A Systematic Analysis of Peptide Linker Length and Liposomal Polyethylene Glycol Coating on Cellular Uptake of Peptide-Targeted Liposomes

Abstract

PEGylated liposomes are attractive pharmaceutical nanocarriers; however, literature reports of ligand-targeted nanoparticles have not consistently shown successful results. Here, we employed a multifaceted synthetic strategy to prepare peptide-targeted liposomal nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities to evaluate the role of liposomal PEG coating, peptide EG-linker length, and peptide valency on cellular uptake in a systematic manner. We analyzed these parameters in two distinct disease models where the liposomes were functionalized with either HER2- or VLA-4-antagonistic peptides to target HER2-overexpressing breast cancer cells or VLA-4-overexpressing myeloma cells, respectively. When targeting peptides were tethered to nanoparticles with an EG45 (∼PEG2000) linker in a manner similar to a more traditional formulation, their cellular uptake was not enhanced compared to non-targeted versions regardless of the liposomal PEG coating used. Conversely, reduction of the liposomal PEG to PEG350 and the peptide linker to EG12 dramatically enhanced cellular uptake by ∼9 fold and ∼100 fold in the breast cancer and multiple myeloma cells, respectively. Uptake efficiency reached a maximum and a plateau with ∼2% peptide density in both disease models. Taken together, these results demonstrate the significance of using the right design elements such as the appropriate peptide EG-linker length in coordination with the appropriate liposomal PEG coating and optimal ligand density in efficient cellular uptake of liposomal nanoparticles.

Significance of the balance between intracellular glutathione and polyethylene glycol for successful release of small interfering RNA from gold nanoparticles

“…While there have been several recent studies on the influence of the PEG chain length and ligand release [6], mixed reports on the effect of PEG density prompted this study, with a shorter chain length and higher density having been considered optimum for both ligand release and maintenance of the PEGylated particles' stealth properties [5].…”

“…Our AuNPs were designed with a view towards exploiting GSH's ability to cleave the thiol-conjugated siRNA. Increasing PEG length and ligand density to saturation can complicate access to the thiol [6], with greater ligand density or higher molecular weight polymers preventing access to the core [30]. Once released, the double-stranded siRNA is loaded into the RISC complex.…”

“…One of the major factors contributing to this inconsistency, along with other ligand issues, is thought to be differences in passivating PEG coatings on the NPs [6]. As discussed in the introduction, the PEG chain length is known to influence the NP-cell interactions; however, it is also well established that the PEG density on an NP surface can also affect the protein repelling capacity of the resultant NPs, and therefore influence the particles' stealth properties [33,34].…”

“…As discussed in the introduction, the PEG chain length is known to influence the NP-cell interactions; however, it is also well established that the PEG density on an NP surface can also affect the protein repelling capacity of the resultant NPs, and therefore influence the particles' stealth properties [33,34]. The PEG density has also been identified as being key in determining cell uptake [6]. In the present study, the AuNPs with 40% PEG coverage demonstrated marginally lower uptake levels compared to the 25% PEG AuNPs (as demonstrated by ICP-MS results), and the TEM images indicated clear availability of the 40% PEG AuNPs in the cytoplasm.…”

“…This protein repulsion tendency of PEG does not depend on the nature of PEG binding to the NP, as both covalently and electrostatically bound PEG show good protein rejection capacity; however, stronger binding can ensure that PEG functionality is not lost [5]. To date, when utilizing PEG, researchers have focused on the importance of the PEG chain length, as it has been established that shorter chains provide enhanced cellular uptake [6]. There are mixed reports with respect to PEG chain density on the surface of an NP, mainly due to both the branching effect and overall molecular weight.…”