Weifeng Lin

Investigation of the Hydration of Nonfouling Material Poly(sulfobetaine methacrylate) by Low-Field Nuclear Magnetic Resonance

The strong surface hydration layer of nonfouling materials plays a key role in their resistance to nonspecific protein adsorption. Poly(sulfobetaine methacrylate) (polySBMA) is an effective material that can resist nonspecific protein adsorption and cell adhesion. About eight water molecules are tightly bound with one sulfobetaine (SB) unit, and additional water molecules over 8:1 ratio mainly swell the polySBMA matrix, which is obtained through the measurement of T(2) relaxation time by low-field nuclear magnetic resonance (LF-NMR). This result was also supported by the endothermic behavior of water/polySBMA mixtures measured by differential scanning calorimetry (DSC). Furthermore, by comparing both results of polySBMA and poly(ethylene glycol) (PEG), it is found that (1) the hydrated water molecules on the SB unit are more tightly bound than on the ethylene glycol (EG) unit before saturation, and (2) the additional water molecules after forming the hydration layer in polySBMA solutions show higher freedom than those in PEG. These results might illustrate the reason for higher resistance of zwitterionic materials to nonspecific protein adsorptions compared to that of PEGs.

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

A comprehensive insight into the interaction between proteins and poly(ethylene glycol) (PEG) is crucial to understand the behavior of PEG, which is widely used in pharmaceutical and medical applications. Although PEG is believed to be an excellent material to resist non-specific protein adsorption, there is a lack of quantitative information about the interactions between proteins and PEG. In this paper the interactions of bovine serum albumin (BSA) and lysozyme (LYZ) with different molecular weight (MW) PEGs were investigated through the T2 relaxation time of PEGs measured by low field nuclear magnetic resonance spectroscopy. The integrated signal intensity of PEGs was quantified under various conditions from the concentrations and MWs of PEG, and ionic strength of solutions, as well as the molar ratios of PEG to protein. The results show that a large number of PEG molecules could associate with protein molecules with association constants in the range ~10(4)-10(5) M(-1). The association constant is insensitive to the ionic strength change in the physiological range and the lowest associate constant occurs at the medium MW PEG with protein. This suggests that the interaction between PEG and protein molecules might not be negligible in investigations of the resistance to non-specific protein adsorption. Long chain PEG coatings might cause modest protein adsorption, which could interfere with any weak specific interaction between ligand and receptor. Thus, it is necessary to reconsider the popular accepted method of protecting nanoparticles (NP) in blood with long chain PEG coatings since these NPs might be surrounded by a layer of weakly adsorbed plasma protein in the circulatory system.

Probing the weak interaction of proteins with neutral and zwitterionic antifouling polymers

Protein-polymer interactions are of great interest in a wide range of scientific and technological applications. Neutral poly(ethylene glycol) (PEG) and zwitterionic poly(sulfobetaine methacrylate) (pSBMA) are two well-known nonfouling materials that exhibit strong surface resistance to proteins. However, it still remains unclear or unexplored how PEG and pSBMA interact with proteins in solution. In this work, we examine the interactions between two model proteins (bovine serum albumin and lysozyme) and two typical antifouling polymers of PEG and pSBMA in aqueous solution using fluorescence spectroscopy, atomic force microscopy and nuclear magnetic resonance. The effect of protein:polymer mass ratios on the interactions is also examined. Collective data clearly demonstrate the existence of weak hydrophobic interactions between PEG and proteins, while there are no detectable interactions between pSBMA and proteins. The elimination of protein interaction with pSBMA could be due to an enhanced surface hydration of zwitterionic groups in pSBMA. New evidence is given to demonstrate the interactions between PEG and proteins, which are often neglected in the literature because the PEG-protein interactions are weak and reversible, as well as the structural change caused by hydrophobic interaction. This work provides a better fundamental understanding of the intrinsic structure-activity relationship of polymers underlying polymer-protein interactions, which are important for designing new biomaterials for biosensor, medical diagnostics and drug delivery applications.

Reducing the Cytotoxity of Poly(amidoamine) Dendrimers by Modification of a Single Layer of Carboxybetaine

The surface primary amines of generation five poly(amido amine) (G5 PAMAM) dendrimer were modified by different amounts of carboxybetaine acrylamide (CBAA). As a result, the fully modified molecules (CBAA-PAMAM-20, obtained from the 20:1 molar ratio of CBAA molecules to amino groups in modification solution) show excellent compatibility with protein and cells. CBAA-PAMAM-20 and fibrinogen (Fg) could coexist in solution without forming aggregation, indicating very weak interaction force between CBAA-PAMAM-20 and fibrinogen. CBAA-PAMAM-20 exhibits almost undetectable hemolytic activity, while other partially modified ones cause severe hemolysis and fibrinogen aggregation. Furthermore, the membrane of human umbilical vascular endothelial cell (HUVEC) remains intact after 24 h incubation with CBAA-PAMAM-20. The cytotoxicity assay of HUVEC cells and KB cells also showed that the CBAA-PAMAM-20 was not cytotoxic up to a 2 mg/mL concentration (>90% cell viability). In short, a thin compact layer of zwitterionic carboxybetaine could reduce the cytotoxicity of PAMAM through minimizing the interaction with protein and cell membranes, which suggest that the carboxybetaine-coated PAMAM could be a useful platform for biocompatible carriers to load contrast agents and drugs.

Binding characteristics between polyethylene glycol (PEG) and proteins in aqueous solution

Polymer-protein interactions are crucial for determining the activity of both polymer and protein for many bio-related applications. Poly(ethylene glycol) (PEG) as a well-known antifouling material is often coated on surfaces to form highly solvated brushes, which exhibit excellent protein-repellent properties. However, unlike surface-induced antifouling effects, little is known about the intrinsic PEG-protein interactions in aqueous solution, which is an important yet neglected problem. Here, we investigate the interactions between PEG and proteins in aqueous solution using fluorescence spectroscopy, atomic force microscopy (AFM), and nuclear magnetic resonance (NMR). Two important characteristics, molecular weight of PEG and mass ratio of PEG : protein, are examined to determine the effect of each on PEG-protein interactions as well as binding characteristics between PEG and proteins. In contrast to too long and too short PEG chains, collective results have shown that PEG with optimal molecular weight (MW) is more capable of interacting with proteins, which induces the conformational change of proteins through more stable binding sites and stronger interactions with long chain PEG. Enhanced PEG-protein interactions are likely due to the change of hydrophilicity to amphiphilicity of PEG with increasing MWPEG. In contrast to almost none or weak interactions of PEG surfaces with proteins, this work provides new evidence to demonstrate the existence of interactions between PEG and proteins in aqueous solution, which is important not only for better understanding of the structure-activity relationship of PEG both in solution and on surfaces, but also for the rational design of new PEG-based materials for specific applications.

Development of Zwitterionic Polymer-Based Doxorubicin Conjugates: Tuning the Surface Charge To Prolong the Circulation and Reduce Toxicity

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.

Development of Nonstick and Drug-Loaded Wound Dressing Based on the Hydrolytic Hydrophobic Poly(carboxybetaine) Ester Analogue

A novel biocompatible polymer is developed for antimicrobial and nonstick coatings of wound dressing. The polymer is formed by copolymerization of carboxybetaine ester analogue methacrylate (CB-ester) and small partial poly(ethylene glycol) methacrylate (PEGMA) for cross-linking by hexamethylene diisocyanate (HDI), which is highly resistant to nonspecific protein adsorption and mammalian cell attachment after a quick hydrolysis. A small hydrophobic drug, aspirin, can be incorporated into the new polymer and slowly released to inhibit microorganism growth while the new polymer shows very low cytotoxicity. Moreover, the wound dressing, the new polymer coated medical gauze, shows good mechanic properties, such as flexibility and strength, for medical application. After all, this new nonfouling polymer offers great potential for an antimicrobial wound dressing and other applications.

Biocompatible long-circulating star carboxybetaine polymers

Polyethylene glycol (PEG) is considered to be the most effective material to prolong the circulation time of nanoparticles by reducing non-specific protein adsorption in blood. However, it is recognized that PEG decomposes in most physiological solutions, and an anti-PEG antibody has been detected in some normal blood donors as a response to injection with PEGylated polymer particles. Zwitterionic polymers are potential alternatives to PEG for biomedical applications because of their super resistance to non-specific protein adsorption. Thus, finding one polymer with a long circulation time and resistance to the immune response is of significant importance. Here, we prepared four star carboxybetaine polymers of different molecular weights via atom transfer radical polymerization (ATRP) from a β-cyclodextrin (β-CD) initiator for investigating the biocompatibility of carboxybetaine polymer, a typical zwitterionic polymer. The circulation half-life of the largest star polymer (123 kDa) in mice was prolonged to 40 h in vivo, with no appreciable damage or inflammation observed in the major organ tissues. Furthermore, the circulation time of repeat injections showed similar results to the first injection, with no obvious increase in the amount of antibody in blood. The internalization of the star carboxybetaine polymers by macrophage cells was a relatively slow process. The high cell viability in the presence of star carboxybetaine polymers up to 2 mg mL-1 was maintained. The hemolytic activity of the star carboxybetaine polymers at 5 mg mL-1 was almost undetectable. In vitro results prove a key prediction of excellent biocompatibility in vivo. All the results suggest that the carboxybetaine polymer, perhaps even most of the zwitterionic ones, might be a good alternative to PEG in the development of a drug delivery system.

A novel zwitterionic copolymer with a short poly(methyl acrylic acid) block for improving both conjugation and separation efficiency of a protein without losing its bioactivity

PEGylation of protein drugs has thus far been the most important method used in improving the stability and the circulation time, while lowering the immunogenicity of protein drugs. However, the loss of bioactivity of PEGylated protein drugs and the low recovery through the complicated separation process are the most challenging issues. To overcome these problems, a zwitterionic block copolymer poly(methyl acrylic acid-b-sulfobetaine methacrylate) (PMAA-b-PSBMA or abbreviated to PMS) has been chosen to modify a protein drug (uricase) since zwitterionic materials are superior to polyethylene glycol (PEG) in resistance to nonspecific protein adsorption, chemical stability and also low cytotoxicity. As indicated by the results, the conjugation between PMS and uricase could be achieved under 0.03 mg mL-1 of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.018 mg mL-1N-hydroxysuccinimide (NHS) through weak charge-induced adsorption of PMS on uricase, which is about 0.3% of the concentration for conventional protein-polymer conjugation. The conjugates, efficiently separated from the reaction solution and re-suspended in physiological solution through pH adjustment from pH 5.35 to 7.4, show about 133% of the original activity and better anti-trypsin digestion than the native uricase. These results demonstrate that the conjugation of a zwitterionic copolymer with a short poly(methyl acrylic acid) block could improve the stability of an enzyme without destructively affecting its bioactivity. This may open a new way for the delivery of therapeutic protein drugs.

Development of robust biocompatible silicone with high resistance to protein adsorption and bacterial adhesion

A new biocompatible silicone comprising a carboxybetaine (CB) ester analogue, 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS) and an organic silicone macromer (bis-α,ω-(methacryloxypropyl) polydimethylsiloxane) has been developed using photo-polymerisation. Following interfacial hydrolysis of the CB ester, the resulting zwitterionic material became significantly more hydrophilic and exhibited high resistance to both non-specific protein adsorption and bacterial adhesion. Moreover, the stability of these non-fouling properties was dramatically improved by using a slow and controlled rate of ester hydrolysis of the original protective hydrophobic matrix. The subsequent ability to maintain the original optical and mechanical properties of the bare silicone following surface activation makes this material an ideal candidate for preparing contact lenses and other medical devices.