Using a Membrane-Penetrating-Peptide to Anchor Ligands in the Liposome Membrane Facilitates Targeted Drug Delivery

Abstract

Antimicrobial peptides (AMPs) are typical cell penetrating peptides (CPPs) that intercalate into biomembranes and exhibit broad activities. We designed a triple fusion protein consisting of an AMP, Ib-AMP4 at the N-terminus, a fluorescent GFP probe in the center, and the tumor-targeting peptide P1c at the other terminus. After purification from E. coli, the interaction between the Ib-AMP4-GFP-P1c fusion protein (IGP) and the lipid membrane was characterized. Experiments using isothermal titration calorimetry (ITC) and quartz crystal microbalance with dissipation (QCM-D) demonstrated that IGP proteins spontaneously bound the lipid bilayer with a maximal molar ratio of 1:52 (protein:lipid). Furthermore, transmission electron microscopy (TEM) confirmed that the IGP protein was present in the liposome membrane. After decoration with IGP proteins, the DOPC:DOPG liposomes were applied to cancer cells. Microscopy and flow cytometry reveal that the decorated liposomes selectively bound integrin αvβ3-positive A549 cells. In addition, compared with the common chemical conjugation method, the reported method seemed to be superior in certain aspects, such as simple sample preparation and costeffectiveness. Next, the IGP protein was applied to decorate red blood cell (RBC) liposomes for targeted delivery in both in vitro and in vivo applications. The IGP-decorated RBC liposomes preferentially targeted integrin αvβ3 expressing A549 cancer cells. The in vivo imaging showed that IGP-decorated RBC liposomes were concentrated in tumor tissue and were primarily metabolized by the liver and kidney.

Cell Penetrating Peptides: Classification, Mechanisms, Methods of Study, and Applications

“…The use of TEM to explore CPPs mechanism usually requires peptide labelling with an electron-dense tag like gold, iron oxide, silica nanoparticles or quantum dots. [186][187][188] Alternatively, TEM images could also be obtained incorporating CPP in fusion protein-like architectures [189] or using self-assembling CPPs molecules that organize in hierarchical supramolecular species. [190,191] Such an example is the study of the branched peptide dendrimers, G3KL, rich in lysine and leucine, that exerts a strong activity against Gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli.…”

Lipophilic Anchors that Embed Bioconjugates in Bilayer Membranes: A Review

“…This type of logic is apparent in work reported by Fan and co-workers who used cell-based biosynthesis to fabricate the three-component construct as a single polypeptide. Moreover, the amino acid sequence of the lipophilic membrane anchor component was based on a cell-penetrating peptide and contained a small number of polar residues that attenuated self-aggregation of the construct after dosing but the anchor component was sufficiently lipophilic for membrane insertion with high affinity. , …”

“…Moreover, the amino acid sequence of the lipophilic membrane anchor component was based on a cellpenetrating peptide and contained a small number of polar residues that attenuated self-aggregation of the construct after dosing but the anchor component was sufficiently lipophilic for membrane insertion with high affinity. 45,46 Synthetically, the simplest choice of a membrane anchor component is one of the readily available hydrocarbon anchors within naturally occurring polar lipids and shown in Scheme 4 are the three most used lipid anchors; double chain diacylglycerol, cholesteryl, and single chain fatty acid. Relatively few studies have attempted to systematically compare the membrane affinities of these three different lipophilic anchors, and most of this work has indirectly assessed relative anchor stability by measuring the loss of fluorescently labeled anchor molecules from liposomes.…”

Real-Time Tracking of Digestive Behavior of Different Structured Triglycerides Based on Ordered Porous Layer Interferometry

“…Analyzing the kinetics and affinity of biomolecular interactions is crucial in the biomedical field because relevant parameters and information can explain their behavior and actions in cells, thereby increasing our understanding of the relevant functions and structures of biomolecules. − Lipids are esters formed by fatty acids and glycerol which are widely used in food, biofuels, pharmaceuticals, chemicals, cosmetics, and other fields. − Therefore, how to identify and distinguish lipids with different structures has become key research. Researchers have focused on the triglycerides (TGs), lipids with relatively simple molecular structures.…”