Polyethylene Glycol Grafting Density Research Articles

Excipient-free lyophilization of block copolymer micelles for potential lung surfactant therapy applications

Polymer lung surfactant (PLS) is a polyethylene glycol (PEG)-brushed block copolymer micelle designed for pulmonary surfactant replacement therapy. Saccharides (e.g., sucrose and (2-hydroxypropyl)-β-cyclodextrin) and water-soluble polymers (e.g., PEG), common excipients for lyophilization, were found to severely impair the surface activity of lyophilized PLS. To investigate the feasibility of excipient-free lyophilization of PLS, we studied the effects of both PLS material parameters and lyophilization operating parameters on the redispersibility and surface availability of reconstituted PLS, all without relying on excipients. We found that the redispersibility was improved by three factors; a faster cooling rate during the freezing stage reduced freezing stress; a higher PEG grafting density enhanced dissipating effects; and the absence of hydrophobic endgroups in the PEG block further prevented micelle aggregation. Consequently, the surface availability of PLS increased, enabling the micelle monolayer at the air/water interface to achieve a surface tension below 10 mN/m, which is a key pharmaceutical function of PLS. Moreover, the lyophilized micelles in powder form could be easily dispersed on water surfaces without the need for reconstitution, which opens up the possibility of inhalation delivery, a more patient-friendly administration method compared to instillation. The successful excipient-free lyophilization unlocks the potential of PLS for addressing acute respiratory distress syndrome (ARDS) and other pulmonary dysfunctions.

In silico prediction of protein binding affinities onto core-shell PEGylated noble metal nanoparticles for rational design of drug nanocarriers.

Polymer-coated nanoparticles (NP) are commonly used as drug carriers or theranostic agents. Their uptake rates are modulated by the interactions with essential serum proteins such as transferrin and albumin. Understanding the control parameters of these interactions is crucial for improving the efficiency of these nanoscale devices. In this work, we perform a multiscale computational study of protein adsorption onto polyethylene glycol (PEG) coated gold and silver NPs, producing protein-NP adsorption rankings as a function of PEG grafting density, which are validated against previously reported experimental protein-NP binding constants. Furthermore, the applied nano-docking method provides information on the preferred orientation of proteins immobilised on the surface of NPs. We propose a method of construction of model core-shell NPs in silico. The presented protocol can provide molecular level insights for the experimental development of biosensors, nanocarriers, or other nanoplatforms where information on the preferred orientation of protein at the bio-nano interface is crucial, and enables fast in silico prescreening of assays of various nanocarriers, i.e., combinations of proteins, NPs, and coatings.

Influence of Molecular Weight and Grafting Density of PEG on the Surface Properties of Polyurethanes and Their Effect on the Viability and Morphology of Fibroblasts and Osteoblasts.

Grafting polyethylene glycol (PEG) onto a polymer's surface is widely used to improve biocompatibility by reducing protein and cell adhesion. Although PEG is considered to be bioinert, its incorporation onto biomaterials has shown to improve cell viability depending on the amount and molecular weight (MW) used. This phenomenon was studied here by grafting PEG of three MW onto polyurethane (PU) substrata at three molar concentrations to assess their effect on PU surface properties and on the viability of osteoblasts and fibroblasts. PEG formed a covering on the substrata which increased the hydrophilicity and surface energy of PUs. Among the results, it was observed that osteoblast viability increased for all MW and grafting densities of PEG employed compared with unmodified PU. However, fibroblast viability only increased at certain combinations of MW and grafting densities of PEG, suggesting an optimal level of these parameters. PEG grafting also promoted a more spread cell morphology than that exhibited by unmodified PU; nevertheless, cells became apoptotic-like as PEG MW and grafting density were increased. These effects on cells could be due to PEG affecting culture medium pH, which became more alkaline at higher MW and concentrations of PEG. Results support the hypothesis that surface energy of PU substrates can be tuned by controlling the MW and grafting density of PEG, but these parameters should be optimized to promote cell viability without inducing apoptotic-like behavior.

Assembly of partially covered strawberry supracolloids in dilute and concentrate aqueous dispersions

HypothesisCore-corona supracolloids can be assembled in aqueous dispersions by controlling the physical interactions between the corona and core colloidal particles. A raspberry corona configuration with full surface coverage of the core can be reached by inducing strong attractive interactions between the individual particles. A controlled partial surface coverage of the core, i.e. strawberry configuration, is however, more difficult to achieve. Supracolloids with different surface coverage ratio exhibit unique and multifunctional surface properties. ExperimentsBy counterbalancing the multiple physical interactions playing a role during the assembly, the configuration and stability of the assemblies could be fine-tuned over a wide range of concentrations. Supracolloids consisting of polyethylene glycol (PEO)-grafted polymer particles covered by silica nanoparticles were assembled with different configurations, by adjusting the pH and ionic strength of the dispersion, the PEO grafting density and the particles concentration. The self-assembly process and resulting configurations were monitored via cryogenic transmission electron microscopy (Cryo-TEM) and light scattering. FindingsThe suitable conditions to assemble supracolloids with partial corona coverage have been established. Stable strawberry supracolloids could be prepared, both for diluted (1 wt%) and concentrated (12 wt%) dispersions. These hybrid supracolloids with well-defined configuration are highly relevant to developing advanced water-borne paints and inks, food dispersions, cosmetic and healthcare products.

Effect of PEGylation on the Drug Release Performance and Hemocompatibility of Photoresponsive Drug-Loading Platform.

Coronary stenosis has been one of the most common heart diseases that drastically increases the risk of fatal disorders such as heart attack. Angioplasty using drug coated balloons (DCB) has been one of the most safe and promising treatments. To minimize the risk of thrombosis of such DCBs during intervention, a different approach that can secure high hemocompatibility under blood flow is necessary. Here we report a method of improving the photoresponsive platform’s hemocompatibility by conjugating polyethylene glycol (PEG), onto the functional groups located at the balloon surface. In this study, latex microbeads were used as models for balloons to enable precise observation of its surface under microscopy. These beads were decorated with PEG polymers of a variety of lengths and grafting densities, along with the Cy5-Photoclevable (PC) linker conjugate to mimic drugs to be loaded onto the platform. Results showed that PEG length and grafting density are both critical factors that alter not only its hemocompatibility, but also the drug load and release efficiency of such platform. Thus, although further investigation is necessary to optimize the tradeoff between hemocompatibility, drug load, and release efficiency, it is safe to conclude that PEGylation of DCB surface is an effective method of enhancing and maintaining high hemocompatibility to minimize the risk of thrombosis during angioplasty.

Effect of PEG grafting density on surface properties of polyurethane substrata and the viability of osteoblast and fibroblast cells

The surface of Tecoflex SG-80A Polyurethane (PU) films was modified by grafting polyethylene glycol (PEG) chains at three different molar amounts (0.05, 0.10, and 0.15 mmol). The resulting substrata were characterized by FTIR-ATR, TGA, AFM, SEM and contact angle to assess the surface modifications occurred during the grafting reactions. Osteoblasts and fibroblasts were cultured with PU extracts for 24 h, and their cell viability and morphology were evaluated by CellTiterBlue assay, Crystal Violet staining and Live/Dead assay. FTIR and TGA results indicated that PEG chains were successfully grafted onto PU surfaces, specifically in the hard segment of PU forming allophanate groups as the PEG grafting density increased. SEM and AFM images suggest that PU substrata were partially covered by PEG, increasing the dispersive and basic components of the PU surface energy. It was found that extracts from PEG-grafted polyurethanes increased the osteoblast viability, although fibroblasts viability remained constant regardless PEG grafting density; in spite of this both cells presented a more spread morphology at the lower PEG grafting density. Our results showed that surface energy of PU substrata can be tuned by PEG grafting density; also, the PEG leached tends to increase the pH of culture medium which leads to a higher viability of osteoblasts; nevertheless, PEG grafting density should be optimized to promote a healthy cell morphology as alterations in its morphology were detected at higher concentrations.Graphical abstract

An Integrated Design of a Polypseudorotaxane‐Based Sea Cucumber Mimic

The development of integrated systems that mimic the multi-stage stiffness change of marine animals such as the sea cucumber requires the design of molecularly tailored structures. Herein, we used an integrated biomimicry design to fabricate a sea cucumber mimic using sidechain polypseudorotaxanes with tunable nano-to-macroscale properties. A series of polyethylene glycol (PEG)-based sidechain copolymers were synthesized to form sidechain polypseudorotaxanes with α-cyclodextrins (α-CDs). By tailoring the copolymers' molecular weights and their PEG grafting densities, we rationally tuned the sizes of the formed polypseudorotaxanes crystalline domain and the physical crosslinking density of the hydrogels, which facilitated 3D printing and the mechanical adaptability to these hydrogels. After 3D printing and photo-crosslinking, the obtained hydrogels exhibited large tensile strain and broad elastic-to-plastic variations upon α-CD (de)threading. These discoveries enabled a successful fabrication of a sea cucumber mimic, demonstrating multi-stage stiffness changes.

An Integrated Design of a Polypseudorotaxane‐Based Sea Cucumber Mimic

AbstractThe development of integrated systems that mimic the multi‐stage stiffness change of marine animals such as the sea cucumber requires the design of molecularly tailored structures. Herein, we used an integrated biomimicry design to fabricate a sea cucumber mimic using sidechain polypseudorotaxanes with tunable nano‐to‐macroscale properties. A series of polyethylene glycol (PEG)‐based sidechain copolymers were synthesized to form sidechain polypseudorotaxanes with α‐cyclodextrins (α‐CDs). By tailoring the copolymers’ molecular weights and their PEG grafting densities, we rationally tuned the sizes of the formed polypseudorotaxanes crystalline domain and the physical crosslinking density of the hydrogels, which facilitated 3D printing and the mechanical adaptability to these hydrogels. After 3D printing and photo‐crosslinking, the obtained hydrogels exhibited large tensile strain and broad elastic‐to‐plastic variations upon α‐CD (de)threading. These discoveries enabled a successful fabrication of a sea cucumber mimic, demonstrating multi‐stage stiffness changes.

Subtle changes in surface-tethered groups on PEGylated DNA nanoparticles significantly influence gene transfection and cellular uptake

PEGylation strategy has been widely used to enhance colloidal stability of polycation/DNA nanoparticles (NPs) for gene delivery. To investigate the effect of polyethylene glycol (PEG) terminal groups on the transfection properties of these NPs, we synthesized DNA NPs using PEG-g-linear polyethyleneimine (lPEI) with PEG terminal groups containing alkyl chains of various lengths with or without a hydroxyl terminal group. For both alkyl- and hydroxyalkyl-decorated NPs with PEG grafting densities of 1.5, 3, or 5% on lPEI, the highest levels of transfection and uptake were consistently achieved at intermediate alkyl chain lengths of 3 to 6 carbons, where the transfection efficiency is significantly higher than that of nonfunctionalized lPEI/DNA NPs. Molecular dynamics simulations revealed that both alkyl- and hydroxyalkyl-decorated NPs with intermediate alkyl chain length exhibited more rapid engulfment than NPs with shorter or longer alkyl chains. This study identifies a new parameter for the engineering design of PEGylated DNA NPs.

Adsorption of plasma proteins onto PEGylated single-walled carbon nanotubes: The effects of protein shape, PEG size and grafting density

Single-walled carbon nanotubes (SWCNTs) covalently functionalized or noncovalently coated with polyethylene glycol (PEG) of different sizes (Mw=2000 and 5000) and grafting densities (5–16 PEGs per SWCNT) are simulated with human fibrinogen (HFG) and serum albumin (HSA). Proteins migrate toward the SWCNT, but their adsorption extents differ. The extent of the HFG-SWCNT binding decreases with increasing PEG size and grafting density because PEGs more completely cover SWCNTs and thus block hydrophobic interactions between HFGs and SWCNTs, which occurs on PEG-functionalized SWCNTs but not on PEG-coated ones. In particular, the HFG-SWCNT binding significantly decreases in the transition region of PEG conformation from mushroom to brush, where PEGs extend like brushes as described in the Alexander-de Gennes theory. While the HFG adsorption is modulated by PEG conformation, the HSA adsorption is much weaker and less influenced by PEG, because spherical HSAs can bind to the restricted area of the SWCNT and thus cannot bind to the SWCNT as tightly as do linear HFGs. These findings agree with experiments showing less adsorption of proteins on the SWCNT functionalized with larger and more PEGs, and support experimental suggestions regarding the dependence of protein adsorption on protein shape and the mushroom-brush transition of PEG conformation.

Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle-Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage.

In this study, interactions of polyethylene glycol (PEG)-coated gold nanoparticles (AuNPs) with cells were investigated with particular focus on the relationship between the PEG layer properties (conformation, grafting density, and hydrodynamic volume) and cell cycle arrest, apoptosis, and DNA damage. Steric hindrance and PEG hydrodynamic volume controlled the protein adsorption, whereas the AuNP core size and PEG hydrodynamic volume were primary factors for cell uptake and viability. At all PEG grafting densities, the particles caused significant cell cycle arrest and DNA damage against CaCo2 and PC3 cells without apoptosis. However, at a particular PEG grafting density (∼0.65 chains/nm(2)), none of these severe damages were observed on 3T3 cells indicating discriminating behavior of the healthy (3T3) and cancer (PC3 and CaCo2) cells. It was concluded that the PEG grafting density and hydrodynamic volume, tuned with the PEG concentration and AuNP size, played an important role in particle-cell interactions.

Adsorption of Plasma Proteins onto PEGylated Lipid Bilayers: The Effect of PEG Size and Grafting Density.

Lipid bilayers grafted with polyethylene glycol (PEG) of different sizes (Mw = 750, 2000, and 5000) and grafting densities (1.6-25 mol % of PEGylated lipid in dipalmitoylphosphatidylcholine (DPPC) lipid molecules) were simulated with human serum albumin (HSA) using coarse-grained force fields. At low enough grafting density, the PEG has a conformation similar to that of an isolated chain in water, and its Flory radius RF is smaller than the distance between the grafting points (d), which is the so-called "mushroom" regime. In contrast, densely grafted PEG chains (RF > d) extend like brushes, with brush layer thickness given by the Alexander-de Gennes theory. A nearly spherical HSA added to this simulation migrates to the bilayer surface because of the charge interactions between anion residues of HSA and cationic cholines of DPPC, but this HSA-bilayer binding can be sterically suppressed by the PEG chains to an extent that depends on the PEG size and grafting density. In particular, regardless of the extent of the coverage of the PEG on the bilayer, the binding between HSAs and bilayers is suppressed by the PEG layer in a brush but not in a mushroom, indicating that the attractive force between proteins and bilayers can overcome the steric effect of the PEG layer in the mushroom state or in the transition region from mushroom to brush. This helps explain and clarify experiments that show much less adsorption of plasma proteins onto the particle or membrane surface when PEGs are in the brush rather than in the mushroom state.

Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods.

Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations.

Dispersion and bilayer interaction of single-walled carbon nanotubes modulated by covalent and noncovalent PEGylation

Single-walled carbon nanotubes (SWNTs) covalently functionalised with polyethylene glycol (PEG) or noncovalently coated with PEGylated lipids were simulated in water and in lipid bilayers at different PEG sizes and grafting densities using coarse-grained force fields. Starting with the random position of three SWNT–PEG complexes in water, larger PEGs at higher grafting densities more significantly inhibit the aggregation of SWNTs because of larger radii of gyration and hydrodynamic radii of the SWNT–PEG complex, which influence the thickness and the wrapping extent of PEG layer. In particular, PEG-functionalised SWNTs, where PEGs are evenly grafted along the SWNT, disperse, while PEG-coated SWNTs aggregate because SWNTs are less covered by randomly adsorbed PEGylated lipids. Simulations of SWNT–PEGs in lipid bilayers show that PEG (Mw = 550 and 2000)-functionalised SWNTs bind to the bilayer surface but do not insert into the bilayer, while PEG-coated SWNTs insert into the bilayer because PEGylated lipids detach from SWNTs and mix with bilayer lipids. These findings support recent experiments at the same PEG size and density, which suggested that PEG-coated SWNTs may form bundles and thus cannot be easily excreted through the renal route, while PEG-functionalised SWNTs may remain individual and thus show more renal excretion.

Polyethylene Glycol Density and Length Affects Nanoparticle Uptake by Cancer Cells

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.

Evading Immune Cell Uptake and Clearance Requires PEG Grafting at Densities Substantially Exceeding the Minimum for Brush Conformation

Coating nanoparticles with polyethylene glycol (PEG), which reduces particle uptake and clearance by immune cells, is routinely used to extend the circulation times of nanoparticle therapeutics. Nevertheless, due to technical hurdles in quantifying the extent of PEG grafting, as well as in generating very dense PEG coatings, few studies have rigorously explored the precise PEG grafting density necessary to achieve desirable "stealth" properties. Here, using polymeric nanoparticles with precisely tunable PEG grafting, we found that, for a wide range of PEG lengths (0.6-20 kDa), PEG coatings at densities substantially exceeding those required for PEG to adopt a "brush" conformation are exceptionally resistant to uptake by cultured human macrophages, as well as primary peripheral blood leukocytes. Less than 20% of these nanoparticles were cleared from the blood after 2 h (t1/2 ∼ 14 h) in BALB/c mice, whereas slightly less densely PEGylated and uncoated control particles were both virtually eliminated within 2 h. Our results suggest that the stealth properties of PEG-coated nanoparticles are critically dependent on achieving PEG grafting at densities exceeding those required for brush conformation.

Molecular Modeling of PEGylated Peptides, Dendrimers, and Single-Walled Carbon Nanotubes for Biomedical Applications

Polyethylene glycol (PEG) has been conjugated to many drugs or drug carriers to increase their solubility and circulating lifetime, and reduce toxicity. This has motivated many experimental studies to understand the effect of PEGylation on delivery efficiency. To complement the experimental findings and uncover the mechanism that cannot be captured by experiments, all-atom and coarse-grained molecular dynamics (MD) simulations have been performed. This has become possible, due to recent advances in simulation methodologies and computational power. Simulations of PEGylated peptides show that PEG chains wrap antimicrobial peptides and weaken their binding interactions with lipid bilayers. PEGylation also influences the helical stability and tertiary structure of coiled-coil peptides. PEGylated dendrimers and single-walled carbon nanotubes (SWNTs) were simulated, showing that the PEG size and grafting density significantly modulate the conformation and structure of the PEGylated complex, the interparticle aggregation, and the interaction with lipid bilayers. In particular, simulations predicted the structural transition between the dense core and dense shell of PEGylated dendrimers, the phase behavior of self-assembled complexes of lipids, PEGylated lipids, and SWNTs, which all favorably compared with experiments. Overall, these new findings indicate that simulations can now predict the experimentally observed structure and dynamics, as well as provide atomic-scale insights into the interactions of PEGylated complexes with other molecules.

Molecular Dynamics Studies of PEGylated Single-Walled Carbon Nanotubes: The Effect of PEG Size and Grafting Density

Single-walled carbon nanotubes (SWNTs) covalently or noncovalently modified with polyethylene glycol (PEG) of different sizes (Mw = 550, 2000, 5000, and 7000) and grafting densities (5–16 PEGs per SWNT) were simulated using coarse-grained force fields. The covalently grafted PEGs are evenly distributed on SWNTs, while the noncovalently PEGylated SWNTs show the random distribution of PEGylated lipids adsorbed to the SWNT, in which the SWNT sidewall is less completely wrapped by PEGs and thus largely exposed to water, as was observed in experiments. For covalently PEGylated SWNTs, longer PEG chains with higher grafting density yield a larger size, more isotropic shape, and lower diffusivity of the SWNT–PEG complex. In particular, at low grafting density, the thickness of the PEG layer on SWNTs nearly equals the size of the mushroom, where the end-to-end distance of PEGs is smaller than the distance between the grafting points, similar to the conformation of an isolated chain in water. However, at high graft...

DEVELOPMENT OF PEG-CONTAINING BRUSH COPOLYMER: THEIR EFFECT ON RESISTANCE TO PROTEIN ADSORPTION BEHAVIORS

Polyethylene glycol (PEG) has been grafted onto surface of many medical devices to reduce or eliminate protein adsorption that often leads to infection or inflammatory responses. Besides commonly used surface activation strategies, here in this work, the novel functional brush copolymer poly (vinyl alcohol)-g-PEG-co-polyurethane (PVA-g-(PEG-co-PU)) was first synthesized, characterized, and evaluated. PVA is used here as a versatile backbone platform for grafting functional molecules. On the one hand, the PU molecules can attach to the surface of desired PU-based implants, on the other hand, the presence of densely grafted PEG at the materials–tissue interface effectively impedes nonspecific protein adsorption. These copolymers with various grafting ratio of PEG and PU were analyzed by Fourier transform infrared spectroscopy and gel permeation chromatography. The brush copolymers that were obtained were further coated on the PU surface and contact angle measurements were performed. It was found that the brush copolymers turn the surface more hydrophilic with increasing PEG grafting density. Furthermore, the effects of PEG ratio on protein adsorption-resistant behavior were investigated. Protein adsorption was found to be the lowest on the surfaces with the highest PEG grafting density on PVA backbone. The study demonstrated unique brush copolymer to work as a potentially useful coating material.

Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I: Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions

Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val4-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media.