Synthesis of linear and cyclic peptide–PEG–lipids for stabilization and targeting of cationic liposome–DNA complexes

Abstract

Because nucleic acids (NAs2) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome–DNA (CL–DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL–DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL–DNA nanoparticles.

Study of the Antimicrobial Activity of Tilapia Piscidin 3 (TP3) and TP4 and Their Effects on Immune Functions in Hybrid Tilapia (Oreochromis spp.)

“…Treatment with TP4 has also been previously reported to reduce bacterial ( Helicobacter pylori ) loads in mice [5]. There are several ways to stabilize TP3 or TP4 in vivo , such as using liposome-packaged peptides, nanoparticles, or D-form peptides [31,32], but these experimental methods are not suitable for aquacultural applications due to cost issues. Therefore, additional analyses are required to determine the best strategy for peptide stabilization and reducing the costs associated with the manufacture of TP3 or TP4 in fish fodder.…”

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides

“…Different synthetic approaches have been explored for addition of iRGD and other peptides to pegylated cationic lipids (49). At least for synthesis, the cyclic azide-alkyne cycloaddition was found to be particularly useful for coupling disulfide-bridge peptides such as iRGD to a PEGylated-lipid.…”

Competition of charge-mediated and specific binding by peptide-tagged cationic liposome–DNA nanoparticles in vitro and in vivo

“…This creates a hydrophilic corona that stabilizes the complexes into nanoparticles (NPs) [22,29–33] and limits opsonization and nonspecific protein binding through a combination of steric repulsion and effective charge screening (Figure 1A) [34–38]. Such a limiting of nonspecific interactions reduces transfection efficiency but opens up possibilities for specific targeting by attachment of affinity ligands (e.g., peptides, antibodies) that selectively interact with molecules expressed on the target cells or tissue (Figure 1) [27,28,39,40]. Specifically, surface functionalization of NPs with homing peptides, by incorporation of peptide-PEG-lipids, provides a combination of affinity targeting and steric stabilization for precise guided delivery.…”

“…Proof of principle has been established that specific, peptide-mediated interactions can enhance internalization of NPs and restore some of the transfection efficiency that is lost when nonspecific electrostatic interactions are suppressed by PEGylation [33,40]. However, little is known about how to select compositional parameters for peptide-targeted CL–DNA NPs to achieve maximum effectiveness in vitro (strong binding to and internalization by target cells but minimal nonspecific binding and internalization) and how these results transfer to in vivo studies.…”