A General Approach to Enzyme-Responsive Liposomes

Liposomes are effective nanocarriers due to their ability to deliver encapsulated drugs to diseased cells. Nevertheless, liposome delivery would be improved by enhancing the ability to control the release of contents at the target site. While various stimuli have been explored for triggering liposome release, enzymes provide excellent targets due to their common overexpression in diseased cells. We present a general approach to enzyme-responsive liposomes exploiting targets that are commonly aberrant in disease, including esterases, phosphatases, and β-galactosidases. Responsive lipids correlating with each enzyme family were designed and synthesized bearing an enzyme substrate moiety attached via a self-immolating linker to a non-bilayer lipid scaffold, such that enzymatic hydrolysis triggers lipid decomposition to disrupt membrane integrity and release contents. Liposome dye leakage assays demonstrated that each enzyme-responsive liposome yielded significant content release upon enzymatic treatment compared to minimal release in controls. Results also showed that fine-tuning liposome composition was critical for controlling release. DLS analysis showed particle size increases in the cases of esterase- and β-galactosidase-responsive lipids, supporting alterations to membrane properties. These results showcase an effective modular strategy that can be tailored to target different enzymes, providing a promising new avenue for advancing liposomal drug delivery.     

Di‐ and tri‐phenyltin chlorides transfer across a model lipid bilayer

A compound's ability to penetrate the plasma membrane of a cell is the critical parameter that determines its potential to become a biologically potent factor. A well‐known group of organotin compounds that exhibit toxic properties in relation to biological systems are phenyltins. There are as yet no studies that in a direct manner have established whether organotin compounds such as diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT) diffuse, or not, through the lipid bilayer, although we know that at least some organotins absorb in both liposome and biological membranes. In this paper we present a series of experiments that show transfer of these compounds across the lipid membrane using the stopped‐flow technique. The results obtained demonstrate that DPhT and TPhT first adsorb onto the lipid bilayer surface, in a diffusion‐controlled manner and within a very short time (0.05 s), whereas the membrane crossing was observed to be on the order of a minute. The adsorption process was easily fitted with a single exponential for both the compounds studied, indicating a single process phenomenon. The longer time kinetics (characteristic of membrane crossing) showed a complex dependence on compound concentration and the presence of cholesterol in the membrane. On passing from the outer to the inner surface of the bilayer, organotins undergo desorption and enter the liposome interior, which has been shown in lipid monolayer desorption studies. In conclusion, it can be stated that amphiphilic DPhT and TPhT permeate the liposome membrane. Copyright © 2005 John Wiley & Sons, Ltd.     

Differential Effects of Polymer‐Surface Decoration on Drug Delivery,Cellular Retention,and Action Mechanisms of Functionalized Mesoporous Silica Nanoparticles

Polymer‐surface decoration has been found to be an effective strategy to enhance the biological activities of nanomedicine. Herein, three different types of polymers with a cancer‐targeting ligand Arg‐Gly‐Asp peptide (RGD) have been used to decorate mesoporous silica nanoparticles (MSNs) and the functionalized nanosystems were used as drug carriers of oxaliplatin (OXA). The results showed that polymer‐surface decoration of the MSNs nanosystem by poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) significantly enhanced the anticancer efficacy of OXA, which was much higher than that of chitosan (CTS). This effect was closely related to the enhancement of the cellular uptake and cellular drug retention. Moreover, PEI@MSNs‐OXA possessed excellent advantages in penetrating ability and inhibitory effects on SW480 spheroids that were used to simulate the in vivo tumor environments. Therefore, this study provides useful information for the rational design of a cancer‐targeted MSNs nanosystem with polymer‐surface decoration.     

Study of PEGylated lipid layers as a model for PEGylated liposome surfaces: molecular dynamics simulation and Langmuir monolayer studies

We have combined Langmuir monolayer film experiments and all-atom molecular dynamics (MD) simulation of a bilayer to study the surface structure of a PEGylated liposome and its interaction with the ionic environment present under physiological conditions. Lipids that form both gel and liquid-crystalline membranes have been used in our study. By varying the salt concentration in the Langmuir film experiment and including salt at the physiological level in the simulation, we have studied the effect of salt ions present in the blood plasma on the structure of the poly(ethylene glycol) (PEG) layer. We have also studied the interaction between the PEG layer and the lipid bilayer in both the liquid-crystalline and gel states. The MD simulation shows two clear results: (a) The Na(+) ions form close interactions with the PEG oxygens, with the PEG chains forming loops around them and (b) PEG penetrates the lipid core of the membrane for the case of a liquid-crystalline membrane but is excluded from the tighter structure of the gel membrane. The Langmuir monolayer results indicate that the salt concentration affects the PEGylated lipid system, and these results can be interpreted in a fashion that is in agreement with the results of our MD simulation. We conclude that the currently accepted picture of the PEG surface layer acting as a generic neutral hydrophilic polymer entirely outside the membrane, with its effect explained through steric interactions, is not sufficient. The phenomena we have observed may affect both the interaction between the liposome and bloodstream proteins and the liquid-crystalline-gel transition and is thus relevant to nanotechnological drug delivery device design.     

Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

ζ-potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0). Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan-decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan-hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.     

Enzymatic Cell-Surface Decoration with Proteins using Amphiphilic Lipid-Fused Peptide Substrates

Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G₃S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

Enhanced Survivin siRNA Delivery Using Cationic Liposome Incorporating Fatty Acid-modified Polyethylenimine

We described a novel approach for survivin siRNA cellular delivery via a cationic liposome incorporating fatty acid-modified polyethylenimine. A linoleic acid derivative of branched polyethylenimine(PEI, M_w=25 kDa), PEI-LA, was synthesized and incorporated into the liposome. The properties of the liposome, cytotoxicity, cellular uptake of cancer cells for survivin siRNA, survivin protein downregulation levels were investigated. PEI-modified liposome showed a lower cytotoxicity and delivered survivin siRNA into HeLa cells and A549 cells efficiently compared with PEI-25kDa.     

Factors affecting responsivity of unilamellar liposomes to 20 kHz ultrasound

Ultrasound is commonly used in the preparation of unilamellar liposome dispersions and is often considered for cell membrane disruption for drug delivery or DNA transfection applications. To better understand the physical and chemical properties of lipid membranes that render them susceptible to ultrasonic permeabilization, the roles of temperature, lipid composition (cholesterol and PEG-lipid content), and liposome size have been studied. The results of these studies suggest that lipid packing is very important to ultrasound responsiveness; surprisingly, cohesive energy and tensile strength are not. Taken together, the experimental results implicate a defect-mediated permeabilization mechanism, rather than pore formation or membrane tearing. The implications of this work for drug release from liposomes and ultrasound-mediated DNA transfection are discussed.     

A study on the characterization and stability of rhenium(III) chloride-incorporated liposomes

Nanoliposomes are important carriers capable of packaging drugs for various delivery applications through passive targeting tumor sites by enhancing permeability and retention effect. Radiolabeled liposomes have potential applications in radiotherapy and diagnostic imaging. However, the physico-chemical instability of liposomes during manufacturing and storage limits their extensive application. Therefore, considerable numbers of studies have been made on the stability of liposomes over the last few years in order to overcome this problem. In this study, we attempted to prepare polymer-coated liposomes using water-soluble chitosan in order to enhance the stability of rhenium(III) chloride-incorporated liposomes. They were characterized by an electrophoretic light-scattering spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), UV–Vis spectrometer, and phase-contrast microscopy. The chitosan-coated liposomes are spherical and the particle size is about 800–850 nm. Incorporation of chitosan into the liposome bilayer decreased rhenium(III) chloride release from the liposome due to an increased rigidity of the liposome membrane structure. Chitosan-coated liposomes showed a higher stability compared with the stability of non-coated liposomes. The release characteristics of rhenium(III) chloride encapsulated in the liposome were taken as a measure of stability of the liposome membrane.     

Enhanced Anticancer Efficacy by ATP‐Mediated Liposomal Drug Delivery

A liposome‐based co‐delivery system composed of a fusogenic liposome encapsulating ATP‐responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP‐mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein–DNA complex core containing an ATP‐responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell‐penetrating peptide‐modified fusogenic liposomal membrane was coated on the core, which had an acid‐triggered fusogenic potential with the ATP‐loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH‐sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo.     

Immunoliposomes assemblied layer by layer from reverse micelles

A method is described for the coupling of the HBsAb antibody on to a liposome by reverse micelles passing through the modified organic/water interface. Incorporation of the fatty acid modified HBsAb into liposomal membranes was studied as a function of the extent of fatty acid coupling. Results show that the incorporation of HBsAb is proportional to the lipid coupling degree. The HBsAb incorporated into the liposomal membrane by this method remains an antigen binding activity shown by the liposome dialysis assay.     

基因载体材料聚乙烯亚胺与磷脂酰胆碱脂质体的相互作用对膜结构的影响

采用动态光散射、荧光光谱、zeta电位测定和等温滴定量热技术分析了分子量分别为25000,10000和1800的聚乙烯亚胺(PEI)与二油酰磷脂酰胆碱(DOPC)脂质体的相互作用及其对脂质体膜内环境极性和膜通透性的影响.结果表明,PEI通过氨基与DOPC的磷脂基团和胆碱基团产生氢键或范德华作用,从而与脂质体结合形成复合物;低浓度PEI(0.075 mg/mL)导致DOPC脂质体的聚集和表面电位的增加,但未引起脂质体膜融合和表面电位反转;进一步增加PEI的浓度对脂质体表面电位的影响很小,而结合在表面的PEI分子链之间的排斥作用阻碍了脂质体聚集.PEI分子与DOPC脂质体的结合降低了脂质分子碳氢链的堆积密度和脂质体膜内环境的疏水性,从而增强了钙黄绿素和槲皮素在脂质体膜中的通透性.PEI与DOPC脂质体的相互作用具有明显的分子尺寸效应,增大PEI的分子量可以增强与脂质体的相互作用及对脂质体膜结构的影响.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

Tumor cell surface modification with immuno-amplified nanoparticles to enhance cancer immunotherapy

The process of cancer immunogenic cell death (ICD) provides adjuvanticity and antigenicity from dying tumor cells, thereby stimulating host immune system and promoting antitumor immunity. However, due to the immune evasion of tumor cells and the immunosuppressive tumor microenvironment formed in the process of cancer progression, it is far from satisfactory in the efficacy of the cancer treatments based on ICD. Herein, we report an immuno-amplified nanoparticle (IANP) that can modify mannose onto the tumor cell surface while delivering ICD-inducing drug doxorubicin (DOX) into the tumor cytoplasm. IANP consists of a DOX-loaded polymer core encapsulated within a mannose modified, fusogenic liposome. After reaching tumor cells, IANP achieved to transfer the mannose groups onto the surface of tumor cells through membrane fusion, and simultaneously transport the polymer core into tumor cells for DOX delivery. With this unique ability, IANP triggered the ICD of tumor cells and facilitated the activation of dendritic cells (DCs) via the mannose-C-type lectin receptors (CLRs) interaction, leading to the enhanced immunogenic effects of chemotherapy-induced tumor cell death. As a result, intratumoral injection of IANP achieved to trigger ICD of tumor cells and enhance the anti-tumor immune responses, thereby suppressing the tumor growth effectively. This work demonstrated a potential strategy towards the development of novel ICD-based cancer immunotherapies.     

Endocytosis-like uptake of surface-modified drug nanocarriers into giant unilamellar vesicles

We had previously developed surface-modified poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for use as a cellular drug delivery system. The cellular uptake of PLGA-NPs was mediated predominantly by endocytosis, and this uptake was increased by surface modifications with polymers, such as chitosan (CS) and polysorbate 80 (P80). In the present study, we prepared a cell-sized giant unilamellar vesicle (GUV) that mimics a cell membrane to investigate the interaction between cell membranes and NPs. Endocytosis-like uptake of NPs into a GUV was observed when the NPs were modified with nonionic surfactant P80 probably due to change in viscoelasticity and enhanced fusion activity of the membrane induced by P80. In contrast, unmodified NPs and those modified with CS were not internalized into a GUV. These results suggest that surface properties of PLGA-NPs are an important formulation parameter for their interaction with lipid membranes.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

We present a critical review of recent work related to the assembly of multicompartment liposome clusters using nucleic acids as a specific recognition unit to link liposomal modules. The asymmetry in nucleic acid binding to its non-self complementary strand allows the controlled association of different compartmental modules into composite systems. These biomimetic multicompartment architectures could have future applications in chemical process control, drug delivery and synthetic biology. We assess the different methods of anchoring DNA to lipid membrane surfaces and discuss how lipid and DNA properties can be tuned to control the morphology and properties of liposome superstructures. We consider different methods for chemical communication between the contents of liposomal compartments within these clusters and assess the progress towards making this chemical mixing efficient, switchable and chemically specific. Finally, given the current state of the art, we assess the outlook for future developments towards functional modular networks of liposomes.     

A Micromechanical Cantilever‐Based Liposome Biosensor for Characterization of Protein‐Membrane Interaction

We have newly evaluated the interaction of lipid membrane with two different proteins of lysozyme and carbonic anhydrase from bovine (CAB) using a micro cantilever‐based liposome biosensor with a new droplet‐sealing structure. Herein 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) liposomes are used as model lipid membrane and are immobilized on the surface of cantilever. The interaction of DPPC liposome with the target protein causes deflection of the micro‐cantilever, which can stably be detected by measuring the resistance change of the strain gauge. The resistance change dependent on time is used to evaluate the characteristic of liposome‐protein interaction. The resistance of the cantilever‐based biosensor increases monotonously with time in both of the two protein solutions. Especially, chronological resistance change depends markedly on both the concentration and species of target proteins. Finally, these results lead us to conclude that the cantilever‐based liposome biosensor with the droplet‐sealing structure facilitates the characterization of protein‐membrane interaction. It also means that this biosensor is a promising candidate device for label‐free detection of concentration and species of different target proteins.     

Calorimetric Evaluation of Glycyrrhetic Acid (GA)- and Stearyl Glycyrrhetinate (SG)-Loaded Solid Lipid Nanoparticle Interactions with a Model Biomembrane

Glycyrrhetic acid (GA) and stearyl glycyrrhetinate (SG) are two interesting compounds from Glycyrrhiza glabra, showing numerous biological properties widely applied in the pharmaceutical and cosmetic fields. Despite these appreciable benefits, their potential therapeutic properties are strongly compromised due to unfavourable physical-chemical features. The strategy exploited in the present work was to develop solid lipid nanoparticles (SLNs) as carrier systems for GA and SG delivery. Both formulations loaded with GA and SG (GA-SLNs and SG-SLNs, respectively) were prepared by the high shear homogenization coupled to ultrasound (HSH-US) method, and we obtained good technological parameters. DSC was used to evaluate their thermotropic behaviour and ability to act as carriers for GA and SG. The study was conducted by means of a biomembrane model (multilamellar vesicles; MLVs) that simulated the interaction of the carriers with the cellular membrane. Unloaded and loaded SLNs were incubated with the biomembranes, and their interactions were evaluated over time through variations in their calorimetric curves. The results of these studies indicated that GA and SG interact differently with MLVs and SLNs; the interactions of SG-SLNs and GA-SLNs with the biomembrane model showed different variations of the MLVs calorimetric curve and suggest the potential use of SLNs as delivery systems for GA.