Patterning poly(organophosphazenes) for selective cell adhesion applications

Five polyphosphazenes with different hydrophilicites were synthesized and screened in vitro. The purpose was to identify unique types of polymeric substrates that distinctly favored or markedly prevented cellular adhesion. The SK-N-BE(2c) human neuroblastoma cell line, utilized for its electrogenic responses, was used to test this differential adhesion. In particular, the objective was to specifically culture this cell line in a highly selective pattern. Each candidate polymer was cast into films and plated with neuroblastoma cells for 3 days. The polyphosphazene materials which showed negative cellular adhesive properties (-CAPs) were poly[bis(trifluoroethoxy)phosphazene] (TFE) and poly[bis(methoxyethoxyethoxy)phosphazene] (MEEP). The polyphosphazenes which showed positive cellular adhesive properties (+CAPs) were poly[(methoxyethoxyethoxy)(1.0)(carboxylatophenoxy)(1.0)phosphazene] (PMCPP), poly[(methoxyethoxyethoxy)(1.0)(cinnamyloxy)(1.0)phosphazene] (PMCP), and poly[(methoxyethoxyethoxy)(1.0)(p-methylphenoxy)(1.0)phosphazene] (PMMP). To test cellular selectivity, films of -CAP and +CAP were copatterned onto glass substrates. The micropatterned films were plated with SK-N-BE(2c) neuroblastoma cells for one week. The results showed that neuroblastoma cells adhere selectively (over 60%) to the +CAP microfeatures. We also showed that multiple properties can be achieved with a single material and that we can use TFE as both a -CAP and an insulation layer and PMCP as a conductive +CAP layer.     

Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

The critical micelle concentrations of 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 microM, respectively, in buffer solutions ([Tris] = 20 mM, [NaCl] = 140 mM, pH 7.4) at 37 degrees C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEG-DSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEG-DPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.     

Dual stimuli-responsive polypeptide prepared by thiol-ene click reaction of poly(l-cysteine) and N,N-dimethylaminoethyl acrylate

Stimuli-responsive polymers that can undergo conformational changes with external triggers have enabled themselves as smart materials for various utilizations, among which biodegradability is of particular importance to be engineered for biomedical application. In this study, a thermo and pH dual responsive polypeptide (N, N-dimethylaminoethyl acrylate-modified poly(l -cysteine)) (PLC-g-DMAEA) was prepared by the combination of N-carboxyanhydride ring-open polymerization and thiol-ene click chemistry. The biodegradable poly(l -cysteine) (PLC) with pendant thiol groups provided an easily clickable backbone for postmodification, which was demonstrated by reacting with a well-known monomer of N, N-dimethylaminoethyl acrylate (DMAEA) to achieve both temperature and pH responsiveness. The irreversible thermo-response of PLC-g-DMAEA could be attributed to the ordered β-sheets formed upon heating, leading to the trapped side groups with poor water accessibility. Moreover, this copolymer precipitated at pH ranging from 7.5 to 9.7, but protonation of tertiary amine groups (pH < 7.5) and salt forming of masked thiol groups (pH > 9.7) rendered it soluble in water. Our results revealed that a ready available vinyl monomer could be easily clicked onto the biodegradable PLC and its stimuli responsiveness would be reserved. Moreover, the primary and secondary structures of PLC might influence the conformation, thus leading to the unique responsive behavior of the resulted copolymer.     

Highly branched poly(N-isopropylacrylamide) for use in protein purification

Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragment directly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned to occur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueous media of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflected in the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition of Cu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added the polymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of the histidine-tagged breast cancer susceptibility protein his6-BRCA1. Cu(II) was found to be better suited to the formation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixtures generated very little purified protein. The polymers were synthesized using a previously reported variation of the reversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.     

Conformational change of poly(L-lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly(L-lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a conformational change of poly(L-lysine) from the random coil to beta-structure in 5 mM Tes, pH 7.0. The fraction of induced beta-structure (F beta) was determined via a procedure of curve fitting of the observed spectra to the reference spectra. F beta increased linearly with the molar ratio, r, of DLPA to lysine residues up to r congruent to 0.7, and reached a saturation value of 1 at r greater than 1. Within the range 0.7 less than or equal to r less than or equal to 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the beta-structure of poly(L-lysine) was also induced by mixed vesicles, the saturation value of F beta decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F beta with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.     

Conformational change of poly( -lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly( -lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a confomiational change Of poly( -lysine) from the random coil to β-structure in 5 mM Tes, pH 7.0. The fraction of induced β-structure (F_β) was determined via a procedure of curve fit the observed spectra to the reference spectra. F_β increased linearly with the molar ratio, r, of DLPA to lysine residues up to r 0.7, and reached a saturation value of 1 at r > 1. Within the range 0.7 r 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the β-structure Of poly -lysine) was also induced by mixed vesicles, the saturation value of F_β decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F_β with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Synthesis of poly(amidoamine) dendron-bearing lipids with poly(ethylene glycol) grafts and their use for stabilization of nonviral gene vectors

Recently, we developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl groups. These dendron-bearing lipids achieved efficient gene transfection of cells through synergetic action of the proton sponge effect and membrane fusion in combination with fusogenic lipid dioleoylphosphatidylethanolamine. Using those dendron-bearing lipids as a base material, we developed in this study a functional component of gene vectors that stabilizes lipoplexes by multiple PEG chains and promotes gene transfection through the proton sponge effect. We combined a poly(ethylene glycol) (PEG, 550 Da) graft to each of four chain ends of the G2 dendron-bearing lipid (P4-DL). An analogous molecule having single PEG graft was also synthesized using the G0 dendron-bearing lipid (P1-DL). Inclusion of P4-DL decreased the size of the G3 dendron-bearing lipid-based lipoplexes more efficiently than P1-DL. In addition, P4-DL-containing lipoplexes exhibited two-orders-higher transfection efficiency than P1-DL-containing lipoplexes with the same PEG graft density. These results indicate the superiority of multiple attachments of PEG graft chains to a lipid for heightened ability to increase colloidal stability of lipoplexes while retaining their transfection activity. The lipoplexes stabilized by P4-DL were small, around 250 nm, and achieved efficient transfection in the presence of serum. Therefore, P4-DL and its analogues will form the basis for production of efficient nonviral vectors for in vivo use.     

Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy

Summary Specimens infused with or suspended in a mixture of 10–30% poly(vinylpyrrolidone) and 2.07–1.61m sucrose can often be more easily frozen-sectioned than those infused with sucrose alone. The pH of such a mixture can be efficiently adjusted to neutrality by using Na₂CO₃. Use of poly(vinylpyrrolidone) causes little or no increase in the background level of immunolabelling. Adsorption staining of ultrathin frozen sections with a mixture of uranyl acetate and poly(vinyl alcohol), i.e. a simple thin-embedding of the sections in such a mixture, produces positive staining effects that are often enough to delineate structures of many organelles. When OsO₄-treated frozen sections are stained with uranyl acetate and further adsorption-stained with a mixture of lead citrate and poly(vinyl alcohol), the overall staining effects are similar to those observed in double-stained conventional sections.A large portion of the findings was reported as a part of the author's presentation in the 11th International Congress on Electron Microscopy, held in Kyoto, Japan, in 1986.     

Novel stimuli-responsive micelle self-assembled from Y-shaped P(UA-Y-NIPAAm) copolymer for drug delivery

A new amphiphilic Y-shaped copolymer, comprised of hydrophobic poly(undecylenic acid) (PUA) and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm), was designed and synthesized. A cytotoxicity study revealed that P(UA-Y-NIPAAm) copolymers did not exhibit apparent inhibition impact on the proliferation of cells when the concentration of the copolymer was below 1000 mg/L. Characterization demonstrated that the P(UA-Y-NIPAAm) copolymer is thermosensitive with a lower critical solution temperature (LCST) of 31 degrees C. In water, the P(UA-Y-NIPAAm) copolymer would self-assemble into micelles with a critical micelle concentration (CMC) of 20 mg/L. Self-assembled P(UA-Y-NIPAAm) micelles exhibited a nanospherical morphology of 40 to approximately 80 nm in size. The controlled drug release behavior of the P(UA-Y-NIPAAm) micelles was further investigated, and self-assembled micelles exhibited improved properties in controlled drug release.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

L-Phe end-capped poly(L-lactide) as macroinitiator for the synthesis of poly(L-lactide)-B-poly(L-lysine) block copolymer

A poly(L-lactide)-b-poly(Nepsilon-(Z)-L-lysine) (PLLA-b-PZLys) block copolymer was synthesized through the ring-opening polymerization of Nepsilon-(Z)-lysine-N-carboxyanhydride using L-Phe-terminated PLLA as a macroinitiator. The L-Phe-terminated PLLA was prepared through a novel three-step process. First, the hydroxyl-terminated PLLA was synthesized through the ring-opening polymerization of L-lactide initiated by n-butanol under the existence of tin(II) ethylhexanoate. Subsequently, the complete capping of the hydroxyl end group of PLLA with BOC-L-Phe was achieved by using a mixed anhydride of BOC-L-Phe under the catalysis of 4-(1-pyrrolidinyl) pyridine. Finally, the free amino end group was obtained by removal of the t-butoxycarbonyl group through trifluoroacetic acid treatment under anhydrous condition. All these treatments were conducted under mild conditions, thus avoiding the breakdown of the PLLA backbone. Poly(L-lactide)-b-poly(L-lysine) block copolymer was produced after deprotection treatment of PLLA-b-PZLys. The structure of the block copolymer was confirmed by 1H NMR, IR, and GPC. Adjustment of the ratio of the NCA monomer to the macroinitiator could control the chain length of the PLys block.     

Proton-NMR study of the interaction of trans-dichloro-diamine-platinum(II) with poly(I) and poly(I).poly(C)

The fixation of trans-(NH₃)₂Cl₂ Pt(II) to poly(I)·poly(C) at low r_b (< 0.05) leads to the formation of two complexed species. The major species (ca. 82% of bound platinum) involves coordination of platinum to a single hypoxanthine base, while the other species involves coordination of two hypoxanthine bases, which are either far apart on the same strand or on separate poly(I) strands, to the platinum. These same two species are found after reaction with poly(I), as are two other species throughout the entire r_b range studied (r_b = 0–0.30). The latter two species are assigned to trans-Pt bound to two bases on a poly(I) strand with (a) one or (b) two free bases between the two bound bases. These two species, (a) and (b), account for ca. 35% of the bound platinum, although the 1:1 species remains dominant (ca. 55%). These two additional species are observed at high r_b (>0.075) after reaction with poly(I)·poly(C) but as very minor species. They are formed by reaction with melted poly(I) loops. Also at high r_b, we have observed a shifted cytidine H⁵ resonance arising from interaction of trans-Pt with a melted loop of poly(C). Most probably, this arises from an intramolecular poly(I) to poly(C) crosslink. Results from the reaction of trans-Pt with poly(C) are presented for comparison.