Helix stabilization of poly(ethylene glycol)-peptide conjugates

Hybrid polymer-peptide conjugates offer the potential for incorporating biological function into synthetic materials. The secondary structure of short helical peptides, however, frequently becomes less stable when expressed independent of longer protein sequences or covalently linked with a conformationally disordered synthetic polymer. Recently, new amphipathic peptide-poly(ethylene glycol) conjugates were introduced (Shu, J., et al. Biomacromolecules 2008, 9, 2011), which displayed enhanced peptide helicity upon polymer functionalization while retaining tertiary coiled-coil associations. We report here a molecular simulation study of peptide helix stabilization by conjugation with poly(ethylene glycol). The polymer oxygens are shown to favorably interact with the cationic lysine side chains, providing an alternate binding site that protects against disruption of the peptide hydrogen-bonds that stabilize the helical conformation. When the peptide lysine charges are neutralized or poly(ethylene glycol) is conjugated with polyalanine, the polymer exhibits a negligible effect on the secondary structure. We also observe the interactions of poly(ethylene glycol) with the amphipathic peptide lysines tends to segregate the polymer away from the nonpolar face of the helix, suggesting no disruption of the interactions that drive tertiary contacts between helicies.     

Synthesis and characterization of poly(ethylene glycol)-insulin conjugates

Human insulin was modified by covalent attachment of short-chain (750 and 2000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-PheB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG(2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were performed using analytical ultracentrifugation to quantitatively determine the association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dimers while Zn(2+)-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This independent method qualitatively suggests that mPEG-insulin conjugates behave similarly to Zn-free insulin in the concentration range studied and complements results from ultracentrifugation studies. The physical stability/resistance to fibrillation of mPEG-insulin conjugates in aqueous solution were assessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates are substantially more stable than controls but the mPEG(750 and 2000)-LysB29-insulin conjugates were only slightly more stable than commercially available preparations. Circular dichroism studies done in the far ultraviolet region confirm insulin's tertiary structure in aqueous solution is essentially conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal that there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000) decreased the bioactivity of the conjugates to about 85% of Lilly's HumulinR formulation. The characterization presented in this paper provides strong testimony to the fact that attachment of mPEG to specific amino acid residues of insulin's B-chain improves the conjugates' physical stability without appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.     

Fluorescent-labeled poly(ethylene glycol) lipid conjugates with distal cationic headgroups

The synthesis of a new class of fluorescent cationic poly(ethylene glycol) lipid conjugates (CPLs) is described. These lipids consist of a hydrophobic distearoyl-phosphatidylethanolamine (DSPE) anchor coupled to a highly fluorescent N(epsilon)-dansyl lysine moiety, which is attached to a hydrophilic poly(ethylene glycol) (PEG) spacer that is linked to a cationic headgroup made of lysine residues. Introduction of the dansyl moiety allows rapid and accurate quantification of CPLs within lipid bilayers using fluorescence techniques. The synthetic scheme is straightforward, using repeated amino-carboxyl coupling reaction steps, with purification by precipitation. A series of dansylated CPLs was synthesized with zero, one, three, and seven lysine residues located at the distal end of the PEG chain, giving rise to CPLs with one, two, four, and eight distal positive charges, respectively. The structures of the CPLs were confirmed by (1)H NMR spectroscopy and chemical analysis. CPLs provide a means of introducing positive charge to a bilayer that is localized some distance from the membrane surface, and are of particular interest for nonviral gene delivery applications. The usefulness of CPLs is demonstrated by the enhanced in vitro cellular binding and uptake of liposomes containing CPL(4).     

Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates

Poly(ethylene glycol) (PEG) with the terminal group of active ester was coupled to the amino group of gelatin to prepare PEG-grafted gelatin (PEG-gelatin). The affinity chromatographic study revealed that the PEG-gelatin with high degrees of PEGylation did not adsorb onto the gelatin affinity column, in remarked contrast to gelatin alone and the PEG-gelatin with low PEGylation degrees. The former PEG-gelatin showed a critical micelle concentration while it had the apparent molecular size of about 100 nm and a surface charge of almost zero. These findings indicate that the PEG-gelatin formed a micelle structure of which the surface is covered with PEG molecules grafted. When the body distribution of 125I-labeled gelatin and PEG-gelatin after intravenous injection was evaluated, the radioactivity of micellar PEG-gelatin was retained in the blood circulation compared with that of gelatin and the PEG-gelatin of no micelle formation. At the same PEGylation degree, the blood concentration was significantly higher for the PEG-gelatin prepared from PEG with a molecular weight of 12 000 than that of molecular weights of 2000 and 5000. It is concluded that the PEG-gelatin is a drug carrier with a micelle structure which retains in the blood circulation.     

Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.     

Multivalent poly(ethylene glycol)-containing conjugates for in vivo antibody suppression

Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of beta(2)GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-beta(2)GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with (125)I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress anti-domain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

The influence of poly(ethylene glycol) 6000 on spermine-induced aggregation of liposomes.

Poly(ethylene glycol) 6000 affected the aggregation of mixed liposomes induced by spermine. It lowered the concentration of spermine causing 50% maximal aggregation, accelerated the rate and increased the extent of aggregation. The effect was inversely proportional to the density of the acidic phospholipid in the vesicles. These effects were not due either to poly(ethylene glycol) 6000-induced permanent structural modification of the liposome or increased binding of spermine to the vesicles. These findings are discussed in relation to a decreased hydration force caused by the ability of poly(ethylene glycol) 6000 to alter the water of hydration of the phospholipid polar groups in the liposome.     

Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.     

Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer

Advances in tissue engineering require biofunctional scaffolds that can provide not only physical support for cells but also chemical and biological cues needed in forming functional tissues. To achieve this goal, a novel RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) (PEG-PLA-PGL/RGD) was synthesized in four steps (1) to prepare diblock copolymer PEG-PLA-OH and to convert its -OH end group into -NH(2) (to obtain PEG-PLA-NH(2)), (2) to prepare triblock copolymer PEG-PLA-PBGL by ring-opening polymerization of NCA (N-carboxyanhydride) derived from benzyl glutamate with diblock copolymer PEG-PLA-NH(2) as macroinitiator, (3) to remove the protective benzyl groups by catalytic hydrogenation of PEG-PLA-PBGL to obtain PEG-PLA-PGL, and (4) to react RGD (arginine-glycine-(aspartic amide)) with the carboxyl groups of the PEG-PLA-PGL. The structures of PEG-PLA-PGL/RGD and its precursors were confirmed by (1)H NMR, FT-IR, amino acid analysis, and XPS analysis. Addition of 5 wt % PEG-PLA-PGL/RGD into a PLGA matrix significantly improved the surface wettability of the blend films and the adhesion and proliferation behavior of human chondrocytes and 3T3 cells on the blend films. Therefore, the novel RGD-grafted triblock copolymer is expected to find application in cell or tissue engineering.     

Probing protein nanopores with poly(ethylene glycol)s

Neutral water-soluble poly(ethylene glycol)s (PEGs) have been extensively explored in protein nanopore research for the past several decades. The principal use of PEGs is to investigate the membrane protein ion channel physical characteristics and transport properties. In addition, protein nanopores are used to study polymer–protein interactions and polymer physicochemical properties. In this review, we focus on the biophysical studies on probing protein ion channels with PEGs, specifically on nanopore sizing by PEG partitioning. We discuss the fluctuation analysis of ion channel currents in response to the PEGs moving within their confined geometries. The advantages, limitations, and recent developments of the approach are also addressed.     

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.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

Interaction of phospholipid membranes with poly(ethylene glycol)s

1. The water-soluble polymer, poly(ethylene glycol), causes concentration-dependent increases in the temperature of the gel--liquid crystalline phase transitions of aqueous dispersions of dipalmitoyl phosphatidylcholine and of dipalmitoyl phosphatidylethanolamine. 2. For dipalmitoyl phosphatidylcholine it has been further demonstrated that poly(ethylene glycol) increases the transition enthalpy and entropy while decreasing the cooperativity of the transition. 3. These results are discussed in relation to the possible modes of action of poly(ethylene glycol) in promoting cell fusion.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M_w=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1±0.8 (mg/m²)/(mg/ml) for 2.5 loops to 78.1±12.2 (mg/m²)/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4±0.1 (mg/m²)/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

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.     

RGD peptides micro-patterning on poly(ethylene terephthalate) surfaces

The aim of this study was to evaluate the impact of RGD micro-patterned poly(ethylene terephthalate) (PET) on human osteoblast progenitor (HOP) cells attachment. Biomimetic modifications were performed by means of a four-step reaction: surface hydrolysis, oxidation in order to create COOH functions, coupling agent grafting (EDC, NHS) and finally immobilization of peptides. In addition to homogeneous or statistically distribution of peptides, micro-patterns of RGD were generated by: optical photolithography and UV excimer laser ablation. Modification steps were validated by physico-chemical techniques: XPS was used to prove covalent grafting at each stage of the surface functionalization, toluidine blue assay and high resolution µ-imager (using [3H]-Lys) to evaluate peptide densities and validate micro-patterns formation. Finally, the efficiency of this biomodification of PET was demonstrated onto homogeneous surfaces by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC.