Combined effect of surface electrostatic charge and poly(ethyl glycol) on the association of liposomes with colon carcinoma cells

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers.     

Combined Effect of Surface Electrostatic Charge and Poly(ethyl glycol) on the Association of Liposomes with Colon Carcinoma Cells

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers.     

Degradation of N5-(2-hydroxyethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain

The rate of degradation of poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG), poly(L-glutamic acid) (PGA) and poly[HEG-co-GA] random copolymers by papain was measured in the pH range 4.0-7.5, employing the gel permeation chromatography method. The effect of the degree of ionization on the polymer conformation was measured by circular dichroism (c.d.). PHEG, which is uncharged, had a random coil conformation and an almost constant degradation rate within the whole pH interval. The ionization of PGA increased with increasing pH and was accompanied by conformational transition from helix to random coil. The hydrolysis of PGA by papain depended on pH with the optimum at about pH 5, indicating that both the high content of helix (at pH less than 5) and increasing charge density (at pH greater than 5), decreased the degradation rate. Contrary to PGA, pH profiles of the degradation rate of poly[HEG-co-GA] copolymers are monotonous and do not decrease at pH less than 5. In the copolymers the HEG residues act as a helix breaker and limit the formation of helical conformation. The role of structural features of a macromolecular substrate, i.e. the charge, helical conformation and the nature of amino acid residues, in the interaction between enzyme and polymer is discussed.     

A novel family of L-amino acid-based biodegradable polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting capacity

The objective of this study was to develop biodegradable polypeptide-lipid conjugates for the design of polymer-coated long-circulating liposomes (LCL). Lipid conjugates of poly(hydroxyalkyl L-asparagine/L-glutamine) were synthesized and incorporated into 0.15 microm dipalmitoyl phosphatidylcholine (DPPC)-cholesterol liposomes. Circulation times and biodistribution were assessed in rats using a radioactive lipid marker. Evaluation of the therapeutic activity of prednisolone phosphate loaded in 0.1 microm PHEA-DPPC-cholesterol liposomes in a rat experimental arthritis model was performed to demonstrate the drug-targeting potential of the polymer-coated liposomes. Coating of liposomes with poly(hydroxyethyl L-asparagine) (PHEA) and poly(hydroxyethyl L-glutamine) (PHEG) extended the circulation half-life to a similar extent as poly(ethylene glycol) (PEG), which is normally used for the preparation of LCL. Glutamine polymers with a hydroxypropyl or a hydroxybutyl group instead of hydroxyethyl group also yield prolonged circulation, however, not to the same extent as PHEA/G. The pharmacokinetic properties of PHEA-liposomes were independent of the lipid dose even at very low lipid doses of around 50 nmol per rat. PLP was successfully entrapped in PHEA-liposomes. These liposomes were shown to be stable in the circulation and equally effective in rat experimental arthritis as PLP encapsulated in PEG-liposomes. PHEA and PHEG are attractive alternative polymers for the design of LCL: their performance is similar to that of PEG-liposomes but they have the advantage of being biodegradable.     

Macrophage activation by PAF incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes

The phospholipid mediator, platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine), was recently reported to activate macrophage-monocyte cells as well as neutrophils or platelets. PAF was incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes, and their effect on guinea pig peritoneal macrophages was examined. PAF incorporated into liposomes was found to activate macrophages much more potently than PAF in the free form, whereas the effect of PAF in liposome on platelets was weaker than that of PAF in the free form. A large difference between PAF in liposomes and PAF in the free form was observed in the rate of degradation of PAF during incubation with macrophages. This rapid degradation of PAF in the free form may partially explain the poor activation by PAF of macrophages.     

Temperature-sensitive poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate)-coated liposomes for triggered contents release

We prepared thermosensitive poly( N-(2-hydroxypropyl)methacrylamide mono/dilactate) (pHPMA mono/dilactate) polymer and studied temperature-triggered contents release from polymer-coated liposomes. HPMA mono/dilactate polymer was synthesized with a cholesterol anchor suitable for incorporation in the liposomal bilayers and with a cloud point (CP) temperature of the polymer slightly above normal body temperature (42 degrees C). Dynamic light scattering (DLS) measurements showed that whereas the size of noncoated liposomes remained stable upon raising the temperature from 25 to 46 degrees C, polymer-coated liposomes aggregated around 43 degrees C. Also, noncoated liposomes loaded with calcein showed hardly any leakage of the fluorescent marker when heated to 46 degrees C. However, polymer-coated liposomes showed a high degree of temperature-triggered calcein release above the CP of the polymer. Likely, liposome aggregation and bilayer destabilization are triggered because of the precipitation of the thermosensitive polymer above its CP onto the liposomal bilayers, followed by permeabilization of the liposomal membrane. This study demonstrates that liposomes surface-modified with HPMA mono/dilactate copolymer are attractive systems for achieving temperature-triggered contents release.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

A comparison of negatively and positively charged liposomes containing entrapped polynosinic · polycytidylic acid for interferon induction in mice

Intravenous injection of negatively charged liposomes containing entrapped poly(I) · poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I) · poly(C). The response did not persist over an extended time period as was observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [¹⁴C]poly(I) · poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7–9% of the dose compared to 4–6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [¹⁴C]poly(I) · poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight [¹⁴C]poly(I) · poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to the free polymer. Similarly, for spleen, seven times and three times as much polymeric [¹⁴C]poly(I) · poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [¹⁴C]poly(I) · poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [¹⁴C]poly(I) · poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.     

Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases

Four intracellular proteases partially purified from liver preferentially degraded the oxidatively modified (catalytically inactive) form of glutamine synthetase. One of the proteases was cathepsin D which is of lysosomal origin; the other three proteases were present in the cytosol. Two of these were calcium-dependent proteases with different calcium requirements. The low-calcium-requiring type (calpain I) accounted for most of the calcium-dependent activity of both mouse and rat liver. The calcium-independent cytosolic protease, referred to as the alkaline protease, has a molecular weight of 300,000 determined by gel filtration. Native glutamine synthetase was not significantly degraded by the cytosolic proteases at physiological pH, but oxidative modification of the enzyme caused a dramatic increase in its susceptibility to attack by these proteases. In contrast, trypsin and papain did degrade the native enzyme and the degradation of modified glutamine synthetase was only 2- to 4-fold more rapid. Adenylylation of glutamine synthetase had little effect on its susceptibility to proteolysis. Although major structural modifications such as dissociation, relaxation, and denaturation also increased the rate of degradation, the oxidative modification is a specific type of covalent modification which could occur in vivo. Oxidative modification can be catalyzed by a variety of mixed function oxidase systems present within cells and causes inactivation of a number of enzymes. Moreover, the presence of cytosolic proteases which recognize the oxidized form of glutamine synthetase suggests that oxidative modification may be involved in intracellular protein turnover.     

Antimicrobial-modified sulfite pulps prepared by in situ copolymerization

Grafting guanidine polymer (PHGH) onto cellulose fibers was conducted via in situ free-radical polymerization using ceric ammonium nitrate (CAN) as an initiator. The optimum reaction conditions were obtained, under which the grafting percentage and the grafting efficiency reached over 20% and 50%, respectively. Atomic force microscopy (AFM) images revealed that the grafted polymer tended to form grains with diameters ranging from 60 to 200 nm. AFM also enabled us to identify the location of the grafts on the surfaces of cellulose fibers by the measurements of the adhesion and attraction forces between a colloid probe and the samples. The cellulose fibers were rendered antimicrobial in the presence of 1.0% (wt) grafted polymer, and an excellent antimicrobial activity (over 99% inhibition) toward Escherichia coli was achieved. The AFM results also demonstrated that the antimicrobial mechanism of PHGH is to destroy the membrane of the cells.     

RGD peptides grafting onto poly(ethylene terephthalate) with well controlled densities

The aim of this study was to graft RGD peptides with well controlled densities onto poly(ethylene terephthalate) (PET) film surfaces. Biomimetic modifications were performed by means of a four-step reaction procedure: surface modification in order to create -COOH groups onto polymer surface, coupling agent grafting and finally immobilization of peptides. The originality of this work is to evaluate several grafted densities peptides. Toluidine blue and high-resolution mu-imager (using [(3)H]-Lys) were used to evaluate densities. Moreover, mu-imager has exhibited the stability of peptides grafted onto the surface when treated under harsh conditions. Benefits of the as-proposed method were related to the different concentrations of peptides grafted onto the surface as well as the capacity of RGD peptide to interact with integrin receptors.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.     

Stabilization of retinol through incorporation into liposomes

Chemical and photochemical processes during storage and preparation rapidly degrade retinol, the most active form of vitamin A. Therefore, the efficacy of incorporation into liposomes in order to modulate the kinetics of retinol degradation was investigated. Retinol was readily incorporated into multilamellar liposomes that were prepared from soybean phosphatidylcholine; the extent of the incorporation was 98.14 +/- 0.93% at pH 9.0 at a ratio of 0.01 : 1 (wt : wt) retinol : phospholipid. It was only marginally lower at higher retinol concentrations. The pH of the hydration buffer had a small effect. The incorporation efficiency ranged from 99.25 +/- 0.47% at pH 3 to 97.45 +/- 1.13% at pH 11. The time course of the retinol degradation in the aqueous solution in liposomes was compared to that of free retinol and free retinol with alpha-tocopherol under a variety of conditions of pH (3, 7, and 11), temperature (4, 25, 37, and 50 degrees ), and light exposure (dark, visible, and UV). The retinol that was incorporated into the liposomes degraded significantly slower than the free retinol or retinol with alpha-tocopherol at pH 7 and 11. At pH 3, where the free retinol degrades rapidly, the degradation kinetics were similar in liposomes and the presence of alpha-tocopherol. At pH 7.0 and 4 degrees in the light, for example, free aqueous retinol was completely degraded within 2 days, while only 20% of the retinol in the liposomes were degraded after 8 days. In general, the protective effect of the liposome incorporation was greater at low temperatures, at neutral and high pH, and in the dark. The results suggest that protection is greater in the solid, gel phase than in the fluid liquid crystalline phase lipids. These results indicate that the incorporation into liposomes can extend the shelf-life of retinol under a variety of conditions of temperature, pH, and ambient light conditions.     

fMLP Receptor Stimulated Activation of Macrophage: Its Effect on Killing of Intracellular Leishmania donovani

The fMLP receptor of peritoneal macrophages stimulated by fMLP graftedliposomes as ligand, was analysed and compared with respective controlsfor its ability to promote killing of intracellular Leishmaniaparasites. fMLP grafted liposomes show greater efficacy in killingintracellular L. donovani (MHOM/IN/1983/AG83) parasites in a timedependent manner than free fMLP. fMLP grafted liposomes also releasemore active oxygen intermediates and reactive nitrogen intermediates(O^– ₂, H₂O₂, NO) than free fMLP. The key enzymes PKCand PTK for the respiratory burst and nitric oxide generation werefound to be important in this fMLP receptor mediated signaling processas the enzyme specific inhibitors viz. staurosporine, genistein andAG126 suppressed the leishmanicidal effect of fMLP graftedliposomes. The above findings suggest that the fMLP receptorof macrophages activates PKC and PTK mediated signalling thatis responsible for the intracellular parasite killing.     

Grafting of oligosaccharides onto synthetic polymer colloids

A new method to form colloidally stable oligosaccharide-grafted synthetic polymer particles has been developed. The oligosaccharides, of weight-average degree of polymerization approximately 38, were obtained by enzymatic debranching of amylopectin. Through the use of a cerium(IV)-based redox initiation process, oligosaccharide chains are grafted onto a synthetic polymer colloid comprising electrostatically stabilized poly(methyl methacrylate) or polystyrene latex particles swollen with methyl methacrylate monomer. Ce(IV) creates a radical species on these oligosaccharides, which then propagates, initially with aqueous-phase monomer, then with the methyl methacrylate monomer inside the particles. Ultracentrifugation, NMR, and total starch analyses together prove that the grafting process has occurred, with at least 7.7 wt % starch grafted and a grafting efficiency of 33%. The surfactant used in latex preparation was removed by dialysis, resulting in particles colloidally stabilized with only linear starch as a steric stabilizer. The debranched starch that comprises these oligosaccharides is found to be a remarkably effective colloidal stabilizer, albeit at low electrolyte concentration, stabilizing particles with very sparse surface coverage.     

Hybrid rigid/soft and biologic/synthetic materials: polymers grafted onto cellulose microcrystals

Rigid nanoscale polymer rods were prepared by grafting preformed amine-terminated poly(styrene) and poly(tert-butyl acrylate) onto oxidized cellulose microcrystals. Low polydispersity polymers, grown using atom transfer radical polymerization, were characterized and purified prior to cellulose attachment. Oxidation of the cellulose microcrystal led to the formation of carboxylic acids on the surface of the microcrystals. Covalent attachment of the polymers onto the cellulose microcrystals was achieved via a carbodiimide-mediated amidation reaction. The length and diameter of the polymer-cellulose composites increased upon surface modification. Typically, polymer-cellulose composites are synthesized by a grafting-from method because it can be difficult to obtain sufficient graft density using a grafting-to preparation. However, the composites reported here comprised 60-64% grafted polymer by mass. This degree of grafting-to allowed the composite to form stable suspensions in organic solvents.     

Enhanced stability of immobilized keratinolytic protease on electrospun nanofibers

Abstract

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100–150?nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72?hr.     

Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.