PEG Grafting Density Research Articles

Decorated Rods: A “Bottom-Up” Self-Assembly of Monomolecular DNA Complexes

Fluorescence correlation spectroscopy (FCS) and gel electrophoresis measurements are performed to investigate both the number and size of complexes of linear double-stranded DNA (dsDNA) fragments with 1:1 diblock copolymers consisting of a cationic moiety, branched polyethyleneimine (bPEI) of 2, 10, or 25 kDa, covalently bound to a neutral shielding moiety, poly(ethylene glycol) (PEG; 20 kDa). By systematically decreasing the bPEI length, the PEG grafting density along the DNA chain can be directly controlled. For 25 and 10 kDa bPEI-PEG copolymers, severe aggregation is observed despite the presence of the shielding PEG. Upon decreasing the bPEI length to 2 kDa, controlled self-assembly of monomolecular DNA nanoparticles is observed. The resulting complexes are in quantitative agreement with a theoretical model based on a single DNA encased in a dense PEG polymer brush layer. The resulting PEGylated complexes show high stability against both salt and protein and hence are of potential use for in vivo gene delivery studies.

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.

Relationship between Interfacial Forces Measured by Colloid-Probe Atomic Force Microscopy and Protein Resistance of Poly(ethylene glycol)-Grafted Poly(l-lysine) Adlayers on Niobia Surfaces

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.

Surface modification of nanoporous alumina surfaces with poly(ethylene glycol).

Nanoporous alumina surfaces have a variety of applications in biosensors, biofiltration, and targeted drug delivery. However, the fabrication route to create these nanopores in alumina results in surface defects in the crystal lattice. This results in inherent charge on the porous surface causing biofouling, that is, nonspecific adsorption of biomolecules. Poly(ethylene glycol) (PEG) is known to form biocompatible nonfouling films on silicon surfaces. However, its application to alumina surfaces is very limited and has not been well investigated. In this study, we have covalently attached PEG to nanoporous alumina surfaces to improve their nonfouling properties. A PEG-silane coupling technique was used to modify the surface. Different concentrations of PEG for different immobilization times were used to form PEG films of various grafting densities. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the alumina surface. High-resolution C1s spectra show that with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of PEG films formed using the XPS intensities of the Al2p peaks. The films formed by this technique are less than 2.5 nm thick, suggesting that such films will not clog the pores which are in the range of 70-80 nm.

On the Nature of Conformational Transition in Poly(ethylene glycol) Chains Grafted onto Phospholipid Monolayers

This study was aimed at understanding the nature of conformational transition(s) that occur in poly(ethylene glycol) chains with molecular weight 5000 (PEG5000) grafted onto phospholipid monolayers. The study was performed with monolayers of PEG−phospholipid, DSPE−PEG5000, at the air/water interface and on solid substrates. Surface pressure and surface potential measurements together with ellipsometry and Brewster angle and atomic force microscopy were used to assess changes in the conformation and hydration of PEG5000 chains with increasing PEG grafting density. A comparative analysis of our experimental data suggests that neither the concept of the first-order pancake-to-brush conformational transition nor the model considering two subsequent transitions, namely, pancake-to-mushroom and mushroom-to-brush transitions, are adequate in describing conformational changes in the DSPE−PEG5000 monolayer at the air/water interface. At low grafting densities, PEG5000 chains did behave as grafted polymeric chains ...

Stability of Poly(ethylene glycol) Graft Coatings

Electrokinetic surface characterization is sensitive to both charge groups (pK and surface density) and neutral polymer coatings (molecular weight, surface density, grafting chemistry). It allows nondestructive, semiquantitative evaluation of coating stability, as regards alteration of the above parameters in relation to various environments. Characterization of surface electroosmosis versus pH (2 to 11) was used to evaluate poly(ethylene glycol) (PEG) coatings of biotechnical significance following a 3-week 22 °C exposure to 0.5 M sodium phosphate solutions of pH 4−11. The coatings involved epoxide-functionalized PEGs of Mr 3400 (E-PEG 3400) covalently grafted to aminosilane-modified fused quartz capillaries. Coatings made from PEGs with an epoxide group at one end (mE-PEG 5000) were stable at pH 4−11 whereas those made from PEGs with epoxide groups at both ends (diE-PEG 3400) were only stable at pH 4−9. During storage at pH 11 the latter exhibited time-dependent generation of surface negative charge groups of pK ≈ 5.3 and surface density (≈0.1/nm2) similar to the PEG grafting density. The effect was significantly reduced by N2 purging of storage solutions to remove dissolved O2 and was mimicked by fresh, carboxyl difunctionalized PEG 3400 (diSPA-PEG 3400) coatings of similar surface density. The mE-, diE-, and diSPA-based coatings studied appeared to exhibit similar grafting densities (≈0.1/nm2) and coating thicknesses (7 nm), suggesting grafting at only one end group. The factors responsible for limiting surface reaction by both end groups aided the ability of the diE-PEG coatings to secondarily tether (≈0.04/nm2) a model amine-containing compound (n-propylamine) at the diE-PEG-coated quartz surface. Results are discussed in relation to practical applications.

Grafting with hydrophilic polymer chains to prepare protein-resistant surfaces

Different ways of grafting poly(ethylene glycol) (PEG) chains to solid polyethylene were compared with respect to grafting density and efficiency in preventing fibrinogen adsorption. Covalent grafting of PEG was performed by attaching a nucleophilic PEG derivative to electrophilic surface groups or by binding electrophilic PEG to nucleophilic groups at the solid surface. Two adsorption procedures were also used. In the first of these an ethylene oxide propylene oxide (EO-PO) block copolymer was adsorbed at unmodified, hydrophobic polyethylene. In the second procedure the surface was made car☐yl-functional by free-radical grafting of tiglic acid and then exposed to a solution of a positively charged copolymer consisting of PEG chains grafted to poly(ethylene imine) (PEI). According to ESCA measurements, all four routes gave proper PEG grafting densities and the difference in the ration of C C O carbon (from PEG) to C C C carbon (from the underlying surface) was relatively small. There was a substantial difference in efficiency in fibrinogen rejection, however. Whereas surface modification with the PEG-PEI graft copolymer gave the lowest, treatment with the EO-PO block copolymer gave the highest amount of protein adsorption. The good effect of the PEG-PEI layer is believed to be related to the large entropy loss associated with protein adsorption on top of this copolymer which is known to be loosely bound in a loops-and-trains configuration. The limited effect of the EO-PO block copolymer may be due to the fact that this polymer is not entirely hydrophilic at the temperature used. Another contributing factor may be that the EO-PO block copolymer, unlike the PEG-PEI graft copolymer, is not irreversibly bound to the surface and may therefore be exchanged by fibrinogen.

Surface modification of polyurethane by plasma‐induced graft polymerization of poly(ethylene glycol) methacrylate

A new approach, plasma-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA), was used to introduce PEG graft chains with hydroxyl end groups onto a polyurethane (Tecoflex) surface. After argon plasma treatment and subsequent exposure to air, graft polymerization onto Tecoflex films was allowed to proceed in deaerated aqueous solutions of PEGMA at 60°C. The virgin, plasma-treated, and grafted films were characterized comparatively by means of attenuated total reflection infrared spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy, measurement of contact angle, and protein adsorption. The Tecoflex film undergoes etching during argon plasma treatment, surface oxidation when exposed to air after plasma treatment, and surface restructuring in response to environment upon storage in air. The plasma-induced graft polymerization of PEGMA proved to be successful in introducing PEG graft chains with reactive hydroxyl end groups onto the surface. Grafted films with different surface grafting density of PEG were prepared. Grafted films with higher PEG content exhibit higher hydrophilicity, smoother topography, and lower fibrinogen adsorption. The hydroxyl end groups built onto the surface offer further possibilities of improving its biocompatibility by immobilizing bioactive molecules.

Surface modification of polyurethane by plasma-induced graft polymerization of poly(ethylene glycol) methacrylate

A new approach, plasma-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA), was used to introduce PEG graft chains with hydroxyl end groups onto a polyurethane (Tecoflex) surface. After argon plasma treatment and subsequent exposure to air, graft polymerization onto Tecoflex films was allowed to proceed in deaerated aqueous solutions of PEGMA at 60°C. The virgin, plasma-treated, and grafted films were characterized comparatively by means of attenuated total reflection infrared spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy, measurement of contact angle, and protein adsorption. The Tecoflex film undergoes etching during argon plasma treatment, surface oxidation when exposed to air after plasma treatment, and surface restructuring in response to environment upon storage in air. The plasma-induced graft polymerization of PEGMA proved to be successful in introducing PEG graft chains with reactive hydroxyl end groups onto the surface. Grafted films with different surface grafting density of PEG were prepared. Grafted films with higher PEG content exhibit higher hydrophilicity, smoother topography, and lower fibrinogen adsorption. The hydroxyl end groups built onto the surface offer further possibilities of improving its biocompatibility by immobilizing bioactive molecules. © 1996 John Wiley & Sons, Inc.