scope and limitations by the application of cell

Review Received: 5 February 2014

Revised: 6 June 2014

Accepted: 10 June 2014

Published online in Wiley Online Library: 11 August 2014

(wileyonlinelibrary.com) DOI 10.1002/psc.2672

Cell penetration: scope and limitations by the application of cell-penetrating peptides Siegmund Reissmanna,b* The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell-penetrating peptides (CPPs) are very promising chemical helpers. Because of their low cytotoxicity and final degradation to amino acids, they are particularly favored in in vivo studies and for clinical applications. Clearly, the future of CPP research is bright; however, the required optimization studies for each drug require considerable individualized attention. Thus, CPPs are not the philosopher’s stone. As of today, a large number of such transporter peptides with very different sequences have been identified. These have different uptake mechanisms and can transport different cargos. Intracellular concentrations of cargos can reach a low micromole range and are able to influence intracellular reactions. Internalized ribonucleic acids such as small interfering RNA (siRNA) and mimics of RNA such as peptide nucleic acids, morpholino nucleic acids, and triesters of oligonucleotides can influence transcription and translation. Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging. This review describes a wide array of differing CPPs, cargos, cell lines, and tissues. The application of CPPs is compared with electroporation, magnetofection, lipofection, viral vectors, dendrimers, and nanoparticles, including commercially available products. The limitations of CPPs include low cell and tissue selectivity of the first generation and the necessity for formation of fusion proteins, conjugates, or noncovalent complexes to different cargos and of cargo release from intracellular vesicles. Furthermore, the noncovalent complexes require a strong molar excess of CPPs, and extensive experimentation is required to determine the most optimal CPP for any given cargo and cell type. Yet to predict which CPP is optimal for any given target remains a complex question. More recently, there have been promising developments: the enhancement of cell specificity using activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, and the transport of drugs through blood–brain barriers, through the conjunctiva of eyes, skin, and into nerve cells. Proteins, siRNA, and mimics of oligonucleotides can be efficiently transported into cells and have been tested for treatment of certain diseases. The recent state of the art in CPP research is discussed together with the overall scope, limitations, and some recommendations for future research directions. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: cell-penetrating peptides; amino acid sequences; conformations; cellular uptake process; intracellular trafficking; activatable CPPs; pH/pO2-sensitive CPPs; cell- and tissue selectivity; maurocalcine; azurin; oncogenase; uptake efficiency; intracellular concentrations; cargos proteins; enzymes; antibodies; siRNA; RNA mimics; coupling to dendrimers; multifunctional nanoparticles; preclinical and clinical studies

Introduction

760

Membranes of live cells are permeable only by small hydrophobic compounds. Strongly polar, hydrophilic, and poorly soluble compounds require transport helpers. Hydrophilic compounds such as proteins and nucleic acids can be internalized through electrically triggered pores in the membrane or surrounded by lipid particles, incorporated into nanoparticles or viral particles, and they can form conjugates, fusion proteins, or noncovalent complexes with cell-penetrating peptides (CPP). In the last two decades, a large group of synthetic peptides have been derived from natural penetrating peptides and proteins. Modified and new designer peptides have been developed. These CPPs can be used for internalization of peptides, proteins, ribonucleic acids, mimics of oligonucleotides, quantum dots, or other nanoparticles into live cells. CPPs can have different names. Thus, CPPs are also known as protein translocation domains, membrane translocation sequences, or Trojan horse peptides. Because in most cases these peptides have a low cytotoxicity and are also able to transport nonpermeable drugs into live cells (i.e. cancer cells, nerve cells, and inflamed tissue), they are of

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significant pharmaceutical interest. It can be concluded that cell penetration is an essential prerequisite for signal transduction therapy, for therapies influencing transcription and translation, and thus for effective tumor therapy, as well as possibly for gene therapy in the future.

* Correspondence to: Siegmund Reissmann, Friedrich Schiller University, Biological and Pharmaceutical Faculty, Institute of Biochemistry and Biophysics, Dornburger Stasse 25, 07743 Jena, Germany. E-mail: siegmund. [email protected] a Friedrich Schiller University, Biological and Pharmaceutical Faculty, Institute of Biochemistry and Biophysics, Dornburger Strasse 25, 07743 Jena, Germany b Jena Bioscience GmbH, Loebstedter Strasse 80, 07749 Jena, Germany Abbreviations:: ACPP, activatable cell-penetrating peptide; AMP, antimicrobial peptide; CPP, cell-penetrating peptide; Cyto-Tox-Glo™, assay for dead protease release; GAG, glycosaminoglycan; GPCR, G protein-coupled receptor; HSP, heat shock protein; LDH, lactate dehydrogenase; NLS, nuclear localization sequence; PEI, polyethylenimine; PKC, protein kinase C; PNA, peptide nucleic acid; siRNA, small interfering RNA; SCON, splice-correcting oligonucleotide.

Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES

Biography Prof. Dr habil. Siegmund Reissmann was born in 1940 in Chemnitz, Germany, and graduated from the Friedrich Schiller University Jena: diploma in Chemistry 1963, PhD in 1968, habilitation in 1977. He is a Professor of Biochemistry since 1985, Director of the Institute of Biochemistry and Biophysics from 1985 to 2005, and Scientific Advisor of Jena Bioscience GmbH since 2005. He has given lectures in Biochemistry at the universities of Jena and Chemnitz. His research of interest includes syntheses of and conformational studies on peptides with nonproteinogenic amino acids and backbone cyclization, design and synthesis of catalytic active peptide complexes with transition metal ions, SAR studies and investigations on the molecular action mechanisms of peptide hormone bradykinin and bradykinin potentiating peptides, and studies on cargo internalization into live cells by formation of noncovalent complexes with cell-penetrating peptides. He has studies abroad with Y. A. Ovchinnikov, V. T. Ivanov and M. P. Filatova (Moscow); K. Blaha and I. Fric (Prague); K. Medzihradszky (Budapest); P. Fromageot (Paris); J. M. Stewart (Denver); and V. Hruby (Tucson).

Beside electroporation, magnetofection, lipofection, or the application of nanoparticles and viral vectors, CPPs have assumed an increasingly important role in the transport of strongly different compounds into live cells. Their application has been mainly focused in the direction of therapeutic applications. In addition to internalization and cargo transport into live cells, CPPs are also able to transport compounds through the skin, the blood–brain barrier, and through the conjunctiva of eyes. Labeled CPPs can detect diseased tissues including tumors and their metastases. Furthermore, they can transport drugs into the diseased tissues. Designer peptides can carry drugs selectively into target tissue or organs. Today numerous specific peptides, conjugates, and fusion proteins are in clinical trials. Already approximately 4000 publications exist, which include more than 400 reviews. Since the first CPP was elucidated only 25 years ago, further rapidly expanding investigations also involving broadened application in preclinical research and clinical therapy can be expected. This review offers my personal reflections on the current state of this field and shall stimulate further development into distinct research fields. I would like to undertake a broad comparison of these peptides to other methods of cell penetration, describing particular advantages and future applications as well as temporal and general limitations. For more detailed information about cellpenetrating peptides recommended books and special issues of journals are listed in references [1–5].

The History of CPPs

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The history of the development of CPPs is discussed from another viewpoint and in more detail in reviews [6] and [7]. The history is mainly based on three fundamental investigations, but other valuable studies also deserve credit. Bienert and his

group first elucidated in 1987 a receptor-independent activation of mast cells by substance P (SP) analogs [8]. Continuing this penetration studies, SP sequences were used for uptake studies [9], and new amphipathic model peptides were developed [10]. The second investigation was by Frankel and Pabo [11], which described in 1988 the cellular uptake of TAT-protein from HIV-1. Two years later, Joliot et al. for consistency [12,13] found that a 60-mer peptide corresponding to the sequence of Drosophila antennapedia gene homeobox could penetrate the cells of neuronal cultures, the third fundamental investigation. Subsequently, approaches were undertaken to develop nonviral gene delivery systems by using recombinant DNA-binding proteins [14]. An important step was the design of chimeric peptides, such as transportan, constructed from sequences of galanin and mastoparan and that was able to transport cargos through the cell membrane [15]. These discoveries in toto the uptake of peptides and proteins into live cells and the transport of cargos through cell membranes, respectively, started the run to research in the field of CPPs. Derived from the cationic HIV-TAT peptide some polyarginine peptides were investigated, leading to octaarginine, nonaarginine, and dodecaarginine and related sequences [16,17]. The internalization of different cargos by formation of noncovalent complexes with designed amphipathic peptides, developed by the group of Heitz and Divita, also represents an important milestone in the history of CPP development [18,19]. Also other observations led to the finding of new peptides for the internalization of cargos. On the basis of a nasal application of human calcitonin, Beck-Sickinger and coworkers [20–22] developed fragments of this hormone without receptor binding, which were able to transport different cargos, mostly anticancer therapeutics. Here, the contribution of Langel is noteworthy. Not only has he participated in the development of new peptides [23,24] but he has also worked tirelessly in the field by coauthoring articles in scientific journals and books on the topic, as well as organizing research conferences. Aiming to reveal the mechanisms of the internalization process, research groups have focussed increasingly on live cells and on artificial lipid membranes. As an efficient uptake requires the formation of nanoparticles, in certain cases, nanostructures were built by coupling to dendrimers or other nanoparticles. Torchilin et al. [25] used CPPs to form multifunctional drug delivery systems from liposomes. Coupling CPPs to dendrimers such as polyethylenimine (PEI) can enhance their efficiency and cell selectivity of the cargo transport [26]. A landmark study in the clinical use of CPPs was the development of activatable CPPs, the so-called ACPPs, by Tsien et al. [27]. These peptides can be converted from an inactive into an active form through cleavage of a linker sequence by cell-specific proteases. In general, sufficiently high cell selectivity is a prerequisite for diagnosis and treatment of inflammation and cancer. Selectivity can also be achieved by pH-sensitive or pO2-sensitive CPPs, integrin ligands, chemokin receptors, scavenger receptors, syndecans, neuropilins, homing domains, and biofunctional transporters such as azurin or oncogenase [28–35]. In the last 20 years, key studies have been performed to uncover the dependency of uptake mechanisms on incubation conditions, cell types, CPP sequences, and cargos [36–40]. Despite the relatively short period since the beginning of research on CPPs, some of these peptides are already being used as fusion proteins, conjugates, or complexes in clinical trials for therapeutic application against cancers, infarct, stroke, pain,

REISSMANN

Figure 1. Chronological arrow in cell-penetrating peptide (CPP) development. The given landmarks in the development are selected by the very personal view of the author. Description and references are given in the first section.

psoriasis, eye diseases, and muscle dystrophy as discussed in the following chapters. The chronological arrow in Figure 1 indicates a few of the major landmarks in the short history of CPP development and provides an outline of the divergent structural and functional types of CPPs, as well as various research directions and applications.

Structural and Functional Classification of CPPs In the previous chapter, important studies marking the history of research on CCPs were outlined; here, a detailed description of the chemical and biological properties that determine classification of CPPs is provided. Since the initial discoveries of the benefits of peptides, huge advances have been made in uncovering, designing, and optimizing the function of new sequences in CPPs. These newer peptides have transduction and selectivity domains and are therefore able to penetrate membranes and to transport cargos selectively. They represent the next generation. For reviews on the subject see ref. [36,38,41–43]. Cell-penetrating peptides can be classified by the functions of their original proteins, their uptake mechanisms, intracellularly evoked reactions, and their chemical properties. They can also be divided according to whether they are receptor mediated or nonreceptor mediated. Classification according to structure

762

Peptides can be classified according to their structure (Table 1). On the basis of thermodynamic binding studies performed on lipid model membranes and on real membranes of live cells, Ziegler and coworkers [37–39] postulated three structural classes of CPPs that were in good agreement to the 2 years later published review from to Madani et al. [36]. They categorized CPPs based upon the length of the chain and distribution of positively charged and hydrophobic amino acids in the sequence of primary amphipathic, secondary amphipathic, and nonamphipathic peptides. Primary amphipathic CPPs contain typically more than 20 amino acids and have sequentially hydrophilic and hydrophobic amino acids in their primary structure. Their transduction occurs mainly by insertion into the surface of lipid membranes. Secondary amphipathic CPPs commonly have less than 20 amino acids, which form their α-helical or β-sheet uptake conformations upon interaction with phospholipid membranes. The third class,


the nonamphipathic peptides, are rather short such as HIV-TAT or nonaarginine and have a high content of positively charged amino acids, i.e. Arg or Lys. At low micromolar concentrations, no interaction of this class of CPPs with artificial lipid membranes occurs. Binding to headgroups of lipids was detected only at higher concentrations. In membranes of live cells, positively charged CPPs can bind electrostatically besides to these headgroups also to other anions at the surface. They bind at low micromolar concentrations to negatively charged sulfated glycosaminoglycans (GAGs) of the surface of mammalian cells followed by clustering and endocytotic uptake. Especially, nonamphipathic peptides lead to internalization through GAG clustering [39]. Receptor-mediated uptake Several receptors were found to be involved in internalization of CPPs and have been uncovered. These include such receptor types as chemokine receptors [31,44], syndecans [33], neuropilins [45,46], the family of integrins [30,34,47,48], homing sequences, and the positively charged scavenger receptors [32]. The number of these ubiquitously distributed receptors was steadily increasing in the last few years, and this is expected to continue. Sequences that enable docking of viruses such as hepatitis B [49] or HIV-1 [50] interact with binding proteins or receptors in the membrane, too. Receptor-mediated uptake depends on the incidence of the corresponding receptor and its density at the target cell, thus indicating to some degree cell and tissue selectivity. Apart from tissue and species specific differences the receptor density varies also with the cell cycle, cell pathology (i.e. cancer) and age of the organism. Recently, a new type of penetrating peptide that has intrinsic tissue-selective properties has been found; the copper-containing transport protein Azurin [35] preferentially targets human cancer cells. Nonreceptor-mediated uptake Cell-penetrating peptides that are not receptor ligands are derived from naturally occurring proteins that are able to influence gene transcription [11–13]. Belonging to the class of membrane active CPPs [52], numerous antimicrobial peptides (AMPs) are not only capable of penetrating membranes but also of transporting cargo into mammalian cells [53–55]. Histones [56,57], histidine-rich peptides [58,59], and prion sequences [60–63] can also act as transporters. Histones and prions are known to be able to migrate from one cell to another. Venoms and toxins can contain components that allow a triggering of very fast toxic reactions via permabilization or the destruction of barriers and membranes. Thus, venoms such as mellitin [64], mastoparan [15], and the rattle snake venom crotamin [65–67] interact directly with the lipid bilayer. The toxin maurocalcine acts as a drug transporter and was first isolated from scorpion maurus palmatus [68]. Its chain from 33 amino acid residues contains six cysteine residues and forms three disulfide bridges [68,69]. Replacement of Cys by 2-aminobutyric acid retains the high uptake efficiency, and a sequence truncation down to peptides of up to nine residues in length is even active [70]. Maurocalcine binds to disialoganglioside GD3 and activates ryanodine receptors. Depending on the type of coupling to the cargo, CPPs can be classified into whether they are covalently or noncovalently bound (Figure 2). By formation of a covalent bond, the CPP


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28 29 30 31 32 33

27

Hydrophobic peptides 26

763

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Amphipathic peptides 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Cationic peptides 1 2 3 4 5 6 7 8

No.

Name

Kaposis sarcoma fibroblast growth factor Kaposi FGF Signal sequence of Ig light chain from Caiiman crocodylus Integrin β3-fragment Grb2 (SH2 domain) Fusion sequence HIV-1 gp41(1–23) Hepatitis B virus translocation motif Sperm-egg fusion protein (89–111) Human calcitonin partial sequence 9–32, hCT(9–32)-br

(Chimeric galanin/mastoparan) Transportan Transportan 10: TP 10 CPP with protease cleavage side YTA2 Pep-1 MPGα MPGβ MPG8 CADY Pepfect6 PepFect14 PepFect 15 NickFect Hel 11–27 InfluencaHA-2 (1–20) KALA sequence KLA sequence sC18 (Vascular endothelial cadherin) pVEC

Penetratin HIV-TAT (47–57) HIV-1 Rev (34–50) FHV coat(35–49) Oligoarginines (R9–R12) CCMV Gag (7–25) Chimeric dermaseptin S4 and SV40 ‘S413-PV’ Herpes simplex virus transcription factor (267–300) VP22

Table 1. Amino acid sequences of selected cell-penetrating peptides

VTVLAGALAGVGVG AAVLLPVLLAAP GALFLGFLGAAGSTMGA PLSSIFSRIGDP SFP23: Ac-KLIATGISSIPPIRALFAAIQIP-amide LGTYTQDFNK(X)FHTFPQTAIGVGAP-amide

MGLGLHLLVLAAALQGAMGLGLHLLLAAALQGA

AAVALLPAVLLALLAP

GWTLNSAGYLLGKINLKALAALAKKIL-amide AGYLLGKINLKALAALAKKIL-amide YTAIAWVKAFIRKLRK-amide Ac-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-NH-CH2-CH2-SH Ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH βAFLGWLGAWGTMGWSPKKRK-NH-CH2-CH2-SH Ac-GLWRALWRLLRSLWRLLWKA-NH-CH2-CH2-SH Stearyl-AGYLLGK(ε-TMQ)INLKALAALAKKIL Stearyl-AGYLLGKLLOOLAAAALOOLL-amide Stearyl-AGYLLGK(K3QN4)LLOOLAAAALOOLL-amide NF61: Stearyl-AGYLLGOINLKALAALAKKIL-amide KLLKLLLKLWKLLLKLLK WEAKLAKALAKALAHLAKALAKALKACEA Acetyl-KLALKLALKALKAALKLA-amide GLRKRLRKFRNKIKEK LLIILRRRIRKQAHAHSK-amide

RQIKIWFQNRRMKWKK YGRKKRRQRRR KQAIPVAK-amide RRRRNRTRRNRRRVR-amide RRRRRRRRR/ RRRRRRRRRRRR KLTRAQRRAAARKNKRNTRGC ALWKTLLKKVLKAPKKKRKVC DAATATRGRSAASRPTERPRAPARSASRPRRPVE

Amino acid sequence

(Continues)

[159,161] [162] [50,51] [49] [173] [102,265]

[50,157]

[311]

[15] [169] [105] [74] [19,73] [19,73] [75] [76,77] [80] [24] [81] [23] [306] [10,78,79,306] [9,10,167,175] [168] [309,310]

[12,13] [51,84] [17] [17] [17,107,158] [147] [176] [316]

Ref.


764

wileyonlinelibrary.com/journal/jpepsci RGD peptides αvβ3, αvβ5 RGD peptide RGD peptides α5β1 AGR (prostata carcinoma) LyP-2 (skin and cervix tumor) REA (prostata, cervix, breast carcinoma) LSD (melanoma, osteosarcoma)

36 37 38

Cell-penetrating homing peptides 39 40 41 42

Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Partial sequences 1–24 Partial sequences 1–30

N-terminal prion peptides 54 55 H1, H2A, H2B, H3, H4 Histidine-rich CPP

Protegrin-1 Lactoferrin sequences

Antimicrobial peptides 52 53

Histones and histidine peptides 56 57 Poly-α-amino acids and Dendrimers

RRIRPRPRLPRPRPRPLPFPRGPRPIPRPLPFP PRPRLPRPRPRPRPLPFPRGPRPIPRPLPFP

Bac7 (1–35) Bac7 (5–35)

Bacterial Peptides 50 51

Single histones or mixtures of them HR9: C-HHHHH-RRRRRRRRR-HHHHH-C

Bovine PrP 1–24: MVKSKIGSWILVLFVAMWSDVGLC Bovine PrP 1–30: MVKSKIGSWILVLFVAMWSDVGLCKKRPKP

RGGRLCYCRRRFCVCVGR VSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQI

VPTLK KLPVM

MCaUF1-9: GDAbuLPHLKL YKQSHKKGGKKGSG

INLKALAALAKKIL-amide GIGAVLKVLTTGLPALISWIKRKRQQ-amide GDAbuLPHLKLAbuKENKDAbuAbuSKKAbuKRRGTNIEKRAbuR

CAGRRSAYC CNRRTKAGC CREAGRKAC CLSDGKRKC

RGD-Temporin-LA, RGD-Dye RGD-bearing PAMAM dendrimers Cilengitide: Cyclo[RGDfNmeV] Cyclo(RGDfK); cyclo(RGDyK) RGD mimic

X: PKKKRKVEDPGVGFA Capr-K(X)FHTFPQTAIGVGAP-amide X:KKRKAPKKKRKFA RPKPQQFGLM-amide

Amino acid sequence

Pentapeptides derived from BAX inhibitory peptides 48 CPP5 49 CPP5

46 47

Mastoparan Mellitin Scorpion toxin MAUROCALCINE with replacement of C by Abu Mini maurocalcine peptides Rattle snake toxin (Crotamine) derived NrTP6

RGD peptides αvβ3

Venoms and toxins 43 44 45

Substance P and analogs

Ligands for the subfamily of integrins 35

hCT(18–32)-br

Name

34

No.

Table 1. (Continued)

[56,57,200,238] [58,59]

[60–63] [60–63]

[54] [53]

[146] [146]

[119] [120]

[70] [65–67]

[15] [64] [68,69]

[34] [34] [34] [34]

[315] [47,48,96] [201]

[30,47,48]

[8,10,113]

[261]

Ref.

REISSMANN

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wileyonlinelibrary.com/journal/jpepsci Pharmaliposome Nanoparticle for multimodality imaging Conjugate of CPP with branched PEI

Multifunctional nanocarriers 80 81 92

67

TAT-containing nanocarriers as drug and DNA delivery systems Integrin targeting paramagnetic quantum dots PEI-PEG-CPP

86-101

Cyt c LRRK 492-507 eNOS

50

Azurin Leu –Gly : LSTAADMQGVVTDMGASG 50 77 Azurin Leu –Asp : LSTAADMQGVVTDMGASGLDKDYLKPDD

Repeat-sequence: EALA WEAALAEALAEALAEHLAEALAEALAE ALEALAA Stearoyl-RRRRRRRR-(EALA)n

HIF-1α lacking oxygen-sensitive degradation domain coupled to gene delivery vector in fibrin (HIV-TAT)-HIF-1α ΔODD-(procaspase 3) Nitroimidazole coupled to sC18

Cationic CPPs masked with negatively charged sulfonamides pHLIP: AEQNPIYWARYADWLFTTPLLLLELALLVDADEGT KKLADap(Me2)AL Dap(Me2)LLALLWLDap(Me2)LADap(Me2) ALKKA-amide ε-Lys succinyl amide

Suc-(D-Glu)8-PLGC(Me)AG-(D-Arg)9-amide Suc-(D-Glu)8-xPLGLAG-(D-Arg)9-D-C-Cy5 Suc-(D-Glu)8-Ox-DPRSFL-(D-Arg)9-amide

Proline-based dendrimers Amphipathic polyproline helix containing modified side chains Poly(Lys), poly(Orn), poly[Lys(Glun,Alam)] (VRLPPP)3 SAP(E): (VELPPP)3 Polyglutamic acid

Amino acid sequence

[136] [47] [90,91]

[118] [117] [117]

[35] [35]

[112]

[111,112]

[219] [221]

[218]

[217]

[29] [214] [174]

[209]

[103]

[94,95] [92,195] [89] [98,152] [98] [96]

Ref.

Abu, 2-aminobutyric acid; Dap, Nβ-dimethyl-diaminopropionic acid; f, D-Phe; O, ornithine; Ox, 5-amino-3-oxopentanoyl residue coupled with PEG; PAMAM, polyamidoamine; PEG, polyethylenglycole; PEI, polyethylenimine; stearyl, CH3―(CH2)16―CO~; TMQ, trifluoromethylquinoline acid; y, D-Tyr.

Bioportides within cytochrome c Leucine-rich repeat kinases Nitric oxide synthase

Viral fusion protein sequence

CPPs with endosomolytic GALA-sequence 73

Bioportides 77 78 79

HIF-1α ΔODD Nitroimidazole–peptide conjugates

71 72

Redox protein azurin ‘p18’ Redox protein azurin ‘p28’

HIF-1α ΔODD

Hypoxia-activatable cell-penetrating peptides 70

pH-sensitive R8-construct

pH-dependent hydrolysis

69

74

Activation by dissociation pH-sensitive conformation pH-sensitive conformation

Partial sequences of tumor selective enzyme 75 76

Thrombin-activatable CPP

65

MMP (2,9)-activatable CPP

Protease activatable cell-penetrating peptides 64

765

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pH-sensitive (activatable) cell-penetrating peptides 66 67 68

Proline-based biodendrimers Cationic amphiphilic polyPro Branched copolymers Sweet arrow peptide (SAP) Amphipathic negative CPP Negative charged polymer

Name

58 59 60 61 62 63

No.

Table 1. (Continued)


REISSMANN

Figure 2. Assembly of complex cell-penetrating peptide (CPP) structures. Inactivating negatively charged sequence fragment; sensors (pH or pO2);

liposome;

CPP;

fluorescent label;

nanoparticle.

sequence is either fused to a recombinant cargo protein or can be conjugated by a linker to the cargo. Certain CPPs, mainly from Pep [19,72–74], MPG [50,75–77], KALA [78,79], KLA [10,175], or PepFect types [24,80,81], are capable of forming noncovalent complexes to cargos. These CPPs envelop the cargos by forming complexes via noncovalent interactions. Interaction with the lipid bilayer can be improved by acylation with hydrophobic residues such as stearyl or cholesterol moieties or by the incorporation of lipoamino acids [82,83].

structures can be somewhat stabilized via acylation of the Nterminus and more effectively by insertion of nonproteinogenic amino acids [101–103], by cyclization, or even by backbone cyclization [104]. The applied excess of CPPs by formation of noncovalent complexes protects peptides and nucleic acids from enzymatic hydrolysis. In contrast, easily degradable CPPs, e.g. YTA2 [105], have also been developed. After internalization, these are able to deliver anticancer drugs free from CPP sequences. Release from endosomes

Biodendrimers Because transport through the membrane requires formation of nanoparticles, preformed nanocarriers [85–87] are also used. In addition, CPPs can be coupled to homogeneous or heterogeneous dendrimers, for example, linear and branched poly amino acids (mainly Lys) [88,89], or dendrimers as polyethyleneimine (Figure 2) [26,90,91]. Biodendrimers with proline and proline-rich peptidomimetics are of special interest [92–95] because they show unique conformational properties. Surprisingly, the negatively charged polymer polyglutamic acid [96,97] is also able to penetrate membranes and to transport drugs. Giralt et al. developed the first anionic CPP [98]. Prediction Attempts were undertaken by Hansen et al. [99] to develop algorithm for predicting CPPs. For this purpose, known sequences of penetrating and nonpenetrating peptides were taken from the literature and analyzed using up to 23 different parameters. Utilizing support vector machines, Sanders et al. [100] predicted and synthesized sequences, which show to 100% penetrating activity. But by my opinion, a multitude of different factors, very different cell types, and controversies about uptake mechanisms makes it difficult to create a general algorithm. Stabilization against proteolytic degradation

766

In addition to such naturally occurring proteins and their peptide fragments, semisynthetic chimeric peptides have also been developed. These contain elements of native penetrating structures and are combined through linkers to sequences with additional functions for more precise targeting. Because native peptides are degraded both extracellularly and intracellularly by proteases, their


Cargo;

selectivity part (receptor ligands, homing sequences, and localization sequences),

Many studies have not only focused on the uptake mechanisms but also on release of the CPP and cargo from endosomes [106–110], leading to the utilization of auxiliaries or even to insertion of endosomolytic sequences (GALA, KALA, and KLA) into the CPPs [79,111,175] or to covalent coupling of the CPPs to endosomolytic compounds [81]. Additional functions Cell-penetrating peptides can have additional properties that are useful to cells. Bovine serum albumin can transport or aid in the transport of cargos into live cells because it acts as a nutrient, especially for hungry tumor cells [114–116]. Bioportides [117,118] are a new class of cell penetrants; their sequences possess physiologically useful bioactive profiles. Classical bioportides are derived from cytochrome c, leucine-rich repeat kinase 2, or endothelial nitric oxide synthase. Cell-penetrating pentapeptides also carry out cargo uptake and have cytoprotective properties as well. They were derived from BAX inhibitory peptides, synthesized, and functionally tested primarily by the group of Matsuyama [119,120]. Further possibilities for classification of CPPs consist in varying preferences for cargos and in evoked intracellular processes. In particular, internalization, replication, and expression of DNA and plasmids seem to be a very difficult task. In contrast to the highly efficient transduction of cells with peptides, proteins, and short nucleic acids, the transfection with linear DNA or plasmids is less efficient, and only certain CPPs function in this application. Table 2 lists such CPPs, which have been more or less successfully used or that were especially developed and recommended for transfection [25,84,122–124,126–129]. Additionally to these CPPs and their conjugates with dendrimers, DNA-binding proteins with or without fused CPP sequences are also recommended [14,130,131].


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CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES Table 2. Cell-penetrating peptides (CPPs), dendrimers, and DNA-binding proteins recommended for transfection with DNA or plasmid DNA (pDNA) No. CPPs 1 2 3 4 5 6 7 8

Compound

Influenca HA-2 (1–20) KALA sequence NickFects Nonaarginine (R9) HIV-TAT (47–57) Stearylated arginine-rich peptides Amphipathic peptide Td3701 Amphipathic helical peptide ppTG1 Human calcitonin partial sequence 9–32, hCT(9–32)-br

Dendrimers 9 Polyethylenimine 10 polyLys 11 Multifunctional pharmaceutical nanocarrier DNA-binding proteins 12 Chimeric multidomain protein 13 Fusion protein GAL4/invasin 14

Cell-penetrating DNA-binding protein

Ref.

WEAKLAKALAKALAHLAKALAKALKACEA NF61: Stearyl-AGYLLGOINLKALAALAKKIL-amide RRRRRRRRR YGRKKRRQRRR Stearyl-RRRRRRRR TRYLRIHPRSWVHQIALRLRYLRIHPRSWVHQIALRS GLPKALLKLLKSLWKLLLKA LGTYTQDFNK(X)FHTFPQTAIGVGAP-amide X: PKKKRKVEDPGVGFA

[78,79] [128] [122–124,264] [16,84] [82] [129] [127] [265]

HIV-TAT Polyethylenimine complexes HIV-TAT coupled to branched polyLys HIV-TAT-containing nanocarrier

[124] [126] [136]

Antibody-(translocation domain)-(DNA-binding domain) GAL4(DNA-binding domain)-flexible liker Invasin (mammalian cell binding) YARVRRRGPRR (Hph-1)-GAL4(DNA-binding domain)

[14] [130]

Physiological differentiation Another physiological difference splits the CPPs into two groups, either elevating or not affecting intracellular Ca2+ concentrations [132]. Enhanced cytosolic calcium levels trigger membrane fusion events. Thus, the membrane break evoked by internalization is resealed with vesicles derived from the endocytotic compartment. This type of differentiation is based on both internalization and membrane repair mechanisms [133]. Multifunctional drug delivery complexes The most efficient cargo transporters have complex structures, i.e. multifunctional dendrimers, liposomes, or nanoparticles. One of the most effective transporters is the multifunctional drug delivery liposome with CPPs at the surface, developed by Torchilin [25,136–140].

Other Cell Penetrators and Permeation Methods Permeation of cell membranes can also be performed with other methods besides those described in the previous discussion. Most are not specific to cell organelles, cell type, tissues, or organs. The intracellular amounts and concentrations achieved are in most cases limited. These methods may be used as helpful tools in research studies, but with only few exceptions, they are not applicable in the clinic. CPPs are often compared in their functional parameters with these methods and compounds. It has been demonstrated that CPPs show, in many cases, improved internalization efficiencies and lower toxicity but do not differ completely in their functional properties. Biophysical Methods

J. Pept. Sci. 2014; 20: 760–784

plant cells from Arabidopsis thaliana), microinjection is often used. Penetration can be facilitated with an external electrical field. Thus, many different cell types are transfected with nucleic acids and even plasmids by electroporation. Optimized conditions have been developed for this commonly used method, including special buffers for each used cell line and each applied type of cargo. Magnetofection uses magnetic power for permeation of membranes and tissues; magneto beads are required for covalent coupling of cargo. In some cases, good results have been obtained. However, only a very limited application of this method is possible in the clinic.

Biochemical Methods Chemical helpers, i.e. amphiphatic detergents, liposomes, dendrimers, nanoparticles, and additionally auxiliary compounds, are used for transporting different cargos. For reviews of the subject see ref. [91,134,136]. The use of detergents from the Lipofectamine series has a long tradition in cell transfection. These cationic detergents have been successfully used for in vitro studies and currently also for in vivo studies (Invivofectamine). Covalently coupled lipids facilitate the transport of cargos through the membrane [135]. Liposomes filled with cargos can be inserted into the membranes and are capable of releasing the cargo into the cytosole. Binding to the membrane and internalization are strongly enhanced by CPPs at their surface [25,136]. Branched polymers from different monomer types have been found to be in many cases more effective in the internalization of cargos than linear polymers. Thus, branched lysine polymers (Lys-loligomers) [88,89] or PEIs [90,91], more recently also polyprolines [92–95] and polyglutamic acid [96,97], have been used to transfect different types of cells. Nanoparticles from different structural sources can be applied as transport vehicles. Their uptake occurs probably via macropinocytosis. Thus, silica nanoparticles have been used



767

Mechanical procedures can help to transport cargos through cell membranes. For relatively large cells (Xenopus oocytes or large

[131]

REISSMANN successfully to deliver anticancer active compounds [86,87]. Until now, the enzyme streptolysine has been used for internalization of nucleotides such as derivatives of guanosine triphosphates [141] into target cells. This enzyme forms pores in the membrane that allow the entry of hydrophilic and negatively charged compounds, but it reduces the survival time of the cells to a few minutes. Auxiliaries can enhance membrane permeability. Dimethyl sulfoxide facilitates the transduction of cargos [142]. In our experience, serum albumin has been shown to have very favorable properties as an auxiliaria. It protects CPPs from proteolytic degradation and enhances the cargo uptake by acting as a nutrient [114–116].

uptake mechanisms depending on the conditions. The route that is used depends on the type of CPP, cell line, cargo, the applied concentration, temperature, and time of incubation. For recommended reviews see ref. [36,38]. Certain CPPs have unknown uptake mechanisms. Furthermore, we have to keep in mind the different repair mechanisms for disturbed membranes of different cell types. As the process is highly complex, studies are performed on model membranes and on live cells. But the mechanisms behind these processes remain to some degree unresolved. Thus, internalization is as mysterious as the internalization of the mythological Trojan horse more than 3000 years ago.

Use of Viral Particles

Membranes protect cells from harmful external influences; they seal the cells off from their surroundings, e.g. from the cell matrix, neighboring cells, and the intercellular fluids. Biological membranes are composed of different lipids and proteins. Depending on the lipid composition and on the intracellularly, transmembranal, or attached proteins, the membranes have a different fluidity. Lipid and protein composition are characterized by microheterogeneity. The shape of cell membranes, their so-called curvature, is formed by interaction with the intracellular cytoskeleton, which leads to the formation of caveolae and lipid rafts. It is assumed that internalization of CPPs and the cargo requires interaction with the glycoprotein skeleton, receptor, and adhesion molecules of different types, and with the lipid bilayer, mainly in microheterogeneous regions such as the caveolae and lipid rafts. Because different cells have varying lipid, phospholipid, and protein compositions in their membranes, they also can have different uptake mechanisms and uptake efficiencies. Despite all the differences in membrane structures and composition, the CPPs can penetrate eukaryotic cells such as mammalian cells, nerve cells, immune cells, and plant cells, as well as certain prokaryotic cells. Whereas AMPs are mainly characterized by the distribution of polar and nonpolar amino acids in their primary sequence, it is assumed that CPPs are characterized by more complexes, socalled bulk classifiers, including net charge, isoelectric point, molecular weight, hydropathicity, percent composition of each amino acid, donated hydrogen bonds, and percent of α-helix, β-sheet, or random coils [36,143]. Furthermore, their conformational flexibility plays, in certain CPPs, an important role in the internalization process. Thus, Deshayes et al. [144] have postulated a conformational polymorphism as a prerequisite for cellular uptake of noncovalent complexes with CPPs of the CADY type. Extracellular, membrane-bound, and intracellular proteases can also degrade CPPs, cargo peptides, and proteins. In the case of long internalization times or animal experiments, it seems to be necessary to estimate and to inhibit the proteolytic degradation.

The use of viruses to transport genes into live cells was performed long before the first CPPs were introduced. Until now, the transfection of live cells with genes has been performed with viruses or viral particles, including mainly Bacculo, Adeno, Herpes (simplex), hepatitis B virus, or the translocation motif of Lenti virus (LentiBrite, Lentiviral Particle gene silencer). These commercially provided systems allow the transport of genes into the nucleus of the target cells. Lentiviral vectors gene silencers were targeted to all human and mouse genes. Both the adenovirus and the lentiviral vectors, respectively, were successfully used for transfection and transduction of cells in vitro and in vivo. However, these viral systems require handling in special biological safety labs. For clinical use, viruses and viral vectors are not allowed because of the potential of dangerous accidents in their application to patients. Viruses can have undesired side effects, for instance, immunogenicity and promiscuous tropism. Furthermore, they are unable to transfect certain types of cells.

Uptake Processes The internalization of CPPs and cargos is a complex process. It begins with the interaction with the membrane surface or directly with the phospholipid bilayer, evoking local membrane disturbance and intracellular processes that can lead to further changes in the membrane shape or structure and to internalization into the cytosole or endosomes (Figure 3). Most CPPs utilize two or more

Cell Membranes

Differences in the Membrane Composition of Prokaryotes and Eukaryotes

768

Figure 3. Uptake mechanisms and intracellular trafficking.


Despite the differences between target membranes of bacteria and the nontarget membranes of mammalian cells, certain AMPs can act as CPPs, and vice versa, CPPs can act as AMPs. For review on the subject see ref. [52]. AMPs can disrupt the integrity of membranes through interaction of their cationic areas with negatively charged phospholipids at the cell surface. Depending on


J. Pept. Sci. 2014; 20: 760–784

CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES the structure and concentration of the AMP and on membrane composition, the disturbance of the membrane structure occurs by diverse processes, either via pore formation, carpet-like perturbations, or the formation of inverted micelles in the membrane bilayer. Membranes of microorganisms and eukaryotes differ in their content of sterols. The membranes of eukaryotes contain distinctly more sterols than membranes from prokaryotes. These sterols are neutral and therefore not able to interact with cationic peptides. But they stabilize the membrane by reducing the fluidity of the lipid bilayer [145]. It is assumed that the reduced membrane flexibility may reduce in eukaryotic cells the membranolytic activity of AMPs [52]. On the other hand, certain AMPs have been found to be able to transport cargos into live mammalian cells. Thus, lactoferrin sequences are able to transport peptide antigens into dendritic cells [53]. Bactenecin 7 fragments are able to transport avidin into murine monocytes [146]. Protegrin-1 can transport doxorubicin [54], and the C-terminal fragment of sC18 is able to internalize cytostatic organometallic complexes [55].

Transport Processes Through Cell Membranes

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769

To elucidate the transport process, many studies have been performed, both on live cells and on artificial phospholipid bilayers (for recommended reviews see refs. [36,147]). The initial peptide–lipid interaction is a fast process driven by electrostatic interactions. This interaction depends on the sequence of the CPP and the lipid composition of the bilayer. It has been found that different conformational shapes of CPPs, e.g. α-helix or β-sheet, can in combination with hydrophobic interactions also trigger different uptake mechanisms of internalization. Helical CPPs can form a barrel with hydrophobic side chains oriented to the lipid chains whereby the hydrophilic residues form a pore. Gräslund and Mäler [143] and Madani et al. [36] assume that primary amphipathic CPPs can act via pore formation, carpet-like perturbations, via membrane thinning or formation of inverted micelles. Kuriyama et al. [129] could show that certain CPPs have high affinity to the lipid phosphatidylserine, which is normally located at the cytosolic side of the plasma membrane and is translocated to the external face only in disordered membrane regions. With freeze-fracture electron microscopy, Afonin et al. [148] found that HIV-TAT (48–60) forms inverted micelles in dimyristoylphosphatidylcholine membranes, which can represent intermediates during translocation across eukaryotic membranes. The group of Ulrich assumes that CPPs induce lipid vesicle fusion by folding and aggregation [151]. For the CPP S4 (13)-PV, Cardoso et al. [149] conducted that lateral phase separation and destabilization of membrane lamellar structure are the basis of lipid-driven and receptor-independent entry mechanism. They assume that this process occurs without compromising the membrane integrity. The penetration into cells can occur very rapidly with halftimes from 5 to 20 min. Large cargos prolong the penetration time significantly. The uptake mechanism depends also on the concentration. If the concentration exceeds a certain threshold, the uptake mechanism may switch to another pathway. How different and contradictory results can be that were obtained on artificial membranes even with the same type of CPP is shown by the following two publications. While BaranyWallje et al. [150] postulate: ‘that under the conditions used

here, penetratin does not cross any of these lipid barriers regardless of bilayer curvature and transmembrane voltage’. Lamaziere et al. [113] published 2 years later a penetratininduced ‘nonmetabolic membrane tubulation’ and postulated ‘the existence of a facilitated physical endocytosis, which represents a new pathway for peptide cellular internalization’. These contradictory findings might result from different lipid compositions of the used model membranes and from different experimental conditions. Contrary to the commonly held theories on uptake mechanisms, surprisingly, even negatively charged polypeptides such as polyglutamic acid, its derivatives, and negatively charged CPPs are able to penetrate membranes and transport cargos [96–98]. Major mechanism for cell entry, the endocytosis can be divided into macropinocytosis, clathrin-mediated endocytosis, caveolae lipid raft-mediated endocytosis, and clathrin/caveolaeindependent endocytosis [153]. However, uptake processes are also attributed to micropinocytosis [154]. The involvement of clathrin, caveolin, and other proteins can be detected or excluded by using specific inhibitors. As shown in the Sections on Structural and Functional Classification of CPPs and Uptake Processes, the uptake into live cells requires according to Ziegler [37,38] the binding to GAGs. By this mechanism, the first binding is followed by formation of inverted micelles with negatively charged lipids. The precise CPP interaction with heparin, heparan sulfates, and phospholipids has been studied by Ghibaudi et al. [155] with electron spin resonance spectroscopy. Verdurmen et al. found besides a chiralitydependent uptake that treatment of cells with heparinase did not reduce the uptake [40]. Thus, they assumed that syndecan-4, a member of the universally expressed syndecan family of transmembrane heparan sulfate proteoglycans, is likely one of the binding partners for cationic CPPs and mediates their internalization [33]. More generally, different types of membrane-bound receptors can be involved in the internalization process. The group of Futaki has identified the chemokine receptor CXC type 4 as the binding partner for arginine-rich CPPs such as the arginine 12mer (R12) peptide [107]. Binding to this receptor type mediates internalization through macropinocytosis by multimerization of the receptor, polymerization of actin, and remodeling of its network [44]. The receptor CXCR4 is one of the major coreceptors for HIV-1. Because tumor cells overexpress the CXCR4 receptor, a tissue-selective internalization occurs [31]. Pooga et al. postulated the initial binding of a negatively charged nanocomplex formed from PepFect 14 and oligonucleotides to the scavenger receptor types SCARA 3 and 5 [32]. Binding is followed by internalization indicating that uptake efficiency depends on distribution patterns of these receptor types on different immune cells. Neuropilins are multifunctional coreceptors. They play an important role in developing immunity and cancer. Neuropilins are expressed in a wide variety of cells, including endothelial cells, neurons, hepatocytes, melanocytes, and osteoblasts. CPPs that bind with their Arg/Lys-rich C-terminal motif to the electronegative pocket of neuropilins are quickly internalized [45]. Selective binding of CPPs to members of the integrin family leads to cell-specific, tissue-specific, and organ-specific uptake [30,34,47,48,163]. As theoretically expected for G protein-coupled receptor (GPCR)-ligands, uptake of their ligand fragments occurs as a lipid raft-mediated endocytosis [164,165]. But we have to

REISSMANN keep in mind that the analogs of calcitonin [20,21] and SP [9,113] were internalized in a receptor-independent way.

Release from Endosomes For this important aspect of CPPs see also the review [172]. Depending upon different factors, the cargos can be internalized by endosomal way and completely or partially located in vesicles [107,108]. Release from these vesicles can be provoked by addition of auxiliaries such as chloroquine [83,109,166,167] or its more strongly active trifluoromethyl quinoline [169], by wortmannin [170], Ca ions [171], and biopolymers such as low molecular PEI [172]. These compounds destabilize endosomes or inhibit their formation. For instance, chloroquine and its derivative trifluoromethyl quinoline can block the pH drop inside the endosomes. Another means has been shown by Langel with his PepFect series, containing intrinsically trifluoromethyl quinoline, covalently coupled to the peptide [169]. Internalization studies with cationic peptides containing D- and L-arginine residues have revealed that the uptake of polypeptide R8 with only L-arginine residues ends in endosomes, whereas a sequence with alternating D-Arg/L-Arg achieves efficient cytosolic delivery [106]. The authors assume that the chimeric cationic peptides enter the cells through direct membrane translocation driven by membrane potential. Furthermore, insertion of GALA, KLA, or KALA sequences into CPPs facilitates endosomal escape [78,79,111,173,175]. Peptides containing 2,3-diamino propionic acid residues in their sequence drive release from endosomes probably by interrupting the intermolecular hydrogen bonding networks [174]. Certain other peptide transporters such as BAX inhibitory pentapeptides [119,120], the noncationic helical Azurin peptides p18 and p28 [35], and S413-PV [176] are thought to enter cells by a nonendocytotic mechanisms. Recently, Mellert et al. [156] described an enhanced endosomal delivery by using photochemical disruption of the endosomal membranes. Furthermore, a photochemical enhanced uptake by addition of the photosentisizer Aluminium phthalocyanine [177] and an endosomal escape of unchanged native cargo triggered by brief photoinduction (545–580 nm, 10% laser power) have been reported [178]. In more detail the most possibilities of endosomal release are discussed and reviewed by Erazo-Oliveras et al. [172].

Selective Intracellular Transport

770

One of the most interesting and potentially very important advantages of CPPs is the directed transport into cell organelles by specific localization sequences. Neither electroporation nor magnetofection or detergents and unmodified nanoparticles are able to transport cargos specifically into different cell organelles. In Table 3, for different organelles, localization sequences are listed, which are taken from the literature. For transport into and staying in the cytosole of cells, the CPPs can be designed with inactivated localization sequences. But fluorescence labels and cargos can alter intracellular sorting as described by Szeto et al. [179]. Use of a nuclear localization sequence is common and firmly established; it is derived from a simian virus [180,181]. Incorporated into the sequences of CPPs and fusion proteins or conjugates, thereby the cargo can be transported into the nucleus. Also, other sorting sequences for the nucleus have been found [182–186]. To improve direct transport into the nucleus, combinations of two


consecutive sequences linked through a spacer are used [187]. Nuclear localization can be enhanced by addition of Glu residues to the nuclear localization sequence (NLS) [188]. In addition to amino acid sequences, oligosaccharides can also function as NLS [189,190]. Using a 15-mer sequence of rattle snake toxin (Table 1, comp. 47), cargos can be transported also into the nucleolus [191]. In addition to the 15-mer peptide, a series of shortened sequences, the NrTP-peptides [66], also have cell-penetrating properties and are derived from this venom. It has also been shown that βoctaarginine, a completely different sequence, appears to penetrate into the nucleolus compartment of the cell nucleus [192]. Schiller’s group have reported an uptake of casomorphine peptide analogs into the mitochondria [179]. Kelley’s group developed a class of CPPs that efficiently enter human cells and specifically localize to mitochondria, the so-called ‘mitochondria-penetrating peptides’ [193,194]. They synthesized and studied systematically series of peptides, controlled and tuned the mitochondrial localization by altering lipophilicity and positive charge. Surprisingly, completely divergent structures such as polyproline oligomers are also capable of internalizing into the mitochondria [195]. Transport of cargos, especially proteins and nucleotides, through the inner membrane of mitochondria opens new possibilities for studies on influencing intramitochondrial signal pathways and mitochondrial replication and transcription. Because the mitochondria are implicated in many human diseases such as diabetes, Parkinson’s disease, and cancer, efficient strategies are important to deliver therapeutics to this intracellular compartment. A CPP with a lysosomal sorting sequence was recently described [196]. This peptide is capable of transporting gold nanoparticles and fluorescent cargos into lysosomes. Using costaining with a specific anti-Golgi antibody, we detected a CPP-internalized fluorescent cargo protein at the Golgi apparatus [197]. This finding indicates that possibly more sorting capabilities may yet be discovered. As the aforementioned studies indicate, an imaging of cell organelles for diagnostic purposes should soon be possible allowing the development of therapeutic treatment of diseases that result from the dysfunction of distinct cell organelles, e.g. mitochondrial or lysosomal dysfunctions. Further development of effective sorting sequences for other organelles remains a challenge for cell biologists and biochemists.

Cell and Tissue Selectivity of CPPs Most CPPs from the first generation do not show any cell specificity. They differ substantially between mammalian and nonmammalian cell membranes. However, for the same organism, the CPPs have a low cell, tissue, and organ selectivity. In contrast to similar uptake efficiencies, the susceptibility of the used cell types differs more strongly for CPPs, auxiliaries, and cargos [199,200]. To overcome this drawback several strategies have been used. For a review to this subject see ref. [34]. Most selective internalization helpers are hormone analogs such as ligands for GPCRs, as well as ligands for more widely distributed integrin, chemokine, and scavenger receptors or syndecans and neuropilins. The adhesion protein integrin forms a subfamily through combining different α-subunits and β-subunits. Because tissues and organs have distinct combinations of these subunits, the integrin receptor ligands are tissue and organ specific. Thus, ligands for the integrin receptors αvβ3 [30], α4β1, α3 [47,48], or α5β1 [201]


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CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES Table 3. Intracellular targeting: localization sequences for cell organelles Name

Structure

Nucleus Simian virus 40 large T-antigen (NLS) Stearylated NLS Homodimeric NLS (C-terminal) Nucleoplasmin NLS, bipartite NLS Ku702-NLS ADAR1 NLS GluOct6 ReIB Dermaseptin-peptide K4-NLS Glycosylated proteins Glycoconjugates Nucleolus Nucleolar targeting peptide

References

PKKKRK

[181] [180] [185] [187] [186] [183]

Stearyl-PKKKRKV (GYGPKKKRKVGGC)2 KRPAATKKAGQAKKKK C-GSKGARPAKKRKPKRGAAHKHAGAKVRKTVTGAKK MMPNVKRKIGELVRYLNTNPVG EEEAAGRKRKKRT 405 424 YGVDKKRKRGMPDVLGELNS 425 446 SDPMGIESKRRKKKPAILDHFL PVK: PKKKRLKVAKWKTLLKKVLKA-amide Galβ4Glc-protein

[312] [190] [184] [182] [190] [189]

YKQCHKKGGXKKGSG Beta-octaarginine

[191] [192]

Mitochondria Opioid peptides Cationic amphiphilic polyproline helix Mitochondria-penetrating peptides

Dimethytyrosine-rFKF-NH2 Polyproline-based scaffold with guanido and carboxy groups F-r-F-K-F-r-F-K

[179] [195] [193,194]

Sperm mitochondria Mitoparan

NLKKLAKL(Aib)KKIL

[282]

Lysosoma Lysosomal sorting sequences L1 Lysosomal sorting sequences L2

YQRLC CNPGY

[196] [196]

Endoplasmatic reticulum Retention sequence

C-terminal fusion sequence405: HDEL

[313]

Direct cytosolic delivery Chimeric peptides containing D- and L-Arg

rRrRrR-RR

[106]

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activation, the released peptide is capable of transporting cargos specifically into target cells, primarily into tumors and their metastasis cells, or the peptide can detect these cells with fluorescent and other markers [198,208–210]. Linkage of ACPPs to dendrimeric nanoparticles can further enhance the selectivity to tumor cells [27,103]. On the other hand, the cleavage of a corresponding ACPP by thrombin provides the possibility for rapid in vivo detection and localization of activated thrombin [209,211]. Also, for other proteases, for example, calpain, cellspecific ACPPs have been developed [212]. Additionally, to upregulated proteases such as matrix metalloproteases, tumor cells and the tumor environment are characterized by an acidic pH value, a lower transmembrane potential, hypoxia, and a preferred uptake of dendrimers and nanoparticles. For effective tumor cell killing, all these special tissue properties have been used. Formation of pH-sensitive CPPs can be achieved in different ways. For tumor targeting, Sethuraman et al. [29] used negatively charged sulfonamides to mask the positively charged HIV-TAT conjugates. When the pH decreases in the vicinity of the tumor, the sulfonamides lose charge, detach, and expose the active CPP. Another means is to use the conformational changes evoked by acidic pH values. Thus, the pH-sensitive CPPs form, only at acidic values, a helical conformation that allows insertion into the lipid membrane [28,121,214–216]. Even the



771

can be used for imaging cancer tissue with fluorescence markers or for internalization of coupled anticancer drugs. Further possibilities for cell-selective and tissue-selective transport were found by insertion of homing sequences into the fusion proteins containing the CPP and cargo. Each normal organ expresses organ-specific sets of molecules on their vasculature. Homing peptides concentrate in the target tissue and are therefore efficient delivery vectors in a cell-specific manner [46]. Some of their sequences have been, for example, taken from phage display peptide libraries [203]. Cell-penetrating homing peptides recognize the target cell and are able to translocate cargos across the cellular membrane. Examples of some homing peptides and their cancer targets have been described [204], collected by Giralt [34], and some of them are and listed in Table 1 (comp. 39–42). Activatable CPPs represent a new and highly sophisticated development. Activation occurs at the target tissue using its specific biochemical properties. An excellent innovation is the development of a protease activatable CPP by the group of the Nobel prize winner Roger Tsien [27,103,205]. Membrane-bound and secreted proteases of tumor cells, mostly matrix metalloproteases [206], plasmin, elastase [207], and proteases in inflamed tissue, activate these CPPs. Activation occurs by proteolytic cleavage of a designed and very sensitive linker between a positively charged CPP and negatively charged capping peptide sequence. After

REISSMANN maurocalcine nonapeptide MCUF1-9 acts in a pH-sensitive mode because of protonation of His residue [70] and can be used for specific transport into tumor tissue [71]. Fast hydrolysis of succinyl amides of lysine residues in acidic tumor tissue has been used to activate Lys containing HIV-TAT peptides and to internalize doxorubicin into cancer cells [217]. Because hypoxia has often been recognized as a tumorspecific microenvironment, the oxygen-dependent degradation domain of the α-chain of hypoxia-inducible factor can be fused to the cargo and HIV-TAT. In the case of β-galactosidase as cargo, only the hypoxic regions showed evidence of this enzyme after intraperitoneal application [218,219]. Another way is the application of multifunctional liposomal nanocarriers, developed by Torchilin and coworkers. These stimuli-responsive carriers can transport cargos in tissuespecific mode [136]. Doxorubicin [137], small interfering RNA (siRNA) [138], and DNA [25] are transported as cargos and used to treat for instance nonsmall-cell lung cancer [137]. The delivery was mediated by HIV-TAT sequences on the surface of these nanocarriers or by selective antitumor antibodies on the surface of immunoliposomes [139]. The group of Neundorf treated successfully cancer tissue with metallorganic compounds coupled to suitable CPPs [168,222] and even an ACPP [223].

Cargos Drugs, Peptides, and Proteins

772

As shown in the Section on Introduction, research on CPPs was primarily motivated by the fight against different diseases, and this aim propelled experimentation on internalization of hydrophilic or badly soluble drugs into the cytosol or distinct cell organelles (Table 4). For instance, in tumor therapy, it is necessary in many cases to transport organometallic compounds such as cisplatin or its derivatives [168,222,223], methotrexate [89], doxorubicin [54,68,103,115,224], anticancer peptide vaccines [225], and other hydrophilic anticancer drugs and chaperons [227,228] into the tumor cells. Cancer-killing [213,226,229,230] and antiAlzheimer [231] antibodies can be transported into the corresponding target cells. Another important field of CPP application involves studies on intracellular processes. Thus, metabolic and pathway antibodies could be useful tools for signal transduction studies and signal transduction therapy. Generally, antibodies, substrates, and inhibitors of intracellular enzymes, ligands for the many different protein binding domains, activators or inhibitors of the transcription factors can be internalized with the aim to study signal transduction and signal pathways and to treat related diseases. Thus, inhibitors of nuclear factor-κB [232,233] can be used to treat inflammatory processes such as rheumatoid arthritis. Internalization of antiapoptotic peptides including BH4 [234], the Bcl-xL-protein [235], or a peptide inhibitor against cJun N-terminal kinase [236] protects the heart and brain against ischemic injuries. CPPs are able to internalize enzymes, from the larger-sized enzymes such as β-galactosidase with 540 kDa [200,237,238], Cre recombinase [125,239] chaperon heat shock protein 70 (HSP-70) [227], and HSP-gp96 [228] to the comparably small antibacterial enzyme lysozyme [240]. Uptake of fluorescent probes including fluorescent-labeled peptides, proteins [200,237,238], nucleoside triphosphates [199], or of quantum dots [241,242] and gold nanoparticles [196,243] allows pathway studies, elucidating intracellular trafficking and fluorescent


imaging of selected cells and tissues. Signal pathways can be studied with transported ligands for protein domains [239,244,245] or loops of GPCRs [246]. The simultaneous inhibition of protein kinase C-δ (PKCδ) and activation of PKCε by internalized peptide regulators protects the ischemic heart [247]. Thus, a recombinant fusion protein, formed from human homeobox B4 and the HIV-TAT peptide can influence hematopoietic stem cells [248]. Nucleotides and Oligonucleotides Transduction of labeled nucleoside phosphates such as GTP analogs allows studies on GTP-dependent intracellular reactions [199]. Transport of nucleic acids such as siRNA, miRNA, or shRNA helps to influence the transcriptional processes. Furthermore, peptide nucleic acids (PNAs), morpholino oligonucleotides, and splicecorrecting oligonucleotides are internalized for research and clinical studies. Because nucleic acid mimics can be easily synthesized with such functional groups, which enable covalent coupling to CPPs, these cargos are often used and have been well investigated. In the literature on internalization, primarily three classes of RNA mimics are described. Covalently to CPPs conjugated or noncovalently complexed PNAs have been utilized as antisense nucleic acids [177,249,250]. This type of nucleic acid mimic was developed by Nielsen [251] and contains no negative charges because the phosphodiester bond is replaced by an amide bond. Morpholino oligonucleotides can be conjugated to CPPs for internalization into live cells [23,252–255]. These peptide– morpholino conjugates have been successfully used for splice correction in certain muscle dystrophies [253,254,256]. Because it is difficult to form conjugates between negatively charged oligonucleotides and positively charged CPPs, noncovalent complexes with CPPs have also been formed to deliver oligonucleotides into cells [23,24,32] and splice-correcting oligonucleotides into muscle tissues [252]. In the last decade, the silencing RNA has become important, because this short RNA is relatively specific and triggers an enzymatic process that magnifies the inhibiting action. Therefore, many attempts have been undertaken to internalize siRNA into many different live cells and tissues. Certain CPPs, mainly from the Pep, MPG, PepFect, and hCT family, have been applied with notable success. They form noncovalent complexes with siRNAs and are able to internalize sufficiently high amounts [77,80,257,259,260]. Hoyer and Neundorf [261] achieved a knockdown of NPY1 receptor by internalization of stable electrostatic complexes that are formed from siRNA and a CPP derived from a branched hCT(18–32) sequence. Also, dendrimers are capable of transporting siRNA into live cells [138]. DNA and Plasmids A necessary prerequisite for classical gene therapy is the reproducible and stable transfection of cells with DNA. Because this transfection is a significantly complex task, much more difficult than the transduction of cells with proteins, short ribonucleic acids and their chemical mimics, it should be discussed separately. For clinical aspects the usefulness of CPPs has been investigated through many approaches. In recent overviews [41,44,91] and some original publications, the transport of linear DNA and plasmid DNA has been described to transfect cells transiently and to express coded proteins. Replication and transcription of DNA require in many cases the formation of noncovalent complexes to avoid covalent coupling of CPPs. An efficient transport of DNA has been


J. Pept. Sci. 2014; 20: 760–784

CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES Table 4. Different types of cargos No.

Cargo

Ref.

Peptides 1 2 3 4 5 6 7 8 9

Peptide ligand for SH2 domain Insulin Fluorescent probes Radiolabeled probes Magnetic probes Inhibitor PKCδ Inhibitor PKCε Cyclosporin A Activating peptide for protein p53

[239,244,245] [300–302] [47,105,199,200,208,210,237,238] [307] [47] [247] [247] [303] [314]

Enzymes 10 11 12

β-galactosidase Cre recombinase Chaperons

[199,200,237,238] [125,239] [227,228,304]

Proteins 13 14 15 16 17 18

Bovine/human serum albumin BH4-domain of antiapoptotic protein Inhibitor NF-κB protein N-terminal kinase binding motif of c-Jun Bcl-xl-protein Peptide vaccines

[114,115,199,200,237,238] [234,248] [232,233] [236] [235] [225]

Antibodies 19 20 21 22

Antiactin Ab Anti-PI3Kinaseϒ-Ab Cancer-killing Ab Anti-Alzheimer’s Ab

[200] [200,237] [213,220,229,230] [231]

Nucleic acids and their mimics 23 24 25 26

siRNA Peptide nucleic acids (PNAs) Morpholino oligonucleotides (MOs) Splice-correcting oligonucleotides (SCONs)

[77,80,257,259–261] [177,249,250] [23,252–256,308] [253,254,256]

DNA and pDNA 27

Linear and cyclic (plasmid) DNA

[16,82,84,122–124,126–129]

Chemotherapeutics 28 29 30 31

Methotrexate Doxorubicin Organometal conjugates Paclitaxel

[89,305] [54,68,103,115,137,224] [168,222,223,258,263] [96]

Nanoparticles 32 33 34

Quantum dots Paramagnetic quantum dots Gold nanoparticles

[241,242] [47] [196,243]

J. Pept. Sci. 2014; 20: 760–784

It may perhaps be a more philosophical question as to how plasmids are internalized into the nucleus by electroporation, magnetofection, detergents, dendrimers, or nanoparticles. From our own experience with plasmid transfection, all attempts in experimentation using noncovalent complexation with CPPs have only lead to very low transcription efficiency. This disappointing result raises questions on the minimum amount of internalized DNA required for the transport through the nucleus membrane and replication in proliferating cells. The literature describes an influence of the size of plasmids on uptake. Smaller plasmids were internalized with a higher number of copies [266]. It could be shown that with PEI polyplexes the number of plasmids per nucleus reaches 75 to 50 000 [267]. A total of 3000 plasmids per nucleus were required for an optimum response [267].



773

described only for certain CPPs. In particular, KALA peptides [122], arginine-rich peptides [84,123,124,126], dendrimers containing guanido groups [126], amphiphilic peptides ppTG1, ppTG20 [127], NickFect [128], the carboxy-terminal fragment Td3701 of the blood coagulation factor VIII [129], and S413-PV [262] have been shown to achieve transfection. Arginine-rich peptides are also capable of transporting genes into plant cells and into microorganisms [123,264]. Human calcitonin derivatives can internalize noncovalently complexed plasmids into primary cells [265]. Another possibility for uptake of DNA and plasmids consists in the coating of DNA with binding proteins, which are derived from different sources [14,19]. These are recombinant proteins, and they can be fused to CPP sequences [131].

REISSMANN Ultrasound-based delivery system can help to overcome several key barriers [268]. One possibility to enhance efficiency may be to consider the synchronization of the cell cycle. It is believed that plasmids enter the nucleus during the division process that is coupled with lysis of its nucleus membrane. Polyethylenimine-based nanoparticles were described as most efficient system in gene delivery. Combining PEI with CPPs enhance efficiency and selectivity [269]. Despite that the transduction of proteins and transient transfection can be performed with CPPs with high efficiency, the stable transfection of proliferating cells with CPPs, necessary for classical gene therapy, will remain to my opinion a big challenge for the future.

Formulation of the Cargo Expression of Fusion Proteins The internalization of recombinant proteins can be performed by fusion to CPP sequences. Thus, enzymes, enzyme substrates, enzyme inhibitors, antibodies, vaccines, and other functional sequences can be expressed as fusion proteins with CPPs [235,240,248]. In this case, the CPPs remain covalently coupled to the functional protein after transport into the cells or organelles. Therefore, it is necessary to check the functional intactness of the construct before using it. In many cases, the enhanced biological activity of these constructs, even with additional coupled fluorescent proteins, has been shown.

[73,74]. The CADY family was derived from PPTG1 [76,77] and is characterized by a conformational polymorphism [144]. The recently invented MPG8 [75] differs from CADY considerably, has a cationic C-terminal sequence, and has a high affinity to siRNA. Thus, peptides from the MPG and CADY series are favorites to transport siRNA [77,257,259]. In high molar ratios from 1 : 125 or 1 : 250, peptides derived from branched hCT(18–32) type form stable complexes with siRNA and are able to suppress expression of a GPCR in HEK-293 cells [261]. The PepFect family, developed by the group of Ülo Langel, is described as being able to transport proteins, mimics of oligonucleotides and siRNA [23,24,77,81,83]. Other CPPs such as KALA [78,79] and KLA peptides [10,175], HIV-TAT [84], or penetratin [199] are also able to form noncovalent complexes with a cargo. But, in contrast to results from the literature, in our own experience, this uptake occurred with HIVTAT and penetratin only with low or very low uptake efficiency [200,237,238]. The newly developed NrTP family, derived from rattle snake venom, is also recommended for formation of noncovalent complexes with proteins and nucleic acids [65]. The advantage of the formation of noncovalent complexes is the easy handling of the transduction procedure. But, the required high excess of CPPs can influence the viability of the cells and should be tested before using. Another disadvantage is the instability of the noncovalent complexes in high concentrations of buffers and other proteins. Combination with Nanocarriers and Dendrimers

Formation of Conjugates Expressed, synthesized, or isolated proteins and peptides can be covalently coupled to CPPs using bivalent linkers or activated CPPs. In most cases, a bridge between an amino group and a SH group or between two amino groups is formed. Thus, the bivalent linker should contain an S-pyridyl or maleinimide moiety and on the other side an activated esters such as the N-hydroxysuccinimide ester. Activated CPPs containing S-pyridyl moieties are able to form disulfide bridges with SH groups of cysteinyl residues in the cargo. Because of the convenient incorporation of corresponding functional groups into PNAs and morpholino oligonucleotides during their chemical synthesis, these chemical mimics of nucleic acids are often and very successfully used for formation of conjugates with CPPs [251,256,275,276]. Fusion proteins and conjugates are stable under transduction conditions and provide high uptake efficiency. Formation of Noncovalent Complexes Between Cargo and CPP

774

This method requires only the mixing of the CPP and cargo to form a complex, followed by incubation of the target cells, tissues, or organs (recommended reviews are reported in ref. [72,274]). For peptides and proteins, a molar ratio from 1 : 10 between cargo and CPP gives optimum yields of internalized cargo. Nucleotides, nucleic acids, and plasmids require about a twofold to fourfold excess of positive charges of the CPPs over negative charges of the cargo. The group of Heitz and Divita [72,77] have developed several CPPs with different sequences suitable for cells with different membrane polarity and for different cargos. The series of MPG peptides were derived from a fusion sequence of HIV protein gp41, a hydrophilic lysine-rich nuclear localization sequence, and a spacer


Cell-penetrating peptides alone are in many cases capable of delivering different cargos into cells, tissues, and organs. However, to enhance uptake efficiency and selectivity to cancer cells, different multifunctional nanocarriers have been constructed. They combine the specific functions of liposomes or other biopolymers in the uptake mechanism with the equivalent internalization power and cell specificity of CPPs. Nanoparticles and biopolymers accumulate passively in tumors because solid tumors tend to present a permeable structure, impaired lymphatic drainage, and sloppy tumor vasculature with poorly aligned endothelial cells. In addition to linear and branched poly amino acids [88,89,92–95], peptide and glycopeptide dendrimers [270,271], linear and branched polymers [87] primarily of the type polyethyleneimine [90,91], as well as nanoparticles of different chemical structure [85–87], have been used. The fruitful attempts of Torchilin et al. to design multifunctional liposomal pharmaceutical nanocarriers for intracellular drug delivery were mentioned in the previous discussion [25]. It can be expected that in the near future, multifunctional carriers will be constructed for certain specific research targets or clinical targets, which will be optimized for cargo-type, celltype, or tissue-type handling. Further developments of CPPs will encompass broad development in the field of biopolymers, dendrimers, and nanoparticles and include research into formation of multifunctional complexes [271–273].

Cells A large number of different cell types have been successfully transduced. To these target cells belong mammalian cell lines and adhesion, suspension and immuno cells, plant cells [125,202], algal cells from Chlamydomonas reinhardtii [278], yeast


J. Pept. Sci. 2014; 20: 760–784

CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES cells from Saccharomyces cerevisae [279] and Candida albicans [280], amoeba Dictyostelium discoideum [277], and the protozoa such as Leishmania tarentolae [200,238] or Paramecium [264]. Using CPPs, several cargos have been transported into stem cells [248,281], primary cells [265], spermatozoa [282], dendritic cells [45], nerve cells [283,284], immune cells [45,285,286], and many different tumor cells. Many attempts have been undertaken to influence intracellular pathways and to program lymphocytes, monocytes, and macrophages [285–287]. Because the cells differ in chemical composition, in transmembranal signaling, and in intracellular pathways, they are also differently susceptible to CPPs. Thus, mammalian CPPs are for some nonmammalian cells highly cytotoxic [279,280] and have been even recommended in patents for treatment of protozoa-mediated diseases such as leishmaniasis and malaria. Despite the effective transduction of cargos into most cells, a few cell types are difficult to transfect. Such cells are not only difficult to transfect with the previously described biophysical or biochemical methods but mostly with CPPs, too. Thus, for each cell type or tissue, the right combination of CPP and cargo has to be estimated experimentally. The current state of knowledge on the properties of cell types and uptake mechanisms does not yet allow clarification or the ability to easily predict the most optimal conditions.

Internalized Amounts of Cargos

J. Pept. Sci. 2014; 20: 760–784

Influence on Intracellular Signaling, Cell Viability, and Membrane Integrity One of the most important advantages of CPPs and an essential prerequisite for therapeutic application is a low cytotoxicity. Nevertheless, cytotoxic effects are concentration dependent, and it can be expected that high amounts of CPPs and also cargos can have side effects and can be cytotoxic. As already shown, influencing intracellular processes requires in most cases sufficiently high amounts of internalized cargos, and their cytotoxicity has to be estimated experimentally. It has also been mentioned in Section on Structural and Functional Classification that CPPs and auxiliaria interact with lipid bilayers enhancing the permeability of the cell membranes. It has been shown that the cytotoxic effect depends not only on the dose but also on the length of the CPPs [292]. Tünnemann and coworker [293] studied short-term and longterm dose-dependent effects of arginine-rich peptides on cell viability and cell cycle progression by biochemical and microscopical methods. They could identify concentration windows with low toxicity and high transduction efficiency. Intactness of the cell membrane can be checked by the release of enzymes from the cytosol such as lactate dehydrogenase (LDH) (LDH leakage assay) [294] or the death protease [199,237,295]. The uptake of propidium iodide by cells as measured by fluorescence-activated cell sorting analysis provides values that agree well to the release of death protease [200,237]. Disturbance of the membrane bilayer by CPPs and auxiliaria can also be detected by measuring the occurrence of phosphatidyl serine in the outer leaflet of the plasma membrane [296]. This translocation indicates induction of apoptosis and has been found mainly in cancer cells. Cell-penetrating peptides and cargos can evoke intracellular reactions. Thus, CPPs themselves act as bioactive molecules interfering with cellular signaling. In an excellent review, Verdurmen and Brok [289] described a wide range of side effects, which can be evoked by cationic CPPs. Thus, cationic CPPs are capable to affect at micromolar concentrations cell metabolism, to internalize TNF and EGF receptors [160] via enhancement of fluid phase of membranes. Furthermore, they are capable to activate the acidic sphingomyelinase [297], to enhance in murine macrophages superoxide generation and to release histamine via degranulation of mast cells by a receptor-independent G protein activation. Nevertheless, it could be shown in animal experiments and clinical studies that by local administration, therapeutic doses of CPPs were well tolerated. Internalization processes require GTPase-dependent actin network remodeling, which is associated with the activation of GTPase Rac1 [298]. Membrane processes and intracellular signaling can influence the functionality of the mitochondria



775

The application of CPPs in research and for therapeutic purposes requires the uptake of a sufficiently high amount of cargo. However, as the precise concentration of the intracellular target molecules is unknown, in most cases, the required cargo amount to achieve complete target blocking or interaction is also unknown. The most optimal intracellular amounts and concentrations must be determined when choosing the most favorable CPP. To achieve this, several methods are possible. The internalized amount can be estimated by fluorescence measurements [199], matrix-assisted laser desorption/ionization mass spectrometry [288], quantitative western blotting [205], or by functional characterization of the transported cargo [289]. For instance, the internalized Cre recombinase enables the expression of enhanced green fluorescent protein from a construct, and thus, the uptake efficiency can be measured by fluorescence intensity [290]. In another example, the measurement of luciferase activity was used to estimate the uptake of a reporter conjugate into both cell cultures and animal models [291]. Both of these examples are only indirect estimations. The staining is achieved after an enzymatic multiplication process. Fluorescent peptide, protein, and nucleotide cargos can be internalized and quantitatively measured that provide the intracellular amounts per cell in the attomole range and concentrations in the low micromolar range [199,200,237,238]. The reached amounts and concentrations seem to be high enough to achieve interaction with enzymes, protein binding domains, transcription complexes, and other intracellular targets. Only in a few cases, with very high amounts of the target or by low affinities to the target molecules, the binding or blocking can remain incomplete. Results from Mussbach et al. [199] show that the internalization of nucleoside triphosphate for studies with adenosine triphosphate or GTPbinding functional proteins are highly superior to application of the commonly used poor forming enzyme streptolysine

[141]. Because in the cases of RNA and DNA, the detection of uptake is mostly performed indirectly through functional characterization; no comparable intracellular values on amount and concentration are available. Thus, we are not yet able to predict the necessary intracellular amount of DNA that is required for transient or stable transfection of a given cell. From the literature, it is also difficult to compare the appropriate intracellular amounts of DNA or plasmids, which were achieved by internalization [265] or by enveloping in multifunctional liposomes [25,136,140].

REISSMANN by reducing their dehydrogenase activity. The test based on the activity of this enzyme is known as the MTT test, which quantitatively measures the formation of colored formazane and also indicates the influence of CPPs and auxiliaries on cell viability. The MTT test [199,237,299] is in most cases more highly sensitive to CPPs and auxiliaria than the membrane integrity test and should be used for detecting toxic effects. For in vivo experiments, in particular for clinical use, the hemolytic effect must be measured on erythrocytes. Using certain other cells such as Leishmania tarentolae, it is possible to monitor the morphology and mobility of the cells as indicators for viability and intactness of the membrane [200,238,295]. Cells can recover from toxic treatment by repair mechanisms and also by proliferation. A large group of CPPs enhance the intracellular calcium concentration followed by membrane repair processes [132,133]. Penetrating oligopeptides derived from BAX inhibitory peptides have only low transport efficiency, but they can stabilize the mitochondria and protect cells from apoptosis [119]. Thus, they can be used as helpful cytoprotective components.

Commercially Available Products As previously mentioned in the Section on Other Cell Penetrators and Permeation Methods, numerous reagents are commercially available that are based on cationic lipids or polymers and on nanoparticles formed from different structural classes. The first commercially available CPP was ‘Chariot’, provided by Active Motif. It belongs to the amphipathic Pep series (Pep-1). The methods and compounds, including CPPs, are listed in Table 5. Many reagents are cocktails of different CPPs or are mixtures of CPPs with polymers. They form noncovalent complexes and are specifically developed for distinct cargos such as peptides, proteins, or RNA species and are recommended for distinct cell types. Cocktails provide a more universal approach for internalization of cargos through compatibility with various cell types, triggering different uptake mechanisms, and allowing complexation with structurally different cargos. Additionally, single CPPs and also activated HIV-TAT are available. Single CPPs can form noncovalent complexes with cargos or can form conjugates to the cargos using suitable bivalent linker molecules as described in the Section on Formation of Conjugates. However, because of the complexity of precisely determining peptide sequences and other components in commercially available cocktails, Table 5 may miss certain peptide compounds or even contain nonpeptide compounds.

Table 5. Commercially available reagents No.

Product name

Transduction and transfection reagents 1 ICA 614 2 ICAFectin

Producer

SanofiPasteur SanofiPasteur

Cell line-specific transfections reagents 3 TransiT-TKO Transfection KMF-Laborchemie Reagent 4 TransiT-293 Transfection KMF-Laborchemie Reagent 5 Transit HeLa Monster KMF-Laborchemie Transfection Kit 6 Transit-CHO KMF-Laborchemie Transfection Kit 7 Transit-Jurkat KMF-Laborchemie Transfection Kit Cocktails for transduction of peptides and proteins 8 ProteoJuice Merck/Novagen 9 JBS-Proteoducin Jena Bioscience siRNA transfection reagent 10 X-tremeGENE

Roche Diagnostics

Cocktail for transduction of nucleotides, oligonucleotides, and DNA 11 JBS-Nucleoducin Jena Bioscience Single CPPs 12 13 14 15 16

Chariot MPGα CAD-2 HIV-TAT (47–57) Penetratin

17 18

CPPP-2 PepFect

Active Motif Jena Bioscience Jena Bioscience BACHEM Jena Bioscience, NeoMPS Jena Bioscience Cepep

Activated CPP 19 HIV-TAT(47–57)-S-Pyridide

BACHEM

CPP for formation of conjugates 20 Cys-HIV-TAT(47–57)

Jena Bioscience

Fluorescent-labeled CPP 21 Cys(maleoimido-FM)[HIV-TAT(47–57)] FM-maleoyl-betaAla-[HIVTAT(47–57)] 22 Cys(maleoimido-Atto488)[HIV-TAT(47–57)] 23 Cys(maleoimido-DY676)[HIV-TAT(47–57)]

Jena Bioscience KeraFast Jena Bioscience Jena Bioscience

Overall Scopes

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Cell-penetrating peptides are a field that is rapidly growing. These peptides can transport into live cells proteins and peptides such as antibodies, enzymes, chaperons, peptide substrates, peptide inhibitors, and nucleic acids such as nucleoside phosphates, oligonucleotides, mimics of them, interfering RNA, but only to some degree DNA. Thus, they can be used as tools for cell research including signal transduction and intracellular signaling. In contrast to electroporation, magnetofection, lipidofection, and viral vectors, the CPPs have their biggest potential in therapeutic application. Because of their low toxicity in mammalian cells, they are able to deliver poorly permeable or impermeable generic drugs into cells and tissues or through the skin, conjunctiva of the eyes, and the


blood–brain barrier. Furthermore, CPPs open completely new therapeutical applications with the internalization of proteins, nucleic acids, and their mimics. CPPs with only natural amino acids are completely degraded after the delivery of the cargo, thus avoiding any accumulation of toxic products, a prerequisite for long-time treatment. Formation of noncovalent complexes between CPPs and cargo is very convenient to handle and represents the easiest method for intracellular release of the native cargo. To overcome the low cell and tissue specificity of the first CPP generation, different strategies were performed. Thus, CPPs from the second generation act as ligands of membrane receptors such as integrins, scavenger, or chemokin receptors and neuropilins.


J. Pept. Sci. 2014; 20: 760–784

CPPS, TYPES, UPTAKE, TRAFFICKING, SELECTIVITY, AND CLINICAL STUDIES Furthermore, partial sequences of certain tumor-specific enzymes are used to enhance cell selectivity. Incorporation of homing domains into CPPs enables a tissue-directed uptake. A third generation of CPPs can be activated by specific conditions in cancer or inflamed tissue, such as by secreted tissue-specific proteases or by the acidic and hypoxic environment. First approaches have shown that CPPs with incorporated localization sequences are capable of a directed transport of cargos into cell organelles. Reinforcement of these studies can help in developing a further generation of CPPs that should be able to achieve organellespecific drug delivery, thus enabling treatment of diseases caused by dysfunctions of cell organelles. The CPP-mediated internalization even allows transfection of very sensitive cells such as stem cells, somatic cells, and spermatozoa. Thus, CPPs are a potentially massive aid in developing effective drugs for treatment of different types of cancer, inflammation, retinal degradation, viral and microbial infections, and for metabolic, neuronal, and genetic disorders. Selective and highly sensitive imaging of cancer cells and inflamed tissues with CPPs improves diagnosis and surgery.

Internalization of peptides, proteins, and impermeable drugs into cells occurs with sufficiently high efficiency into many different cells. However, the so-called ‘difficult to transfect cells’ are also hard to transfect with the help of CPPs. In contrast to peptides, proteins, drugs, and short RNAs, the transport and incorporation of linear and plasmid DNA into chromosomes remains a practical and theoretical problem, a challenge to be resolved in the future.

Recommendations for Further Research •

• •

•

Limitations

•

J. Pept. Sci. 2014; 20: 760–784

• •

Acknowledgements The author gives thanks to his research and diploma students for engaged experimental research on CPPs and many stimulating discussions about technical and philosophical problems. Andrea Anneliese Keller is thanked for technical help by preparing Figure 3 and Elizabeth Kley from English Editing for proofreading and correcting. The Jena Bioscience GmbH (http://www.jenabioscience.com) supported for more than one decade my research on cell penetration.

References 1 Handbook of Cell-penetrating Peptides, Langel Ü (ed.). CRC Taylor & Francis: Boca Raton, London, New York, 2007. 2 Cell-penetrating peptides. Methods and protocols. Methods in Molecular Biology, Series Editor Johm M. Walker, Vol. 683, Langel Ü (ed.). Springer: New York, Dodrecht, Heidelberg, London 2011. 3 Special Issue “Arginine-rich Peptides”. Guest Editor: Shiroh Futaki. Curr. Protein Pept. Sci. 2003; 4(2). 4 Special Issue “Delivery of therapeutic molecules - from bench to bedside”. Biochimica et Biophysica Acta (BBA) Biomembranes 2010; 1798(12): 2177–2314. 5 Special Issue “Cell penetrating Peptides”. Guest Editor: Vladimir P. Torchilin, Pharmaceuticals, 2010-2013. 6 Heitz F, Morris MC, Divita G. Twenty years of cell-penetrating peptides: from molecular mechanism to therapeutics. Brit. J. Pharmacol. 2009; 157: 195–206. 7 Brasseur R, Divita G. Happy birthday cell penetrating peptides: already 20 years. Biochim. Biophys. Acta 2010; 1798: 2177–2181. 8 Repke A, Bienert M. Mast cell activation – a receptor –independent mode of substance P action. FEBS Lett. 1987; 221: 236–240. 9 Oehlke J, Lorenz D, Wiesner B, Bienert M. Review: studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides. J. Mol. Recognit. 2005; 18: 50–59.



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Despite the many advantages of CPPs, it should be noted that there are also many limitations, resulting from their chemical and functional properties. The large number of first generational CPPs have low cell, tissue, and organ specificity. Therefore, these CPPs must be administered directly to the target tissue for therapeutic use. Development of cell-selective, organ-selective, or disease-selective peptide carriers began only a few years ago and will require time until successful clinical use. The transport efficiency, the cytotoxicity of each CPP cargo complex and required auxiliaries, must be estimated for each target because these properties differ from CPP to CPP, from cargo to cargo, and from cell type to cell type. Thus, CPPs are not the philosopher’s stone. Each application must be carefully optimized with regard to internalization effectivity and cytotoxicity. One of the biggest drawbacks results from the uptake into intracellular endosomes. Release from these cell organelles requires their destabilization by addition of auxiliary compounds or charged polymers such as PEIs. All these auxiliary compounds may be cytotoxic. To avoid this endosomal uptake, new CPPs with nonendosomal uptake mechanisms should be developed. The advantage of easy handling by the formation and internalization of noncovalent complexes between CPPs and cargos has some time-consuming disadvantages requiring extended studies in order to be able to determine the correct CPP for the given cargo and cell type. High molar excess of CPPs (5- to 20-fold over cargo) used by the noncovalent method enhances the danger of cytotoxic effects. Conjugates or fusion proteins with CPPs are more stable than noncovalent complexes against high ionic concentrations and by-products in the transduction medium, but they require in some cases removal of CPP sequences after uptake. Although the uptake process requires in many cases only 5 to 30 minutes, in slow processes (large cargos) and in animal experiments, secreted, membrane-bound, intracellular, and serum proteases are able to inactivate CPPs. To avoid inactivation by proteases, the CPPs must be stabilized by the incorporation of nonproteinogenic amino acids or by cyclization. The transport of cargos through the blood–brain barrier may be an advantage but can become a disadvantage by whole body administration.

Focus on uptake mechanisms to explore the relationship between properties of distinct CPPs, cell types, and cargos, which allow a theoretical prediction of optimal partners and conditions for a given task. Develop and improve localization sequences for all cell organelles. Focus on investigating the internalization and expression process of linear and plasmid DNA. Application of CPPs in gene therapy requires enhancement of their transfection effectivity and of incorporation. Enhancement of cell, tissue, and organ specificity by more specific activation or incorporation of highly selective sequences. Incorporation of CPPs into multifunctional dendrimeric or liposomal nanocarriers to improve selectivity, efficiency, and capacity of cargo transport, mainly into cancer cells. Research in improving medical diagnosis, especially tumor and metastases, by using highly selective and near-infrared fluorescent-labeled CPPs or nanocarriers. Develop new therapeutics by using the special probabilities of CPPs for internalization of antibodies, enzymes, chaperons, and other large functional proteins into live cells.

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