Posted by Cyclodextrins are commonly used as excipients in modern drug delivery, but they also have a less common role as active pharmaceutical ingredients. Due to their ability to form complexes with various lipids, CDs are used in treating lysosomal storage disorders, which are diseases that impair lipid metabolism. A drawback of free CDs is their low cellular uptake. Moreover, a disadvantage mainly associated with β-CD and its derivatives is that, at the concentrations used, they can cause increased toxicity or damage to cell membranes by extracting lipids, particularly cholesterol.
An elegant solution for both issues is the use of polyrotaxanes, where CDs are threaded onto polymer chains and the dissociation of this supramolecular structure is prevented by bulky stopper molecules. The cellular uptake of polyrotaxanes is significantly higher than that of free macrocycles, and because the hydrophobic cavity of the CDs is occupied, lipid extraction from the cell membrane is also hindered. Although research in this field is ongoing and intense, no studies have yet been published on how polyrotaxane functionality influences toxicity and cellular uptake.
Randomly methylated, along with 2-hydroxypropyl β-CDs (RAMEB and HPβCD, respectively), were used to form polyrotaxanes with a molar mass of approximately 45 kDa and a threading efficiency of about 40% through rotaxa-polymerization. The HPβCD-based polyrotaxane was further modified, resulting in thiolated and mercaptosuccinate (MSA) derivatives. The RAMEB- and HPβCD-based polyrotaxanes exhibited significantly lower cell toxicity and hemolytic effects compared to free CDs, while the thiolated and MSA-functional polyrotaxanes showed a less pronounced toxicity-reducing effect. The cellular uptake of all four functional polyrotaxanes was determined and compared to that of free HPβCD in HEK293 and Caco-2 cell lines. In both cases, the highest increase, over 57-fold, was observed with the RAMEB-based polyrotaxane, likely due to its amphiphilic nature, which mimics cell membranes. The performance of the HPβCD-based structure was also notable, exhibiting 33- and 62-fold higher cellular uptake compared to free CD in Caco-2 and HEK293 cells, respectively. Although thiolated polyrotaxanes were internalized slightly better than HPβCD-based ones into Caco-2 cells, their uptake in HEK293 cells fell far short of that. This difference is likely due to the different primary uptake mechanisms of the two cell lines. In HEK293 cells, dynamin- and clathrin-independent pathways—meaning no CLIC/GEEC endocytosis—are observed, which typically results in lower cellular uptake of thiol-functionalized excipients. MSA functional products showed the lowest cellular uptake among the polyrotaxanes, as these presented anionic substructures. Their performance was limited by electrostatic repulsion between the negatively charged acidic groups and the cell membrane.
Besides these direct effects on cellular uptake, some indirect effects are also considered. The free sulfhydryl groups of thiolated, as well as MSA-modified, polyrotaxanes may be directly oxidized to disulfide during storage or application, increasing the particle size from around 30 nm to 100 nm, which significantly influences cellular uptake.
Figure 1. Cellular uptake of free HP-βCD, as well as mercaptosuccinate-modified, thiolated-, HPβCD-, and RAMEB-polyrotaxanes at 37 °C. The relative mean fluorescence intensity (RMFI) values are shown for Caco-2 (in red) and HEK293 (in blue) cell lines.
These results demonstrate that the functionality of polyrotaxanes plays a crucial role in cellular uptake; however, they also highlight that many aspects of this complex drug delivery process still require detailed investigation.