Antibiofouling polymer interfaces: poly(ethylene glycol) and other promising candidates

Non-specific protein adsorption has a major confounding influence on the in vitro and in vivo performances of synthetic polymer scaffolds for tissue engineering. For control over cell phenotype to be realised, non-specific protein adsorption needs to be constrained and surfaces modified by immobilisation of specific cell attachment and bioactive molecules. Our aim was to develop biodegradable scaffolds incorporating specific bioactive functionalities, whilst at the same time limiting non-specific protein adsorption by the polymer. The approach we developed was to functionalise PLGA scaffolds with diamino-terminated poly(ethylene glycol) (diamino-PEG), followed by the grafting of heparin to the free –NH2group at the PEG chain end using a coupling agent. The covalent grafting of poly(ethylene glycol) chains both suppressed non-specific protein attachment and provided a substrate for grafting of heparin, onto which growth factors could be tethered and presented to cells in a bioactive configuration. This approach offers a widely applicable solution to overcome the serious and confounding problem of serum fouling of scaffold surfaces, whilst also providing a generic route for specific biofunctionalisation.

Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors.

Modification of the surface with densely packed poly(ethylene glycol) (PEG) brush layer was studied to improve the protein repellent ability of the surface. A PEG-brushed layer was constructed on a gold substrate using a PEG possessing a mercapto group at the chain end. The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the gold substrate, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved high density brush and almost complete prevention of nonspecific protein adsorption. On the other hand, surface modification with only long PEG chain (5 kDa) showed lower PEG brush density regardless of repetitive immobilization. Detailed characterization of the PEGylated surface was done from the physicochemical (QCM, contact angle, and SPR) as well as the biological (protein adsorption) point of view to highlight the relation between the PEG brush density and the protein repellent ability. Densely packed PEG surface which showed great protein repellent ability, presented in this study, suggests promising utility as engineered biomaterials including high-throughput screening and clinical diagnostics.

Minocycline enhances the mesenchymal stromal/stem cell pro-healing phenotype in triple antimicrobial-loaded hydrogels.

The strong surface hydration layer of nonfouling materials plays a key role in their resistance to nonspecific protein adsorption. Poly(ethylene glycol) (PEG) is an effective example of materials that can resist nonspecific protein adsorption and cell adhesion. Thus, the strong interaction between water molecules and PEG was investigated through each T(2) component in water/PEG mixtures using multiexponential inversion of T(2) relaxation time measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence of low-field nuclear magnetic resonance (LF-NMR). Results show that about one water molecule is tightly bound with one ethylene glycol (EG) unit, and additional water molecules over 1:1 ratio mainly swell the PEG matrix and are not tightly bound with PEG. This result was also supported by the endothermic behavior of water/PEG mixtures measured by differential scanning calorimetry (DSC). It is believed that the method developed could be also applied to investigate various interactions between macromolecules and other small molecules without using deuterium samples, which might open a novel route to quantitatively measure guest-host interactions in the future.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTSelf-Assembled Monolayers of Alkanethiolates Presenting Tri(propylene sulfoxide) Groups Resist the Adsorption of ProteinLi Deng, Milan Mrksich, and George M. WhitesidesView Author Information Department of Chemistry, Harvard University Cambridge, Massachusetts 02138 Cite this: J. Am. Chem. Soc. 1996, 118, 21, 5136–5137Publication Date (Web):May 29, 1996Publication History Received13 February 1996Published online29 May 1996Published inissue 1 January 1996https://pubs.acs.org/doi/10.1021/ja960461dhttps://doi.org/10.1021/ja960461drapid-communicationACS PublicationsCopyright © 1996 American Chemical SocietyRequest reuse permissionsArticle Views1058Altmetric-Citations109LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (2)»Supporting Information Supporting Information SUBJECTS:Adsorption,Alcohols,Alkyls,Oxides,Peptides and proteins Get e-Alerts

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.

A wide variety of cells usually integrate and respond to the microscale environment, such as soluble protein factors, extracellular matrix proteins, and contacts with neighboring cells. To gain insight into cellular microenvironment design, we investigated two-dimensional microarray formation of endothelial cells on a micropatterned poly(ethylene glycol) (PEG)-brushed surface, based on the relationship between PEG chain density and cellular attachment. The patterned substrates consisted of two regions: the PEG surface that acts as a cell-resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A PEG-brushed layer was constructed on a gold substrate using PEG with a mercapto group at the end of the chain. The density of the PEG-brushed layer increased substantially with repetitive adsorption/rinse cycles of PEG on the gold substrate, allowing marked reduction of nonspecific protein adsorption. These repeated adsorption/rinse cycles were further regulated by using longer (5 kDa) and shorter (2 kDa) PEG to construct PEG layers with different chain density, and subsequent micropatterning was achieved by plasma etching through a micropatterned metal mask. The effects of PEG chain density on pattern formation of cell attachment were determined on micropatterning of endothelial cells. The results indicated that cell pattern formation was strongly dependent on the PEG chain density and on the extent of protein adsorption. Notably, a PEG chain density high enough to inhibit outgrowth of endothelial cells from the cell-adhering region in the horizontal direction could be obtained only by employing formation of a short filler layer of PEG in the preconstructed longer PEG-brushed layer, which prevented nonspecific protein adsorption almost completely. In this way, a completely micropatterned array of endothelial cells with long-term viability was obtained. This clearly indicated the importance of a short underbrushed PEG layer in minimizing nonspecific protein adsorption for long-term maintenance of the active cell pattern. The strategy for cell patterning presented here can be employed in tissue engineering to study cell-cell and cell-surface interactions. It is also applicable for high-throughput screening and clinical diagnostics, as well as interfacing cellular and microfabricated components of biomedical microsystems.

Polymer-drug conjugates are commonly used as nano drug vehicles (NDVs) to delivery anticancer drugs. Zwitterionic polymers are ideal candidates to conjugate drugs because they show higher resistance to nonspecific protein adsorption in complex media than that of nonionic water-soluble polymers, such as poly(ethylene glycol). However, the charge balance characteristics of zwitterionic polymers used as NDVs will be broken from the inclusion of additional charged groups brought by conjugated drugs or functional groups, leading to the loss of resistance to protein adsorption. Consequently, the nonspecific protein adsorption on drug carriers will cause fast clearance from the blood system, an immune response, or even severe systemic toxicity. To overcome this drawback, a model zwitterionic polymer, poly(carboxybetaine methacrylate) (pCBMA), was modified by the introduction of a negatively charged component, to neutralize the positive charge provided by the model drug, doxorubicin (DOX). A DOX-conjugated NDV which possesses excellent resistance to nonspecific protein adsorption was achieved by the formation of a strongly hydrated pCBMA shell with a slightly negative surface charge. This kind of DOX-conjugated NDV exhibited reduced cytotoxicity and prolonged circulation time, and it accelerated DOX release under mild acid conditions. In tumor-bearing mouse studies a 55% tumor-inhibition rate was achieved without causing any body weight loss. These results indicate the importance of charge tuning in zwitterionic polymer-based NDVs.

Investigation of the interaction between poly(ethylene glycol) and protein molecules using low field nuclear magnetic resonance

The goal of this study was to develop a gene delivery vehicle that can specifically target cell surface receptors with low nonspecific protein adsorption and low cytotoxicity. Toward this goal, four-arm poly(ethylene glycol) vehicles were functionalized with DNA-binding peptides (DBPs) and integrin-binding (RGD) peptides. We have previously described a novel PEG-based gene delivery vehicle functionalized with DBPs that successfully transfected Chinese hamster ovary (CHO) cells with low toxicity and low protein adsorption. This work investigated whether incorporating RGD peptides onto PEG-DBP vehicles could target specific cell surface receptors and increase transfection efficiency of HEPG2 cells. DBP and RGD peptides were coupled onto PEG-tetraacrylate (PEG-TA) in three combinations (molar ratios of DBP:RGD of 1:3, 2:2, and 3:1) and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of charge ratio (peptide amine groups:DNA phosphate). Nonspecific protein adsorption and cytotoxicity of PEG-DBP-RGD vehicles were also measured. Dynamic light scattering showed that PEG-DBP-RGD vehicles condensed DNA into particles having mean diameters of 250-300 nm and zeta potentials ranging from -10 to 7 mV. It was found that coupling two RGD peptides to the PEG-DBP 2 vehicle increased the transfection efficiency at a polymer/DNA charge ratio of 5:1 (+/-) and 6:1 (+/-) and that these vehicles had transfection efficiencies similar to those of polyethylenimine (PEI)/DNA particles. However, coupling one or three RGD peptides to PEG-DBP vehicles did not increase the transfection efficiency. Additionally, the PEG-DBP-RGD/DNA particles adsorbed less protein than PEI particles and were less toxic to HEPG2 cells.

A high-density poly(ethylene glycol) (PEG)-coated Si(111) surface is used for the immobilization of polyhistidine-tagged protein molecules. This process features a number of properties that are highly desirable for protein microarray technology: (i) minimal nonspecific protein adsorption; (ii) highly uniform surface functionality; (iii) controlled protein orientation; and (iv) highly specific immobilization reaction without the need of protein purification. The high-density PEG-coated silicon surface is obtained from the reaction of a multi-arm PEG (mPEG) molecule with a chlorine terminated Si(111) surface to give a mPEG film with thickness of 5.2 nm. Four out of the eight arms on each immobilized mPEG molecule are accessible for linking to the chelating iminodiacetic acid (IDA) groups for the binding of Cu(2+) ions. The resulting Cu(2+)-IDA-mPEG-Si(111) surface is shown to specifically bind 6x histidine-tagged protein molecules, including green fluorescent protein (GFP) and sulfotransferase (ST), but otherwise retains its inertness towards nonspecific protein adsorption. We demonstrate a particular advantage of this strategy: the possibility of protein immobilization without the need of prepurification. Surface concentrations of relevant chemical species are quantitatively characterized at each reaction step by X-ray photoelectron spectroscopy (XPS). This kind of quantitative analysis is essential in tuning surface concentration and chemical environment for optimal sensitivity in probe-target interaction.

This paper describes a simple method for preparing protein microarrays that is compatible with high throughput manufacturing. The microarrays were formed by maskless photolithography and pin spotting to study protein adsorption on a fluorinated/methoxy-poly(ethylene glycol) (PEG) self-assembled monolayer (SAM). The mixed nonionic surfactants, Tween20 and PEG200, were utilized to control nonspecific protein adsorption on both of the SAMs. Measurements using double-antibody sandwich quantum dots-linked immunosorbent assay (DAS-QDLISA) showed that the fluorinated SAM could effectively minimize nonspecific adsorption in the presence of Tween 20 and PEG 200 (inhibitors of nonspecific protein adsorption (INSPAs)) while the PEGylated surface was biofouling. Additionally, pin spotting was used to fabricate high-throughput protein arrays on the fluorinated SAM. The results displayed that fluorinated SAM could not only effectively immobilize protein by hydrophobic interactions, but could also resist the other nonspecific adsorption with the INSPA. In this way, protein microarrays would be formed more conveniently and environmentally friendly. It is believed that simple, practical, and high-throughput protein immunosensing could be established with these mixed nonionic surfactants.

Nonspecific adsorption of proteins is a crucial problem in the detection of analytes in complex biological media by affinity sensors operating with label-free detection. We modified the gold surface of surface plasmon resonance (SPR) sensors with three types of promising antifouling coatings: self-assembled monolayers (SAM)s of alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) (PEG) grafted onto the SAMs, and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phosphorylcholine methacrylate). Using SPR, we compared the efficacy of the coatings to reduce nonspecific adsorption from human blood plasma and from single-protein solutions of human serum albumin, immunoglobulin G, fibrinogen, and lysozyme. There was no direct relationship between values of water contact angles and plasma deposition on the coated surfaces. A rather high plasma deposition on SAMs was decreased by grafting PEG chains. Fouling on PEG was observed only from plasma fractions containing proteins with molecular mass higher than 350 000 Da. The adsorption kinetics from plasma collected from different healthy donors differed. Poly(carboxybetaine methacrylate) completely prevented the deposition from plasma, but the other more hydrophilic zwitterionic polymers prevented single-protein adsorption but did not prevent plasma deposition. The results suggest that neither wettability nor adsorption of the main plasma proteins was the main indicator of deposition from blood plasma.

The prospective cancer therapy applications of inorganic nanoparticles (NPs) have been encouraged by surface modifications with polyethylene glycol (PEG). When grafted at high densities, PEG reduces NP surface interaction with its environment, hence minimizing its detection by the immune system. The drawback of high density PEG surfaces is its reduced uptake by cancer cells. This is disadvantageous for applications that favor intracellular localization of such NPs. On the other hand, reduced PEG density leads to nonspecific adsorption of proteins on NP surface. Adsorbed proteins may either facilitate cancer cell entry or mark inorganic NPs for clearance from the body. This trade-off between intracellular localization and in vivo residency time was observed in this study. Two grafting densities of 1 PEG/nm 2 and 1 PEG/2 nm 2 for two PEG chain lengths with molecular weights of 2kDa and 5kDa were used to coat gold NPs (GNPs). Spherical GNPs with a diameter of 50 nm were used as a model system to investigate the effect of PEG characteristics on NP nonspecific protein adsorption and uptake by cancer cells in vitro in HeLa, MDA-MB-231 and MCF-7 cells. Higher grafting densities resulted into less protein adsorption and lower NP uptake by all cell lines. Longer PEG chain lengths also resulted into less cancer cell entry in all cell lines. Quantitative results were confirmed qualitatively via hyperspectral imaging of NPs in cancer cells. Hence, lower PEG grafting densities and shorter chain lengths lead to higher cellular uptake at the cost of greater nonspecific protein adsorption. Higher density grafting of shorter PEG chain lengths was found most favorable. This combination resulted into reduced nonspecific protein adsorption and higher uptake when compared to its lower density and longer chain length counterparts, respectively. To maximize cancer therapy improvement via inorganic NP platforms, using such PEG grafting characteristics may be beneficial.