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PubMed2000

Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.

Most solid tumors possess unique pathophysiological characteristics that are not observed in normal tissues or organs, such as extensive angiogenesis and hence hypervasculature, defective vascular architecture, impaired lymphatic drainage/recovery system, and greatly increased production of a number of permeability mediators. The phenomenon now known as the enhanced permeability and retention (EPR) effect for lipid and macromolecular agents has been observed to be universal in solid tumors. Primarily, enhanced vascular permeability will sustain an adequate supply of nutrients and oxygen for rapid tumor growth. The EPR effect also provides a great opportunity for more selective targeting of lipid- or polymer-conjugated anticancer drugs, such as SMANCS and PK-1, to the tumor. In the present review, the basic characteristics of the EPR effect, particularly the factors involved, are described, as well as its modulation for improving delivery of macromolecular drugs to the tumor. Tumor-specific vascular physiology is also described.

Animals↗
PubMedDate not supplied

Gastrointestinal host defence: importance of gut closure in control of macromolecular transport.

An important adaptation of the gastrointestinal tract to the extrauterine environment is its development of a mucosal barrier against the penetration of harmful substances (bacteria, toxins and antigens) present within the intestinal lumen. At birth, the newborn infant must be prepared to deal with bacterial colonization of the gut, with formation of toxic byproducts of bacteria and viruses (enterotoxins and endotoxins) and with the ingestion of antigens (milk proteins). These potentially noxious substances if allowed to penetrate the mucosal epithelial barrier under pathological conditions can cause inflammatory and allergic reactions which may result in gastrointestinal and systemic disease states. To combat the potential danger of invasion across the mucosal barrier the infant must develop an elaborate system of defence mechanisms within the lumen and on the luminal mucosal surface which act to control and maintain the epithelium as an impermeable barrier to uptake of macromolecular antigens. These defences include a unique immunological system adapted to function in the complicated milieu of the intestine as well as other non-immunological processes such as a gastric barrier, intestinal surface secretions, peristaltic movement and natural antibacterial substances (lysozyme, bile salts) which also help to provide maximum protection for the intestinal surface. Unfortunately, during the immediate postpartum period, particularly for premature and small-for-dates infants, this elaborate local defence system is incompletely developed. As a result of the delay in the maturation of the mucosal barrier newborn infants are particularly vulnerable to pathological penetration by harmful intraluminal substances. The consequences of altered defence are susceptibility to infection and the potential for hypersensitivity reactions and for formation of immune complexes. With these reactions comes the potential for developing life-threatening diseases such as necrotizing enterocolitis, sepsis and hepatitis. Fortunately, 'nature' has provided a means for passively protecting the 'vulnerable' newborn against dangers of a deficient intestinal defence system, namely human milk. It is now increasingly apparent that human milk contains not only antibodies and viable leucocytes but many other substances which can interfere with bacterial colonization and prevent antigen penetration.

Animals↗
PubMed1981

Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2.

During protein synthesis the interaction with ribosomes of elongation factors Tu (EF-Tu), G (EF-G) and initiation factor 2 (IF-2) is associated with the hydrolysis of GTP which is directly related to the functions of the factors. In this article we review systematically the properties of these GTPase activities in the presence and absence of protein synthesis, and by examining the characteristics of the different minimal systems for the expression of these activities we point to the role of the various effectors and to the enzymological aspects of the systems. For EF-Tu, it has been possible to eliminate any requirement for macromolecular effectors, showing that the factor itself is a GTPase. For EF-G, the presence of at least the 50S ribosomal subunit has remained a requirement, whereas IF-2 needs both the 50S and 30S subunits to exhibit GTPase activity. Between the GTPase activities of the three factors there are some striking similarities, but important differences prevail as a consequence of the specificity of the different functions. This can also be seen by examining the respective ribosomal regions implicated in these reactions. When coupled with protein synthesis, the three GTPase activities reveal characteristics differing from those observed in partial systems.

Anti-Bacterial Agents↗
PubMed2001

Investigation on a novel core-coated microspheres protein delivery system.

Among the different approaches to achieve protein delivery, the use of polymers, specifically biodegraded, holds great promise. In this work, a new microsphere delivery system composed of alginate microcores surrounded by a biodegradable poly-DL-lactide-poly(ethylene glycol (PELA) was designed to improve the loading efficiency and stability of proteins. Alginate was solidified by calcium (MS-1), polylysine (MS-2) and chitosan (MS-3), respectively, to form different microcores. Human Serum Albumin (HSA), used as a model protein, was efficiently entrapped within the alginate microcores using a high-speed stirrer and then microencapsulated into PELA copolymer using a w/o/w solvent extraction method. DSC analysis of the microspheres revealed the efficient encapsulation of the alginate microcores, while the microcores were dispersed in the PELA matrix. SDS-PAGE results showed that HSA kept its structural integrity during encapsulation and release procedure. Microspheres were characterized in terms of morphology, size, loading efficiency, in vitro degradation and protein release. The degradation profiles were characterized by measuring the loss of microsphere mass, the decrease of polymer intrinsic viscosity and the reduction of PEG content of PELA coat. The release profiles were investigated from the measurement of protein presented in the release medium at various intervals. The results were that the degradation rate of these core-coated microspheres was MS-2>MS-1>MS-3. The extent of burst release from the core-coated microspheres in the initial protein release was lower than the 27% burst release from the conventional microspheres. In conclusion, the work presents a new approach for macromolecular drugs (such as protein, peptide drugs) delivery. The core-coated microspheres system may have potential use as a carrier for drugs that are poorly absorbed after oral administration.

Alginates↗
PubMed2004

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier.

Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.

Cell Count↗
PubMed1998

Implications of macromolecular crowding for signal transduction and metabolite channeling.

The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux-response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of approximately 1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux-response coefficient, in one case to values of approximately 1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux-response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.

Cell-Free System↗
PubMed1994

In situ-forming gels for ophthalmic drug delivery.

Poor bioavailability of ophthalmic solutions caused by dilution and drainage from the eye can be overcome by using in situ-forming ophthalmic drug delivery systems prepared from polymers that exhibit reversible phase transitions. Joshi et al. (1), have demonstrated that aqueous compositions that reversibly gel in response to simultaneous variations in at least two physical parameters, such as temperature, pH, and ionic strength, can be formed by appropriate combinations of macromolecular polymers which exhibit reversible gelation properties. In the present study, the rheological characterization of such a system, prepared by a combination of Carbopol (C) and methyl cellulose (MC), was carried out at two different pH (4.0 and 7.4) and temperatures (25 and 37 degrees C) by rotational cone and plate viscometry. The shear stress (tau) vs. shear rate (D) flow curves of the aqueous polymer solutions indicated a pseudoplastic behavior, with a yield point. An increase in pH from 4.0 to 7.4, or temperature from 25 to 37 degrees C, resulted in an increase in viscosity (eta), tau, and yield point, the magnitude of changes being highest when both the parameters were altered simultaneously. An increase in concentration of either C or MC, or an increase in MC molecular weight results in an increase in eta, tau, and yield point. Among the compositions studied, a solution containing 1.5% MC 0.3% C was found to have low eta, and formed a strong gel under simulated physiological conditions. Such a system can be formulated as drug containing liquid suitable for administration by instillation into the eye, which upon exposure to physiological conditions will shift to the gel (semi-solid) phase, thus increasing the precorneal residence time of the delivery system and enhancing ocular bioavailability.

Acrylic Resins↗
PubMed1995

Hydrostatic and osmotic pressure as tools to study macromolecular recognition.

Clearly, hydrostatic and osmotic pressure techniques offer unique potential in the study of fundamental problems of molecular recognition in biological systems. With the recent advances in technology such investigations are rapidly becoming commonplace. We look forward to further advances and their report in succeeding compendiums such as this volume.

DNA↗
PubMed2004

Thiomers for oral delivery of hydrophilic macromolecular drugs.

In recent years thiolated polymers (thiomers) have appeared as a promising new tool in oral drug delivery. Thiomers are obtained by the immobilisation of thio-bearing ligands to mucoadhesive polymeric excipients. By the formation of disulfide bonds with mucus glycoproteins, the mucoadhesive properties of thiomers are up to 130-fold improved compared with the corresponding unmodified polymers. Owing to the formation of inter- and intramolecular disulfide bonds within the thiomer itself, matrix tablets and particulate delivery systems show strong cohesive properties, resulting in comparatively higher stability, prolonged disintegration times and a more controlled drug release. The permeation of hydrophilic macromolecular drugs through the gastrointestinal (GI) mucosa can be improved by the use of thiomers. Furthermore, some thiomers exhibit improved inhibitory properties towards GI peptidases. The efficacy of thiomers in oral drug delivery has been demonstrated by various in vivo studies. A pharmacological efficacy of 1%, for example, was achieved in rats by oral administration of calcitonin tablets comprising a thiomer. Furthermore, tablets comprising a thiomer and pegylated insulin resulted in a pharmacological efficacy of 7% after oral application to diabetic mice. Low-molecular-weight heparin embedded in thiolated polycarbophil led to an absolute bioavailability of > or = 20% after oral administration to rats. In these studies, formulations comprising the corresponding unmodified polymer had only a marginal or no effect. These results indicate drug carrier systems based on thiomers appear to be a promising tool for oral delivery of hydrophilic macromolecular drugs.

Administration, Oral↗
PubMed1995

Influence of phosphoenolpyruvate and magnesium ions on the quaternary structure of enzyme I of the phosphotransferase system from gram-positive bacteria.

Solution X-ray scattering patterns of enzyme I of the phosphotransferase system from Staphylococcus carnosus indicate an increase in radius of gyration and molecular mass in the presence of Mg2+ or both Mg2+ and phosphoenolpyruvate, indicating a partial dimerization of enzyme I. Mg2+ ions are essential for both the dimerization and the activation, whereas the substrate phosphoenolpyruvate shifts the monomer--dimer equilibrium to the enzymatically active dimer by decreasing the dissociation rate of the phosphorylated dimer.

Enzyme Activation↗
PubMed2001

The distinct roles that Gln-192 and Glu-217 of factor IX play in selectivity for macromolecular substrates and inhibitors.

In this paper, we report functional characterization of positions 192 and 217 (chymotrypsinogen numbering system) in human factor IX and discuss the distinction and similarity of these two sites among the blood coagulation factors. Recombinant factor IXQ192E (residue glutamine at position 192 replaced by glutamic acid), IXQ192K, IXE217D, and IXE217R proteins exhibited 11%, 46%, 39%, and 2% of the wild-type factor IX's clotting activity, respectively. Binding of these variants to factor VIIIa (FVIIIa) was inefficient compared to that of wild-type factor IX, and the dissociation constants doubled for IXQ192E, 3-fold higher for IXQ192K and 4-fold higher for both IXE217D and IXE217R. In the presence of FVIIIa, all variant factor IX hydrolyzed factor X at the catalytic efficiencies correlating with respective clotting activities. However, FVIIIa greatly enhanced the catalytic efficiency of both IXE217 variants to a greater extent (approximately 7 x 10(4)-fold) as compared to its effect on the wild-type factor IXa and the other two IXQ192 variants [by a factor of (1-2) x 10(4)]. Moreover, while both IXQ192 variants demonstrated small substrate selectivity similar to that of wild-type factor IXa, the selectivity of both IXE217 variants was greatly altered. Mutations at position 192 disturbed the interaction of factor IXa with physiological inhibitors. Although all variants formed an SDS-stable complex with antithrombin III (ATIII) equally well in the presence of heparin and were readily inhibited by ATIII in the absence of heparin, activated IXQ192K exhibited a slower stable complex formation with ATIII without heparin. On the other hand, only IXQ192E showed decreased interaction with TFPI. Our results demonstrate that positions 192 and 217 play different roles unique to factor IX in specifying the interaction of factor IX with substrates and inhibitors.

Antithrombin III↗
PubMed2004

Oligonucleotide delivery to tumours using macromolecular carriers.

Microparticles have been used for delivery of chemo- and radio-therapeutic agents to tumours in patients for more than a decade, whereas cationic liposomes have been used for introducing nucleic acids into mammalian cells for the same period of time. This minireview discusses the potential of delivering therapeutic ONs (oligonucleotides) to tumours using cationic liposomes and the major obstacles to such delivery. It also proposes how microparticles can be utilized for delivery of ONs to solid tumours. Prospects for improved ON delivery using these carriers and ways to achieve this are discussed.

Animals↗
PubMed1995

Comparison of recombination in vitro and in E. coli cells: measure of the effective concentration of DNA in vivo.

Despite the extremely high concentration of DNA in nucleoid/nuclear regions, chromosomal dimerization and entanglement are avoided. To help understand this, we measured the effective concentration of DNA in E. coli, a value that reflects the functional impact of the cellular milieu on DNA site reactivity. We used as probes plasmid fusion reactions by two site-specific recombinases. The normalized extents and rates of fusion in these systems were much lower in vivo than in analogous in vitro reactions. We calculate that the effective concentration of plasmid DNA is about one order of magnitude lower than the chemical concentration. We suggest that in bacterial cells DNA accessibility is highly restricted and that this dominates the forces that increase DNA activity, such as macromolecular crowding.

Cell-Free System↗
PubMed1994

The thermodynamic and hydrodynamic properties of macromolecules that influence the hydrodynamics of porous systems.

The water flow across porous, semipermeable membranes associated with osmosis and filtration under a variety of conditions is analysed and compared to macromolecular diffusion across free-liquid boundaries, diffusion and sedimentation in the ultracentrifuge, and tracer diffusion of water. This study establishes that osmosis can be explained in terms of the irreversible thermodynamics of diffusion. For macromolecular osmotically active solutes in the semidilute concentration regime the water flows across semipermeable porous membranes are interpreted in terms of a rate-limiting solute-solvent exchange layer that exists on the solution side of the membrane adjacent to the membrane pore; both osmosis and filtration will be governed by these exchange layers. These exchange layers also yield unique properties of their constituent molecules in systems where there is osmotic equilibration between solutions of different solutes. This study also establishes the need to consider the internal osmotic pressure of membranes in the pressure balance associated with the flow across the membrane. The complex situation of partially permeable membranes is analysed for the simple case where there are no mechanical gradients and there is only one osmotically active solution that creates a rate-limiting exchange layer. This treatment predicts that the flow will be governed primarily by the osmotic pressure difference associated with the partitioning of the solute at the membrane-solution interface.

Animals↗
PubMed1997

Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.

Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens have also been prepared by platinum-carbon shadowing, following freeze-cleavage. The two hemocyanins reassociate to produce characteristic oligomeric and polymeric forms. Subunits of purified KLH1 reassociate to produce a small number of didecamers, short multidecamers (ca 33 nm diameter) and much larger quantity of a ca 25 nm diameter flexible/undulatory tubular form of varying length. These tubules exhibit characteristic oblique features, indicative of an 'open' helical structure which appears to be a loosly or incompletely annealed twisted ribbon of subunits. After a period of days the tubules aggregate in parallel to produce large paracrystalline bundles, which do not have a tendency to associate end-to-end. Following transfer of this reassociated KLH1 to low calcium magnesium stabilizing buffer, the tubular bundles are unstable; they slowly break down into shorter lengths, fragments, subunit groups and individual subunits, which subsequently regenerate decamers, didecamers, and some multidecamers. Subunits of purified KLH2 reassociate to produce ca 25 nm diameter 'closed' tubules, which do not exhibit the oblique 'open' features shown by the KLH1 tubules; however, the ends of the these 'closed' tubules are often oblique. In addition to the tubular form, KLH2 reassociation also generates a somewhat small proportion of ca 33 nm diameter multidecamers, often containing many decamers and more than one 'nucleating' didecamer. On transfer to low calcium magnesium stabilizing buffer the KLH2 tubules are remarkably stable, but the number of multidecamers slowly increases with time. There is a significant structural difference between the short KLH1 multidecamers (only detected following in-vitro reassociation) and those of KLH2 quite apart from their length. Study of the metal shadowed specimens confirmed the difference between the KLH1 and KLH2 tubular forms, with relatively smooth helical surface ridges and a rougher internal surface, indicating internalization of subunit domains that are not required for the construction of the tubular wall, in accord with current understanding of the subunit organization within the native molecules.

Calcium Chloride↗
PubMed2000

Lectin-mediated drug delivery: the second generation of bioadhesives.

This paper reviews some recent developments in the area of bioadhesive drug delivery systems. The area of bioadhesion in drug delivery had started some 20 years ago by using so-called mucoadhesive polymers. Many of these polymers were already used as excipients in pharmaceutical formulations. This has facilitated the development of the first bioadhesive drug products, which are now commercially available. A major disadvantage of the hitherto known mucoadhesives, however, is their non-specificity with respect to the substrate. In particular for gastro-intestinal applications, this may cause some premature inactivation and moreover limits the duration of mucoadhesive bonds to the relatively fast mucus turnover. Nevertheless, for some mucoadhesive polymers other interesting functionalities were discovered, such as their ability to modulate epithelial permeability and to inhibit proteolytic enzymes. In contrast to the mucoadhesive polymers, lectins and some other adhesion molecules specifically recognize receptor-like structures of the cell membrane and therefore bind directly to the epithelial cells themselves ("cytoadhesion") rather than to the mucus gel layer. Furthermore, when bioadhesion is receptor-mediated, it is not only restricted to mere binding, but may subsequently trigger the active transport of large molecules or nanoscalic drug carrier systems by vesicular transport processes (endo-/transcytosis). Rather than only acting as a platform for controlled release systems, the concept of lectin-mediated bioadhesion therefore bears the potential for the controlled delivery of macromolecular biopharmaceuticals at relevant biological barriers, such as the epithelia of the intestinal or respiratory tract.

Adhesives↗
PubMed1999

Topographic Imaging of Chromium-Coated Frozen-Hydrated Cell and Macromolecular Complexes by In-Lens Field Emission Scanning Electron Microscopy.

: An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification (>/=200,000x) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be </=2-3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.

Journal Article↗
PubMed2004

Calculation of electrostatic interaction energies in molecular dimers from atomic multipole moments obtained by different methods of electron density partitioning.

Accurate and fast evaluation of electrostatic interactions in molecular systems is still one of the most challenging tasks in the rapidly advancing field of macromolecular chemistry, including molecular recognition, protein modeling and drug design. One of the most convenient and accurate approaches is based on a Buckingham-type approximation that uses the multipole moment expansion of molecular/atomic charge distributions. In the mid-1980s it was shown that the pseudoatom model commonly used in experimental X-ray charge density studies can be easily combined with the Buckingham-type approach for calculation of electrostatic interactions, plus atom-atom potentials for evaluation of the total interaction energies in molecular systems. While many such studies have been reported, little attention has been paid to the accuracy of evaluation of the purely electrostatic interactions as errors may be absorbed in the semiempirical atom-atom potentials that have to be used to account for exchange repulsion and dispersion forces. This study is aimed at the evaluation of the accuracy of the calculation of electrostatic interaction energies with the Buckingham approach. To eliminate experimental uncertainties, the atomic moments are based on theoretical single-molecule electron densities calculated at various levels of theory. The electrostatic interaction energies for a total of 11 dimers of alpha-glycine, N-acetylglycine and L-(+)-lactic acid structures calculated according to Buckingham with pseudoatom, stockholder and atoms-in-molecules moments are compared with those evaluated with the Morokuma-Ziegler energy decomposition scheme. For alpha-glycine a comparison with direct "pixel-by-pixel" integration method, recently developed Gavezzotti, is also made. It is found that the theoretical pseudoatom moments combined with the Buckingham model do predict the correct relative electrostatic interactions energies, although the absolute interaction energies are underestimated in some cases. The good agreement between electrostatic interaction energies computed with Morokuma-Ziegler partitioning, Gavezzotti's method, and the Buckingham approach with atoms-in-molecules moments demonstrates that reliable and accurate evaluation of electrostatic interactions in molecular systems of considerable complexity is now feasible.

Algorithms↗