Review
Biological responses towards cationic peptides and drug carriers

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In drug development, major resources are invested into the development of cellular delivery systems to increase the effectiveness of a large array of potential therapeutics, such as proteins and oligonucleotides. These carriers comprise cell-penetrating peptides (CPPs), cationic lipids and cationic polymers. In recent years, evidence has been accumulating that these carriers not only act as mere pharmacokinetic modifiers but also interfere with cellular processes in various ways. In this review, we present an overview of the biological side effects associated with carrier systems. The focus will be on CPPs, which have been explored for a diverse set of cargos. Reported activities range from an induction of receptor internalization to the generation of reactive oxygen species. Ultimately, cell-penetrating molecules with such biological side effects might evolve into new bioactive agents that combine delivery capacity and pharmacophore in a single molecular entity. First examples for such molecules will be presented.

Introduction

As research provides an ever greater understanding of disease at a molecular level, the number of potential therapeutic targets is increasing steadily. The challenge is to translate these targets into therapies [1]. Progress with classical, small-molecule based drug development has been slow. Underlying reasons include the need to target protein–protein interactions, reach targets inside cells and to achieve specificity 2, 3.

Therefore, attention is shifting towards alternative approaches that enable a rapid rational design of active and specific molecules. Oligonucleotides, including siRNA, proteins and peptidomimetics are prominent examples 4, 5, 6. However, for these molecules, a rapid and rational design is hampered by poor bioavailability, which prevents them from reaching their intended targets in vivo. The successful implementation of these molecules into new therapies therefore crucially depends on delivery and targeting strategies [7], which generally involve the use of peptides, proteins, lipids or polymers. Although the specific molecular design underlying these delivery strategies varies in detail, the incorporation of positive charge is a common denominator 8, 9, 10. The positive charge mediates the interaction with negatively charged sugar moieties and lipids on the outer leaflet of the plasma membrane, which promotes cellular uptake.

Ideally, these carriers act exclusively as pharmacokinetic modifiers with no biological activity of their own. However, an increasing body of evidence indicates that these carriers can influence cellular activity in multiple ways 11, 12, 13, 14, 15, 16, 17, 18, 19. Hence, testing of carriers should extend beyond mere cytotoxicity and delivery efficiency. In this review, we will therefore summarize recent results on biological responses towards cationic carriers. The focus will be on cationic cell-penetrating peptides (CPPs) and related transporters.

Research into the biological side effects of cationic and amphiphilic membrane-active peptides has a long tradition in the area of peptide toxins, with melittin and mastoparan being paradigmatic examples. Extensive studies on structure–activity relationships have been conducted for induction of mast cell degranulation [20] and hemolysis [21]. Mast cell degranulation has been related to the interaction of the positive charges of the cationic peptides with sialic acids present on the plasma membrane and a G-protein-coupled receptor (GPCR)-independent G protein activation [20].

Many membrane-active peptides also have cell-penetrating activity. Therefore, it is difficult to make a clear distinction between both types of peptides [22]. CPPs might be considered membrane-active peptides with little toxicity. As a consequence, CPP research has focused on mechanisms of uptake and biomedical applications rather than potential side effects. However, because the biological side effects were reported to occur in the absence of acute toxicity, they could be very relevant in future clinical applications. In this review, we will present an overview of the in vitro and in vivo biological responses reported for the interaction of cells and organisms with cationic carrier systems, especially cationic CPPs. Frequently, these responses directly relate to uptake. Finally, we will provide examples of CPPs that have an intended intrinsic biological activity.

Section snippets

Cell-penetrating peptides: promising molecules for the intracellular delivery of therapeutics

CPPs are characterized by their ability to promote the receptor-independent cellular uptake of membrane-impermeable macromolecules, such as peptides, proteins, nucleic acids and nanoparticles [23]. CPPs contain fewer than 30 amino acids and are mostly cationic or cationic and amphipathic. Cationic and amphipathic CPPs show some differences in their internalization mechanisms [24], which might be attributed to the ability of amphipathic CPPs to more directly interact with membrane lipids [25].

Biological activity of cationic CPPs

Cationic CPPs were originally considered as ‘Trojan horse’ delivery vehicles that enter cells without eliciting a cellular response [41]. However, CPPs can induce a wide range of side effects that might be more subtle than cell death. These side effects have been related to the uptake itself as well as to interactions inside the cell.

CPPs with intrinsic bioactivity

In addition to the traditional cationic CPPs that are applied as vectors for cargo delivery, CPPs with intrinsic activities are rapidly gaining interest (Figure 2). This class of peptides demonstrates that cell entry and intracellular function can be combined effectively (for examples, see Table 1).

One example of a bioactive CPP is a seven arginine residue-containing CPP derived from the p14 ARF protein [54]. Internalization of this peptide is as efficient as uptake of the CPP TP10 and is

Concluding remarks

The data reviewed in this article indicate that side effects of cationic delivery agents can be diverse and are not necessarily directly related to cellular uptake. Therefore, when screening for side effects, a comprehensive analysis of the interactions between the delivery agent and the biological system is required. Global gene expression profiling is the most straightforward screen. Ideally, such a screen should be complemented with a more directed search for side effects based on available

Glossary

Amphipathic peptide
peptide that contains both polar and nonpolar regions.
Acid sphingomyelinase (ASMase)
an enzyme that converts sphingomyelin to ceramide and phosphorylcholine.
Cationic peptide
peptide with a high positive net charge and few acidic amino acid residues.
Caveolae-dependent endocytosis
endocytosis emerging from membrane microdomains (lipid rafts) rich in the protein caveolin; sensitive to cholesterol depletion.
Cell-penetrating peptide (CPP)
a short peptide that facilitates cellular

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