Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.

Reversible Bioconjugation: Biodegradable Poly(phosphate)-Protein Conjugates.

Protein-polymer conjugates are widely used to improve the pharmacokinetic properties of therapeutic proteins. Commercially available conjugates employ poly(ethylene glycol) (PEG) as the protective polymer; however, PEG has a number of shortcomings, including non-biodegradability and immunogenicity, that call for the development of alternatives. Here, the synthesis of biodegradable poly(phosphate), that is, poly(ethyl ethylene phosphate) (PEEP), by organo-catalyzed anionic ring-opening polymerization exhibiting dispersity values Ð < 1.3 is reported. Polymers with molecular weights between 2000 and 33 200 g mol-1 are then ω-functionalized with a succinimidyl carbonate group and subsequently conjugated to model proteins. These are the first conjugates based on polyphosphates which degraded upon exposure to phosphodiesterase. As is the case for PEGylated therapeutics, residual in vitro activity of the PPEylated conjugates depends on the extent of protein modification. These results suggest that PEEP exhibits the desired properties of a biopolymer for use in next generation, fully degradable drug delivery systems.

Degradable Polyphosphoester-Protein Conjugates: "PPEylation" of Proteins.

Pharmacokinetic properties determine the efficacy of protein therapeutics. The covalent attachment of poly(ethylene glycol) (PEG) extends the half-life of such biologicals to maintain a therapeutically effective concentration over a prolonged period of time and improves administration and compliance. A major obstacle of these polymer-protein conjugates is the chemical stability of the PEG preventing its metabolism and leading to side effects. Instead, we propose the PPEylation, that is, the conjugation of degradable poly(phosphoester)s (PPE) to proteins, in order to generate fully biodegradable polymer-protein conjugates. The structure of the PPEylated protein conjugates was verified with mass spectrometry and size exclusion chromatography. They were compared to structural analogues, except classical, PEGylated proteins, and exhibit comparable bioactivity, but avoiding any nondegradable polymer in the conjugate. We proved the degradation of the protective polymer shell surrounding the conjugate in aqueous environments at physiological conditions by online triple detection size exclusion chromatography and gel electrophoresis. We believe that this research will provide an attractive alternative for future drug design with implications for the clinical use of biologicals.

Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers.

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

Investigation into the Relaxation Dynamics of Polymer-Protein Conjugates Reveals Surprising Role of Polymer Solvation on Inherent Protein Flexibility.

Fully biodegradable protein-polymer conjugates, namely, MBP-PMeEP (maltose binding protein-poly methyl-ethylene phosphonate), have been investigated in order to understand the role of polymer solvation on protein flexibility. Using elastic and quasi-elastic incoherent neutron scattering, in combination with partially deuterated conjugate systems, we are able to disentangle the polymer dynamics from the protein dynamics and meaningfully address the coupling between both components. We highlight that, in the dry state, the protein-polymer conjugates lack any dynamical transition in accordance with the generally observed behavior for dry proteins. In addition, we observe a larger flexibility of the conjugated protein, compared to the native protein, as well as a lack of polymer-glass transition. Only upon water hydration does the conjugate recover its dynamical transition, leading to the conclusion that exclusive polymer solvation is insufficient to unfreeze fluctuations on the picosecond-nanosecond time scale in biomolecules. Our results also confirm the established coupling between polymer and protein dynamics in the conjugate.

Poly(phosphoester)s: A New Platform for Degradable Polymers.

Poly(phosphoester)s (PPEs) play an important role in nature. They structure and determine life in the form of deoxy- and ribonucleic acid (DNA and RNA), and, as pyrophosphates, they store up chemical energy in organisms. Polymer chemistry, however, is dominated by the nondegradable polyolefins and degradable poly(carboxylic ester)s (PCEs) that are produced on a large scale today. Recent studies have illustrated the potential of PPEs for future applications beyond flame retardancy, and provided a coherent vision to implement this classic biopolymer in modern applications that demand biocompatibility and degradability as well as the possibility to adjust the properties to individual needs.

Water-Soluble Poly(phosphonate)s via Living Ring-Opening Polymerization.

A small difference brings high control: In poly(phosphonate)s a stable carbon-phosphorus linkage attaches a side chain to a degradable poly(phosphoester)-backbone. A novel cyclic phosphonate monomer was developed to generate water-soluble aliphatic poly(ethylene methylphospho-nate)s. The monomer is accessible via a robust three-step protocol that can be easily scaled-up. Polymerization was initiated by a primary alcohol, mediated by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in less than 2 h at 0 °C. The molecular weight distributions were monomodal and very narrow (below 1.1) in all cases and molecular weights up to about 20000 g/mol have been prepared, proving the living nature of this polymerization. The resulting polymers were characterized in detail via NMR spectroscopy, size exclusion chromatography, and differential scanning calorimetry. Also, the reaction kinetics have been evaluated for several monomer/initiator ratios and found to guarantee a living behavior in all cases superior to other poly(phosphate)s reported earlier. The polymers are all highly water-soluble without a lower critical solution temperature and are nontoxic against HeLa cells.

One-pot squaric acid diester mediated aqueous protein conjugation.

A water-soluble squaric acid dialkyl diester derivative is presented, which enables one-pot, two-step amine-selective protein conjugation reactions with (functional) amines in water. This reagent not only allows all-aqueous protein modifications, but also tolerates e.g. hydroxyl groups and can also be used for the modification of proteins with water-insoluble amines.

Universal concept for the implementation of a single cleavable unit at tunable position in functional poly(ethylene glycol)s.

Poly(ethylene glycol) (PEG) with acid-sensitive moieties gained attention particularly for various biomedical applications, such as the covalent attachment of PEG (PEGylation) to protein therapeutics, the synthesis of stealth liposomes, and polymeric carriers for low-molecular-weight drugs. Cleavable PEGs are favored over their inert analogues because of superior pharmacodynamic and/or pharmacokinetic properties of their formulations. However, synthetic routes to acetal-containing PEGs published up to date either require enormous efforts or result in ill-defined materials with a lack of control over the molecular weight. Herein, we describe a novel methodology to implement a single acetaldehyde acetal in well-defined (hetero)functional poly(ethylene glycol)s with total control over its position. To underline its general applicability, a diverse set of initiators for the anionic polymerization of ethylene oxide (cholesterol, dibenzylamino ethanol, and poly(ethylene glycol) monomethyl ether (mPEG)) was modified and used to synthesize the analogous labile PEGs. The polyether bearing the cleavable lipid had a degree of polymerization of 46, was amphiphilic and exhibited a critical micelle concentration of 4.20 mg·L(-1). From dibenzylamino ethanol, three heterofunctional PEGs with different molecular weights and labile amino termini were generated. The transformation of the amino functionality into the corresponding squaric acid ester amide demonstrated the accessibility of the cleavable functional group and activated the PEG for protein PEGylation, which was exemplarily shown by the attachment to bovine serum albumin (BSA). Furthermore, turning mPEG into a macroinitiator with a cleavable hydroxyl group granted access to a well-defined poly(ethylene glycol) derivative bearing a single cleavable moiety within its backbone. All the acetal-containing PEGs and PEG/protein conjugates were proven to degrade upon acidic treatment.