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.

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.

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.

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.

Tracking the bioreduction of disulfide-containing cationic dendrimers.

Disulfides enhance the transfection efficacy and reduce the toxicity of cationic gene delivery polymers. A quantitative analysis is provided of the bioreduction of a dynamic bioreducible dendritic polycationic probe in four cell lines. Such knowledge is indispensible for understanding and optimizing bioreducible drug and gene delivery systems.

Arginine-specific modification of proteins with polyethylene glycol.

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ "kinetic" selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on "thermodynamic" selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an α-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.

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.

Bile acids as constituents for dental composites: in vitro cytotoxicity of (meth)acrylate and other ester derivatives of bile acids.

Methacrylic derivatives of bile acids have been synthesized for use as monomers in dental composites. Polymeric dental materials are known to leach cytotoxic unreacted monomers and degradation products. In this study, the in vitro cytotoxicity of bile acids and their derivatives towards 3T3 fibroblasts has been evaluated by colorimetric MTT assay and compared with that of the common dental monomers BisGMA, UDMA and TEGDMA. In general, the bile acids and their derivatives induced mitochondrial dysfunction at similar or higher concentrations than the commercial dental monomers. Certain monomers did not influence MTT response over their entire range of solubility.

Site-Specific Polymer Conjugation Stabilizes Therapeutic Enzymes in the Gastrointestinal Tract.

The site-specific conjugation of polymers to multiple engineered cysteine residues of a prolyl endopeptidase leads to its stabilization in the gastrointestinal tract of rats, without compromising the activity relative to the native enzyme. The importance of polymer attachment sites is investigated, as well as the significance of polymer structure.

A new method for quantifying the intensity of the C=C band of dimethacrylate dental monomers in their FTIR and Raman spectra.

The degree of conversion (DC) of methacrylate dental resins is typically determined by spectroscopically measuring the decrease of the vinyl (C=C) stretching band at 1640 cm(-1), ratioed before and after polymerization to an internal standard (aromatic ring quadrant stretching vibration (Ph)) at around 1609 cm(-1). While standard methods exist for measuring the intensity of the C=C and Ph peaks from the FTIR or Raman spectrum, these methods either fail under certain circumstances, or lack a physical basis, being purely based on spectral features. In this study, we present a rigorous method (named rotational isomerism method) for determining the intensity of the vinyl and aromatic bands from the FTIR and Raman spectra of dental monomer mixtures, and compare it to a standard baseline method (SBM) and a standard curve fitting method. Model triethyleneglycol dimethacrylate (TEGDMA)/2,2-bis(4-hydroxyphenyl)propane (Bisphenol-A) and TEGDMA/2,2-bis(4-(2-hydroxy-3-methacryloxypropoxy)phenyl)propane (Bis-GMA) mixtures with a series of known C=C/Ph molar ratios were prepared in order to simulate the effect of curing. The accuracy of the new method for measuring the DC was found to be as good as the commonly used SBM. The standard curve fitting method was shown to be inappropriate for measuring C=C/Ph ratios by FTIR spectroscopy due to its inability to realistically simulate the features of the spectra. The insight gained through the use of this new method may be useful for the characterization of other methacrylate biomaterials.

Twin disulfides as opportunity for improving stability and transfection efficiency of oligoaminoethane polyplexes.

The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure-activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine-arginine-cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency.

Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo.

In comparison to neutral linear polymers, functional and architecturally complex (that is, non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. Here we present an in vivo demonstration that modification of a protein with a polymer of appropriate architecture can impart low immunogenicity, with a commensurably low loss of therapeutic activity. These combined properties are inaccessible by conventional strategies using linear polymers. For the model protein L-asparaginase, a comb-polymer bio-conjugate significantly outperformed the linear polymer control in terms of lower immune response and more sustained bioactivity. The semi-permeability characteristics of the coatings are consistent with the phase diagram of the polymer, which will facilitate the application of this strategy to other proteins and with other therapeutic models.

Modular design of redox-responsive stabilizers for nanocrystals.

Many potent drugs are difficult to administer intravenously due to poor aqueous solubility. A common approach for addressing this issue is to process them into colloidal dispersions known as "nanocrystals" (NCs). However, NCs possess high-energy surfaces that must be stabilized with surfactants to prevent aggregation. An optimal surfactant should have high affinity for the nanocrystal's surface to stabilize it, but may also include a trigger mechanism that could offer the possibility of altering size distribution and uptake of the NC. This study presents a modular and systematic strategy for optimizing the affinity of polymeric stabilizers for drug nanocrystals both before and after oxidation (i.e., the selected trigger), thus allowing for the optimal responsiveness for a given application to be identified. A library of 10 redox-responsive polymer stabilizers was prepared by postpolymerization modification, using the thiol-yne reaction, of two parent block copolymers. The stabilizing potential of these polymers for paclitaxel NCs is presented as well as the influence of oxidation on size and dissolution following exposure to reactive oxygen species (ROS), which are strongly associated with chronic inflammation and cancer. Owing to the versatility of postpolymerization modification, this contribution provides general tools for preparing triggered-sheddable stabilizing coatings for nanoparticles.

Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates.

Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

PEG nanocages as non-sheddable stabilizers for drug nanocrystals.

Many potent drugs are difficult to administer intravenously due to poor aqueous solubility. One validated approach for addressing this issue is to process them into colloidal dispersions known as "nanocrystals" (NCs). However, NCs possess high-energy surfaces that must be stabilized with surfactants to prevent aggregation. In addition, the stabilizer provides a means of anchoring targeting moieties to the NCs for achieving deposition or uptake at specified locations. Nevertheless, a critical challenge is that the surfactant (and consequently the targeting agents) can be shed upon high dilution. This work demonstrates successful cross-linking by click chemistry of stabilizers around paclitaxel NCs to form polymeric "nanocages". Cross-linking does not cause aggregation, as evidenced by transmission electron microscopy, and the nanocages retained the particulate drug through a combination of physical entrapment and physisorption. Size measurements by dynamic light scattering showed that nanocages act as sterically stabilizing barriers to particle-particle interactions and aggregation. The nanocages were shown to be less shed from the NCs than comparable non-cross-linked stabilizers. This contribution provides crucial general tools for preparing poorly sheddable stabilizing coatings to NCs and potentially other classes of nanoparticles for which covalent attachment of the stabilizer to the particle is undesirable (e.g., a drug) or impossible (chemically inert). The presented approach also offers the possibility of more stably attaching targeting moieties to the latter by use of heterotelechelic PEG derivatives, which may favor active targeting and internalization by cells.

New dental composites containing multimethacrylate derivatives of bile acids: a comparative study with commercial monomers.

We have prepared multifunctional methacrylate derivatives of bile acids as cross-linkable monomers for use in dental composites. By modifying the chemical structure of the monomers, we were able to vary the viscosity, hydrophobicity, and reactivity and have studied the effect of these parameters on the conversion of the monomers, the shrinkage during polymerization, and the mechanical properties of the resulting polymers and composites. Materials containing these new monomers generally had physical, thermal, and mechanical properties comparable to those containing the commonly used dental monomers BisGMA or UDMA and had lower polymerization shrinkage. The multimethacrylate derivatives of cholic acid, which are known to be less cytotoxic than BisGMA and UDMA, are shown to be promising materials for dental applications.

Thiol-functionalized polymeric micelles: from molecular recognition to improved mucoadhesion.

Surface-modified colloids which can selectively interact with biological species or surfaces show promise as drug delivery systems. However, the preparation of such targeted devices remains challenging, especially when considering polyion complex micelles for which side reactions with the ionic core components (typically carboxylic acid or amino groups) can occur. To solve this issue, an innovative synthetic strategy is proposed and used to prepare an asymmetric poly(ethylene glycol)-block-poly(2-(N,N-dimethylamino)ethyl methacrylate) copolymer presenting a thiol group at the end of the poly(ethylene glycol) chain. Thiol groups are highly appealing given that they react almost exclusively and quantitatively with maleimides under physiological conditions, thereby facilitating the chemical functionalization of the copolymer. The simplicity of the derivatization procedure is illustrated by preparing model biotin-capped copolymers. The biotinylated copolymers are shown to self-assemble with an oligonucleotide in aqueous media to form polyion complex micelles with biotin groups at their outer surface. These micelles are capable of molecular recognition toward streptavidin. Alternatively, thiol-decorated (nonderivatized) micelles are prepared and show improved mucoadhesion through the formation of disulfide bonds with mucin. Finally, intermicellar disulfide bonds are generated under oxidative conditions to promote the formation of stimuli-responsive micellar networks.