Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

‘Stealth’ liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (< 1 μmol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 μmol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Enzymatic degradation of liposome-grafted poly(hydroxyethyl L-glutamine)

Liposomes coated with poly(hydroxyethyl L-glutamine) (PHEG) show prolonged circulation times and biodistribution patterns comparable to PEG-coated liposomes. While PEG is a nondegradable polymer, PHEG is expected to be hydrolyzed by proteases. In this study the enzymatic degradability of PHEG both in its free form and grafted onto liposomes was investigated, using the proteases papain, pronase E, and cathepsin B. Enzymatic action was monitored with a ninhydrin assay, which quantifies amine groups formed due to hydrolysis of amide bonds, and the degradation products were characterized by MALDI-ToF mass spectrometry. PHEG, both in its free form and when grafted onto liposomes, showed degradation into low molecular weight peptides by the enzymes. Thus, we present a polymer-coated long-circulating liposome with an enzymatically degradable coating polymer, avoiding the risk of cellular accumulation.     

1H NMR spectroscopy as a tool for determining the composition of poly(hydroxyethyl-L-asparagine)-coated liposomes

Liposomes coated with the poly(amino acid) poly(hydroxyethyl-L-asparagine) (PHEA) show long-circulation properties comparable to the frequently used PEG-liposomes. The pharmacokinetic characteristics of long-circulating liposomes are dependent on the density of the shielding polymer on the liposome surface. Therefore, it is necessary to know the exact composition of the liposomes including the amount of coating polymer present on the liposome surface. In this study, a 1H NMR method to establish the composition of liposomes coated with PHEA was developed and validated.     

Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.     

Effect of Repeated Intravenous Administration on the Circulation Kinetics of Poly(Ethyleneglycol)-Liposomes in Rats

Abstract

The use of sterically stabilized poly(ethyleneglycol)-coated liposomes (PEG-liposomes) is becoming increasingly important and several preparations based on long-circulating liposomes are already commercially available. From a clinical point of view, it is of importance to study the effect of multiple i.v. administration of PEG-liposomes on their pharmacokinetic behavior. Sterically stabilized liposomes were obtained by incorporation of PEG conjugated to distearoylethanolamine (DSPE) into the liposomal bilayers. Rats received 4 i.v. injections of small (0.12 um) PEG-liposomes at 24 or 48 h dosing intervals. Blood levels of liposomal label were determined at several time-points after injection. Our findings demonstrate that, under the chosen conditions, i.v. injection of PEG-liposomes has no effect on the blood circulation kinetics of subsequent doses of similar liposomes given at 24 or 48 h dosing intervals. These findings suggest that PEG-liposomes are suitable as drug carriers for diagnostic and therapeutic applications that require repeated i.v. injections.     

Preclinical and Clinical Evidence for Efficient Opsonization of Poly(Ethyleneglycol)-Liposomes

Abstract

The clinical use of poly(ethyleneglycol)-coated liposomes (PEG-liposome) is becoming increasingly important. Here we summarize our recent preclinical and clinical evidence pointing to the possibility of efficient opsonization of intravenously injected PEG-liposomes under certain circumstances. Drastically enhanced blood clearance of PEG-liposomes was observed at low lipid dose and in case of multiple injection schemes. Further clinical evaluation of the observed “anti-stealth” phenomenon is indicated.     

Photoinitiated destabilization of sterically stabilized liposomes

A considerable effort has been devoted to the development of liposomes for the transport and buffering of drugs in the body. Several research groups have reported the increased localization of sterically stabilized liposomes (PEG-liposomes) at tumor sites. If PEG-liposomes are to be effective carriers of therapeutic agents, their drug permeability must be sufficiently low that little passive release occurs during the circulation time of the PEG-liposomes. However, once PEG-liposomes reach tumor sites, it may be desirable to accelerate the release of the encapsulated drug. The use of light to stimulate the release of encapsulated compounds from liposomes is attractive, because it is possible to control the spatial and temporal delivery of the radiation. PEG-liposomes composed in part of the photosensitive lipid, bis-SorbPC, can be prepared in a manner that effectively encapsulates water soluble compounds, yet releases them upon exposure to ultraviolet light in the presence of oxygen. The observed increase in liposome permeability is about 200-fold at high photoconversion of the monomeric bis-SorbPC. The increase in permeability is dependent on the extent of photolysis, but independent of both the charge on the PEG-lipid and the mole fraction of PEG-lipid included in the liposome. Therefore the photoinitiated destabilization of these PEG-liposomes is not a consequence of micellization of the PEG-lipid, but probably due to the formation of defects in the bilayer during crosslinking of the bis-SorbPC. The photoinduced increase in liposome permeability is great enough to make it possible to release therapeutic agents from PEG-liposomes at specific sites in a manner of tens of minutes to hours.     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.     

Steric stabilization of lipid/polymer particle assemblies by poly(ethylene glycol)-lipids

Biocompatible and biodegradable assemblies consisting of spherical particles coated with lipid layers were prepared from sub-micrometer poly(lactic acid) particles and lipid mixtures composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-3-trimethylammonium-propane. These original colloidal assemblies, named LipoParticles, are of a great interest in biotechnology and biomedicine. Nevertheless, a major limitation of their use is their poor colloidal stability toward ionic strength. Indeed, electrostatic repulsions failed to stabilize LipoParticles in aqueous solutions containing more than 10 mM NaCl. By analogy with the extensive use of poly(ethylene glycol) (PEG)-lipid conjugates to improve the circulation lifetime of liposomes in vivo, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] with various PEG chain lengths was added to the lipid formulation. Here, we show that LipoParticle stabilization was enhanced at least up to 150 mM NaCl (for more than 1 year at 4 degrees C). To determine the structure of PEG-modified LipoParticles as a function of the PEG chain length and the PEG-lipid fraction in the lipid formulation, a thorough physicochemical characterization was carried out by means of many techniques including quasi-elastic light scattering, zeta potential measurements, transmission electron microscopy, 1H NMR spectroscopy, and small-angle X-ray scattering. Finally, an attempt was made to link the resulting structural data to the colloidal behavior of PEG-modified LipoParticles.     

Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis

The aim of this study was to investigate whether long-circulating liposomes can improve the anti-inflammatory activity of superoxide dismutase (SOD). Small-sized poly(ethyleneglycol) (PEG)-liposomes containing SOD were prepared via different preparation protocols and characterized in terms of encapsulation efficiency (EE), size, enzymatic activity and protein structure, to establish conditions where high EE can be combined with preservation of enzyme activity and structure. It was observed that structural information from circular dichroism analyses does not correlate with data on enzyme activity. SOD-containing PEG-liposomes prepared by the dehydration-rehydration method appeared to represent the most attractive formulation for in vivo evaluation. The therapeutic potential of selected SOD-containing PEG-liposomes was established and compared with SOD entrapped in stearylamine (SA)-liposomes and 'free' SOD upon intravenous (i.v.) injection in an arthritic rat model. Both small PEG-liposomes and SA-liposomes showed a superior therapeutic activity compared to 'free' SOD, with PEG-liposomes inducing stronger anti-inflammatory effects than SA-liposomes.     

Fully degradable hydrophilic polyals for protein modification

Modification of proteins with hydrophilic polymers is an effective strategy for regulation of protein pharmacokinetics. However, conjugates of slowly or non-biodegradable materials, such as poly(ethylene glycol), are known to cause long-lasting cell vacuolization, in particular in renal epithelium. Conjugates of more degradable polymers, e.g., polysaccharides, have a significant risk of immunotoxicity. Polymers that combine complete degradability, long circulation in vivo, and low immuno and chemical toxicity would be most beneficial as protein conjugate components. This study explores new fully biodegradable hydrophilic polymers, hydrophilic polyals. They are nontoxic, stable at physiological conditions, and undergo proton-catalyzed hydrolysis at lysosomal pH. The model enzyme-polyal conjugates were prepared with 61-98% yield using conventional and novel conjugation techniques and retained 90-95% of specific activity. The model conjugates showed a significant prolongation of protein circulation in rodents, with a 5-fold reduction in the renal accumulation. The data suggests that hydrophilic polyals may be useful in designing protein conjugates with improved properties.     

Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis

The aim of this study was to investigate whether long-circulating liposomes can improve the anti-inflammatory activity of superoxide dismutase (SOD). Small-sized poly(ethyleneglycol) (PEG)-liposomes containing SOD were prepared via different preparation protocols and characterized in terms of encapsulation efficiency (EE), size, enzymatic activity and protein structure, to establish conditions where high EE can be combined with preservation of enzyme activity and structure. It was observed that structural information from circular dichroism analyses does not correlate with data on enzyme activity. SOD-containing PEG-liposomes prepared by the dehydration-rehydration method appeared to represent the most attractive formulation for in vivo evaluation. The therapeutic potential of selected SOD-containing PEG-liposomes was established and compared with SOD entrapped in stearylamine (SA)-liposomes and ‘free’ SOD upon intravenous (i.v.) injection in an arthritic rat model. Both small PEG-liposomes and SA-liposomes showed a superior therapeutic activity compared to ‘free’ SOD, with PEG-liposomes inducing stronger anti-inflammatory effects than SA-liposomes.     

POLY(HPMA)-COATED LIPOSOMES DEMONSTRATE PROLONGED CIRCULATION IN MICE

Surface modification of liposomes with amphiphilic flexible polymers significantly prolongs their circulation time in blood and reduces uptake by cells of the reticuloendothelial system (RES). Several polymers have already been shown to provide steric protection to liposomes. Still more polymers are expected to serve this purpose, thus broadening the variability of properties of long-circulating liposomes. Poly[N-(2-hydroxypropyl)methacrylamide] (poly (HPMA)) seems to have some properties similar to polyethylene glycol (PEG), the most widely used polymer in liposome surface modification, including flexibility, hydrophilicity and low immunogenicity, which suggest that it may also function as an efficient steric protector of liposomes. Semitelechelic poly(HPMA) with single- or double-oleic acid hydrophobic terminus were synthesized and incorporated into the surface of liposomes composed of phosphatidylcholine and cholesterol. These poly(HPMA)-modified liposomes provided strong steric protection for liposomes, increasing their circulation time and decreasing liver accumulation in experimental mice. Poly(HPMA)-modified liposomes may become a useful addition to a family of long-circulating liposomes with potential to be used as a drug delivery system.     

Synthesis, characterization, and degradation behavior of amphiphilic poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(epsilon-caprolactone)

A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(epsilon-caprolactone) (PCL) sequences onto a water-soluble poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) backbone. The graft copolymers were prepared through the ring-opening polymerization of epsilon-caprolactone (CL) initiated by the macroinitiator PHEA with pendant hydroxyl groups without adding any catalyst. By controlling the feed ratio of the macroinitiator to the monomer, the copolymers with different branch lengths and properties can be obtained. The successful grafting of PCL sequences onto the PHEA backbone was verified by FTIR, 1H NMR, and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The hydrolytic degradation and enzymatic degradation of these graft copolymers were investigated. The results show the hydrolytic degradation rate increases with increasing content of hydrophilic PHEA backbone. While the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. In situ observation of the degradation under polarizing light microscope (PLM) demonstrates the different degradation rates of different regions in the polymer samples.     

Transport of Asparagine by Rat Brain Synaptosomes: An Approach to Evaluate Glutamine Accumulation

Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 microM and a Vmax value of 3.7 nmol/mg of protein/min at 28 degrees C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas alpha-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparagine was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20-30, and this value was reduced to 5-6 by withdrawal of sodium or addition of high [KCI]. A plot of log [Na+]o/[Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to I, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [Gln]i maintained by the brain in vivo.     

Self-exploding lipid-coated microgels

Self-exploding microparticles show potential for advanced delivery of certain therapeutics. This study evaluates (1) whether degrading biodegradable dextran hydroxyethyl methacrylate (dex-HEMA) microgels can be coated by a lipid membrane and (2) whether the surrounding membrane can be ruptured by the increasing swelling pressure of the degrading microgel. We found that adsorption of charged liposomes to oppositely charged dex-HEMA microgels provides efficient coating of the microgels, whereby microparticles with a "core-shell" structure were clearly obtained. Especially, we could confirm experimentally that the swelling pressure increase of degrading dex-HEMA microgels can destroy the lipid membrane surrounding the microgels.     

The Structural State and Form of Free and Biopolymer-Encapsulated Phosphatidylcholine Liposomes in the Absence and Presence of Natural Plant Antioxidants

Electron spin resonance (ESR) and atomic force microscopy (AFM) were used to study liposomes that were prepared from soybean phosphatidylcholine (PC); they incorporated plant antioxidants (ginger, allspice, and black-pepper extracts; clove oil; etc.) that were encapsulated in biopolymers (sodium caseinate or sodium caseinate–maltodextrin covalent conjugates). Plant antioxidants were shown to cause a 15–25% decrease in the microviscosity of deep-lying regions of the liposome lipid bilayer by ESR with a 16-doxylstearic acid spin probe. A ginger extract exerted the greatest effect (24%). Sodium caseinate and its covalent conjugates with maltodextrins (dextrose equivalents (DEs) 2 and 10) increased the microviscosity by 30–35% as compared with free and antioxidant-incorporating liposomes. AFM showed that antioxidants increased the cross-sectional area and volume of liposomes and that the polymers made liposomes denser and their structure more compact.     

The stimulus-secretion coupling of amino acid-induced insulin release

Summary Aminooxyacetate, an inhibitor of cytosolic transamination reactions, inhibited insulin release evoked by either 2-ketoisocaproate or L-leucine in rat pancreatic islets incubated in the presence of L-glutamine or L-asparagine. As a rule, aminooxyacetate also inhibited the oxidation of these nutrient secretagogues and impaired the respiratory response of the islets to the combinations of nutrients. However, the oxidative and secretory response to the combination of L-leucine and L-glutamine was less severely affected by aminooxyacetate than that evoked by the three other combinations of exogenous nutrients. These findings reinforce the view that the stimulus-secretion coupling of insulin release in response to L-leucine and 2-ketoisocaproate in association with either L-glutamine or L-asparagine tightly depends on the oxidation of these nutrient secretagogues, on their effect upon O₂ uptake and, within limits, on the intracellular site of generation of reducing equivalents in the pancreatic islet cells.This paper is the 16th in a series.     

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.