Fluorescent-labeled Liposomes Research Articles

Development of the observation of membrane fusion with label-free liposomes by calcium imaging

Liposomes are artificial vesicles composed of lipid bilayers that have enabled drugs to be encapsulated and delivered to tumor tissue. Membrane-fusogenic liposomes fuse with the plasma membranes of cells to deliver encapsulated drugs directly to the cytosol, which makes it a promising method for rapid and highly efficient drug delivery. In a previous study, liposomal lipid bilayers were labeled with fluorescent probes, and colocalization of labeled lipids with plasma membrane was observed under a microscope. However, there was concern that fluorescent labeling would affect lipid dynamics and cause liposomes to acquire membrane fusogenic ability. In addition, encapsulation of hydrophilic fluorescent substances in the inner aqueous phase sometimes requires an additional step of removing unencapsulated substances after preparation, and there is a risk of leakage. Herein, we propose a new method to observe cell interaction with liposomes without labeling. Our laboratory has developed two types of liposomes with different cellular internalization pathways, i.e., endocytosis and membrane fusion. We found that cytosolic calcium influx would be triggered following the internalization of cationic liposomes, and different cell entry routes led to different calcium responses. Thus, the correlation between cell entry routes and calcium responses could be utilized to study liposome-cell interactions without fluorescent labeling lipids. Briefly, liposomes were added to phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells, and calcium influx was measured by time-lapse imaging using a fluorescent indicator (Fura 2-AM). Liposomes with high membrane fusogenic ability elicited a strong transient calcium response immediately after adding liposomes, whereas those taken up mainly by endocytosis elicited multiple weak calcium responses. In order to verify the cell entry routes, we also tracked the intracellular distribution of fluorescent-labeled liposomes in PMA-primed THP-1 cells using a confocal laser scanning microscope. It was shown that for fusogenic liposomes, colocalization with plasma membrane occurred at the same time as calcium elevation, whereas for liposomes with a high endocytosis potential, fluorescent dots were observed in the cytoplasm, suggesting the cell internalization by endocytosis. These results suggested that the calcium response patterns correspond to cell entry routes, and membrane fusion can be observed by calcium imaging.

Quantitative Detection of Biological Nanoparticles in Solution via Their Mediation of Colocalization of Fluorescent Liposomes

Detection of biomolecules and biological nanoparticles by means of induced aggregation of larger nanoparticles using light scattering as readout was first accomplished in the middle of the last century. Since then, technical advances together with novel nanomaterials have enabled more sophisticated readout schemes, paving the way for methods exploiting dual-probe hybridization for biomolecular or nonoparticle recognition that today can compete with established bioanalytical methods. Herein, we present a quantitative assay, with single-nanoparticle readout, utilizing receptor-containing cell-membrane mimics in the shape of approximately 100-nm lipid liposomes rather than conventional antibody-modified nanoparticles to enable detection of virus particles in solution. Specifically, the method is based on virus-mediated aggregation of differently fluorescent-labeled liposomes that contain the ganglioside GM1 receptor for the Simian Virus 40 (SV40). The aggregation kinetics of the differently colored liposomes is studied by monitoring the spatial colocalization level of the liposomes and a theoretical model that successfully represents aggregation kinetics versus virus concentration is proposed. The limits of detection are identified experimentally for our current setup and theoretically in a more general context. The consistency between theory and experiments suggests that the approach will be generically applicable for similar biosensing applications or for the study of related systems where natural interactions with cell-membrane components can be used to induce liposome aggregation.

Efficient targeted tumor imaging and secreted endostatin gene delivery by anti-CD105 immunoliposomes

BackgroundAnti-CD105 mAb-conjugated immunoliposomes, loaded with secreted mouse endostatin gene, were developed for targeted tumor imaging and antiangiogenic gene therapy.MethodsThe liposomes were investigated for size, zeta-potential, lipid content, antibody binding ability, and pcDNA loading capacity. The ability of immunoliposomes to target tumor-derived endothelial cells and perform gene transfer in vitro was measured and their basic biocompatibility was evaluated. A nude mouse/breast cancer xenograft model was used to examine the tumor internalization of fluorescent-labeled liposomes and the clinical potential of immnuoliposomes loaded with pcDNA3.1-CSF1-endostatin. ResultsLoaded immunoliposomes were homogenously distributed with a well-defined spherical shape and bilayer, diameter of 122 ± 11 nm, and zeta potential + 1.40 mV. No significant differences were observed in body weight, liver index, oxidative stress, or liver and kidney function in mice after liposomes exposure. The addition of CD105 mAb to liposomes conferred the ability to target tumor-derived endothelial cells in vitro and in vivo. Systemic intravenous administration of fluorescent immunoliposomes in the xenograft model resulted in selective and efficient internalization in tumor vasculature. Treatment of mice with pcDNA3.1-CSF1-endostatin-loaded immunoliposomes suppressed tumor growth by 71%.ConclusionsThese data demonstrate the advantages of using anti-CD105 mAb-conjugated immunoliposomes to enhance tumor targeting, imaging, and gene transfer applications.

Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes.

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats

Background and aimsAlthough the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain.Materials and methodsIn order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS® Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography.ResultsWhereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood–brain barrier, when administered either as a free drug or encapsulated into liposomes.ConclusionThis study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects.

Ultrasound-mediated delivery of gadolinium and fluorescent-labeled liposomes through the blood-brain barrier

The main objective of this study was to examine liposome extravasation across the BBB as a function of size after disruption via ultrasound and microbubbles. The liposomes were labeled with gadolinium (Gd) and fluorophore, thus enabling detection of extravasated liposomes via MRI in vivo and fluorescence methods in tissue, respectively. Liposomes labeled with gadolinium and fluorophore were prepared using lipid film hydration and extrusion to two different sizes (~80 nm and ~140 nm). Animals were divided into two groups based on the use of particle sizes. FUS–BBB disruption was produced in one hemisphere in 10 mice. Particles were injected before sonication. Sonications (0.69 MHz at 0.68 MPa) were performed in two locations combine with Definity (10 μl/kg). Acoustic emissions were recorded during FUS. T1 & T2*-weighted MRI were used to confirm Gd leakage and damage detection respectively. Mice were euthanized 5–24 h after FUS and post-process for fluorescence measurement. In sonicated area, Gd-leakage was detected in both groups at 5–24 after FUS but not on non-sonicated area. Fluorescence measurements from brain tissue homogenates suggest enhanced accumulation of liposomes in FUS versus non-FUS brain regions. These findings reveal that liposomes were able to extravasate via FUS-BBB disruption.

Multimodality imaging demonstrates trafficking of liposomes preferentially to ischemic myocardium.

Nanoparticles may serve as a promising means to deliver novel therapeutics to the myocardium following myocardial infarction. We sought to determine whether lipid-based liposomal nanoparticles can be shown through different imaging modalities to specifically target injured myocardium following intravenous injection in an ischemia-reperfusion murine myocardial infarction model. Mice underwent ischemia-reperfusion surgery and then either received tail-vein injection with gadolinium- and fluorescent-labeled liposomes or no injection (control). The hearts were harvested 24h later and underwent T1 and T2-weighted ex vivo imaging using a 7 Tesla Bruker magnet. The hearts were then sectioned for immunohistochemistry and optical fluorescent imaging. The mean size of the liposomes was 100nm. T1-weighted signal intensity was significantly increased in the ischemic vs. the non-ischemic myocardium for mice that received liposomes compared with control. Optical imaging demonstrated significant fluorescence within the infarct area for the liposome group compared with control (163±31% vs. 13±14%, p=0.001) and fluorescent microscopy confirmed the presence of liposomes within the ischemic myocardium. Liposomes traffic to the heart and preferentially home to regions of myocardial injury, enabling improved diagnosis of myocardial injury and could serve as a vehicle for drug delivery.

Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonumaviculare L. extract

In this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150nm. Whereas the zeta potential of typical liposomes was −45mV, that of CPP-conjugated liposomes was +42mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics.

Adjuvant Liposomal Doxorubicin Markedly Affects Radiofrequency Ablation–induced Effects on Periablational Microvasculature

PurposeTo evaluate the effects of radiofrequency (RF) ablation without and with adjuvant intravenous (IV) liposomal doxorubicin (Doxil) on microvessel morphology and patency and intratumoral drug delivery and retention. Materials and MethodsThere were 133 tumors/animals used in this experiment. First, single subcutaneous tumors (R3230 in Fischer rats and 786-0 in nude mice) were randomly assigned to receive RF ablation alone or no treatment and sacrificed 0–72hours after treatment. Next, combined RF ablation and liposomal doxorubicin (1mg given 15min after RF ablation) was studied in R3230 tumors at 0–72hours after treatment. Histopathologic assessment, including immunohistochemical staining for cleaved caspase-3, heat-shock protein 70, and CD34, was performed to assess morphologic vessel appearance, vessel diameter, and microvascular density. Subsequently, tumors were randomly assigned to receive RF ablation alone, RF ablation and liposomal doxorubicin, or no treatment (control tumors), followed by IV fluorescent-labeled liposomes (a surrogate marker) given 0–24hours after RF ablation to permit qualitative assessment. ResultsRF ablation alone resulted in enlarged and dysmorphic vessels from 0–4hours, peaking at 12–24hours after RF ablation, occurring preferentially closer to the electrode. The addition of doxorubicin resulted in earlier vessel contraction (mean vessel area, 47,539μm2±9,544 vs 1,854μm2±458 for RF ablation alone at 15min; P<.05). Combined RF ablation and liposomal doxorubicin produced similar fluorescence 1hour after treatment (40.88AU/μm2±33.53 vs 22.1AU/μm2±13.19; P = .14) but significantly less fluorescence at 4 hours (24.3AU/μm2±3.65 vs 2.8AU/μm2±3.14; P<.002) compared with RF ablation alone denoting earlier reduction in microvascular patency. ConclusionsRF ablation induces morphologic changes to vessels within the ablation zone lasting 12–24hours after treatment. The addition of liposomal doxorubicin causes early vessel contraction and a reduction in periablational microvascular patency. Such changes would likely need to be considered when determining optimal drug administration and imaging paradigms.

280 LIPOSOMAL TREATMENT OFFERS PROTECTION AGAINST ACROLEIN-INDUCED UROTHELIAL CELL DAMAGE

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology (I)1 Apr 2013280 LIPOSOMAL TREATMENT OFFERS PROTECTION AGAINST ACROLEIN-INDUCED UROTHELIAL CELL DAMAGE Jayabalan Nirmal, Amanda Wolf-Johnston, Michael Chancellor, Pradeep Tyagi, Jonathan Kaufman, Michele Anthony, and Lori Birder Jayabalan NirmalJayabalan Nirmal Royal Oak, MI More articles by this author , Amanda Wolf-JohnstonAmanda Wolf-Johnston Pittsburgh, PA More articles by this author , Michael ChancellorMichael Chancellor Royal Oak, MI More articles by this author , Pradeep TyagiPradeep Tyagi Pittsburgh, PA More articles by this author , Jonathan KaufmanJonathan Kaufman Pittsburgh, PA More articles by this author , Michele AnthonyMichele Anthony Pittsburgh, PA More articles by this author , and Lori BirderLori Birder Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1664AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES A growing body of evidence supports the use of intravesical therapies for treatment of urinary bladder dysfunctions. Recent data has revealed that topical nanoparticle liposome treatment has a beneficial effect on reducing inflammation and irritation-induced bladder hyperactivity by forming a protective, urothelial coating. This study examined the mechanism underlying liposome uptake in urothelial cells and whether liposome treatment can reduce acrolein induced urothelial cell damage. METHODS Primary urothelial cells were cultured as previously described and protective effect of liposomes against acrolein (2 μM) exposure was assessed by: a) BrdU cell proliferation, b) cytokine production and c) trypan blue staining. This was determined in control cells (unexposed to acrolein), control cells pretreated with liposomes (2 mg/ml), cells exposed to acrolein (2 μM) and in urothelial cells treated with liposomes prior to acrolein exposure. The mechanisms underlying this cytoprotection were assessed by confocal microscopy of fixed urothelial cells treated with fluorescent-labeled liposomes. RESULTS Acrolein induced epithelial toxicity caused a reduction in cellular integrity (increased trypan blue staining), proliferation of urothelial cells (mean 43% decrease as compared with untreated control cells) and increased cytokine (IFN-γ) production, that was blocked or prevented by liposome pretreatment. Confocal microscopy was able to localize fluorescent-labeled liposomes outside the urothelial cell membrane as well as within the cytoplasm. CONCLUSIONS We demonstrated that liposomes increased the survival of urothelial cells exposed to acrolein and cell surface bound liposomes are internalized via endocytosis. Taken together, our findings suggest that liposomes may act to repair the urothelial barrier function and reduce inflammation. This could be important in pathologies such as ulcerative cystitis, a severe form of painful bladder syndrome that causes severe, chronic pelvic pain. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e114 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jayabalan Nirmal Royal Oak, MI More articles by this author Amanda Wolf-Johnston Pittsburgh, PA More articles by this author Michael Chancellor Royal Oak, MI More articles by this author Pradeep Tyagi Pittsburgh, PA More articles by this author Jonathan Kaufman Pittsburgh, PA More articles by this author Michele Anthony Pittsburgh, PA More articles by this author Lori Birder Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Composition and Properties of Complexes between Spherical Polycationic Brushes and Anionic Liposomes

A spherical polycationic brush (SPB) is made by graft-polymerizing a cationic monomer onto the surface of a 100 nm polystyrene bead. It is possible to adsorb anionic liposomes (40-60 nm diameter) onto the SPBs while maintaining the liposome integrity. The liposomes were constructed with phosphatidyl choline (PC) admixed with 0.05-0.4 mol fraction of an dianionic lipid, cardiolipin (CL(2-)). As shown by electrophoretic mobility measurements, SPB-to-liposome complexation leads to a conversion from the initial positive charge of the copolymer to a negative charge. The higher the CL(2-) content of the liposomes, the lower the concentration needed for charge neutralization. Dynamic light scattering (DLS) revealed that multicomplex aggregates are formed with a maximum size at the SPB/liposome charge-equivalence point. Experiments with fluorescent-labeled liposomes show that at low CL(2-) content about 80 liposomes are adsorbed per SPB. As the mole fraction of CL(2-) increases from 0.05 to 0.4, fewer liposomes adsorb owing to electrostatic repulsion among neighboring liposomes. The effect of added NaCl also depends upon the CL(2-) content. With 0.05 mol fraction CL(2-), the SPB/liposome complex dissociates into its components at 0.15 M NaCl. With a mole fraction of >0.1, complexes fail to dissociate even at 1.2 M NaCl. Additional information about the SPB/liposome morphology was obtained from cryo-TEM. For example, cryo-TEM data confirm liposome integrity upon complexation, a behavior that contrasts with the liposome destruction as found with adsorption to many other types of surfaces.

Fluorescent liposomes for intravital staining of Kupffer cells to aid in vivo microscopy in rats

The potential of newly formulated fluorescent-labeled liposomes for the intravital staining of Kupffer cells was evaluated in rats. Fluorescently labeled phosphatidylcholine (PC) was incorporated into liposomes consisting of PC and phosphatidylserine. After intravenous injection, Kupffer cells in the rat liver were intravitally stained and were clearly delineated under the fluorescence image of both confocal laser scanning microscopy and in vivo microscopy. Specificity of the staining was confirmed by immunohistochemistry using the anti-rat macrophage antibody Ki-M2R, which suggested that the liposomes were selectively entrapped by the hepatic reticuloendothelial system. A time-course study revealed that the suitable observation window was between 16 and 24 h after the injection. Phagocytic activity of Kupffer cells after the administration of liposomes was examined by measuring the amount of hepatic uptake of intravenously administered fluorescent microspheres; no detrimental influence of the liposomes on the phagocytic activity was observed. Additionally, no histopathologic changes were found in the livers from liposome-treated rats. Therefore, the fluorescent-labeled liposomes appear to be a useful research tool for labeling Kupffer cells for in vivo microscopic observation of the liver.

Transmembrane Domains of Hepatitis C Virus Envelope Glycoproteins: Residues Involved in E1E2 Heterodimerization and Involvement of These Domains in Virus Entry

The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins.

A common pathway for the uptake of surfactant lipids by alveolar cells.

The uptake of different surfactant lipids-dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)-and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red-labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages.

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.

Design of a short membrane-destabilizing peptide covalently bound to liposomes

We characterized the physical and biological properties of a 14-residue amphipathic sequence called SFP (for short fusogenic peptide). At acidic pH, this short synthetic peptide interacts with various phospholipidic monolayers. These interactions were correlated with a pH-dependent conformational transition of SFP resulting in a hydrophobic α-helical structure. The hemolysis assay showed a pH-dependent weak membrane destabilizing activity of SFP. However, membrane anchoring of SFP through a covalently bound myristic acid enhanced by 1000-fold its membrane-destabilizing power. Moreover, SFP covalently bound to fluorescent-labeled liposomes induced a pH-dependent mixing of both membranes. SFP, a small synthetic peptide, is thus able to mimick many aspects of viral protein-induced membrane fusion: conformational change, membrane destabilization, membrane anchoring and finally pH-dependent lipid mixing.

Characterization of the pH-induced fusion of liposomes with the plasma membrane of rye protoplasts

The authors present evidence that at acidic pH, liposomes composed of soybean lipids fuse with the plasma membrane of protoplasts isolated from rye leaves. Using the resonance energy transfer assay (RET), they determined the rate and extent of liposome and protoplast plasma membrane lipid mixing. The fluorescent donor-acceptor pair was N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidyl-ethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE). Fusion was substantial below pH 5, and the half-time of lipid mixing was fast (t/sub 1/2/ on the order of minutes) and pH, concentration, and temperature dependent. The extent of liposome and protoplast fusion from the total amount of liposomes associated with the protoplasts was also determined by the RET assay. Protoplasts were incubated with fluorescent-labeled liposomes (5 min at 30/sup 0/C) at different pH values and then washed twice by centrifugation. The fluorescence spectra of the protoplast suspension permitted determination of the ratio of N-NBD-PE emission at 530 nm to the N-Rh-PE emission at 590 nm, which is a measure of the degree of lipid mixing. The amount of liposomes associated (fused and unfused) with protoplasts at pH 3.9 was approximately 9 times greater than that at pH 5.6. The transfer of liposome contents to the protoplast interior was studied withmore » a method based on the fluorescence enhancement of a solution of calcein, initially confined in the liposomes at self-quenching concentrations. The kinetics of calcein release were very similar to those of lipid mixing. Fluorescence microscopy showed that after fusion with liposomes containing calcein, the protoplasts exhibited a strong diffuse fluorescence in the interior.« less