Thermoresponsive behavior and in vitro investigations of random copolymers of ethylene oxide and glycidyl methyl ether (rPEGs)
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Abstract
Polyethers are crucial materials in modern medicine, with polyethylene glycol (PEG) being the most prominent member of this class with a global production of 2.5 million metric tons in 2022. Derived from the polymerization of ethylene oxide (EO) via anionic ring-opening polymerization (AROP), PEG presents exceptional characteristics: it is water soluble, biocompatible and can be excreted through the human renal system up to a molecular weight of 30 kDa. PEGylation – the process of functionalizing active pharmaceutical ingredients (APIs) with PEG – has revolutionized drug delivery research in medical science since its discovery in the 1970s. By enveloping APIs with a corona of PEG molecules, the immune reaction to these compounds is significantly reduced, thanks to the inhibition of opsonin adsorption. This phenomenon, known as the "stealth effect",
prolongs the circulation time of PEGylated compounds in the bloodstream significantly compared to their non-modified counterparts. In March 1990 the first PEGylated protein (Adagen® by Enzon Pharmaceuticals) was approved by the food and drug administration (FDA) for application and since then dozens more have entered the market. As of 2023, 38 PEGylated drugs have earned approval by the FDA. However, the discovery of the stealth effect was soon met with the detection of anti-PEG antibodies (APAs). These antibodies can induce chronic adverse reactions to PEG, ranging from accelerated blood clearance (ABC), which counteracts the stealth effect, to hypersensitive reactions similar to anaphylactic symptoms. Recent studies suggest that APAs are present in 83 % of the German general population, likely due to extensive exposure to PEG in everyday household products. This underscores the need for alternatives to PEG that can mitigate immune responses while maintaining its beneficial properties.
To counteract the increasing problem of immunogenic reactions to PEG, extensive research is performed to develop alternatives for PEGylation. We have recently developed a synthesis for random copolymers of EO and glycidyl methyl ether (GME), which have the potential to be a viable alternative for PEG in case of severe immunogenic reactions. These “rPEGs” are structurally isomeric to PEG but possess randomly distributed side chains at the polymer backbone to prevent antibody binding. Previously, copolymers with GME could not be synthesized under GMPcertified conditions, excluding them from biomedical applications. However, we have successfully synthesized random copolymers of EO and GME under the same GMP compliant AROP conditions that are used for the synthesis of PEG, granting the possibility of medical usage.
This thesis covers the synthesis of rPEGs via AROP, functionalization of the copolymers through post-polymerization reactions, investigations of physicochemical properties as well as behavior towards antibodies and proteins under in vitro conditions. A theoretical framework for the synthesis, properties and application of polyethers with a focus on PEG is provided in Chapter 1. Additionally, general concepts of PEGylation and the phase behavior of polyethers in aqueous solutions are discussed.
Chapter 2 covers the synthesis of the monomer GME and its copolymerization procedure with EO. Several copolymers with constant chain length and varying molar amount of GME were synthesized and characterized via NMR spectroscopy, MALDI TOF mass spectrometry and GPC. The results show a reproducible synthesis for rPEGs of a molar GME content up to 80 mol %. A homopolymer of GME with a chain length of Dp = 103 could be synthesized and used for further measurements, but GME homopolymerization still provides limited reaction control compared to the copolymers. Furthermore, the thermoresponsive behavior of rPEGs in aqueous solution was investigated by turbidimetry and ESR. Turbidimetric measurements were performed at concentrations of 5 mg mL-1 and 100 mg mL-1, while ESR measurements were performed at 100 mg mL-1. At a concentration of 5 mg mL-1, cloud points could be measured turbidimetrically for copolymers of molar fractions above 40 mol % and ranged between 96 °C and 70 °C. At
concentrations of 100 mg mL-1, cloud points were determined between 86 °C and 68 °C by turbidimetry and show good accordance with the onset of chain collapse and the formation of hydrophobic segments observed in the EPR measurements. Even at high concentrations, all polymers showed cloud points above 60 °C, providing no indication of macroscopic phase separation in the range of physiologically relevant temperatures.
Chapter 3 examines the functionalization of the copolymers with a fluorescent dye, specifically to enable studies via fluorescence correlation spectroscopy (FCS) and microscale thermophoresis, (MST) regarding the binding affinity of monoclonal backbone-specific anti-PEG Immunoglobulin G (IgG) antibodies to rPEGs of constant molar mass and varying molar GME content under in vitro conditions. The obtained results showed a steadily decreasing antigenicity of the copolymers with increasing molar content of GME substantiated by a decreasing change in hydrodynamic radius when exposing rPEGs to anti-PEG IgG up to concentrations of 5 μM as well as a strong increase in the dissociation constant Kd for higher molar contents of GME in the copolymer backbone. While reduced binding affinity was observed for copolymers with molar GME contents up to 42 mol%, a copolymer with a molar GME content of 52 mol% yielded no detectable antibody binding even at high ligand concentration.
The focus of Chapter 4 is placed on the synthesis of rPEGs with lipid functionalization and analysis of the protein corona formed by particles coated with these copolymers upon exposure to human serum. To achieve full functionalization, a lipid initiator was synthesized, and AROP was performed under adapted conditions to accommodate for reactivity and solubility of this initiator. The prepared lipids of constant chain length and varying molar amounts of GME were coated onto nanocarriers so the effect of the rPEG backbone on the formation of the protein corona could be observed. Although overall protein concentration in the corona was found to be significantly lower for the particles coated with the synthesized samples compared to Lutensol AT50, the relative abundance of anti-PEG IgG in total IgG was higher for all synthesized samples and remained approximately constant for varying molar contents of GME in the copolymers. The lowest total concentration of IgG in the protein corona was found for the sample with the highest molar content of GME. At the same time, this sample showed increased concentrations of opsonins in the corona, while dysopsonins were most abundant in samples with low molar GME content. It was concluded that the unexpectedly low packing density of the synthesized rPEGs on the particle surface had a strong influence on the formation of the protein corona so that only general trends in the composition could be observed while the precise influence of the molar GME content on the composition of the corona could not be conclusively determined.
Appendix Chapter 1 introduces the fundamental concept of rPEGs and provides the basis for the investigations carried out in this work. Successful synthesis of rPEGs under AROP conditions was shown for copolymers up to a GME content of 74 mol% through analysis via 1H NMR and DOSY NMR spectroscopy, SEC and MALDI TOF. Statistical calculations provided insight into the diminished occurrence of epitopes containing 16 or more consecutive EO units for rPEGs with increasing GME content. First turbidimetric measurements showed no phase separation for any of the polymers in aqueous solution below 70 °C for concentrations of up to 10 mg mL-1. Cell viability and B cell activation as well as hemocompatibility in human blood in vitro were demonstrated for the rPEG samples and showed comparable values to mPEG. Competitive backbone-selective and
end group-selective APA ELISAs were able to demonstrate a stark reduction in relative antibody affinity to rPEGs with increasing molar GME content compared to mPEG.