PEG Spacer Length Research Articles

The chemoenzymatic synthesis of glycan-terminated oligo(Leu)x

Introduction: Glycopeptides contain carbohydrate moieties (glycans) covalently attached to the side chain and/or terminal peptide units. Since glycans are present on cell surfaces, these constructs can potentially address a wide array of therapeutic functions. To overcome the deficiencies associated with current synthetic routes to glycopeptides, such as costly processes and toxic reagents, this work aimed to develop versatile environmentally friendly protease-catalyzed peptide synthesis routes to peptides decorated with a glycan at their N-terminus.Methods: “Grafters” were first synthesized that consist of a glycan conjugated directly, or through a spacer, to the amine group of L-Phe-ethyl ester (Phe-OEt). The role of Phe-OEt is to increase the conjugate’s recognition by the protease (papain) catalytic active site. A series of grafters were synthesized with variation of the glycan structure, linkage-chemistry, and presence of an oligo (ethylene glycol) “spacer” of varied length between the glycan and Phe-OEt moiety. High grafter efficiency will result by the successful acceptance of the grafter at the enzymes S1/S2 subsites, formation of an acyl enzyme complex and subsequent conversion to glycan-terminated oligo(Leu)x (x ≥ 1), as opposed to construction of non-glycan N-terminated oligo(Leu)x.Results and discussion: While glycan-Phe-OEt grafters without a spacer between the glycan and Phe-OEt resulted in low grafter efficiency (8.3% ± 2.0%), insertion of a short oligo (ethylene glycol) spacer between the glycan and Phe-OEt moieties (glycan-PEGn-Phe-OEt, n ≥ 3) increased the grafter efficiency by 3-fold–24.5% ± 1.8%. In addition, computational modeling was performed using Rosetta software provided insights on a molecular level of how grafter efficiency is influenced by the PEG spacer length.

Influence of Membrane-Fusogen Distance on the Secondary Structure of Fusogenic Coiled Coil Peptides.

Liposomal membrane fusion is an important tool to study complex biological fusion mechanisms. We use lipidated derivatives of the specific heterodimeric coiled coil pair E: (EIAALEK)3 and K: (KIAALKE)3 to study and control the fusion of liposomes. In this model system, peptides are tethered to their liposomes via a poly(ethylene glycol) (PEG) spacer and a lipid anchor. The efficiency of the fusion mechanism and function of the peptides is highly affected by the PEG-spacer length and the lipid anchor type. Here, the influence of membrane–fusogen distance on the peptide–membrane interactions and the peptide secondary structures is studied with Langmuir film balance and infrared reflection absorption spectroscopy. We found that the introduction of a spacer to monolayer-tethered peptide E changes its conformation from solvated random coils to homo-oligomers. In contrast, the described peptide–monolayer interaction of peptide K is not affected by the PEG-spacer length. Furthermore, the coexistence of different conformations when both lipopeptides E and K are present at the membrane surface is demonstrated empirically, which has many implications for the design of effective fusogenic recognition units and the field of artificial membrane fusion.

Controlled Fab installation onto polymeric micelle nanoparticles for tuned bioactivity

Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm −2 and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM −1 cm −2. The kinetic parameters Michaelis–Menten constant (K M app) and maximum current density ( j max) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm −2 increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm −2, the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml −1.

Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate‐mediated targeting in vitro and prolonged plasma circulation in vivo

Gene delivery vectors based on poly(L-lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non-specifically. In this study we have attempted to form folate-targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers. pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da. Polymer-coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000-fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA. We show increased systemic circulation, decreased blood cell and protein binding, and folate-targeted transgene expression using pLL/DNA complexes surface-modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma-circulating targeted vectors for in vivo applications.