Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.     

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.     

Synthesis and characterization of triblock copolymers of methoxy poly(ethylene glycol) and poly(propylene fumarate)

Amphiphilic block copolymers were synthesized by transesterification of hydrophilic methoxy poly(ethylene glycol) (mPEG) and hydrophobic poly(propylene fumarate) (PPF) and characterized. Four block copolymers were synthesized with a 2:1 mPEG:PPF molar ratio and mPEGs of molecular weights 570, 800, 1960, and 5190 and PPF of molecular weight 1570 as determined by NMR. The copolymers synthesized with mPEG of molecular weights 570 and 800 had 1.9 and 1.8 mPEG blocks per copolymer, respectively, as measured by NMR, representing an ABA-type block copolymer. The number of mPEG blocks of the copolymer decreased with increasing mPEG block length to as low as 1.5 mPEG blocks for copolymer synthesized with mPEG of molecular weight 5190. At a concentration range of 5-25 wt % in phosphate-buffered saline, copolymers synthesized with mPEG molecular weights of 570 and 800 possessed lower critical solution temperatures (LCST) between 40 and 45 degrees C and between 55 and 60 degrees C, respectively. Aqueous solutions of copolymer synthesized with mPEG 570 and 800 also experienced thermoreversible gelation. The sol-gel transition temperature was dependent on the sodium chloride concentration as well as the mPEG block length. The copolymer synthesized from mPEG 570 had a transition temperature between 40 and 20 degrees C with salt concentrations between 1 and 10 wt %, while the sol-gel transition temperatures of the copolymer synthesized from mPEG molecular weight 800 were higher in the range 75-30 degrees C with salt concentrations between 1 and 15 wt %. These novel thermoreversible copolymers are the first biodegradable copolymers with unsaturated double bonds along their macromolecular chain that can undergo both physical and chemical gelation and hold great promise for drug delivery and tissue engineering applications.     

Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.     

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.     

Block copolymerization of vinyl compounds by the terminal-group activation of poly(α-amino acids) and the characterization of block copolymers

The terminal amino groups of polysarcosine, poly(γ-benzyl l-glutamate), and poly(ε-benzyloxycarbonyl-l-lysine) were haloacetylated. The mixture of the terminally haloacetylated poly(α-amino acid) and styrene or methyl methacrylate was photoirradiated in the presence of Mn₂(CO)₁₀, or heated with Mo(CO)₆, yielding A-B-A-type block copolymers consisting of poly(α-amino acid) (the A component) and vinyl polymer (the B component). The block copolymers were characterized, and the present investigation revealed that the thermally initiated polymerization of vinyl compounds by the trichloroacetyl poly(α-amino acid)/Mo(CO)₆ system was the most suitable for the synthesis of the α-amino acid/vinyl compound block copolymers. The A-B-A type block copolymers showed higher antithrombogenicity than the corresponding homopolymers. In particular, a film of the A-B-A-type block copolymer of poly[Glu(OBzl)] and polystyrene possessed a microphase-separated structure and did not induce a conformational change of fibrinogen adsorbed, leading to a high antithrombogenicity.     

Temperature-dependent aggregation of pH-sensitive phosphatidyl ethanolamine-oleic acid-cholesterol liposomes as measured by fluorescent spectroscopy.

pH-sensitive liposomes made of phosphatidyl ethanolamine-oleic acid-cholesterol (4:2:4 molar ratio) at neutral pH values aggregate at approximately 40 degrees C. The aggregation is accompanied by liposome destabilization and by the release of intraliposomal fluorescent marker (calcein). Both aggregation and calcein leakage start at the temperature corresponding to the lipid phase transition into hexagonal phase. In the system studied the phase transition temperature interval is within 45 to 55 degrees C as estimated with the use of the fluorescent probe 1,6-diphenylhexatriene. The presence of cell cultivation medium RPMI 1640 decreases liposome aggregation temperature. The addition of 10% serum to the system decreases the temperature at which the aggregation proceeds still further. The conclusion that serum-free media should be used for cell experiments involving pH-sensitive liposomes is made.     

Serum-induced leakage of liposome contents

Efflux of contents from small unilamellar vesicles of various compositions, containing a highly quenched fluorescent compound (calcein, 175 mM) was determined as a function of temperature in the presence and absence of human serum. Efflux of calcein from the liposomes was monitored as an increase in fluorescence as calcein became dequenched upon release from the liposomes. The presence of serum significantly increased liposome leakage in all cases. Incorporation of increasing molar ratios of cholesterol into liposomes reduced leakage of calcein from liposomes incubated with buffer and with serum. Leakage was significantly faster from liposomes with an osmotic gradient across the membrane (higher inside) than from equiosmolar liposomes. The leakage of [¹⁴C]sucrose from egg lecithin liposomes at 37°C was also dramatically increased in the presence of serum.     

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.     

Sulfonamide-based pH- and temperature-sensitive biodegradable block copolymer hydrogels

Novel pH- and temperature-sensitive biodegradable poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol) (PCLA-PEG) block copolymers were synthesized with oligomeric sulfamethazine (OSM) end groups (OSM-PCLA-PEG-PCLA-OSM). Aqueous solutions of these block copolymers have shown sol-gel transition behavior upon both temperature and pH changes under physiological conditions (37 degrees C, pH 7.4). The sol-gel transition of these block copolymer solutions was fine-tuned by controlling the PEG length, the hydrophobic to hydrophilic block ratio (PCLA/PEG), and the molecular weight of the sulfamethazine oligomer. Since changes in temperature do not induce gel formation in this pH- and temperature-sensitive block copolymer solution, this hydrogel can be employed as an injectable carrier using a long guide catheter into the body. In addition, the pH of the block copolymer solution showed no change following PCLA degradation over 1 month, and no indication of gel collapse was observed on addition of buffer solution. As such, these properties make the OSM-PCLA-PEG-PCLA-OSM hydrogel an ideal candidate for use as an injectable carrier for certain protein-based drugs known to denature in low-pH environments.     

Block versus random amphiphilic copolymers as antibacterial agents

We examined the antibacterial and hemolytic activities in a series of amphiphilic block and random copolymers of poly(vinyl ether) derivatives prepared by base-assisting living cationic polymerization. Block and random amphiphilic copolymers with similar monomer compositions showed the same level of activity against Escherichia coli . However, the block copolymers are much less hemolytic compared to the highly hemolytic random copolymers. These results indicate that the amphiphilic copolymer structure is a key determinant of activity. Furthermore, the block copolymers induced dye leakage from lipid vesicles consisting of E. coli -type lipids, but not mammalian lipids, while the random copolymers disrupted both types of vesicles. In addition, both copolymers displayed bactericidal and hemolytic activities at concentrations 1 or 2 orders of magnitude lower than their critical (intermolecular) aggregation concentrations (CACs), as determined by light scattering measurements. This suggests that polymer aggregation or macromolecular assembly is not a requisite for the antibacterial activity and selectivity against bacteria over human red blood cells (RBCs). We speculate that different single-chain conformations between the block and random copolymers play an important role in the antibacterial action and underlying antibacterial mechanisms.     

Evaluation of acrylate-based block copolymers prepared by atom transfer radical polymerization as matrices for paclitaxel delivery from coronary stents

Acrylate-based block copolymers, synthesized by atom transfer radical polymerization (ATRP) processes, were evaluated as drug delivery matrices for the controlled release of paclitaxel from coronary stents. The polymers were multiblock copolymers consisting of poly(butyl acrylate) or poly(lauryl acrylate) soft blocks and hard blocks composed of poly(methyl methacrylate), poly(isobornyl acrylate), or poly(styrene) homo- or copolymers. Depending on the ratio of hard to soft blocks in the copolymers, coating formulations were produced that possessed variable elastomeric properties, resulting in stent coatings that maintained their integrity when assessed by scanning electron microscopy (SEM) imaging of overexpanded stents. In vitro paclitaxel release kinetics from coronary stents coated with these copolymers typically showed an early burst followed by sustained release behavior, which permitted the elution of the majority of the paclitaxel over a 10-day time period. It was determined that neither the nature of the polyacrylate (n-butyl or lauryl) nor that of the hard block appeared to affect the release kinetics of paclitaxel at a loading of 25% drug by weight, whereas some effects were observed at lower drug loading levels. Differential scanning calorimetry (DSC) analysis indicated that the paclitaxel was at least partially miscible with the poly(n-butyl acrylate) phase of those block copolymers. The copolymers were also evaluated for sterilization stability by exposing both the copolymer alone and copolymer/paclitaxel coated stents to e-beam radiation at doses of 1-3 times the nominal dose used for medical device sterilization (25 kGy). It was found that the copolymers containing blocks bearing quaternary carbons within the polymer backbone were less stable to the radiation and showed a decrease in molecular weight as determined by gel-permeation chromatography. Conversely, those without quaternary carbons showed no significant change in molecular weight when exposed to 3 times the standard radiation dose. There was no significant change in drug release profile from any of the acrylate-based copolymers after exposure to 75 kGy of e-beam radiation, and this was attributed to the inherent radiation stability of the poly(n-butyl acrylate) center block.     

HER2-specific affibody-conjugated thermosensitive liposomes (Affisomes) for improved delivery of anticancer agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z(HER2:342)-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as "Affisomes" were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80-100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 degrees C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16-20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were approximately 70% at a protein-lipid ratio of 20 microg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, lambdaex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41 degrees C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm approximately 0 degrees C) under similar conditions released only 5-10% calcein at 41 degrees C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.     

In vitro characterization of a novel polymeric-based pH-sensitive liposome system

This study demonstrates rapid and pH-sensitive release of a highly water-soluble fluorescent aqueous content marker, pyranine, from egg phosphatidylcholine liposomes following incorporation of N-isopropylacrylamide (NIPA) copolymers in liposomal membranes. The pH-sensitivity of this system correlates with the precipitation of the copolymers at acidic pH. In vitro release can be significantly improved by increasing the percentage of anchor in the copolymer and thus favoring its binding to the liposomal bilayer. In the case of liposomes containing a poly(ethylene glycol)-phospholipid conjugate, the insertion of the pH-sensitive copolymer in the liposomal membrane appears to be sterically inhibited. Dye release from these formulations at acidic pH can still be achieved by varying the anchor molar ratio and/or molecular mass of the polymers or by including the latter during the liposome preparation procedure. Removal of unbound polymer results in decreased leakage only when the copolymer is inserted by incubation with preformed liposomes, but can be overcome by preparing liposomes in the presence of polymer. Aqueous content and lipid mixing assays suggest contents release can occur without membrane fusion. The results of this study indicate that the addition of pH-sensitive copolymers of NIPA represents promising strategy for improving liposomal drug delivery.     

Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique

Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.     

Thiol-yne and thiol-ene "click" chemistry as a tool for a variety of platinum drug delivery carriers, from statistical copolymers to crosslinked micelles

Statistical and block copolymers based on poly(2-hydroxyethyl methacrylate) (PHEMA) and poly[oligo(ethylene glycol) methylether methacrylate] (POEGMEMA) were modified with 4-pentenoic anhydride or 4-oxo-4-(prop-2-ynyloxy)butanoic anhydride to generate polymers with pendant vinyl or acetylene, respectively. Subsequent thiol-ene or thiol-yne reaction with thioglycolic acid or 2-mercaptosuccinic acid leads to polymers with carboxylate functionalities, which were conjugated with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) to generate a drug carrier for Pt-drugs. Only the polymers modified with 2-mercaptosuccinic acid resulted in the formation of soluble well-defined polymers with gel formation being prevented. Due to the hydrophobicity of the drug, the block copolymers took on amphiphilic character leading to micelle formation. The micelles were in addition crosslinked to further stabilize their structure. Pt-containing statistical copolymer, micelles, and crosslinked micelles were then tested regarding their cellular uptake by the A549 lung cancer cell line to show a superior uptake of crosslinked micelles. However, due to the better Pt release of the statistical copolymer, the highest cytotoxicity was observed with this type of polymer architecture.     

Small, but not large, unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by human plasma

Small unilamellar liposomes, composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA), prepared by sonication, were incubated in the presence of human plasma at 37 degrees C. The release of entrapped calcein after 8-h incubation was about 15% in plasma, compared with about 70% in phosphate-buffered saline under the same conditions. In contrast, dioleoylphosphatidylcholine (DOPC)/OA liposomes under the same conditions release about 70% in plasma and only 10% in PBS. Total release of calcein from the DOPE/OA liposomes was observed in a PBS solution containing bovine serum albumin, and the release was completely blocked by preincubation of the liposomes with plasma. These results indicate that the unstable DOPE/OA liposomes are stabilized by incubation with plasma. The stabilization process was very fast, being completed within 1 min. Only relatively small liposomes (d less than or equal to 200 nm) were completely stabilized by plasma; larger liposomes were progressively less stabilizable. SDS-polyacrylamide gel electrophoresis of liposomes which had been incubated with plasma and then washed indicated that several proteins were tightly associated with liposomes. Using liposomes containing [14C]OA, it was found that about 70% of the original OA was extracted after 1-h incubation with human plasma at 37 degrees C. Thin-layer chromatographic analysis of the plasma-treated liposomes showed the presence of the plasma lipids in the liposomes. These results suggest that liposomes composed of DOPE/OA are stabilized by protein and/or lipid components from human plasma and that the composition of the liposomes is altered. The mechanism of stabilization is discussed in terms of the surface pressure of small vesicles with a high degree of curvature.(ABSTRACT TRUNCATED AT 250 WORDS)