Juneyoung Lee

Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors

Herein, we report the synthesis of trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. The glycomonomer 4,6-O-(4-vinylbenzylidene)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing the trehalose pendent groups were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization using two different thiol-reactive chain transfer agents (CTAs) for subsequent conjugation to proteins through disulfide linkages. The resulting glycopolymers were well-defined, and a range of molecular weights from 4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers when added or covalently attached to protein significantly increased stability toward lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) (PEG) and found to be superior at stabilizing the protein to lyophilization and heat. In addition, the protein-glycopolymer conjugates exhibited significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the protein.

Synthesis of Glycopolymers by Controlled Radical Polymerization Techniques and Their Applications

Natural saccharides are involved in numerous biological processes. It has been shown that these carbohydrates play a role in cell adhesion and proliferation, as well as protein stabilization, organization, and recognition. Certain carbohydrates also serve as receptors for viruses and bacteria. They are over expressed in diseases such as cancer. Hence, a lot of effort has been focused on mimicking these sugars. Polymers with pendent saccharide groups, also known as glycopolymers, are studied as oligo‐ and polysaccharide mimics. Controlled radical polymerization (CRP) techniques such as atom transfer radical polymerization (ATRP), reversible addition–fragmentation chain transfer (RAFT) polymerization, and nitroxide‐mediated polymerization (NMP), as well as cyanoxyl‐mediated free radical polymerization have allowed chemists to synthesize well‐defined glycopolymers that, in some cases, have particular end‐group functionalities. This review focuses on the synthesis of glycopolymers by these methods and the applications of glycopolymers as natural saccharide mimics.

Trehalose Glycopolymers as Excipients for Protein Stabilization

Herein, the synthesis of four different trehalose glycopolymers and investigation of their ability to stabilize proteins to heat and lyophilization stress are described. The disaccharide, α,α-trehalose, was modified with a styrenyl acetal, methacrylate acetal, styrenyl ether, or methacrylate moiety resulting in four different monomers. These monomers were then separately polymerized using free radical polymerization with azobisisobutyronitrile (AIBN) as an initiator to synthesize the glycopolymers. Horseradish peroxidase and glucose oxidase were incubated at 70 and 50 °C, respectively, and β-galactosidase was lyophilized multiple times in the presence of various ratios of the polymers or trehalose. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. Different molecular weights (10 kDa, 20 kDa, and 40 kDa) were tested, and all were equivalent in their stabilization ability. However, some subtle differences were observed regarding stabilization ability between the different polymer samples, depending on the stress. Small molecules such as benzyl ether trehalose were not better stabilizers than trehalose, and the trehalose monomer decreased protein activity, suggesting that hydrophobized trehalose was not sufficient and that the polymeric structure was required. In addition, cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells, RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and human umbilical vein endothelial cells (HUVECs) were conducted with polymer concentrations up to 8 mg/mL. The data showed that all four polymers were noncytotoxic for all tested concentrations. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors.

Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography

Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein we present a new resist that protects proteins during electron beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively cross-link to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometer and nanometer scale without requiring cleanroom conditions.

Trehalose hydrogels for stabilization of enzymes to heat.

Enzymes can catalyze various reactions with high selectivity and are involved in many important biological processes. However, the general instability of enzymes against high temperature often limits their application. To address this, we synthesized a trehalose-based hydrogel in two steps from commercial starting materials with minimal purification procedures. Mono- and multi-functional trehalose monomers were cross-linked by redox-initiated radical polymerization to form a hydrogel. Phytase, an important enzyme utilized in animal feedstock, was employed to study the effectiveness of the trehalose hydrogel to stabilize proteins against heat. Addition of the phytase solution to the hydrogel resulted in enzyme internalization as confirmed by confocal microscopy. The phytase in the hydrogel retained 100% activity upon heating at 90 °C compared to 39% when the hydrogel was absent. The enzyme could also be recovered from the hydrogel. The trehalose hydrogel synthesis reported herein should be readily scalable for thermal stabilization of a wide variety of enzymes.

Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography

Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nanoscale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates, allowing for micro- and nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark-field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications.

Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein

Biocompatible polymers such as poly(ethylene glycol) (PEG) have been successfully conjugated to therapeutic proteins to enhance their pharmacokinetics. However, many of these polymers, including PEG, only improve the in vivo lifetimes and do not protect proteins against inactivation during storage and transportation. Herein, we report a polymer with trehalose side chains (PolyProtek) that is capable of improving both the external stability and the in vivo plasma half-life of a therapeutic protein. Insulin was employed as a model biologic, and high performance liquid chromatography and dynamic light scattering confirmed that addition of trehalose glycopolymer as an excipient or covalent conjugation prevented thermal or agitation-induced aggregation of insulin. The insulin-trehalose glycopolymer conjugate also showed significantly prolonged plasma circulation time in mice, similar to the analogous insulin-PEG conjugate. The insulin-trehalose glycopolymer conjugate was active as tested by insulin tolerance tests in mice and retained bioactivity even after exposure to high temperatures. The trehalose glycopolymer was shown to be nontoxic to mice up to at least 1.6 mg/kg dosage. These results together suggest that the trehalose glycopolymer should be further explored as an alternative to PEG for long circulating protein therapeutics.

Enhancing the conjugation yield of brush polymer–protein conjugates by increasing the linker length at the polymer end-group

Polymers with oligoethylene glycol side chains are promising in therapeutic protein-polymer conjugates as replacements for linear polyethylene glycol (PEG). Branched PEG polymers can confer additional stability and advantageous properties compared to linear PEGs. However, branched PEG polymers suffer from low conjugation yields to proteins, likely due to steric interactions between bulky side chains of the polymer and the protein. In an effort to increase yields, the linker length between the protein-reactive functional end-group of the polymer chain and branched PEG side chain was systematically increased. This was accomplished by synthesizing four well-defined poly(poly(ethylene glycol methyl ether) acrylates) (pPEGA) with pyridyl disulfide end-groups by reversible addition-fragmentation chain transfer (RAFT) polymerization mediated by chain transfer agents (CTAs) with different linker lengths. These, along with linear PEG and poly(N-isopropylacrylamide) (pNIPAAm), were conjugated to two model proteins, bovine serum albumin (BSA) and beta-lactoglobulin (βLG). The conjugation yields were determined by gel electrophoresis. The length of the linker affected conjugation yield for both proteins. For BSA, the conjugation yield step increased from 10% to 24% when the linker was altered from 1 ethylene glycol (EG) unit to 3, with no additional increase for 4 and 6 EG units. In the case of βLG, the yield gradually increased from 9% to the 33% when the linker length was increased from 1 to 6. PEG and pNIPAAm reacted with yields as high as 75% further emphasizing the effect of steric hindrance in lowering conjugation yields.

In Situ Catalyst Modification in Atom Transfer Radical Reactions with Ruthenium Benzylidene Complexes

Ruthenium benzylidene complexes are well-known as olefin metathesis catalysts. Several reports have demonstrated the ability of these catalysts to also facilitate atom transfer radical (ATR) reactions, such as atom transfer radical addition (ATRA) and atom transfer radical polymerization (ATRP). However, while the mechanism of olefin metathesis with ruthenium benzylidenes has been well-studied, the mechanism by which ruthenium benzylidenes promote ATR reactions remains unknown. To probe this question, we have analyzed seven different ruthenium benzylidene complexes for ATR reactivity. Kinetic studies by (1)H NMR revealed that ruthenium benzylidene complexes are rapidly converted into new ATRA-active, metathesis-inactive species under typical ATRA conditions. When ruthenium benzylidene complexes were activated prior to substrate addition, the resulting activated species exhibited enhanced kinetic reactivity in ATRA with no significant difference in overall product yield compared to the original complexes. Even at low temperature, where the original intact complexes did not catalyze the reaction, preactivated catalysts successfully reacted. Only the ruthenium benzylidene complexes that could be rapidly transformed into ATRA-active species could successfully catalyze ATRP, whereas other complexes preferred redox-initiated free radical polymerization. Kinetic measurements along with additional mechanistic and computational studies show that a metathesis-inactive ruthenium species, generated in situ from the ruthenium benzylidene complexes, is the active catalyst in ATR reactions. Based on data from (1) H, (13)C, and (31)P NMR spectroscopy and X-ray crystallography, we suspect that this ATRA-active species is a RuxCly(PCy3)z complex.

Glucose‐Responsive Trehalose Hydrogel for Insulin Stabilization and Delivery

Effective delivery of therapeutic proteins is important for many biomedical applications. Yet, the stabilization of proteins during delivery and long-term storage remains a significant challenge. Herein, a trehalose-based hydrogel is reported that stabilizes insulin to elevated temperatures prior to glucose-triggered release. The hydrogel is synthesized using a polymer with trehalose side chains and a phenylboronic acid end-functionalized 8-arm poly(ethylene glycol) (PEG). The hydroxyls of the trehalose side chains form boronate ester linkages with the PEG boronic acid cross-linker to yield hydrogels without any further modification of the original trehalose polymer. Dissolution of the hydrogel is triggered upon addition of glucose as a stronger binder to boronic acid (Kb = 2.57 vs 0.48 m-1 for trehalose), allowing the insulin that is entrapped during gelation to be released in a glucose-responsive manner. Moreover, the trehalose hydrogel stabilizes the insulin as determined by immunobinding after heating up to 90 °C. After 30 min heating, 74% of insulin is detected by enzyme-linked immunosorbent assay in the presence of the trehalose hydrogel, whereas only 2% is detected without any additives.