Saturated Fatty Acid Analogues of Cell-Penetrating Peptide

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Saturated fatty acid analogues of cell-penetrating peptide PepFect14: Role of fatty acid modification in complexation and delivery of splice-correcting oligonucleotides Tõnis Lehto, Luis Vasconcelos, Helerin Margus, Ricardo Figueroa, Margus Pooga, Mattias Hällbrink, and Ulo Langel Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00680 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Bioconjugate Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 2 of 29

Page 3 of 29





Page 4 of 29

Page 5 of 29

A Complexes/Hoechst

C2-14

Transmition

C22-14

B 120

N o . o f c o m p le x e s

100

N o . o f c e lls ( + /- ) N o . o f c e lls ( + )

80 60 40 20


T U

O

0

C

0 2

L

F

S

0

4

4 C

2

2

0 2

-1

-1

4

4 1

8

-1

-1

1

6 C

C

4

4

-1 4

1 C

1 C

C

4

-1 2

-1 0

1 C

C

8

-1

4

4

4

-1

C

6

-1 4

C

2 C

0

-1

-1

4

4

0

C

T o ta l c o u n t

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acylated peptide

Bioconjugate Oligonucleotide Chemistry 100

Page 6 of 29

50

0


C 0C 14 2C 14 4C 14 6C 14 8C 14 10 C -14 12 C -14 14 C -14 16 C -14 18 C -14 20 C -14 22 -1 4

1 2 3 4 5 6 7Cytoplasm

Transfection

Complex

Nucleus

Hydrophobicity

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Saturated fatty acid analogues of cell-penetrating peptide PepFect14: Role of fatty acid modification in complexation and delivery of splice-correcting oligonucleotides Tõnis Lehto,*,† Luis Vasconcelos,† Helerin Margus,‡ Ricardo Figueroa,† Margus Pooga,‡ Mattias Hällbrink,† Ülo Langel†,§ Affiliations † Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrhenius väg 16B, 10691 Stockholm, Sweden ‡ Institute of Molecular and Cell Biology, University of Tartu, Riia 23a, 51010 Tartu, Estonia § Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia *Corresponding author. E-mail address: [email protected] (Tõnis Lehto) Keywords Cell-penetrating peptide, PepFect14, fatty acid, oligonucleotide delivery, splice-correction, acylation, non-covalent complexes Abstract Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were non-covalently complexed with a splicecorrecting oligonucleotide (SCO) and tested in the HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors how the transfection efficacy of these complexes is dependent on hydrophobicity we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay a ground for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing in efficiency. Introduction Splice-correcting oligonucleotides (SCOs) are a group of antisense oligonucleotides (asONs) that specifically target and modulate the splicing of mRNA and display remarkable potential for providing potential cure for many splice mutation-relate disorders, including for example different neuromuscular disorders, such as Duchenne’s muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, due to their low stability in blood and very limited ability to cross biological membranes, their translation to the clinics has been held back due to their



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

poor bioavailability. It is accepted that for potential clinical application most of the SCOs require to be used in combination with effective delivery vectors. For the delivery of genetic material, viral vectors are undoubtedly most efficient, but they are not compatible with the delivery of short synthetic antisense oligonucleotides, such as SCOs. Hence, for SCOs, different non-viral vectors have been studied most intensely. Non-viral vectors can arise from the variety of chemical classes of molecules and are most often based on cationic lipids, polymers, dendrimers and peptides. These cationic molecules bind negatively charged nucleic acids due to electrostatic interactions and condense them into particles with varying size in the nanometer range that are able to cross the cell membrane. Cell-penetrating peptides (CPPs) are a class of short cationic and/or amphipathic peptides that are able to condense different kinds of nucleic acid cargoes into nanoparticles and facilitate their delivery both in vitro and in vivo. Alternatively, CPPs can also be used via direct covalent conjugation with cargo molecules, for example with peptide and proteins and also charge neutral oligonucleotides chemistries, making CPP platform a highly versatile group of transport vectors. To harness the potential of CPPs via nanoparticle-based approach, most of the CPPs, however, do not provide stable and active enough nanoparticles and most of the successful examples carry further chemical modifications in their structure. One type of modifications that has exhibited excellent potential in this context are different modification that render CPPs more hydrophobic. In this line, different fatty acid modifications have been used to great effect. For example, to improve the delivery efficiency of plasmid DNA, Futaki et al modified various polyarginine-based CPPs in the N-terminus of the peptides with stearic acid showed that these stearoylated polyarginines induced around 100-fold increase in transfection for octaarginine.1 Since then, several groups, including ours, have successfully used acylation by stearic acid to improve their delivery vectors.2–4 In another study Khalil and co-workers showed that the mechanism behind how stearoylation of octaarginine improves the transfection efficiency is due to increased uptake and binding of pDNA complexes to the cells.5 Furthermore, we have reported that stearoylated transportan10 (also termed PepFect3 peptide) in complex with splice-correcting oligonucleotides lead to efficient splice correction in the in vitro models, by enhancing endosomal release compared to its parent peptide.2 Later, upon optimizing the length and structure of acyl chain for PepFect3 stearoylation was found optimal for maximal delivery efficiency whereas longer acyl chains were more toxic.6 In another line of development, the peptide sequence of PepFect3 was modified, lysines were changed to ornithines and one additional charge was added, whereas the stearic acid modification was conserved, yielding a CPP named PepFect14. This peptide was shown to have superior efficiency over its parent peptide in delivering both SCO and pDNA.3,7 Furthermore, PepFect14 has been recently shown to also be able to deliver charge neutral oligonucleotides of phosphordiamidate morpholino chemistry (PMOs), further indicating the importance of the hydrophobic interactions in the CPP nanoparticle-based delivery process.8 To rationally develop efficient CPP-type carriers for splice-correcting oligonucleotide it is necessary to elucidate the mechanism behind the functional oligonucleotide delivery. As mentioned above, it has been long known that acylation of CPPs enhances their delivery efficiency. However, the current knowledge on the mechanism by which acyl chain modification of CPPs enhances their delivery efficiency has been obtained either with very narrow choice of acyl chain lengths or structurally very different fatty acid modifications. Thus it is difficult to extract the mechanism that lies behind it. To decide whether it is due to nucleic acid binding and the consequent differences in size or zeta potential of the peptide/oligonucleotide complexes, their binding to cell membrane, cellular uptake, endosomal release, the release of cargo in the site of action or all of them together all these steps have to be systematically characterized. In the context of non-viral delivery, it has


Page 8 of 29

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


previously been shown that for PEI/oligonucleotide polyplexes above certain molar ratio all oligonucleotide is bound by the polymer and further addition of PEI will lead to unbound fraction of carrier termed free fraction. This free fraction in the PEI/oligonucleotide polyplexes has been reported to contribute to the delivery efficacy for nucleic acids, whereas it does not increase the uptake.9 Moreover, it has been indicated that amount of this free fraction of PEI could be responsible for the toxicity both in vitro and in vivo.10 We hypothesized that this potential free peptide fraction can also play a role in the transfection process of CPP/nucleic acid nanoparticles. In this study we have unraveled the potential mechanism by which acylation improves the capability of CPPs to deliver functional splice-correcting oligonucleotides. To gain a systematic insight and establish the structure-activity relationships for fatty acid-modified CPP-mediated delivery process of splice correcting antisense oligonucleotides, we synthesized a small library of N-terminally acylated CPPs with varying hydrocarbon chain length on the basis of previously characterized PepFect14 peptide. We used these PepFect14 analogues to extensively screen and characterize the properties of peptides and their ability for formulating splice-correcting oligonucleotides. We show that the free fraction of CPP, which is not associated with the CPP/SCO complexes, plays and important role in the delivery process, modulating both delivery efficiency/membrane activity, but also toxicity. Moreover, we demonstrate that effective delivery of SCOs increases linearly with the growing acyl chain length from the threshold of 12 carbons, while shorter acyl chains remain completely inactive. Furthermore, epifluorescence microscopy studies indicate that transfection efficiency of more hydrophobic analogues correlates with the ability of these nanoparticles to associate with cells. Finally, physicochemical characteristics of these nanoparticles, i.e. hydrodynamic size and surface charge, remain very similar, thus we concluded that the hydrophobic interactions between the complexes and the cell membrane are the key contributor in mediating efficient SCO delivery.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 29

Results and discussion Self-association of designed CPP library To have a systematic understanding of the impact of the fatty acid modifications and the hydrophobicity of the CPPs on the delivery process of antisense splice correcting oligonucleotides (SCOs), we designed and synthesized a small library of CPPs based on previously established PepFect14 peptide by introducing saturated linear acyl chains with the length of 0 to 22 carbons to the backbone of this peptide. The peptides were named based on their acyl chain length (C0-C22) and their peptide backbone origin (14 according to the PepFect14). For example the parent PepFect14 peptide according to this nomenclature is designated C18-14 (Table 1). Furthermore, peptide C0-14, serves as a non-acyl-modified control, with free N-terminus and one additional net positive charge. Table 1. Structures of peptides used in the study Name Sequence C0-14a NH2-AGYLLGKLLOOLAAAALOOLL-CONH2 C2-14 CH3-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C4-14 CH3-(CH2)2-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C6-14 CH3-(CH2)4-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C8-14 CH3-(CH2)6-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C10-14 CH3-(CH2)8-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C12-14 CH3-(CH2)10-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C14-14 CH3-(CH2)12-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C16-14 CH3-(CH2)14-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C18-14b CH3-(CH2)16-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C20-14 CH3-(CH2)18-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 C22-14 CH3-(CH2)20-CONH-AGYLLGKLLOOLAAAALOOLL-CONH2 O – Ornithine a – also known in literature as non-stearylated PepFect14 b – also known in literature as PepFect14

Charge +6 +5 +5 +5 +5 +5 +5 +5 +5 +5 +5 +5

To understand the role of acylation in complex formation between the peptides and SCO, we firstly sought to examine the properties of the free peptide analogues. As expected, RP-HPLC purification chromatograms revealed that the retention times of pure peptides increased linearly with an increasing acyl chain length (Figure S1). It has been shown that in some cases the process of nano-complex formation with non-viral vectors, for example with cationic lipids, starts with self-associated vector particles, which bind avidly to nucleic acids and rearrange.11 Also, amphiphilic peptides, such as PepFects, are expected to have a tendency to self-associate. For example, we have previously shown that the predecessor of PepFect peptides, TP10 peptide (which is structurally very close to the C0-14 peptide backbone), has a critical micelle concentration of around 40 nM.12 Additionally, we hypothesized that the pKa values of primary amines can be much lower in acylated peptides due to hydrophobic environment as is shown for folded proteins where pKa values can shift up to 5 units meaning that bases can become acids dependent on the microenvironment.13 Lowered degree of protonation of primary amino groups would decrease the charge repulsion between peptide molecules, which would further induce the self-association of the peptides. To find evidence for the self-association of the peptides and its dependence on the level of protonation the peptides were studied at pH 4.0, 6.0, 8.0 and in MQ water. First of all it was found by dynamic light scattering that all studied peptides self-associate already at 10 µM concentration in all tested conditions. Secondly, the size of the aggregates increased roughly by 1 order of magnitude with every 2 pH units while the sizes of the aggregates ranged between 10-2000 nm (Figure 1A). Interestingly, the analogues with shortest fatty acid were


Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


more prone to aggregation even at pH 4.0. In MQ water, which is the media for complex formation with oligonucleotides, the shorter analogues behave similarly to pH 4.0 whereas the longer analogues behaved similarly to pH 6.0. In conclusion these results showed that it was possible to reduce the size of the peptide aggregates by lowering the pH, but it was not possible to dissolve the aggregates even at pH 4.0.

Figure 1. Characterization of the self-assembly for designed peptides. The peptides were diluted in 10 mM phosphate buffer at pH 4.0 (red), 6.0 (green), 8.0 (blue) and MQ (black) to final concentration of 10 µM. (A) Then number mean size and (B) zeta potential were measured. The values represent the mean of at least three independent experiments (mean ± SEM, n=3). Additionally, the structure of selfassembled peptides were studied by negative staining transmission electron microscopy at 1 mM concentration in MQ for (C) C2-14, (D) C12-14, (E) C18-14 and (F) C22-14. The scale bar indicates 200 nm. Both methods show larger continuous aggregates for peptides with shorter acyl chains while peptides with longer acyl chains show smaller more discrete aggregates and/or micelles.

The zeta potential values measured for the same samples confirm that when pH was increased from 6.0 to 8.0 the zeta potential for analogues with longer acyl chain (C6-14 to C22-14)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dropped from +15 mV to +10 mV (Figure 1B). These results coincided with 1 order of magnitude increase in size due to lowered colloidal stability caused by decreased protonation. Interestingly at pH 4.0 peptides from C0-14 to C12-14 showed almost neutral zeta potential while peptides C14-14 to C22-14 were between +10 mV and +15 mV indicating that the latter have higher colloidal stability. The different behavior of the shorter acyl chain analogues can be partially explained by the differences in secondary structure of the peptides as shown by circular dichroism (Figure S2). The shorter acyl chain analogues form random coiled structure whereas the longer analogues adopt α-helical conformation, with characteristic minima at 208 and 222 nm. The CD results correlate well with previous findings on the secondary structures of acylated CPPs that longer acyl chains induce more α-helical structure.14 After confirming that these peptides self-associate into nanoparticles we next sought to study the morphological features of these formed aggregates by transmission electron microscopy (TEM). Negative staining TEM was used to visualize the differences between the selfassociation and supramolecular structures of peptides with different acyl chain lengths. For initial analysis, peptides C2-14, C12-14, C18-14 and C22-14 were chosen and tested at 1 mM concentration. TEM analysis of these peptides indicate that longer acyl chain length (C18-14 and C22-14) improves the association into smaller more discrete particles (Figure 1C,D) whereas peptides with shorter acyl chains (C2-14 and C12-14) form more continuous structures (Figure 1E,F). Interestingly, for C12-14 fibrillar structures are formed (Figure 1D). At 100 µM concentration self-association was still present for all the tested peptides (Figure S3) further confirming that in case of complex formation with oligonucleotides these peptides should be considered as a mixture of peptide aggregates, oligomers and monomers. In conclusion all the tested peptides self-associated, suggesting that they behave similarly to cationic lipids, and that aggregation needs to be taken into account when describing and optimizing for nanoparticle formulations with oligonucleotides. Additionally, our data indicates that the pKa values of the primary amino groups in all tested peptides in their selfassociated form might be much lower due to their microenvironment compared to that reported for respective amino acids in solution (10.53 for lysine and 10.76 for ornithine).

Characterization of peptide/oligonucleotide complexes Physicochemical characteristics of nanoparticles, such as size, surface charge and shape, are known to play important role in their delivery pattern in vivo. For example, it has been shown that size of the nanoparticles has a pronounced influence on their biodistribution.15,16 Moreover, it has been demonstrated that particles with zeta potential outside of the range of 30 mV to +30 mV are prone to opsonization leading to recognition and clearance by mononuclear phagocyte system.17 To analyze the formation of complexes between the peptides and oligonucleotide first DLS studies were carried out. Complexes were formed in MQ water at four different molar ratios of 3, 5, 7 and 10 (corresponding to peptide/oligonucleotide charge ratios N/P 0,8; 1,4; 1,9; 2,8); and already from MR3 all peptides form nanoparticles with negative zeta potential and with sizes varying between 50 and 100 nm (Figure 2A,B). At MR5 and higher the shortest 2 analogues show large aggregates with neutral charge. For the intermediate and long analogues from MR3 to 7 all the peptides are able to complex the oligonucleotide into positively charged nanoparticles with sizes ranging between 20-200 nm. The zeta potential profiles for both MR5 and MR7 showed high similarity whereas the zeta potential increased with the acyl chain length from 0 mV to +22 mV. All particles reached the plateau level of zeta potential at MR5 indicating that all the oligonucleotide molecules are complexed by the peptide. Interestingly, at MR10, the zeta potential could not be reliably measured as the zeta potential distribution plots showed peaks above +60 mV (data not shown). We assume that this was due to very


Page 12 of 29

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


high concentration of non-complexed peptide particles or molecules pointing to difficulties in interpreting the DLS results in heterogeneous mixtures in general.

Figure 2. Characterization of peptide/oligonucleotide complexes. The complexes were formed at peptide/SCO molar ratios (MR) 3, 5, 7 and 10 in MQ water at 1 µM of oligonucleotide. After 1h of incubation the complexes were diluted 10X in MQ and (A) number mean size and (B) zeta potential were measured from the same sample. The values represent the mean of at least three independent experiments (mean ± SEM, n=3). (C) To describe the formation of the complexes Alexa568 labelled SCO was used. The complexes were formed as described above and consequent quenching of AlexaSCO fluorescence upon complexation was measured. The values represent the mean of at least three independent experiments done in triplicates (mean ± SEM, n=3).

To further confirm at which MR all oligonucleotide is complexed by the peptide an Alexa568SCO quenching assay was performed. In this assay Alexa labelled SCO is complexed with peptide at different peptide/oligonucleotide MRs. Upon complexation the fluorescence from the dye is reduced due to quenching caused by electrostatic and hydrophobic interactions with the peptide. We titrated the Alexa-568 labelled SCO with peptides at different MRs and at MR3 around 70-80% of the fluorescence was quenched, which can be related to the percent of SCO complexed by the peptide (Figure 2C). At MR5 the quenching is approaching the maximum (around 95%) as for the longest analogues all oligonucleotide molecules are completely bound while for the intermediate analogues it ranges between 80-95%. For the shortest analogues it should be noted that C0-14 has an extra positive charge and thus it complexes the oligonucleotide better than C2-14 which is the least efficient among the tested peptides in complexing SCO (around 60% quenching at MR5). The difference between the binding properties of C0-14 and C2-14 also indicates that the mechanism behind quenching the fluorescence of labelled SCO is due to both electrostatic and hydrophobic interactions. At MR7 and MR10 maximal quenching is also achieved in case of intermediate analogues (together with C4-14 and C6-14). The dependence of Alexa568-SCO quenching on the MR was found to be very similar in a more qualitative agarose gel retardation assay (Figure S4), where in all cases the migration of free Alexa568-SCO disappeared at MR5 due to binding of the oligonucleotide by the peptide. Together these data suggest that all the oligonucleotide is complexed roughly between MR3 and MR5 and rest of the peptide remains in solution as a free fraction, which has very similar



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

size and size distribution as complexes themselves (Figure 1). Additionally, we have validated the superiority of the Alexa568-SCO quenching assay for the analysis of non-covalent complex formation over gel retardation assay both in terms of sensitivity and labour consumption. Yet it should be noted that this assay should be used in combination with size and zeta potential measurements in order to draw any solid conclusions. Role of acylation and free fraction of CPP in membrane activity The C0-14 peptide is a secondary amphiphile. In case of acylation the peptides should change towards primary amphiphiles with peptide backbone acting as a charged head group and acyl chain as hydrophobic tail. As primary amphiphiles can be highly membrane active we assessed the role of lipid tail in the hemolytic activity as lysis of blood cells would be the first sign of toxicity of these complexes after systemic administration. Thus, lysis of 2% bovine red blood cells (RBCs) in suspension caused by peptides and complexes was analyzed. Peptides were applied at concentrations from 0.008 to 5 µM and complexes at MR3, 5, 7 and 10 (the corresponding total peptide concentrations during the treatment would be 0.3; 0.5; 0.7 and 1.0 µM, respectively). Analysis of naked peptides shows that all of them caused dose dependent hemolysis and the hemolytic activity reached maximum at acyl chain length of 16 carbons, and plateaued for longer derivatives (Figure 3A).

Figure 3. Membrane activity studies for peptides and peptide/oligonucleotide complexes. To evaluate the membrane activity of the peptides and complexes in biologically more relevant model their hemolytic activity was studied on 2% bovine RBCs diluted in 1X PBS. (A) The peptides were studied at various concentrations ranging from 0,008 to 5 µM. (B) To characterize the hemolytic activity of peptide/SCO complexes the complexes were formed at MRs 3, 5, 7 and 10 corresponding to peptide concentrations 0,3; 0,5; 0,7 and 1,0 µM, respectively. The values represent the mean of at least two independent experiments done in triplicates (mean ± SEM, n=2).

Shorter analogues (C0-14 to C4-14) have very little or no hemolytic activity below 5 µM (020% of hemolysis). Analogous trend in the increase of hemolytic activity with acyl chain length has also been shown for antimicrobial peptides, which also are cationic and α-helical.18 As maximal hemolysis was reached already at 5 µM peptide concentration. It should be considered that the concentration of RBCs in our assays was 50 times lower than it is in blood, and the absence of the plasma proteins turns RBCs even more vulnerable to lysis as shown in (Figure S5) suggesting that in relevant biological conditions hemolysis is not induced at such low concentrations of CPP. The hemolytic effects of the complexes confirmed the dependence of membrane activity on the peptide concentration as the hemolytic activity increased in parallel with peptide/oligonucleotide molar ratio. In addition, the hemolytic activity correlated well with the length of acyl chain and with the fraction of free peptide (Figure 3B). At MR3 none of the


Page 14 of 29

Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptides had detectable hemolytic activity in our system, whereas at MR5 the intermediate and longest analogs showed up to 20% hemolysis; and at MR7 and MR10 hemolysis reached 60% and 85% accordingly. Together these data support the concept that above a certain molar ratio all oligonucleotide is complexed by peptide and the excess of peptide stays in the solution as so called free fraction that causes membrane activity, toxicity and interferes with measurements like in DLS. This is in accordance with previous studies about the role of a free fraction in solution of polyplexes where it was shown that removal of free PEI reduced the toxicity both in vitro and in vivo, however, on the other hand the efficiency of gene induction was also reduced.10 To further confirm the role of free fraction in hemolysis we conducted a control experiment where we titrated a constant concentration of peptide (in this case 1 µM) with oligonucleotide to MR3, 5, 7 and 10 (peptide/oligonucleotide). The hemolytic activity of the peptide was abolished with the increasing oligonucleotide concentration at MR3 and 5 (Figure S6) indicating that the free fraction of peptide is responsible for the membrane activity and toxicity. In conclusion the reduction of peptide to oligonucleotide MR is the simplest way for decreasing the membrane activity and even highly membrane active peptides completely lost their hemolytic activity in complex with SCO.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Transfection efficiency and cytotoxicity Multiple studies have demonstrated that delivery efficiency of nucleic acid cargos by CPPs is significantly improved by N-terminal acylation of carrier with saturated hydrocarbon chains up to 18 carbons.1,2,7,14 Peptides with longer acyl chains start to become toxic, as shown for fatty acid analogs of TP10 by our group before,6 but recent developments of CPPs have reduced the required doses for optimal biological effect, thus widening the therapeutic window. To optimize the acyl chain length for PepFect14, we chose to screen the formulations in the widely applied splice correction model, HeLa pluc705 cells. Peptides were complexed with SCO at four different molar ratios of 3, 5, 7 and 10 and the cells were treated at 100 nM concentration of SCO, which has been previously shown to be the EC50 value for mRNA splicing with the parent peptide PepFect14 at MR5.3 The luciferase expression after 24 h of transfection (Figure 4A) shows that the complexes of peptides with acyl chain shorter than 12 carbons do not induce splice correction activity. Furthermore, for all tested MRs the splicing correction effect starts to linearly increase from the acyl chain length of 12 carbons. This strongly supports the significance of acyl modification of CPPs for the transfection of nucleic acids, and also points to the minimal acyl chain length of 12 carbons.

Figure 4. Influence of acyl chain length on SCO transfection efficiency and toxicity in HeLa pLuc705 cells. Cells were seeded 24 h before prior to experiment in 96-well plates. Cells were


Page 16 of 29

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


transfected with peptide/SCO complexes at different MRs at 100 nM final SCO concentration for 24h. (A) The luminescence from functional splice-correction of luciferase gene expression is presented as fold increase of relative light units (RLU) over untreated cells. Toxicity was evaluated by MTT assay which measures the metabolic activity of cells. The results are normalized to untreated cells and shown as viability. Complexes were formed as described above. The viabilities for complexes (B) and peptides alone (C) are shown. All the values represent the mean of at least three independent experiments done in triplicates (mean ± SEM, n=3).

Comparison of the functionally active peptides at different MRs revealed the lowest transfection efficiency for MR3, which was most probably due to insufficient complexation of SCO as the quenching assay demonstrated (Figure 2C), or insufficient neutralization of SCO charge (Figure 1B). MR5 was the most efficient for formulation of complexes whereas from MR7 the splice-correction effect started to decrease, which is in accordance with previously published results on PepFect14 (in this study named as C18-14).3 When the slopes of the transfection efficiency curves at different MRs were compared in the linear range (C12-14 to C22-14) MR5 showed approximately 3-fold higher slope than MR3. In order to clarify whether the increased membrane activity of longer acyl chain analogues, observed in hemolysis assay, also plays a role in increased transfection efficiency, we measured the cell viability in the same setup as applied for transfection. Viability of the cells was assessed using MTT assay, which estimates the number of live cells and/or their metabolic activity. Complexes at MR3 and MR5 showed viabilities above 80% for all peptides whereas at MR7 and MR10 the viabilities were lower in general but did not drop below 75% (Figure 4B) suggesting that mostly the free fraction of peptide causes the toxicity. We assume that the observed decrease in transfection at higher MRs is most probably due to the increased toxicity caused by the free fraction of peptide, which is not incorporated into oligonucleotide complexes. Additionally, a slight increase in toxicity upon increasing the acyl chain length of CPP was observed. In order to analyze the impact of peptides themselves on cell viability, cells were treated with peptides at 0.01; 0.1; 1.0 and 10 µM for 24 h. All used CPPs showed dose dependent toxicity starting from 0.1 µM concentration. Surprisingly, the toxicity profile over the acyl chain length at 1 µM peptide concentration was different from that for hemolysis (Figure 3A). Cleraly the shorter and longer analogs were less toxic than analogs with intermediate–length acyl chains (Figure 4C). Together these results suggest that the length of acyl chain and consequent hydrophobicity are the key characteristics of CPPs in efficient and functional SCO delivery. Furthermore, there is a requirement for a minimal carbon chain length at 12 carbons and below that no transfection occurs. As the physicochemical properties of oligonucleotide complexes with the six most hydrophobic peptides were quite similar we hypothesized that the reason behind increased delivery efficiency upon enhancement of hydrophobicity were the hydrophobic interactions between the cell membrane core and the complexes. Association of CPP-SCO complexes with cells In further search for the reasons behind the linear increase in functional SCO delivery starting from 12 carbon acyl chain on CPPs, we harnessed live cell imaging using fluorescently labelled complexes. As it has been shown before that stearoylation of octaarginine improves the association of CPP/pDNA complexes with the cells proposedly due to increased hydrophobic interactions with the cell membrane,5 we assumed that the same might apply for CPP/SCO complexes although the nucleic acid cargoes are of drastically different size. Furthermore the higher acyl chain length could favor the increased association with the plasma membrane of cell and consequent uptake.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The association of complexes with the cells was analyzed after treatment for 1 h with Alexa568-SCO-containing complexes at MR10. In order to remove the non-associated complexes the cells were washed 2 times with media and followed by imaging under epifluorescence microscopy. As expected, behenoylation (C22-14) of CPP increased the association of complexes with the cells significantly compared to acetylation (C2-14) (Figure 5A), although the background fluorescence had also risen. We assume that the higher background fluorescence was due to more hydrophobic complexes binding better to the cell culture plastic. To exclude the background fluorescence from the equation, the signal of complexes was quantified only in the close proximity of the cell nucleus. The images were recorded for all the peptide analogs, and two characteristic examples are presented in Figure


Page 18 of 29

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5A.

Figure 5. Longer acyl chains improve the association of peptide/SCO complexes with cells. Cells were seeded 24h before prior to experiment in 96-well microscopy plates. Complexes were formed with Alexa568-SCO (red) at MR10. 15 min before adding the complexes cells were treated with Hoechst for nuclear staining (blue). Cells were then transfected with peptide/SCO complexes for 1 h at 100 nM final SCO concentration after which cells were washed 2 times with full media. (A)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Association of complexes with cells was visualized under epifluorescence microscope at 10X magnification. (B) Quantification of Alexa568 signal complexes from the close proximity of the cell nucleus was carried out at 20X magnification from the same cells. Blue bars correspond to total number of complexes identified on the image, black bars to the total number of cells and red bars to the number of cells containing associated with complexes (n=1).

Quantification of Alexa568-SCO fluorescence in images revealed that the amount of complexes started to increase from C8-14 (Figure 5B). These results are in good accordance with the transfection results where the efficient transfection started to increase from acyl chain length of 12 carbons (C12-14). The difference in the minimal acyl chain length for biological activity and cell association indicates that the association with cells on the minimal threshold level is insufficient for complexes to induce splice-correction. Interestingly, complexes with C2-14 peptide showed higher cell association than other shorter acyl chain analogs. We suggest that this is perhaps caused by the lower quenching of the Alexa dye on the oligonucleotide by this particular peptide as shown in Figure 2C rather than due to increased cell association. As for the rest of the peptides the quenching of Alexa568 on SCO at MR10 was identical, we considered it to be constant for all the used peptides at quantification of cell association. Since the microscopy images did not reveal whether the complexes were associated with the outer membrane of the cells or were taken up, we performed a control experiment to release the complexes from the endosomes using the endosomolytic reagent chloroquine. For that the cells were first treated with CPP-SCO complexes at MR3, 5, 7 and 10 for 4 h; and the residual complexes were removed by changing the media to chloroquine-containing one. Cells were left with chloroquine for 2 h and the media was replaced again. No effect of endosomal release induction by chloroquine at any of the tested MRs was observed for complexes with CPPs having shorter acyl chains than 12 carbons suggesting that all such complexes were completely removed by changing the media (Figure S7). At the same time peptides with 12 carbons acyl chains and longer showed higher splicing after chloroquine treatment at all tested MRs, indicating that these complexes induce higher cellular uptake but they are still suffering from endosomal entrapment. Thus, the increased activity of the longer-chain CPP analogs in nucleic acid transfection could hardly be caused by increased endosomal escape. We thus suggest that the efficiency of functional SCO delivery was dependent on the association of complexes with the cells, which in turn correlates with the hydrophobicity of the peptides in the complexes issuing from the acyl chain length. More importantly, our data show that there is a lower threshold for hydrophobicity that is required for the complexes to associate with the cells, inducing the uptake and delivery of functional SCO. For PepFect14type CPPs, this threshold is 10 carbons, though this might greatly vary for different peptides. Therefore we hypothesize that hydrophobic interactions between the nucleic acid-carrying complexes and the plasma membrane of cells play the key role in the functional delivery of oligonucleotides by cell-penetrating peptides, and we assume that this could also be extended to other non-viral delivery vectors as well.


Page 20 of 29

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusions In this study we demonstrate that enhancement of the hydrophobicity of cell-penetrating peptides by N-terminal acylation, increases the functional delivery of splice-correcting oligonucleotides, proportionally to the length of acyl chain starting from 12 carbons. We suggest that the reason behind this turning-point is the higher association of peptide/oligonucleotide complexes to the cell membrane due to increased hydrophobic interactions between the complexes and the plasma membrane of cells. Characterization of the CPP/oligonucleotide complexes at different molar ratios (MRs) demonstrated that starting from a certain MR, all oligonucleotide molecules were incorporated in complexes, and further increasing of MR only lead to higher excess of peptide, which was not incorporated into complexes, termed free fraction. The membrane activity and toxicity studies of the CPP/oligonucleotide complexes showed that at higher molar ratios, both the membrane activity and toxicity mostly originated from the free fraction of peptide. Furthermore this free fraction might also form aggregates of similar size, thus making it difficult to distinguish these from the functional CPP-SCO complexes in used assays. Together these data lay solid ground for designing CPP/oligonucleotide formulations for efficient splice-correcting oligonucleotide delivery without suffering from toxicity sideeffects. By increasing the hydrophobicity of CPP, higher delivery efficiency of SCO can be achieved, and in order to avoid cell lysis and reduce toxicity, lower amounts of free fraction of CPP in the transfection complexes solution should be aimed. As these factors are among the key determinants in broadening the therapeutic window for cell-penetrating peptides further insights into the impact of these factors both in vitro and in vivo are needed.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Materials and Methods Reagents For delivery experiments PS-2′-OMe splice-correcting oligonucleotide (5′-CCU CUU ACC UCA GUU ACA-3′) was used (RiboTask, Denmark). The luciferin assay buffer was freshly prepared before the measurements according to the protocol developed by Helmfors et al.19 Briefly, the final concentrations of assay buffer constituents are as follows: DTT 25 mM, Dluciferin 1 mM, ATP 1 mM, CoA 25 µM, EDTA 1 mM, Tricine 20 mM, MgCO3 1 mM and MgSO4 5 mM. The working solution concentration for peptides and oligonucleotides used in the studies were 100 µM and 10 µM. Peptide synthesis Peptide backbone for the analogues was synthesized by standard Fmoc chemistry on a Biotage Initiator + Alstra peptide synthesizer (Biotage, Sweden) using Rink Amide ChemMatrix resin (PCAS Biomatrix, Canada), N,N′-diisopropylcarbodiimide (DIC) and OxymaPure as coupling reagents. The synthesis was carried out in one batch after which the peptidyl resin was divided into 12 syringes where acylation was carried out manually. Acetylation was carried out with acetic anhydride and N,N-Diisopropylethylamine (DIEA) 1:1 (v/v) while rest fatty acids were coupled in 2 eq excess over peptide using Cl-HOBT and HCTU in DMF as activators and DIEA as base. Peptides were cleaved using trifluoroacetic acid/water/trisopropylsilane (95%/2.5%/2.5% (v/v)) and after 3h precipitated in cold diethyl ether. Peptides were purified on preparative RP-HPLC with Biobasic C8 column (ThermoFisher, Sweden) using AcN/water (0.1% TFA) gradient from 20-100% in 40 minutes. The purified products were identified using ESI–TOF MS (micrOTOF, Bruker Daltonics). After purification peptides were lyophilized resulting in white powder in all of the cases. CD spectroscopy To determine the secondary structure of the peptides circular dichroism measurements were carried out according to protocol published by our group before.20 CD spectra were recorded between 185 nm and 260 nm with bandwidth of 0.5 nm on a Chirascan CD spectrometer. Temperature was adjusted to 20 °C and the measurements were carried out in quartz cuvette with optical path length 1 mm. Peptides were diluted in 10 mM phosphate buffer at pH 7.4 to final concentration of 10 µM. Background spectra from the buffer was subtracted from the peptide spectra. Spectra of 10 measurements were averaged and converted from CD units (mdeg) to Mean Residue Ellipticity (deg cm2 dmol−1) by using Pro-Data™ software (Applied Photophysics, Leatherhead, UK). Preparation of non-covalent complexes 2ÓMe SCO and peptide complexes were formed in MQ water at different peptide/oligo MRs 3:1-10:1. After mixing the oligo and the peptide the complexes were let to incubate at RT for 1h. Complexes were added to the cells in 1/10 of the final volume of the cell media (100 µl). The final concentration of oligo at the treatment was 100nM in the well. For fluorescence assays Alexa568-labelled 2ÓMe SCO was used at the same concentration as normal SCO. As a positive control commercial transfection reagent LipofectamineTM2000 (Invitrogen, Sweden) was used according to protocol where 10 µl of 10 µM SCO was dilute in 40 µl of


Page 22 of 29

Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


OptiMem and 2.5 µl of LF2000 in 47.5 µl of OptiMem were mixed 15 min before transfection and 10 µl was added to the cells. Dynamic Light Scattering Size and zeta potential of peptide and peptide/oligonucleotide particles was determined by dynamic light scattering using a Zetasizer Nano ZS apparatus (Malvern Instruments, United Kingdom). For peptides 100 µM solution was diluted 10X with 10 mM phosphate buffer at pH 4.0; 6.0 and 8.0 to final volume of 500 µl. Peptide/oligonucleotide complexes were formulated according to the protocol for in vitro transfection in 50 µl volume. Prior to the measurement complexes were diluted 10X with MQ. Measurements were performed over a period of time at 22°C. 3 measurements were made and 1 measurement was set to 8 runs (1 run was 8 s). Both size and zeta potential measurements were carried out in zeta potential cuvettes. The size is presented as number mean size of the particles due to the heterogeneous nature of the system and for making future correlations with electron microscopy easier. TEM Negative staining analysis was performed as described before.21 Briefly, copper grids were covered with formvar film. Thereafter, a thin layer (5 nm) of carbon was precipitated onto the grids followed by glow discharge treatment. 5 µl of each sample was absorbed to the grids for 2 minutes followed by washing with MQ water. Samples were then exposed to 2% aqueous uranyl acetate solution for 1 minute. After removing excess stain with filter paper the samples were allowed to air dry. The samples were examined at 120 kV accelerating voltage on FEI Tecnai G2 Spirit BioTwin electron microscope (FEI, Netherlands). Hemolysis The hemolysis experiments were carried out with bovine red blood cells (Håtunalab, Sweden). The cells were washed 3 times with 1X PBS and then diluted to final concentration of 2% of RBCs in PBS.22 10 µl of peptides were added to 190 µl of cells in PCR plates and shaken at 37 °C for 1h. Then the cells were spun down at 1000G and then 100 µl of supernatant was transferred to transparent 96-well tissue culture plates. The absorbance of released hemoglobin was measured at 450 nm on plate reader (Tecan Sunrise™, Switzerland). As a positive control 20% Triton X-100 was used while PBS served as a negative control. The percentage of hemolysis was calculated by the formula: Hemolysis (%) = [(AS – AN) / (APAN)] × 100, where AS is the absorbance for the sample, AP for positive control and AN for negative control. All samples were measured in triplicates. Alexa568-SCO quenching assay Alexa568-SCO quenching assay is an alternative to EtBr and PicoGreen exclusion assays for studying the complex formation between the SCO and peptides. The assay works based on the fact that fluorescence emission of Alexa568 label is dependent on its environment. When peptides bind to the oligo through electrostatic and hydrophobic interactions they also bind to the Alexa568 label and this causes the quenching of fluorescence of the dye. As additional information this same effect is commonly used in fluorescent dye based critical micelle concentration measuring assays.23,24 The decrease in fluorescence caused by increasing amount of peptide can be measured with spectrofluorometer. Upon maximum quenching all



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oligo is bound by the peptides and this corresponds to optimal peptide/oligo MR where there is not much free peptide in solution which would cause for example toxicity9,10 or potential interference with assays. For the experiments complexes were formed as described for normal SCO with final concentration of 100 nM SCO. 100 µl of complexes were added to black 96-well plates (Corning) and the fluorescence of free Alexa568-SCO was measured (Flexstation, Molecular Devices) at wavelengths of 580 nm for excitation and 605 nm for emission with a cutoff at 590 nm for recording the emission. The results were normalized to the fluorescence of 100 nM free Alexa568-SCO in MQ. The separately prepared samples were measured in triplicates and the experiment was repeated 3 times. As a qualitative control experiment, all complexes were all tested on gel shift assay. For that 10 µl of complexes were mixed with 6X loading dye (Thermo Scientific) and loaded on a 1% agarose gel in 1X TAE buffer. Electrophoresis was carried out for 10 min at 70 V. Alexa568SCO shift was visualized on a UV transilluminator with ImageQuant 150 imager (GE Healthcare). Cell cultures HeLa pLuc 705 cells, provided by Prof. R. Kole, were cultivated at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) containing 100 U/ml penicillin, and 100 µg/ml streptomycin, glutamax, 1 mM sodium pyruvate, 10% fetal bovine serum (FBS, Life Technologies). When seeding the cells for experiments the cells were split every second day to maintain stable luminescence values in untreated cells. Splice-correction assay For the splice-correction activity measurements 7 × 104 HeLa pLuc 705 cells were seeded 24h prior to treatment into tissue culture-treated transparent bottom 96-well white plates (Corning) in the media volume of 100 µl. Before the treatment media was replaced with 90 µl of serum containing media and 10 µl peptide/SCO complexes was added giving 100 nM for the final SCO concentration. After 24 h of treatment the media was removed and cells were lysed in 20 µl of 0.1% Triton X-100 for 30 minutes at room temperature. For measuring the luciferase activity 80 µl of freshly prepared luciferin assay buffer was added and total luminescence from each well was measured on GLOMAXTM 96 microplate luminometer (Promega, Sweden). It should be noted that due to cross-talk of luminescence between the wells of 96well plate firstly luciferin was added to negative controls and untreated cells and then luminescence was measured followed by not active complexes after which the highest expressing cells were measured. Resultant luminescence was measured as relative light units (RLU). For the chloroquine induced endosomal release assay cells were seeded 24h before the experiment. Media was changed before treating the cells with complexes for 4 h followed by changing the media to either 100 µM chloroquine containing media or normal media and incubated for additional 2 h. Thereafter the media was changed again and cells incubated for 18 h in cell incubator after which luciferase expression was measured. Viability assay To evaluate the influence of the peptides and complexes to cell viability MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used. This assay measures the metabolic activity of the cells which can be correlated with the viability. For the


Page 24 of 29

Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


experiments cells were prepared and treated as described before. Before the treatment media was changed to 90 µl of full media and then 10 µl of peptides or complexes were added followed by 24 h incubation in the cell incubator. Thereafter the media was changed again to 90 µl of fresh serum containing media to avoid unwanted interactions between the MTT reagent (Sigma-Aldrich) and peptides or complexes. This was followed by addition of 10 µl of MTT reagent (5 mg/ml in serum containing DMEM) followed by 4 h incubation. The formed formazan crystals were dissolved by adding 100 µl of 10 % (w/v) SDS in 10 mM HCl into each well and let to dissolve at the cell incubator over-night. HCl was added to avoid the absorbance from phenol red in the cell media. The results were normalized to untreated cells (UT) and DMSO was used as a positive control. All the measurements were done in triplicates. The absorbance was measured in total volume of 200 µl on Multiskan™ GO Microplate Spectrophotometer at 570 nm and at reference wavelength 690 nm, which was subtracted from the prior, and results were normalized to untreated (UT) and presented as a percent of UT. Epifluorescence microscopy HeLa pLuc 705 (10 × 104) cells were seeded in 96-well microscopy plates (Ibidi GmbH, Germany) 24h before treatment in 200 µl final volume in full media. Prior to adding the complexes cells were stained with Hoechst (1:1000) in full media for 15 minutes followed by media exchange. Then 20 µl of media was removed and replaced with 20 µl of complexes at MR10 making the final concentration of Alexa568-SCO 100 nM. The cells were treated for 1 h and then washed 2X with serum containing media. Imaging was carried out in serum containing media with a Leica DM/IRBE 2 epi-fluorescence microscope using 10 × 0.3 NA and 20 × 0.6 NA dry objectives. The system was equiped with a Ludin Box and Cube climate system set at 37°C and automated DMIRES X/Y stage and a Hamamatsu ER orca CCD, controlled by Micro-Manager.25 Images were automatically processed to quantify the number of aggregates associated with cells. In short, the 20X magnification Z-stack images of cells treated with complexes were automatically merged and thresholded using FIJI and further processed in combination with images of nuclei using CellProfiler to segment the complexes associated with cells. Briefly, the cells were identified by nuclear Hoechst staining and limited to 30-60 pixel diameter. Then the nuclear area was expanded by 30 pixels and the nuclei was excluded from analysis leaving only close proximity of the nucleus from where the complexes were quantified for each identified cell on the image. The size limitation for detecting the complexes surrounding the nucleus was set to 2-20 pixels in diameter. The CellProfiler pipeline and FIJI macro used to process the images are in supplementary method 1 and 2 respectively.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abbreviations CPP, cell-penetrating peptide; SCO, splice-correcting oligonucleotide; asON, antisense oligonucleotide; pDNA, plasmid DNA; PEI, polyethyleneimine; PMO, phosphorodiamidate morpholino oligomer; MR, molar ratio; CD, circular dichroism; TEM, transmission electron microscopy; RBCs, red blood cells; TP10, transportan 10; MTT, 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide; DLS, dynamic light scattering; DTT, dithiothreitol; ATP, adenosine triphosphate; CoA, coenzyme A; EDTA, ethylenediaminetetraacetic acid. Acknowledgements This work was supported by the Innovative Medicine Initiative Joint Undertaking under grant agreement no. 115363 resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2207-2013) and EFPIA companies in kind contribution; by the Swedish Research Council (VR-NT) and the Estonian Ministry of Education and Research (0180019s11). Supporting Information Figure S1. Characterization of the hydrophobicity of the peptides by RP-HPLC. Figure S2. Role acylation in the secondary structure of the peptides. Figure S3. Negative staining TEM of peptides at 100 µM concentration. Figure S4. Characterization of peptide/Alexa568-SCO complexes by gel retardation assay. Figure S5. Hemolytic activity of peptides with and without plasma proteins. Figure S6. Hemolytic activity of peptide/SCO complexes at constant peptide concentration. Figure S7. Chloroquine induced endosomal release of peptide/SCO complexes. Supplementary method 1. ImageJ macro. Supplementary method 2. Cell Profiler Command Pipeline.


Page 26 of 29

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reference list (1) Futaki, S., Ohashi, W., Suzuki, T., Niwa, M., Tanaka, S., Ueda, K., Harashima, H., and Sugiura, Y. (2001) Stearylated arginine-rich peptides: A new class of transfection systems. Bioconjug. Chem. 12, 1005–1011. (2) Mäe, M., EL Andaloussi, S., Lundin, P., Oskolkov, N., Johansson, H. J., Guterstam, P., and Langel, Ü. (2009) A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy. J. Control. Release 134, 221–227. (3) Ezzat, K., Andaloussi, S. E. L., Zaghloul, E. M., Lehto, T., Lindberg, S., Moreno, P. M. D., Viola, J. R., Magdy, T., Abdo, R., Guterstam, P., et al. (2011) PepFect 14, a novel cellpenetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Res. 39, 5284–98. (4) Arukuusk, P., Pärnaste, L., Hällbrink, M., and Langel, Ü. (2015) PepFects and NickFects for the intracellular delivery of nucleic acids, in Cell-Penetrating Peptides: Methods and Protocols, pp 303–315. (5) Khalil, I. A., Futaki, S., Niwa, M., Baba, Y., Kaji, N., Kamiya, H., and Harashima, H. (2004) Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation. Gene Ther. 11, 636–644. (6) Langel, K., Lindberg, S., Copolovici, D., Arukuusk, P., Sillard, R., and Langel, Ű. (2010) Novel Fatty Acid Modifications of Transportan 10. Int. J. Pept. Res. Ther. 16, 247–255. (7) Veiman, K.-L., Mäger, I., Ezzat, K., Margus, H., Lehto, T., Langel, K., Kurrikoff, K., Arukuusk, P., Suhorutšenko, J., Padari, K., et al. (2013) PepFect14 Peptide Vector for Efficient Gene Delivery in Cell Cultures. Mol. Pharm. 10, 199–210. (8) Järver, P., Zaghloul, E. M., Arzumanov, A. A., Saleh, A. F., McClorey, G., Hammond, S. M., Hällbrink, M., Langel, Ü., Smith, C. I. E., Wood, M. J. A., et al. (2015) Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides. Nucleic Acid Ther. 25, 65–77. (9) Klauber, T. C. B., Søndergaard, R. V., Sawant, R. R., Torchilin, V. P., and Andresen, T. L. (2016) Elucidating the role of free polycations in gene knockdown by siRNA polyplexes. Acta Biomater. 35, 248–259. (10) Boeckle, S., von Gersdorff, K., van der Piepen, S., Culmsee, C., Wagner, E., and Ogris, M. (2004) Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J. Gene Med. 6, 1102–1111. (11) Karmali, P. P., and Chaudhuri, A. (2007) Cationic liposomes as non-viral carriers of gene medicines: Resolved issues, open questions, and future promises. Med. Res. Rev. 27, 696–722. (12) Eiríksdóttir, E., Konate, K., Langel, Ü., Divita, G., and Deshayes, S. (2010) Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochim. Biophys. Acta - Biomembr. 1798, 1119–1128. (13) Isom, D. G., Castañeda, C. A., Cannon, B. R., and García-Moreno, B. (2011) Large shifts in pKa values of lysine residues buried inside a protein. Proc. Natl. Acad. Sci. U. S. A. 108, 5260–5. (14) Niidome, T., Urakawa, M., Takaji, K., Matsuo, Y., Ohmori, N., Wada, A., Hirayama, T., and Aoyagi, H. (1999) Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells. J. Pept. Res. 54, 361–367. (15) Tang, L., Yang, X., Yin, Q., Cai, K., Wang, H., Chaudhury, I., Yao, C., Zhou, Q., Kwon, M., Hartman, J. A., et al. (2014) Investigating the optimal size of anticancer nanomedicine. Proc. Natl. Acad. Sci. U. S. A. 111, 15344–9. (16) Alexis, F., Pridgen, E., Molnar, L. K., and Farokhzad, O. C. (2008) Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles. Mol. Pharm. 5, 505–515.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(17) Roser, M., Fischer, D., and Kissel, T. (1998) Surface-modified biodegradable albumin nano- and microspheres. II: Effect of surface charges on in vitro phagocytosis and biodistribution in rats. Eur. J. Pharm. Biopharm. 46, 255–263. (18) Radzishevsky, I. S., Rotem, S., Zaknoon, F., Gaidukov, L., Dagan, A., and Mor, A. (2005) Effects of acyl versus aminoacyl conjugation on the properties of antimicrobial peptides. Antimicrob. Agents Chemother. 49, 2412–20. (19) Helmfors, H., Eriksson, J., and Langel, Ü. (2015) Optimized luciferase assay for cellpenetrating peptide-mediated delivery of short oligonucleotides. Anal. Biochem. 484, 136– 142. (20) Regberg, J., Vasconcelos, L., Madani, F., Langel, Ü., and Hällbrink, M. (2016) pHresponsive PepFect cell-penetrating peptides. Int. J. Pharm. 501, 32–38. (21) Margus, H., Arukuusk, P., Langel, Ü., and Pooga, M. (2016) Characteristics of CellPenetrating Peptide/Nucleic Acid Nanoparticles. Mol. Pharm. 13, 172–179. (22) Evans, B. C., Nelson, C. E., Yu, S. S., Beavers, K. R., Kim, A. J., Li, H., Nelson, H. M., Giorgio, T. D., and Duvall, C. L. (2013) Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs. J. Vis. Exp. e50166. (23) Goddard, E. D., Turro, N. J., Kuo, P. L., and Ananthapadmanabhan, K. P. (1985) Fluorescence probes for critical micelle concentration determination. Langmuir 1, 352–355. (24) Patist, A., Bhagwat, S. S., Penfield, K. W., Aikens, P., and Shah, D. O. (2000) On the measurement of critical micelle concentrations of pure and technical-grade nonionic surfactants. J. Surfactants Deterg. 3, 53–58. (25) Edelstein, A., Amodaj, N., Hoover, K., Vale, R., and Stuurman, N. (2010) Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol. Chapter 14, Unit14.20.


Page 28 of 29

Page 29 of 29



Saturated Fatty Acid Analogues of Cell-Penetrating Peptide

Recommend Documents