenic transmission electron microscopy (cryo-TEM) observations of DNA complexes with mixed liposomes revealed that the lamellar packing of lipid molecules was typical for the complexes formed from the cationic lipid-enriched mixtures, while inverted hexagonal arrays were found for the neutral lipid-enriched complexes. The microstructures of the complexes were also examined with the use of the small-angle X-ray scattering (SAXS) technique, which confirmed the results obtained by cryo-TE microscopy and enabled the quantitative characterization of lipid packaging in the complexes with DNA macromolecules. We also found that the introduction of the neutral lipid into the complexes between DNA and oppositely charged lipids, DODAB and DOTAP, moderately increased the thermal stability of the complexes and changed the quantitative characteristics of the melting profiles of the complexes.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Mechanism of Membrane Permeation Induced by Synthetic β-Hairpin Peptides

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ∼50% of the liposomes that contain small (R_h <1.5 nm) markers, only ∼15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 β-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.     

Size distribution of spontaneously formed liposomes by the alcohol injection method

A dynamic light scattering study of the size distribution of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes formed by the injection method is presented. By this method, an aliquot of methanol stock solution containing the surfactant is injected into water. The main aim of the present work was to determine under which conditions a monomodal and narrow size distribution could be obtained. The influence of several parameters on the size distribution was investigated. Firstly, we examined the influence of the POPC concentration in the initial stock methanol solution, when the POPC concentration in the final aqueous solution remains constant; secondly, the influence of POPC concentration in the aqueous phase, while the lipid concentration in the stock methanol remains constant. In both cases narrow monomodal size distributions of liposomes, centered between 40 and 70 nm, are obtained at low concentrations of POPC, in the stock methanol solution (相似文献   

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNA^Phe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNA^Phe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.     

Preparation of Irinotecan-Loaded Folate-Targeted Liposome for Tumor Targeting Delivery and Its Antitumor Activity

The purpose of this study was to investigate the in vivo distribution and antitumor activity of irinotecan (camptothecin (CPT)-11)-loaded folate-targeted liposome (F-Lip) in tumor-bearing mice following i.v. administration. Folate–poly(ethylene glycol)–distearoylphosphatidylcholine (FA–PEG–DSPE) was synthesized by amide reaction of DSPE–PEG–NH₂ and FA. F-Lip modified by FA–PEG–DSPE was prepared by an ammonium sulfate gradient. The mean particle size and entrapment efficiency of F-Lip with negative charge were 197.8 ± 4.58 nm and 91.39 ± 2.34 %, respectively. The distributions of CPT-11 and SN-38 in the tumor after i.v. administration of F-Lip, CPT-11-loaded liposomes (C-Lip), and CPT-11 injection (C-Inj) were far greater with the F-Lip group in comparison to the C-Inj and C-Lip, which might contribute to folate-meditated targeting uptake by the folate receptor on the surface of the tumor cells. The uptake of CPT-11 in the liver and rectum for two liposome groups were all markedly increased as compared to the C-Inj. Moreover, F-Lip exhibited a dose-dependent tumor growth inhibition and superior anticancer activity to C-Lip and C-Inj after i.v. administration. It also showed no significant body weight loss and much lower toxicity on the center immune organs. Therefore, F-Lip may be presented as potential candidates for tumor targeting drug delivery.KEY WORDS: cancer targeting, CPT-11, folate, liposomes, SN-38     

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

Calorimetric study of the interaction of binary DMTAP/DOTAP cationic liposomes with plasmid DNA

Cationic liposomes have been suggested as possible agents for nonviral gene transfer. The interaction of plasmid DNA (pDNA) with dispersions of stable unilamellar cationic liposomes based on the binary lipid system 1,2-dimyristoyl-3-trimethyl-ammonium-propane (DMTAP):1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) has been studied by using isothermal titration calorimetry (ITC), high-precision differential scanning calorimetry (DSC), dynamic light scattering (DLS), and circular dichroism (CD). Systematic calorimetric and DLS exploration of the DMTAP:DOTAP binary system reveals that single-bilayer liposomes are stable at the 4:1 molar ratio, exhibiting the main lipid-phase transition temperature at ~25.3°C, and a total enthalpy change δH = 8.5 ± 0.4 kcal/mol. The interaction of pDNA with unilamellar DMTAP:DOTAP vesicles was investigated by ITC experiments, which clearly distinguished endothermic binding between the phosphate and the ammonium groups from exothermic processes, driven by slow kinetics, corresponding to interliposomal, DNA-triggered aggregation that leads to the formation of large multilamellar liposome/pDNA assemblies. Lipid-added-to-pDNA and pDNA-added-to-lipid experiments have been carried out in order to systematically explore the interaction mechanisms. Complex ITC profiles are revealed, which may be linked to packing rearrangements of the pDNA molecules bound at the outer liposomal surface, possibly due to binding to more than one liposome or due to p-DNA-enhanced heterogeneity in the local lipid concentration. DNA-mediated aggregation effects are detected at high [ammonium]/[phosphate] molar ratios in the case of lipid-added-to-pDNA interactions and at relatively low [phosphate]/[ammonium] molar ratios in the case of pDNA-added-to-lipid.     

Fluorescence cross-correlation spectroscopy as a valuable tool to characterize cationic liposome-DNA nanoparticle assembly

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.