Cytomimetic large-scale vesicle aggregation and fusion based on host-guest interaction

Herein, we have shown a large-scale cell-mimetic (cytomimetic) aggregation process by using cell-sized polymer vesicles as the building blocks and intervesicular host-guest molecular recognition interactions as the driving force. We first prepared the hyperbranched polymer vesicles named branched polymersomes (BPs) around 5-10 μm through the aqueous self-assembly of a hyperbranched multiarm copolymer of HBPO-star-PEO [HBPO = hyperbranched poly(3-ethyl-3-oxetanemethanol); PEO = poly(ethylene oxide)]. Subsequently, adamantane-functionalized BPs (Ada-BPs) or β-cyclodextrin-functionalized BPs (CD-BPs) were prepared through the coassembly of HBPO-star-PEO and Ada-modified HBPO-star-PEO (HBPO-star-PEO-Ada), or of HBPO-star-PEO and CD-modified HBPO-star-PEO (HBPO-star-PEO-CD), respectively. Macroscopic vesicle aggregates were obtained by mixing CD-BPs and Ada-BPs. The intervesicular host-guest recognition interactions between β-CD units in CD-BPs and Ada units in Ada-BPs, which were proved by (1)H nuclear Overhauser effect spectroscopy (NOESY) spectrum and the fluorescence probe method, are responsible for the vesicle aggregation. Additionally, the vesicle fusion events happened frequently in the process of vesicle aggregation, which were certified by double-labeling fluorescent assay, real-time observation, content mixing assay, and component mixing assay.     

Directed aggregation and fusion of lipid vesicles induced by DNA-surfactants

We investigated DNA-directed aggregation of vesicles using DNA-surfactants. Following tethering of single-stranded DNA oligonucleotides to vesicles using DNA-surfactant, the tethered vesicles were assembled with other vesicles bearing complementary strands. The vesicle aggregation was strongly affected by the salt concentration and by temperature according to the characteristics of DNA hybridization. Restriction enzyme, which can hydrolyze the double-stranded DNA used in the present study, dissociated the vesicle aggregates. Exploration using fluorescently labeled vesicles suggested that the DNA-directed vesicle aggregation took place in a sequence-specific manner through DNA-duplex formation. Interestingly, the DNA-directed aggregation using short DNA-surfactant induced the fusion of vesicles to produce giant vesicles, resulting in an enzymatic reaction in the giant vesicle.     

Dynamics of vesicle self-assembly and dissolution

The dynamics of membranes is studied on the basis of a particle-based meshless surface model, which was introduced earlier [Phys. Rev. E 73, 021903 (2006)]. The model describes fluid membranes with bending energy and-in the case of membranes with boundaries-line tension. The effects of hydrodynamic interactions are investigated by comparing Brownian dynamics with a particle-based mesoscale solvent simulation (multiparticle collision dynamics). Particles self-assemble into vesicles via disk-shaped membrane patches. The time evolution of assembly is found to consist of three steps: particle assembly into discoidal clusters, aggregation of clusters into larger membrane patches, and finally vesicle formation. The time dependence of the cluster distribution and the mean cluster size is evaluated and compared with the predictions of Smoluchowski rate equations. On the other hand, when the line tension is suddenly decreased (or the temperature is increased), vesicles dissolve via pore formation in the membrane. Hydrodynamic interactions are found to speed up the dynamics in both cases. Furthermore, hydrodynamics makes vesicle more spherical in the membrane-closure process.     

Synthesis of amphiphilic biodegradable glycocopolymers based on poly(ε‐caprolactone) by ring‐opening polymerization and click chemistry

Amphiphilic, biodegradable block glycopolymers based on poly(ε‐caprolactone) (PCL) with various pendent saccharides were synthesized by combination of ring‐opening polymerization (ROP) and “click” chemistry. PCL macroinitiators obtained by ROP of ε‐caprolactone were used to initiate the ROP of 2‐bromo‐ε‐caprolactone (BrCL) to get diblock copolymers, PCL‐b‐PBrCL. Reaction of the block copolymers with sodium azide converted the bromine groups in the PBrCL block to azide groups. In the final step, click chemistry of alkynyl saccharides with the pendent azide groups of PCL‐b‐PBrCL led to the formation of the amphiphilic block glycopolymers. These copolymers were characterized by ¹H NMR spectroscopy and gel permeation chromatography. The self‐assembly behavior of the amphiphilic block copolymers was investigated using transmission electron microscopy and atomic force microscope, spherical aggregates with saccharide groups on the surface were observed, and the aggregates could bind reversibly with Concanavalin A. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3583–3594, 2009     

Photoinduced increase in vesicle size and role of photoresponsive malachite green leuconitrile derivative in vesicle fusion

The influence of photoirradiation on vesicles containing a Malachite Green leuconitrile derivative carrying a long alkyl chain, affording photogenerated amphiphilicity, was investigated. The photoresponsive Malachite Green leuconitrile derivative was embedded in the vesicle bilayer of two single-tailed amphiphiles with oppositely charged head groups consisting of cetyltrimethylammonium chloride (CTAC) and sodium octyl sulfate (SOS). Transmission electron microscopy, which was used for observing photoinduced structural change in the vesicles, demonstrated that photoirradiation of the vesicles containing the Malachite Green leuconitrile derivative increased the average size of the vesicle diameter from 116 to 243 nm in the [CTAC]/[SOS] = 0.48 system. The mechanism for vesicle enlargement was studied with fluorescent probe molecules. The photoinduced change in the vesicle size can be explained by the destabilization of the vesicle bilayer, which is perturbed by photogenerated amphiphilicity. In addition, it was shown that the fusion process arising from the destabilized bilayer contributed to the increase in vesicle size.     

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.     

Temperature-controlled vesicle aggregation in the mixed system of sodium n-dodecyl sulfate/n-dodecyltributylammonium bromide

Temperature-controlled vesicle aggregation was investigated in a catanionic surfactant system of sodium n-dodecyl sulfate/n-dodecyltributylammonium bromide. Vesicle aggregation took place as the temperature reached the critical value (Tc). Tc can be adjusted by the variations of the total surfactant concentration and the mixed molar ratio. It was also found that the temperature variation above Tc can greatly influence the vesicle aggregation rate. The vesicle aggregation process was irreversible as long as T >/= Tc, whereas the vesicle disaggregation process occurred only below Tc.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

Polyion-induced liposomal vesicle aggregation: a radiowave dielectric relaxation study

The radiowave dielectric properties of aqueous heterogeneous systems during the complexation of charged polyions and oppositely charged liposomal particles have been measured in a wide frequency range, between 100 Hz and 2 GHz. The formation of a polyion-liposome complex driven by the correlated polyion adsorption at the particle surface implies two concomitant effects referred to as reentrant condensation and charge inversion. Both of them are governed by electrostatic interactions and there is now strong evidence, based on experiments and simulations, that counterion release is the driving force of the aggregation process. From this point of view, dielectric technique may offer a suitable tool in the investigation of the structural properties of these aggregates. In spite of the fact that interaction of polyions with oppositely charged surfaces was extensively experimentally investigated, there are no papers concerning the dielectric properties during the polyion-induced aggregation. To get an insight into this important topic, the authors present here an extensive set of radiowave dielectric measurements of liposomal vesicle aqueous suspensions where the liposome aggregation was induced by an oppositely charged polyion. The aggregation was followed from the beginning, when most of the isolated liposomes predominate, up to the formation of polyion-coated liposomes of inverted charge, crossing the isoelectric condition, where large, almost neutral, aggregates appear. The authors describe the observed dielectric dispersions as due to counterion polarization in the adjacency of the liposome and liposome aggregate surface, primarily governed by the zeta potential, according to the standard electrokinetic model.     

Mn(Ⅲ)-mediated radical reaction of 2-isocyano-6-alkenyl(alkynyl)benzonitriles with arylboronic acids:Synthesis of pyrroloisoquinoline derivatives

A Mn(Ⅲ) mediated radical reaction of new designed multi-functionalized 2-isocyano-6-alkenyl(alkynyl)benzonitriles with arylboronic acids has been developed.This reaction provides a method for the synthesis of pyrroloisoquinoline derivatives through the formation of two C-C bonds and one C-N bond via radical cascade cyclization in one step.     

双分子膜上染料的吸附性质及对膜结构的影响

系统地研究了染料甲基橙和达旦黄在含席夫碱基囊泡双分子膜阳离子表面上的吸附和聚集，以及由此产生的对膜聚集结构的影响，染料的吸附和聚集可经电子吸收光谱的因吸附产生的沉淀证实，并且ＭＯ的聚集结构的Ｈ－聚集，ＴＹ的聚集结构为Ｊ－聚集。改变ｐＨ的研究表明ＭＯ在变色ｐＨ值以及因形成阳离子而解聚，ＴＹ在酸性条件下亦因离子化程度减弱和部分质子化而使聚集被削弱，但在碱性条件下其聚集产生更大的红移。温度变化的结果显示     

Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization

A new methodology for the preparation of well-defined core-shell nanoparticles was developed, based upon the employment of a multifunctional crosslinker to coincidently stabilize supramolecular polymer assemblies and imbed into the shell unique chemical functionalities. Amphiphilic diblock copolymers of poly(acrylic acid)(80)-b-poly(styrene)(90) that had been assembled into micelles and partially functionalized throughout the corona with alkynyl groups were utilized as Click-readied nanoscaffolds for the formation of shell Click-crosslinked nanoparticles (SCCs). Divergently grown dendrimers of the zero, first, second, and third generations having increasing numbers of azide terminating groups ((N(3))(2)-[G-0], (N(3))(4)-[G-1], (N(3))(8)-[G-2], and (N(3))(16)-[G-3], respectively) were investigated as crosslinkers via Click reactions with the alkynyl groups to form covalent linkages throughout the block copolymer micelle corona, thus forming a crosslinked shell. The crosslinking reactions were characterized by (1)H NMR and IR spectroscopies, differential scanning calorimetry (DSC), and dynamic light scattering (DLS) measurements. Only the first generation dendrimer ((N(3))(4)-[G-1]) possessed a sufficient balance of polyvalency and water solubility to achieve crosslinking and establish a robust nanostructure. The resulting SCC was further characterized with atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). The dendritic crosslinker is important as it also allows for the incorporation of excess functionality that can undergo complementary reactions. Within the shell of this SCC the remaining azide termini of the dendrimer crosslinker were then consumed in a secondary Click reaction with an alkynyl-functionalized fluorescein to yield a fluorescently labeled SCC that was characterized with DLS, AFM, TEM, AU, UV-vis, and fluorescent measurements as a function of pH.     

A Versatile and Robust Vesicle Based on a Photocleavable Surfactant for Two‐Photon‐Tuned Release

A small amphiphile that contains a coumarin unit and alkynyl groups, as a two‐photon‐cleavable segment and polymerizable groups, respectively, was designed and synthesized. The amphiphile showed a critical aggregation concentration of about 4.6×10^?5 M and formed a vesicle‐type assembly. The formed vesicles were stabilized by in situ “click” polymerization without altering their morphology. Hydrophobic and hydrophilic guests can be encapsulated within the vesicle membrane and inside the aqueous core of the vesicle, respectively. The loaded guests can be released from the vesicle by using UV or near‐IR stimuli, through splitting up the amphiphilic structure of the amphiphile. Distinguished dose‐controlled photorelease of the polymeric vesicle is achieved with the maintenance of vesicular integrity, which makes the guest release dependent on the amount of cleavage of the amphiphilic structure during irradiation. This study provides a potential strategy for the development of versatile and stable drug‐delivery systems that offer sustained and photo‐triggered release.     

Tethering hydrophobic peptides to functionalized self-assembled monolayers on gold through two chemical linkers using the Huisgen cycloaddition

Gold surfaces functionalized with an α-helical peptide have been generated by reacting an azide-terminated self-assembled monolayer with structured peptides containing two cyanophenylalanines through a Huisgen cycloaddition. Mixed monolayers of a reactive bromine-terminated thiol and inert alkane thiol were prepared at various concentrations of the Br-terminated moiety. These were reacted with sodium azide to form azide-terminated monolayers with controlled concentration of the reactive azide. These surfaces were studied through ellipsometry and X-ray photoelectron spectroscopy, which demonstrated that the concentration of the reactive azide group on the surface is controlled by the chemical conditions under which the monolayer is prepared. Grazing incident angle surface infrared spectroscopy (GRAS-IR) of the azide-terminated surface demonstrated that the azide is approximately perpendicular to the plane of the surface, as expected. These surfaces were then exposed to an α-helical peptide composed of alternating leucine and lysine residues, with two residues replaced with cyanophenylalanine to react with two neighboring surface-bound azide groups to bind the peptide to the surface through two covalent bonds. The yield of this reaction was quantified through monitoring the absorption of the azide group by GRAS-IR. Despite damage to the monolayer during the reaction, reaction yields of 80-98% were determined for optimized reaction conditions. Although the peptide retains its α-helical configuration under the reaction conditions, GRAS-IR analysis of the amide I and II modes of the surface-bound peptide showed that it is probably randomly oriented on the surface.     

Composition and asymmetry in supported membranes formed by vesicle fusion

The structure and formation of supported membranes at silica surfaces by vesicle fusion was investigated by neutron reflectivity and quartz crystal microbalance (QCM-D) measurements. The structure of equimolar phospholipid mixtures of DLPC-DPPC, DMPC-DPPC, and DOPC-DPPC depends intricately on the vesicle deposition conditions. The supported bilayer membranes exhibit varying degrees of compositional asymmetry between the monolayer leaflets, which can be modified by the deposition temperature as well as the salt concentration of the vesicle solution. The total lipid composition of the supported bilayers differs from the composition of the vesicles in solution, and the monolayer proximal to the silica surface is always enriched in DPPC compared to the distal monolayer. The results, which show unambiguougsly that some exchange and rearrangement of lipids occur during vesicle deposition, can be rationalized by considering the effects of salt screening and temperature on the rates of lipid exchange, rearrangement, and vesicle adsorption, but there is also an intricate dependence on the lipid-lipid interactions. Thus, although both symmetric and asymmetric supported bilayers can be prepared from vesicles, the optimal conditions are sensitive to the lipid composition of the system.     

Study on size growth in the vesicle formation upon detergent removal from phospholipid/ detergent mixed micelles

When 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) was removed from the mixed CHAPS/EggPC micelles, large vesicles were prepared by dialysis or by slow step-by-step dilution, but small vesicles were prepared by fast one-step dilution. When sodium cholate was removed from the sodium cholate/EggPC micelles, small vesicles formed either by dialysis or by dilution; however, in the presence of 5 mM Ca²⁺ large vesicles were produced by dialysis, while small vesicles were prepared by dilution. The size growth was related to a detergent-induced fusion of the vesicles containing a large amount of detergent. Using spectrophotometry, quasielastic light scattering and freeze–fracture electron microscopy the fusion events were investigated both through the process of vesicle solubilization by adding detergent and through the process of vesicle formation by diluting a mixed micelle. The results suggest that a rapid CHAPS-induced fusion of the vesicles led to the large resultant vesicles and that no fusion of vesicles containing sodium cholate is responsible for the formation of small vesicles. Furthermore, the ultimate vesicle size related to rapid or slow detergent removal is dependent on the kinetic aspects of the fusion. Received: 19 August 1999 Accepted: 18 February 2000     

Solvent replacement to thermo‐responsive nanoparticles from long‐subchain hyperbranched PSt grafted with PNIPAM for encapsulation

Long‐subchain hyperbranched polystyrene (lsc‐hp PSt) with uniform subchain length was obtained through copper‐catalyzed azide‐alkyne cycloaddition click chemistry from seesaw macromonomer of PSt having one alkynyl group anchored at the chain centre and two azido group attached to both chain ends [alkynyl‐(PSt‐N₃)₂]. After precipitation fraction, different portions of lsc‐hp PSt having narrow overall molecular weight distribution were obtained for further grafting with alkynyl‐capped poly(N‐isopropylacrylamide) (alkynyl‐PNIPAM), which was obtained via single‐electron transfer living radical polymerization of NIPAM with propargyl 2‐bromoisobutyrate as the initiator and grafted onto the peripheral azido groups of lsc‐hp PSt via click chemistry. Thus, amphiphilic lsc‐hp PSt grafted with PNIPAM chains (lsc‐hp PSt‐g‐PNIPAM) was obtained and would have star‐like conformation in tetrahydrofuran (THF). By replacing THF with water, lsc‐hp PSt‐g‐PNIPAM was dissolved at molecular level in aqueous solution due to the hydrophilicity of PNIPAM and exhibited thermal induced shrinkage of PNIPAM arms. The water‐insoluble lsc‐hp PSt would collapse densely and could be served as a reservoir to absorb hydrophobic chemicals in aqueous solution. The influence of overall molecular weight of lsc‐hp PSt on the absorption of pyrene was studied. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Preparation of ethynylpyridine macrocycles by oxidative coupling of an ethynylpyridine trimer with terminal acetylenes

Macrocycles consisted of pyridine rings and acetylene bonds were prepared by Eglinton coupling from a tandem precursor bearing two terminal alkynyl groups. The composition of molecular size in the cyclized products changed by the reaction solvent. In pyridine, 9-meric and bigger macrocycles were obtained, while that of 6-mer was not. On the other hand, in pyridine/THF mixed solvent, the 6-mer was obtained as a major product.     

Regioselective trapping of terminal di-, tri-, and tetraynes with benzyl azide

[Structure: see text] The reaction of benzyl azide with terminal di-, tri-, and tetraynes appended with a range of functional groups has been explored. Standard reaction conditions for BnN3 catalyzed by CuSO4.5H2O gave alkynyl, butadiynyl, and hexatriynyl triazoles in moderate to good yields. The reaction proceeds regioselectively as determined by the X-ray crystallographic analysis of three derivatives (1c, 1d, and 3c), and no evidence of multiple azide addition to the polyyne framework is observed.     

Vesicle aggregation by multivalent ligands: relating crosslinking ability to surface affinity

In an effort to improve the stability of our tissue-mimetic vesicle aggregates, we have investigated how increasing the valency of our multivalent crosslinking ligand, poly-l-histidine, affected both the extent of vesicle aggregation and the affinity of the multivalent ligand for the synthetic receptor Cu(1) embedded in the vesicle membranes. Although increasing ligand valency gave the anticipated increase in the size of the vesicle aggregates, isothermal calorimetric studies did not show the expected increase in the valence-corrected binding constant for the embedded receptors. To explain both observations, we have developed a simple new binding model that encompasses both multivalent binding to receptors on a single vesicle surface (intramembrane binding) and vesicle crosslinking (intermembrane binding).