S T R U C T U R E S , T O X I C I T Y A N D I N T E R N A L I Z A -

T I O N O F C E L L - P E N E T R A T I N G P E P T I D E S

Emelía Eiríksdóttir

Structures, toxicity and internalization of cell-penetrating peptides

Emelía Eiríksdóttir

Cover picture: Lavafall in Fimmvörðuháls in Iceland (©Kristján Freyr Þrastarson)

©Emelía Eiríksdóttir, Stockholm 2010

ISBN 978-91-7447-058-1

Printed in Sweden by Universitetsservice, Stockholm 2010

Distributor: Department of Neurochemistry, Stockholm University

To my favorite person

List of publications

This thesis is based on the following publications, referred to in the text by their corresponding Roman numerals: I Külliki Saar, Maria Lindgren, Mats Hansen, Emelía Eiríksdóttir, Yang

Jiang,Katri Rosenthal-Aizman, Meeri Sassian, Ülo Langel

Cell-penetrating peptides: A comparative membrane toxicity study

Anal. Biochem. (2005) 345, 55–65

II Emelía Eiríksdóttira, Karidia Konate

a, Ülo Langel, Gilles Divita,

Sébastien Deshayes

Secondary Structure of Cell-Penetrating Peptides Controls Mem-

brane Interaction and Insertion

Biochim. Biophys. Acta - Biomembranes (2010) 1798, 1119–1128

III Emelía Eiríksdóttir, Ülo Langel, Katri Rosenthal-Aizman

An improved synthesis of releasable luciferin-CPP conjugates

Tetrahedron Lett. (2009) 50, 4731–4733

IV Emelía Eiríksdóttira, Imre Mäger

a, Taavi Lehto, Samir EL Andaloussi,

Ülo Langel

Cellular Internalization Kinetics of Luciferin-(Cell-Penetrating

Peptide) Conjugates

Bioconjug. Chem. (2010) Submitted

aThese authors contributed equally to this work

Additional publications

Emelía Eiríksdóttir, Helena Myrberg, Mats Hansen, Ülo Langel

Cellular Uptake of Cell-Penetrating Peptides

Drug Delivery Reviews - Online (2004) 1(2), 161–173

Maria Lindgren, Katri Rosenthal-Aizman, Külliki Saar, Emelía

Eiríksdóttir, Yang Jiang, Meeri Sassian, Pernilla Östlund, Mattias

Hällbrink, Ülo Langel

Overcoming methotrexate resistance in breast cancer tumour cells

by the use of a new cell-penetrating peptide

Biochem. Pharmacol. (2006) 71(4), 416–525

Tiina Peritz, Fanyi Zeng, Theresa J Kannanayakal, Kalle Kilk, Emelía

Eiríksdóttir, Ülo Langel & James Eberwine

Immunoprecipitation of mRNA-protein complexes

Nat. Prot. (2006) 1(2), 577–580

Jennifer Zielinski, Kalle Kilk, Tiina Peritz, Theresa Kannanayakal,

Kevin Y. Miyashiro, Emelía Eiríksdóttir, Jeanine Jochems, Ülo Langel,

and James Eberwine

In vivo identification of ribonucleoprotein–RNA interactions

Proc. Nacl. Acad. Sci. USA (2006) 103(5), 1557–1562.

Fanyi Zeng, Tiina Peritz, Theresa J Kannanayakal, Kalle Kilk, Emelía

Eiríksdóttir, Ülo Langel & James Eberwine

A protocol for PAIR: PNA-assisted identification of RNA binding

proteins in living cells

Nat. Prot. (2006) 1(2), 920–927

Imre Mäger, Emelía Eiríksdóttir, Kent Langel, Samir EL Andaloussi,

Ülo Langel

Assessing the uptake kinetics and internalization mechanisms of

cell-penetrating peptides using a quenched fluorescence assay

Biochim. Biophys. Acta - Biomembranes (2010) 1798, 338–343

Abstract

Cellular internalization is a highly regulated process controlled by proteins in the plasma membrane. Large and hydrophilic compounds, in particular, face difficulties conquering the plasma membrane barrier in order to gain access to intracellular environment. This puts serious constrains on the drug industry since many drugs are hydrophilic. Several methods aiming at aiding the cellular internalization of other-wise impermeable compounds have therefore been developed. One such class, so-called cell-penetrating peptides (CPPs), emerged around twenty years ago. This group constitutes hundreds of peptides that have shown a remarkable ability in translocating diverse molecules, ranging from small molecules to large proteins, over the cell mem-brane. The internalization mechanism of CPPs has been questioned ever since the first peptides were discovered. Initially, the consensus in the field was direct translocation but endocytosis has gradually gained ground. The confusion and the disunity within this research field through the years proceeds from divergent results between re-search groups that hamper comparison of the peptides.

This thesis aims at characterizing several well-established CPPs with comprehensive studies on cellular toxicity, secondary structure and cellular internalization kinetics.

The results demonstrate that CPPs act in general in a low or non-toxic way, but the apparent toxicity is both peptide- and cell line-dependent. Structural studies show that the CPPs have a diverse po-lymorphic behavior ranging from random coil to structured β-sheet or α-helix, depending on the environment. The ability to change second-ary structure could be the key to the internalization property of the CPPs. Internalization kinetic studies of CPP conjugates reveal two sorts of internalization profiles, either fast curves that cease in few minutes or slow curves that peak in tens of minutes. Furthermore, im-proved synthesis of CPP conjugates is demonstrated.

In conclusion, the studies in this thesis provide useful information about cytotoxicity and structural diversity of CPPs, and emphasize the importance of kinetic measurements over end-point studies in order to give better insights into the internalization mechanisms of CPPs.

Contents

Introduction .................................................................................................. 1 Plasma membrane..................................................................................................... 1 Membrane transport ................................................................................................. 2

Clathrin-mediated endocytosis .......................................................................... 3 Caveolae-mediated endocytosis ........................................................................ 4 Clathrin- and caveolae-independent endocytosis .......................................... 4 Macropinocytosis .................................................................................................. 4

Artificial membranes and vesicles .......................................................................... 6 Unilamellar vesicles ............................................................................................. 6 Monolayer membranes ........................................................................................ 7

Evaluation of cellular internalization ...................................................................... 8 Fluorescence-based methods............................................................................. 8 Mass spectroscopy ............................................................................................. 10 Luminescence and functional assays .............................................................. 10

Cell-penetrating peptides ....................................................................................... 12 A brief history ..................................................................................................... 12 Structural features ............................................................................................. 13 Cytotoxicity ......................................................................................................... 17 Internalization mechanisms ............................................................................. 18 Cargo delivery .................................................................................................... 20

Aims of the study ...................................................................................... 22

Methodological considerations ................................................................ 23 Selection of CPPs and CPP-conjugates. ............................................................... 23 Peptide synthesis ..................................................................................................... 25 Selection of a cargo and coupling to peptides (papers III and IV) ................ 26

Selection of a cargo ........................................................................................... 26 Conjugation through a disulfide bridge .......................................................... 27

Cell cultures .............................................................................................................. 28 Cancer cells (paper I) ........................................................................................ 28 Endothelial cells (paper I) ................................................................................ 28 HeLa cells (paper IV) ......................................................................................... 28

Membrane leakage studies .................................................................................... 29 LDH leakage (papers I and IV) ........................................................................ 29 DiBAC4(3) assay (paper I) ................................................................................ 30

Hemolysis assay (paper I)................................................................................ 31 Glutathione leakage (paper IV) ....................................................................... 31 Leakage from liposomes (paper II) ................................................................ 31

Determining peptide secondary structure (paper II) ........................................ 32 CPP-lipid interaction (paper II) ............................................................................. 33 Kinetic studies and evaluation of CPP internalization ....................................... 34

Analysis of peptide translocation by mass spectrometry (paper I) .......... 34 Kinetic studies with luciferin-luciferase system (paper IV) ........................ 34

Results and discussion ............................................................................. 36 Membrane leakage caused by CPPs (paper I).................................................... 36 Membrane interaction and insertion of CPPs (paper II) ................................... 37 Synthesis of luciferin–CPP conjugates (paper III) ............................................ 38 Internalization kinetics of luciferin-CPP conjugates (paper IV) ...................... 40

Conclusions ................................................................................................. 42

Acknowledgements ................................................................................... 44

References .................................................................................................. 47

Abbreviations

AEC Aortic endothelial cell asON Antisense oligonucleotide CCV Clathrin-coated vesicle CD Circular dichroism CLIC Clathrin-independent carrier CMC Critical micellar concentration CME Clathrin-mediated endocytosis CPI Critical pressure of insertion CPP Cell-penetrating peptide Cys Cysteine DCC N,N′-dicyclohexylcarbodiimide DiBAC4(3) bis-(1,3-dibutylbarbituric acid)trimethine oxonol DIEA N,N′-diisopropylethylamine DMPC Dimyristoylphosphatidylcholine DMPG Dimyristoylphosphatidylglycerol DOPC Dioleoylphosphatidylcholine DOPG Dioleoylphosphatidylglycerol DPPC Dipalmitoylphosphatidylcholine DPPG Dipalmitoylphosphatidylglycerol DTNB 5,5′-Dithiobis-(2-nitrobenzoic acid) Fmoc Fluorenylmethyloxycarbonyl FRET Fluorescence resonance energy transfer FTIR Fourier transform infra-red spectroscopy GAG Glycosaminoglycan HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid GPI-AP Glycosylphosphatidylinositol-anchored protein GUV Giant unilamellar vesicle HF Hydrogen fluoride HOBt Hydroxybenzotriazole IRRAS Infrared reflection absorption spectroscopy LDH Lactate dehydrogenase LPC Palmitoyl-hydroxy-glycerophosphocoline LUV Large unilamellar vesicle MALDI-TOF Matrix-assisted laser desorption/ionization-time of

flight

MAP Model amphipathic peptide MBHA 4-Methylbenzhydrylamine MIP Maximum insertion pressure MRR Membrane repair response MS Mass spectrometry NLS Nuclear localization signal NMR Nuclear magnetic resonance NPys 3-Nitro-2-pyridylsulfenyl OCD Oriented circular dichroism pAntp Penetratin PBS Phosphate buffered saline PG Proteoglycan PIP2 Phosphatidylinositol-4,5-bisphosphate PIP3 Phosphatidylinositol-3,4,5-trisphosphate PI3P Phosphatidylinositol-3-phosphate PM Plasma membrane PNA Peptide nucleic acid POPC Palmitoyloleoylphosphatidylcholine POPG Palmitoyloleoylphosphatidylglycerol PTD Protein transduction domain RP-HPLC Reversed phase high performance liquid chromato-

graphy SDS Sodium dodecyl sulfate SPPS Solid phase peptide synthesis SUV Small unilamellar vesicle t-Boc tert-Butyloxycarbonyl TBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-

tetramethyluronium tetrafluoroborate TFE Trifluoroethanol TP10 Transportan 10

1

Introduction

Plasma membrane

All living organisms are either unicellular or multicellular entities. Cells of multicellular organisms vary in size, shape and specialized function but are generally microscopic, take up nutrients, transduce signals from messenger molecules, and interact with several other types of substances. Cells also secrete a variety of molecules. By this, cells communicate with each other and contribute to the survival of the organism, all in a highly regulated manner.

Cells are categorized as either eukaryotic or prokaryotic where the former contain a nucleus but the latter don’t. Eukaryotic cells will only be discussed in this thesis, but all higher organisms are composed of eukaryotic cells.

Eukaryotic cells are commonly 5–100 µm in diameter (with the ex-ception of neurons, which can reach meters in length) and consist of various organelles, all with their specific function. These organelles are shielded with a double lipid membrane, which are then enclosed by an outer membrane, the plasma membrane (PM). The PM, also a lipid bilayer, separates the interior of the cell (the cytoplasm) from the extracellular environment and regulates the trafficking of substances in and out of the cell. The hydrophobic cell membrane is selectively permeable. It is quite an obstacle for free diffusion of inorganic ions (e.g. Na

+, K

+, Cl

-) and most other charged or polar substances, but it is

permeable to small, nonpolar compounds [1]. Cellular processes are highly regulated by thousands of proteins, both soluble and membrane bound. Cellular entry and intracellular signaling is also directed by proteins, i.e. transport proteins, receptor proteins, and membrane en-zymes that “float” around in a flexible lipid cell membrane [2]. The lipids and the proteins in the cell membrane are mainly non-covalently bound and are able to move laterally in the fluid bilayer, although this movement is subjected to restriction. PMs of various cell types have distinct lipid and protein contents but share some common characteris-tics. Steroids and phospholipids are the major structural components in membranes. Cholesterol, a rigid and bulky molecule, is the main steroid in animal cells and is required to establish proper membrane permeability and fluidity [3]. Phospholipids are composed of fatty

2

acids and phosphatidyl groups joined in a glycerol or sphingosine. In the plasma bilayer membrane, the phospholipids arrange in a tail to tail fashion with hydrophobic interactions, exposing the charged phosphatidyl groups to the extra- and intracellular aqueous environ-ment. The lipid bilayer is typically 3 nm thick but extends to 5–8 nm when the protruding proteins are accounted for, and the distribution of the various lipids and proteins between the inner and outer monolayers is asymmetric [1].

Lipids serve more functions than acting as a physical barrier. They are used for energy storage, they can act as messengers, and they pos-sess an important property in allowing the membrane to bud [4]. This last quality is essential for intracellular membrane trafficking, which will be discussed further herein.

Membrane transport

Small, hydrophobic molecules, such as oxygen, nitric oxide, and car-bon dioxide are able to cross the PM freely while the transport of small, polar or charged molecules is mediated by various membrane-bound proteins, i.e. channels, pores and carriers. Larger substances, however, are internalized through endocytic processes called phagocy-tosis or pinocytosis, which can be subdivided into several pathways. Despite of the diversity in the underlying mechanisms of the endocy-tosis pathways, they all share some basic steps, which start with inva-gination of the membrane that converts into a vesicle called an endo-some, release of the endosome from the membrane, and finally an intracellular trafficking of the endosome. These endocytic mechan-isms differ substantially in their recognition of extracellular molecules and the fate of corresponding endosomes.

Large particles (mainly microbes) and cell debris are internalized through phagocytosis, which is mainly carried out by macrophages, neutrophils, and dendritic cells [5, 6]. Phagocytosis is mediated by Fc receptors, i.e. receptors that bind to the Fc domain on immunoglobulin molecules, which activate actin polymerization to extend the PM over the particle to be engulfed [7]. Phagocytosis is a more complicated process that will not be described further here (for review see [7]).

Smaller compounds and big amount of fluid is internalized through pinocytosis process. Pinocytosis is therefore also called fluid-phase endocytosis. It is generally subcatagorized into four main groups, which all have their distinct endocytosis processes; clathrin-mediated endocytosis, caveolae-mediated endocytosis, clathrin- and caveolae-independent endocytosis, and macropinocytosis. More precise

3

classification such as lipid-raft-, flotillin-, CDC42-, or Arf6-dependent endocytosis have emerged last years [8, 9].

Clathrin-mediated endocytosis

Receptors on the PM have a certain tendency to concentrate at some spots. The binding of the ligand to the receptors triggers invagination of the membrane and internalization of the receptor-bound ligand in clathrin-coated vesicles (CCV). This process is called clathrin-mediated endocytosis (CME) and substances that are known to be internalized by this pathway are for example nutrients, transferrin, receptor tyrosine kinases (RTKs), G-protein-coupled receptors, and growth factors [10, 11].

Several proteins are involved in CME but clathrin and dynamin are certainly best studied. Clathrins have a so called triskelion structure and are comprised of three heavy chains and two light chains, which rearrange themselves in a highly organized manner on the exterior of the nascent vesicle. The clathrins thereby build a cage around the ve-sicle, which resembles the structure of a football with hexagonal and pentagonal facets [11]. The assembly of clathrins is facilitated by AP2, the main endocytosis clathrin adaptor, AP180, and epsins, which also promote membrane invagination at phosphatidylinositol-4,5-bisphosphate (PIP2) rich domains [10, 11]. AP2 is a heterotetrameric protein that links the cargo, the PM, the clathrins and accessory pro-teins [10]. When the vesicle has reached a certain size (which is con-trolled by AP180 for instance), ranging from 40 nm to 160 nm inner diameter, the accessory proteins amphiphysin, intersectin, and sorting nexin 9 recruit the GTPase dynamin to the neck of the vesicle (i.e. the lipid leash between the vesicle and PM) to perform the scission of the CCV from the PM [10-12]. The dynamins wrap around the neck in a spiral way (ca. 20 dynamins per circle), which is thought to extend lengthwise upon GTP hydrolysis until the vesicle and the PM are se-parated [12, 13]. Immediately after endocytosis, the clathrin cage is disintegrated by auxilin, synaptojanin, and the ATPase HSC70, and fuses with traditional early endosomal compartment, which can be recognized by Rab5, phosphatidylinositol-3-phosphate (PI3P) and early endosomal antigen 1 (EEA1) [5, 10]. From there, the membrane and fluid components are sorted to the trans-Golgi network, late endo-some and lysosome, or recycled to the PM [5].

There are several more proteins involved in the CME process, in-cluding actin, which may even be essential for some CME [11]. Dif-ferent subtypes of CCVs (with different cargo content), which could even have distinct trafficking routs, should therefore not be ruled out [8].

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Caveolae-mediated endocytosis

Caveolae is the most common non-clathrin-mediated endocytosis. It facilitates uptake of cholera toxin, SV40 virus, and glycosylphosphatidylinositol-anchored proteins (GPI-APs), to name a few cargos. It is a flask-shaped bud, 60–80 nm in diameter, and enriched in caveolin-1 protein (ca. 100–200 caveolin-1 molecules per caveolae), which is considered to be necessary and perhaps sufficient for the caveolar mechanism. Caveolin-1 is a palmitoylated hairpin-structured integral protein that binds cholesterol and glycosphingolipids in a complex with 14–16 cavolin-1 molecules.

The scission of the caveolar bud is dynamin-dependent and leads to free vesicles, tubules or large structures (caveosomes) with neutral pH. These caveolae are dependent on the GTPase Rab5 and are capa-ble of fusing with other organelles as well with each other [8].

Clathrin- and caveolae-independent endocytosis

There are several endocytic pathways that belong to the clathrin- and caveolae-independent group; for instance the flotillin-associated endocytic mechanism and clathrin-independent carrier/GPI-AP enriched early endosomal compartment (CLIC/GEEC) pathway. The CLIC/GEEC pathway is dynamin-independent and takes up fluid phase markers, choleratoxin and GPI-APs, while the flotillin mechanism, which internalizes choleratoxin and proteoglycans (PGs), can both proceed with or without dynamin.

The fate of the tubular/ring-like CLIC/GEEC vesicles varies; they do not fuse with traditional Rab5-positive endocytic compartments, but fusion with lysosomal and recycling compartments has been estab-lished.

The flotillins are hairpin-structured integral proteins and bear there-fore some similarity to caveolin-1. Flotillin proteins are however not enriched in caveolae, but oligomerize in distinct cholesterol-rich mi-crodomains. Some flotillin-positive vesicles end up in late endosomes [8].

Macropinocytosis

Macropinocytosis, which can occur constitutively or upon stimuli, form the biggest cavities of pinocytosis (0.2–10 µm in diameter) and resemble in a way phagocytosis [5, 7]. Here, ruffled PM extends from the cell surface, folds back upon the membrane and encapsulates cargos such as fluid phase markers and RTKs [8].

5

PIP2 and phosphatidylinositol-3,4,5-trisphosphate (PIP3) act as platforms for protein recruitment, and Bar-domain (an actin-associated protein) influences the curvature of the PM, which leads to PM protrusions or invaginations [6]. PAK1 kinase is necessary and sufficient to induce macropinocytosis, but PAK1 binds Rac1 (a small G-protein), which is recruited by cholesterol. Actin is also one of the crucial components in macropinocytosis, along with phosphatidylinositol-3-kinase, Ras GTPase, Src kinase, and Arf6 GTPase. The fission of macropinocytotic vesicles is independent of dynamin but the CtBP1/BARS protein is implicated in the process [8]. The Ras-, Src-, and Arf6-associated micropinocytosis are induced by different growth factors; all of the pathways recruit Rab5 onto the macropinosomes but do not fuse with the traditional early endosomes. Most of the fluid and membrane of the macropinosomes are then re-turned to the PM [5].

A few markers and inhibitors for different endocytic mechanism are listed in Table 1. It should be noted that these molecules may not al-ways be specific for a certain endocytic pathway [14-17].

Table 1. A selection of markers and inhibitors for endocytic mechanisms.

Marker Ref. Inhibitor Ref.

Clathrin-mediated endocytosis

Transferrin [18, 19] Wortmannin [15, 20]

α-Macroglobulin [19] Chloropromazine [15, 21]

Receptors for low-density-lipoprotein (LDL)

[19]

Caveolae-mediated endocytosis

Caveolin-1 [8, 22]

Clathrin- and caveolin-independent endocytosis

Flotillin-1 [8, 22]

Cholera toxin B subunit [15, 17]

Macropinocytosis

Dextran [15] 5-(N-ethyl-N-isopropyl)amiloride [15, 18]

Cytochalasin D [20]

Wortmannin [20]

Lipid-raft-dependent endocytosis

Methyl-β-cyclodextrin [15, 16]

Early endocytosis

GTPase Rab5 [17]

PI3P [17]

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Artificial membranes and vesicles

The PM is a dynamic entity with constant endo- and exocytosis. The composition of the PM is extremely complex with a forest of proteins, carbohydrates and lipids, which renders it difficult to measure interac-tions between cell membranes and applied molecules [2]. Simplified experimental conditions with model membranes are therefore fre-quently used. These artificial membranes may sometimes not resem-ble PMs very well and have often been questioned. However, lipids are the basic building blocks of cell membranes and model mem-branes may therefore provide useful information at membrane level that otherwise would be inaccessible [23].

Various natural and synthetic lipids are commercially available with their distinct length, charge and other properties that may be es-sential for certain experimental procedures [24]. Phosphatidylcholine is the most common phospholipid in eukaryotic membranes and is frequently used in studies with artificial membranes. Other naturally widespread lipids are for instance phosphatidylethanolamine, phos-phatidylserine, phosphatidylinositol, phosphatidic acid, sphingomye-lin, and cholesterol [4]. These lipids can be mixed in various propor-tions to increase the virtuality of artificial lipid membranes. If even more realistic membrane system is required, the lipid layers can be enriched with membrane proteins, peripheral proteins or enzymes [2, 4].

Unilamellar vesicles

Unilamellar vesicles (or liposomes) consist of one bilayer and can be of various sizes and composition. These vesicles can be filled with variety of substances, for example with a fluorophore and a quencher to study leakage of the vesicles. PM potential can even be mimicked with Na

+-K

+ chemical gradients across the vesicle membranes [25].

Vesicles with acidic interior are also of interest since many intracellu-lar organelles have pH gradients across their membranes [26], e.g. the mitochondria, endosomes and lysosomes. Consequently, studies of these vesicles, with for example drugs or peptides, could imitate intra-cellular distribution of these compounds.

Four popular models that are commonly utilized to study all kinds of interaction of peptides and lipids are micelles, small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and giant unila-mellar vesicles (GUVs) [27]. Out of the four abovementioned models, micelles are the simplest ones and resemble least biological membrane systems. Sodium dodecyl sulfate (SDS) micelles are not the only mi-celles available but they are frequently used [28]. They are only com-

7

posed of negatively charged SDS molecules and are therefore some-times used as models for bacterial membranes, which are rich in nega-tively charged lipids [28, 29]. SDS micelles are small (ca. 4 nm in diameter), have high curvature and are monolayers with a hydrophob-ic core and hydrophilic surface. Hence, studies on encapsulated hy-drophilic substances cannot be carried out. SDS micelles have though been utilized with good results in for instance X-ray crystallography and nuclear magnetic resonance (NMR) studies, especially due to their small size [28-30]. Even though SDS micelle studies may not be rea-listic, they are very useful and can be complementary to other lipid methods. It should though be kept in mind that SDS has the ability to induce α-helical structures [28, 29].

The SUVs, LUVs and GUVs are all bilayer vesicles with water-filled cores. The lipids are arranged as in a biomembrane, tail to tail with the lipid head-groups extra- and intravesicularly. All of the ve-sicles can be stored for days after preparation but LUVs are the most stable liposomes. The diameter of SUVs depends on the lipid compo-sition and preparation conditions but is typically 30–70 nm. The small size renders SUVs compatible with optical spectroscopy and NMR. Hence, SUVs have been studied in numerous biophysical studies [31].

LUVs are 50 nm up to 10 µm in diameter with a less curvature than SUVs [27] and resemble the PM more closely. Typical membrane pressure for 100 nm LUVs is 32 mN/m, while for 30 nm SUVs and 4 nm micelles it is around 23 mN/m and 10 mN/m, respectively [32]. Lateral pressure for biological membranes is close to 35 mN/m [24, 32]. LUVs are compatible with some optical spectroscopy methods, such as CD, but light scattering can cause a problem.

GUVs are probably the best PM-mimicking model, at least regard-ing size. GUVs resemble eukaryotic cells with their 10–100 µm di-ameter [23] and the capacity to form intra- and extravesicular buds [33]. GUVs are even available with a variety of lipid and protein con-tents, which increases their “virtuality” [34].

Monolayer membranes

Monolayer membranes are very practical tools to gain information on conformation and interaction of molecules in presence of membranes. These monolayer membranes are extensively used because several physical parameters, such as temperature, membrane area and surface pressure, are easily controlled, as opposed to the vesicle systems de-scribed above. Various experimental methods are compatible with monolayer membranes, such as CD and fluorescence spectroscopy [2, 35].

8

One approach, when evaluating physico-chemical properties of pep-tides, is adsorption to air/water (hydrophobic/hydrophilic) interface where the peptide is injected into the subphase and the surface pres-sure that it exerts is measured. Here, the surface pressure may be measured with a commonly used method called Wilhelmy where a thin platinum or glass plate connected to electromicrobalance is par-tially immersed in the liquid phase [35]. These measurements give information on the surfactant activity of the peptides, i.e. peptides that exert with a higher saturation of surface pressure are generally more amphipathic.

When assessing penetration of peptides into lipid monolayers, the lipid monolayer is spread at air/water interface and the peptide solu-tion is injected in the subphase. Here, the maximum insertion pressure (MIP) (also known as critical pressure of insertion (CPI) in paper II) of peptides in lipid monolayer is evaluated by recording the increase of surface pressure for different initial lipid surface pressures [24, 35]. This is done to specify the degree of peptide/lipid interaction because MIP of the monolayer is the maximum surface pressure at which the peptide is capable of penetrating. High MIP for a certain peptide cor-responds therefore to strong penetration into the lipid membrane. No labeling (fluorophores or other tags) is necessary when carrying these experiments out. It should be noted that the lateral pressure for biolog-ical membranes has been estimated to be 30–35 mN/m, which means that peptides with MIP below this value are thought to be unable to penetrate these membranes [24, 32].

Evaluation of cellular internalization

Several methods are available to study cellular internalization of vari-ous substances [36]. A few of the techniques will be discussed here.

Fluorescence-based methods

Fluorescence occurs when a molecule emits light after being excited with a photon of higher frequency (i.e. lower wavelength). Fluores-cence occurs over wide wavelength spectra but each fluorophore has a distinct absorption and emission spectra, with either broad or narrow peaks where some overlap while others are completely separated. Several other intrinsic properties of the fluorophores have led to the wide variety of commercially available fluorophores to choose from. Fluorescence quantum yield is a measure of the efficiency of the fluo-rescence process, i.e. the ratio of emitted and absorbed photons. High quantum yield for fluorophores, for example, help to increase the sig-

9

nal to noise ratio in experiments. Stability of the fluorophores is al-ways a concern. Some fluorophores have shown considerable photob-leaching, which is normally unwanted under experimental conditions but can be a preferred quality, for example in photobleaching studies [37]. Other features, like pH dependence can also be devastating in some methods while crucial in others. Autofluorescence from intracel-lular proteins (mainly flavinoids, which are excitable below 500 nm) and/or extracellular solvents can give additional concerns due to lo-wered signal to noise ratio. Autofluorescence can therefore be avoided by choosing fluorophores that operate at higher wavelengths, but other means of action are also possible [38].

Fluorophores are frequently used as tags for cellular experiments, either to visualize the localization of fluorescently labeled delivery vector or to quantify the amount of internalized delivery vector. Quan-tification can be assessed with, for example, a regular fluorometer or with fluorescence-activated cell sorter, which sorts cells based on their fluorescent characteristics. Fluorescently labeled compounds can in principle reside anywhere within cells, in cell membranes or com-partments. Quantification of internalized fluorescent compounds can therefore be problematic since it does not discriminate between fluo-rophores retained in the PM, endosomally entrapped fluorophores, cytosolic or nuclear fluorophores. Different routes have been taken to minimize and circumvent these factors. Extracellular degradation of a delivery vector can minimize molecules stuck in the PM. This has been extensively used when quantifying peptide vehicles [39]. Similar results of evaluation of intracellular fluorophore signal should be reached with extracellular quenching of fluorescence [40]. Subcellular fractionation approach, i.e. separation of subcellular components by series of centrifugations, could certainly be used in order to quantify the intracellular distribution of fluorophore-labeled compounds in dif-ferent organelles. However, this method is laborious and time con-suming and therefore not often used. Confocal microscopy has proven useful when exact intracellular location of the fluorescently labeled compounds is preferred, and is often used in conjunction with fluoro-metric methods [41]. Confocal microscopy can on the other hand not provide a quantitative measure of fluorescence.

A disadvantage with these fluorometric assays is that they are only applicable as end-point studies and cannot be used to evaluate real-time kinetics.

Some of the problems mentioned above can be avoided by adding a quenching molecule to the fluorophore labeled substance. If a quench-ing molecule is in close proximity to the fluorophore (usually less than 10 nm), it receives the excitation energy from the fluorophore before the probe is able to fluoresce [41]. Thereby, no fluorescence can be

10

detected upon excitation. This process is called fluorescence reson-ance energy transfer (FRET) and can occur if the absorption spectrum of the acceptor overlaps the emission spectrum of the donor. If, on the other hand, these two molecules drift apart, an increase in fluores-cence is noted. With FRET, real-time kinetics can be recorded, but only if the fluorophore and the quencher can be separated. Proteolysis is an option if proteins or peptides are the subjects of analysis [42]. Disulfide bridges are also often used when cleavable bonds are re-quired.

Mass spectroscopy

Using mass spectrometry (MS) to evaluate internalization efficacy of carrier molecules is a promising tool that has an advantage over many other methods. The compounds do not need a reporter such as a fluo-rophore tag, i.e. native compounds can be detected. In addition to quantifying the amount of internalized peptide carriers for example, MS studies can provide information on the degree of membrane bound peptides and peptide degradation, both which occurs intra- and extra-cellularly. This works well, in principle, but in order to retrieve all this information, the peptide vectors need to be separated from the ubi-quitous cellular peptides and proteins. One possibility is micropurifi-cation which has given good results in several studies [43, 44]. Anoth-er way to fish out the molecule of interest is to attach a marker to it, e.g. biotin [45, 46]. However, the MS approach has its limitations. MS measurements are merely end-point studies and do not provide infor-mation on intracellular localization unless they are combined with subcellular fractionation [47].

Luminescence and functional assays

Several organisms, such as fireflies, possess a phenomenon called bioluminescence, where chemical energy is converted into light. This occurs when the luciferase enzyme catalyzes the conversion of D-luciferin substrate into oxyluciferin and a photon in the presence of ATP, oxygen and magnesium ions. The firefly luminescence generates light in the 530–640 nm range, has high quantum yield, and low back-ground (low signal to noise ratio) because there is no need for excita-tion light that can excite unwanted molecules (which then emit light) in the experimental zone [48, 49]. Luminescence is therefore often a preferred choice over fluorescence. Indeed, the firefly luciferase is a widely used reporter enzyme in biochemical assays [49]. The lucife-rin-luciferase reaction is a multi-step process that can be described as follows:

11

Functional assays are great tools to explore internalization because these assays only give response if the substrates end up in the right intracellular compartment. Thus, these methods have a clear advan-tage over techniques using marker molecules, which cannot discrimi-nate between intracellular localization. It should though be kept in mind that functional assays with positive readout are usually more suitable than assays with negative readout.

Two methods have been developed that combine the luciferin-luciferase system and biological functionality.

A so-called splice correction assay makes use of HeLa pLuc 705 cells that have been stably transfected with a faulty luciferase plasmid [50]. The luciferase plasmid sequence contains an intron from β-globin mRNA that causes an aberrant splicing of the luciferase mRNA, which results in an inactive luciferase protein. Correction of this aberrant splicing is, though, possible by introducing into cells a specific antisense oligonucleotide (asON) that masks the aberrant splice site on β-globin intron. By attaching the asON to a vector mole-cule, the splice correction assay can be used to evaluate the efficacy of vectors [51].

A more recent assay that also relies on luciferin-luciferase reaction utilizes luciferin as a cargo molecule and luciferase transfected cells [52]. Both assays give positive biological response, but are otherwise very different. The splice correction assay is only capable of giving end-point results since the cells need to be lysed before evaluation, but real-time kinetics is measurable with the luciferin cargo assay. Fur-thermore, the splice correction assay gives only a response if the asON reaches the nucleus, while readout for the latter assay occurs in the cytosol where the luciferase enzyme resides. The cargos for both as-says are commonly attached via a disulfide bridge, and in principle, any carrier should work. Furthermore, the positive readout from the splice correction and luciferin carrier assays minimizes biased effects due to for example cell toxicity, which functional assays with down-regulation of cell proliferation do not.

12

Cell-penetrating peptides

A brief history

In 1988, trans-activator of transcription (Tat) from the human immu-nodeficiency virus type 1 (HIV-1) protein was reported to traverse cells. Shortly after, in 1991, a transcription factor from Drosophila’s Antennapedia homeodomain protein was also shown to possess trans-location ability [53]. This lead to the discovery of sequences within the proteins which were responsible for their translocation, so called protein transduction domains (PTDs) or cell-penetrating peptides (CPPs). These specific peptide sequences from segments 48–60 in Tat protein and 43–58 Antennapedia protein are commonly called Tat and penetratin (or pAntp), respectively [54, 55].

Now a snowball started to roll and a new field was established, which focused on finding more peptide sequences with cell penetrat-ing properties and the ability to carry various cargo molecules into cells. This led to the next generation of CPPs, i.e. synthesized peptides that were predicted to have cell-penetrating properties and/or chimeric peptides that contained parts from proteins with additional features such as nuclear localization signal (NLS) or endosomal escaping property. CPPs like transportan [56], model amphipathic peptide (MAP) [57], MPG-β [58], Pep-1 [59], polyarginine [60], and pVEC [44] can be mentioned in this context.

A small setback hit the field with the discovery of intracellular re-localization of CPPs upon cell fixation, thereby jeopardizing previous data describing their penetration ability [39, 61]. This, however, led to new experimental approaches that avoided fixation, such as functional assays or measurements of real-time uptake, which only strengthened the field. Binding of positively charged peptides to plastic and glass surfaces was also awakening of possible overestimation of the effica-cy of CPPs, at least at nM concentration [62].

Today, hundreds of peptides have joined the CPP category and extensive experminents have been carried out within the CPP field for years (for recent reviews see [63, 64]). Hence, the definition of CPPs has changed a little bit over the years. Nowadays, CPPs are thought to be short cationic and/or amphipathic peptides that are able to internal-ize cells and to promote internalization of cargos. Generally, endocytosis seems to be the main route here, but the originally proposed direct translocation mechanism is still on the table [63, 65, 66]. Both mechanisms are even sometimes thought to work simultaniously. But even though the mechanism of entry for many CPPs is still debated, their cellular internalization is not. Only more

13

research will help to solve this important issue which is the basis for a peptide specificity and cargo delivery. In context with this, structural features of CPPs, their cytotoxicity, internalization mechanisms and the influence of cargos are of concern and interest and will therefore be adressed here.

Structural features

CPPs do not display a common structure. Some are random coils in hydrophobic and hydrophilic environment while others are fully or partially stable α-helical or β-sheet structures. Structural diversity of CPPs is worth to consider and evaluate because this could turn out to be the key to the cell-penetrating ability of CPPs or perhaps explain their carrier function [67]. A moderate hydrophilicity could help to concentrate the peptides on the polar cell membrane and a minimal hydrophobicity could be needed to traverse the lipid interior of the cell membrane. Too much hydrophobic property might retain the peptides in the lipid environment and could give solubility problems. The line between these two qualities might be thin and difficult to predict. Even though the secondary structure of peptides is an outcome based on their primary sequence, modeled structures deviate sometimes too much from experimental structures to be fully reliable. It is therefore still necessary to carry out measurements in distinct conditions to gather structural information. However, it should be kept in mind that the nature of the solvent and other environmental factors, which the experiments are carried out in, can be crucial for the secondary structure findings [68].

Tat has been shown to be unstructured in PBS buffer [69, 70], however, left-handed 310-helical structure (also known as PPII helix) has also been observed in Tris buffer, SDS micelle [71], and in the presence of lipids [69]. In the 310-helix the hydrophobic amino acid residues are faced on one side along the helix while the charged residues are on another side [71]. This finding may not be surprising, unfolded proteins are thought to be, and often mistaken for, random coils but several of them can adopt PPII helical structure [72-74]. Polar amino acids are favored over non-polar in PPII helices, but proline residues are predominant [74]. Additionally, the majority of PPII helices are only composed of 4–5 residues and the prevalence of prolines is 0–2 residues per helix. These critera are met in Tat, which contains two prolines and is highly cationic (Table 4).

Only limited structural observations are available for Arg9, but it has been shown to be unordered in buffer solution [75]. Arg9 is deduced from Tat peptide and, hence, expected to possess similar structural features.

14

Penetratin has a poor self-stabilizing ability, which results in a highly polymorphic peptide with an ability to change structural state. Penetratin is generally a random coil in water solutions but rearranges into α-helix and β-sheet depending on the environmental conditions [67, 76, 77]. α-helical structure is sometimes favoured at low peptide/lipid ratio while higher ratio may result in aggregation of negatively charged liposomes and a conformational switch of penetratin to β-sheet [77-80]. Electrostatic forces are likely the explanation for the lipid binding [79].

MAP has been reported to be a random coil in buffer but changes its conformation to an α-helix in the presence of lipids [81]. Also, various lipid/peptide ratios (10–230) do not contribute to further conformational change from α-helix to β-sheet as shown to occur in other publications [82, 83]. This induction of β-sheet formation increased with higher peptide/lipid ratio and was attributed to the change of peptide orientation from being surface bound to being tilted in the lipid bilayer [82]. This plasticity ability might explain the membrane perturbing effect and cell-penetrating function for MAP.

The structure of TP10 has not been determined, at least not directly. However, its parent peptide, transportan, has been extensively studied. Transportan is reported to be unstructured in phosphate buffer (or par-tially α-helical) and to adopt α-helical conformation in presence of lipids [77, 79, 84]. No conformational change from α-helix to β-sheet has been observed, even for lipid/peptide ratio ranging from 10 to 100 [77, 79]. The helical structure is pronounced in the mastoparan part of transportan, i.e. the C-terminus, but the N-terminus is less structured although an α-helix has been detected [84, 85]. Furthermore, interac-tion of TP10 with phospholipids is suggested to be independent of the lipid charge and rather controlled by hydrophobic association [79]. Since TP10 is a truncated form of transportan with deletion of 6 resi-dues in the N-terminus [86], it is highly likely that TP10 behaves simi-larly as the mastoparan part.

MPG-β (also known as Pβ or MPG) and MPG-α (also known as Pα) have a similar primary sequence but show distinct difference in struc-

Figure 1. An example of peptides with α-helical and β-sheet (antiparallel) conforma-tions; structures of transportan (PDB code 1SMZ) [85] and the antimicrobial pep-tide PG-1 (PDB code 1SMZ) [87], respectively, determined by NMR.

15

Table 2. Structural diversity of CPPs in various environments

Experimental data Structurea Ref.

Tat

CD in PBS buffer rc [69, 70]b

CD in Tris buffer (very low ionic strength) and SDS

lefthanded 310-helix (also known as PPII helix)

[71]

CD in TFE rc and partially α-helix [69, 71]

CD in SDS, POPG/POPC (25:75) rc [69, 70]

CD in LPC PPII helix [69]

Arg9

CD in HEPES buffer rc [75]

Penetratin

CD in PBS and phosphate buffers rc [69, 78]

CD in phosphate buffer, DMPC mainly rc and partially β-sheet [77, 79]

NMR in TFE non-ideal helix (residues 4–12) that looks like a 310 helix with β-turns at both ends

[88]

NMR in SDS α-helix (residues 3–9) [80]

CD in SDS partially helical [40, 79]

CD in TFE, SDS, POPG/POPC (70/30), POPG/POPC (30/70) (L/P=100), LPC

rc and partially α-helix [69, 77, 80]

CD in DOPG α-helix at high L/P ratio [78]

CD in DOPG (L/P=8), DMPG (L/P=10), POPG (L/P=100)

antiparallel β-sheet [77-79]

MAP

CD in Tris buffer rc [81]

CD in TFE α-helix [81]

CD in POPG (L/P=10–230), POPC (L/P=10–500, POPG/POPC (1/3)

α-helix [81]

IRRAS on air/water interface α-helix at low peptide concentration (i.e. large surface area per peptide) and antiparallel β-sheet at high concentra-tion

[83]

NMR in DMPC conformational change from α-helix to β-sheet at L/P=156 (lowered L/P ratio increased β-sheet content)

[82]

OCD in DMPC α-helix at L/P=100 and β-sheet at L/P=15

[82]

MPG-β

CD in water and phosphate buffer rc [89]

CD in SDS and TFE α-helix [58, 89]

FTIR in DOPC, DOPG, DPPC, DPPG β-sheet [89]

CD in PBS, DOPG β-sheet [58]

16

Table 2. (continued)

Experimental data Structurea Ref.

MPG-α

CD in water and phosphate buffer partially α-helix at high concentration [89]

FTIR in DOPC, DOPG, DPPC, DPPG α-helix [89]

Pep-1

CD in water rc or poorly ordered structure at 100 µM concentration but partially α -helical at 1 mM concentration

[90]

CD in SDS partially helical [90]

NMR in water at 1 mM concentration α-helix (4–13) [90]

NMR in SDS α-helix (4–13) with a 310 helix at N-terminus

[90]

CD in DOPC, DOPG, DOPC/DOPG (80/20), L/P = 7

α-helix [90]

FTIR in DOPC, DOPG, L/P = 20 α-helix and 310 helix [90]

CD in water, POPC and POPC/Chol mainly rc but partially α-helix at 69 µM concentration

[91]

CADY

CD in water rc [92]

CD in DOPC, DOPG α-helix [92, 93] a The main secondary structure is reported if not otherwise stated. rc denotes random coil. L/P denotes Lipid/Peptide ratio. b Tat sequence used in ref. [69] was YGRKKRRQRRRG-NH2.

tural plasticity where MPG-β adopts β-sheet structure and MPG-α forms an α-helix in the presence of phospholipids [89]. MPG-α is un-structured in water but remains helical in all other environments, which is in agreement with the predicted characteristics of this peptide sequence. The structure of MPG-β is more variable and is highly de-pendent on the environment. MPG-β is random coil in water but changes the conformation to α-helix in trifluoroethanol (TFE) or SDS and β-sheet in the presence of various phospholipids [58, 89]

Pep-1 behaves differently from other CPPs by adopting an α-helical structure with increasing concentration [90]. NMR studies reveal that the helical structure extends from residue 4–13 in water and residue 1–13 in SDS micelles. Phospholipids induce an α-helical conforma-tion in Pep-1 and the hydrophobic interaction between them seems to be a result of Trp residues that align on one side.

CADY peptide is a random coil in water and adopts α-helical con-formation in the presence of lipids as predicted from its sequence [92, 93].

The structural conformation of penetratin is dependent on environ-mental factors and lipid/peptide ratio. However, since penetratin is the most studied CPP from Table 2 (at least with regard to lipid interac-tions), it is not excluded that other CPPs may behave in a similar

17

manner. MAP shows the same trend as penetratin; α-helical conforma-tion at low peptide/lipid concentration but β-sheet at high concentra-tion, an indication of aggregation [82]. Similar conformational transi-tion has been noticed for transmembrane domains of SNARE proteins, and several parameters were found to influence this, e.g. lipid struc-ture, peptide/lipid ratio, membrane fluidity, and peptide length. A possible explanation given here is that the repulsion of dipole mo-ments of the α-helices at high peptide concentration drives the con-formational change to β-sheet, resulting in a termination of the dipole [94]. The same assumption could apply to CPPs.

It should be mentioned that structural data for pVEC, M918, EB1, and TP10 are only available in paper II.

Cytotoxicity

Even though all CPPs do not seem to take the same internalization route, they are thought to share common initial steps. Electrostatic interaction with glycosaminoglycans (GAGs) on the cell membrane and phosphogroups of the lipid heads concentrate the peptides close or onto the PM [70, 95, 96]. CPPs that possess enough hydrophobicity may then be partially or fully embedded in the hydrophobic part of the lipid membrane. Structural conformational change of the peptides can occur in the vicinity of the lipids or in the lipid layer, which may be the main reason for the membrane perturbing ability of the CPPs.

Certain CPPs bear some resemblance to antimicrobial peptides, which are cationic, amphipathic, and are able to adopt a secondary structure. Antimicrobial peptides are surface-active molecules (surfac-tants) that act in a membrane-lytic way by forming pores or by disin-tegrating the membranes. Acidic lipids, which are particularly abun-dant in bacterial membranes, are especially targeted by these peptides [30, 35]. Nonetheless, a general consensus of CPPs is that they are non-toxic or act in a mild way even though many CPPs have shown considerable antimicrobial activity [97, 98] or pore formation [99]. Interaction with negatively charged lipids is also favored for many CPPs [32, 81, 92].

Like with internalization studies, measurement of cytotoxicity is af-fected by the experimental conditions. Each cell line has its unique properties and expresses special proteins in the PM, which affects the cell’s resistance to alien substances. Several techniques are available to measure cytotoxicity (see chapter 32 in ref. [100]). Many approach the short term membrane integrity, but some estimate long term ef-fects on the cells. A number of studies have also been directed to-wards disturbance of artificial lipid membranes, which in many cases show higher effect on membrane integrity than studies on biological

18

membranes [21, 81, 101, 102]. This difference can be attributed to the membrane repair response (MRR), which is the ability of cells to re-pair the PM upon local calcium ion influx due to membrane disrup-tion. The MRR mechanism acts within seconds and induces the fusion of intracellular vesicles (especially lysosomes) with the PM [103, 104]. Thus, the MRR could mask real membrane disturbing effect exerted by CPPs, leading to underestimation of membrane disturbance with methods that measure membrane integrity [104].

Production of peptide fragments due to intra- and extracellular pep-tide degradation [105] could also contribute to cytotoxicity. Further-more, the influence of cargos on cytotoxicity has been demonstrated in numerous articles [102, 106].

By the abovementioned reasons, it is apparent that CPPs affect cells directly or indirectly through peptide fragments or cargos. Minor membrane disturbance may be experienced which may not be detected with commonly used cytotoxicity assays.

Internalization mechanisms

The proposed internalization mechanism for CPPs has changed during the years. Direct translocation was first proposed since the internaliza-tion occurred in an energy-independent way and also with all D-amino acid residues, indicating that chiral receptors were not necessary for uptake [60, 107-109].

Lately, however, various endocytic routes have been pointed out as the main means of internalization but no consensus in internalization mechanism of CPPs has been reached. CPPs vary in size, structure, and amino acid composition and it is therefore highly unlikely that all CPPs use exactly the same mechanisms of cellular entry. Discrepan-cies in uptake routes of a particular CPP are though concerning. This can partially be attributed to use of various cell lines, which all have their unique protein and lipid contents. Divergent experimental setup and the use of different cargos could also explain a large deal. Lack of comprehensive understanding and characterization of endocytic path-ways is also a part of the cause [19].

Amino acid composition of CPPs is of great interest, especially since the common denominator with CPPs is their high content of positively charged amino acids. Arginine-rich peptides have been im-plicated to be better cell-penetrating peptides than lysine-, histidine- and ornithine-rich peptides [109]. The difference in uptake was attri-buted to the unique bidentate guanidine group on arginine, which was supported by abolished uptake of polycitrulline peptides [109].

19

Figure 2. Chemical structures of the basic amino acids arginine, citrulline, lysine, orthinine, and histidine at physiological pH.

As mentioned earlier, before entering cells, CPPs first need to interact with the cell surface, i.e. the extracellularly attached sugars, protrud-ing proteins, and/or phospholipids. GAGs, particularly heparan sul-fates, have gained special attention in this regard since they have been shown to bind to CPPs and strongly influence their internalization [19, 95, 96]. CPPs bind even more strongly to GAGs than to negatively charged phospholipids [70]. The GAGs are tethered to two families of membrane proteins; syndecans, which are transmembrane signal transducing proteins with a cytosolic domain that can be phosphory-lated, and glypicans, which are GPI-APs [19, 110]. The interaction of CPPs and GAGs can therefore lead to complexing of the GAGs and rearrangement of actin filaments, which are important in some endo-cytic mechanisms [19, 110]. Indeed, MPG-β, MPG-α, and Tat have been demonstrated to induce actin network remodeling by increasing the activity of Rac1 GTPase, which regulates lamellipodia formation [111]. Furthermore, a recent study on Tat, penetratin and Arg8, has demonstrated that CPP uptake is mediated by syndecans (especially syndecan-4) or other PGs, and is dependent on protein kinase C alpha (PKCα) and ATP [18]. Glypican-bound CPP complexes could, on the other hand, activate endocytosis by interacting with cell-surface pro-teins [19].

It should be noted that not all CPPs necessarily depend on GAGs. This has been shown for M918 where heparinase III treatment in-creased the internalization [21].

Internalization mechanisms of CPPs have also been noticed to be concentration- and cargo-dependent. Most of those studies show en-docytosis-dependent internalization to take place at low peptide con-centrations or when large cargos are attached to the peptides, while direct translocation is implicated with high concentration or small car-

20

gos [15, 16, 98, 112-114]. The concentration thresholds are likely to be cell-type dependent and vary with peptides. Translocation at low concentration and combination of translocation and endocytosis at high concentration has also been noted [46, 114]. Interestingly, inhibi-tion of endocytic mechanisms has been seen to increase the internali-zation of TP10, Tat and Arg9, indicating that inhibition of one endo-cytic route can induce another internalization pathway [15, 21].

When assessing the internalization efficacy of CPPs or when de-signing new CPPs, it is good to keep in mind that by nature, peptides are susceptible to both extracellular and intracellular degradation which can affect dramatically the internalization efficacy of a particu-lar peptide. Peptide degradation is difficult to predict and has been demonstrated to be highly peptide- and cell line-dependent; ranging from completely intact to fully degraded peptides [45, 46, 98, 105, 115].

Internalization mechanisms of a selection of CPPs are summarized in Table 3 and illustrations of proposed internalization mechanisms for various CPPs can be viewed in numerous reports [17, 64, 99, 111]. However, many of the proposed internalization mechanisms for CPPs are results from the use of endocytosis inhibitors and/or marker mole-cules. Even though endocytosis inhibitors are great tools that can give good insight into cellular function, they seldom fully block a distinct endocytic mechanism and may even act on more than one mechanism simultaneously [15-17]. Marker molecules can also lack specificity [17].

Cargo delivery

Experiments have demonstrated that CPPs are potent carrier tools that could be used to bring drug molecules into cells [63]. Several studies have reported evaluations on CPP internalization. Most of these stu-dies are based either on a reporter group that can be directly measured or a biologically active cargo [21, 47]. The cargos can be linked with a covalent bond or non-covalent complexes held together via electros-tatic and/or hydrophobic interaction [102, 116].

The influence of the cargo for the internalization of the CPPs has been reported in numerous articles where both the efficacy of the CPP and the mechanism of entry can be affected [69, 102, 117]. These me-chanism-transitions can be attributed to conformational change in the structure of the CPPs due to the attached cargo, as has been shown for Tat [69].

A few examples of cargos that have been internalized with aid from CPPs can be viewed in Table 3.

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Table 3. Internalization mechanisms of a selection of CPPs and cargo-CPPs.

Cargo Mechanism Ref.

Tat

- ATP-dependent, endocytic process (either mediated by SDCs or other PGs), with partial macropinocytosis

Rac1 GTPase mediated actin network remodelling

[18]

[111]

PNA Predominantly endocytic pathways (fluid-phase is in-volved)

[51]

Fluorescein Chloropramazine-sensitive pathway (peptide concentration dependent), macropinocytosis, and caveolae/lipid-raft-mediated endocytosis. Above a certain concentration threshold, the internalization occurs through nonendocytic pathway.

[15]

Biotin Translocation and endocytosis. Reduced GAGs content reduced endocytosis. Fluid-phase endocytosis not in-volved.

[46]

Arg9

- (Arg8 used, not Arg9)

ATP-dependent, endocytic process (either mediated by SDCs or other PGs), with partial macropinocytosis

[18]

Fluorescein Chloropramazine-sensitive pathway (peptide concentration dependent), macropinocytosis, caveolae/lipid-raft-mediated endocytosis. Above a certain concentration threshold, the internalization occurs through nonendocytic pathway.

[15]

Biotin Translocation and endocytosis. Reduced GAGs content reduced endocytosis. Fluid-phase endocytosis not in-volved.

[46]

Penetratin

- ATP-dependent, endocytotic process (either mediated by SDCs or other PGs), with partial macropinocytosis

[18]

PNA Predominantly endocytic pathways (fluid-phase is in-volved)

Macropinocytosis (not clathrin-mediated endocytosis)

[21, 51]

Fluorescein Clathrin-mediated endocytosis and macropinocytosis. Above a certain concentration threshold, the internaliza-tion occurs through nonendocytic pathway.

[15]

Biotin Translocation and endocytosis. Reduced GAGs content reduced endocytosis. Fluid-phase endocytosis not in-volved.

[46]

M918

PNA Mainly macropinocytosis but also clathrin-mediated endo-cytosis to some extent

[21]

TP10

PNA Clathrin-mediated endocytosis (not macropinocytosis) [21]

Protein (avidin, neutravidin)

Macropinocytosis, clathrin- or caveolae-mediated endocy-tosis, and clathrin- and caveolin-independent endocytosis. Flotillin-mediated pathway is not utilized.

[22, 118]

MPG-β

- Rac1 GTPase mediated actin network remodelling [111]

MPG-α

- Rac1 GTPase mediated actin network remodelling [111]

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Aims of the study

The main goal of this thesis was a comprehensive study on several well established cell-penetrating peptides to facilitate comparison of their properties. Specific aims for each paper are listed below.

Paper I Study CPP-induced cell-membrane toxicity with different membrane leakage assays.

Paper II Determine the secondary structures of a number of CPPs in different environments and their affinity for phospholipid membranes. Paper III Improve the synthesis of a luciferin-linker and introduce a new lucife-rin-linker for studies of CPP internalization mechanisms. Paper IV Assess the internalization kinetics of several luciferin-CPP conjugates using a functional luciferin-luciferase assay.

23

Methodological considerations

The methods and materials used in this thesis are described in each paper. In this section some theoretical and practical aspects of the me-thods will be discussed and the protocols briefly explained.

Selection of CPPs and CPP-conjugates.

In this thesis, several well-characterized and frequently used CPPs have been utilized in comprehensive studies on their cytotoxicity, membrane interaction, and cellular internalization kinetics (Table 4).

In paper I, cell-membrane toxicity was established for five well-documented CPPs (penetratin, Tat, pVEC, MAP, and TP10) on three cell lines. The effect of the peptides was studied on the leakage of lactate dehydrogenase (LDH) and on the fluorescence of PM potentiometric dye bis-oxonol. The hemolytic effect on bovine erythrocytes for these five CPPs was investigated as well.

In paper II, a comparative analysis of the physico-chemical properties of several CPPs (all peptides in Table 4) was used in conjuction with their ability to enter cells (data taken from the literature) in order to catagorize the peptides into “biophysical” sub-groups. In this comprehensive study, the alteration in the peptides structural state was assessed by circular dichroism (CD) spectroscopy. The affinity of the CPPs for model membranes, i.e. the interaction and insertion into phospholipids membranes (consisting of DOPC, DOPG, SM, and Chol), was evaluated with surface physic methods.

Paper III contains improved synthesis of previously published luciferin-linker as well as the synthesis of a new luciferin-linker. The conjugation of three CPPs (TP10, pVEC, and M918) with the luciferin-linkers is also demonstrated.

In paper IV, the comparison of internalization kinetics of eight luci-ferin-CPP conjugates is assessed using the luciferin releasable assay in order to shed light on the uptake mechanism of these CPPs. Here, the luciferin-CPP conjugate has a disulfide bridged cargo that becomes active upon reduction in the cytosolic environment. Cytosolic lucife-rase then converts the free luciferin to oxyluciferin and light.

24

Table 4. Peptides studied in this thesis

Peptide Sequence Origin Ref.

Protein derived

Tat GRKKRRQRRRPPQ-NH2 HIV-1 TAT transactiva-tor (48–60)

[54]

Penetratin RQIKIWFQNRRMKWKK-NH2 Antennapedia homeodomain (43–58)

[55]

pVEC LLIILRRRIRKQAHAHSK-NH2 Murine vascular endothelial cadherin (615–632)

[44]

Retro-

pVEC

KSHAHAQKRIRRRLIILL-NH2 Reversed sequence of pVEC

[44]

M918 MVTVLFRRLRIRRACGPPRVRV-NH2

p14ARF (1–22) [20]

M1073

or

[Ser15]M918

MVTVLFRRLRIRRASGPPRVRV-NH2 [Ser15]p14ARF (1–22) [20]

Designed/Chimeric

EB1 LIRLWSHLIHIWFQNRRLKWKKK-NH2

Secondary amphipathic peptide derived from pe-netratin

[119]

TP10 AGYLLGKINLKALAALAKKIL-NH2 Chimeric peptide of ga-lanin(7–12) and masto-paran

[86]

MAP KLALKLALKALKAALKLA-NH2 Model amphipathic pep-tide (secondary amphipathic peptide)

[57]

Arg9 RRRRRRRRR-NH2 Arginine rich peptide [109]

MPG-β GALFLGFLGAAGSTMGAWSQPKKKRKV-Cyaa

Primary amphipathic peptide with hydrophobic domain (derived from HIV gp41) and NLS

[58]

MPG-α GALFLAFLAAALSLMGLWSQPKKKRKV-Cyaa

Primary amphipathic peptide derived from MPG-β

[89]

Pep-1 KETWWETWWTEWSQPKKKRKV-Cyaa

Primary amphipathic peptide with Trp rich hydrophobic motif and NLS

[59]

CADY GLWRALWRLLRSLWRLLWKA-Cyaa Secondary amphipathic peptide with distinct sides of positively charged, Trp rich, and hydrophobic residues

[93]

YDEGE YDEEGGGE-NH2 Anionic peptide which does not possess cell-penetrating properties

[20]

Numbers in parenthesis refer to the segment of the protein where the peptide is derived from. NLS, nuclear localization signal. a Peptides containing cysteamidated C-terminus.

25

Peptide synthesis

The first synthesized peptide saw daylight in 1901 when Emil Fischer successfully prepared the dipeptide glycyl-glycine. Peptide synthesis progressed over the years with introduction of amino protecting groups, side-chain protecting groups and coupling reagents. The labo-rious and time-consuming isolations of intermediates between each amino acid coupling as well as solubility problems set some restric-tions to the solvent peptide synthesis [120]. It was therefore a reason for joy when in 1963, Bruce Merrifield published the solid phase pep-tides synthesis (SPPS) method, a pioneering work that earned him the Nobel prize later on (for review on SPPS, see ref. [121]). In SPPS, the peptide chain is assembled in a stepwise manner on a polymeric solid support in a C→N terminal direction with repeating cycles of coupling and deprotection of α-amino protected amino acids. Due to the solid phase (the resin), any remaining reagents, which are generally in an excess, can easily be removed by washing and filtration. This en-hances the reaction speed and decreases the total synthesis time consi-derably compared to solution synthesis. The two predominant strate-gies used in SPPS today are based on the tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) α-amino protecting groups. The main difference between these two systems lies in the chemicals used for deprotecting the α-amino group and cleaving the peptide from the solid support (and concurrently the side-chain pro-tecting groups). The t-Boc chemistry is a bit harsher with removal of the t-Boc group with trifluoroacetic acid (TFA) and cleavage of the peptide from the resin with hydrogen fluoride (HF) acid while piperi-dine base and TFA, respectively, are used in the Fmoc method.

30 amino acid long peptides are normally not a difficult task (de-pending on the amino acid sequence of course) with SPPS and pep-tides containing over 50 amino acids have been synthesized. If longer peptides or even proteins are required, an alternative method, such as the native chemical ligation, can be used in conjunction.

All peptides presented in this thesis were synthesized with SPPS on an automated peptide synthesizer (Applied Biosystems model 431A (paper I) or model 433A (papers II–IV), USA). t-Boc chemistry was applied for all peptides where amino acids were coupled on p-methylbenzylhydrylamine (MBHA) resin as hydroxybenzotriazole (HOBt) esters by using N,N′-dicyclohexylcarbodiimide (DCC) to gen-erate C-terminally amidated peptides.

Manual coupling (paper V) of cysteine (Cys) residues to the ε-amino group on Lys

7 of TP10 was achieved with HOBt, 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and N,N'-diisopropylethylamine (DIEA) activation.

26

All peptides were cleaved from the resin for 1 h at 4°C under anhydr-ous HF supplemented with p-cresol or p-cresol/p-thiocresol scaven-gers. The role of these scavengers is to trap reactive carbocations and thereby prevent side reactions with the amino acids side chains. p-Thiocresol prevents alkylation of methionine and Cys, and p-cresol protects other amino acids.

The peptides were purified with C-18 reverse-phase high-performance liquid chromatography (RP-HPLC) columns (Supelco

®,

Sigma-Aldrich, MO, USA), utilizing Dionex HPLC system with ace-tonitrile (0.1% TFA) and water (0.1% TFA) solvents.

Molecular weights of the peptides were analyzed on Voyager-DE STR Biospectrometry Workstation (MALDI-TOF by Applied Biosys-tems, Foster City, CA) (paper I) or prOTOF 2000 MALDI O-TOF (Perkin Elmer, Sweden) (papers II–IV) mass spectrometer in α-cyano-4-hydroxycinnamic acid (CHCA) matrix.

Selection of a cargo and coupling to peptides (papers III and IV)

Selection of a cargo

In paper IV a luciferin releasable technique was applied when measur-ing internalization rate of CPP-mediated cargo uptake. This method relies on a release of a cargo into cellular cytosol upon reduction of a disulfide bridge between the cargo and CPP (Figure 3). Luciferin was used as the releasable cargo based on a previously published lucife-rin/luciferase assay [52]. Luciferin, like other luminescing molecules, has an intrinsic light-producing property and therefore does not re-quire excitation to emit light like fluorescing chemicals do. Luciferin does, on the other hand, need luciferase enzyme to be able to lumi-nesce and this is accomplished in the luciferin/luciferase assay by transfecting cells with luciferase gene. The luminescence measured in this method accounts for the luciferin that has reached the cytosol (where the luciferase enzyme is located) and corresponds thereby to the amount of cytosolic CPP as well. The advantage of luminometric assays over, for example, fluorometric methods is the higher sensitivi-ty, i.e. higher signal to noise, due to low or no background.

27

Figure 3. Upon cytosolic entry of a luciferin-CPP conjugate, a cascade of reactions occurs, which leads to an emission of a photon.

Conjugation through a disulfide bridge

Conjugation of CPPs to a cargo via a disulfide bridge is a commonly used strategy within the CPP-field. The constructs are relatively easy to prepare and the cargo is released in the reducing cytosolic environ-ment of the cells, which minimizes the influence of the CPP in down-stream mechanisms of the cargo [21, 122-124]. An obvious disadvan-tage of introducing a disulfide bond is the possibility of premature reduction, i.e. this kind of a covalent bond is fairly labile and can be reduced before reaching the target compartment. This should specially be taken into consideration when applying these conjugates in vivo since blood-circulating disulfide compounds are gradually reduced [125].

3-Nitro-2-pyridylsulfenyl (Npys) group has been widely used when preparing asymmetric disulfides; it serves as an orthogonal thiol protecting group during synthesis and acts as an activator at the same time, controling regioselective disulfide bond formation with a free thiol group [126]. Disulfide bonds can be formed with Npys over a wide pH range and the reaction can be monitored spectrophotometrically by the formation of the yellow compound 3-nitro-2-thiopyridone [126-128]. The 2-pyridylsulfenyl group has been

28

used for the same purpose as Npys [52, 123]. Even though the asym-metric reaction rate increases with increasing pH [127], the pH range below 7 may be preferred to prevent symmetrical disulfide resulting from air oxidation or mixed disulfide interchange [129, 130].

In paper IV, luciferin-linkers and Cys-containing peptides were mixed in a 1:1 or a 2:1 ratio at a final peptide concentration of 0.88 mM in either DMF or DMF/acetic acid buffer (pH 5, 50 mM) at room temperature, under nitrogen. A 30 min reaction time proved sufficient.

In paper V, luciferin-linker and Cys-CPPs were mixed in 1:1 molar ratio at 0.88 mM concentration in DMF/acetic acid buffer (pH 5, 50 mM) for 1 h under nitrogen.

All mixtures were purified on RP-HPLC and the correct molecular weight verified by MALDI-TOF.

Cell cultures

Several malignant and immortalized human cell lines were used in this thesis. The choice of cancer cells and “normal” cells in paper I was based on the increasing field of cell-selective CPPs, which especially concerns cancer-selectivity for medical application of CPPs. A widely used cell line in paper IV was chosen in order to facilitate comparison with published data.

Cancer cells (paper I)

The K562 cell line dates back to 1970 as the first human immortalised erythroleukemia cells [131]. K562 cells are non-adherent and have been widely used in differentiation and anticancer drug studies [132].

MDA-MB-231 cells are methotrexate (an antifolate) resistant hu-man breast adenocarcinoma cells, which lack the expression of re-duced folate and antifolate transporter [133]. These cells are therefore often used as models in the context of anticancer drug transport.

Endothelial cells (paper I)

The AEC cell line (SV40-immortalized human aortic endothelial cells) was used in this thesis as an in vitro culture model for normal endothelial cells.

HeLa cells (paper IV)

HeLa cells are the first human immortal cell-line, derived from cervic-al cancer tissue of Henrietta Lack in 1951. Since then, these cells have

29

been widely used in laboratories all over the world, particularly in cancer research. The popularity of HeLa cells can in part be attributed to their known robust and rapid proliferation properties. Another im-portant quality is that the massive data behind the application of HeLa cells attracts further use.

The HeLa pLuc 705 cell line [50] (paper IV) was used in this thesis to facilitate comparison of data. In our hands, the HeLa pLuc 705 cells proliferated better and were transfected to higher extent than regular HeLa cells, which were the main factors in choosing the HeLa pLuc 705 cell line over the latter. The HeLa pLuc 705 cells were transiently transfected with luciferase encoding pGL3 plasmid resulting in the expression of functional luciferase protein which is the basis of the luciferin-luciferase method used in paper IV.

Membrane leakage studies

Membrane integrity is an important element for cell viability. A pre-ferable quality of CPPs is low cellular disturbance, both extra- and intracellularly. The role of CPPs is mainly to transfer cargos into cells and if these peptides are suppose to be compatible with clinical trials, the cytotoxicity exerted by them must be fully explored. Elevated cy-totoxicity may obscure experimental results and must therefore be clarified. All the toxicity methods described in this thesis address short-term membrane disturbance.

LDH leakage (papers I and IV)

CytoTox-ONETM

Homogeneous Membrane Integrity assay (Promega, USA) (Figure 4) measures the release (or leakage) of cytosolic LDH enzyme from cells with damaged membrane. LDH catalyzes the oxi-dation of lactate to pyrovate with concomitant conversion of NAD

+ to

NADH, which subsequently reduces resazurin to a fluorescent resoru-fin with aid from diaphorase enzyme. The quantification of resorufin was performed on a fluorometer with excitation/emission wavelengths at 560/590 nm. LDH is a relatively large molecule (MW~132 kDa) and the LDH assay might therefore be suspected to be unable to detect early events in membrane integrity. However, membrane leakage measurements of the considerably smaller molecule 2-deoxy-D-[1-H

3]glucose-6-phosphate (MW~221 Da) gave identical results.

30

Figure 4. Schematic representation of chemicals and reactions involved in the Cyto-Tox-ONE

TM Homogeneous Membrane Integrity assay.

DiBAC4(3) assay (paper I)

Membrane potential arises from ionic gradients with K+, Na

+, and Cl

-

playing a pivotal role regarding PM potential in eukaryotic cells. Damaged membrane loses its ability to maintain the ionic gradients resulting in depolarization.

In paper I, the change in membrane potential after CPP application was measured with the DiBaC4(3) assay on a fluorometer with excita-tion/emission wavelengths at 494/518 nm. Bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) (Figure 5) is an anionic voltage-sensitive fluorescent probe that enters depolarized cells and binds to hydrophobic parts of proteins and membranes with enhanced fluores-cence and red spectral shift [134, 135]. DiBAC4(3) is a slow respond-ing dye that reaches a plateau of fluorescence signal in seconds or minutes after voltage change.

Figure 5. The chemical structure of DiBAC4(3), which was used to study membrane depolarization.

31

Hemolysis assay (paper I)

One way to gain access into the human body is intraveneous administration. Several intraveneous CPP studies have been reported, which calls for investigation of hemolysis effect provoked by CPPs. This was dealt with in paper I when hemoglobin leakage of bovine blood-cells after CPP treatment was quantified with absorption analysis at 540 nm.

Glutathione leakage (paper IV)

Glutathione is a cytosolic tripeptide that maintains the intracellular reducing environment. The leakage of glutathione into culture media could also have a reducing effect on extracellularly applied substances containing a disulfide bond, e.g. a cargo-containing CPP. This leakage could then lead to biased outcome; either enhanced or lowered results, depending on the experimental assay.

The disulfide conjugates in paper IV result in higher signalling when the disulfide bridge between the CPP and its cargo is broken, whether it occurs outside or inside the cells. It was therefore appropriate to view the glutathione leakage exerted by CPPs in this paper. This was done by monitoring (with absorption at 405 nm) the release of the yellow dianion 2-nitro-5-thiobenzoate (TNB

2−) which is

produced when Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB) reacts with free thiols [136]. Ellman’s reagent, introduced in 1959, is a popular reagent for spectrophotometric measurements of sulfhydryls.

Figure 6. The fluorophore and quencher pair (ANTS and DPX, respectively) used to determine leakage from liposomes.

Leakage from liposomes (paper II)

Liposomes are not identical to cell membranes; they are much simp-ler, comprised mainly of phospholipids, but can be useful tools for

32

clarification of complicated cell membrane interactions and cellular internalization.

Liposomal pore formation induced by CPPs was evaluated in paper II with a fluorometric assay. The increase in fluorescence (λex 355 nm, λem 512 nm) from the polyanionic dye 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS) was measured to correlate with the CPP-induced leakage of liposomally-encapsulated fluorophore/quencher pair ANTS/p-xylene-bis-pyridinium bromide (DPX) (Figure 6).

Figure 7. CD spectra of poly-L-lysine in α-helical, β-sheet and random coil confor-mation that are used as reference spectra when determining conformations of pep-tides measured with the CD technique. The figure is adapted from [137].

Determining peptide secondary structure (paper II)

Peptides do not always adapt single and well defined structures in solution like proteins are known to. Even though peptides can be highly flexible with random coil tails, some possess structural polymorphism, i.e. the ability to adapt different secondary structures in distinct environment.

33

In paper II, the frequently applied circular dichroism (CD) spectrosco-py was used to evaluate the secondary structure of several CPPs in water and different lipid environments. CD spectra are measured in the far-UV spectral region (190–250 nm) and with this technique it is possible to differentiate between α-helix, β-sheet, and random coil in proteins and peptides (Figure 7) [137, 138]. Since CD spectra reflect the averaged molecular population, it is not possible to locate specific secondary structures within a peptide or protein with CD spectrosco-py. CD can on the other hand display the proportion of these structures for each peptide. CD is therefore a valuable tool, especially regarding conformational changes. The CD technology, discovered in 1898, measures the difference in absorbance of left- and right-handed circularly polarized light. The CD property is therefore only exhibited by chiral and optically active substances. This criteria is met in pep-tides since all the natural amino acids are chiral (except of course gly-cine) with the chiral center located at the α-carbon.

CPP-lipid interaction (paper II)

Adsorption experiments indicate the degree of amphipathicity of sur-factants but give a greater perspective when combined with structural information, from for example CD.

In paper II, the peptide adsorption at the air-water interface and pe-netration into a phospholipids monolayer were measured by the Wil-helmy platinum plate method [35]. Affinity of the peptides for an air-water interface, i.e. hydrophobic/hydrophilic interface, was evaluated in order to assess their amphipathicity. This was done by measuring the variation of the surface pressure as a function of the peptide con-centration in the subphase with a thin platinum plate connected to a tensiometer (practically a balance). The saturating surface pressure (πsat) values, i.e. the surface pressure plateau, for the CPPs was then retrieved from the profiles because πsat is a measure of the amphipath-ic character of the surfactant, i.e. the higher the πsat, the more amphi-pathic is the surfactant. The profile led also to the determination of critical micellar concentration (CMC) for the peptides, which is the concentration at πsat. CMC is also defined as the concentration of a surfactant at which aggregates start to form in the solution, i.e. at CMC the surface is loaded with surfactant molecules and further in-crease in surfactant concentration will lead to aggregates in the solu-tion. The CMC values are important because they are taken into ac-count in the lipid penetration experiments, which were all carried out at slightly lower concentration than the CMC for each peptide. Here, variation in surface pressure (Δπ) for different initial surface pressure

34

(πi) is measured for each peptide. Finally, Δπ is plotted against πi to provide information on peptide-lipid interaction. Extrapolation to the x-axis (for Δπ=0) gives the critical pressure of insertion (CPI), i.e. the maximum surface pressure of the lipid monolayer in which a peptide can penetrate. Hence, CPI is a measure of the affinity of the peptide towards the lipid type used. Extrapolation to the y-axis (for πi=0) gives the highest surface pressure variation (Δπmax) that a peptide in-duces in a lipid monolayer, a measure of the peptide-lipid interaction. Generally, a Δπmax that is higher than the πsat (from the adsorption ex-periments) suggests a strong interaction between the peptide and the phospholipids.

Kinetic studies and evaluation of CPP internalization

The internalization of CPPs and CPP-conjugates can be monitored and quantified in order to compare their uptake and delivery efficacy. Dif-ferent methods for these tasks are available, each with its advantages and limitations.

Analysis of peptide translocation by mass spectrometry (paper I)

The quantification and intracellular localization of untagged peptides can be challenging since these molecules lack a specific label or a functional probe.

In paper I, the uptake of five unlabelled CPPs was assessed with mass spectrometry (MS) by measuring the amount of peptides in cell lysates and media after a certain incubation time. Peptide quantifica-tion was achieved with an internal standard. ZipTipC18 device was applied for the cleanup and concentration of peptide mixtures to cir-cumvent the interference from the enormous amount of intracellular proteins and peptides.

MS is essentially a qualitative method that provides the possibility of evaluating the internalization of unlabeled peptides with clues to their degradation. There are however some drawbacks to this method. Rapid degradation will obviously lead to underestimated internaliza-tion. This method can therefore not discriminate between low interna-lization and degradation of the peptides. Also, measurements of lysed cells do not differentiate between cytosolic and endosomal peptides.

Kinetic studies with luciferin-luciferase system (paper IV)

Uptake of CPPs has frequently been estimated with fluorescent car-gos. As with the MS assay above, it is not possible to discriminate

35

between cytosolic or compartmentalized CPPs. Fluorescent degrada-tion products might also be a problem for the evaluation. In functional assays, on the other hand, the cargo only gives a biological response if it is delivered to the desired intracellular location. Functional assays therefore possess a quality that fluorescence assays do not have.

One such method, the luciferin-luciferase assay [52], was used in paper IV to evaluate the delivery of luciferin into cytosol by eight CPPs. This was accomplished by measuring the luminescence stem-ming from the conversion of luciferin to oxyluciferin by the cytosolic luciferase enzyme. The luciferin-luciferase assay gives a positive rea-dout and allows for real-time kinetics measurements.

A possible flaw encountered with this method is if the disulfide bridge between the CPP and the luciferin is reduced before the conju-gate reaches the cytosol because free luciferin is capable of entering cells without any aid. This reduction would then overrate the interna-lization of the CPPs.

36

Results and discussion

The articles that this thesis is based on describe different features of CPPs. The first article focuses on cytotoxicity exerted by CPPs through PM disturbance. Measurements on cytotoxicity can also be viewed in the fourth article. In the second article, structural plasticity and phospholipid membrane interaction of CPPs were determined. The third article deals with synthesis of luciferin-linkers used in the synthesis of luciferin-CPP conjugates. In the fourth article, the deli-very kinetics of CPPs is quantified with a real-time luminescing luci-ferin-luciferase assay. Here, the main results of the four articles will be presented and discussed.

Membrane leakage caused by CPPs (paper I)

Cellular toxicity is an important issue that has to be considered when CPPs are applied to cells. Here, two malignant cells (K562 and MDA-MB-231) and immortalized cells (AEC) were used in order to examine the toxicity caused by five CPPs. This was done by measuring mem-brane disturbance with the LDH leakage assay and the DiBAC4(3) membrane depolarization assay. The findings were in principle the same for all cell lines and both assays. 10 µM Tat, pVEC and penetra-tin induced no membrane disturbance while MAP and TP10 showed up to 50% leakage. The effect of MAP and TP10 was similar within each cell line but most pronounced for the cancer cells K562 and MDA-MB-231. Unfortunately, experiments for TP10 were not in-cluded in the DiBAC4(3) assay because the peptide induced an in-crease in DiBAC4(3) fluorescence in the absence of cells.

It should be pointed out that several other membrane leakage me-thods were included during preliminary experiments; calcein AM lea-kage assay and ethidium homodimer-1 entry assay were not suitable due to unstable baseline. Interestingly, the LDH leakage assay gave identical results as the 2-deoxy-D-[1-H

3]glucose-6-phosphate leakage

assay despite a 600-fold difference of the marker molecules, which should correlate with the size of membrane pores formed due to mem-

37

brane disturbance. Since the latter assay is much more cumbersome than the former, it was decided to proceed with the LDH assay.

The hemolytic effect exerted by 50 µM CPPs was also investigated by monitoring hemoglobin release from bovine erythrocytes. The re-sults demonstrate that there is an overall low hemolytic activity pro-voked by the CPPs. Tat showed only background-level effect while other peptides showed statistically significant but low leakage.

Additionally, the quantity of cellular internalized CPPs was ana-lysed by mass spectrometry. MAP and TP10 were detected in high amounts in the intracellular fraction, while penetratin and pVEC were detected to a lesser extent. Surprisingly, Tat was undetectable in both the extracellular fraction and the lysate. A possible explanation for this is a rapid intra- and/or extracellular degradation of Tat and/or low intracellular level.

Membrane interaction and insertion of CPPs (paper II)

Whether CPPs are endocytosed or internalized into cells via direct translocation, interaction with the PM is inevitable. The structural plasticity of CPPs, i.e. their ability to change conformation, may be one of the explanations to their interaction with the cell membrane and the specific route of internalization mechanism. Hence, structural features of several CPPs were evaluated in water and in the presence of phospholipid vesicles with CD spectroscopy (see Table 2 in paper II). Further, interaction and insertion of these CPPs into phospholipid membranes was assessed with surface pressure measurements (see Table 3 in paper II).

All of the CPPs formed random coil structures in water, but pene-tratin, TP10, MAP, EB1, pVEC, and M918 might adopt a low level of secondary structure. Aggregation or auto-association of the peptides is not involved at the concentration used herein (50–150 μM).

Most of the peptides adopted a specific secondary structure when the environment was changed from water to SUV or LUV phospholi-pid vesicles. All three structures were observed however, i.e. random coil, α-helix and β-sheet, and the CPPs were classified into three sub-groups based on their secondary structure. Tat and Arg9 remained ran-dom coils in all environments (various lipids and lipid/peptide ratios) tested. On the other hand, pVEC, penetratin, and M918 adopted a β-structure in the presence of negatively charged DOPG phospholipids. Here, increased concentration of the phospholipids induced β-structure conformation. Finally, TP10, MAP and EB1 exhibited an α-helical structure in the presence of DOPG and DOPC/DOPG (80/20), which was also triggered by increased concentration of the phospholipids.

38

An aggregation of all of the helical peptides probably occurs in the presence of fully negatively charged lipids (DOPG) while isolated helices are predominant in the presence of DOPC/DOPG mixture. All of the peptides remained unstructured in the presence of DOPC zwit-terionic liposome or DOPC/sphingomyelin/cholesterol (40/40/20) LUVs. These results suggest that electrostatic interaction is the major factor in the lipid-mediated CPP conformational transitions.

The air/water interface adsorption experiments revealed that the α-helical peptides (TP10, MAP, and EB1) display more amphipathic character than the β-sheet (M918, penetratin, and pVEC) and random coil peptides (Tat and Arg9). Tat, Arg9, penetratin and pVEC did not display any interfacial properties while TP10, MAP, EB1 and M918 had clear amphipathic characteristics with the following order TP10>MAP>EB1>M918.

In the phospolipid monolayer penetration experiments, TP10, MAP, EB1, and M918 peptides were measured in the presence of DOPG and DOPC lipids. Tat, Arg9, penetratin and pVEC were left out of these experiments since they did not show any interfacial properties in the air/water experiments. The α-helical peptides (EB1, TP10, and MAP) penetrated to a greater extent into DOPC lipid monolayer than the β-sheet peptide M918. The insertion of all the peptides was higher into the negatively charged DOPG monolayer than the zwitterionic DOPC. The results indicate that EB1 interacts strongly with DOPC and DOPG lipids, and that M918 interacts strongly with DOPG. The order of interaction with DOPC was EB1>TP10>MAP>M918, and the order with DOPG was M918>TP10>EB1>MAP. These results further indi-cate that electrostatic interactions play a big role when the peptides insert into a lipid monolayer and that these four peptides should be able to insert spontaneously into biological membranes.

The structural features and parameters retrieved for the CPPs meas-ured in this article were compared to earlier studies from the same laboratory for MPG-β (denoted MPG in paper II), MPG-α, CADY and Pep-1 peptides. This was done in order to associate the structural and interaction properties of the peptides with internalization mechanisms (see Figure 6 in paper II).

Synthesis of luciferin–CPP conjugates (paper III)

The synthesis of previously published luciferin-linker [52], used to prepare luciferin-CPP conjugates, was examined in this paper. The original synthesis steps were improved, new synthesis steps were also added into the original steps, and a new approach was presented which lead to a new luciferin-linker. Steps a–c in Figure 8 illustrate the orig-

39

inal steps, which start by the reaction of hydroxyl-thiol 1 with 2,2ʹ-dithiopyridine 2. The production of intermediate disulfide product 3 was followed by flash-chromatographic purification in order to re-move the by-product mercaptopyridine 10. The modified approach to product 3 included two reaction steps (aʹ–aʹʹ) in one-pot. Product 3 was obtained in nearly quantitative yield and needed no chromato-graphic purification for subsequent reaction steps. If needed, simple washes with ammonium carbonate ((NH4)2CO3) were sufficient to remove traces of 10. Step b in Figure 8 was originally carried out at room temperature with 3:1 ratio of reactants 3 and triphosgene 4. This yielded chloroformate derivative 5, which was subsequently reacted with luciferin 6 in order to obtain the end-product luciferin-linker 7. The procedure in step b was improved substantially in this article. The original approach resulted in very unpure and low yield of the end-product 7. Lowering the reaction temperature to -10°C and changing the ratio of 3 and 4 to 1:1 resulted in very clean synthesis of 5, which could be used in subsequent steps without purification.

New luciferin-linker was synthesized (Figure 9) by simplifying the new approach in Figure 8, i.e. a new leaving group was introduced to the reducible luciferin-linker by omitting step aʹʹ. The purity of the new luciferin-linker 12 was superior to the purity of the original luci-ferin-linker 7, despite the improved approach presented in this article.

Figure 8. Synthetic route to luciferin-linker 7.

40

Figure 9. Synthetic route to luciferin-linker 12.

Both the original luciferin-linker 7 and the new luciferin-linker 12 were equally efficient in conjugating to CPPs. All the conjugation reactions were finished within 30 min and gave complete conversion into luciferin-CPP conjugates.

Internalization kinetics of luciferin-CPP conjugates (paper IV)

The luciferin-luciferase assay [52] used in this paper provides useful information on the real-time internalization kinetics of luciferin that reflects the cytosolic internalization kinetics of corresponding CPPs. This functional assay proved to be highly reproducible and sensitive enough to distinguish between different luciferin-CPP conjugates. Two major types of internalization profiles for the CPPs were ob-served; a fast exponential profile that reached a maximum within mi-nutes and a slow sigmoidal profile that peaked between 30 and 60 min. The internalization for all the peptides belonging to the slow in-ternalization category was still considerable after 2 h. Luciferin, Tat, and higher concentrations of MAP and TP10 belong to the former profile while EB1, penetratin, TP10(Cys), M918, pVEC, and lower concentrations of MAP and TP10 fall into the latter group. Luciferin is known to penetrate cell membranes [139] and it was therefore sug-gested that Tat, MAP and TP10, which all closely resemble the kinetic profile of the membrane-permeable free luciferin, might also internal-

41

ize in a similar manner. Endocytosis was a proposed internalization mechanism for the peptides from the “slow” category.

The membrane-permeable property of luciferin could put some strains on the assay if the luciferin-CPP conjugate is reduced before it enters the cytosol. This could occur if the applied CPPs would disturb the cell membrane and cause a leakage of glutathione. Extracellular sulfhydryl groups were therefore quantified with Ellman’s reagent, which revealed that extracellular reduction of disulfide bonds of 5 µM luciferin-CPP conjugates does not seem to take place in general after 30 min incubation. It is noticable that all the peptides with the fast kinetics (Tat, MAP, TP10) show detectable level of free sulfhydryl groups after 1 h incubation. However, free sulfhydryls were not detected 10 min after the application of these three peptides, i.e. within the time-frame of their maximum internalization.

Cellular toxicity, which was assessed with LDH leakage assay, was in agreement with the glutathione experiments. None of the peptide concentrations used herein (1–10 µM) compromised the membrane integrity after 35 min incubation. MAP, TP10 and EB1 were the only peptides that affected the morphology of the HeLa pLuc 705 cells. LDH leakage was therefore further evaluated for these peptides. EB1 had no influence on the membrane permeability after 2 h incubation, however, MAP and TP10 induced a mild LDH leakage (13.0 and 8.2%, respectively).

Experiments carried out in buffer were quite different from those in media. No sigmoidal profiles were detected here, but the internaliza-tion was up to 28 fold higher.

It should be noted that all the experiments were carried out at room temperature (25°C).

42

Conclusions

The findings in this thesis provide information about cytotoxicity, structural diversity, and internalization kinetic measurements of CPPs. The main conclusions of this thesis, based on findings in each of the four papers included, are described below.

Paper I Tat, penetratin, and pVEC had no or low cytotoxic effect on the stu-died cell lines. MAP and TP10, however, had more membrane per-turbing effect, which was most pronounced in malignant cells.

Paper II All the peptides tested adopted random coil structure in water but most of them adopted an α-helical or β-sheet conformation in the presence of negatively charged phospholipid vesicles. However, all of the peptides remained unstructured in zwitterionic phospholipid envi-ronment. Only a part of the CPPs adsorbed at hydrophobic/hydrophilic (air/water) interface, which reflects their amphipathic character. The order of the affinity for the lipid free interface was following: TP10>MAP>EB1>M918. The order of interaction of the CPPs with phospholipid monolayers did not correlate with their order of amphi-pathicity.

Paper III The synthesis steps of the previously published luciferin-linker were improved in this paper. The greatest change was reached by increasing the phosgene content and lowering the temperature in the phosgene reaction (step b in Figure 8) from room temperature to ca. -10°C. The improvements both increased the purity and yields of each interme-diate product as well as the end-product. Furthermore, synthesis of a new luciferin-linker, which was much cleaner than for the original luciferin-linker, was presented. Both luciferin-linkers were equally efficient in conjugation with Cys-CPPs. They both reacted completely in 30 min, yielding luciferin-CPPs in high purity.

43

Paper IV The cell-penetrating peptides tested here (Tat, MAP, TP10, EB1, penetratin, pVEC, TP10(Cys), and M918) have distinct internalization (cytosolic) kinetics. The kinetic profiles can be divided into fast and slow catagories where the former reaches maximum within minutes and the latter in 30–60 min. The internalization profiles depend also on the peptide concentrations. It was concluded that Tat, and higher concentrations of MAP and TP10 internalized with direct translocation but the other peptides mainly with endocytosis, at least at the concentrations used herein (1–10 µM).

44

Acknowledgements

When I am writing this, I am not sure I really comprehend that this long Ph.D. journey is soon over. There is a certain relief in my heart but also a big grief.

Dear Professor Ülo. Thank you so much for giving me the opportunity to become your student. I have certainly learned a lot in your group, not only science-related matters. I have realized that Ph.D. students deserve strawberries and champagne once in a while. Thank you for your generosity, and for the trust and free hands you have given me in the lab.

The administration staff: Siv, Birgitta, Ulla and Marie-Louise. Thank you all for the tremendous help during the years, it was not less the last days than in the beginning.

The teachers at Neurochemistry department: Anders, Anna, Kerstin, Mattias and Tiit. Thank you for all the talk and help through the years.

Now to my co-authors and collaborators.

Külliki, the first person whom I learned to know at Neurochemistry department. Thank you for getting me started and for all the fantastic get-to-gather outside work. And thank you for introducing me to Mårten, which I had such great discussions with.

Katri, always busy and energetic, it was so great to do all this chemistry with you, but all the talk was much more fun.

Imre, luckily your sarcasm and humor resonances with mine, which made the time I spent with you seem like on a playground.

Sébastien Deshayes, thank you for your immense tolerance for my endless questions, which you answered so extensively.

Jim Eberwine, it was a great pleasure to join your lab the summer 2007, getting to know you and your family, your crew, and of course USA. Thank you for that opportunity.

And my dearest former room mates

Mats. Well, well, well. I miss our endless talks and the time when you translated the Estonian news for me, it is much more diffucult to do it on my own.

45

Maarja. The golden girl. I wish I could clone you. It has been wonderful to have you as a room mate, a neighbor and a baby-sitter. And with your other half (Farid) by your side, I think you are complete. Thank you both so much for all the help and friendship.

Yang, you constantly surprised me with your humor. It was great to get to know you and Ling.

Kalle, you stayed to short time in the nest, but the talking during that time was though fun.

Helena M, Ulla, and Tina. You are all so wonderful, funny, and with great, positive spirit. Keep that up, the world needs it.

A lot of hugs and kisses to everybody from Neurochemistry department that I had so many laughs with during the years: Maria, Samir, Johanna, Henke, Peter J, Anders F, Karolina, Caroline, Johan, Marie, Tom, Rania, Peter G, Marie-Louise, Helena G, Rannar, Ursel, Meeri, Linda L, Pontus, Linda T, Andrés, Tom, Jessica, Anna-Lena.

And everybody else from Neurochemistry department that I did not mention, thank you for being there and making my days.

Special thanks go to Auður, Maarja, Külliki, Ruairi, Katri and Ülo for taking their time to comment and correct my thesis.

When I started playing billjard, I never imagined that we would spend so much time outside the billjard hall. Andreas, Tony, Eva, Yvonne and Henke, thank you for all the billjard lessons, but moreover all the fun elsewhere.

Our small Iceland association in Sweden: Arna & Karvel, Hrönn & Georg, Sigrún & Snævar, and all the heirs. Thank you for all the parties and other activities.

My best greetings to the Icelandic chicks in my former knitting club (yes, I can knit!). It was a fantastic time for me to recharge the batteries.

My family in Iceland: Mom, dad, Haukur (my little brother), mothers-in-law, fathers-in-law, brothers-in-law, sisters-in-law, grandmothers, grandfathers, aunts, uncles and cousins. Now I have probably counted everybody in Iceland. I do not know where to start to thank you. You have always supported me in everything I have done. Most of all I am grateful for the time you took to visit us through the years in Sweden. It was never too long and I always missed you when you walked out of the door. Our endless Skype talks also helped keeping me relatively sane.

46

Hlín & Sigga, Biggi and Gilli. You are the greatest, it is always fun

when you are around, especially with a little liquor and cards or board

games. I am so glad to see how big our family is growing; we have

managed to double ourselves, which must mean double fun ;) Hlín &

Biggi, thank you for all the great Christmas times we spent together in

Stockholm and Copenhagen.

Anna Eir and Maggý Nóa. Can anyone be luckier than me, to have

two such beautiful and smart girls. This is how god must have felt

when she created her first people.

And last but definitely not least. Aujan mín, my precious. It is partial-

ly your fault that I am graduating. You dragged me over to Sweden

and you introduced me to Ülo. You have been my biggest support

through the years. You inspire me with your wisdom. I would never

have made it so far without you. Thank you for being.

47

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