The 60-year evolution of lipid nanoparticles for nucleic acid delivery.

Delivery of genetic information to the interior of target cells in vivo has been a major challenge facing gene therapies. This barrier is now being overcome, owing in part to dramatic advances made by lipid-based systems that have led to lipid nanoparticles (LNPs) that enable delivery of nucleic acid-based vaccines and therapeutics. Examples include the clinically approved COVID-19 LNP mRNA vaccines and Onpattro (patisiran), an LNP small interfering RNA therapeutic to treat transthyretin-induced amyloidosis (hATTR). In addition, a host of promising LNP-enabled vaccines and gene therapies are in clinical development. Here, we trace this success to two streams of research conducted over the past 60 years: the discovery of the transfection properties of lipoplexes composed of positively charged cationic lipids complexed with nucleic acid cargos and the development of lipid nanoparticles using ionizable cationic lipids. The fundamental insights gained from these two streams of research offer potential delivery solutions for most forms of gene therapies.

Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Structural Properties of Inverted Hexagonal Phase: A Hybrid Computational and Experimental Approach.

Inverted/reverse hexagonal (HII) phases are of special interest in several fields of research, including nanomedicine. We used molecular dynamics (MD) simulation to study HII systems composed of dioleoylphosphatidylethanolamine (DOPE) and palmitoyloleoylphosphatidylethanolamine (POPE) at several hydration levels and temperatures. The effect of the hydration level on several HII structural parameters, including deuterium order parameters, was investigated. We further used MD simulations to estimate the maximum hydrations of DOPE and POPE HII lattices at several given temperatures. Finally, the effect of acyl chain unsaturation degree on the HII structure was studied via comparing the DOPE with POPE HII systems. In addition to MD simulations, we used deuterium nuclear magnetic resonance (2H NMR) and small-angle X-ray scattering (SAXS) experiments to measure the DOPE acyl chain order parameters, lattice plane distances, and the water core radius in HII phase DOPE samples at several temperatures in the presence of excess water. Structural parameters calculated from MD simulations are in excellent agreement with the experimental data. Dehydration decreases the radius of the water core. An increase in hydration level slightly increased the deuterium order parameter of lipids acyl chains, whereas an increase in temperature decreased it. Lipid cylinders undulated along the cylinder axis as a function of hydration level. The maximum hydration levels of PE HII phases at different temperatures were successfully predicted by MD simulations based on a single experimental measurement for the lattice plane distance in the presence of excess water. An increase in temperature decreases the maximum hydration and consequently the radius of the water core and lattice plane distances. Finally, DOPE formed HII structures with a higher curvature compared to POPE, as expected. We propose a general protocol for constructing computational HII systems that correspond to the experimental systems. This protocol could be used to study HII systems composed of molecules other than the PE systems used here and to improve and validate force field parameters by using the target data in the HII phase.

Efficacy of antireflux surgery in children with or without neurological impairment: a systematic review.

BACKGROUND: Antireflux surgery is commonly performed in children, yet evidence for its efficacy is limited. The aim of this review was to determine the effect of antireflux surgery with regard to objective measures of quality of life (QoL) and value of upper gastrointestinal investigations in neurologically normal (NN) and neurologically impaired (NI) children. METHODS: A systematic review was conducted of articles reporting children undergoing antireflux surgery in whom preoperative and postoperative objective testing was performed. Primarily, Embase, CINAHL, MEDLINE and PubMed were searched from inception to April 2019. Methodological Index for Non-randomized Studies (MINORS) criteria were used to assess article quality. RESULTS: Of 789 articles, 14 met the eligibility criteria, 12 prospective observational and 2 retrospective studies. The median MINORS score was 59·4 (i.q.r. 39 to 62·5) per cent. Seven studies reported assessment of validated QoL measures before and after antireflux surgery in 148 children. Follow-up ranged from 1 to 180 months. All studies confirmed significant improvements in QoL measures among NN and NI children at all follow-up points. Eleven studies reported on preoperative and postoperative investigations in between 416 and 440 children children. Follow-up ranged from 0·5 to 180 months. Nine studies confirmed improvements in gastro-oesophageal reflux using 24-h oesophageal pH monitoring with or without manometry, but conflicting results were identified for four studies reporting gastric emptying. No studies reported fluoroscopy or endoscopy adequately. CONCLUSION: Based on the results of studies of low-to-moderate quality, antireflux surgery improved QoL and reduced oesophageal acid exposure in NN and NI children in the short and medium term. Although antireflux surgery is a common elective operation, the lack of rigorous preoperative and postoperative evaluation(s) in the majority of patient-reported studies is striking.

ANTECEDENTES: La cirugía antirreflujo (antireflux surgery, ARS) se realiza con frecuencia en niños, aunque la evidencia sobre su eficacia es limitada. Nos propusimos determinar la eficacia de la ARS con respecto a medidas objetivas de calidad de vida (quality of life, QoL) y la utilidad de las exploraciones del tracto gastrointestinal superior en niños con funciones neurológicas normales (neurologically normal, NN) y con discapacidad neurológica (neurologically impaired, NI). MÉTODOS: Se llevó a cabo una revisión sistemática (de acuerdo con la normativa PRISMA) de artículos que describiesen series de niños sometidos a ARS en los que se realizaron pruebas objetivas preoperatorias y postoperatorias. Principalmente, se efectuó una búsqueda en EMBASE, CINAHL, Medline y Pubmed desde un comienzo hasta 04/19. La calidad de los artículos se evaluó siguiendo los criterios MINORS. RESULTADOS: De 789 artículos, 14 reunían los criterios de elegibilidad - 12 estudios observacionales prospectivos y 2 estudios retrospectivos. La mediana de la puntuación MINORS fue de 59,4 % (rango intercuartílico 23,4%). Siete estudios describieron la evaluación de medidas de QoL validadas antes y después de ARS en 148 niños. El seguimiento varió de 1 a 180 meses. Todos los estudios confirmaron mejorías significativas en medidas de QoL en los niños NN y NI en todos los puntos del seguimiento. Once estudios describieron sobre las investigaciones preoperatorias y postoperatorias en 507 niños. El seguimiento oscilaba de 0,5 a 180 meses. Nueve estudios confirmaron mejorías en el reflujo gastroesofágico utilizando la monitorización del pH esofágico de 24 horas +/- manometría, pero los resultados eran contradictorios en 3 estudios que evaluaron el vaciamiento gástrico. Ningún estudio describió de forma adecuada los datos de la fluoroscopia o endoscopia. CONCLUSIÓN: De los estudios identificados de baja o moderada calidad, en un seguimiento a corto y medio plazo, la ARS mejora la QoL y reduce la exposición del esófago al ácido en niños NN y niños NI. A pesar de que la ARS es una cirugía electiva habitual, es llamativa la falta de evaluaciones preoperatorias y postoperatorias rigurosas en la mayoría de los estudios de pacientes.

Coating of PLA-nanoparticles with cyclic, arginine-rich cell penetrating peptides enables oral delivery of liraglutide.

Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.

Ionizable amino lipid interactions with POPC: implications for lipid nanoparticle function.

Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.

Lipid nanoparticle delivery systems for siRNA-based therapeutics.

Therapeutics based on small interfering RNA (siRNA) have a huge potential for the treatment of disease but requires sophisticated delivery systems for in vivo applications. Lipid nanoparticles (LNP) are proven delivery systems for conventional small molecule drugs with over eight approved LNP drugs. Experience gained in the clinical development of LNP for the delivery of small molecules, combined with an understanding of the physical properties of lipids, can be applied to design LNP systems for in vivo delivery of siRNA. In particular, cationic lipids are required to achieve efficient encapsulation of oligonucleotides; however, the presence of a charge on LNP systems can result in toxic side effects and rapid clearance from the circulation. To address these problems, we have developed ionizable cationic lipids with pKa values below 7 that allow oligonucleotide encapsulation at low pH (e.g., pH 4) and a relatively neutral surface at physiological pH. Further optimization of cationic lipids to achieve maximized endosomal destabilization following uptake has resulted in LNP siRNA systems that can silence genes in hepatocytes at doses as low as 0.005 mg siRNA/kg body weight in mouse models. These systems have been shown to be highly effective clinically, with promising results for the treatment of hypercholesterolemia and transthyretin-induced amyloidosis among others. More LNP siRNA therapeutics, targeting different tissues and diseases, are expected to become available in the near future.

Nineteenth century mortality at the Royal Hospital for Sick Children, Glasgow.

BACKGROUND AND AIMS: Since Glasgow's Royal Hospital for Sick Children opened in 1882, significant advances have been made in child health. Our aim was to investigate the hospital mortality that occurred in the last decade of the 19th century at Royal Hospital for Sick Children. METHODS AND RESULTS: Hospital mortality records for the decades, 1890-1899, were collected from the Archives of the Royal Hospital for Sick Children. Data were extracted from the hospital inpatient records and the pathology records. In the decade 1890-1899, there were 731 hospital deaths. The main cause of death at that time in the 19th Century was infection, particularly of the respiratory tract. The age at which death occurred was analysed and the recorded cause were analysed as was the distance patients travelled to the hospital. The ratio of boys to girls and length of inpatient stay was similar to that seen in the hospital currently. CONCLUSION: This study records the mortality in a children's hospital in a large developing industrial city in the 19th century as experienced in a children's hospital.

Dipyridamole and paracetamol overdose resulting in multi-organ failure.

Dipyridamole intoxication is rare and few reports exist amongst the current literature. A case of dipyridamole and paracetamol overdose is described in a previously healthy 58-year-old woman, which resulted in multi-organ failure requiring dialysis, inotropic support, ventilation and extensive surgical intervention for small bowel ischaemia. This case highlights the dangers of an unusually large overdose of a commonly prescribed drug, and reviews current knowledge of dipyridamole intoxication.

Biologically active contaminants of intravenous saline in PVC packaging: Australasian, European, and North American samples.

We have previously found evidence of contaminants in 0.9% saline packaged in polyvinyl chloride (PVC) for clinical use. For this current study we asked two questions: (1) what are the organic chemical contaminants in solutions packaged in PVC? and (2) do the contaminants vary in bags manufactured in different countries? We studied samples of 0.9% saline packaged in PVC from Australasia, Europe, and North America. We analysed the saline solutions with high-resolution capillary gas chromatography - mass spectrometry. Components of the Australasian and European bags were also analysed using thermodesorption followed by gas chromatography and mass spectrometry. In three pairs of samples we found twenty-four different organic contaminants of the saline solutions. Diethylhexyl phthalate (DEHP) was found in all solutions; the concentrations were greater than 10 microg/l in the Australasian and European samples. All samples contained 2-ethyl hexanol (>50 microg/l), a DEHP breakdown product. The Australasian and North American samples contained cyclohexanone at concentrations of about 1000 microg/l. The cyclohexanone probably originated in joints at the bases of the bags. Both the Australasian and European bags contained t-butyl cyclohexanol (>500 microg/l) and t-butyl cyclohexanone (>50 microg/l). Printing ink on the outside of the bags was the most likely source for both of these contaminants. Several of the contaminants are toxic to animals. Little is known about the toxicity of combinations of contaminants.

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression.

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.

An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression.

The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.

The use of extended amino acid motifs for focussing on toxic peptides in coeliac disease.

Cereal prolamins of wheat, rye and barley are the major proteins that have been implicated in toxicity in patients with coeliac disease. The gliadins of wheat are the best characterised with the identification of toxic peptides from rye and barley not as well advanced. This study has employed extended motifs, based on the known toxic motifs are derived from the sequence of A-gliadin, to search protein databases for matches with coeliac-toxic cereals. The results obtained have provided pointers to specific regions in rye and barley prolamins, which have received little attention in in vitro and in vivo studies of toxicity in coeliac disease. The results obtained in this study indicate that the size of the extended motif is critical when searching for coeliac-toxic cereals using protein databases. Extended motifs that are common to all three coeliac-toxic cereals and found in active wheat gliadin peptides are QQPYP, PQQPY and QQQPFP.

Extraction of cereal prolamins and their toxicity in coeliac disease.

Simple extraction of prolamins from cereal flour using 70% aqueous ethanol leads to co-extraction of lipids and other secondary products. Treatment of the crude extract with an excess of pure ethanol resulted in the removal of the majority of these compounds. Prolamin extracts obtained following ethanol precipitation showed little difference to products of more complex, multi stage, selective extractions, when compared using MALDI-TOF mass spectrometry. Toxicity tests on enzymic digests of ethanol-precipitated prolamins from coeliac-toxic cereals, coeliac-non-toxic cereals and oats were carried out using a rat liver lysosome assay. The prolamins from the ethanol-precipitated extracts showed greater activity than those extracted using only 70% ethanol for extraction. As the ethanol precipitation method is simple and provides a prolamin extract of sufficient purity for further evaluation, this procedure has been adopted as an alternative to more tedious procedures for preparation of cereal prolamins.

Developments in liposomal drug delivery systems.

Liposomes are the leading drug delivery systems for the systemic (iv.) administration of drugs. There are now liposomal formulations of conventional drugs that have received clinical approval and many others in clinical trials that bring benefits of reduced toxicity and enhanced efficacy for the treatment of cancer and other life-threatening diseases. The mechanisms giving rise to the therapeutic advantages of liposomes, such as the ability of long-circulating liposomes to preferentially accumulate at disease sites including tumours, sites of infection and sites of inflammation are increasingly well understood. Further, liposome-based formulations of genetic drugs such as antisense oligonucleotides and plasmids for gene therapy that have clear potential for systemic utility are increasingly available. This paper reviews the liposomal drug delivery field, summarises the success of liposomes for the delivery of small molecules and indicates how this success is being built on to design effective carriers for genetic drugs.

Cervical dystonia severity scale reliability study.

Cervical dystonia (CD) is characterized by sustained contractions of the neck musculature, resulting in abnormal head postures. The Cervical Dystonia Severity Scale (CDSS) was developed to provide a reliable measure of treatment response in patients with CD. The CDSS uses a protractor and wall chart to rate the severity of the head's deviation from neutral in each of three planes of motion (rotation, laterocollis, anterocollis/retrocollis), which is then scored in 5 degree intervals (1 degree to 5 degrees deviation = 1; 86 degree to 90 degrees deviation = 18). To test the reliability of the CDSS, four centers, each with two independent examiners, evaluated 42 patients with CD. At each site, each of the two examiners used the CDSS to evaluate the head position of each patient twice, on the same day, for a total of four evaluations. The kappa value for intra-examiner agreement was 0.94 (95% confidence limit of 0.900-0.972), indicating excellent intra-examiner reliability. The kappa value for interexaminer reliability was 0.79 for the first evaluation and 0.86 for the second evaluation (95% confidence limits of 0.668-0.920 and 0.790-0.920) indicating excellent interexaminer reliability. Thus, the CDSS was highly reliable in both intra-examiner and interexaminer scoring comparisons.

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids.

The mechanism whereby cationic lipids destabilize cell membranes to facilitate the intracellular delivery of macromolecules such as plasmid DNA or antisense oligonucleotides is not well understood. Here, we show that cationic lipids can destabilize lipid bilayers by promoting the formation of nonbilayer lipid structures. In particular, we show that mixtures of cationic lipids and anionic phospholipids preferentially adopt the inverted hexagonal (H(II)) phase. Further, the presence of 'helper' lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipid-mediated transfection of cells also facilitate the formation of the H(II)phase. It is suggested that the ability of cationic lipids to promote nonbilayer structures in combination with anionic phospholipids leads to disruption of the endosomal membrane following uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating cytoplasmic release of the plasmid or oligonucleotide.

Mechanism and stereochemistry of diphosphate formation from dioxaphosphorinanes: a critical reassessment.

The mechanism of diphosphate formation from (R)-2-chloro-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorinane (5a) and 2-hydroxy-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorinane (6) has been investigated. The products formed are the ax-ax diphosphate 7a and the ax-eq diphosphate 7b, with no evidence in the 31P NMR spectrum for pentacoordinate chlorooxyanionic phosphoranes 9. The structure of 7bhas been established unambiguously by NMR spectroscopy, mass spectrometry, and elemental analysis, and the structures of 5a and 7a have been confirmed by X-ray crystallography. The mechanism of the crucial diphosphate-forming reaction has been probed using 18O-labeling studies. The 18O-labeling patterns are consistent with the unsymmetric ax-eq diphosphate 7b arising from selective nucleophilic attack of the axial oxygen of 6 on the chloride 5a with inversion of configuration at phosphorus. The symmetric ax-ax diphosphate 7a can be formed directly, as a result of selective nucleophilic attack of the axial oxygen of 6 on the chloride 5a with retention of configuration, but the majority arises indirectly by isomerization of the ax-eq diphosphate 7b. The isomerization apparently involves intermolecular exchange, with nucleophilic attack of the phosphate anion 6 on the equatorially substituted phosphorus atom of 7b with inversion of configuration at phosphorus.

Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells.

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer (M(r) 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the headgroup (referred to as CPL(1)-CPL(4), respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios < or =0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60 degrees C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca(2+). The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL(2) displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL(3), while for CPL(4)-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.