PEGylation of recombinant human deoxyribonuclease I decreases its transport across lung epithelial cells and uptake by macrophages

Abstract

Conjugation to high molecular weight (MW) polyethylene glycol (PEG) was previously shown to largely prolong the lung residence time of recombinant human deoxyribonuclease I (rhDNase) and improve its therapeutic efficacy following pulmonary delivery in mice. In this paper, we investigated the mechanisms promoting the extended lung retention of PEG-rhDNase conjugates using cell culture models and lung biological media. Uptake by alveolar macrophages was also assessed in vivo. Transport experiments showed that PEGylation reduced the uptake and transport of rhDNase across monolayers of Calu-3 cells cultured at an air-liquid interface. PEGylation also decreased the uptake of rhDNase by macrophages in vitro whatever the PEG size as well as in vivo 4 h following intratracheal instillation in mice. However, the reverse was observed in vivo at 24 h. The uptake of rhDNase by macrophages was dependent on energy, time, and concentration and occurred at rates indicative of adsorptive endocytosis. The diffusion of PEGylated rhDNase in porcine tracheal mucus and cystic fibrosis sputa was slower compared with that of rhDNase. Nevertheless, no significant binding of PEGylated rhDNase to both media was observed. In conclusion, decreased transport across lung epithelial cells and uptake by macrophages appear to contribute to the longer retention of PEGylated rhDNase in the lungs.

Advances in Pulmonary Protein Delivery Systems

“…[ 49 ] They investigated the potential mechanism of the prolonged retention of PEGylated proteins using 40 kDa PEGylated rhDNase and concluded that decreased transport across lung epithelial cells and uptake by macrophages are the main reasons. [ 58 ] The MW of PEG also affects the enzymatic stability of PEGylated proteins as PEG creates steric barriers of protein–enzyme interactions. Karuri et al showed that the enzymatic stability of PEGylated fibronectin is positively correlated with the MW of PEG (2–10 kDa).…”

Protein Engineering Strategies for Improved Pharmacokinetics

“…[21] Finally, the hydrophilic nature of PEG decreases protein interactions with the cell membrane and thereby, decreases protein endocytosis by epithelial cells and alveolar macrophages. [22]…”

Optimization of a Liposomal DNase I Formulation with an Extended Circulating Half-Life

“…For instance, Guichard et al have recently developed a mono-PEGylated form of DNase I by conjugating high-molecular-weight PEG chains (20–40 kDa) to the N-terminus of the enzyme, while preserving its activity and secondary structure . Additionally, the conjugation of 30 kDa PEG to DNase I prolonged its in vivo mucolytic activity up to 24 h in lung homogenates, increased lung accumulation up to 7 days versus 24 h for native DNase I, reduced transportation across lung epithelial cells, and diminished uptake and systemic elimination by alveolar macrophages after a single intratracheal inhalation in mouse models of cystic fibrosis. − Gene therapy has also been used to optimize the PK parameters of DNase I, with the IV administration of a single dose of a hepatotropic adeno-associated virus (AAV) vector encoding DNase I increasing serum levels for at least 21 days in a dose-dependent manner. This expression of DNase I was found to be positively correlated with the reduced accumulation of NETs in tumors and in serum, factors associated with reduced liver metastasis of colorectal cancer .…”