Functional Characterization of an Endosome-disruptive Peptide and Its Application in Cytosolic Delivery of Immunoliposome-entrapped Proteins

Abstract

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH 2 -terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.To exert an optimal therapeutic effect, an administered drug must safely reach not only its target cell but also the appropriate location within that cell. Many biotherapeutic agents (e.g. peptides, proteins, and nucleic acids) act at sites in the cytosol or nucleus that are difficult to reach due to poor membrane permeation characteristics. Therefore, these biotherapeutic agents rely on drug carriers that allow cytosolic delivery of these agents into diseased cells.One attractive strategy to obtain cytosolic delivery is the development of fusion-competent antibody-directed liposomes (immunoliposomes) that, after receptor binding, can fuse with the plasma-membrane of target cells or, after endocytosis of liposomes, with the endosomal membranes. This membrane fusion process should result in cytosolic access of liposomeentrapped drug molecules. Such fusion-competent liposomes can be obtained by disassembling enveloped viruses, isolating their fusion-promoting components, and reassembling these into vesicles to obtain so-called virosomes (1-4). Another, more refined approach involves the use of synthetic peptides designed to resemble the putative fusion peptide of viruses (5-8).It is hypothesized that destabilization of endosomal membranes is less damaging than plasma membrane destabilization. The latter may result in cytotoxicity through eradication of the electrochemical potential generated by the asymmetric distribution of ions across the plasma membrane. Therefore, drug delivery into the cytosol of target ce...

Cytosolic Delivery of Liposomally Targeted Proteins Induced by Photochemical Internalization

“…PEGliposomes (PEG-L) without an antibody attached were used as a control. Due to the PEG-layer surrounding these liposomes, the cellular association and uptake by OVCAR-3 cells is low as shown previously (9,19).…”

“…This delivery system mimics the intracellular invasion strategy of the pathogen Listeria monocytogenes which uses LLO to escape from the endosomes (6)(7)(8). Coencapsulation of a fusogenic peptide derived from the influenza virus hemagglutinin resulted in efficient cytosolic delivery of liposomal diphtheria toxin A-chain as reported by Mastrobattista et al (9). Kakuda et al demonstrated that the insertion of cholesterol derivatized GALA-peptide into the liposomal bilayer also stimulated release of liposomal contents into the cytosol (10).…”

On the formulation of pH-sensitive liposomes with long circulation times

“…A similar strategy has been explored by Mastrobattista et al [7], who have demonstrated that coencapsulation of a pH-dependent fusogenic peptide (diINF-7) and diphtheria toxin A chain (DTA) in nonpH-sensitive immunoliposomes promotes cytosolic delivery of the encapsulated macromolecule. This peptide (resembling the NH 2 -terminal domain of influenza virus hemagglutinin HA-2 subunit) was used because functional characterization studies showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH.…”

“…This peptide (resembling the NH 2 -terminal domain of influenza virus hemagglutinin HA-2 subunit) was used because functional characterization studies showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. Moreover, co-encapsulation of diINF-7 into DTA-containing immunoliposomes resulted in drastically increased cytotoxicity toward ovarian carcinoma cells, indicating that this peptide can be used to obtain cytosolic delivery of liposomeentrapped drugs with poor membrane permeation capacities [7].…”

“…carboxylic group) that act as a stabilizer at neutral pH [4][5][6]. More recently, the use of novel pH-sensitive lipids, synthetic fusogenic peptides/proteins either encapsulated [7,8] or incorporated in the lipid bilayer [9][10][11][12][13], and association of pH-sensitive polymers with liposomes [14 -16] have been reported.…”