Hsiang-Chieh Hung

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

Significance A protein modification technology has been developed in this study to overcome current problems faced by protein therapy. After being individually encapsulated in a super-hydrophilic zwitterionic gel, a therapeutic protein showed exceptional stability and long in vivo circulation half-life. More importantly, no immune response against either the protein or the polymer was observed following repeated injections. This technology will benefit patients by reducing administration frequency and mitigating adverse reactions and could allow more immunogenic proteins to enter into human therapeutic or protective applications. 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.

Probing the Surface Hydration of Nonfouling Zwitterionic and PEG Materials in Contact with Proteins

Zwitterionic polymers and poly(ethylene glycol) (PEG) have been reported as promising nonfouling materials, and strong surface hydration has been proposed as a significant contributor to the nonfouling mechanism. Better understanding of the similarity and difference between these two types of materials in terms of hydration and protein interaction will benefit the design of new and effective nonfouling materials. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied for in situ and real-time assessment of the surface hydration of the sulfobetaine methacrylate (SBMA) and oligo(ethylene glycol) methacrylate (OEGMA) polymer brushes, denoted as pSBMA and pOEGMA, in contact with proteins. Whereas a majority of strongly hydrogen-bonded water was observed at both pSBMA and pOEGMA surfaces, upon contact with proteins, the surface hydration of pSBMA remained unaffected, but the water ordering at the pOEGMA surface was disturbed. The effects of free sulfobetaine, free PEG chains with two different molecular weights, and PEG coated gold nanoparticles on the surface hydration of proteins were investigated. The results indicated that free sulfobetaine could strengthen the protein hydration layer, but free PEG chains greatly disrupt the protein hydration layer and likely directly interact with the protein molecules. In contrast to free PEG, the PEG chains anchored on the nanoparticles behave similarly to the pOEGMA surface and could induce strong hydrogen bonding of the water molecules at the protein surfaces.

Stealth surface modification of surface-enhanced Raman scattering substrates for sensitive and accurate detection in protein solutions.

Reliable surface-enhanced Raman scattering (SERS) based biosensing in complex media is impeded by nonspecific protein adsorptions. Because of the near-field effect of SERS, it is challenging to modify SERS-active substrates using conventional nonfouling materials without introducing interference from their SERS signals. Herein, we report a stealth surface modification strategy for sensitive, specific and accurate detection of fructose in protein solutions using SERS by forming a mixed self-assembled monolayer (SAM). The SAM consists of a short zwitterionic thiol, N,N-dimethyl-cysteamine-carboxybetaine (CBT), and a fructose probe 4-mercaptophenylboronic acid (4-MPBA). The specifically designed and synthesized CBT not only resists protein fouling effectively, but also has very weak Raman activity compared to 4-MPBA. Thus, the CBT SAM provides a stealth surface modification to SERS-active substrates. The surface compositions of mixed SAMs were investigated using X-ray photoelectron spectroscopy (XPS) and SERS, and their nonfouling properties were studied with a surface plasmon resonance (SPR) biosensor. The mixed SAM with a surface composition of 94% CBT demonstrated a very low bovine serum albumin (BSA) adsorption (∼3 ng/cm(2)), and moreover, only the 4-MPBA signal appeared in the SERS spectrum. With the use of this surface-modified SERS-active substrate, quantification of fructose over clinically relevant concentrations (0.01-1 mM) was achieved. Partial least-squares regression (PLS) analysis showed that the detection sensitivity and accuracy were maintained for the measurements in 1 mg/mL BSA solutions. This stealth surface modification strategy provides a novel route to introduce nonfouling property to SERS-active substrates for SERS biosensing in complex media.

Hierarchical zwitterionic modification of a SERS substrate enables real-time drug monitoring in blood plasma.

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive analytical technique with molecular specificity, making it an ideal candidate for therapeutic drug monitoring (TDM). However, in critical diagnostic media including blood, nonspecific protein adsorption coupled with weak surface affinities and small Raman activities of many analytes hinder the TDM application of SERS. Here we report a hierarchical surface modification strategy, first by coating a gold surface with a self-assembled monolayer (SAM) designed to attract or probe for analytes and then by grafting a non-fouling zwitterionic polymer brush layer to effectively repel protein fouling. We demonstrate how this modification can enable TDM applications by quantitatively and dynamically measuring the concentrations of several analytes—including an anticancer drug (doxorubicin), several TDM-requiring antidepressant and anti-seizure drugs, fructose and blood pH—in undiluted plasma. This hierarchical surface chemistry is widely applicable to many analytes and provides a generalized platform for SERS-based biosensing in complex real-world media.

Butyrylcholinesterase nanocapsule as a long circulating bioscavenger with reduced immune response.

Butyrylcholinesterase (BChE) is the most promising bioscavenger candidate to treat or prevent organophosphate (OP) poisoning. However, the clinical application of BChE is limited by two obstacles: an inadequate circulation half-life and limited sources for production. Although several modification technologies including glycosylation and PEGylation have been developed to improve its pharmacokinetics, none of them have been able to outperform blood-derived native BChE. In this work, we designed a long-circulating bioscavenger nanogel by coating equine serum-derived BChE with a zwitterionic polymer gel layer. This zwitterionic gel coating protected BChE from denaturation and degradation under harsh conditions. Notably, the nanocapsule exhibited a long circulation half-life of ~45h, a three-fold increase from the unmodified native version, enabling both therapeutic and prophylactic applications. In addition, the gel coating reduced the immunogenicity of equine BChE, unlocking the possibility to use non-human derived BChE as an OP bioscavenger in humans.

Functionalized plasmonic nanostructure arrays for direct and accurate mapping extracellular pH of living cells in complex media using SERS

The extracellular pH (pHe) of living cells is one of the major factors that influence cell behaviors including cycle progression, migration, and proliferation, as well as metastasis and invasion of tumor cells. Thus, accurate sensing and mapping of the pHe is still a critical yet challenging task in the study of pHe-dependent cell behaviors. In this work, we present a method to map pHe of living cells based on surface-enhanced Raman spectroscopy (SERS). We immobilized a pH probe molecule, 4-mercaptobenzoic acid (4-MBA), on a gold quasi three-dimensional plasmonic nanostructure array (Q3D-PNA) to enable an exceptionally sensitive and reproducible pH measurement. We prudentially investigated the influences of cations and complexity of detecting solutions on the responses of 4-MBA SERS spectra to pH variations to ensure the accuracy. Herein, a normal cell line (NIH/3T3) and a tumor cell line (HepG2) were cultured on the 4-MBA modified SERS substrates. Localized pHe was detected and mapped with good spatial resolution and pH sensitivity showing pHe domains on both cells. Moreover, the averaged pHe of tumor cells was shown to be more acidic compared with that of normal cells.

Achieving Ultralow Fouling under Ambient Conditions via Surface-Initiated ARGET ATRP of Carboxybetaine

We achieved ultralow fouling on target surfaces by controlled polymerization of carboxybetaine under ambient conditions. The polymerization process for grafting polymer films onto the surfaces was carried out in air and did not require any deoxygenation step or specialized equipment. This method allows one to conveniently introduce a nonfouling polymer network onto large substrates.

Zwitterionic Nanocages Overcome the Efficacy Loss of Biologic Drugs

For biotherapeutics that require multiple administrations to fully cure diseases, the induction of undesirable immune response is one common cause for the failure of their treatment. Covalent binding of hydrophilic polymers to proteins is commonly employed to mitigate potential immune responses. However, while this technique is proved to partially reduce the antibodies (Abs) reactive to proteins, it may induce Abs toward their associated polymers and thus result in the loss of efficacy. Zwitterionic poly(carboxybetaine) (PCB) is recently shown to improve the immunologic properties of proteins without inducing any antipolymer Abs against itself. However, it is unclear if the improved immunologic profiles can translate to better clinical outcomes since improved immunogenicity cannot directly reflect amelioration in efficacy. Here, a PCB nanocage (PCB NC) is developed, which can physically encase proteins while keeping their structure intact. PCB NC encapsulation of uricase, a highly immunogenic enzyme, is demonstrated to eradicate all the immune responses. To bridge the gap between immunogenicity and efficacy studies, the therapeutic performance of PCB NC uricase is evaluated and compared with its PEGylated counterpart in a clinical-mimicking gouty rat model to determine any loss of efficacy evoked after five administrations.

A Coating-Free Nonfouling Polymeric Elastomer

Medical devices face nonspecific biofouling from proteins, cells, and microorganisms, which significantly contributes to complications and device failure. Imparting these devices with nonfouling capabilities remains a major challenge, particularly for those made from elastomeric polymers. Current strategies, including surface coating and copolymerization/physical blending, necessitate compromise among nonfouling properties, durability, and mechanical strength. Here, a new strategy is reported to achieve both high bulk mechanical strength and excellent surface nonfouling properties, which are typically contradictory, in one material. This is realized through a nonfouling polymeric elastomer based on zwitterionic polycarboxybetaine derivatives. By hiding both charged moieties of the zwitterionic compounds with hydrocarbon ester and tertiary amine groups, the bulk polymer itself is elastomeric and hydrophobic while its superhydrophilic surface properties are restored upon hydrolysis. This coating-free nonfouling elastomer is a highly promising biomaterial for biomedical and engineering applications.

Achieving low-fouling surfaces with oppositely charged polysaccharides via LBL assembly.

UNLABELLED The aim of this work is to understand and achieve low fouling surfaces by mixing two oppositely charged polysaccharides through layer-by-layer (LBL) assembly. Diethylaminoethyl-dextran hydrochloride and alginate were employed as a model system to build LBL films. A surface plasmon resonance (SPR) biosensor was used to measure quantitatively the adsorption behavior of charged macromolecules during LBL buildup and the protein adsorption behavior of each deposited bilayer in situ in real time accordingly. Results show that LBL films have lower protein adsorption as the films are constructed above the substrate surface. These LBL films eventually reach very low fouling when they are sufficiently far from the substrate surface, where the substrate surface effect is minimized and bilayers consisting of positively and negatively charged marcromolecules are uniformly mixed. Single proteins, undiluted human blood serum and plasma and cells were tested for adsorption to LBL films with similar trends. To verify the generality of these findings, alginates of low and high molecular weights and carboxymethylcellulose as a substitute to alginate were studied with similar trends observed. These results demonstrate that oppositely charged polymers, when uniformly mixed, are able to achieve low fouling properties. Findings from this work will provide a fundamental understanding of and design principles on how to build nonfouling LBL films. STATEMENT OF SIGNIFICANCE We demonstrate that protein adsorption decreases with the increase of bilayer numbers. Results indicate that oppositely charged components tend to be uniformly mixed and distinct change layers in classical layer-by-layer (LBL) theories no longer exist as LBL films are sufficiently far from the substrate surface. Findings from this work provide a fundamental understanding of and design principles on how to build nonfouling LBL films.