Strong Surface Hydration and Salt Resistant Mechanism of a New Nonfouling Zwitterionic Polymer Based on Protein Stabilizer TMAO.

Zwitterionic polymers exhibit excellent nonfouling performance due to their strong surface hydrations. However, salt molecules may severely reduce the surface hydrations of typical zwitterionic polymers, making the application of these polymers in real biological and marine environments challenging. Recently, a new zwitterionic polymer brush based on the protein stabilizer trimethylamine N-oxide (TMAO) was developed as an outstanding nonfouling material. Using surface-sensitive sum frequency generation (SFG) vibrational spectroscopy, we investigated the surface hydration of TMAO polymer brushes (pTMAO) and the effects of salts and proteins on such surface hydration. It was discovered that exposure to highly concentrated salt solutions such as seawater only moderately reduced surface hydration. This superior resistance to salt effects compared to other zwitterionic polymers is due to the shorter distance between the positively and negatively charged groups, thus a smaller dipole in pTMAO and strong hydration around TMAO zwitterion. This results in strong bonding interactions between the O- in pTMAO and water, and weaker interaction between O- and metal cations due to the strong repulsion from the N+ and hydration water. Computer simulations at quantum and atomistic scales were performed to support SFG analyses. In addition to the salt effect, it was discovered that exposure to proteins in seawater exerted minimal influence on the pTMAO surface hydration, indicating complete exclusion of protein attachment. The excellent nonfouling performance of pTMAO originates from its extremely strong surface hydration that exhibits effective resistance to disruptions induced by salts and proteins.

Zwitterionic Nanoconjugate Enables Safe and Efficient Lymphatic Drug Delivery.

The lymphatic system provides a major route for the dissemination of many diseases such as tumor metastasis and virus infection. At present, treating these diseases remains a knotty task due to the difficulty of delivering sufficient drugs into lymphatics. After subcutaneous (SC) injection, the transferring of drugs to lymphatic vessels is significantly attenuated by physiological barriers in the interstitial space. Moreover, SC injection represents a highly challenging administration route for biological drugs, as it increases the risk of undesirable immune responses. Here, we demonstrate a simple and effective strategy to address this dilemma by conjugating protein therapeutics with zwitterionic poly(carboxy betaine) (PCB) polymers. PCB conjugation to l-asparaginase (ASP), a highly immunogenic enzyme drug, manifests to significantly promote the diffusion of ASP into the lymphatic system while mitigating its immunogenicity. This platform will facilitate the development of new therapies against diverse lymph-related diseases by enabling safe and efficient lymphatic drug delivery.

Zwitterionic Polymer Conjugated Glucagon-like Peptide-1 for Prolonged Glycemic Control.

Glucagon-like peptide-1 (GLP-1) is of particular interest for treating type 2 diabetes mellitus (T2DM), as it induces insulin secretion in a glucose-dependent fashion and has the potential to facilitate weight control. However, native GLP-1 is a short incretin peptide that is susceptible to fast proteolytic inactivation and rapid clearance from the circulation. Various GLP-1 analogs and bioconjugation of GLP-1 analogs have been developed to counter these issues, but these modifications are frequently accompanied by the sacrifice of potency and the induction of immunogenicity. Here, we demonstrated that with the conjugation of a zwitterionic polymer, poly(carboxybetaine) (pCB), the pharmacokinetic properties of native GLP-1 were greatly enhanced without serious negative effects on its potency and secondary structure. The pCB conjugated GLP-1 further provided glycemic control for up to 6 days in a mouse study. These results illustrate that the conjugation of pCB could realize the potential of using native GLP-1 for prolonged glycemic control in treating T2DM.

Strong Hydration at the Poly(ethylene glycol) Brush/Albumin Solution Interface.

Albumin molecules are extensively used as biocompatible coatings, and poly(ethylene glycol) (PEG) materials are widely used for antifouling. PEG materials have excellent antifouling property because of their strong surface hydration. Our previous research indicates that hydration at the PEG/bovine serum albumin solution interface is stronger than that at the PEG/water interface. This research shows that this observation is general for different types of albumin molecules. Different albumins including bovine, porcine, rat, rabbit, and sheep serum albumins were studied in this research. It was found that the hydration at the PEG methacrylate (pOEGMA)/albumin solution interface is always stronger than that at the pOEGMA/water interface. Here, we define "strong interfacial hydration" as "ordered strongly hydrogen-bonded interfacial water". We believe that such a strong hydration is because of the strong hydration on the albumin surface, leading to its biocompatible property. All of the albumin molecules demonstrated stronger hydration on the pOEGMA surface compared to other protein molecules such as lysozyme and fibrinogen. The strong hydration on albumin molecules is related to the high surface coverage of glutamic acid and lysine with similar amounts.

Zwitterionic Hydrogels Based on a Degradable Disulfide Carboxybetaine Cross-Linker.

We report the synthesis of a zwitterionic carboxybetaine disulfide cross-linker (CBX-SS) and biodegradable poly(carboxybetaine) (PCB) hydrogels and nanocages (NCs) made using this cross-linker. The structure of CBX-SS combines zwitterionic carboxybetaine to confer nonfouling properties and a disulfide linkage to facilitate degradation. The physical, mechanical, and fouling characteristics of PCB hydrogels cross-linked with CBX-SS were investigated. Then, the degradation characteristics of CBX-SS-cross-linked hydrogels were evaluated through their weight loss and release of an encapsulated protein in a reducing environment. Furthermore, CBX-SS was applied to prepare degradable PCB NCs. Results show that encapsulating the highly immunogenic enzyme uricase in degradable PCB NCs eliminates or prevents an in vivo immune response to both the protein and polymer.

Ultralow Fouling and Functionalizable Surface Chemistry Based on Zwitterionic Carboxybetaine Random Copolymers.

Here, we report a simple yet effective surface-modification approach to imparting hydrophobic surfaces with superhydrophilicity using ultralow fouling/functionalizable carboxybetaine (CB) copolymers via a dip-coating technique. A new series of CB random copolymers with varying amphiphilicities were synthesized and coated on hydrophobic polypropylene (PP) and polystyrene (PS) surfaces. The nonfouling capability of each coating was screened by an enzyme-linked immunosorbent assay (ELISA) and further comprehensively assessed against 100% human serum by a Micro BCA protein assay kit. The random copolymer containing ∼30 mol % CB units showed superhydrophilicity with the highest air contact angle of more than 165° in DI water and the best nonfouling capability against 100% human blood serum. Surfaces of a 96-well plate coated with the optimal CB random copolymer had a significantly better nonfouling capability than those of a commercial 96-well plate with an ultralow attachment surface. The adhesion of mouse embryonic fibroblast cells (NIH3T3) was completely inhibited on surfaces coated with CB random copolymers. Furthermore, the optimal nonfouling CB copolymer surface was functionalized with an antigen via covalent bonding where its specific interactions with its antibody were verified. Thus, this CB random copolymer is capable of imparting both ultralow fouling and functionalizable capabilities to hydrophobic surfaces for blood-contacting devices.

Effect of Surface Hydration on Antifouling Properties of Mixed Charged Polymers.

Interfacial water structure on a polymer surface in water (or surface hydration) is related to the antifouling activity of the polymer. Zwitterionic polymer materials exhibit excellent antifouling activity due to their strong surface hydration. It was proposed to replace zwitterionic polymers using mixed charged polymers because it is much easier to prepare mixed charged polymer samples with much lower costs. In this study, using sum frequency generation (SFG) vibrational spectroscopy, we investigated interfacial water structures on mixed charged polymer surfaces in water and how such structures change while being exposed to salt solutions and protein solutions. The 1:1 mixed charged polymer exhibits excellent antifouling property whereas other mixed charged polymers with different ratios of the positive/negative charges do not. It was found that on the 1:1 mixed charged polymer surface, SFG water signal is dominated by the contribution of the strongly hydrogen bonded water molecules, indicating strong hydration of the polymer surface. The responses of the 1:1 mixed charged polymer surface to salt solutions are similar to those of zwitterionic polymers. Interestingly, exposure to high concentrations of salt solutions leads to stronger hydration of the 1:1 mixed charged polymer surface after replacing the salt solution with water. Protein molecules do not substantially perturb the interfacial water structure on the 1:1 mixed charged polymer surface and do not adsorb to the surface, showing that this mixed charged polymer is an excellent antifouling material.

Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity.

Advances in protein therapy are hindered by the poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity of many therapeutic proteins. Polyethylene glycol conjugation (PEGylation) is the most successful strategy to date to overcome these shortcomings, and more than 10 PEGylated proteins have been brought to market. However, anti-PEG antibodies induced by treatment raise serious concerns about the future of PEGylated therapeutics. Here, we demonstrate a zwitterionic polymer network encapsulation technology that effectively enhances protein stability and PK while mitigating the immune response. Uricase modified with a comprehensive zwitterionic polycarboxybetaine (PCB) network exhibited exceptional stability and a greatly prolonged circulation half-life. More importantly, the PK behavior was unchanged, and neither anti-uricase nor anti-PCB antibodies were detected after three weekly injections in a rat model. This technology is applicable to a variety of proteins and unlocks the possibility of adopting highly immunogenic proteins for therapeutic or protective applications.

Polypeptides with High Zwitterion Density for Safe and Effective Therapeutics.

The commonly used "stealth material" poly(ethylene glycol) (PEG) effectively promotes the pharmacokinetics of therapeutic cargos while reducing their immune response. However, recent studies have suggested that PEG could induce adverse reactions, including the emergence of anti-PEG antibodies and tissue histologic changes. An alternative stealth material with no or less immunogenicity and organ toxicity is thus urgently needed. We designed a polypeptide with high zwitterion density (PepCB) as a stealth material for therapeutics. Neither tissue histological changes in liver, kidney, or spleen, nor abnormal behavior, sickness or death was induced by the synthesized polymer after high-dosage administration for three months in rats. When conjugated to a therapeutic protein uricase, the uricase-PepCB bioconjugate showed significantly improved pharmacokinetics and immunological properties compared with uricase-PEG conjugates.

Sensitive and Quantitative Detection of Anti-Poly(ethylene glycol) (PEG) Antibodies by Methoxy-PEG-Coated Surface Plasmon Resonance Sensors.

Pre-existing and induced anti-poly(ethylene glycol) (PEG) antibodies (abs) have been shown to be related with limitation of therapeutic efficacy and reduction in tolerance of several therapeutic agents. However, the current methods to detect anti-PEG abs are tedious and usually lack quantification. A facile, rapid, sensitive, and reliable technique to detect anti-PEG abs is highly desired in both research and clinic settings. In this work, we have presented a surface plasmon resonance (SPR) biosensor technique for the detection of anti-PEG abs and compared three PEG surface chemistries. Methoxy-PEG (mPEG) 5k was found to have the best performance. The detection of anti-PEG abs directly from diluted blood serum was achieved within 40 min. Detection sensitivity is as good as or better than enzyme-linked immunosorbent assay (ELISA). Furthermore, different antibody isotypes can be quantitatively differentiated by adopting secondary antibodies. A pilot study has been performed to analyze clinical blood samples using this technology, demonstrating its potential as a convenient and powerful method to prescreen and monitor anti-PEG abs in the patients before or after they receive treatment with PEG-containing drugs.

Paper Sensor Coated with a Poly(carboxybetaine)-Multiple DOPA Conjugate via Dip-Coating for Biosensing in Complex Media.

Cellulose paper is an ideal diagnostic platform for low-cost, easily disposable and lightweight implementation, but requires surface modification to achieve detection with high sensitivity and specificity in complex media. In this work, a polymer-catechol conjugate containing a superhydrophilic nonfouling poly(carboxylbetaine) (pCB) and four surface-binding l-3,4-dihydroxyphenylalanine (DOPA) groups, pCB-(DOPA)4, were applied onto a paper-based sensor surface via a simple "graft-to" immersion process to render the surface with both nonfouling and protein functionalizable properties. This dip-coating technique is effective, convenient and robust as compared to the "graft-from" techniques reported previously with similar nonfouling properties. The coated paper sensor showed both increased analyte diffusion rate and improved sensitivity of glucose detection in human blood serum. The capability of pCB-(DOPA)4-modified paper sensor for specific antigen-antibody detection was demonstrated via the covalent immobilization of bovine serum albumin antibody (anti-BSA) and fibrinogen antibody (anti-Fg) onto the pCB-coated surface via simple 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry.

Poly(ectoine) Hydrogels Resist Nonspecific Protein Adsorption.

The development of nonfouling zwitterionic materials has a wide range of biomedical and engineering applications. This work delineates the design and synthesis of a new zwitterionic material based on a naturally occurring compatible solute, ectoine, which is known to possess additional protective properties that stabilize even whole cells against ultraviolet radiation or cytotoxins. These properties and applications of ectoine inspire us to design a functional monomer containing the natural zwitterion moiety of ectoine imparting nonfouling properties and the methacrylate moiety for polymerization. The synthesis route designed for the ectoine methacrylate monomer is simple with a high yield, which is characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. After monomer synthesis, we have prepared a poly(ectoine) hydrogel via thermal polymerization. The equilibrium water content, degree of cross-linking, mechanical strength, and nonfouling properties are determined for polyectoine hydrogels with different cross-linking conditions. Poly(ectoine) hydrogels are shown to have highly hydrated and excellent nonfouling properties and can be considered to be a promising biomaterial.

Fluorescent porous silicon biological probes with high quantum efficiency and stability.

We demonstrate porous silicon biological probes as a stable and non-toxic alternative to organic dyes or cadmium-containing quantum dots for imaging and sensing applications. The fluorescent silicon quantum dots which are embedded on the porous silicon surface are passivated with carboxyl-terminated ligands through stable Si-C covalent bonds. The porous silicon bio-probes have shown photoluminescence quantum yield around 50% under near-UV excitation, with high photochemical and thermal stability. The bio-probes can be efficiently conjugated with antibodies, which is confirmed by a standard enzyme-linked immunosorbent assay (ELISA) method.

High-Strength and Nonfouling Zwitterionic Triple-Network Hydrogel in Saline Environments.

Zwitterionic hydrogels have received great attention due to their excellent nonfouling and biocompatible properties, but they suffer from weak mechanical strength in the saline environments important for biomedical and engineering applications due to the "anti-polyelectrolyte" effect. Conventional strategies to introduce hydrophobic or non-zwitterionic components to increase mechanical strength compromise their nonfouling properties. Here, a highly effective strategy is reported to achieve both high mechanical strength and excellent nonfouling properties by constructing a pure zwitterionic triple-network (ZTN) hydrogel. The strong electrostatic interaction and network entanglement within the triple-network structure can effectively dissipate energy to toughen the hydrogel and achieve high strength, toughness, and stiffness in saline environments (compressive fracture stress 18.2 ± 1.4 MPa, toughness 1.62 ± 0.03 MJ m-3 , and modulus 0.66 ± 0.03 MPa in seawater environments). Moreover, the ZTN hydrogel is shown to strongly resist the attachment of proteins, bacteria, and cells. The results provide a fundamental understanding to guide the design of tough nonfouling zwitterionic hydrogels for a broad range of applications.

Inhibition of oral biofilm formation by zwitterionic nonfouling coating.

Inhibition of oral biofilm formation is critical to prevent and treat dental caries and periodontal diseases. In this study, we synthesized zwitterionic poly(carboxybetaine) (pCB) based polymer as a nonfouling coating to provide anti-bacterial properties to tooth surfaces. Four catechol derived l-3,4-dihydroxyphenylalanine (DOPA) groups were conjugated to pCB to serve as a surface anchoring group. The pCB-(DOPA)4 polymer was coated on the hydroxyapatite (HA) and enamel samples by simple immersion and characterized by Raman spectroscopy. The nonfouling effectiveness of the pCB based coating was determined by protein adsorption and bacterial adhesion assays. The coating was transparent on sample surfaces. The protein adsorption was significantly reduced to 8.2% and 6.9%, respectively, on pCB-(DOPA)4 coated HA and enamel samples. The pCB-(DOPA)4 -coated samples also demonstrated significantly fewer adhered Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutants compared to the control. This novel coating material provides an innovative approach to resist biofilm formation on tooth surfaces and has great potential in future dental clinical applications.

High-strength and fibrous capsule-resistant zwitterionic elastomers.

The high mechanical strength and long-term resistance to the fibrous capsule formation are two major challenges for implantable materials. Unfortunately, these two distinct properties do not come together and instead compromise each other. Here, we report a unique class of materials by integrating two weak zwitterionic hydrogels into an elastomer-like high-strength pure zwitterionic hydrogel via a "swelling" and "locking" mechanism. These zwitterionic-elastomeric-networked (ZEN) hydrogels are further shown to efficaciously resist the fibrous capsule formation upon implantation in mice for up to 1 year. Such materials with both high mechanical properties and long-term fibrous capsule resistance have never been achieved before. This work not only demonstrates a class of durable and fibrous capsule-resistant materials but also provides design principles for zwitterionic elastomeric hydrogels.

Surface hydration for antifouling and bio-adhesion.

Antifouling properties of materials play crucial roles in many important applications such as biomedical implants, marine antifouling coatings, biosensing, and membranes for separation. Poly(ethylene glycol) (or PEG) containing polymers and zwitterionic polymers have been shown to be excellent antifouling materials. It is believed that their outstanding antifouling activity comes from their strong surface hydration. On the other hand, it is difficult to develop underwater glues, although adhesives with strong adhesion in a dry environment are widely available. This is related to dehydration, which is important for adhesion for many cases while water is the enemy of adhesion. In this research, we applied sum frequency generation (SFG) vibrational spectroscopy to investigate buried interfaces between mussel adhesive plaques and a variety of materials including antifouling polymers and control samples, supplemented by studies on marine animal (mussel) behavior and adhesion measurements. It was found that PEG containing polymers and zwitterionic polymers have very strong surface hydration in an aqueous environment, which is the key for their excellent antifouling performance. Because of the strong surface hydration, mussels do not settle on these surfaces even after binding to the surfaces with rubber bands. For control samples, SFG results indicate that their surface hydration is much weaker, and therefore mussels can generate adhesives to displace water to cause dehydration at the interface. Because of the dehydration, mussels can foul on the surfaces of these control materials. Our experiments also showed that if mussels were forced to deposit adhesives onto the PEG containing polymers and zwitterionic polymers, interfacial dehydration did not occur. However, even with the strong interfacial hydration, strong adhesion between mussel adhesives and antifouling polymer surfaces was detected, showing that under certain circumstances, interfacial water could enhance the interfacial bio-adhesion.

Enhanced pulmonary systemic delivery of protein drugs via zwitterionic polymer conjugation.

Pulmonary delivery of protein drugs into the systemic circulation is highly desirable as the lung provides a large absorption surface area and a more favorable environment for biologics compared to other delivery routes. However, pulmonary systemic delivery of proteins presents several challenges such as poor protein stability and limited bioavailability, especially for large proteins (molecular weight > 50 kDa), which exhibit an average bioavailability of 1% to 5% when delivered via the pulmonary route. Here, we demonstrated that with the conjugation of zwitterionic poly(carboxybetaine) (pCB) polymer, the bioavailability of organophosphate hydrolase (OPH) was significantly increased from 5% to 53%. OPH conjugated with pCB delivered through intubation-assisted intratracheal instillation (IAIS) into the lung exhibited improved pharmacokinetic properties and prophylactic efficacy against organophosphate poisoning, showing its application potential. Zwitterionic polymer conjugation provides the possibility for a favorable, non-invasive delivery of biological therapeutics into the systemic circulation.

De novo design of functional zwitterionic biomimetic material for immunomodulation.

Superhydrophilic zwitterionic polymers are a class of nonfouling materials capable of effectively resisting any nonspecific interactions with biological systems. We designed here a functional zwitterionic polymer that achieves a trade-off between nonspecific interactions providing the nonfouling property and a specific interaction for bioactive functionality. Built from phosphoserine, an immune-signaling molecule in nature, this zwitterionic polymer exhibits both nonfouling and immunomodulatory properties. Its conjugation to uricase is shown to proactively eradicate all unwanted immune response, outperforming the zwitterionic polymers. On the other hand, this polymer could significantly prolong the half-life of protein drugs in vivo, overcoming the innate drawback of phosphoserine in inducing accelerated clearance. Our demonstration of a nonfouling zwitterionic material with built-in immunomodulatory functionality provides new insights into the fundamental design of biomaterials, as well as far-reaching implications for broad applications such as drug delivery, implants, and cell therapy.

Photoreactive Carboxybetaine Copolymers Impart Biocompatibility and Inhibit Plasticizer Leaching on Polyvinyl Chloride.

Protein and cell interactions on implanted, blood-contacting medical device surfaces can lead to adverse biological reactions. Medical-grade poly(vinyl chloride) (PVC) materials have been used for decades, particularly as blood-contacting tubes and containers. However, there are numerous concerns with their performance including platelet activation, complement activation, and thrombin generation and also leaching of plasticizers, particularly in clinical applications. Here, we report a surface modification method that can dramatically prevent blood protein adsorption, human platelet activation, and complement activation on commercial medical-grade PVC materials under various test conditions. The surface modification can be accomplished through simple dip-coating followed by light illumination utilizing biocompatible polymers comprising zwitterionic carboxybetaine (CB) moieties and photosensitive cross-linking moieties. This surface treatment can be manufactured routinely at small or large scales and can impart to commercial PVC materials superhydrophilicity and nonfouling capability. Furthermore, the polymer effectively prevented leaching of plasticizers out from commercial medical-grade PVC materials. This coating technique is readily applicable to many other polymers and medical devices requiring surfaces that will enhance performance in clinical settings.