Functionalization of Liposomes with Hydrophilic Polymers Results in Macrophage Uptake Independent of the Protein Corona

Abstract

Liposomes are established drug carriers that are employed to transport and deliver hydrophilic drugs in the body. To minimize unspecific cellular uptake, nanocarriers are commonly modified with poly(ethylene glycol) (PEG), which is known to minimize unspecific protein adsorption. However, to date, it has not been studied whether this is an intrinsic and specific property of PEG or if it can be transferred to hyperbranched polyglycerol (hbPG) as well. Additionally, it remains unclear if the reduction of unspecific cell uptake is independent of the “basic” carrier at which a surface functionalization with polymers is usually applied. Therefore, we studied the protein corona of differently functionalized liposomes (unfunctionalized vs PEG or hbPG-functionalized) using PEGylated and PGylated lipids. Their cellular uptake in macrophages was compared. For all three liposomal samples, rather similar protein corona compositions were found, and also—more importantly—the total amount of proteins adsorbed was very low compared to other nanoparticles. Interestingly, the cellular uptake was then significantly changed by the surface functionalization itself, despite the adsorption of a small amount of proteins: although the PEGylation of liposomes resulted in the abovementioned decreased cell uptake, functionalization with hbPG lead to enhanced macrophage interaction—both in the media with and without proteins. In comparison to other nanocarrier systems, this seems to be a liposome-specific effect related to the low amount of adsorbed proteins.

Polymeric Nanoparticles with Neglectable Protein Corona

“…Since most single proteins in blood plasma are in the size range of 6–10 nm, [ 55 ] thus smaller than the investigated nanoparticles, they are eluting prior to the nanoparticles in the AF4 channel (Figure 2). [ 19,26 ]…”

“…Moreover, these results further indicate that there is no major adsorption of proteins on the nanoparticles leading to a significant size increase of the complexes, which is in accordance to findings on liposomes as determined by AF4. [ 19 ] On the other hand, a previous study with solid nanoparticles demonstrated corona formation induced changes in particle size as detected by dynamic light scattering (DLS) or retention time shifts in AF4. [ 26 ] This is in agreement with a recent study from Couffin and coworkers, which proved that with AF4 it is possible to detect small size differences (5–10 nm) between different nanoparticles.…”

“…Except for liposomes, other types of nano‐sized drug delivery systems, such as polymeric micelles or more complex polymer constructs like cylindrical polymer brushes [ 17 ] have been so far hardly investigated with respect to protein corona formation (for comparison between the structures of these nanoparticles and the colloidal nanoparticles discussed above, see Figures S1 and S2 in the Supporting Information). [ 8,18–20 ] However, the former are interesting, since polymeric micelles are in advanced stages of clinical testing (e.g., CPC634 (phase II) [ 21 ] and NC‐6004 Nanoplatin (phase III) [ 22 ] ).…”

“…[ 24 ] Only recently, size exclusion techniques such as size exclusion chromatography and asymmetrical flow field‐flow fractionation (AF4) have been employed for this purpose. [ 19,25,26 ] AF4 is a separation technique, which can be applied for the separation of particle and protein mixtures in the size range between 1 nm and 1 µm. [ 27,28 ] It consists of a separation channel with a flow gradient in which the particles are separated by an additional vertical force field depending on their diffusion coefficient.…”

“…[ 26,31 ] Based on this method, Landfester and coworkers recently fully characterized the protein corona of Lutensol AT50‐coated polystyrene nanoparticles and PEG functionalized liposomes, identifying all adsorbed proteins. [ 19,26 ]…”