Interaction of Liposomal Formulations of Meta-tetra(hydroxyphenyl)chlorin (Temoporfin) with Serum Proteins: Protein Binding and Liposome Destruction

mTHPC is a non polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy; however, neither was reported before. We studied drug binding to human serum proteins using size‐exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip^®) and PEGylated (Fospeg^®) liposomes does not affect equilibrium serum protein binding compared with solvent‐based mTHPC. At short incubation times the redistribution of mTHPC from Foslip^® and Fospeg^® proceeds by both drug release and liposomes destruction. At longer incubation times, the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared with conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long‐circulation times the pharmacokinetic behavior of Fospeg^® could be influenced by a combination of protein‐ and liposome‐bound drug. The study highlights the modes of interaction of photosensitizer‐loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in preclinical models, as well as the novel application of NTA to assess the destruction of liposomes.     

Biodegradable poly(ethylene carbonate) nanoparticles as a promising drug delivery system with "stealth" potential

The goal of this study was to investigate the suitability of poly(ethylene carbonate) (PEC) nanoparticles as a novel drug delivery system, fulfilling the requirements for a long circulation time. Particles were obtained with a narrow size distribution and nearly neutral zeta potential. Adsorption studies with human plasma proteins revealed that PEC nanoparticles bind much less proteins in comparison to polystyrene (PS) nanoparticles. Cell experiments with fluorescently labeled PEC showed no uptake of the nanoparticles by macrophages. These novel PEC nanospheres with their unique surface properties are a promising candidate for long circulating drug delivery systems in vivo.     

Bacteria‐Based Production of Thiol‐Clickable,Genetically Encoded Lipid Nanovesicles

Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high‐yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria‐based proteoliposomes are an attractive eco‐friendly alternative that can outperform currently used liposomes.     

Bacteria‐Based Production of Thiol‐Clickable,Genetically Encoded Lipid Nanovesicles

Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high‐yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria‐based proteoliposomes are an attractive eco‐friendly alternative that can outperform currently used liposomes.     

Synthetic polyion-counterion transport systems in polymersomes and gels

Transport across the membranes of polymersomes remains difficult in part due to the great thickness of the polymer bilayers. Here, we report that dynamic polyion-counterion transport systems are active in fluorogenic polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PDMS-PMOXA). These results suggest that counterion-activated calf-thymus DNA can act as cation carrier that moves not only across lipid bilayer and bulk chloroform membranes but also across the "plastic" membranes of polymersomes. Compared to egg yolk phosophatidylcholine (EYPC) lipsosomes, activities and activator scope in PDMS-PMOXA polymersomes are clearly reduced. Embedded in agar gel matrices, fluorogenic PDMS-PMOXA polymersomes respond reliably to polyion-counterion transporters, with high contrast, high stability and preserved selectivity. Compared to standard EYPC liposomes, it cannot be said that PDMS-PMOXA polymersomes are better. However, they are different, and this difference could be interesting for the development of sensing devices.     

Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation

In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.     

Salt,Shake, Fuse—Giant Hybrid Polymer/Lipid Vesicles through Mechanically Activated Fusion

Large (200 nm) poly(ethylene oxide)‐b‐poly(butadiene) polymer vesicles fuse into giant (>1 μm) vesicles with mild agitation in dilute aqueous NaCl solutions. This unusual effect is attributed to the salt‐induced contraction of the poly(ethylene oxide) corona, reducing steric resistance between vesicles and, with agitation, increasing the probability of contact between the hydrophobic cores of adjacent membranes. In addition, NaCl and agitation facilitated the creation of giant hybrid vesicles from much smaller homogeneous polymersomes and liposomes. Whereas lipid vesicles do not readily fuse with each other under the same circumstances, they did fuse with polymersomes to produce hybrid polymer/lipid vesicles.     

Salt,Shake, Fuse—Giant Hybrid Polymer/Lipid Vesicles through Mechanically Activated Fusion

Large (200 nm) poly(ethylene oxide)‐b‐poly(butadiene) polymer vesicles fuse into giant (>1 μm) vesicles with mild agitation in dilute aqueous NaCl solutions. This unusual effect is attributed to the salt‐induced contraction of the poly(ethylene oxide) corona, reducing steric resistance between vesicles and, with agitation, increasing the probability of contact between the hydrophobic cores of adjacent membranes. In addition, NaCl and agitation facilitated the creation of giant hybrid vesicles from much smaller homogeneous polymersomes and liposomes. Whereas lipid vesicles do not readily fuse with each other under the same circumstances, they did fuse with polymersomes to produce hybrid polymer/lipid vesicles.     

Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface

Covalent grafting of poly(ethylene glycol) chains to proteins ("PEGylation") is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surface-induced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface.     

Superhydrophilic zwitterionic polymers stabilize liposomes

Nonionic polyethylene glycol (PEG) as a stealth polymer destabilizes liposomes due to its amphiphilic property. As a result, PEGylated liposomes have to be further stabilized, such as by using a large amount cholesterol. This is a long existing dilemma faced by PEG. In this work, we show that zwitterionic poly(carboxybetaine) (PCB) stabilizes liposomes because of its superhydrophilic nature, thus solving this dilemma. Specifically, PCB-modified liposomes without cholesterol exhibited good retention of hydrophilic drug and long blood circulating characteristics in vivo. To further validate this new PCB chemistry, PCB liposomal doxorubicin without cholesterol was compared with DOXIL for their antitumor therapeutic efficacies.     

Solid‐State NMR of PEGylated Proteins

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic‐level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid‐state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.     

Alterations in the Detergent-Induced Membrane Permeability and Solubilization of Saturated Phosphatidylcholine/Cholesterol Liposomes: Effects of Poly(ethylene glycol)-Conjugated Lipid

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.     

Structural Features of Interacting Complementary Liposomes Promoting Formation of Multicompartment Structures

The structural features of complementary liposomes and factors favoring formation of multicompartment systems are investigated. Specifically, liposomal formulations consisting of PEGylated unilamellar liposomes with guanidinium moieties located at the distal end of polyethylene glycol (PEG) chains interact with complementary multilamellar liposomes bearing phosphate moieties. Furthermore, the number of PEG chains attached to the unilamellar interface of the liposomes is enhanced by incorporating PEGylated cholesterol in their bilayer. While molecular recognition of the liposomes is the driving force for initiating multicompartmentalization, it is the enhanced PEGylation at the liposomal interface that synergistically promotes fusion resulting in large and well‐formed multicompartment systems. A mechanism is proposed according to which initial adhesion of the liposomes, followed by reorganization of their membrane lipids, leads to giant bilayer aggregates incorporating large liposomes.     

pH and reduction dual-bioresponsive polymersomes for efficient intracellular protein delivery

pH and reduction dual-bioresponsive nanosized polymersomes based on poly(ethylene glycol)-SS-poly(2-(diethyl amino)ethyl methacrylate) (PEG-SS-PDEA) diblock copolymers were developed for efficient encapsulation and triggered intracellular release of proteins. PEG-SS-PDEA copolymers with PDEA-block molecular weights ranging from 4.7, 6.8, to 9.2 kg/mol were synthesized in a controlled manner via reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(diethyl amino)ethyl methacrylate (DEAEMA) using PEG-SS-CPADN (CPADN = 4-cyanopentanoic acid dithionaphthalenoate; M(n) PEG = 1.9 kg/mol) as a macro-RAFT agent. These copolymers existed as unimers in water at mildly acidic pH (<7.2) conditions, but readily formed monodisperse nanosized polymersomes (54.5-66.8 nm) when adjusting solution pH to 7.4. These polymersomes were highly sensitive to intracellular pH and reductive environments, which resulted in fast dissociation and aggregation of polymersomes, respectively. Notably, both fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and cytochrome C (FITC-CC) proteins could facilely be encapsulated into polymersomes with excellent protein-loading efficiencies, likely as a result of electrostatic interactions between proteins and PDEA. The in vitro release studies showed that protein release was minimal (<20% in 8 h) at pH 7.4 and 37 °C. The release of proteins was significantly enhanced at pH 6.0 due to collapse of polymersomes. Notably, the fastest protein release was observed under intracellular-mimicking reductive environments (10 mM dithiothreitol, pH 7.4). MTT assays in RAW 264.7 and MCF-7 cells indicated that PEG-SS-PDEA (9.2 k) polymersomes had low cytotoxicity up to a polymer concentration of 300 μg/mL. Confocal laser scanning microscope (CLSM) observations revealed that FITC-CC-loaded PEG-SS-PDEA (9.2 k) polymersomes efficiently delivered and released proteins into MCF-7 cells following 6 h of incubation. Importantly, flow cytometry assays showed that CC-loaded PEG-SS-PDEA (9.2 k) polymersomes induced markedly enhanced apoptosis of MCF-7 cells as compared to free CC and CC-loaded PEG-PDEA (8.9 k) polymersomes (reduction-insensitive control). These dual-bioresponsive polymersomes have appeared to be highly promising for intracellular delivery of protein drugs.     

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins

It is now well‐established that the surface chemistry and “stealth” surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised.     

Hydrogen-bonding-driven self-assembly of PEGylated organosilica nanoparticles with poly(acrylic acid) in aqueous solutions and in layer-by-layer deposition at solid surfaces

PEGylated organosilica nanoparticles have been synthesized through self-condensation of (3-mercaptopropyl)trimethoxysilane in dimethyl sulfoxide into thiolated nanoparticles with their subsequent reaction with methoxypoly(ethylene glycol) maleimide. The PEGylated nanoparticles showed excellent colloidal stability over a wide range of pH in contrast to the parent thiolated nanoparticles, which have a tendency to aggregate irreversibly under acidic conditions (pH < 3.0). Due to the presence of a poly(ethylene glycol)-based corona, the PEGylated nanoparticles are capable of forming hydrogen-bonded interpolymer complexes with poly(acrylic acid) in aqueous solutions under acidic conditions, resulting in larger aggregates. The use of hydrogen-bonding interactions allows more efficient attachment of the nanoparticles to surfaces. The alternating deposition of PEGylated nanoparticles and poly(acrylic acid) on silicon wafer surfaces in a layer-by-layer fashion leads to multilayered coatings. The self-assembly of PEGylated nanoparticles with poly(acrylic acid) in aqueous solutions and at solid surfaces was compared to the behavior of linear poly(ethylene glycol). The nanoparticle system creates thicker layers than the poly(ethylene glycol), and a thicker layer is obtained on a poly(acrylic acid) surface than on a silica surface, because of the effects of hydrogen bonding. Some implications of these hydrogen-bonding-driven interactions between PEGylated nanoparticles and poly(acrylic acid) for pharmaceutical formulations are discussed.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Monosaccharide-responsive release of insulin from polymersomes of polyboroxole block copolymers at neutral pH

We synthesized a boroxole-containing styrenic monomer that can be polymerized by the reversible addition-fragmentation and chain transfer (RAFT) method. Poly(styreneboroxole) (PBOx) and its block copolymers with a poly(ethylene glycol) (PEG) as a hydrophilic block displayed binding to monosaccharides in phosphate buffer at neutral pH, as quantified by Wang's competitive binding experiments. By virtue of a controlled radical polymerization, we were able to adjust the degree of polymerization of the PBOx block to yield sugar-responsive block copolymers that self-assembled into a variety of nanostructures including spherical and cylindrical micelles and polymer vesicles (polymersomes). Polymersomes of these block copolymers exhibited monosaccharide-responsive disassembly in a neutral-pH medium. We demonstrated the possibility of using these polymersomes as sugar-responsive delivery vehicles for insulin in neutral phosphate buffer (pH 7.4). Encapsulated insulin could be released from the polymersomes only in the presence of sugars under physiologically relevant pH conditions.     

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.     

Gelatin Coating for the Improvement of Stability and Cell Uptake of Hydrophobic Drug-Containing Liposomes

Purpose: Most therapeutic agents have limitations owing to low selectivity and poor solubility, resulting in post-treatment side effects. Therefore, there is a need to improve solubility and develop new formulations to deliver therapeutic agents specifically to the target site. Gelatin is a natural protein that is composed of several amino acids. Previous studies revealed that gelatin contains arginyl-glycyl-aspartic acid (RGD) sequences that become ligands for the integrin receptors expressed on cancer cells. Thus, in this study, we aimed to increase the efficiency of drug delivery into cancer cells by coating drug-encapsulating liposomes with gelatin (gelatin-coated liposomes, GCLs). Methods: Liposomes were coated with gelatin using electrostatic interaction and covalent bonding. GCLs were compared with PEGylated liposomes in terms of their size, zeta potential, encapsulation efficiency, stability, dissolution profile, and cell uptake. Results: Small-sized and physically stable GCLs were prepared, and they showed high drug-encapsulation efficiency. An in vitro dissolution study showed sustained release depending on the degree of gelatin coating. Cell uptake studies showed that GCLs were superior to PEGylated liposomes in terms of cancer cell-targeting ability. Conclusions: GCLs can be a novel and promising carrier system for targeted anticancer agent delivery. GCLs, which exhibited various characteristics depending on the coating degree, could be utilized in various ways in future studies.