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.

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.

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.

Sustained blood glutamate scavenging enhances protection in ischemic stroke.

Stroke is a major cause of morbidity, mortality, and disability. During ischemic stroke, a marked and prolonged rise of glutamate concentration in the brain causes neuronal cell death. This study explores the protective effect of a bioconjugate form of glutamate oxaloacetate transaminase (hrGOT), which catalyzes the depletion of blood glutamate in the bloodstream for ~6 days following a single administration. When treated with this bioconjugate, a significant reduction of the infarct volume and a better retention of sensorimotor function was observed for ischemic rats compared to those treated with saline. Moreover, the equivalent dose of native hrGOT yielded similar results to the saline treated group for some tests. Targeting the bioconjugate to the blood-brain-barrier did not improve its performance. The data suggest that the bioconjugates draw glutamate out of the brain by displacing homeostasis between the different glutamate pools of the body.

Surface vs. core N/S/Se-heteroatom doping of carbon nanodots produces divergent yet consistent optical responses to reactive oxygen species.

Carbon nanodots (CNDs) have attracted substantial scientific curiosity because of their intriguing stimuli-responsive optical properties. However, one obstacle to the more widespread use of CNDs as transducers for e.g., biodetection systems is incomplete knowledge regarding the underlying chemical changes responsible for this responsiveness, and how these chemical features can be engineered via the precursors chosen for CND synthesis. This study demonstrates that the precursor's functional groups play a key role in directing N/S/Se heteroatom dopants either towards the surface of the CNDs, towards the aromatic core, or towards small organic fluorophores in the core. Divergent optical properties, which were consistent amongst groups of CNDs prepared with similar precursors, were obtained including either a decrease or increase of fluorescence intensity in the presence of hydrogen peroxide. Moreover, CNDs were identified with orthogonal responsiveness to radical (hydroxyl radicals, ˙OH; down to 2.5 µM) vs. non-radical oxidants (H2O2; down to 50 µM), which suggests that control of the chemistry of CNDs via the choice of precursor could yield probes that are specific to certain sub-species of reactive oxygen species or entirely different molecules altogether, based on the way they chemically-modify the surface (respond faster) and core functional groups (respond slower) associated with chromophores/fluorophores of which the CNDs are composed.

Accelerated inactivation of M13 bacteriophage using millijoule femtosecond lasers.

Irradiation of femtosecond (fs) pulse lasers in the visible and near-infrared ranges have been proposed as a promising approach for inactivating viruses. However, in order to achieve significant virus inactivation, past works have required relatively long irradiation times (1 hour or longer), even for small volumes. Given its advantages compared with other techniques, there is an urgent need to shorten the time required to inactivate viruses using fs laser technology. In this study, we investigate the inactivation of purified M13 bacteriophage in phosphate-buffered saline with large active volume (1 cm3 ), and short exposure time (several minutes), using lasers with 20 mJ/pulse energy at various wavelengths (800, 400 nm or both 800 and 400 nm combined). For an exposure time of 15 and 2 minute, the use of a 400 nm wavelength laser results in a high load reduction of 5.8 ± 0.3 and 2.9 ± 0.15, respectively, on the log10 scale of viability. We show that virus inactivation using the 400 nm laser is much more efficient compared with that using an 800 nm laser, or the simultaneous irradiation of 400 and 800 nm lasers. Higher pathogen inactivation is observed for lasers with shorter pulse duration, whereas at longer pulse durations, the inactivation is reduced. For millijoule-energy fs laser irradiation, the M13 bacteriophage inactivation, via the reduction of the functionality of M13 bacteriophages, is accompanied with relatively small amounts of genetic damage.

Determination of the degree of PEGylation of protein bioconjugates using data from proton nuclear magnetic resonance spectroscopy.

The average number of methoxy poly(ethylene glycol) (mPEG) chains grafted to a protein - also known as the degree of PEGylation - is a fundamental parameter for characterizing a bioconjugate. The degree of PEGylation is typically determined by chromatographic or electrophoretic methods, which are subject to certain biases. This contribution describes an analytical approach alongside technical precautions for quantitatively determining the degree of PEGylation of protein bioconjugates by 1H NMR spectroscopy. An accompanying dataset, corresponding to the raw 1H NMR spectra of thirteen bioconjugates with different degrees of PEGylation and different mPEG molecular weights, is provided for the reader to become familiar with the analysis. The exemplary bioconjugate system used in this Data article is the enzyme glutamate dehydrogenase (GDH) modified with multiple copies of mPEG (0.5-20 kDa). These bioconjugates correspond to those discussed in-depth in the article "Mechanisms of activity loss for a multi-PEGylated protein by experiment and simulation" by Zaghmi et al., 2019 The described approach to calculate degree of PEGylation is quantitative, applicable to other proteins, and can be adapted to other types of polymers.

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.

Evidence, Manipulation, and Termination of pH 'Nanobuffering' for Quantitative Homogenous Scavenging of Monoclonal Antibodies.

This study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, a phenomenon termed "nanobuffering". This can be exploited to dissociate local nanoscale pH from bulk solution pH. Herein, a series of pH-responsive polymers were conjugated to Protein-A to rationally manipulate the latter's binding affinity toward antibodies via nanobuffering ( i. e., this interaction is pH dependent), independently of bulk solution pH. Moreover, the nanobuffering effect could be terminated using low concentrations of strong ion-pairing salts, to achieve quantitative release of the antibodies from the bioconjugate. These complementary discoveries are showcased in the context of the development of a homogeneous affinity precipitation agent ( i. e., a scavenger) for the purification of polyclonal immunoglobulin G and two monoclonal antibodies from cell culture supernatant. Indeed, while bulk solution pH was used to induce precipitation of the scavenger, maintaining local nanoscale pH via nanobuffering maximized binding interaction with the antibodies. A 2:1 binding stoichiometry was observed, which was similar to that observed for native protein. The scavenger could be recycled multiple times, and the purification protocol circumvented lengthy/tedious physical purification processes typically associated with mAb manufacturing. Overall, this study provides perspectives on the local nanoscale pH near pH-responsive polymers and establishes lines of thought for predictably manipulating or even terminating nanobuffering, to control the activity of proteins.

Reprogramming the assembly of unmodified DNA with a small molecule.

The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA 'alphabet' by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.

Controlled growth of DNA structures from repeating units using the vernier mechanism.

In this report, we demonstrate the assembly of length-programmed DNA nanostructures using a single 16 base sequence and its complement as building blocks. To achieve this, we applied the Vernier mechanism to DNA assembly, which uses a mismatch in length between two monomers to dictate the final length of the product. Specifically, this approach relies on the interaction of two DNA strands containing a different number (n, m) of complementary binding sites: these two strands will keep binding to each other until they come into register, thus generating a larger assembly whose length (n × m) is encoded by the number of binding sites in each strand. While the Vernier mechanism has been applied to other areas of supramolecular chemistry, here we present an application of its principles to DNA nanostructures. Using a single 16 base repeat and its complement, and varying the number of repeats on a given DNA strand, we show the consistent construction of duplexes up to 228 base pairs (bp) in length. Employing specific annealing protocols, strand capping, and intercalator chaperones allows us to further grow the duplex to 392 base pairs. We demonstrate that the Vernier method is not only strand-efficient, but also produces a cleaner, higher-yielding product than conventional designs.

Intercalators as molecular chaperones in DNA self-assembly.

DNA intercalation has found many diagnostic and therapeutic applications. Here, we propose the use of simple DNA intercalators, such as ethidium bromide, as tools to facilitate the error-free self-assembly of DNA nanostructures. We show that ethidium bromide can influence DNA self-assembly, decrease the formation of oligomeric side products, and cause libraries of multiple equilibrating structures to converge into a single product. Using a variety of 2D- and 3D-DNA systems, we demonstrate that intercalators present a powerful alternative for the adjustment of strand-end alignment, favor the formation of fully duplexed "closed" structures, and create an environment where the smallest, most stable structure is formed. A new 3D-DNA motif, the ninja star, was self-assembled in quantitative yield with this method. Moreover, ethidium bromide can be readily removed using isoamyl alcohol extractions combined with intercalator-specific spin columns, thereby yielding the desired ready-to-use DNA structure.

The role of organic linkers in directing DNA self-assembly and significantly stabilizing DNA duplexes.

We show a simple method to control both the stability and the self-assembly behavior of DNA structures. By connecting two adjacent duplexes with small synthetic linkers, factors such as linker rigidity and DNA strand orientation can increase the thermal denaturation temperature of 17 base-pair duplexes by up to 10 °C, and significantly increase the cooperativity of melting of the two duplexes. The same DNA sequence can thus be tuned to melt at vastly different temperatures by selecting the linker structure and DNA-to-linker connectivity. In addition, a small rigid m-triphenylene linker directly affects the self-assembly product distribution. With this linker, changes in the orientation of the linked strands (e.g., 5'3' vs 3'3') can lead to dramatic changes in the self-assembly behavior, from the formation of cyclic dimer and tetramer to higher-order oligomers. These variations can be readily predicted using a simple strand-end alignment model.