Size dependent biodistribution and SPECT imaging of (111)In-labeled polymersomes.

Polymersomes, self-assembled from the block copolymer polybutadiene-block-poly(ethylene glycol), were prepared with well-defined diameters between 90 and 250 nm. The presence of ~1% of diethylene triamine penta acetic acid on the polymersome periphery allowed to chelate radioactive (111)In onto the surface and determine the biodistribution in mice as a function of both the polymersome size and poly(ethylene glycol) corona thickness (i.e., PEG molecular weight). Doubling the PEG molecular weight from 1 kg/mol to 2 kg/mol did not change the blood circulation half-life significantly. However, the size of the different polymersome samples did have a drastic effect on the blood circulation times. It was found that polymersomes of 120 nm and larger become mostly cleared from the blood within 4 h, presumably due to recognition by the reticuloendothelial system. In contrast, smaller polymersomes of around 90 nm circulated much longer. After 24 h more than 30% of the injected dose was still present in the blood pool. This sharp transition in blood circulation kinetics due to size is much more abrupt than observed for liposomes and was additionally visualized by SPECT/CT imaging. These findings should be considered in the formulation and design of polymersomes for biomedical applications. Size, much more than for liposomes, will influence the pharmacokinetics, and therefore, long circulating preparations should be well below 100 nm.

Cell surface clustering of heparan sulfate proteoglycans by amphipathic cell-penetrating peptides does not contribute to uptake.

For arginine-rich cell-penetrating peptides (CPPs), an association with heparan sulfate (HS) chains is considered the first step in the stimulation of uptake for many cells. Much less is known about the role of HS chains in the cell-association and internalization of arginine-free amphipathic CPP such as transportan-10 (TP10). Here, we report that various TP10 analogs differ in their capacity to accumulate on HS-rich plasma membranes in an HS-dependent manner. No accumulation was observed on HS-poor plasma membranes or when HS was removed by enzymatic cleavage. The TP10 analog that strongly clustered on the cell surface, also showed a pronounced capacity to form clusters with HS chains in solution. However, aggregation occurred in a thermodynamically different way compared to the interaction of arginine-rich CPP with HS. To monitor the impact of the peptide on the aggregation of the glycocalyx by time-lapse microscopy, sialic acids were visualized by metabolic labeling using copper-free click chemistry to attach fluorophores to metabolically incorporated azido sugars. Strikingly, a highly enhanced HS-mediated accumulation on the plasma membrane of a particular TP10 analog did not correlate with a better uptake. These findings illustrate that the mode of interaction between cell-penetrating peptides and HS chains has important functional consequences regarding peptide internalization and that there is no direct coupling of interaction, accumulation and uptake.