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-<i>O</i>-(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

Aminooxy and Pyridyl Disulfide Telechelic Poly(poly(ethylene glycol) acrylate) by RAFT Polymerization

An efficient method to synthesize telechelic, bioreactive polymers is described. Homotelechelic polymers were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization in one step by employing bifunctional chain transfer agents (CTAs). A bis-carboxylic acid CTA was coupled to <i>N</i>-Boc-aminooxy ethanol or pyridyl disulfide ethanol, resulting in a bis-<i>N</i>-Boc-aminooxy CTA and a bis-pyridyl disulfide CTA, respectively. RAFT polymerization of poly­(ethylene glycol) (PEG) acrylate in the presence of both CTAs resulted in a series of polymers over a range of molecular weights (∼8.4–35.2 kDa; polydispersity indices, PDIs, of 1.11–1.44) with retention of end-groups postpolymerization. The polymers were characterized by ¹H NMR spectroscopy and gel permeation chromatography (GPC). Conjugations of small molecules and peptides resulted in homotelechelic polymer conjugates

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 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

Correction to Trehalose Glycopolymers as Excipients for Protein Stabilization

Correction to Trehalose Glycopolymers as Excipients for Protein Stabilizatio

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

Body Mass Index and Mortality in the General Population and in Subjects with Chronic Disease in Korea: A Nationwide Cohort Study (2002-2010)

<div><p>Background</p><p>The association between body mass index (BMI) and mortality is not conclusive, especially in East Asian populations. Furthermore, the association has been neither supported by recent data, nor assessed after controlling for weight changes.</p><p>Methods</p><p>We evaluated the relationship between BMI and all-cause or cause-specific mortality, using prospective cohort data by the National Health Insurance Service in Korea, which consisted of more than one million subjects. A total of 153,484 Korean adults over 30 years of age without pre-existing cardiovascular disease or cancer at baseline were followed-up until 2010 (mean follow-up period = 7.91 ± 0.59 years). Study subjects repeatedly measured body weight 3.99 times, on average.</p><p>Results</p><p>During follow-up, 3,937 total deaths occurred; 557 deaths from cardiovascular disease, and 1,224 from cancer. In multiple-adjusted analyses, U-shaped associations were found between BMI and mortality from any cause, cardiovascular disease, and cancer after adjustment for age, sex, smoking status, alcohol consumption, physical activity, socioeconomic status, and weight change. Subjects with a BMI < 23 kg/m² and ≥ 30 kg/m² had higher risks of all-cause and cause-specific mortality compared with the reference group (BMI 23–24.9 kg/m²). The lowest risk of all-cause mortality was observed in subjects with a BMI of 25–26.4 kg/m² (adjusted hazard ratio [HR] 0.86; 95% CI 0.77 to 0.97). In subgroup analyses, including the elderly and those with chronic diseases (diabetes mellitus, hypertension, and chronic kidney disease), subjects with a BMI of 25–29.9 kg/m² (moderate obesity) had a lower risk of mortality compared with the reference. However, this association has been attenuated in younger individuals, in those with higher socioeconomic status, and those without chronic diseases.</p><p>Conclusion</p><p>Moderate obesity was associated more strongly with a lower risk of mortality than with normal, underweight, and overweight groups in the general population of South Korea. This obesity paradox was prominent in not only the elderly but also individuals with chronic disease.</p></div

Association between body mass index and all-cause mortality according to disease status.

<p>The association between body mass index (BMI) and mortality was presented separately by those presenting with and without prevalent diabetes mellitus (DM) (A), hypertension (HTN) (B), and chronic kidney disease (CKD) (C). All analyses were adjusted for age, sex, smoking status, alcohol intake, physical activity, socioeconomic status, and body weight change.</p

Baseline characteristics according to body mass index category.

<p>Data are presented by mean ± standard deviation (SD) or %.</p><p>Baseline characteristics according to body mass index category.</p

Association between body mass index category and all-cause mortality.

<p>In the multivariable adjusted model, data was adjusted for age, sex, smoking status, alcohol intake, physical activity, socioeconomic status, and body weight change. In the analyses of stratified subgroups, the variable used in stratification was excluded.</p><p>Association between body mass index category and all-cause mortality.</p