Fluidized or not fluidized? Biophysical characterization of biohybrid lipid/protein/polymer liposomes and their interaction with tetracaine

BackgroundNanomedicine and the pharmaceutical industry demand the investigation of new biomaterials to improve drug therapies. Combinations of lipids, proteins, and polymers represent innovative platforms for drug delivery. However, little is known about the interactions between such compounds and this knowledge is key to prepare successful drug delivery systems.MethodsBiophysical properties of biohybrid vesicles (BhVs) composed of phospholipids, proteins, and amphiphilic block copolymers, assembled without using organic solvents, were investigated by differential scanning calorimetry and dynamic light scattering. We studied four biohybrid systems; two of them included the effect of incorporating tetracaine. Thermal changes of phospholipids and proteins when interacting with the amphiphilic block copolymers and tetracaine were analyzed.ResultsLysozyme and the copolymers adsorb onto the lipid bilayer modifying the phase transition temperature, enthalpy change, and cooperativity. Dynamic light scattering investigations revealed relevant changes in the size and zeta potential of the BhVs. Interestingly, tetracaine, a membrane-active drug, can fluidize or rigidize BhVs.ConclusionsWe conclude that positively charged regions of lysozyme are necessary to incorporate the block copolymer chains into the lipid membrane, turning the bilayer into a more rigid system. Electrostatic properties and the hydrophilic-lipophilic balance are determinant for the stability of biohybrid membranes.General significanceThis investigation provides fundamental information associated with the performance of biohybrid drug delivery systems and can be of practical significance for designing more efficient drug nanocarriers.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

Reactive carriers of immobilized compounds.

Sphericanl macroporous reactive carriers capable of forming covalent bonds with amino acids and proteins were prepared by the suspension copolymerization of 2-hydroxyethyl methacrylate, ethylene dimethacrylate and p-nitrophenyl esters of methacrylic acid and methacryloyl derivatives of glycine, beta-alanine and epsilon-aminocaproic acid. The effect of the spacer length, pH and the type of the buffer used, concentration of reactive groups in the copolymer, concentration of the ligand and the participation of the hydrolytic and aminolytic reaction of p-nitrophenyl functional groups in the attachment of glycine, D,L-phenylalanine and serumalbumin was studied. Macroporous copolymers containing reactive functional groups can be used as active enzyme carriers, if their activity is not blocked by the presence of p-nitrophenol split off in the attachment reaction.     

Controlled release of macromolecules and pharmaceuticals from synthetic polypeptides based on glutamic acid

Biocompatible, biodegradable copolymers of glutamic acid and ethyl glutamate were evaluated for their permeability to proteins ranging in molecular weight from 12,300 to 69,000. The results showed that the copolymers were sufficiently permeable that they could be used for the preparation of implantable, controlled-release systems capable of releasing therapeutic amounts of high-molecular-weight drugs.     

Synthesis and characterization of self-assembling block copolymers containing bioadhesive end groups

3,4-Dihydroxyphenyl-L-alanine (DOPA) is an unusual amino acid found in mussel adhesive proteins (MAPs) that is believed to lend adhesive characteristics to these proteins. In this paper, we describe a route for the conjugation of DOPA moieties to poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers. Hydroxyl end groups of PEO-PPO-PEO block copolymers were activated by N,N'-disuccinimidyl carbonate and then reacted with DOPA or its methyl ester with high coupling efficiencies from both aqueous and organic solvents. DOPA-modified PEO-PPO-PEO block copolymers were freely soluble in cold water, and dye partitioning and differential scanning calorimetry analysis of these solutions revealed that the copolymers aggregated into micelles at a characteristic temperature that was dependent on block copolymer composition and concentration in solution. Oscillatory rheometry demonstrated that above a block copolymer concentration of approximately 20 wt %, solutions of DOPA-modified PEO-PPO-PEO block copolymers exhibited sol-gel transitions upon heating. The gelation temperature could be tailored between approximately 23 and 46 degrees C by changing the composition, concentration, and molecular weight of the block copolymer. Rheological measurement of the bioadhesive interaction between DOPA-modified Pluronic and bovine submaxillary mucin indicated that DOPA-modified Pluronic was significantly more bioadhesive than unmodified Pluronic.     

Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin

The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions. It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2-n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected. Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate). The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased. The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics. It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface. We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.     

A study of the antioxidant and membranotropic activities of luteolin using different model systems

Luteolin, a water-insoluble 3′,4′,5,7-tetrahydroxyflavon, is one of the best-studied representatives of bioflavonoids. Luteolin is an essential food component for humans and other mammals that possesses a wide spectrum of biological activities by affecting the activities of various metabolic enzymes, target receptors, and signal transduction pathways. In this study, we conducted a comparative study of the antioxidant (free-radical scavenging) properties of luteolin in 2,2′-azobis(2-methylpropionamidine) dihydrochloride–luminol and hemoglobin–hydrogen peroxide–luminol systems and assessed its effect on the permeability of planar lipid bilayer membranes. Trolox was used as a reference antioxidant, while ascorbic acid and dihydroquercetin were taken as standards. Luteolin shows moderate antioxidant activity, exhibiting a higher antioxidant capacity than trolox and ascorbic acid, but it was less effective than dihydroquercetin in tests for antioxidant activity in the studied systems. The studied compounds can be ranked according to the effectiveness of their antioxidant action: dihydroquercetin > luteolin > trolox > ascorbic acid. It should be noted that the antioxidant activity of a water-soluble form of luteolin, luteolin disulfate, is comparable with that of luteolin. Luteolin does not cause significant changes in the permeability of planar bilayer membranes in the dose range from 1.5 to 30 μM. Our findings indicate the presence of a high level of free-radical scavenging activity and the absence of a primary membranotropic effect for luteolin. It can be assumed that the multiple pleiotropic nature of luteolin activity towards a variety of biological systems is associated not only with a neutralizing effect in regard to reactive oxygen species, but also with the ability of luteolin to block and modulate different cell-signaling processes and biochemical pathways. The presumed mechanisms of the biological activity of luteolin and luteolin disulfate are discussed.     

Ionophoric activity of pluronic block copolymers

Pluronic block copolymers (triblock copolymers of poly(ethylene oxide) and poly(propylene oxide)) exhibit a chemosensitizing effect on multidrug resistant cell lines. Changes in membrane permeability are hypothesized to be responsible because inhibition of drug transport mediated by both the multidrug-resistance-associated protein and the P-glycoprotein drug efflux system has been observed. To test this hypothesis, we now have studied the ion conductivity mediated by Pluronic L61. Besides a detergent-like action, the copolymer was able to form regular channels and to exhibit carrier activity. Long living ion channels were formed by polymer oligomerization. Aggregate equilibrium was shifted toward L61 monomers and dimers, which operated as mobile carriers. Copolymer-induced membrane permeability for potassium ions (1 M KCl) was less than 10(-8) cm s(-1), whereas the permeability for uncharged doxorubicin molecules (1 mM) was equal to 5 x 10(-4) cm s(-1). The results are consistent with reports about an increased doxorubicin accumulation in cells (Venne, Li, S., Mandeville, R., Kabanov, A., and Alakhov, V. Y. (1996) Cancer Res. 56, 3626-3629). However, the increased permeability contrasts with the polymer-mediated decrease of drug efflux from cells. Preferential polymer binding to membrane proteins may mask the unspecific effect of L61 observed on lipid bilayers.     

Ion transport systems as targets of free radicals during ischemia reperfusion injury

Oxidative stress is a recognized pathogenic factor in ischemia/reperfusion injury (IRI). Iron induced generation of reactive oxygen species (ROS) in vitro reduces both the Na+K+-ATPase activity and Na+-Ca2+ exchanger of synaptosomal membranes, concomitantly with alteration of physical state of membranes. Oxidative insult also leads to the loss of ability of endoplasmic reticular membranes (ER) to sequester Ca2+ as well as to the increase of Ca2+ permeability. Furthermore, ROS induces both lipid peroxidation and lipid-independent modifications of membrane proteins. Acute in vivo ischemia alters kinetic parameters of Na+K+-ATPase affecting mainly the dephosphorylation step of ATPase cycle with parallel changes of Na+-Ca2+ exchanger and alterations of physical membrane environment. Subsequent reperfusion after ischemia is associated with decrease of immuno signal for PMCA 1 isoform in hippocampus. In addition, incubation of non-ischemic membranes with cytosol from ischemic hippocampus decreases level of PMCA 1 in non-ischemic tissues. Loss of PMCA 1 protein is partially protected both by calpain- and by non-specific protease inhibitors which suggest possible activation of proteases in the reperfusion period. On the other hand, ischemia does not affect the level of Ca2+ pump (SERCA 2b) and calreticulin of intracellular Ca2+ stores. However, IRI resulted in a decrease of IP3 receptor I and altered active Ca2+ accumulation into the ER. A non-specific alteration of physical properties of total membranes such as the oxidative modifications of proteins as well as the content of lipoperoxidation products can also be detected after IRI. ROS can alter physical and functional properties of neuronal membranes. We discuss our results suggesting that ischemia-induced disturbation of ion transport systems may participate in or follow delayed death of neurons after ischemia.     

Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization

Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer. The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability. The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization. The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI. The storage conditions for PANI/PP membranes containing immobilized HRP were studied. HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5 degrees C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.     

Dye-ligand affinity systems.

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Water structure changes induced by ceramics can be detected by increased permeability through aquaporin

Aquporins are intrinsic membrane proteins that function as water channel to transport water and/or mineral nutrients across biological membranes. In this study, we aimed to clarify whether water structure can be changed by the presence of ceramics and whether such a change can be determined by aquaporin. First, we confirmed that ceramics could transform tap water into active tap water by increasing water permeability through aquaporin. We also found that this change in water permeability by treatment with ceramics occurred in distilled water. The distilled water was determined to exhibit the same aquaporin permeability as the original tap water. Our data indicate that the aquaporin permeability of water can be changed by severe physical shocks, such as slapping and sonication, which is consistent with the implication that the aquaporin permeability is closely related to the structure of the water. In this study, using aquaporins, we first reported that the treatment of water with ceramics can affect the structure of water, and the water can retain the structure for a given period under certain condition     

Protein-based block copolymers

Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers.     

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.     

Proteins of Excitable Membranes

Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment.     

Modification with polysialic acid–PEG copolymer as a new method for improving the therapeutic efficacy of proteins

A new protein derivatization method was developed with a block copolymer to reduce the immunogenicity of therapeutic proteins. The block copolymer consisted of polyethylene glycol (PEG) and polysialic acid (PSA), a nonimmunogenic and biodegradable biopolymer. Uricase was used as a model protein. Molecular weight analysis results indicated that the uricase–PEG–PSA conjugate was linked with 2.5 copolymers for each uricase unit. The residual enzyme activity of the uricase with modification by the PEG–PSA copolymer was 72.4%. The tolerance and stability to heat, acid, alkaline, and trypsin treatments significantly improved compared with the native uricase. The immunogenicity of uricase modified with PEG–PSA copolymer was remarkably reduced. The transmission electron microscopy results of the uricase–PEG–PSA conjugate showed a spherical hydrated shell with a larger particle size. These findings proved that the PSA–PEG–protein conjugate is a formulation that can potentially be used to deliver the protein and peptide-based drugs.     

Permeability of composite chondrocyte-culture-millipore membranes to solutes of varying size and shape.

A model connective-tissue system was developed that is amenable to the determination of permeability coefficients of diffusing solutes. The system involves the culture of 13-day chick-embryo chondrocytes on a Millipore filter (HA:0.45 micron pore size) to form what is, in effect, a confluent, extremely thin cartilage slice of uniform thickness. These cultured chondrocyte membranes were used to measure the steady-state flux of radioactively labelled low-molecular-weight solutes and micro-ions. Similarly, the permeability coefficients of either radioactively labelled or enzymically active proteins across the membranes were determined. The membrane was found to have no marked effects on the diffusional behaviour of low-molecular-weight non-electrolytes (water, proline, ribose, glucose, sorbitol, raffinose). For micro-ions (Na+, SO42-, Cl-, glutamate, glucuronate,), the diffusive behaviour was found to be markedly affected by the ionic strength of the solvent used in a manner which was consistent with a Donnan distribution resulting from the immobilized proteoglycans. Globular proteins permeated the membrane at rates which decreased as the molecular size of the diffusing solute increased. The apparent diffusion rates of fibrinogen and of collagen through the membranes were greater than would be expected on the basis of their diffusion coefficients in free solution. Reasons for this behaviour are discussed.     

Self-assembly of reactive amphiphilic block copolymers as mimetics for biological membranes

Over the years, polymers have attracted a great deal of interest because they offer a unique platform for the development of materials in fields as diverse as biomedicine and packaging. Many of these purposes use polymers that had been developed for totally different applications. Recently, however, chemical tailoring and molecular and supramolecular control of the chemistry and, thus, the physical and biological response have become a key interest of many researchers. In particular, systems that operate in aqueous media have become an intensely researched field. This is mostly because many devices must be biocompatible, which implies that they have to function in aqueous solutions. Over the past few years, new approaches for mimicking cell surfaces, for generating biocompatible and bioactive drug delivery systems, and for directed targeting have been developed. One recent development is polymeric systems with an enhanced biofunctionality, such as amphiphilic block copolymers that can act as mimetics for biological membranes. Because there are virtually no limits to combinations of monomers, biological and synthetic building blocks, ligands, receptors, and other proteins, polymer hybrid materials show a great promise for applications in biomedicine and biotechnology.     

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.