Advances and challenges in liposome digestion: Surface interaction, biological fate, and GIT modeling.

During the past 50 years, there has been increased interest in liposomes as carriers of pharmaceutical, cosmetic, and agricultural products. More recently, much progress has been made in the use of surface-modified formulas in experimental food matrices. However, before the viability and the applications of nutrients in liposomal form in the edible field can be determined, the digestion behavior along the human gastrointestinal tract (GIT) must be clarified. In vitro digestion models, from static models to dynamic mono-/bi-/multi-compartmental models, are increasingly being developed and applied as alternatives to in vivo assays. This review describes the surface interactions of liposomes with their encapsulated ingredients and with external food components and updates the biological fate of liposomes after ingestion. It summarizes current models for the human stomach and intestine that are available and their relevance in nutritional studies. It highlights limitations and challenges in the use of these models for liposomal colloid system digestion and discusses crucial factors, such as enzymes and bile salts, that affect liposomal bilayer degradation.

Interactions Between Silver Nanoparticles/Silver Ions and Liposomes: Evaluation of the Potential Passive Diffusion of Silver and Effects of Speciation.

Silver nanoparticles, used mainly for their antibacterial properties, are among the most common manufactured nanomaterials. How they interact with aquatic organisms, especially how they cross biological membranes, remains uncertain. Free Ag+ ions, released from these nanoparticles, are known to play an important role in their overall bioavailability. In this project, we have studied the uptake of dissolved and nanoparticulate silver by liposomes. These unilamellar vesicles, composed of phospholipids, have long been used as models for natural biological membranes, notably to study the potential uptake of solutes by passive diffusion through the phospholipid bilayer. The liposomes were synthesized using extrusion techniques and were exposed over time to dissolved silver under different conditions where Ag+, AgS2O3-, or AgCl0 were the dominant species. Similar experiments were conducted with the complexes HgCl 2 0 and Cd(DDC) 2 0 , both of which are hydrophobic and known to diffuse passively through biological membranes. The uptake kinetics of Ag+, HgCl 2 0 , and Cd(DDC) 2 0 show no increase in internalized concentrations over time, unlike AgS2O3- and AgCl0, which appear to pass through the phospholipid bilayer. These results are in contradiction with our initial hypothesis that lipophilic Hg and Cd complexes would be able to cross the membrane, whereas silver would not. Encapsulated tritiated water inside the liposomes was shown to rapidly diffuse through the lipid bilayer, suggesting a high permeability. We hypothesize that monovalent anions or complexes as well as small neutral complexes with a strong dipole can diffuse through our model membrane. Finally, liposomes were exposed to 5-nm polyvinylpyrrolidone-coated silver nanoparticles over time. No significant uptake of nanoparticulate silver was observed. Neither disruption of the membrane nor invagination of nanoparticles into the liposomes was observed. This suggests that the main risk caused by AgNPs for nonendocytotic biological cells would be the elevation of the free silver concentration near the membrane surface due to adsorption of AgNPs and subsequent oxidation/dissolution.

Efavirenz oral delivery via lipid nanocapsules: formulation, optimisation, and ex-vivo gut permeation study.

Present investigation aimed to prepare, optimise, and characterise lipid nanocapsules (LNCs) for improving the solubility and bioavailability of efavirenz (EFV). EFV-loaded LNCs were prepared by the phase-inversion temperature method and the influence of various formulation variables was assessed using Box-Behnken design. The prepared formulations were characterised for particle size, polydispersity index (PdI), zeta potential, encapsulation efficiency (EE), and release efficiency (RE). The biocompatibility of optimised formulation on Caco-2 cells was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Then, it was subjected to ex-vivo permeation using rat intestine. EFV-loaded LNCs were found to be spherical shape in the range of 20-100 nm with EE of 82-97%. The best results obtained from LNCs prepared by 17.5% labrafac and 10% solutol HS15 when the volume ratio of the diluting aqueous phase to the initial emulsion was 3.5. The mean particle size, zeta potential, PdI, EE, drug loading%, and RE during 144 h of optimised formulation were confirmed to 60.71 nm, -35.93 mV, 0.09, 92.60, 7.39 and 55.96%, respectively. Optimised LNCs increased the ex vivo intestinal permeation of EFV when compared with drug suspension. Thus, LNCs could be promising for improved oral delivery of EFV.

Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes.

Fast hyperthermia (i.e. 39-42 °C) triggered doxorubicin release from lysolipid-containing thermosensitive liposomes (LTSL) in the tumor vasculature has been demonstrated to result in considerable enhancement of bioavailable drug levels in heated tumor tissue in preclinical tumor models. However, there is also significant leakage of doxorubicin already at 37 °C in the bloodstream, making these LTSL less efficient and increasing the risk for systemic toxicity. In conventional liposomes, cholesterol is incorporated in the bilayer to increase the stability of the liposomes. Here, we investigate the effect of cholesterol inclusion on the doxorubicin release characteristics of LTSL at 37 °C and hyperthermic temperatures. For this purpose, three LTSL formulations with 0, 5 and 10 mol% cholesterol were prepared. Inclusion of cholesterol reduced the undesired doxorubicin leakage at 37 °C in Hepes-buffered saline (HBS) as well as in fetal bovine serum (FBS). The incorporation of cholesterol in the LTSL bilayers did not influence the hyperthermia-triggered release property of the LTSL. These results were supported by DSC measurements. Therefore, in conclusion, our data indicate that cholesterol inclusion in LTSL offers a simple solution to the problem of significant leakage of doxorubicin from LTSL already at 37 °C in the bloodstream.

PEGylated lipid bilayer-supported mesoporous silica nanoparticle composite for synergistic co-delivery of axitinib and celastrol in multi-targeted cancer therapy.

UNLABELLED: Small-molecule drug combination therapies are an attractive approach to enhancing cancer chemotherapeutic responses. Therefore, this study aimed to investigate the potential of axitinib (AXT) and celastrol (CST) in targeting angiogenesis and mitochondrial-based apoptosis in cancer. Therefore, we prepared AXT/CST-loaded combination nanoparticles (ACML) with CST loaded in the mesoporous silica nanoparticles (MSN) and AXT in PEGylated lipidic bilayers. We showed that ACML effectively inhibited angiogenesis and mitochondrial function and was efficiently internalized in SCC-7, BT-474, and SH-SY5Y cells. Furthermore, hypoxia-inducible factor (HIF)-1α expression, which increased under hypoxic conditions in all cell lines exposed to ACML, markedly decreased, which may be critical for tumor inhibition. Western blotting showed the superior anticancer effect of combination nanoparticles in different cancer cells. Compared to the cocktail (AXT/CST), ACML induced synergistic cancer cell apoptosis. The AXT/CST-based combination nanoparticle synergism might be mediated by AXT, which controls vascular endothelial growth factor receptors while CST acts on target cell mitochondria. Importantly, ACML-treated mice showed remarkably higher tumor inhibition (64%) than other groups did in tumor xenograft models. Tumor xenograft immunohistochemistry revealed elevated caspase-3 and poly (ADP-ribose) polymerase and reduced CD31 and Ki-67 expression, clearly suggesting tumor apoptosis through mitochondrial and antiangiogenic effects. Overall, our results indicate that ACML potentially inhibited cell proliferation and induced apoptosis by blocking mitochondrial function, leading to enhanced antitumor efficacy. STATEMENT OF SIGNIFICANCE: In this research, we formulated an anticancer drug combination nanoparticle loaded with axitinib (AXT) in the lipidic bilayer of PEGylated liposomes and celastrol (CST) in mesoporous silica nanoparticles. The anticancer effects of the AXT/CST-loaded combination nanoparticle (ACML) were synergistic and superior to the other formulations and involved more efficient drug delivery to the tumor site with enhanced effects on angiogenesis and mitochondrial function. Therefore, our study demonstrated that the inhibition of cell proliferation and induction of apoptosis by ACML, which was mediated by blockade of mitochondrial function and anti-angiogenesis, led to enhanced antitumor efficacy, which may be potentially useful in the clinical treatment of cancer.

Interaction of C60 fullerene complexed to doxorubicin with model bilipid membranes and its uptake by HeLa cells.

With an aim to elucidate the effects of C60 fullerene complexed with antibiotic doxorubicin (Dox) on model bilipid membranes (BLM), the investigation of the electrical properties of BLM under the action of Dox and C60 fullerene, and of their complex, C60+Dox,was performed. The complex as well as its components exert a clearly detectable influence on BLM, which is concentration-dependent and also depends on phospholipid composition. The mechanism of this effect originates either from intermolecular interaction of the drug with fatty-acid residues of phospholipids, or from membranotropic effects of the drug-induced lipid peroxidation, or from the sum of these two effects. By fluorescence microscopy the entering of C60 + Dox complex into HeLa cells was directly shown.

Multimodal targeted high relaxivity thermosensitive liposome for in vivo imaging.

Liposomes are spherical, self-closed structures formed by lipid bilayers that can encapsulate drugs and/or imaging agents in their hydrophilic core or within their membrane moiety, making them suitable delivery vehicles. We have synthesized a new liposome containing gadolinium-DOTA lipid bilayer, as a targeting multimodal molecular imaging agent for magnetic resonance and optical imaging. We showed that this liposome has a much higher molar relaxivities r1 and r2 compared to a more conventional liposome containing gadolinium-DTPA-BSA lipid. By incorporating both gadolinium and rhodamine in the lipid bilayer as well as biotin on its surface, we used this agent for multimodal imaging and targeting of tumors through the strong biotin-streptavidin interaction. Since this new liposome is thermosensitive, it can be used for ultrasound-mediated drug delivery at specific sites, such as tumors, and can be guided by magnetic resonance imaging.

Biointerfacing polymeric microcapsules for in vivo near-infrared light-triggered drug release.

Seeking safe and effective water-soluble drug carriers is of great significance in nanomedicine. To achieve this goal, we present a novel drug delivery system based on biointerfacing hollow polymeric microcapsules for effectively encapsulating water-soluble antitumor drug and gold nanorod (GNR) functionalization for triggered release of therapeutic drugs on-demand using low power near-infrared (NIR) radiation. The surface of polymeric microcapsules is covered with fluidic lipid bilayers to decrease the permeability of the wall of polymeric capsules. The temperature increase upon NIR illumination deconstructs the structure of the lipid membrane and polyelectrolyte multilayers, which in turn results in the rapid release of encapsulated water-soluble drug. In vivo antitumor tests demonstrate that this microcapsule has the effective ability of inhibiting tumor growth and preventing metastases. Real time in vivo fluorescence imaging results confirm that capsules can be excreted gradually from the animal body which in turn demonstrates the biocompatibility and biodegradation of these biointerfacing GNR-microcapsules. This intelligent system provides a novel anticancer platform with the advantages of controlled release, biological friendliness and credible biosafety.

Gadolinium oxide nanoplates with high longitudinal relaxivity for magnetic resonance imaging.

Molecular-based contrast agents for magnetic resonance imaging (MRI) are often characterized by insufficient relaxivity, thus requiring the systemic injection of high doses to induce sufficient contrast enhancement at the target site. In this work, gadolinium oxide (Gd2O3) nanoplates are produced via a thermal decomposition method. The nanoplates have a core diameter varying from 2 to 22 nm, a thickness of 1 to 2 nm and are coated with either an oleic acid bilayer or an octylamine modified poly(acrylic acid) (PAA-OA) polymer layer. For the smaller nanoplates, longitudinal relaxivities (r1) of 7.96 and 47.2 (mM s)(-1) were measured at 1.41 T for the oleic acid bilayer and PAA-OA coating, respectively. These values moderately reduce as the size of the Gd2O3 nanoplates increases, and are always larger for the PAA-OA coating. Cytotoxicity studies on human dermal fibroblast cells documented no significant toxicity, with 100% cell viability preserved up to 250 µM for the PAA-OA coated Gd2O3 nanoplates. Given the 10 times increase in longitudinal relaxivity over the commercially available Gd-based molecular agents and the favorable toxicity profile, the 2 nm PAA-OA coated Gd2O3 nanoplates could represent a new class of highly effective T1 MRI contrast agents.

Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe.

We have rationally designed and synthesized a novel near-infrared (NIR) photoactivating probe, designated by iDOPE, in which an indocyanine green (ICG) fluorophore is covalently conjugated with a phospholipid moiety, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), to incorporate into liposome bilayers. NIR irradiation showed that iDOPE retained the optical and fluorescence properties of ICG and demonstrated photoactivator characteristics: fluorescence emission at around 820 nm in a solvent, singlet oxygen production, and concentration-dependent heat generation. Additionally, iDOPE was incorporated into liposome bilayers and maintained stable liposomally formulated iDOPE (LP-iDOPE) over 1week under physiological conditions. We also observed the tumor-specific biodistribution of LP-iDOPE of in vivo xenografts. These findings suggest that LP-iDOPE might be a promising tool for NIR optical imaging, photodynamic therapy, and photothermal therapy.

The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo.

Triggered release of liposomal contents following tumor accumulation and mild local heating is pursued as a means of improving the therapeutic index of chemotherapeutic drugs. Lysolipid-containing thermosensitive liposomes (LTSLs) are composed of dipalmitoylphosphatidylcholine (DPPC), the lysolipid monostearoylphosphatidylcholine (MSPC), and poly(ethylene glycol)-conjugated distearoylphosphatidylethanolamine (DSPE-PEG(2000)). We investigated the roles of DSPE-PEG(2000) and lysolipid in the functional performance of the LTSL-doxorubicin formulation. Varying PEG-lipid concentration (0-5 mol%) or bilayer orientation did not affect the release; however, lysolipid (0-10 mol%) had a concentration-dependent effect on drug release at 42 degrees C in vitro. Pharmacokinetics of various LTSL formulations were compared in mice with body temperature controlled at 37 degrees C. As expected, incorporation of the PEG-lipid increased doxorubicin plasma half-life; however, PEG-lipid orientation (bilayer vs. external leaflet) did not significantly improve circulation lifetime or drug retention in LTSL. Approximately 70% of lysolipid was lost within 1 h postinjection of LTSL, which could be due to interactions with the large membrane pool of the biological milieu. Considering that the present LTSL-doxorubicin formulation exhibits significant therapeutic activity when used in conjunction with mild heating, our current study provided critical insights into how the physicochemical properties of LTSL can be tailored to achieve better therapeutic activity.

Coaxial electrohydrodynamic spraying: a novel one-step technique to prepare oligodeoxynucleotide encapsulated lipoplex nanoparticles.

This study investigates coaxial electrohydrodynamic spraying (electrospray for short) as a novel, rapid, real time and single-step method to produce oligodeoxynucleotide (ODN) encapsulated lipoplex nanoparticles for either intravenous injection or, potentially, pulmonary delivery. Using a coaxial needle setup, we produced G3139 (oblimerson sodium, or Genasense) encapsulated lipoplex nanoparticles, and investigated the effects of production parameters on nanoparticle size and structure. Careful control of production parameters yielded lipoplex nanoparticles 190 +/- 39 nm in diameter with unilamellar structure and 90 +/- 6% encapsulation efficiency of G3139. Both nontargeted and transferrin-targeted G3139 lipoplex nanoparticles were efficiently delivered to K562 cells and downregulated the bcl-2 protein expression by 34 +/- 6% and 57 +/- 3% respectively.

Dissecting the membrane binding and insertion kinetics of a pHLIP peptide.

When it is bound to lipid bilayers, the conformation and location of the membrane pH (low) insertion peptide (pHLIP) depend on pH. This unique feature allows us to explicitly measure the kinetics leading to different membrane-bound states of pHLIP using a model membrane and stopped-flow technique. Our results show that the membrane association kinetics of pHLIP are multiexponential and are consistent with a parallel membrane interaction mechanism. Interestingly, our results also show that the overall rate at which the membrane-inserted state is formed is almost identical to that of formation of the surface-bound state, while prebinding slows the rate of peptide insertion.

Lipid-quantum dot bilayer vesicles enhance tumor cell uptake and retention in vitro and in vivo.

We report the construction of lipid-quantum dot (L-QD) bilayer vesicles by incorporation of the smallest (2 nm core size) commercially available CdSe/ZnS QD within zwitterionic dioleoylphosphatidylcholine and cationic 1,2-dioleoyl-3-trimethylammonium-propane lipid bilayers, self-assembling into small unilamellar vesicles. The incorporation of QD in the acyl environment of the lipid bilayer led to significant enhancement of their optical stability during storage and exposure to UV irradiation compared to that of QD alone in toluene. Moreover, structural characterization of L-QD hybrid bilayer vesicles using cryogenic electron microscopy revealed that the incorporation of QD takes place by hydrophobic self-association within the biomembranes. The L-QD vesicles bound and internalized in human epithelial lung cells (A549), and confocal laser scanning microscopy studies indicated that the L-QD were able to intracellularly traffick inside the cells. Moreover, cationic L-QD vesicles were injected in vivo intratumorally, leading to enhanced retention within human cervical carcinoma (C33a) xenografts. The hybrid L-QD bilayer vesicles presented here are thought to constitute a novel delivery system that offers the potential for transport of combinatory therapeutic and diagnostic modalities to cancer cells in vitro and in vivo.

New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid-DNA complexes for gene delivery.

BACKGROUND: Gene carriers based on lipids or polymers-rather than on engineered viruses-constitute the latest technique for delivering genes into cells for gene therapy. Cationic liposome-DNA (CL-DNA) complexes have emerged as leading nonviral vectors in worldwide gene therapy clinical trials. To arrive at therapeutic dosages, however, their efficiency requires substantial further improvement. METHODS: Newly synthesized multivalent lipids (MVLs) enable control of headgroup charge and size. Complexes comprised of MVLs and DNA have been characterized by X-ray diffraction and ethidium bromide displacement assays. Their transfection efficiency (TE) in L-cells was measured with a luciferase assay. RESULTS: Plots of TE versus the membrane charge density (sigmaM, average charge/unit area of membrane) for the MVLs and monovalent 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) merge onto a universal, bell-shaped curve. This bell curve leads to the identification of three distinct regimes, related to interactions between complexes and cells: at low sigmaM, TE increases with increasing sigmaM; at intermediate sigmaM, TE exhibits saturated behavior; and unexpectedly, at high sigmaM, TE decreases with increasing sigmaM. CONCLUSIONS: Complexes with low sigmaM remain trapped in the endosome. In the high sigmaM regime, accessible for the first time with the new MVLs, complexes escape by overcoming a kinetic barrier to fusion with the endosomal membrane (activated fusion), yet they exhibit a reduced level of efficiency, presumably due to the inability of the DNA to dissociate from the highly charged membranes in the cytosol. The intermediate, optimal regime reflects a compromise between the opposing demands on sigmaM for endosomal escape and dissociation in the cytosol.

A novel gene delivery system for mammalian cells.

Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

UV-induced drug release from photoactive REV sensitized by suprofen.

In order to achieve local administration of drugs, calcein (CAL) encapsulated reverse phase evaporation vesicles (REV) carrying photoactive destabilization agent suprofen (SPF) in the lipid bilayer were prepared. Effect of both UV-A and UV-B photoactivation of liposomal membrane incorporated SPF on the destabilization of the liposome bilayer and the release of encapsulated CAL was investigated. Standard REV of phosphatidylcholine (PC):cholesterol (CHOL) in 7:3 molar ratio, and photoactive REV of PC:CHOL:SPF, and DPPC:CHOL:SPF in 7:3:3 molar ratio were prepared. CAL encapsulation efficiency (EE (%)) and in situ release was studied. SPF incorporation in the PC REV membrane led to approximately 5% increase in the EE (34%) in comparison to standard REV (29%). EE decreased (21%) when DPPC was used to replace PC. Exposure to UV-B caused the highest CAL release. The lowest release was from the unexposed REV. DPPC led to a higher liposomal membrane stability (lower CAL release) than PC. A linear relationship was observed between UV-B exposure duration and REV permeability. This study revealed that membrane destabilization of SPF incorporated REV was best achieved upon photoactivation of the membrane-localized SPF by a 40 min exposure to UV-B.

High ceramide content liposomes with in vivo antitumor activity.

BACKGROUND: The pro-apoptotic activity of ceramide lipids has made this an exciting new target for therapeutic manipulation. While approaches using exogenous application of short-chain ceramides and modulation of endogenous ceramide levels via manipulation of metabolic pathways have been explored, controlled delivery of natural ceramide has not been previously reported. In this paper we describe the formulation of novel liposomes containing high levels of natural ceramide in the lipid bilayer for the purpose of controlled ceramide delivery. MATERIALS AND METHODS: Liposomes were characterized by cryo-transmission electron microscopy, quasi-elastic light scattering and trapped volume analysis. Pharmacokinetic analysis was performed following i.v. bolus dosing, and antitumor activity was evaluated following i.v. bolus and i.p. dosing in the J774 ascites tumor model. RESULTS: Stable ceramide-containing liposomes exhibited pharmacokinetic properties suitable for in vivo applications and resulted in an increase in lifespan of greater than 20% compared to control liposomes following i.v. bolus and i.p. administration. CONCLUSION: This work demonstrates proof-of-concept that delivery of exogenous natural ceramide lipid has antitumor activity in vivo, and highlights the potential utility of ceramide-based liposomes as a novel strategy for cancer chemotherapy based on controlled ceramide delivery.

Poloxamer gel as vehicle for transdermal iontophoretic delivery of arginine vasopressin: evaluation of in vivo performance in rats.

The present study describes the formulation and evaluation for pharmacokinetic and pharmacodynamic activity of arginine vasopressin (AVP), a nanopeptide with antidiuretic activity on being delivered by transdermal iontophoresis. Poloxamer 407 was used to form stable gels that did not reduce the release of AVP. The release rate from the gel followed Higuchi kinetics indicating that the dominant mechanism of release is diffusion. Iontophoresis alone and in combination with chemical enhancers was used to augment the transdermal permeation of AVP. The results of both pharmacokinetic and pharmacodynamic studies emphasize the dimension of 'rapid onset' achieved by iontophoresis. The correlation between pharmacokinetic data and pharmacodynamic activity was only qualitative. Histopathological studies revealed that skin toxicity caused by either iontophoresis or chemical enhancers when used alone could be reduced by using a combination of both the techniques in tandem.

Three-dimensional imaging of lipid gene-carriers: membrane charge density controls universal transfection behavior in lamellar cationic liposome-DNA complexes.

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.