Microwave-Induced Transient Heating Accelerates Protein PEGylation.

PEGylation is one of the most widely employed strategies to increase the circulatory half-life of proteins and to reduce immune responses. However, conventional PEGylation protocols often require excess reagents and extended reaction times because of their inefficiency. This study demonstrates that a microwave-induced transient heating phenomenon can be exploited to significantly accelerate protein PEGylation and even increase the degree of PEGylation achievable beyond what is possible at room temperature. This can be accomplished under conditions that do not compromise protein integrity. Several PEGylation chemistries and proteins are tested, and mechanistic insight is provided. Under certain conditions, extremely high levels of PEGylation were achieved in a matter of minutes. Moreover, considering the significantly reduced reaction times, the microwave-induced transient heating concept was adapted for continuous flow manufacturing of bioconjugates.

PEGylation of a Peptide-Based Amphiphilic Delivery Agent and Influence on Protein Delivery to Cells.

The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain. Amphiphilic peptides with hydrophobic properties, such as S10, can interact with lipids to destabilize the cell membrane, thus promoting cargo internalization or escape from endosomal entrapment. However, acute membrane destabilization can result in a dose-limiting cytotoxicity. In this context, the partial or transient deactivation of S10 by modification with methoxy poly(ethylene glycol) (mPEG; i.e., PEGylation) may provide the means to alter membrane destabilization kinetics, thereby attenuating the impact of acute permeabilization on cell viability. This study investigates the influence of PEGylation parameters (molecular weight, architecture, and conjugation chemistry) on the delivery efficiency of a green fluorescent protein tagged with a nuclear localization signal (GFP-NLS) and cytotoxicity on cells in vitro. Results suggest that PEGylation mostly interferes with adsorption and secondary structure formation of S10 at the cell membrane, and this effect is exacerbated by the mPEG molecular weight. This effect can be compensated for by increasing the concentration of conjugates prepared with lower molecular weight mPEG (5 to ∼20 kDa) but not for conjugates prepared with higher molecular weight mPEG (40 kDa). For conjugates prepared with moderate-to-high molecular weight mPEG (10 to 20 kDa), partial compensation of inactivation could be achieved by the inclusion of a reducible disulfide bond, which provides a mechanism to liberate the S10 from the polymer. Grafting multiple copies of S10 to a high-molecular-weight multiarmed PEG (40 kDa) improved GFP-NLS delivery efficiency. However, these constructs were more cytotoxic than the native peptide. Considering that PEGylation could be harnessed for altering the pharmacokinetics and biodistribution profiles of peptide-based delivery agents in vivo, the trends observed herein provide new perspectives on how to manipulate the membrane permeabilization process, which is an important variable for achieving delivery.

Consecutive Alkylation, "Click", and "Clip" Reactions for the Traceless Methionine-Based Conjugation and Release of Methionine-Containing Peptides.

"Click" reactions have revolutionized research in many areas of science. However, a disadvantage of the high stability of the Click product is that identifying simple treatments for cleanly dissociating the latter under the same guiding principles, i.e., a "Clip" reaction, remains a challenge. This study demonstrates that electron-deficient alkynes, conveniently installed on methionine residues, can participate in well-known Click (nucleophilic thiol-allene addition) and subsequent Clip reactions (radical thiol-ene addition). To illustrate this concept, a variety of bioconjugates (peptide-peptide; peptide-fluorophore; peptide-polymer; and peptide-protein) were prepared. Interestingly, the Clip reaction of these bioconjugates releases the original peptides concurrent with regeneration of their unmodified methionine residue, in minutes. Moreover, the conjugates demonstrate substantial stability toward endogenous levels of reactive species in bacteria, illustrating the potential for this chemistry in the biosciences. The reaction conditions employed in the Click and Clip steps are compatible with the preservation of the integrity of biomolecules/fluorophores and involve readily accessible reagents and the natural functional groups on peptides/proteins.

Overcoming PEG─Protein Mutual Repulsion to Improve the Efficiency of PEGylation.

Bioconjugation reactions, such as protein PEGylation, generally require excess reagents because of their inefficiency. Intriguingly, few reports have investigated the fundamental causes of this inefficiency. This study demonstrates that the excluded volume effect (EVE)─caused by the mutual repulsion of methoxy poly(ethylene glycol) (mPEG) and proteins under typical PEGylation conditions─causes proteins and protein-reactive mPEG (5 kDa) to self-associate into separate "protein-rich" and "mPEG-rich" nano-domains (i.e., soluble self-assemblies). To overcome this obstacle to reaction, "unreactive" low-molecular-weight mPEG was added as a co-solvent to promote the association between the larger protein and the reactive mPEG molecules by harnessing the same EVE. The near complete PEGylation of lysozyme could be achieved with close to stoichiometric amounts of reactive mPEG, and beneficial effects were observed for other proteins. Considering the general nature of the EVE (e.g., salting-out and PEGying-out), this study provides important perspectives on enhancing bioconjugation reactions, which are relevant to many nanoscale systems.

New Perspectives for Developing Therapeutic Bioconjugates of Metabolite-Depleting Enzymes: Lessons Learned Combating Glutamate Excitotoxicity.

Glutamate, the main excitatory neurotransmitter in the central nervous system, plays an essential role in several cognitive activities such as memorizing and learning. Excessive glutamate release and disturbance of glutamate homeostasis participates in multiple neuronal pathologies including cerebral ischemia (inadequate blood supply), traumatic brain injury (e.g., from a fall or an accident), multiple sclerosis, epilepsy, migraine, fetal hypoxia, or Alzheimer's disease. Attenuating excitotoxicity by, for example, targeting glutamate receptors has proved to be beneficial in animal models but has largely failed in clinical trials because of toxic side effects. New therapeutic concepts have been explored to reduce the excitotoxic effect caused by the excessive glutamate release by using or stimulating glutamate-depleting enzymes in the bloodstream. These enzymes indirectly act upon the brain by depleting glutamate in the bloodstream, which is believed to siphon it out of the brain. Recent studies have shown that bioconjugate approaches applied to such enzymes exacerbate this therapeutic effect but raise additional questions for future research. This Perspective provides an overview of lessons learned by our group when exploring bioconjugate approaches for combatting glutamate excitotoxicity as an illustration of how research on therapeutic bioconjugates is evolving.

Noninvasive, label-free, and quantitative monitoring of lipase kinetics using terahertz emission technology.

Enzymes catalyze chemical transformations of great importance in many fields, and analysis of the rate of these transformations is equally important. The latter are typically monitored using surrogate substrates that produce quantifiable optical signals, owing to limitations associated with "label-free" techniques that could be used to monitor the transformation of original substrate molecules. In this study, terahertz (THz) emission technology is used as a noninvasive and label-free technique to monitor the kinetics of lipase-induced hydrolysis of several substrate molecules (including the complex substrate whole cow's milk) and horseradish peroxidase-catalyzed oxidation of o-phenylenediamine in the presence of H2 O2 . This technique was found to be quantitative, and kinetic parameters are compared to those obtained by proton NMR spectroscopy or UV/Vis spectroscopy. This study sets the stage for investigating THz emission technology as a tool for research and development involving enzymes, and for monitoring industrial processes in the food, cosmetic, detergent, pharmaceutical, and biodiesel sectors.

The phage display of Bacillus subtilis Lipase A significantly enhances catalytic activity due to altered nanoscale distribution in colloidal solution.

Screening libraries of mutant proteins by phage display is now relatively common. However, one unknown factor is how the bacteriophage scaffold itself influences the properties of the displayed protein. This communication evaluates the effect of solution parameters on the catalytic activity of phage displayed Bacillus subtilis Lipase A (BSLA), compared to the free enzyme in solution. While the pH- and temperature-activity profiles of BSLA were not intrinsically affected by phage display, the nanoscale distribution of BSLA within the micellar assay buffer was. This lead to a pronounced increase of activity of phage-BSLA relative to the free enzyme, owing to the accumulation of phage-BSLA at the substrate-rich micelles. Considering this result obtained for BSLA, caution is warranted and similar effects should be considered when selecting other enzymes/proteins by phage display, as the activity of the displayed protein may differ from that of the free protein.

Homogenous Scavenging Resolves Low-Purification Yield/Selectivity Caused by Secondary Binding of Protein-A to Antigen-Binding Antibody Fragments.

Antigen-binding fragments of antibodies are biotechnologically useful agents for decorating drug delivery systems, for blocking cell-surface receptors in cell culture, for recognizing analytes in biosensors, and potentially as therapeutics. They are typically produced by enzymatic digestion of full antibodies and isolated from the undesirable fragment crystallizable (Fc) by affinity chromatography using Protein-A columns. However, while Protein-A has a strong "classical" interaction with Fc fragments, it can also more weakly bind to an "alternative" site on the heavy chain variable region of antigen-binding fragments. As such, purifying small amounts of antibody fragments by Protein-A chromatography can result in low yield. Moreover, loading larger amounts of antibody fragments onto a Protein-A column can result in poor separation, because of competition of Fc and antigen-binding fragments for immobilized Protein-A. This study demonstrates that Protein-A-based homogeneous scavenging resolves this issue by precisely controlling the stoichiometry of Protein-A to Fc fragments, something that is not possible for conventional flow-type systems, such as affinity chromatography.

Plasmonic Enhancement of Two-Photon Excitation Fluorescence by Colloidal Assemblies of Very Small AuNPs Templated on M13 Phage.

In this study, an engineered M13 bacteriophage was examined as a biological template to create a well-defined spacing between very small gold nanoparticles (AuNPs 3-13 nm). The effect of the AuNP particle size on the enhancement of the nonlinear process of two-photon excitation fluorescence (2PEF) was investigated. Compared to conventional (one-photon) microscopy techniques, such nonlinear processes are less susceptible to scattering given that the density of background-scattered photons is too low to generate a detectable signal. Besides this, the use of very small AuNPs in 2PEF microscopy becomes more advantageous because individual "isolated" AuNPs of this size do not sufficiently enhance 2PEF to produce a detectable signal, resulting in even less background signal. To investigate the 2PEF of the AuNP-M13 assemblies, a variety of sample preparation approaches are tested, and surface-enhanced Raman spectroscopy (SERS) is employed to study the strength of plasmon coupling within the gaps of AuNPs assembled on the M13 template. Results indicate that assemblies prepared with 9-13 nm AuNP were able to clearly label Escherichia coli cells and produce a 2PEF signal that was orders of magnitude higher than the isolated AuNP (below the threshold of detection). This study thus provides a better understanding of the opportunities and limitations relevant to the use of such small AuNPs within colloidal plasmonic assemblies, for applications in biodetection or as imaging contrast agents.

Room-Temperature and Selective Triggering of Supramolecular DNA Assembly/Disassembly by Nonionizing Radiation.

Recent observations have suggested that nonionizing radiation in the microwave and terahertz (THz; far-infrared) regimes could have an effect on double-stranded DNA (dsDNA). These observations are of significance owing to the omnipresence of microwave emitters in our daily lives (e.g., food preparation, telecommunication, and wireless Internet) and the increasing prevalence of THz emitters for imaging (e.g., concealed weapon detection in airports, skin cancer screenings) and communication technologies. By examining multiple DNA nanostructures as well as two plasmid DNAs, microwaves were shown to promote the repair and assembly of DNA nanostructures and single-stranded regions of plasmid DNA, while intense THz pulses had the opposite effect (in particular, for short dsDNA). Both effects occurred at room temperature within minutes, showed a DNA length dependence, and did not affect the chemical integrity of the DNA. Intriguingly, the function of six proteins (enzymes and antibodies) was not affected by exposure to either form of radiation under the conditions examined. This particular detail was exploited to assemble a fully functional hybrid DNA-protein nanostructure in a bottom-up manner. This study therefore provides entirely new perspectives for the effects, on the molecular level, of nonionizing radiation on biomolecules. Moreover, the proposed structure-activity relationships could be exploited in the field of DNA nanotechnology, which paves the way for designing a new range of functional DNA nanomaterials that are currently inaccessible to state-of-the-art assembly protocols.

Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization.

Monoclonal antibodies (mAb) are a major focus of the pharmaceutical industry, and polyclonal immunoglobulin G (IgG) therapy is used to treat a wide variety of health conditions. As some individuals require mAb/IgG therapy their entire life, there is currently a great desire to formulate antibodies for bolus injection rather than infusion. However, to achieve the required doses, very concentrated antibody solutions may be required. Unfortunately, mAb/IgG self-assembly at high concentration can produce an unacceptably high viscosity for injection. To address this challenge, this study expands the concept of "dynamic covalent chemistry" to "dynamic bioconjugation" in order to reduce viscosity by interfering with antibody-antibody interactions. Ultra-sub-stoichiometric amounts of dynamic PEGylation agents (down to the nanomolar) significantly reduced the viscosity of concentrated antibody solutions by interfering with oligomerization.

In Vitro and In Vivo Evaluation of PEGylated Layer-by-Layer Polyelectrolyte-Coated Paclitaxel Nanocrystals.

Drug nanocrystals (NCs) are colloidal dispersions composed almost entirely of drug. As such, there is substantial interest in targeting them to diseased tissues, where they can locally deliver high doses of the therapeutic. However, because of their uncontrolled dissolution characteristics in vivo and uptake by the monomolecular phagocyte system, achieving tumor accumulation is challenging. To address these issues, a layer-by-layer approach is adopted to coat paclitaxel NCs with alternating layers of oppositely charged polyelectrolytes, using a PEGylated copolymer as the top layer. The coating successfully slows down dissolution in comparison to the noncoated NCs and to Abraxane (an approved paclitaxel nanoformulation), provides colloidal stability in physiologically relevant media, and has no intrinsic effect on cell viability at the concentrations tested. Nevertheless, their pharmacokinetic and biodistribution profile indicates that the NCs are rapidly cleared from the bloodstream followed by accumulation in the mononuclear phagocyte system organs (i.e., liver and spleen). This is hypothesized to be a consequence of the shedding of the PEGylated polyelectrolyte from the NCs' surface. While therapeutic efficacy was not investigated (due to poor tumor accumulation), overall, this work questions whether approaches that rely solely on electrostatic interactions for retaining coatings on the surfaces of NCs are appropriate for use in vivo.

Releasable Conjugation of Polymers to Proteins.

Many synthetic strategies are available for preparing well-defined conjugates of peptides/proteins and polymers. Most reports on this topic involve coupling methoxy poly(ethylene glycol) to therapeutic proteins, a process referred to as PEGylation, to increase their circulation lifetime and reduce their immunogenicity. Unfortunately, the major dissuading dogma of PEGylation is that, in many cases, polymer modification leads to significant (or total) loss of activity/function. One approach that is gaining momentum to address this challenge is to release the native protein from the polymer with time in the body (releasable PEGylation). This contribution will present the state-of-the-art of this rapidly evolving field, with emphasis on the chemistry behind the release of the peptide/protein and the means for altering the rate of release in biological fluids. Linkers discussed include those based on the following: substituted maleic anhydride and succinates, disulfides, 1,6-benzyl-elimination, host-guest interactions, bicin, ß-elimination, biodegradable polymers, E1cb elimination, ß-alanine, photoimmolation, coordination chemistry, zymogen activation, proteolysis, and thioesters.

The Interplay of Disulfide Bonds, α-Helicity, and Hydrophobic Interactions Leads to Ultrahigh Proteolytic Stability of Peptides.

The contribution of noncovalent interactions to the stability of naturally occurring peptides and proteins has been generally acknowledged, though how these can be rationally manipulated to improve the proteolytic stability of synthetic peptides remains to be explored. In this study, a platform to enhance the proteolytic stability of peptides was developed by controllably dimerizing them into α-helical dimers, connected by two disulfide bonds. This platform not only directs peptides toward an α-helical conformation but permits control of the interfacial hydrophobic interactions between the peptides of the dimer. Using two model dimeric systems constructed from the N-terminal α-helix of RNase A and known inhibitors for the E3 ubiquitin ligase MDM2 (and its homologue MDMX), a deeper understanding into the interplay of disulfide bonds, α-helicity, and hydrophobic interactions on enhanced proteolytic stability was sought out. Results reveal that all three parameters play an important role on attaining ultrahigh proteolytic resistance, a concept that can be exploited for the development of future peptide therapeutics. The understanding gained through this study will enable this strategy to be tailored to new peptides because the proposed strategy displays substantial tolerance to sequence permutation. It thus appears promising for conveniently creating prodrugs composed entirely of the therapeutic peptide itself (i.e., in the form of a dimer).

Targeting of injectable drug nanocrystals.

"Nano" drug delivery carriers are established technologies for improving the therapeutic index of chemotherapeutic drugs and overcoming formulation challenges of poorly water-soluble compounds. Two important remaining challenges, however, are the need to formulate drugs on a case-by-case basis (due to the specific chemistry of each drug) and the difficulty associated with transporting large amounts of drug specifically to the site of the tumor (in part because of moderate to poor drug loadings). One of the most valuable "nano" opportunities in this field is to address these challenges by creating nanocarriers composed of the drug itself, in the form of so-called nanocrystals. However, "nano" creates both opportunities and challenges for targeted drug delivery, which are critically discussed in both in vitro and in vivo settings in this contribution.

Bio-reduction of redox-sensitive albumin conjugates in FcRn-expressing cells.

Disulfide-containing IgG-, Fc-, or albumin-based prodrugs that rely on FcRn-trafficking by endothelial cells for prolonged circulation in the body might be hampered by premature bio-reduction processes during FcRn-mediated recycling events. A detailed bio-reduction analysis of redox-sensitive albumin conjugates in two FcRn-expressing cell lines has been performed. The obtained results indicate that the FcRn-mediated recycling pathway is not (or is only poorly) bio-reducing.

Ion-retention properties of graphene oxide/zinc oxide nanocomposite membranes at various pH and temperature conditions.

Laminar graphene oxide (GO) is a promising candidate material for next-generation highly water-permeable membranes. Despite extensive research, there is little information known concerning GO's ion-sieving properties at high acidic/basic pH and temperatures. In this study, the ion-blockage properties of the pristine GO and GO/zinc oxide (ZnO) nanocomposite membranes were tested using a non-pressure-driven filtration setup over a wide range of pH and temperatures. The ZnO nanoparticles within the composite membranes were synthesized via the room-temperature oxidation of zinc acetate and zinc acrylate precursors and were uniformly distributed across the composite membrane. It is observed that partially replacing the zinc acetate precursor with zinc acrylate improves the blockage performance of the composite membranes under extreme basic conditions by 42%. Moreover, photocatalytically-reduced composite membranes blocked copper sulfate ions 28% more than as-prepared composite membranes. Further, it was discovered that the composition of the membrane plays a vital role in its ion blockage performance at higher temperatures.

Broad control of disulfide stability through microenvironmental effects and analysis in complex redox environments.

Disulfide bonds stabilize the tertiary- and quaternary structure of proteins. In addition, they can be used to engineer redox-sensitive (bio)materials and drug-delivery systems. Many of these applications require control of the stability of the disulfide bond. It has recently been shown that the charged microenvironment of the disulfide can be used to alter their stability by ∼3 orders of magnitude in a predictable and finely tunable manner at acidic pH. The aim of this work is to extend these findings to physiological pH and to demonstrate the validity of this approach in complex redox milieu. Disulfide microenvironments were manipulated synergistically with steric hindrance herein to control disulfide bond stability over ∼3 orders of magnitude at neutral pH. Control of disulfide stability through microenvironmental effects could also be observed in complex redox buffers (including serum) and in the presence of cells. Such fine and predictable control of disulfide properties is not achievable using other existing approaches. These findings provide easily implementable and general tools for controlling the responsiveness of biomaterials and drug delivery systems toward various local endogenous redox environments.

Lipase is essential for the study of in vitro release kinetics from organogels.

In vitro drug release studies remain indispensable in the development of drug delivery systems, even if correlations between in vitro and in vivo results are often imperfect. In this work, an improved in vitro analysis method for studying in situ-forming lipid-based implants was developed. More specifically, lipase was found to be an essential additive for evidencing differences in drug release kinetics from organogels of different amino acid-based organogelators, organogelator concentrations, drug loadings, and volumes. Lipases are thought to participate in the degradation of and release from amino acid-based organogel implants in vivo. Our experimental conditions allowed for the rapid and reliable screening of in vitro parameters that may be optimized to slow or accelerate drug release, once preliminary in vivo data are available.