Previous Highlights

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

Liu, Y.; Lee, J. ; Mansfield, K. M.; Ko, J. H.; Sallam, S.; Wesdemiotis, C.; Maynard, H. D., Bioconj. Chem., 2017, published online. [LINK]

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

A Rationally Designed Block Copolymer Both Stabilizes and Increases the Activity of Fibroblast Growth Factor 2 (FGF2)

Paluck, S. J.; Nguyen, T. H.; Lee, J. P.; Maynard, H. D., Biomacromolecules, 2016, published online. [LINK]

Fibroblast growth factor 2 (FGF2) is a protein involved in cellular functions in applications such as wound healing and tissue regeneration. Stabilization of this protein is important for its use as a therapeutic since the native protein is unstable during storage and delivery. Additionally, the ability to increase the activity of FGF2 is important for its application, particularly in chronic wound healing and the treatment of various ischemic conditions. Here we report a heparin mimicking block copolymer, poly(styrene sulfonate-co-poly(ethylene glycol) methyl ether methacrylate)-b-vinyl sulfonate) (p(SS-co-PEGMA)-b-VS, that contains a segment that enhances the stability of FGF2 and one that binds to the FGF2 receptor. The FGF2 conjugate retained activity after exposure to refrigeration (4 ºC) and room temperature (23 ºC) for seven days, while unmodified FGF2 was inactive after these standard storage conditions. A cell study performed with a cell line lacking native heparan sulfate proteoglycans. indicated that the conjugated block copolymer facilitated binding of FGF2 to its receptor similar to the addition of heparin to FGF2. A receptor based enzyme-linked immunosorbant assay confirmed the results. The conjugate also increased the migration of endothelial cells by 80% compared to FGF2 alone. Additionally, the FGF2-p(SS-co-PEGMA)-b-VS stimulated endothelial cell sprouting 250% better than FGF2 at low concentration. These data verify that this rationally designed protein-block copolymer conjugate enhances receptor binding, cellular processes such as migration and tube-like formation, and stability and suggest that it may be useful for applications in biomaterials, tissue regeneration and wound healing.

Visible Light-Induced Olefin Activation using 3D Aromatic Boron-Rich Cluster Photooxidants

Marco S. Messina, Jonathan C. Axtell, Yiqun Wang, Paul Chong, Alex Ian Wixtrom, Kent O. Kirlikovali, Brianna M. Upton, Bryan M. Hunter, Oliver S. Shafaat, Saeed I. Khan, Jay R. Winkler, Harry B. Gray, Anastassia N. Alexandrova, Heather D. Maynard, and Alexander M. Spokoyny, J. Am. Chem. Soc., 2016, published online. [LINK]

We report a discovery that perfunctionalized icosahedral dodecaborate clusters of the type B12(OCH2Ar)12 (Ar = Ph or C6F5) can undergo photo-excitation with visible light, leading to a new class of metal-free photooxidants. Excitation in these species occurs as a result of the charge transfer between low-lying orbitals located on the benzyl substituents and an unoccupied orbital delocalized throughout the boron cluster core. Here we show how these species, photo-excited with a bench-top blue LED source, can exhibit excited-state reduction potentials as high as 3 Volts and can participate in electron-transfer processes with a broad range of styrene monomers, initiating their polymerization. Initiation is observed in cases of both electron-rich and electron-deficient styrene monomers at cluster loadings as low as 0.005 mol%. Furthermore, photo-excitation of B12(OCH2Ar)12 in the presence of a less activated olefin such as isobutylene results in the production of highly branched poly(isobutylene). This work introduces a new class of air-stable metal-free photoredox reagents capable of mediating chemical transformations.

Encapsulated Hydrogels by E-Beam Lithography and Their Use in Enzyme Cascade Reactions

Mancini, R. J.; Paluck, S. J.; Bat, E.; Maynard, H. D., Langmuir, 2016, published online. [LINK]

Electron beam (e-beam) lithography was employed to prepare one protein encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 µm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complimentary reactivity, and the three dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/ AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions.

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

Lau, U. Y.; Saxer, S. S.; Lee, J.; Bat, E.; Maynard, H. D. "Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography," ACS Nano, [LINK]

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 nano- scale, 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 nano-scale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and anti-tumor necrosis factor alpha (TNF-alpha) 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 TNF-alpha 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.

Poly(vinyl sulfonate) Facilitates bFGF-Induced Cell Proliferation

Nguyen, T. H.; Paluck, S. J.; McGahran, A. J.; Maynard, H. D., "Poly(vinyl sulfonate) Facilitates bFGF-Induced Cell Proliferation," Biomacromolecules, 2015, DOI: 10.1021/acs.biomac.5b00557 [LINK]

Heparin is a highly sulfated polysaccharide and is useful because of its diverse biological functions. However, because of batch-to-batch variability and other factors, there is significant interest in preparing biomimetics of heparin. To identify polymeric heparin mimetics, a cell-based screening assay was developed in cells that express fibroblast growth factor receptors (FGFRs) but not heparan sulfate proteoglycans. Various sulfated and sulfonated polymers were screened, and poly(vinyl sulfonate) (pVS) was identified as the strongest heparin-mimicking polymer in its ability to enhance binding of basic fibroblast growth factor (bFGF) to FGFR. The results were confirmed by an ELISA-based receptor-binding assay. Different molecular weights of pVS polymer were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The polymers were able to facilitate dimerization of FGFRs leading to cell proliferation in FGFR-expressing cells, and no size dependence was observed. The data showed that pVS is comparable to heparin in these assays. In addition, pVS was not cytotoxic to fibroblast cells up to at least 1 mg/mL. Together this data indicates that pVS should be explored further as a replacement for heparin.

Grafting From siRNA as an Alternative Synthesis Route to siRNA-Polymer Conjugates

Lin, E. W.; Maynard, H. D., "Grafting From siRNA as an Alternative Synthesis Route to siRNA-Polymer Conjugates," Macromolecules, 2015, DOI: 10.1021/acs.macromol.5b00846 [LINK]

Small interfering ribonucleic acids (siRNAs) are important therapeutic agents and are challenging to deliver efficiently. To address this, covalent attachment of synthetic polymers to siRNA has become of great interest. In this paper, we present a synthetic route to siRNA-polymer conjugates by the grafting from method, meaning that the polymerization of monomers occurs from an initiating site that is attached to siRNA.

Imine Hydrogels with Tunable Degradability for Tissue Engineering

Boehnke, N.; Cam, C.; Bat, E.; Segura, T.; Maynard, H. D., "Imine Hydrogels with Tunable Degradabilityfor Tissue Engineering," Biomacromolecules, 2015, DOI: 10.1021/acs.biomac.5b00519. [LINK]

A shortage of available organ donors has created a need for engineered tissues. In this context, polymer-based hydrogels that break down inside the body are often used as constructs for growth factors and cells. In this paper we reported imine cross-linked gels where degradation is controllable by the introduction of mixed imine cross-links. Specifically, hydrazide-functionalized poly(ethylene glycol) (PEG) reacts with aldehyde-functionalized PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly in media. The time to degradation can be controlled by changing the structure of the hydrazide group or by introducing hydroxylamines to form non-reversible oxime linkages. Hydrogels containing adipohydrazide-functionalized PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone cross-linked gels further stabilized the hydrogels. This imine cross-linking approach should be useful for modulating the degradation characteristics of 3D cell culture supports for controlled cell release.

Trehalose hydrogels for stabilization of enzymes to heat

Lee, J.; Ko, J. H.; Lin, E.-W.; Wallace, P.; Ruch, F.; Maynard, H. D., "Trehalose hydrogels for stabilization of enzymes to heat," Polymer Chemistry, 20156, 3443-3448. [LINK]

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.

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

Bat, E.; Lee, J.; Lau, U. Y.; Maynard, H. D., "Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography," Nature Communications, DOI:10.1038/ncomms7654. [LINK]

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 crosslink 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 micrometre and nanometre scale without requiring cleanroom conditions.

Dual pH- and Temperature-Responsive Protein Nanoparticles

Matsumoto, N. M.; Buchman, G. W.; Rome, L. H.; Maynard, H. D., "Dual pH- and Temperature-Responsive Protein Nanoparticles," European Polymer Journal, DOI: 10.1016/j.eurpolymj.2015.01.043. [LINK]

Multiply responsive protein nanoparticles are interesting for a variety of applications.  In this paper we describe the synthesis of a vault nanoparticle that responds to both temperature and pH.  Specifically, poly(N-isopropylacrylamide-co­-acrylic acid) with a pyridyl disulfide end group was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization.  The polymer had a lower critical solution temperature (LCST) of 31.9 °C at pH 5, 44.0 °C at pH 6 and above 60 °C at pH 7.  The polymer was conjugated to human major vault protein (hMVP), and the resulting nanoparticle was analyzed by UV-Vis, dynamic light scattering (DLS) and electron microscopy.  The data demonstrated that poly(N-isopropylacrylamide-co­-acrylic acid)-vault conjugate did not respond to temperatures below 60 °C at pH 7, while the nanoparticles reversibly aggregated at pH 6.  Furthermore, it was shown that the vault nanoparticle structure remained intact for at least three heat and cooling cycles.  These dually responsive nanoparticles may serve as a platform for drug delivery and other applications. 

Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization

Decker, C. G.; Maynard, H. D., "Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization," European Polymer Journal, 2015, 65, 305-312. [LINK]

Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body.  Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone.  In this paper we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Đ)of1.34 and 1.71, respectively. Hydrolytic degradation of the polymers and reversible covalent attachment of these polymers to lysozyme was analyzed.  Following cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity.  The research described in this paper represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable. 

Amphiphilic/Fluorous Random Copolymers as a New Class of Biocompatible Polymeric Materials for Protein Conjugation

Koda, Y.; Terashima, T.*; Sawamoto, M.; Maynard, H. D., “Amphiphilic/Fluorous Random Copolymers as a New Class of Biocompatible Polymeric Materials for Protein Conjugation,”Polymer Chemistry2015, 6, 240-247. [LINK]

In this paper, amphiphilic/fluorous random copolymers bearing poly(ethylene glycol) (PEG) chains and perfluorinated alkane pendants were developed as novel biocompatible polymers for protein conjugation.  Three kinds of random copolymers with different initiating terminals (carboxylic acid, pyridyl disulfide, N-hydroxysuccinimide ester) were prepared by reversible addition-fragmentation chain transfer (RAFT) copolymerization of a PEG methyl ether methacrylate and a perfluorinated alkane methacrylate with corresponding functional chain transfer agents.  All of the polymers were soluble in water to form nanostructures with perfluorinated compartments via fluorous interaction: large aggregates from the intermolecular multi-chain association and compact unimer micelles from the intramolecular single-chain folding.  Such a PEGylated and perfluorinated random copolymer was non-cytotoxic to NIH 3T3 mouse embryonic fibroblast cells and human umbilical vein endothelial cells (HUVECs).  Additionally, a random copolymer with a pyridyl disulfide terminal was also successfully conjugated with a thiolated lysozyme.  This paper describes a new polymer for protein conjugate that could have far reaching applications in the biomedical field.  

Protein-Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione S-Transferase

Lin, E.-W.; Boehnke, N.; Maynard, H. D. "Protein−Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione S‐Transferase" Bioconjugate Chem. 2014, 25, 1902-1909.* [LINK] *ACS Editor's choice

A photoactivated, site-selective conjugation of poly(ethylene glycol) (PEG) to the glutathione (GSH) binding pocket of glutathione S-transferase (GST) is described. To achieve this, a GSH analogue (GSH-BP) was designed and chemically synthesized with three functionalities: (1) the binding affinity of GSH to GST, (2) a free thiol for polymer functionalization, and (3) a photoreactive benzophe- none (BP) component. Different molecular weights (2 kDa, 5 kDa, and 20 kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed conjugation efficiencies between 52% and 76% to GST. Diazirine (DA) PEG were also prepared but gave conjugation yields lower than for GSBP-PEGs. PEGs with different end-groups were also synthesized to validate the importance of each component in the end group design. End- groups included glutathione (GS-PEG) and benzophenone (BP-PEG). Results showed that both GSH and BP were crucial for successful conjugation to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins were investigated, including bovine serum albumin (BSA), lysozyme (Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure specific binding to GST. By combining noncovalent and covalent interactions, we have developed a new phototriggered protein−polymer conjugation method that is generally applicable to GST-fusion proteins.

Therapeutic Protein-Polymer Conjugates: Advancing Beyond PEGylation

Pelegri-O'Day, E. M.; Lin, E.-W.; Maynard, H. D.; "Therapeutic Protein-Polymer Conjugates: Advancing Beyond PEGylation," J. Am. Chem. Soc. 2014, published online. [LINK]

Protein-polymer conjugates are widely used as therapeutics. All Food and Drug Administration (FDA) approved protein conjugates are covalently linked to poly(ethylene glycol) (PEG). These PEGylated drugs have longer half-lives in the blood stream leading to less frequent dosing, a significant advantage for patients. However, there are some potential drawbacks to PEG that are driving the development of alternatives. Polymers that display enhanced pharmacokinetic properties along with additional advantages such as improved stability or degradability will be important to advance the field of protein therapeutics. This perspective presents a summary of protein-PEG conjugates for therapeutic use and alternative technologies in various stages of development, as well as suggestions for future directions. Established methods of producing protein-PEG conjugates and new approaches utilizing controlled radical polymerization are also covered.

Morphing Hydrogel Patterns by Thermo-Reversible Fluorescence Switching

Bat, E.; Lin, E. -W.; Saxer, S.; Maynard, H. D., “Morphing Hydrogel Patterns by Thermo-Reservsible Fluorescence Switching,” Macro. Rapid Commun., DOI: 10.1002/marc.2014000160.[LINK]

Stimuli responsive surfaces that show reversible fluorescence switching behavior in response to temperature changes are presented. Fluorophore conjugated hydrogel thin films are bright when the gels are swollen; upon collapsing of the gels, self-quenching of fluorophores leads to significant attenuation of fluorescence. Morphing surfaces are obtained by patterning multiple stimuli responsive polymers using electron beam lithography.  The polymers change shape several times upon heating.  These materials may be useful for applications such as (bio)sensors, encryption, biomedical microdevices, and self-reporting surfaces.

Nanoparticle Growth via Concentration Gradients Generated by Enzyme Nanopatterns

de la Rica, R.; Bat, E.; Herpoldt, K. L.; Xie, H.-n.; Bertazzo, S.; Maynard, H. D.; Stevens, M. M., "Nanoparticle Growth via Concentration Gradients Generated by Enzyme Nanopatterns," Adv. Funct. Mater.201424, 3692-3698. [LINK]

Biomineralizing organisms can grow nanomaterials with unexpected morphologies in an organic matrix where temporal and vectorial gradients of crystal growth precursors are established. Here, concentration gradients for the crystallization of gold nanoparticles are generated and applied on silicon substrates. Gradients of crystal growth precursors are generated by enzymes patterned as lines that are separated by distances ranging from the micro- to the nanoscale. The concentration of crystallization precursors around the lines separated by nanometric distances is not only determined by mass transport and enzyme activity but also by the nanoscale organization of biocatalysts. This nanoscale organization favors non-classical crystal growth conditions that lead to the formation of nanoparticle clusters containing nanocrystals that are highly crystallographically aligned. The combination of bottom-up crystal growth with top-down electron beam lithography enables the fabrication of micrometric patterns containing gold nanoparticles of different size, shape, and surface density. These are all critical parameters that determine the physical properties of these nanomaterials.

Chemoselective Immobilization of Proteins by Microcontact Printing and Bio-orthogonal Click Reactions

Tolstyka, Z. P.; Richardson, W.; Bat, E.; Stevens, C. J.; Parra, D. P.; Dozier, J. K.; Distefano, M. D.; Dunn, B.; Maynard, H. D., “Chemoselective Immobilization of Proteins by Microcontact Printing and Bioorthogonal Click Reactions,” ChemBioChem201314, 2464-2471. [LINK]

In this work, a combination of microcontact printing of functionalized alkanethiols and site-specific modification of proteins is utilized to chemoselectively immobilize proteins onto gold surfaces either by oxime or copper catalyzed alkyne-azide click chemistry. Two molecules capable of click reactions, an aminooxy-functionalized alkanethiol and an azide-functionalized alkanethiol, were synthesized, and self-assembled monolayer (SAM) formation on gold was confirmed by IR spectroscopy. The alkanethiols were then individually patterned onto gold surfaces by microcontact printing. Site-specifically modified proteins, horse heart myoglobin (HHMb) containing an N-terminal α-oxoamide and a red-fluorescent protein (mCherry-CVIA) with a C-terminal alkyne, respectively were immobilized by incubation onto the stamped functionalized alkanethiol patterns. Pattern formation was confirmed by fluorescence microscopy. This platform can facilitate the oriented immobilization of proteins and hence should be useful for a wide range of biomedical and biotechnology applications.

Trehalose Glycopolymers as Excipients for Protein Stabilization

Lee, J.; Lin, E.-W.; Lau, U. Y.; Hedrick, J. L.; Bat, E.; Maynard, H. D., “Trehalose Glycopolymers as Excipients for Protein Stabilization,” Biomacromolecules201314, 2561-2569. [LINK]

Four different trehalose glycopolymers were synthesized and shown to stabilize proteins to heat and lyophilization stress. 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.  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 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 two human cell lines and two mouse cell lines showed that the polymers were non-cytotoxic up to at least 8 mg/mL. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors.

Synthesis of Photodegradable Macromers for Conjugation and Release of Bioactive Molecules

Griffin, D. R.; Schlosser, J. L.; Lam, S. F.; Nguyen, T. H.; Maynard, H. D.; Kasko, A. M., “Synthesis of Photodegradable Macromers for Conjugation and Release of Bioactive Molecules,”Biomacromolecules201314, 1199-1207. [LINK]

Hydrogel scaffolds are used in biomedicine to study cell differentiation and tissue evolution, where it is critical to control the delivery of chemical cues both spatially and temporally. While large molecules can be physically entrapped in a hydrogel, moderate molecular weight therapeutics must be tethered to the hydrogel network through a labile linkage to allow controlled release. In this paper is reported the synthesis of a library of polymerizableortho-nitrobenzyl (o-NB) macromers with different functionalities at the benzylic position (alcohol, amine, BOC-amine, halide, acrylate, carboxylic acid, activated disulfide, N-hydroxysuccinyl ester, biotin). This library of polymerizable macromers containing o-NB groups should allow direct conjugation of nearly any type of therapeutic agent and its subsequent controlled photorelease from a hydrogel network.  As a proof-of-concept, phenylalanine, a cell-adhesive peptide (GCGYGRGDSPG), a protein that exhibits enzymatic activity (bovine serum albumin), and a growth factor (transforming growth factor-β1) were incorporated into the hydrogels.  Their release was controlled with light, and verification the bioactivity of the photoreleased molecules was demonstrated.  This versatile approach can be used to sequester peptides and proteins into hydrogel depots and release them in an externally controlled, predictable manner without compromising biological function.  

Synthesis of Biotinylated Aldehyde Polymers for Biomolecule Conjugation

Alconcel, S. N. S.; Kim, S. H.; Tao, L.; Maynard, H. D., “Synthesis of Biotinylated Aldehyde Polymers for Biomolecule Conjugation,” Macro. Rapid Commun.2013 DOI: 10.1002/marc.201300205 [LINK]

Biotinylated polymers with side chain aldehydes were prepared for use as multifunctional scaffolds.  Two different biotin-containing chain transfer agents (CTAs) and an aldehyde-containing monomer, 6-oxohexyl acrylate (6OHA), were synthesized.  Poly(ethylene glycol) methyl ether acrylate (PEGA) and 6OHA were copolymerized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of the biotinylated CTAs. The resulting polymers contained a disulfide bond, which could be readily reduced in solution to remove the biotin.  Reactivity of the aldehydes was demonstrated by oxime and hydrazone formation at the polymer side chains, and conjugate formation of fluorescently labeled polymers with streptavidin was investigated by gel electrophoresis.   

Synthesis of Nanogel-Protein Conjugates

Matsumoto, N. M.; González-Toro, D. C.; Chacko, R. T.; Maynard, H. D.; Thayumanavan, S., "Synthesis of Nanogel-Protein Conjugates," Polymer Chem. 20134, 2464-2469. [LINK]

Protein-functionalized, degradable polymeric nanogel precursors are reported.  The cross-linked polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the resulting polymer p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels containing residual activated thiols. The cross-linking reaction was conducted in the presence of a lipophilic dye, DiI, which was sequestered within the nanogels. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via disulfide bond formation between the BSA and the surface exposed nanogel pyridyl disulfides. We expect that this methodology is generally applicable to the preparation of protein-nanogel therapeutics.

A Heparin-Mimicking Polymer Conjugate Stabilizes Basic Fibroblast Growth Factor (bFGF)

Thi H. Nguyen, Sung-Hye Kim, Caitlin G. Decker, Darice Y. Wong, Joseph A. Loo, and Heather D. Maynard, "A Heparin-Mimicking Polymer Conjugate Stabilizes Basic Fibroblast Growth Factor," Nature Chemistry2013, 5, 221-227. [LINK]

Basic fibroblast growth factor (bFGF) is an important protein that plays a crucial role in diverse cellular functions from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Herein, we describe stabilization of bFGF by covalent conjugation of a heparin-mimicking polymer. The bFGF conjugate of this polymer was stable to a variety of environmentally and therapeutically relevant stressors such as heat, mild and harsh acidic conditions, storage, and proteolytic degradation, compared to native bFGF. After applied stress, the conjugate was also significantly more active than the analogous poly(ethylene glycol)ylated-conjugate system  This research has important implications for the clinical use of bFGF and for stabilization of heparin-binding growth factors in general.

Smart Vaults: Thermally-Responsive Protein Nanocapsules

Nicholas M. Matsumoto, Panchami Prabhakaran, Leonard H. Rome, and Heather D. Maynard  ACS Nano, 2013, 7, 867-874. [LINK]

Vaults are naturally-occurring ubiquitous ribonucleoprotein particles 41 x 41 x 72.5 nm composed of a protein shell enclosing multiple copies of two proteins and multiple copies of one or more small untranslated RNAs. Recombinant vaults are structurally identical but lack the vault content proteins. Poly(N-isopropylacrylamide) (pNIPAAm), a polymer responsive to heat, was conjugated to recombinant vaults that were composed of ~78 copies of the major vault protein (MVP) modified to contain a cysteine rich region at the N-terminus (CP-MVP). The polymer was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization to have a dansyl group at the alpha end and modified to have a thiol-reactive pyridyl disulfide at the omega end, which readily coupled to CP-MVP vaults. The resulting vault nanocapsules underwent reversible aggregation upon heating above the lower critical solution temperature (LCST) of the polymer, and the vault structure remained entirely intact throughout the phase transition. We envision that these smart vault conjugates may be used for drug delivery applications in conjunction with previously reported vault-based hydrophobic drug delivery methods and emerging in vivo localized heating techniques, to build up depots of therapeutic drugs in tumors for a sustained release of drugs to tumor cells, and other interesting medical and biotechnology applications.

Keto-Functionalized Polymer Scaffolds as Versatile Precursors to Polymer Side-Chain Conjugates

Jingquan Liu, Ronald C. Li, Gregory J. Sand, Volga Bulmus, Thomas P. Davis, and Heather D. Maynard  Macromolecules., 2013, 46, 8-14. [LINK]

A new methacrylate monomer with a reactive ketone side-chain, 2-(4-oxo-pentanoate) ethyl methacrylate (PAEMA), was synthesized and subsequently polymerized by reversible addition-fragmentation chain transfer (RAFT) polymerization to give a polymer with a narrow molecular weight distribution (PDI = 1.25). The polymer was chain extended with poly(ethylene glycol methyl ether acrylate) (PEGMA) to yield a block copolymer. Aminooxy containing small molecules and oligoethylene glycol were conjugated to the ketone functionality of the side chain in high yields. Cytotoxicity of the oxime-linked tetra(ethylene glycol) polymer to mouse fibroblast cells was investigated; the polymer was found to be non-cytotoxic up to 1 mg/mL. The ease with which this polymer is functionalized, suggests that it may be useful in forming tailored polymeric medicines.

Biocompatible Hydrogels by Oxime Click Chemistry

Gregory N. Grover, Jonathan Lam, Thi H. Nguyen, Tatiana Segura, and Heather D. Maynard  Biomacromolecules., 2012, 13, 3013-3017. [LINK]

Hydrogels are important for a wide range of applications including tissue engineering and regeneration. Fast gelling systems are advantageous for point of care applications. For this reason, click chemistries including thiol-ene, Huisgens cycloaddition, and Diels Alder reaction have been widely exploited to make these materials. However, there are some disadvantages that include bioreactivity or instability of the starting materials and requirement for the addition of toxic metals. For the first time, oxime Click chemistry was used to form hydrogels that support cell adhesion. Oxime click chemistry has advantages that gelation is fast, the reactive parters are stable and the byproduct is benign. We demonstrated that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenechymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells showed biocompatibility of the material.

Shape-Shifting Micro and Nano-Patterns Controlled by Temperature

Christopher M. Kolodziej and Heather D. Maynard  J. Am. Chem. Soc., 2012, DOI: 10.1021/ja304860q [LINK]

Features that alter their shape to form a different pattern upon an external trigger were created. Electron-beam lithography was used to fabricate micron- and nanometer-sized surface immobilized poly(triethylene glycol methacrylate) (pTEGMA) that exhibit significant thermal responsivity; the resulting hydrogels collapsed by up to 95% of their height upon addition of heat. Multicomponent features composed of contiguous thermoresponsive polymer and non-responsive poly(ethylene glycol) (PEG) were then prepared. Upon increase in temperature, only the thermally responsive component of the pattern collapsed, causing a substantial and predictable alteration in the overall pattern. Reversible micron- and nanometer-sized square-to-triangles, squares-to-checkerboards, smiles-to-neutral face, and zeros-to-ones shapes were shown. This strategy should allow for a large number of different forms that change shape on cue leading to a myriad of applications such as encryption and miniature cell culture substrates to name a few.

Aminooxy and Pyridyl Disulfide Telechelic Polymers via RAFT

Gregory N. Grover, Juneyoung Lee, Nicholas Matsumoto, and Heather D. Maynard  Macromolecules, 2012, 45, 4958-4965.

An efficient method to synthesize telechelic, bio-reactive polymers is described in this paper. Homotelechelic polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in one step by employing bifunctional chain transfer agents (CTAs). Bis functionalized N-BOC-aminooxy and pyridyl disulfide CTAs were synthesized. RAFT polymerization of polyethylene glycol (PEG) acrylate in the presence of both CTAs resulted in a series of polymers over a range of molecular weights with retention of end-groups post-polymerization. Conjugations of small molecules and peptides resulted in homotelechelic polymer conjugates.

Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors

Rock J. Mancini, Juneyoung Lee, and Heather D. Maynard  J. Am. Chem. Soc., 2012, DOI: 10.1021/ja2120234 [LINK]

In this paper are reported trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. Polymers containing trehalose pendant groups were prepared via reversible addition-fragmentation chain transfer polymerization using thiol-reactive chain transfer agents for subsequent conjugation to proteins through disulfide linkages. The glycopolymers when added or covalently attached to protein significantly increased stability towards lyophilization and heat relative to wild-type protein. The glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) and found to be superior. 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. The results show that the polymer is promising as a stabilizer of proteins to environmental stressors as an excipient and conjugate.

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Christopher M. Kolodziej, Sung Hye Kim, Rebecca M. Broyer, Sina S. Saxer, Caitlin G. Decker, and Heather D. Maynard  J. Am. Chem. Soc.., 2011, DOI: 10.1021/ja205524x [LINK]

We have successfully demonstrated production of a surface which presents cell-adhesion factors and growth factors at subcellular length scales, and have used this platform to demonstrate the roles of these two factors both alone and together. This was accomplished by first fabricating poly(ethylene glycol) hydrogel features presenting aminooxy groups and styrene sulfonates using e-beam lithography. Human endothelial cells not only adhered and spread, but also formed focal adhesion assemblies in the presence of both components. Basic fibroblast growth factor (bFGF) remained active on the heparin mimic polymer in culture media and synergistically promoted cell adhesion with integrin binding ligand RGD.

Dual Click Reactions to Micropattern Proteins

Rebecca M. Broyer, Eric Schopf, Christopher M. Kolodziej, Yong Chen, and Heather D. Maynard  Soft Matter., 2011, 7, 9972-9977 DOI: 10.1039/b000000x [LINK]

In this report orthogonal Click reactions were utilized to immobilize two different proteins on surfaces side-by-side and in multilayer constructs. Alkyne- and azide-functionalized poly(ethylene glycol) hydrogel features were fabricated Copper-catalyzed Huisgen 1,3 dipolar cycloaddition and oxime chemistry were employed to conjugate an azide-functionalized ubiquitin and oxoamide-modified myoglobin, respectively. Multicomponent patterning was verified by fluorescence imaging.

Review: FDA-approved PEG-protein conjugates

Steevens N. S. Alconcel, Arnold S. Baas and Heather D. Maynard; Polym. Chem., 2011, DOI: 10.1039/C1PY00034A [LINK]

PEGylation or covalent attachment of poly(ethylene glycol) improves the pharmacokinetic properties of protein drugs. In vivo circulation lifetimes are increased and dosages are decreased, resulting in improved patient quality of life. PEG may be attached to proteins using a variety of different chemical reactions. This review discusses currently available FDA-approved PEGylated protein drugs, their intended use and target, and the PEG attachment chemistry utilized.