Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Crowded Environment Regulates the Coacervation of Biopolymers via Nonspecific Interactions.

The membrane-less organelles (MLOs) with subcompartments are formed via liquid-liquid phase separation (LLPS) in the crowded cell interior whose background molecules are up to 400 mg/mL. It is still a puzzle how the background molecules regulate the formation, dynamics, and functions of MLOs. Using biphasic coacervate droplets formed by poly(l-lysine) (PLL), quaternized dextran (Q-dextran), and single-stranded oligonucleotides (ss-oligo) as a model of MLO, we online monitored the LLPS process in Bovine Serine Albumin (BSA) solution up to 200.0 mg/mL. Negatively charged BSA is able to form complex or coacervate with positively charged PLL and Q-dextran and thus participates in the LLPS via nonspecific interactions. Results show that BSA effectively regulates the LLPS by controlling the phase distribution, morphologies, and kinetics. With increasing BSA concentration, the spherical biphasic droplets evolve in sequence into phase-inverted flower-like structure, worm-like chains, network structures, and confined coacervates. Each kind of morphology is formed via its own specific growth and fusion pathway. Our work suggests that MLOs could be controlled solely by the crowded environment and provides a further step toward understanding the life process in cell.

Coacervate Formed by an ATP-Binding Aptamer and Its Dynamic Behavior under Nonequilibrium Conditions.

Although numerous protocell models have been developed to explore the possible pathway of the origin of life on the early earth, few truly fulfill the roles of the DNA/RNA sequence and ATP molecules, which are keys to cell replication and evolution. The ATP-binding aptamer offers an opportunity to combine sequence and energy molecules. In this work, we choose the coacervate droplet as the protocell model and investigate the interaction of the DNA aptamer, poly(l-lysine)(PLL), and ATP under varying conditions. PLL and aptamers form solid precipitates, which spontaneously transform to coacervate droplets as ATP is introduced. The selective uptake and sequestration of exogenous molecules is achieved by the ATP-containing coacervates. As an electric field is applied to expel ATP, the portion of the droplet deficient in ATP becomes solid. The solid/liquid phase ratio is tunable by varying the electric field strength and excitation time. Together with the vacuolization process, a solid head with a soft mouth periodically opening and closing is created. Moreover, the composite coacervate droplet gradually grows as it is treated with an electric field and cannot recover to the original liquid phase after the power is turned off and replenished with ATP. Our work highlights that the proper integration of the DNA sequence, ATP, and energy input could be a powerful strategy for creating a protocell with certain living features.

PEGylated gene carriers in serum under shear flow.

The behaviour of drug/gene carriers in the blood stream under shear is still a puzzle. In this work, using the complexes formed by 21 bp DNA and poly(ethylene glycol)-b-poly(l-lysine) (PEG-PLL) of varying PEG lengths, we studied the dynamic behaviour of the complexes in the presence of fetal bovine serum (FBS) and under flow at different shear rates, a condition mimicking the internal physical environment of blood vessels. The PEG5k-PLL/DNA complex possesses a dense DNA/PLL core and a loose PEG5k protecting layer. The PEGylated DNA complexes exhibit multiple responses to external shear in the presence of FBS. The loose PEG5k layer is firstly disturbed at a shear rate below 30 s-1. The exposure of the charged core to the environment results in a secondary aggregation of the complex with FBS. The size of the aggregate is limited to a certain range as the shear rate increases to 50 s-1. The dense DNA/PLL core starts to withstand the shear force as the shear rate reaches 500 s-1. The reorganization of the core to accommodate more serum molecules leads to tertiary aggregation of the complexes. If PEG cannot form a valid layer around the complex, as in PEG2k-PLL/DNA, the complex forms an aggregate even without shear, and the first shear dependent region is missing. If the PEG layer is too stable around the complex, as in PEG10k-PLL/DNA, no tertiary aggregation occurs. The mechanism of shear on the behaviour of delivery particles in serum helps to design gene carriers with high efficacy.

Mass Transport in Coacervate-Based Protocell Coated with Fatty Acid under Nonequilibrium Conditions.

Construction of protocell models from prebiotically plausible components to mimic the basic features or functions of living cells is still a challenge. In this work, we prepare a hybrid protocell model by coating sodium oleate on the coacervate droplet constituted by poly(l-lysine) and oligonucleotide and investigate the transport of different molecules under electric field. Results show that sodium oleate forms a layered viscoelastic membrane on the droplet surface, which is selectively permeable to small, polar molecules, such as oligolysine. As the droplet is stimulated at 10 V cm-1, the oleate membrane slips along the direction of electric field while maintaining its integrity. Most of the molecules are still excluded under such conditions. As repetitive cycles of vacuolization occur at 20 V cm-1, all molecules are internalized and sequestrated in the droplet through their specific pathways except enzyme, which anchors in the oleate membrane and is immune to electric field. Cascade enzymatic reactions are then carried out, and the product generated from the membrane exhibits a time-dependent concentration gradient across the droplet. Our work makes a step toward the nonequilibrium functionalization of synthetic protocells capable of biomimetic operations.

Electric field-induced circulation and vacuolization regulate enzyme reactions in coacervate-based protocells.

Artificial protocells operating under non-equilibrium conditions offer a new approach to achieve dynamic features with life-like properties. Using coacervate micro-droplets comprising polylysine (PLL) and a short single-stranded oligonucleotide (ss-oligo) as a membrane-free protocell model, we demonstrate that circulation and vacuolization can occur simultaneously inside the droplet in the presence of an electric field. The circulation is driven by electrohydrodynamics and applies specifically to the major components of the protocell (PLL and ss-oligo). Significantly, under low electric fields (E = 10 V cm-1) the circulation regulates the movement of the vacuoles, while high levels of vacuolization produced at higher electric fields can deform or reshape the circulation. By taking advantage of the interplay between vacuolization and circulation, we achieve dynamic localization of an enzyme cascade reaction at specific droplet locations. In addition, the spatial distribution of the enzyme reaction is globalized throughout the droplet by tuning the coupling of the circulation and vacuolization processes. Overall, our work provides a new strategy to create non-equilibrium dynamic behaviors in molecularly crowded membrane-free synthetic protocells.

Axial growth and fusion of liposome regulated by macromolecular crowding and confinement.

The endomembrane system, including the endoplasmic reticulum, Golgi apparatus, lysosomes, and endosomes, is located in the crowded intracellular environment. An understanding of the cellular structure and functions requires knowledge of how macromolecular crowding and confinement affect the activity of membrane and its proteins. Using negatively charged liposome and the peptide K3L8K3 as a model system, we studied the aggregation behavior of liposome in a matrix of polyacrylamide and hyaluronic acid. Without matrix, the liposomes form spherical aggregates in the presence of K3L8K3. However, they orient in one dimension and fuse into a tube up to 40 µm long in the matrix. The growth of the tube is via end-to-end connection. This anisotropic growth is mainly due to the macromolecular confinement provided by the polymer network. The study of the interactions between liposome and peptide in the crowded environment helps to reveal the mechanism of membrane-related processes in vivo.

Complete dissociation and reassembly behavior as studied by using poly(ethylene glycol)-block-poly(glutamate sodium) and kanamycin A.

Kanamycin A, an amino modified sugar, can interact with poly(ethylene glycol)-block-poly(glutamate sodium) (PEG114-PGlu64) via electrostatic interactions (with PGlu) and hydrogen bonding (with PEG). The interplay of these two forces determines the assembly process and the resulting structure. In deionized water, kanamycin A and PEG114-PGlu64 form a spherical structure at [+]/[-] = 3.5. This structure dissociates instantly and completely in the presence of 30 mM NaCl. However, a new structure is reassembled in about 2 hours. A similar phenomenon is observed when the buffer pH is increased from 7.8 to 8.3. We attribute the distinct dissociation/reassembly process to the reestablishment of the balance between electrostatic interactions and hydrogen bonding. The dissociation/reassembly process in response to environmental changes offers a novel approach to release the loaded cargo in a controlled manner.

Peptides containing blocks of different charge densities facilitate cell uptake of oligonucleotides.

Polyelectrolyte complexes (PECs) are of great importance in drug delivery and gene therapy. The density and the distribution of the charges are key parameters of a polyelectrolyte, determining the structure of the complex and the kinetics of the complexation. Using peptides of precisely-controlled charge density as model molecules, we showed that the presence of weakly-charged peptides, (KGGG)5 or (KGKG)5, did not affect the complexation of highly-charged peptides (KKKK)5 with 21 bp oligonucleotides. However, peptide containing blocks of different charge densities, such as (KKKK)5-b-(KGGG)5 or (KKKK)5-b-(KGKG)5, exhibited superior performance during complexation. With a relatively uniform small size, the complex was also stable in serum. More importantly, the cellular uptake of the complex was greatly enhanced by a ratio of 40-60%, compared to that of the complex formed by uniformly-charged peptides. We attributed the improvement to the structure of the complex, in which the highly-charged blocks form the core with the oligonucleotide whilst the weakly-charged blocks dangle outside, preventing the complexes from further aggregation.

Aggregation, fusion, and leakage of liposomes induced by peptides.

Biological membranes are heterogeneous systems. Their functions are closely related to the lipid lateral segregation in the presence of membrane proteins. In this work, we designed two peptides, amphiphilic cationic peptides K3L8K3 and nonamphiphilic peptides K20, and studied their interactions with binary liposomes in different phases (Lα, Lß', and Lα/Lß'). As mimics of membrane proteins, both K3L8K3 and K20 can cause the liposomes to aggregate, fuse, or leak. These processes were closely related to the phases of liposomes. For the liposomes in Lα phase, heavy aggregation, fusion, and leakage were observed in the presence of either K20 or K3L8K3. For the liposomes in Lß' phase, neither K3L8K3 nor K20 can induce fusion or leakage. For the liposomes in Lα/Lß' phase, K3L8K3 caused the liposomes to aggregate, fuse, and leak, while K20 only led to aggregation. The kinetics of aggregation, fusion, and leakage in each phase were recorded, and they were related to the lipid demixing in the presence of the peptide. Our work not only gained insight into the effect of the lipid demixing on the interactions between peptide and membrane, but also helped in developing drug delivery vehicles with liposomes as the platform.

Liposomes physically coated with peptides: preparation and characterization.

Physically coating liposomes with peptides of desirable functions is an economic, versatile, and less time-consuming approach to prepare drug delivery vehicles. In this work, we designed three peptides-Ac-WWKKKGGNNN-NH2 (W2K3), Ac-WWRRRGGNNN-NH2(W2R3), Ac-WWGGGGGNNN-NH2(W2G3)-and studied their coating ability on negatively charged liposomes. It was found that the coating was mainly driven by the electrostatic interaction between the peptides' cationic side groups and the acidic lipids, which also mediated the "anchoring " of Trp residuals in the interfacial region of lipid bilayers. At the same conditions, the amount of the coated W2R3 was more than that of W2K3, but the stability of the liposome coated with W2R3 was deteriorated. This was caused by the delocalized charge of the guanidinium group of arginine. The coating of the peptide rendered the liposome pH-responsive behavior but did not prominently change the phase transition temperature. The liposome coated with peptides displayed appropriate pH/temperature dual responsive characteristics and was able to release the content in a controlled manner.

Fibril aggregates formed by a glatiramer-mimicking random copolymer of amino acids.

Amyloid formation is now considered a universal and intrinsic property of all proteins, irrespective of their sequences. Therefore, it is interesting to see whether random copolymers of amino acids can also form amyloid aggregates. Here we use a copolymer of 4 amino acids, mimicking the clinically used drug Glatiramer, and demonstrate that it does form amyloid-like fibrils in the aqueous solution despite its random sequence structure. The fibrillar aggregates show an alanine-rich ß-sheet secondary structure, proving the high tolerance of amyloid aggregates to the sequence irregularity in poly(amino acid)s, and suggesting the potential application of random copolymers as amyloid materials.

Polymersomes from dual responsive block copolymers: drug encapsulation by heating and acid-triggered release.

A series of well-defined thermoresponsive diblock copolymers (PEO45-b-PtNEAn, n=22, 44, 63, 91, 172) were prepared by the atom transfer radical polymerization of trans-N-(2-ethoxy-1,3-dioxan-5-yl) acrylamide (tNEA) using a poly(ethylene oxide) (PEO45) macroinitiator. All copolymers are water-soluble at low temperature, but upon quickly heating to 37 °C, laser light scattering (LLS) and transmission electron microscopy (TEM) characterizations indicate that these copolymers self-assemble into aggregates with different morphologies depending on the chain length of PtNEA and the polymer concentration; the morphologies gradually evolved from spherical solid nanoparticles to a polymersome as the degree of polymerization ("n") of PtNEA block increased from 22 to 172, with the formation of clusters with rod-like structure at the intermediate PtNEA length. Both the spherical nanoparticle and the polymersome are stable at physiological pH but susceptible to the mildly acidic medium. Acid-triggered hydrolysis behaviors of the aggregates were investigated by LLS, Nile red fluorescence, TEM, and (1)H NMR spectroscopy. The results revealed that the spherical nanoparticles formed from PEO45-b-PtNEA44 dissociated faster than the polymersomes of PEO45-b-PtNEA172, and both aggregates showed an enhanced hydrolysis under acidic conditions. Both the spherical nanoparticle and polymersome are able to efficiently load the hydrophobic doxorubicin (DOX), and water-soluble fluorescein isothiocyanate-lysozyme (FITC-Lys) can be conveniently encapsulated into the polymersome without using any organic solvent. Moreover, FITC-Lys and DOX could be coloaded in the polymersome. The drugs loaded either in the polymersome or in the spherical nanoparticle could be released by acid triggering. Finally, the DOX-loaded assemblies display concentration-dependent cytotoxicity to HepG2 cells, while the copolymers themselves are nontoxic.

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition.

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Polymer mixtures with enhanced compatibility and extremely low viscosity used as DNA separation media.

Poor compatibility was the major drawback of polymer mixtures when used as DNA separation media. Using poly(ethylene oxide)-b-poly(N, N-dimethylacrylamide) (PEO(44)-b-PDMA(88)) and PEO (M(w): 1.3 MDa) as an example, we demonstrated the concept that the compatibility was significantly improved when mixing a homopolymer with its copolymer. Laser light scattering indicated that the major interaction between PEO and PEO-b-PDMA in dilute solution was the weak hydrodynamic interaction, which showed almost no effect on the viscosity and the static scattering pattern. In semidilute or concentrated solution, viscosity measurement also suggested good compatibility between the two components. When tested as DNA separation medium by CE, the viscosity of the mixture was extremely low, only 5 cP for 5.0 m/v% PEO-b-PDMA+0.1 m/v% PEO at 25 degrees C. The performance on DNA separation could be tuned by varying the concentration of each component as well as the component ratio. Good separation on both dsDNA and ssDNA was achieved.

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems.

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.

Functional electrospun nanofibrous scaffolds for biomedical applications.

Functional nanofibrous scaffolds produced by electrospinning have great potential in many biomedical applications, such as tissue engineering, wound dressing, enzyme immobilization and drug (gene) delivery. For a specific successful application, the chemical, physical and biological properties of electrospun scaffolds should be adjusted to match the environment by using a combination of multi-component compositions and fabrication techniques where electrospinning has often become a pivotal tool. The property of the nanofibrous scaffold can be further improved with innovative development in electrospinning processes, such as two-component electrospinning and in-situ mixing electrospinning. Post modifications of electrospun membranes also provide effective means to render the electrospun scaffolds with controlled anisotropy and porosity. In this article, we review the materials, techniques and post modification methods to functionalize electrospun nanofibrous scaffolds suitable for biomedical applications.

In vitro non-viral gene delivery with nanofibrous scaffolds.

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (beta-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1x TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis.

Polyacrylamide (PAM) was used as a model polymer to build up an empirical model that relates polymer molecular weight, polymer concentration and solution viscosity. The desired random copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) used as DNA separation media for different specifications were synthesized under the guidance of the empirical model. The separation performances of rationally designed copolymers were tested in a 1.2 cm long separation channel, simulating microchip-based capillary electrophoresis. pBR322/HaeIII digest was successfully separated with good separation resolution and fast speed. Validation of the sieving ability of our polymers was performed in the Agilent 2,100 Bioanalyzer. The results of the 10 bp (base pair) DNA ladder separation demonstrate the potential of our approach for the sieving matrix in microchip-based electrophoresis.

The growth of filaments under macromolecular confinement using scaling theory.

Quantitatively describing macromolecular confinement is still a challenge. Using the assembly of DNA tiles in a polyacrylamide network as a model, we studied the effect of macromolecular confinement on the growth of the filament by scaling theory. The results show that the confinement regulates the morphology, the initial growth rate v, and the eventual length of the filament Nm. The initial growth rate is dependent on the medium viscosity η as ν∝η(-0.94), and the filament adjusts its length in the given confined space as Nm∝ (ξ/Rg)(1.8), with ξ being the mesh size of the polyacrylamide solution and Rg being the radius of gyration of polyacrylamide.