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[email protected] Current Pharmaceutical Biotechnology, 2014, 15, 000-000
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Rational Design of CPP-based Drug Delivery Systems: Considerations from Pharmacokinetics Arite Mickana, Dikran Sarkob, Uwe Haberkorna and Walter Miera,* a
Department of Nuclear Medicine, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany; bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Hawash Private University (HPU), Homs, Syria Abstract: Therapeutics are restricted from cellular internalization due to the biological barrier formed by the cell membrane. Especially for therapeutics with high molecular weight, strategies are required to enable delivery to intracellular targets. Cell-penetrating peptides (CPPs) represent a powerful tool to mediate the entry of large cargos such as proteins, siRNA and nanoparticles. The high diversity of CPPs is the prerequisite to use this class of carriers for various applications. However, therapies based on CPPs are hampered by their unfavorable pharmacokinetics, mainly dominated by their rapid renal clearance and their lack of specificity. Rational design is required to overcome these disadvantages and thereby exploits the actual potential of CPPs. We summarize and highlight the current state of knowledge with special emphasis on pharmacokinetics. The unclear internalization pathways of CPPs remain one of the main obstacles and therefore have been in the focus of research. In this review, several promising strategies such as the combination with targeting sequences, activatable CPPs and adjustment of the molecular weight are described. In addition, new absorption pathways such as nasal, pulmonary or transdermal uptake expand the applicability of CPPs and may be a promising prospect for clinical application.
Keywords: Absorption, biodistribution, cell-penetrating peptide, internalization, pharmacokinetics, targeting, topical application. INTRODUCTION The substantial progress in modern medicine, particularly at the cellular, molecular and genetic level, has enabled the rational design of biologicals. However, their rapidly increasing impact on the pharmaceutical sector cannot belie the disadvantages of such oversized therapeutics. Many drugs with high molecular weight do not attain successful clinical application due to their inability to reach their target within the cell. New strategies of pharmaceutical technology like nanoparticles as well as many small molecules like cytostatic drugs and contrast agents encounter a similar problem: Their non-targeting pharmacokinetics and their insufficient ability to penetrate the cell membrane do not let them reach their target within the cell. Cell-penetrating peptides (CPPs) as vehicles for intracellular delivery provide solutions for all these pharmaceutical challenges. Based on the discovery that the amino acid section 43-58 of the homeodomain of Drosophila melanogaster is able to penetrate biological membranes a new class of “cellpenetrating peptides“ was established. This first member was named penetratin [1]. The CPPs enter the cells by various mechanisms and thereby provide the possibility to transfer coupled or uncoupled cargos [1]. The relatively short peptides consisting of up to 40 amino acids are shown to carry several times larger proteins, liposomes, nanoparticles, siRNAs, nucleotides, radioisotopes or hormones without impact on their *Address correspondence to this author at the Department of Nuclear Medicine, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany; Tel: 0049-6221-567720; E-mail:
[email protected] 1389-2010/14 $58.00+.00
membrane penetration ability [2]. These promising attributes result in the increasing use of cell-penetrating peptides in modern drug delivery systems. This review focuses on the invasion phase of the pharmacokinetics of CPPs consisting of application, resorption and distribution. Unmodified cell-penetrating peptides show a pharmacokinetic behavior that is not suited to enhance the therapeutic effect of drugs [3]. However, it is essential to consider the impact of CPPs when coupled and even when mixed with distinct therapeutics. Thus, the addition of CPPs to oral, nasal, pulmonary or transdermal formulations is a promising strategy for the pharmaceutical technology. In contrast, the lack of targeting represents one of the main difficulties. Consequently, the possibilities to provide specificity of uptake are the basis for several recent publications and various approaches. Further understanding of the CPPs internalization is important to exploit their resorption pharmacokinetics and thus, the basis to a targeted design of CPPs. CLASSES OF CPPs After 25 years of research on CPPs, hundreds of sequences have been described. Depending on their future area of application, researchers can choose from various characteristics. Consequently, it is helpful to have a classification system as shown in (Fig. 1). CPPs can be classified as cationic CPPs with a high positive net charge, hydrophobic CPPs consisting of a majority of apolar residues and amphipathic CPPs containing both polar and nonpolar regions [4]. The latter mentioned group often shows an α-helical structure with a highly hydrophobic region whereas the other part © 2014 Bentham Science Publishers
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Fig. (1). Classification system for cell-penetrating peptides. The upper part represents groups assigned according to biochemical characteristics. The lower part shows frequently used subgroups with specific biological properties.
can be cationic, anionic, or polar. CPPs can consist of βsheet structures with one hydrophobic and one hydrophilic section, too [5]. Furthermore, there are three additional subgroups to be mentioned. Bipartite peptides have a chimeric origin and consist of at least two of these characteristics listed above. The proline rich or polyproline amphipathic CPPs show a high diversity appearing in many families. Their common nature is a proline pyrrolidine template [6]. Some CPPs additionally show antimicrobial activity and are summarized in a separate group [7]. Additionally, Table 1 lists sequences and origins of important cell-penetrating peptides mentioned in this review. ABSORPTION OF CPPS INTO THE BLOOD CIRCULATION Large-sized therapeutics are unsuitable for most repeated long term therapies because they require intravenous application to obtain effective plasma levels. Alternative application methods such as oral or nasal application represent an obstacle as the molecules have to pass the cell membrane. Aside from absorption enhancers [8], protease inhibitors [9], carrier systems and mucoadhesive polymers [10], cell-penetrating peptides are one of the most promising co-agents. The oral route remains most suitable, because it is non-invasive, patient-friendly and requires no application by medical staff. However, it also involves substantial problems, especially for protein therapeutics. For an efficient absorption, their high molar masses, hydrophobicity, vulnerability and enzymatic degradation must be evaded. For this purpose, Morishita et al. [11] tested various cell-penetrating peptides e.g. polyarginines in combination with insulin. The area under the curve (AUC) of insulin was increased from 12.6 µU h/ml to 464 µU h/ml upon addition of 25 mg/kg D-R6. Lower
doses showed significant improvements as well. It is remarkable that no chemical conjugation is required to achieve the enhanced uptake values. Kamei et al. [12] compared ten different CPPs for their usefulness to improve intestinal insulin absorption. Although the D-forms promise a higher stability in presence of intestinal enzymes, L-penetratin, L-pVEC and L-RRL showed better results than their D-analogs. Lpenetratin provided the most promising results by increasing the AUC of 50 IU/kg insulin from 5.7 to 163.8 µU h/ml (0.5 mM CPP used). Penetratin also caused significantly higher uptake of glucagon-like peptide-1 and exendin-4, [13] demonstrating the great potential of CPPs for absorption enhancement. Unfortunately, it is not possible to simply enhance the CPP percentage to the amount required to reach an ideal uptake rate. It was observed that at a certain concentration, aggregates were formed which exceeded the maximal size for intestinal absorption. This could explain the better results of L-penetratin compared to D-penetratin, because the L-form is degraded by intestinal enzymes preventing formation of aggregates, however, the decomposition products still retain their permeation ability [14]. The mechanisms of internalization are explained below, however, the CPP-induced intestinal absorption of unlinked cargos is still not understood in detail. Cell surface proteoglycans seem to play an important role, as they mediate the absorption to the cellsurface of the cationic CPPs [15]. A permeation study of FLD-R6 comparing 4 °C and 37 °C suggested an energy dependent pathway [15a]. The characteristics of the transported drug have a great impact as well, since for example the same CPP can significantly increase the insulin uptake but has no influence on IFN-β [11]. This might be explained by varying electrostatic interactions between the CPP and the cargo. Examples are the two tryptophan residues of penetratin that seem to be the key for delivering a hydrophobic cargo [14].
Rational Design of CPP-based Drug Delivery Systems
Table 1.
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Sequences and origins of important CPPs.
CPP
Sequence
Origin
Ref.
Penetratin
RQIKIWFQNRRMKWKK
Antennapedia Drosophila melanogaster
[64]
Polyarginine
Rn
Model peptide
[42b]
pVEC
LLIILRRRIRKQAHAHSK
Murine vascular endothelial cadherin
[65]
TAT
YGRKKRRQRRR
HIV-1 transcriptional activator
[66]
SynB
RGGRLSYSRRRFSTSTGR
Protegrin
[67]
RGD
GRGDSY
Integrin binding proteins
[68]
Nasal administration could gain closer attention due to several aspects. The mucosa provides a big surface area, a porous endothelium and a highly vascular subepithelial layer avoiding the first pass effect. The high bioavailability and absorption rate provide values that are comparable to those obtained by intravenous application. Consequently, several projects pursuing nasal insulin administration have reached clinical trials [16]. However, for molecules of this size, the nasal mucosa provides a low permeability. Khafagy et al. [10a] could achieve a significantly enhanced absorption rate for insulin by co-administration of insulin with CPPs. Lpenetratin was found to lead to the highest uptake enhancement followed by D-penetratin, D-R8 and L-R8. Increasing concentrations could markedly intensify this effect. The same research group could prove that penetratin enhances the nasal as well as the intestinal absorption of other peptides. For example, L-penetratin increases the absolute bioavailability of GLP-1 to 15.9% via nasal and 5% via intestinal administration [13]. A different in vivo transfection efficiency study showed that the uptake of polyethylenimine (PEI) was four times higher when combined with TAT [17]. The pulmonary pathway represents an alternative approach. The major advantages are the alveoli, providing a large absorptive surface of about 100 m², very good vascularization and a low enzymatic activity. Unfortunately, additional to the peptide size the self-clearance of the lungs can impede the uptake. Patel et al. showed that crosslinking insulin to cationic CPPs resulted in a significantly improved blood glucose level reduction, although the insulin-R9 conjugate does not bind to insulin receptors in vitro. Presumably, the disulfide bonds are cleaved under in vivo conditions [2f]. Transdermal application of biologicals has rarely been applied due to the challenge to cross the stratum corneum, the major barrier of dermal or transdermal absorption. However, the possible achievement of transdermal transfer of biologicals is very attractive for many applications such as wound healing factors like IGF-1, TGF-β or leptin, antiviral or antibacterial agents, for example interferon α and bacitracin, and many more.18 Chemical and physical enhancers all encounter limitations such as skin toxicity or the intricateness to use electrical apparatuses (e.g. ultrasound) at home [19]. Recent development in this field presents significant progress in conjunction with cell-penetrating peptides, not only increasing cell penetration but also lowering systemic side-effects and enhancing patient compliance [19]. In
2000, Rothbard et al. [20] were the first to show transdermal inclusion of R7-cyclosporine A conjugates resulting in therapeutically effective doses and were followed by several promising publications. Desai et al. [21] proved penetration of TAT, R8 and R11 through the skin by 31P magic angle spinning (MAS) solid-state NMR recording chemical shifts by interaction of CPPs with skin lipids. Three different skin depths were tested, 0-60 µm for middle epidermis, 61-120 µm for lower epidermis and 121-180 µm for initial dermal layers. Increasing the peptide concentration leads to negative shifts in NMR-spectra until reaching a saturation effect over 100 mg/ml. TAT and R11 treated skin showed larger shifts than R8, however, quantitative differences could be due to dissimilar peptide effects on lipid structures. The control peptide YKA leads to significantly reduced structural changes in comparison to the application of CPPs. In a time dependent experiment with R11, most of the observed effect had already occurred at 30 min post injection indicating a rapid and effective transport. These results lead to the hypothesis that polyarginine CPPs may form complexes with the lipid phosphate groups causing defects in the lipid bilayer and making it accessible to topically applied molecules. For example, Cohen-Avrahami et al. [22] used penetratin to enhance transdermal delivery of diclofenac. For these experiments with porcine skin, a reversed hexagonal mesophase enclosed the CPP and served as a solubilization reservoir. Instrumental analysis confirmed that penetratin induced efficient drug absorption by accelerating the structural transition of the skin lipids. Furthermore, several nanoparticles used for efficient cutaneous drug delivery were improved with CPPs. For instance, TAT enhanced the epidermal permeation of celecoxib nanoparticles, used for the treatment of chronic joint irritation during arthritis [23]. Moreover, this strategy could gain importance for various skin disorders such as psoriasis, infections, and skin cancers [24]. Summarizing, novel strategies for the development of non-invasive delivery systems, including therapeutic peptides and proteins, could be established using new research results about different application pathways. As a consequence, the application of CPPs in pharmaceutical formulations is emerging as a promising future option. BIODISTRIBUTION AND TARGETING Studying the biodistribution of CPPs causes problems because several aspects like the cargo have a great influence.
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A study from 2006 showed the distribution of an enzymatically active CPP-fusion protein: the coupled β-galactosidase was active in various tissues, in particular in the liver (32%) and bowel (48%), but also in the spleen (17%), lung (31%) and brain (12%) after i.v. administration [25]. Other studies showed that CPP-mediated delivery can target virtually any tissue as well, unsurprisingly most localize to the liver, spleen, and kidneys [26]. Sarko et al. [3] performed biodistribution studies by labeling ten of the most commonly applied CPPs with 111In or 68Ga. To minimize the cargo influence, the macrocyclic chelator DOTA known for excellent in vivo stability [27] was used for labeling. All tested CPPs showed a rapid blood clearance resulting in values of approximately 1 %ID/g at 4 h post injection. (Fig. 2) shows a typical average area under the curve of cell-penetrating peptides. Their rapid renal excretion interferes with targeting efforts and hinders the cargo’s ability to interact with the target. This curve is indicative for the poor biodistribution properties of cell-penetrating peptides as a result of the slow interaction with cellular membranes. The possibility to influence the AUC in order to approximate it to the ideal curve shall be exemplified in the section concerning sequence analyses. A further aspect in biodistribution becomes clear by studies in tumor-bearing mice. Generally, the tumor showed the lowest values of uptake among all organs. Considering the protein degradation, arginine-arginine-bonds (RR) seem to be a crucial factor for the stability. Virtually, all highly efficient CPPs containing RR bonds (penetratin, TAT, SynB1, pVEC) were degraded faster than the argininefree CPPs. Considering penetratin in particular, it was already predominantly found in liver (23.6 %ID/g), kidney (25.5 %ID/g) and spleen (20.2 %ID/g) at 10 min post injection. This is representative for all CPPs, which have a transient accumulation in highly vascularized organs. Increasing the systemic toxicity, this might be one of the major problems for in vivo application, leading to unpredictable side effects and lowering the potential effects in the desired tissue. On the other hand, CPP sequences can be modified with targeting motifs [28]. There are several possibilities examined, for example the targeting ligand utilized by Tan et al. [29]. They combined the TAT CPP with an anti-Her-2/neu peptide mimetic targeting ErbB2, a receptor over-expressed in 30% of breast cancers. The cargo was delivered more efficiently into cancer cells that overexpress ErbB2 in vitro and therefore present a good basis for future targeting CPP delivery systems. Using the active targeting with direct ligandreceptor-interactions, it was discovered that the affinity of folate to the folate receptors overexpressed on epithelial cancer types can be exploited [30]. On this basis, several publications reported a combination of CPP and folic acid on cationic polymer vehicles [31]. Similarly, a targeting option with hyaluronic acid (HA) was explored. (Fig. 3) illustrates the HA-mediated pathway. The receptors of hyaluronic acid, e.g. CD44, have been found on the cell surface of several malignant tumors. Drug carrying micelles conjugated with hyaluronic acid and CPPs could induce a selective, HAreceptor-mediated attachment and CPP-induced endocytosis [32]. Furthermore, the benefit of homing domains was shown by coupling the CPP pVEC to PEGA, which accumulates in breast tumor vasculature binding to aminopeptidase P and is cell impermeable unless conjugated to a CPP [33].
Mickan et al.
Fig. (2). A hypothetical concentration-time-diagram observed for cell-penetrating peptides. CPPs below the renal excretion limit are rapidly excreted. Prolonging the circulation time and thus enable specific interactions is one of the primary aims of CPPs design.
Fig. (3). Tumor-targeting of nanoparticles modified with hyaluronic acid. In the first step, selective extravasation occurs preferentially in tumor tissue as a result of the enhanced permeability and retention (EPR) effect. Subsequently, interaction of the cell surface receptor CD44 with hyaluronic acid enables the CPP to induce endocytosis.
Rational Design of CPP-based Drug Delivery Systems
Alternatives are activatable cell-penetrating peptides (ACPP), polycationic CPPs whose cellular uptake is restricted by combination with a polyanionic domain resulting in a net charge close to zero (Fig. 4). The release of the CPP is induced by specific tissue associated proteases, which cleave the linker and remove the polyanionic domain. Jain et al. [34] used penetratin and TAT to improve the tumor uptake of single-chain Fv antibody fragments, an alternative to IgG. The CPPs were not covalently bound but added to the protein by coninjection. Biodistribution in tumor-bearing mice was significantly improved by both CPPs. The doses retained in tumor (24 hours post administration) could be increased to 79.8% with penetratin and 48.5% with TAT as compared to 27.2% without peptide addition. The increased residence time provides enormous therapeutic benefits and is expected to be harmless to normal tissues. A second study concerning the tumor uptake of antibodies used the conjugation of TAT to ScFv(L19)-Cys [35]. Unfortunately, the tumor targeting performance was clearly deteriorated compared to the uncoupled antibody. The TAT-free antibody reached 11.8 %ID/g as compared to the ScFv(L19)-Cys-TAT with only 4.1 %IG/g at 1 hour post injection. The poor in vivo efficiency reported by different authors [35, 36] illustrates that all positive results of in vitro studies have to be considered carefully as a conjugation could cause negative effects on the pharmacokinetics. Aguilera et al. [37] described the combination of R9 with the polyanionic polyglutamate, whose affinity to the CPP is strong enough to form the required hairpin structure but sufficiently instable to dissociate after linker cleavage in the presence of the tumor associated matrix metalloproteases. These ACPPs showed improved controllable pharmacokinetics as they did not remain at the injection site and showed prolonged circulation. Additionally, they reduced the systemic toxicity noticed for many CPPs - probably due to their strong positive charge. A disadvantage of all CPPs might be the instability of peptides in the circulation leading to a short duration of action. Substituting L-amino acids with their respective Danalogs or non-proteinogenic amino acids [38] or combining the CPPs and their cargos on stable nanoparticles leads to
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significant improvements [26]. Pang et al. [39] prolonged the half-life of the CPP iRGD from 8 to 22 min by addition of a cysteine residue which forms a disulfide bond to serum albumin. The rapid renal clearance can be reduced for example by association with carrier proteins and thereby limiting the amount of free peptide [40]. In summary, many scientists see the solution of all these CPP drawbacks in the design of ‘smart delivery platforms’. The ideal delivery system shields CPPs during the first step of transfer and is subsequently presented to the cell membrane triggered by unique local conditions [4]. CELLULAR PHARMACOKINETICS For the internalization of CPP-complexes, two types with substantial differences can be distinguished as shown in (Fig. 5). Large cargos mainly follow endosomal pathways whereas for some peptides and smaller molecules nonendosomal internalization modes have been developed. However, apart from the effects caused by the size a high variability of physical, biophysical and chemical properties of the peptide, their concentration [41] and the composition of the cell membrane [7] cause an influence on the cell entry. The direct transduction including the inverted micelle model, the carpet model and the pore model is shown in (Fig. 5 E-G). Direct transduction is mainly based on electrostatic interactions and hydrogen bonding with components of the cell membrane as shown in studies with penetratin, R7 and TAT [42]. The direct passage should be preferred because the cargo has direct access to its targeted compartment but is unfortunately only the minor part of internalization pathways. Well investigated CPPs can be influenced in this direction as a publication of Takeuchi et al. [43] demonstrated by linking negatively charged counteranions with high hydrophobicity to arginine-rich peptides. For example the counteranion pyrenebutyrate was used showing insertion into the plasma membrane and generated an electrostatic interaction with arginine residues, leading to a direct transduction [44]. Additionally, all known types of endocytotic pathways except that of phagocytosis are reported to be the parts of the
Fig. (4). Activatable CPPs as exemplified for a conjugate of the CPP R9 linked to the inhibitory domain E8 via a cleavable metalloproteinase substrate sequence.
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Fig. (5). Overview of possible internalization pathways of cell-penetrating peptides comparing endocytotic and direct translocation pathways. Following endocytotic internalization, CPPs are located endosomally. In some cases, CPPs provide the possibility to release the delivery systems by endosomal escape.
cellular uptake. They include macropinocytosis, clathrinmediated, caveolae/lipid raft-mediated and clathrin/caveolaeindependent endocytosis (Fig. 5 A-D) [45]. Macropinocytotic vesicles are formed by an overlapping plasma membrane supported by actin and kinase activity (e.g. PI3K, PKC, Pak1, Rac1) [46]. According to several publications, it could be a major internalization pathway [47]. The interaction of positively charged CPP with negatively charged heparan sulfate proteoglycans (HSPG) triggers this mechanism [48]. The importance of caveolae-dependent endocytosis was shown by a down-regulation of caveolin-1 resulting in a 40% decrease of CPP-cargo uptake [49]. The clathrinmediated endocytosis might be the least significant pathway because it normally requires a specific ligand binding to the receptor. However, it has been presented several times as an uptake route [50]. In contrast to these various entrance scenarios, the fate of CPP-complexes after internalization has rarely been investigated. Some hints give reason to presume that the first internalization step determines the intracellular target. Furthermore, the CPP and cargo characteristics and also their concentration can influence the cellular destination [46]. As many large therapeutics target compartments outside endosomes, e.g. the nucleus or mitochondrion, it is of great importance that a delivered cargo can escape prior to recycling or lysosome fusion. Therefore, the possibility of a CPP-guided enhanced escape from endosomes was reported. Wadia et al. [47d] presented a TATp-mediated escape. Furthermore, it is possible to bypass the lysosomal degradation [51] or to use the low pH in the lysosome by applying polymers with a buffering capacity between pH 5.2 and pH 7.0 (proton sponge effect) [52]. However, endosomal escape is very ineffective and therefore no alternative for the direct membrane passage [53]. This could be improved by a higher peptide concentration as shown by fluorescence microscopy analysis of Fretz et al.
[54]. The authors used R8-Alexa488 on leukemia cells which were incubated with 2, 5 or 10 µM of the CPP. Low concentrations created punctuate structures as expected for an endocytotic pathway. With increasing concentration diffuse fluorescence in the cytoplasm overlaid the punctuate structures, which indicates independence of endocytosis [54, 55]. A similar phenomenon was observed with TAT and R9 treated HeLa cells when raising the concentration from 10 µM to 20 µM. In contrast, penetratin was still found in vesicles at high concentrations [56]. Due to the short 30 min time slot, endosomal escape can be excluded as a causal factor. Thus, it has been suggested that cationic CPPs have a stronger tendency to translocate across the plasma membrane. This is also supported by the membrane damaging effect of CPPs at high concentrations [55]. STUCTURAL ANALYSIS Fig. (6) points out that the fast renal excretion is one of the major determinants of the pharmacokinetics of CPPs. Targeted changes in their sequence that increase cell membrane or blood protein interactions or other structure modifications can reduce this phenomenon. Analysis of the detailed sequences of CPPs reveals some recurring sections that significantly improve the internalization efficiency. Hydrophobic terminals (N- or C-terminal) can enhance the membrane interaction as shown by Takayama et al. [57] for a penetration accelerating sequence (FFLIPKG) to R8 to form the penetration accelerating sequence (PasR8). This sequence clearly increased the uptake directly across plasma membranes. Furthermore, tryptophan or tyrosine residues, that constitute many amphiphilic CPPs, seem to play a key role. Originally, they were inserted because of their common occurrence within the membrane and transmembrane protein regions [55]. When assembled on one side of a helical structure, the peptide forms a pore, possibly supporting the translocation process [58]. A third enhancement strategy is lipida-
Rational Design of CPP-based Drug Delivery Systems
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Specific cellular uptake _ improved cell membrane interactions F W I K R Q
I
Q
N R R M
K
Prolonged circulation _ protein interaction _ cargo size above renal excretion limit
K
K W
Renal excretion
Fig. (6). Possible fates of a CPP circulating in the cardiovascular system, as exemplified for the penetratin sequence. To control renal excretion, sequence modifications leading to a more specific cellular uptake or a prolonged circulation are inserted.
tion, which supports the membrane penetration but also reduces the tendency to cross it. It was shown both, an increasing penetration with stearyl, lauryl or cholesterol groups [59] and decreased cytosolic labeling by testing the addition of a myristoyl moiety [60]. The great potential of arginine-rich CPPs like R8 and R9, for example shown by Nakase et al. [61] in tumor tissue, can also be transferred to a macrocyclic scaffold as demonstrated for calyx [4] arene macrocycles by Bagnacani et al. [62]. The clustering of only four units of arginine remarkably boosts the cell penetrating properties. For example, tetraargininocalix [4] arene could reach a DNA transfection efficiency of about 75% (at 48 h) to rhabdomyosarcoma cells. Analyzing the sequence-response relationship remains an important part in current CPP research and could possibly lead to cell-penetrating peptides that evade the currently known problems. CONCLUSIONS The growing use of CPPs indicates their potential as effective delivery vectors. However, despite many years of research, the system is still in a maturing phase. The same characteristics that enable CPPs to cross the cell membrane, such as their positive charges or lipophilic residues, are also responsible for their nonspecific interaction with all tissue types. [63]. Approaches might be the sterical shielding of CPP moieties or their reversible inactivation by polyanions using the unique local conditions when reaching the target. Activatable CPPs represent a highly plausible example; however, their practical application often unveils the lack of efficiency and specificity [7]. The high cell penetration efficiency of CPPs raised hopes to develop drug delivery systems with intracellular targeting capacity. For the design and development of new pharmaceuticals, CPPs could be a valuable tool. However, it is inevitable to implement pharmacokinetic knowledge into this process. The low plasma half-life and the rapid excretion of CPPs must be improved to exploit their excellent resorption properties. Balancing the structural changes for enhanced kinetics without affecting the optimal internalization represents a challenging task. To facilitate the targeted design of
CPPs, further investigations of the cargo impact, the uptake mechanisms and the influence of local conditions are mandatory. New absorption methods for biologicals might represent the biggest potential of CPPs from the pharmaceutical point of view. Nasal, pulmonary or transdermal applications all avoid first-pass metabolism and can be applied without medical staff assistance to increase patient acceptance and compliance. With increasing knowledge about the exact uptake mechanisms, several of these techniques are likely to reach clinical application. There are already several promising approaches for the clinical applications of CPPs. Interestingly, the different methods described exploit numerous aspects of medical opportunities of CPPs. The next step is the translation of welltolerated systems that pass cellular or physiological barriers for clinical application. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2]
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Received: April 08, 2014
Revised: April 28, 2014
Accepted: May 27, 2014
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