Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

PEGylation of bovine serum albumin using click chemistry for the application as drug carriers

Monomethyl poly(ethylene glycol) (mPEG)-modified bovine serum albumin (BSA) conjugates (BSA-mPEG) were obtained by the mild Cu(I)-mediated cycloaddition reaction of azided BSA (BSA-N(3) ) and alkyne-terminated mPEG. The structure and characteristics of BSA-mPEG conjugates were thoroughly investigated. There were about two PEG chains conjugated onto each BSA molecule as determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. The intrinsic nonspecific binding ability of BSA was used for adsorption and sustained release of both rifampicn and 5-fluorouracil (5-FU). The helical structures of BSA were preserved to a large extent after modification and drug adsorption on BSA was confirmed via circular dichroism spectroscopy. Drugs adsorbed onto the conjugated formulation to a lesser extent than on BSA due to mPEG modification. The in vitro release of both rifampicin and 5-FU, however, indicated that BSA-mPEG can function as a drug carrier. Overall, the click reaction provided a convenient tool for the pegylation of BSA. The biological activity of the BSA-mPEG conjugates, including the drug transportation capacity and biocompatibility, were largely retained.     

Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces

Poly(ethylene) glycol (PEG) is an excellent material to modify surfaces to resist non-specific protein adsorption. Linear PEG has been extensively studied both theoretically and experimentally and it has been found that resistance of PEG-coated surfaces to protein adsorption depends mainly on the molecular weight of the polymer and the surface grafting density. End-functionalized star-shaped PEGs allow for interpolymer crosslinking to form a dense layer. An excellent example of such a system consists of a 6-arm PEG/PPG (4 : 1) star polymer functionalized with isocyanate using IPDI. The end functionalization may be further biofunctionalized to recognize specific biomolecules such as streptavidin, His-tagged proteins, amino-terminated oligonucleotides and cell receptors. This functionalization may be patterned into specific geometries using stamping techniques or randomly distributed by statistical reaction of the end group with the biofunctional molecule in solution. The surface preparation uses simple spin-, dip- or spray-coating and produces smooth layers with low background fluorescence. These properties, together with the advantageous chemical properties of PEG, render the surfaces ideal for immobilizing proteins on surfaces with detection limits down to the single molecule level. Proteins immobilized on such surfaces are able to maintain their folded, functional form and are able to completely refold if temporarily exposed to denaturing conditions. Immobilized enzyme molecules were able to perform their function with the same activity as the enzyme in solution. Future directions of using surfaces coated with such crosslinked star polymers in highly sensitive and robust biotechnology applications will be discussed.     

Patterned biofunctional poly(acrylic acid) brushes on silicon surfaces

Protein patterning was carried out using a simple procedure based on photolithography wherein the protein was not subjected to UV irradiation and high temperatures or contacted with denaturing solvents or strongly acidic or basic solutions. Self-assembled monolayers of poly(ethylene glycol) (PEG) on silicon surfaces were exposed to oxygen plasma through a patterned photoresist. The etched regions were back-filled with an initiator for surface-initiated atom transfer radical polymerization (ATRP). ATRP of sodium acrylate was readily achieved at room temperature in an aqueous medium. Protonation of the polymer resulted in patterned poly(acrylic acid) (PAA) brushes. A variety of biomolecules containing amino groups could be covalently tethered to the dense carboxyl groups of the brush, under relatively mild conditions. The PEG regions surrounding the PAA brush greatly reduced nonspecific adsorption. Avidin was covalently attached to PAA brushes, and biotin-tagged proteins could be immobilized through avidin-biotin interaction. Such an immobilization method, which is based on specific interactions, is expected to better retain protein functionality than direct covalent binding. Using biotin-tagged bovine serum albumin (BSA) as a model, a simple strategy was developed for immobilization of small biological molecules using BSA as linkages, while BSA can simultaneously block nonspecific interactions.     

Surface PEGylation via native chemical ligation

Native chemical ligation (NCL) is an emerging chemoselective chemistry that forms an amide bond by trans-thioesterification followed by intramolecular nucleophilic rearrangement between thioester and cysteine. The reaction is simple, occurs in a mild aqueous solution, and gives near-quantitative yields of a desired product. Since the first report in 1994, most studies involving the use of NCL have focused on the total synthesis of proteins to address fundamental questions pertaining to many aspects of protein science, such as folding, mirror images, and site-specific labeling of proteins, but applications of the NCL reaction for other areas remain largely unexplored. Herein, we present a facile strategy for surface immobilization of poly(ethylene glycol) (PEG) utilizing the NCL reaction. Surface immobilization of PEG (i.e., PEGylation) plays a key role in preventing nonspecific protein adsorption on surfaces, which is crucial in a wide variety of medical devices. Using cysteine-PEG and thioester-containing phosphonic acid conjugates, we achieved efficient surface PEGylation on titanium surfaces. Ellipsometry, goniometry, and X-ray photoelectron spectroscopy (XPS) unambiguously confirmed the presence of PEGs, which provided nonfouling effects of surfaces. This study indicates that the NCL reaction will be a useful toolkit for surface bioconjugation and functionalization.     

Immobilization of oligonucleotides on poly(ethylene glycol) brush-coated Si surfaces

High-density poly(ethylene glycol) (PEG) molecules are grafted onto Si surfaces in a brush-like configuration. We demonstrate that this surface is an excellent substrate for oligonucleotide immobilization. p-Maleimidophenyl isocyanate is used as a heterobifunctional cross-linker to tether thiol-modified oligonucleotides to terminal OH groups on the PEG brush. This approach gives excellent immobilization specificity and low background. The immobilized oligonucleotides show high sensitivity for the detection of complementary targets.     

Immobilization by Adsorption of Hydrophobic Lipase Derivatives to Porous Polymer Beads for Use in Ester Synthesis

A novel procedure for attaching lipase to certain kinds of hydrophobic surfaces is described. The procedure involves covalent derivatization of the protein molecule by reaction in solution with a hydrophobic imidoester, aldehyde or activated polyethylene glycol. The resulting protein derivative is then allowed to adsorb onto an insoluble hydrophobic surface. Quantitative adsorption is observed and the enzyme is bound very strongly on the support The number and nature of the hydrophobic substituents introduced in the chemical derivatization step can be easily controlled. The adsorption step occurs spontaneously upon exposure of the modified protein to a variety of hydrophobic materials. The hydrophobic lipase derivative obtained by reaction with PEG activated with p-nirrophenyl chloroformate, for example, adsorbs readily onto polyacrylate and polystyrene beads, with most of its esterification activity in organic solvent intact. Its thermostability is also greatly enhanced. Derivatization of lipase with hydrophobic groups greatly enhances its esterification activity in organic solvent, and its immobilization in this manner enables the preparation of a highly reactive biocatalyst for biotechnological application.     

Dual-functional biomimetic materials: nonfouling poly(carboxybetaine) with active functional groups for protein immobilization

We introduce a dual-functional biocompatible material based on zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), which not only highly resists protein adsorption/cell adhesion, but also has abundant functional groups convenient for the immobilization of biological ligands, such as proteins. The dual-functional properties are unique to carboxybetaine moieties and are not found in other nonfouling moieties such as ethylene glycol, phosphobetaine, and sulfobetaine. The unique properties are demonstrated in this work by grafting a polyCBMA polymer onto a surface or by preparing a polyCBMA-based hydrogel. PolyCBMA brushes with a thickness of 10-15 nm were grafted on a gold surface using the surface-initiated atom transfer radical polymerization method. Protein adsorption was analyzed using a surface plasmon resonance sensor. The surface grafted with polyCBMA very largely prevented the nonspecific adsorption of three test proteins, that is, fibrinogen, lysozyme, and human chorionic gonadotropin (hCG). The immobilization of anti-hCG on the surface resulted in the specific binding of hCG while maintaining a high resistance to nonspecific protein adsorption. Transparent polyCBMA-based hydrogel disks were decorated with immobilized fibronectin. Aortic endothelial cells did not bind to the polyCBMA controls, but appeared to adhere well and spread on the fibronectin-modified surface. With their dual functionality and biomimetic nature, polyCBMA-based materials are very promising for their applications in medical diagnostics, biomaterials/tissue engineering, and drug delivery.     

Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers

A two-step approach is described to chemically camouflage the inert surface of model polystyrene nanospheres of 60 nm in diameter against recognition by the body's defenses. The first step was based on the strong protein adsorbing potency of polystyrene, and the second step utilized the chemical reactivity of the adsorbed proteins for conjugation with cyanuric chloride-activated methoxypoly(ethyleneglycol)5000, mPEG5000. Bovine serum albumin (BSA) and rat IgG were used as models of non-immune and immune proteins, respectively. The maximum adsorbance values for both proteins were near expectation for a close-packed monolayer. Adsorption isotherms studies and analysis of the hydrodynamic thickness of the adsorbed protein layer confirmed the close-packed side-on mode of adsorption for BSA and the end-on mode of adsorption for IgG, respectively. Nucleophiles on the adsorbed proteins were then reacted with cyanuric chloride activated mPEG5000. The average poly(ethyleneglycol) (PEG) content for a 60-nm nanospheres was found to be 13.7+/-0.4 micromol PEG/micromol BSA and 3.6+/-0.3 micromol PEG/micromol IgG. The interaction of both PEG-bearing nanospheres with the hydrophobic column material octyl-agarose indicated surface heterogeneity among the nanospheres. Only nanospheres with the most hydrophilic phenotype (approximately 70% of the total population) exhibited stealth properties after intravenous injection to rats. In contrast to the described approach, incubation of uncoated nanospheres with preformed BSA-mPEG5000 conjugates failed to produce long circulating entities. For design of splenotropic particles cyanuric chloride-activated mPEG5000 was conjugated to BSA-coated polystyrene beads of 225 nm in diameter. Despite their stealth property to hepatic Kupffer cell recognition, these nanospheres were cleared by the splenic red pulp macrophages.     

Controlling the reactivity of ampiphilic quantum dots in biological assays through hydrophobic assembly of custom PEG derivatives

Modifications of the quantum dot (QD) surface are routinely performed via covalent biomolecule attachment, and poly(ethylene glycol) (PEG) derivatization has previously been shown to limit nonspecific cellular interactions of QD probes. Attempts to functionalize ampiphilic QDs (AMP-QDs) with custom PEG derivatives having a hydrophobic terminus resulted in self-assembly of these PEG ligands to the AMP-QD surface in the absence of covalent coupling reagents. We demonstrate, via electrophoretic characterization techniques, that these self-assembled PEG-QDs exhibit improved passivation in biological environments and are less susceptible to unwanted protein adsorption to the QD surface. We highlight the artifactual fluorescent response protein adsorption can cause in biological assays, and discuss considerations for improved small molecule presentation to facilitate specific QD interactions.     

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

Patterning of proteins and cells on functionalized surfaces prepared by polyelectrolyte multilayers and micromolding in capillaries

A method for protein and cell patterning on polyelectrolyte-coated surfaces using simple micromolding in capillaries (MIMIC) is described. MIMIC produced two distinctive regions. One contained polyethylene glycol (PEG) microstructures fabricated using photopolymerization that provided physical, chemical, and biological barriers to the nonspecific binding of proteins, bacteria, and fibroblast cells. The second region was the polyelectrolyte (PEL) coated surface that promoted protein and cell immobilization.

The difference in surface functionality between the PEL region and background PEG microstructures resulted in simple patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin, E. coli expressing green fluorescence protein (GFP), and fibroblast cells were successfully bound to the exposed PEL surfaces at micron scale. Compared with the simple adsorption of protein, fluorescence intensity was dramatically improved (by about six-fold) on the PEL-modified surfaces. Although animal cell patterning is prerequisite for adhesive protein layer to survive on desired area, the PEL surface without adhesive proteins provides affordable microenvironment for cells.

The simple preparation of functionalized surface but universal platform can be applied to various biomolecules such as proteins, bacteria, and cells.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Comparison of coatings from reactive star shaped PEG-stat-PPG prepolymers and grafted linear PEG for biological and medical applications

Grafting of poly(ethylene glycol) (PEG) is a common strategy for reducing nonspecific interactions of surfaces with proteins. We have used grafting at "cloud point" solution conditions that ensures maximum grafting density of linear methoxy terminated PEG-aldehyde (mPEG-ald, M(w) = 5000 and 30000). In an alternative approach, surfaces were modified with layers prepared from isocyanate terminated, star shaped poly(ethylene glycol-stat-propylene glycol) prepolymers (80% ethylene glycol, six arms, M(w) = 3000, 12,000, and 18,000; this compound will be referred to as "Star PEG" in the text). Due to the highly reactive endgroups, these molecules form a dense network on the substrate with a high polymer surface coverage. The two systems were compared regarding their ability to prevent unspecific adsorption of insulin and lysozyme. The layers were analyzed by ellipsometry, contact angle measurements, and XPS. Protein adsorption was monitored by surface MALDI-TOF MS and fluorescence microscopy. No protein adsorption could be detected on Star PEG coatings and on mPEG-ald 5000, whereas mPEG-ald 30,000 could only prevent adsorption of lysozyme but not of the smaller insulin.     

Covalent attachment of three derivatives of pegylated RGD peptides on the NH2-terminated silicon surfaces: Impact on fibroblast cell behavior

This paper describes a simple strategy for covalent immobilization of the NHS-PEG-RGD peptide with the three different PEG lengths (8, 13, and 22) onto the amine-terminated monolayers with the subsequent investigation of fibroblast cellular response to the three derivatives of pegylated RGD peptides-modified substrates. First, acetamide-terminated monolayers were prepared on the hydride terminated silicon surface to protect NH2-terminated monolayers. This was followed by the removal of the protective groups, and the reaction of NHS-PEG8-RGD, NHS-PEG13-RGD and NHS-PEG22-RGD peptides with the NH2-terminated monolayers while reducing nonspecific protein adsorption. Analysis of X-ray photoelectron spectroscopy (XPS), Fourier Transform Infrared (ATR-FTIR) spectroscopy, and Ellipsometry measurements demonstrated that PEG13-RGD peptide forms relatively a more homogenous, thicker and stable structure compared with those of PEG8-RGD and PEG22-RGD peptide. The quantitative and qualitative assessment of cell adhesion, spreading, and proliferation indicated that relatively further elongated fibroblast cells attached on the PEG13-RGD peptide relative to those on the PEG8-RGD and PEG22-RGD peptide. The results presented here may offer a developed strategy based on the length of the spacer to regulate cellular behavior on the surface substrates.     

Development of affinity chromatography using a bioactive peptide as a ligand

By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.     

A new metal chelate affinity adsorbent for cytochrome C

We have prepared a novel metal-chelate adsorbent utilizing N-methacryloyl-L-histidine methyl ester (MAH) as a metal-chelating ligand. MAH was synthesized by using methacryloyl chloride and l-histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu(2+) ions were chelated directly on the chelating beads. Cu(2+)-chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu(2+)-chelated beads (658.2 micromol/g Cu(2+) loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu(2+) loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris-HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.     

Zwitterionic polymers exhibiting high resistance to nonspecific protein adsorption from human serum and plasma

This study examined six different polymer and self-assembled monolayer (SAM) surface modifications for their interactions with human serum and plasma. It was demonstrated that zwitterionic polymer surfaces are viable alternatives to more traditional surfaces based on poly(ethylene glycol) (PEG) as nonfouling surfaces. All polymer surfaces were formed using atom transfer radical polymerization (ATRP) and they showed an increased resistance to nonspecific protein adsorption compared to SAMs. This improvement is due to an increase in the surface packing density of nonfouling groups on the surface, as well as a steric repulsion from the flexible polymer brush surfaces. The zwitterionic polymer surface based on carboxybetaine methacrylate (CBMA) also incorporates functional groups for protein immobilization in the nonfouling background, making it a strong candidate for many applications such as in diagnostics and drug delivery.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

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.