Peptide-Based Nanoparticles to Rapidly and Efficiently âWrap 'n Roll

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Peptide-based nanoparticles to rapidly and efficiently “Wrap’n Roll" siRNA into cells Karidia Konate, Marion Dussot, Gudrun Aldrian, Anaïs Vaissière, Véronique Viguier, Isabel Ferreiro Neira, Franck Couillaud, Eric Vivès, Prisca Boisguerin, and Sebatien Deshayes Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00776 • Publication Date (Web): 26 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Bioconjugate Chemistry

1

Peptide-based nanoparticles to rapidly and

2

efficiently “Wrap’n Roll" siRNA into cells

3 4

Karidia Konate1, Marion Dussot1, Gudrun Aldrian2, Anaïs Vaissière1, Véronique Viguier3,

5

Isabel Ferreiro Neira4, Franck Couillaud4, Eric Vivès1, Prisca Boisguerin1, Sébastien

6

Deshayes1*

7 8

1

9

Montpellier, 1919 Route de Mende, 34293 Montpellier Cedex 5, France

Centre de Recherche en Biologie cellulaire de Montpellier, CNRS UMR 5237, Université de

10

2

11

CEDEX 4, France

12

3

Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier, France

13

4

EA 7435 IMOTION (Imagerie moléculaire et thérapies innovantes en oncologie), Université

14

de Bordeaux, 146 rue Leo Saignat, 33076 Bordeaux, France

Sys2Diag, UMR 9005-CNRS/ALCEDIAG, 1682 Rue de la Valsière, 34184 Montpellier

15 16 17

Corresponding author: *[email protected]

18 19 20 21

Keywords:

22

siRNA delivery, cell penetrating peptides, nanoparticle, gene knock-down, cancer.

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ACS Paragon Plus Environment

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

1

ABSTRACT. Delivery of small interfering RNA (siRNA) as a therapeutic tool is limited due

2

to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule and

3

its instability in serum. Several siRNA delivery systems have been constructed using cell-

4

penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide

5

delivery into the cells, especially by forming nanoparticles. In this study, we have developed a

6

new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic

7

Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells.

8

The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as

9

characterized by biophysical methods. Furthermore, they have several benefits as

10

oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA

11

delivery in several cell types and the high gene silencing activity, even in the presence of serum.

12

In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA

13

delivery trough nanoparticle formation. Our results indicate that the WRAP family has

14

significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.

15 16 17 18 19

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1

RNA interference (RNAi) in mammalian cells has opened a very exciting research field

2

by taking advantage of this mechanism for the treatment of various diseases. Short interfering

3

RNAs (siRNAs) are 21–23 base-pair RNA duplexes including a 2-nucleotide overhang at the 3

4

prime end. The antisense strand of siRNA with sequence complementarity to a target mRNA

5

can suppress gene expression with the highest sequences specificity (silencing). siRNA has

6

been demonstrated to be a potential drug candidate for many difficult-to-treat diseases such as

7

viral infections and cancers 1. Many clinical trials have also been initiated over the last decade,

8

but only very limited siRNA treatments have been approved by the FDA (e.g., topical

9

administration of naked siRNA in ocular and retinal diseases) 2.

10

The design versatility and the highly specific nature of these oligonucleotides highlight their

11

potential as drugs of the future. However, limitations such as the weak cell penetration as well

12

as the rapid endo/lysosomal degradation once inside the cell could be overcome by the use of

13

various carriers. A number of delivery technologies based on cationic lipids, polymers,

14

dendrimers or peptides have been developed 3. Among these non-viral carrier materials, cell

15

penetrating peptides (CPP) have gained increasing popularity because of their sequence and

16

function diversity. CPPs are usually short peptides (up to 30 amino acids) that originate from a

17

wide variety of sources (e.g., humans, mice, viruses or purely synthetic) 4. Based on their

18

structural characteristics, CPPs can be divided into two classes: arginine-rich CPPs and

19

amphipathic CPPs 5.

20

Amphipathic CPPs contain both hydrophilic and hydrophobic domains necessary for

21

interaction with the cargo and cellular internalization. In primary amphipathic CPPs, these

22

domains are distributed according to their position along the peptide chain as shown for pVEC

23

6,

24

the separation between hydrophilic and hydrophobic domains occurs due to the secondary

25

structure formation, such as α helices or β sheets 7. Many of the most commonly used CPPs are

MPG and Pep-1 7. Secondary amphipathic CPPs are another large class of peptides in which

3



1

members of this class such as penetratin 8, transportan 9, hCT-variants (human calcitonin) 10,

2

RICK 11 or C6M1 12.

3

There are two main strategies to vectorize oligonucleotides (ONs) using CPPs: by directly

4

conjugating ONs to the CPPs (covalent conjugation approach) or by forming non-covalent

5

complexes between the CPPs and ONs (nanoparticle-based approach). The formation of non-

6

covalent nanoparticles (NPs) has been particularly efficient in the CPP-mediated delivery of

7

oligonucleotides carrying multiple negative charges and therefore able to form electrostatic

8

complexes with cationic peptides. In addition, formation of peptide-based nanoparticles (PBN)

9

is a one-step process very easy to perform since no chemical modification is required to allow

10

NP assembly. Therefore formation of electrostatic complexes remain the most popular

11

application for CPP-mediated siRNA delivery, and some significant progresses have been made

12

both in vitro and in vivo by several groups using either natural or synthetic CPPs 13,14. Moreover

13

such stable complexes should provide greater ON-protection against nucleases.

14

Keystone of formation of non-covalent complexes is based on the balance of electrostatic and

15

hydrophobic interactions between peptides and oligonucleotide. Although the contribution of

16

arginine residues in the electrostatic interactions with nucleic acids is well described, the role

17

of tryptophan has been also emphasized as a main important feature in the design of CPPs.

18

Indeed, it has been well established that tryptophan residue play a key role in the interaction

19

with lipids because the tryptophan residue is able to insert at hydrophilic/hydrophobic interface

20

of lipid bilayer 15–17. Interaction with lipids is often related with cell penetrating mechanism and

21

the tryptophan residue seems to be largely involved in this process 18,19. In the same way, some

22

studies highlight the implication of GAG (Glycosaminoglycans) in cellular peptide mechanism

23

of entry through interaction with tryptophan residues 19,20. Furthermore, it has been shown for

24

other peptides that tryptophan residues could also play a role in siRNA complexation by

25

hydrophobic interactions with the minor groove of an oligonucleotide 21. For all these reasons

4


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1

many recent studies have focused on the numbers and on the position of these residues within

2

the CPP sequence 22,23.

3

In this context our last development investigating structure and activity of the RICK peptide

4

and its PEGylated form 11,24 pointed out the necessity of the presence of a tryptophan cluster.

5

Based on these analyses, two new peptide families were designed in order to investigate

6

modulations in their tryptophan and arginine residues content. These new peptides, also called

7

WRAP, for W- and R- rich Amphipathic Peptides, might have tryptophan residues either

8

scattered all along the sequence or clustered in one domain of the sequence. All the peptides

9

were then analyzed and tested for their abilities to “wrap’n roll” siRNA into peptide-based

10

nanoparticles and to induce efficient gene silencing. A combination of several biophysical and

11

cellular approaches allowed the identification of two WRAP peptides as new peptide leaders

12

for further peptide-based nanoparticles development for in vivo applications.

13 14

RESULTS AND DISCUSSION

15

Design of new amphipathic peptides for peptide-based nanoparticles formation.

16

Cell-penetrating peptides were discovered more than 20 years ago and have been extensively

17

studied for their innate properties and their way to interact with membranes and enter cells 25.

18

Although most of them are cationic and display a high propensity to involve electrostatic

19

interactions with negatively charged membranes, the involvement of other residues, such as

20

tryptophan, has also been described. In addition analyses of the amphipathic cell-penetrating

21

peptides revealed the ability to interact and associate with different cargoes to form peptide-

22

based nanoparticles. The primary/secondary structure of these peptides play a crucial role in

23

the distribution of hydrophilic/hydrophobic domains, resulting in self-assembly features 7.

24

Recently the role of secondary structure was also emphasized with the design of the RICK

25

peptide, a retro-inverso version of the CADY-K peptide and PEG-RICK, its PEGylated form

26

11,24,26.

In these works, structural investigations indicated the formation of a tryptophan cluster, 5



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1

highlighting the important role of the tryptophan residue. Indeed, tryptophan residues play a

2

key role in (1) peptide:oligonucleotide interactions by intercalation of the indole ring with the

3

minor groove of a double-stranded oligonucleotide

4

interaction between the arginine residues), (2) hydrophilic/hydrophobic interface of lipid

5

bilayer 15–17 and in cellular internalization 23,29.

27,28

(in complement to the electrostatic

6

Taking these observations into account, new peptides with various tryptophan

7

distributions, either scattered or clustered, were designed. Moreover these peptides were

8

conceived with the same number of Arginine residues in order to keep the same content of

9

positive charges. Sequences of the resulting W- and R-rich Amphipathic Peptides, or WRAP,

10

are reported in Table 1.

11 12

Table 1: Sequences used in this study. ID

SEQUENCES

AA

CHARGES

W

WRAP1

LLWRLWRLLWRLWRLL

16

5

4

WRAP2

AAWRAWRAAWRAWRAA

16

5

4

WRAP3

GGWRGWRGGWRGWRGG

16

5

4

WRAP4

LLRLLRWWRLLRLL

14

5

2

WRAP5

LLRLLRWWWRLLRLL

15

5

3

WRAP6

LLRLLRWWWWRLLRLL

16

5

4

13 14

WRAP1 to WRAP3 were newly designed based on our previous work

11,26,

by taking

15

into account their secondary amphipathic properties; i.e. helical conformation and structural

16

polymorphism. They consist in “scattered tryptophan” peptides where only hydrophobic

17

residues were varied from glycine to leucine, while maintaining the same W and R positions,

18

enabling adoption of an amphipathic α-helix. WRAP4 to WRAP6 were conceived from a

19

tryptophan-rich motif identified as a membrane penetrating domain 22,30. The central tryptophan

20

domain involved 2 to 4 tryptophan residues while flanking regions were maintained identical. 6


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1

As for WRAP1 to WRAP3, the number of arginine residues was maintained to keep the same

2

final number of positive charges. All the designed peptides were first investigated for their

3

structural properties.

4 5

Biophysical characterization of new amphipathic peptides.

6

To check structural properties of WRAPs, an in silico prediction of the 3D structure of each

7

peptide was undergone. Sequences of WRAP1 to WRAP6 were submitted to PEPstrMOD

8

server in a hydrophilic environment 31,32. After computation, the peptides models generated by

9

PEPstrMOD revealed that most of the peptides adopted α-helical structure, with the exception

10

of WRAP3 (Figure 1). WRAP1 and WRAP5 seemed to present a more pronounced helicity

11

than the other models. Moreover helices were clearly secondary amphipathic in the case of

12

WRAP1 and WRAP2. In contrary, only WRAP3 was computed in a random coil, which is in

13

agreement with the fact that glycine is known to prevent folding and to disturb helical

14

conformation abilities 33.

15 16

Figure 1. Prediction of the 3D structure of the used amphipathic peptides by PEPstrMOD

17

under hydrophilic condition. Surface and ribbon representation of the peptides. For the

18

surface representation two views are given to determine their amphipathic character: along the

19

N-terminal to C-terminal axis and after a 90° turn. Tryptophan residues are given in red,

20

arginine in blue and the others in yellow. 7



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1 2

The predicted structures were then compared to experimental investigations. Circular

3

dichroism spectra (CD) were recorded for peptides in solution, for peptides in the presence of

4

siRNA and then after addition of liposomes (Table 2, Figure S1). The first observation

5

concerned WRAP3 whose CD signals were too low to identify any secondary structure (Figure

6

S1). This was mainly due to the absence of asymmetric carbon in glycine residues representing

7

50% of the sequence and lacking any polarization signature and CD signals whatever the

8

environment; i.e. solution, presence of siRNA or liposomes. In contrast the other peptides gave

9

characteristic CD profiles. Spectra of WRAP1 and WRAP2 free in solution were mainly

10

characterized by a minimum at 198 nm, suggesting a main random coiled configuration, even

11

if the presence of helical contributions cannot be excluded for WRAP1 (Figure S1). WRAP4,

12

WRAP5 and WRAP6 displayed spectra with two minima; i.e. at 202 nm and 227nm, supporting

13

the existence of a turn conformation with a cluster of tryptophan

14

negative contribution at 227 nm was observed for WRAP5 and WRAP6, suggesting a slightly

15

different orientation of the tryptophan residues due to their increased number within the cluster,

16

compared to WRAP4 (Figure S1) 36.

17

8


34,35.

In addition, a stronger

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1


Table 2: Conformational analyses of amphipathic peptides by circular dichroism. ID

PEPTIDE +siRNA +neutral LUVs

+charged LUVs

WRAP1

rc/helix

helix

helix

helix

WRAP2

rc

helix

helix

helix

WRAP3

low signal

low signal

low signal

low signal

WRAP4

turn/rc

helix

helix

helix

WRAP5

turn

helix

helix

helix

WRAP6

turn

helix

helix

helix

2

Footnotes: Random coiled (rc) conformation has a positive band at 212 nm and a negative one

3

around 195 nm. Helical conformation is characterized by two minima at 207 nm and 222 nm

4

and one maximum around 191 nm. Turn conformation has two minima at 202 nm and 227 nm.

5

CPP:siRNA molar ratio of 20 (R = 20) and Lipid:CPP of 10 (r = 10) with a CPP concentration

6

of 40 µM.

7 8

In the presence of siRNA, at the exception of WRAP3, all WRAP underwent a

9

conformational change and adopted a more or less pronounced α-helical structure, characterized

10

by CD spectra with a maximum at 191 nm and two minima at 207 and 222 nm, respectively

11

(Table 2, Figure S1). However, while WRAP1, WRAP5 and WRAP6 showed clear helical

12

profiles, those of WRAP2 and WRAP4 were less defined (Figure S1). Finally two types of

13

liposomes were added to the peptide/siRNA mixtures in order to check the effect of membrane

14

mimicking environment on peptides structure. Whereas neutral LUVs (Large Unilamellar

15

Vesicles) did not significantly modify peptides structure, negatively charged LUVs induced a

16

deformation of the CD α-helical spectra characterized by a modification of the 222/207 nm

9



1

ratio, suggesting an increase in helical content and probably helix aggregation as already

2

observed 18. This effect was not observed for WRAP2 and WRAP4.

3 4

Colloidal characterization of the amphipathic peptides.

5

The conformational changes detected by CD suggest that nearly all WRAP peptides interact

6

with siRNA. To assess complexation of siRNA, the formation of WRAP:siRNA complexes

7

was investigated by agarose shift assay (Figure 2). siRNA alone migrated into the agarose gel

8

(=100% signal) but when complexed with CPPs, the peptides prevent oligonucleotide migration

9

in a molar ratio-dependent manner. WRAP1, WRAP4, WRAP5 and WRAP6 clearly complexed

10

siRNA in a similar manner with an optimal peptide:siRNA molar ratios of R = 20 and R = 40.

11

WRAP2 and WRAP3 showed low and no complexation properties, respectively, even at R =

12

40. This observation might be correlated to the slight conformational changes in the presence

13

of siRNA as observed in CD.

14

To become biologically active, WRAP:siRNA nanoparticles should be stable enough

15

for the transport into the cells but, subsequently, the siRNA must be released from the

16

complexes once delivered into cells. To have an insight into these characteristics in the case of

17

WRAP1:siRNA and WRAP5:siRNA complexes, nanoparticles were formed at R = 20, and then

18

incubated with varying amounts of a negatively charged heparin sodium (heparin) as siRNA

19

competitor molecule (Figure S2). Heparin was able to displace siRNA from both WRAP

20

nanoparticles, indicated by the observed free siRNA fraction. However, WRAP1 nanoparticles

21

release the siRNA at a lower heparin ratio compared to WRAP5 suggesting lower electrostatic

22

forces between the WRAP1:siRNA complex.

10


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1 2

Figure 2. Evaluation of the capacity of the amphipathic peptides to form complexes in the

3

presence of siRNA by gel shift assay. (A) Pre-formed WRAP:siRNA complexes (constant

4

siRNA concentration of 10 µM, CPPs at indicated molar ratio (= R)) were analyzed by

5

electrophoresis on agarose gel (1% wt/vol) stained with GelRed. Data represent: mean ± SD,

6

with n = 3 independent experiments. (B) Transmission electron microscopy (TEM) images of

7

WRAP1 and WRAP5-based nanoparticles. Images of WRAP:siRNA nanoparticles in ultra-

8

pure water (R = 20, 40 µM WRAP). Scale bars correspond to 1 µm and 100 nm for the

9

magnified images. Corresponding DLS Spectra are shown in Figure S3A.

10 11

Colloidal features of WRAP:siRNA complexes (5 % glucose, R = 20) were

12

characterized in terms of nanoparticle size and homogeneity by dynamic light scattering (DLS).

13

Intensity measurements (%) revealed that WRAP1, WRAP4, WRAP5 and WRAP6 formed

14

peptide-based nanoparticles with diameters between 80 - 200 nm and with polydispersity index 11



Page 12 of 35

1

(PDI) around 0.3 (Table 3). The nanoparticle size distribution based on number (%) suggests

2

the presence of smaller nanoparticles in addition to those found by the intensity-based size

3

distribution. These smaller particles have diameters between 20 - 50 nm for the four

4

WRAP:siRNA (Table 3). More importantly, both measured size values did not change

5

significantly even after 72 h (or more) storage at 4°C (Figure S3B), which is in agreement with

6

previous experiments for other PBNs formulated with CADY-K or RICK

7

WRAP2 and WRAP3 showed mean sizes higher than 1,000 nm, both in intensity (%) and

8

number (%) confirming that these two peptides were not able to form stable nanoparticles. For

9

that reason, they were excluded for further evaluations.

11,26.

In contrast,

10 11

Table 3: WRAP characterization by DLS measurements. Mean size (nm) PdI

ZP (mV)

I (%)

Nb (%)

WRAP1

73.3 ± 7.4

26.5 ± 4.1

0.38 ± 0.08

42.2 ± 4.5

WRAP2

>1,000

>1,000

0.48 ± 0.15

n.d.

WRAP3

>1,000

>1,000

0.68 ± 0.12

n.d.

WRAP4

193.5 ± 34.9

47.2 ± 2.8

0.38 ± 0.05

n.d.

WRAP5

80.0 ± 4.9

35.7 ± 7.3

0.29 ± 0.05

28.8 ± 0.9

WRAP6

122.0 ± 71.5

23.6 ± 7.9

0.44 ± 0.11

n.d.

12

Footnotes: All WRAP:siRNA complexes were formed at R = 20 using a siRNA concentration

13

of 500 nM in an aqueous solution of 5 % glucose for mean size acquisition and in an aqueous

14

solution of 5% glucose with 5 mM NaCl for WRAP1 and 1 mM NaCl for WRAP5 for zeta

15

potential (ZP) evaluation. n ≥ 2 independent formulations (3 measures per run). n.d.: not

16

determine, I: Intensity; Nb: Number; PdI: Polydispersity Index.

17

12


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1

Nanoparticle surface charge which is quantified by zeta potential (ZP) measurements is

2

a good indicator to predict its behavior in biological environment, (Table 3). WRAP1:siRNA

3

and WRAP5:siRNA exhibit a positive charge surface of 42.2 ± 4.5 mV and 28.8 ± 0.9 mV,

4

respectively. These values were in agreement with previous observation with CADY-K 26 and

5

RICK peptides 11. To further address nanoparticle shape, we carried out transmission electronic

6

microscopy (TEM) measurements as exemplified for WRAP1:siRNA and WRAP5:siRNA

7

(Figure 2B). TEM images enabled identification of large branching “beads necklace”-like

8

particles around 100 nm a well as smaller globular nanoparticles with a mean size < 100 nm.

9

These latter could be correlated to the nanoparticles with diameters between 20 - 50 nm as

10

proposed by the size distribution based on number (%). Both TEM profiles are quite similar to

11

what we observed for other nanoparticle forming CPPs such as RICK

12

Considering experimental differences between DLS (solvated sample) and TEM imaging (dried

13

sample), both observed size distributions are in agreement for both WRAP nanoparticles.

11

or Pepfect14

37.

14 15

Characterization of the cellular activity of WRAP:siRNA nanoparticles in a luciferase

16

screening.

17

Cellular activity of WRAP-based nanoparticles was evaluated in a luciferase assay 11,24.

18

screening, as previously described

The knock-down efficiency of the different

19

WRAP:siRNA nanoparticles was performed on U87 cell line stably transfected for constitutive

20

expression of FLuc/NLuc reporter genes (U87-FRT-CMV/Fluc-CMV/iRFP-IRES-NLuc).

21

First of all, we investigated the optimal molar ratio between each peptide and the siRNA

22

(constant concentration of 20 nM) required for an effective luciferase knock-down (Figure 3A).

23

For WRAP1, WRAP5 and WRAP6, we observed a significant luciferase knock-down >70 %

24

at R = 20 which was slightly increased at R = 40. In parallel no relative cytotoxicity was

25

observed with the nanoparticle treatments. However because WRAP4:siRNA revealed a knock13



1

down efficiency only at R = 40 with a great variability, this nanoparticle was excluded from the

2

further evaluations.

3

Thereafter, the optimal siRNA concentration was elucidated by using the optimal

4

peptide:siRNA molar ratio of R = 20 (Figure 3B). The control conditions such as the peptides

5

alone, the siRNA-Fluc alone did not induced any luciferase silencing. In contrast, the three

6

nanoparticles (WRAP1, WRAP5 and WRAP6) displayed a clear dose-response relationship

7

showing a relevant knock-down efficiency with an estimated IC50 value of around 7.5 nM. By

8

looking at the siRNA-FLuc transfection using RNAiMAX, it seems that the silencing efficiency

9

is more pronounced compared to the WRAP conditions. However, this effect is in part due to a

10

cytotoxic effect (20%) whereas no cytotoxicity was detected for all WRAP:siRNA condition

11

(>5% relative cytotoxicity). For that reason, further experiments were performed using a siRNA

12

concentration of 20 nM if not otherwise mentioned.

13 14

14


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1 2

Figure 3: Evaluation of WRAP:siRNA nanoparticles delivery on cells. (A) Relative Luc

3

activity (% FLuc/NLuc) and relative toxicity (LDH quantification) after transfection with

4

WRAP:siFLuc complexes in U87 cells at different molar ratios R and for a siFLuc

5

concentration of 20 nM. (B) Relative Luc activity (% FLuc/NLuc) and relative toxicity (LDH

6

quantification) after transfection with WRAP:siFLuc nanoparticles at different siFLuc

7

concentrations on U87 cells. WRAP1, WRAP5 and siFLuc correspond to peptides and siRNA

8

alone at 400 nM and 20 nM, respectively. RNAimax is associated to siFLuc at 20 nM.

9

Abbreviations: R = molar ratio, siFLuc = FLuc siRNA, siSCR = scrambled siRNA, N.T. = non-

15



1

treated cells, Ctrl = Controls, n.d. = not determined. Data represent: mean ± SD, with n = 2

2

independent experiments in triplicates.

3 4

Selection of WRAP1 and WRAP5 nanoparticles as novel transfection tools.

5

Despite a rather equivalent biological effect of WRAP1, WRAP5 and WRAP6

6

(Figure 3), WRAP1 and WRAP5 were selected for further cellular investigations and

7

developments, since these peptides showed a lower mean size and polydispersity index

8

compared to WRAP6 (Table 3). We believe that small and homogeneous nanoparticles could

9

be an important issue in a future in vivo development.

10

In this context, to simulate an in vivo administration, silencing efficiency of both

11

peptides was evaluated in the presence of serum. Using the optimal siRNA-FLuc concentration

12

of 20 nM, strong FLuc inhibitions (up to 60%) were observed for both peptides (Figure S4)

13

which were similar to the condition without serum due to the overlapping error bars (p = ns),

14

proving that their cellular activity was not completely aborted by serum. The slide reduction in

15

luciferase silencing could be due to a masking phenomenon by the serum proteins reducing the

16

interaction of the nanoparticle with the plasma membrane. On the other hand, another

17

possibility could be a destabilization of the WRAP nanoparticle by the serum proteins. To avoid

18

such phenomenon, the WRAP peptides could be synthesized as retro-inverso isoforms as

19

reported for the RICK peptide 11.

20

Both peptides were then tested on different cell lines in order to estimate their potential

21

as ubiquitous transfection agents. A FLuc gene silencing of 50 % to 80 % was detected at 20

22

nM siRNA for U87, KB, MCF7, HuH7, Neuro2a and MDA-MB-231 cells, at 50 nM siRNA

23

for CMT93, HT29 and RM1 cells and at 100 nM siRNA for GL261 cells. In parallel, measured

24

cytotoxicity (LDH) revealed no relevant effect of the nanoparticles on the viability of all used

25

cells. All together, these results suggest that some cell lines required higher siRNA doses to 16


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1

reach a comparable knock-down (Figure 4A), probably because of different membrane

2

composition or different level of Firefly luciferase expression. In addition, the observed

3

cytotoxicity ( 2 independent experiments in triplicates. (B) CDK4 silencing after WRAP:siRNA

6

nanoparticle transfection in U87 cells at 20 nM siRNA-CDK4 concentration (R = 20) for

7

different incubation times in min. Abbreviations: siSCR = scrambled siRNA, N.T. = non-

8

treated cells, Ctrl = Controls. Data represent: mean ± SD, with n = 2 independent experiments

9

in duplicates. (C) Visualization of cellular distribution of Cy5-labelled siRNA vectorized by

10

WRAP1 or WRAP5 nanoparticle formulation (siRNA-Cy5 =20 nM, R = 20) in living U87 cell

11

line after an incubation of 1 h. Nuclei were labeled using Hoechst 33342 dye. White bars

12

represent 20 μm. (D) Comparison of WRAP1:siRNA-Cy3b and WRAP5:siRNA-Cy3b

13

internalization kinetics by spinning disk (siRNA-Cy3b = 20 nM, R = 20) with image recorded

14

every 5 s for 1200 s. Thereafter, the percentage of mean grey values was plotted against the

15

time (Data represent: mean ± SD; 2–5 individual counted cells from 2 to 3 independent

16

experiments).

17

20


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1

To obtain more information about the internalization of the WRAP-based nanoparticles,

2

we performed confocal microscopy experiments. A first control experiment with siRNA alone

3

at the same concentration revealed no observable siRNA internalization (Figure S5A). In

4

contrast, representative images of the siRNA cellular distribution revealed a punctuated pattern

5

(Figure 5C) comparable to that previously observed after 1 h incubation for other peptide-

6

based nanoparticles such as CADY-K:siRNA or RICK:siRNA

7

the CADY-based nanoparticles a punctuated pattern in the cellular cytoplasm even if the

8

internalization mechanism was reported to be endocytosis independent 39. In the same way, we

9

do not observe any co-localization with any endocytosis molecular markers (data not shown

10

11,26.

Indeed, it was shown for

and ongoing works).

11

Afterwards, we performed experiments using a Spinning Disk confocal microscope

12

(Figure 5D) to evaluate the internalization kinetics of both WRAP-based nanoparticles relied

13

on the intracellular accumulation of the Cy3b-labelled siRNA (z-scan acquisition). However,

14

using this method, it is possible that we slightly overestimate the fluorescence signal, because

15

it is difficult to avoid the quantification of Cy3b-labelled siRNA at the cell surface.

16

Representative images of the siRNA internalization over the time are shown in Figure S5B

17

exemplified by the WRAP1:siRNA-Cy3b incubation. As shown in the graphical representation,

18

we observe that the siRNA delivered by both nanoparticles reached the maximal fluorescence

19

(95 %  5 %) after ~15 min of incubation (900 s to 945 s). The IC50 values of cell-associated

20

and cell-internalized Cy3b-siRNA (50 %) were obtained after 240 s and 185 s incubation for

21

WRAP1 and WRAP5 nanoparticles, respectively. This internalization is very fast compared to

22

previously analyzed NPs, such as RICK:siRNA or 20 % PEG-RICK:siRNA reaching the

23

internalization maximum after 120 min of incubation 11,24. Altogether, the observed fast siRNA

24

internalization kinetics for both WRAP-based nanoparticles identified by confocal microscopy

25

could be an explanation for the knock-down efficacy obtained for 5 min of WRAP:siRNA

26

incubation (Figures 5A and 5B). 21



1 2

CONCLUSION

3

We designed two new families of cell-penetrating peptides for the cellular siRNA

4

delivery, differing in the position of tryptophan residues in the primary peptide sequence:

5

scattered distribution for the WRAP1, WRAP2 and WRAP3 peptides or clustered for the

6

WRAP4, WRAP5 and WRAP6 peptides. By modeling or experimental determination all

7

peptides except WRAP3 are able to adopt a specific structure. They mainly fold in an

8

amphipathic helical conformation in the presence of siRNA or lipids which is a requirement for

9

membrane interaction as demonstrated for other cell-penetrating or anti-microbial peptides.

10

Based on fine-tuned screening implicating biophysical and molecular biological

11

methods, we rapidly selected WRAP1 and WRAP5 as the peptide leads of each family. Both

12

siRNA-loaded nanoparticles induced an optimal luciferase silencing at very low siFLuc

13

concentration (20 nM) without any cytotoxicity on U87 glioblastoma cells, but also in various

14

cells lines even if the siRNA concentration needed to be adjusted for some cell lines.

15

Internalization process of both siRNA-loaded nanoparticles is very fast and kinetics obtained

16

with spinning disk experiments showed that estimated IC50 values of cell-associated and cell-

17

internalized siRNA were obtained after 240 s and 185 s of incubation for WRAP1 and WRAP5

18

nanoparticles, respectively. This is in agreement with the fact that luciferase gene silencing

19

could be observed in a relevant manner even after only 5 min of nanoparticle incubation.

20

Altogether, WRAP1 and WRAP5 are cell-penetrating peptides composed of

21

combinations of three amino acids (W, R and L) with length of 15 to 16 amino acids and with

22

a net positive charge of 5. This new series of peptides shows a rather similar transfection

23

efficacy compared to CADY or RICK, but they are much shorter (25%) and made of 3 amino

24

acids only (versus 7 amino acids for CADY-K or RICK), making easier their synthesis. For

25

siRNA transfection tryptophan residues are important: they must be clustered such in WRAP5 22


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1

along the primary sequence or clustered through secondary structure adoption such as in

2

WRAP1. This is further confirmed by WRAP3 which is not able to form a secondary structure

3

resulting in the absence of nanoparticle formation (Figure 1, Table 3). Therefore, both

4

peptides, WRAP1 and WRAP5, are efficient for delivering siRNA into cells without any

5

cytotoxicity.

6

In addition, we are now working on their mechanism of cellular internalization, strongly

7

suggesting a direct passage through the plasma membrane. We are also investigating as well as

8

on their potential in vivo applications upon their functionalization with both cell-targeting

9

sequences and PEG entities.

10 11

MATERIALS AND METHODS

12

Materials. Dioleylphosphatidylglycerol (DOPG) and dioleylphosphatidylcholine

13

(DOPC) phospholipids, cholesterol (Chol), sphingomyelin (SM) were purchased from Avanti

14

Polar Lipids. Large Unilamellar Vesicles (LUVs) were prepared by the extrusion method from

15

a lipid mixture of DOPC/SM/Chol (2:2:1) as previously reported 11. Peptides were purchased

16

from LifeTein (sequences in Table 1). The different siRNA sequences (unlabeled and Cy5-

17

labeled) were purchase from Eurogentec. Cy3b-labeled siRNA was purchased from

18

BioSynthesis. The following siRNA were used: anti-firefly luciferase (siFLuc): 5’-CUU-ACG-

19

CUG-AGU-ACU-UCG-AdTdT (sense strand) and the corresponding scrambled version of the

20

anti-luciferase (siSCR): 5’-CAU-CAU-CCC-UGC-CUC-UAC-UdTdT-3’ (sense strand) as

21

well as the anti-cyclin dependent kinase 4 (siCDK4) siRNA based on 40: 5′-CAG-AUC-UCG-

22

GUG-AAC-GAU-GdTdT-3′ (anti-sense strand) and the scrambled version: 5′-AAC-CAC-

23

UCA-ACU-UUU-UCC-CAA-dTdT-3′ (anti-sense strand). The siRNA stock solutions were

24

prepared in RNase-free water. Lipofectamine ® RNAiMAX was purchased by Life

25

Technologies. 23



1

The following stably transfected Firefly luciferase cell lines were used: Human

2

glioblastoma cell line (U87 MG) 24 and murine prostate carcinoma cell line (RM1) 41; Human

3

ectum carcinoma cell line (CMT93) (ATCC) and murine gliomablastoma cell line (GL261)

4

(generously provided by Prof L. Zitvogel, Villejuif, France) were genetically modified using

5

the Flip-In system (Invitrogen) with pcDNA5/FRT/CMV/FLuc; Murine neuroblastoma cell line

6

(Neuro2a) 42, keratin forming cell line derived from Hela (KB) 43, and human hepato carcinoma

7

cell line (HuH7) 44 were kindly provided by Prof. E. Wagner (Ludwig Maximilian University

8

of Munich); Human breast carcinoma cell line (MCF7) and human breast adenocarcinoma cell

9

line (MDA-MB-231) both generated using a CMV-FLuc-neomycin were kindly provided by P.

10 11 12

Balaguer (IRCM). Peptide structure prediction. PEPstrMOD server was used to predict the secondary structure of WRAPs (http://osddlinux.osdd.net/raghava/pepstrmod/) 31,32.

13

Nanoparticle formation: Stock solutions of WRAP peptides were prepared in pure

14

water. Nanoparticle formation was performed in pure water supplemented by 5% glucose

15

(Sigma-Aldrich) by adding first the peptide and then the corresponding amount of siRNA for

16

the desired molar ratio (R) at room temperature. As an example, a molar ratio of 20 (R 20)

17

corresponds to 20-fold higher molar concentration of peptides compared to the siRNA.

18

Phospholipid liposome preparations: Large Unilamellar Vesicles (LUV) were

19

prepared by the extrusion method using a lipid mixture of DOPC/SM/Chol (40/40/ 20,

20

mol/mol/mol) for neutral liposomes and using only DOPG for charged liposomes. All lipids

21

were dissolved in a chloroform/methanol mixture (3/1, v/v) and gently mixed. The solvent was

22

evaporated under high vacuum for at least 1 h to remove residual solvents. The lipids were

23

hydrated in pure water through vortex mixing and extruded through a 0.1 mm polycarbonate

24

membrane (Avanti Polar Lipids). For DOPC/SM/Chol LUVs, lipids concentration was checked

25

with phospholipid assay from WIKO by evaluating choline from DOPC and Sphingomyelin.

24


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1

Concentrations of DOPG lipids were obtained by calculation. Finally, LUV preparations were

2

equilibrated overnight at 4°C and used the day after.

3

Circular dichroism (CD) measurements. CD spectra were recorded on a Jasco 810

4

(Japan) dichrograph in quartz suprasil cells (Hellma) with an optical path of 1 mm for peptide

5

in solution or in the presence of liposomes vesicles. Same concentrations of peptide (40 µM)

6

were used for each condition. siRNA solution was first added to reach CPP:siRNA molar ratio

7

of 20 (R = 20) and in a second step LUVs of DOPG or of DOPC/SM/Chol were added to reach

8

lipid:CPP molar ratio of 10 (r = 10). Spectra were obtained from 3 accumulations between 190

9

and 260 nm with a data pitch of 0.5 nm, a bandwidth of 1 nm and a standard sensitivity.

10

Agarose gel shift assay. CPPs:siRNA complexes [siRNA = 10 µM in 20 µL of an

11

aqueous solution containing 5% glucose] were formed at different ratios and pre-incubated for

12

30 min at room temperature. Then 20 µL were loaded on an agarose gel (1 % w/v, Sigma-

13

Aldrich) and electrophoresis was performed at 50 V for 25 min. To visualize the siRNA the

14

agarose gel was stained with GelRed (Interchim) for UV detection. The signal intensities were

15

quantified after background subtraction using Image J software (gel analyze tool). Each band

16

intensity corresponding to a distinguished condition is then normalized to the band intensity of

17

the siRNA alone (= 100%): Relative fluorescence (%) = fluorescence intensity

18

(condition x)/fluorescence intensity (siRNA alone) x 100.

19

Dynamic light scattering (DLS) and Zeta potential (ZP). CPPs:siRNA nanoparticles

20

(CPP = 10 µM, siRNA = 500 nM, R = 20) were evaluated with a Zetasizer NanoZS (Malvern)

21

in terms of mean size (Z-average) of the particle distribution and of homogeneity (PdI). Zeta

22

potential was determined in 5% glucose with 1 mM or 5 mM NaCl. All results were obtained

23

from three independent measurements (three runs for each measurement at 25°C).

24

Transmission Electron Microscopy (TEM). For transmission electron microscopy

25

(TEM), a drop of 5 µL of suspension of WRAP:siRNA (in pure water) was deposited on a

26

carbon coated 300 mesh grid for 1 minute, blotted dry by touching filter paper and then placed 25



1

on a 2% uranyl acetate solution drop. After 1 minute the excess stain was removed by touching

2

the edge to a filter paper, the grid was dried at room temperature for few minutes and examined

3

using using a Jeol 1400Plus Transmission Electron Microscope operating at 100 kV

4

accelerating voltage. Data were collected with a High sensitivity sCMOS JEOL Matataki Flash

5

camera. Culture conditions. U87 MG, human glioblastoma stably transfected with firefly and

6

24

7

Nanoluc luciferases (FLuc-NLuc) encoding plasmid

were grown in a complete medium:

8

DMEM with GlutaMAX™ (Life Technologies), penicillin/streptomycin (Life Technologies),

9

10 % heat-inactivated fetal bovine serum (FBS, PAA), non-essential amino acids NEAA 1 X

10

(LifeTechnologies). Furthermore, hygromycine B (Invitrogen, 50 µg/mL) was added as

11

selection antibiotic. Keratin forming cell line derived from Hela (KB), murine neuroblastoma

12

cell line (Neuro2a), human hepato carcinoma cell line (HuH7), human breast carcinoma cell

13

line (MCF7) were grown in the same complete medium without the addition of selection

14

antibiotics. Murine prostate carcinoma cell line (RM1) was grown in the same complete

15

medium with the addition of blasticidin S (Invitrogen, 10 µg/mL) and human colon carcinoma

16

cell line (HT29), rectum carcinoma cell line (CMT93) and murine gliomablastoma cell line

17

(GL261) with hygromycine B (Invitrogen, 1,400 µg/mL, 500 µg/mL and 150 µg/mL,

18

respectively) as selection antibiotics. Human breast adenocarcinoma cells (MDA-MB-231)

19

were grown in the same complete medium with the addition of G418 (Invitrogen, 100 µg/mL)

20

as selection antibiotic. All the cells were maintained in a humidified incubator with 5% CO2 at

21

37°C.

22

Transfection experiments. For Luciferase assay, the different cells (5,000 cells per

23

well) were seeded 24 h before experiment into 96-well plates using the corresponding medium

24

as described above. The next day, nanoparticles were formed by mixing siRNA and CPPs (equal

25

volumes, “siRNA solution added to the CPP solution”) in 5 % glucose water, followed by an

26

incubation of 30 min at 37 °C. In the meantime, the growth medium covering the cells was 26


Page 26 of 35

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1

replaced by 70 µL of fresh pre-warmed serum-free DMEM. 30 µL of the nanoparticle solutions

2

were added directly to the cells and after 1.5 h of incubation, 100 μL DMEM supplemented

3

with 20 % FBS (final FBS concentration = 10 %) were added to each well without withdrawing

4

the transfection reagents. Cells were then incubated for another 36 h and finally lysed for the

5

luciferase detection. For transfection in the presence of serum, cells were incubated with

6

nanoparticles directly diluted in DMEM supplemented with 10 % FBS. Lipofectamine®

7

RNAiMAX was used as positive control following the manufacture protocol recommendations.

8

For Western blot assay, 75,000 U87 cells were seeded 24 h before experiment into 24-

9

well plates. For standard incubation, the cells were incubated with 175 µL of fresh pre-warmed

10

serum-free DMEM + 75 µL of the nanoparticle solutions. After 1.5 h of incubation, 250 μL

11

DMEM supplemented with 20 % FBS (final FBS concentration = 10 %) were added to each

12

well without withdrawing the transfection reagents. Cells were then incubated for another 24 h

13

and finally lysed for CDK4 western blotting detection.

14

For microscopy experiments, 300,000 U87 cells were seeded 24 h before imaging into

15

glass bottom dishes (FluoroDish, World Precision Instruments). Before experiments, cells were

16

washed twice with D-PBS and covered with 1,600 μl of complete medium. Afterwards, 400 μL

17

of nanoparticles (peptide:siRNA; R = 20, siRNA = 20 nM), formulated in an aqueous solution

18

of 5% glucose were directly added on the cells as previously described 11.

19

Measurement of cell cytotoxicity. Evaluation of cytotoxicity induced by the

20

nanoparticles was performed using Cytotoxicity Detection KitPlus (LDH, Roche Diagnostics)

21

on 50 µL of supernatant, following the manufacturer instructions. After the nanoparticle

22

incubation (after 36 h for the luciferase assay or 20 h for the Western blot evaluation), at least

23

one well is used as LDH positive control (100% toxicity) by adding Triton X-100 (Sigma-

24

Aldrich) to a final concentration of 0.1% (~15 min incubation at 37°C). Afterwards, 50 µL

25

supernatant of each well were transferred in a new clear 96-well plate. 50 µL of the “dye

26

solution/catalyst” mixture were added to the supernatant and incubated in the darkness for 30 27



1

min at room temperature. Reaction was stopped by adding 25 µL of HCl (1 N) to each well

2

before measuring the absorption at 490 nm. Relative toxicity (%) = [(exp. value – value non-

3

treated cells) / (value triton – value non-treated cells)] x 100.

4

Luciferase reporter gene silencing assay. The evaluation of siRNA delivery using the

5

different vectors was carried out by measuring the remaining luciferase firefly (FLuc) activity

6

in cell lysates. Briefly, after 36 h, the medium covering the cells was carefully removed and

7

replaced by 50 µL of 0,5 X Passive Lysis Buffer (PLB; Promega). After 30 min of shaking at

8

4 °C, plates containing the cells were centrifuged (10 min, 1,800 rpm, 4°C) and 5 µL of each

9

cell lysate supernatant were finally transferred into a white 96-well plate. For the U87-FLuc-

10

NLuc cells, both luciferase activities were quantified using a plate-reading luminometer

11

(POLARstar Omega, BMG Labtech) using half-diluted Dual Luciferase Assay Reagents

12

(Promega) as reported in

13

(RLU) for each luciferase normalized first to non-treated cells (%FLuc and %NLuc) and then

14

normalized to the value of %NLuc to obtain the Relative Luc Activity (%FLuc/%NLuc).

15

For the other cell lines having only the FLuc expression, the luciferase activity was quantified

16

using a plate-reading luminometer using half-diluted Luciferase Assay Reagents (Promega).

17

The results were expressed as percentage of relative light units (RLU) normalized to the non-

18

treated cells (%FLuc).

24.

The results were expressed as percentage of relative light units

19

Western blotting. Transfected cells washed in PBS, and lysed in RIPA buffer [50 mM

20

Tris pH 8.0, 150 mM sodium chloride, 1% Triton X-100, 0.1% SDS (sodium dodecyl sulfate,

21

Sigma-Aldrich), including protease inhibitors (SigmaFAST, Sigma-Aldrich)]. Cells were

22

incubated for 5 min on ice with 130 µL/24-well lysis buffer. Thereafter, cells were scrapped

23

and transferred in a 1.5 mL tube. After 5 min on ice, the cell lysates were centrifuged (10 min,

24

16,100 g, 4 °C), supernatants were collected and protein concentrations were determined using

25

the Pierce BCA Protein Assay (ThermoFisher). Cell extracts were separated by 4-20% Mini-

26

PROTEAN® TGXTM Precast Gel (Bio-Rad). After electrophoresis, samples were transferred 28


Page 28 of 35

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1

onto Trans-Blot® Turbo™ Mini PVDF Transfer membrane (Bio-Rad). As antibodies (all from

2

Cell Signaling), we used anti-CDK4 rabbit mAb D9G3E, anti-Vinculin rabbit mAb E1E9V,

3

anti-mouse IgG HRP and anti-rabbit IgG HRP. Blots were revealed with the Pierce ECL plus

4

Western blotting substrate (ThermoFisher) on an Amersham imager 600 (GE Healthcare Life

5

Science). The signal intensities of the blots were quantified using ImageJ software. Each band

6

intensity corresponding to a distinguished condition is then normalized to the band intensity of

7

non-treated

8

(condition x)/intensity (N.T.) x 100.

cells

(N.T.)

(=

100%):

Relative

Signal

Intensity

(%)

=

intensity

9

Confocal microscopy. Cell imaging by confocal microscopy was performed on an

10

inverted Leica SP5-SMD microscope. Nanoparticles were formulated in an aqueous ultra-pure

11

solution containing 5% glucose to obtain a final siRNA-Cy5 concentration of 20 nM (with R

12

20, this corresponds to CPP = 400 nM). After the addition of the nanoparticles, cells were

13

incubated for 1 h in a humidified incubator with 5% CO2 at 37 °C. 10 min before the end of the

14

incubation, Hoechst 33342 dye (Sigma-Aldrich) was added to the cell for nucleus labeling.

15

Afterwards, cells were washed twice with D-PBS and covered with FluoroBrite DMEM

16

medium (Life Technologies). Images were acquired using a lens 63x/1.4 NA oil. For all

17

acquisition settings, the main excitation sources for confocal mode was a diode 405 nm and a

18

helium/neon laser 633nm. The parameters speciﬁc for each fluorophore were: Hoechst dye,

19

excited at 405 nm, detected between 415 nm - 485 nm; Cy5 excited at 633 nm and detected

20

between 655 nm - 685 nm. Image acquisition was done sequentially to minimize crosstalk

21

between the ﬂuorophores. Each confocal image was merged and adjusted with the same

22

brightness and contrast parameters using the ImageJ software.

23

Spinning disk experiments were performed on an inverted microscope (Nikon Ti

24

Eclipse) coupled to a spinning disk (ANDOR) system to study the internalization kinetics of

25

the nanoparticles inside living cells. Nanoparticles were formulated in an aqueous ultra-pure

26

solution containing 5% glucose to obtain a final siRNA-Cy3b concentration of 20 nM (with R 29



1

20, this corresponds to a CPP concentration of 400 nM). After the addition of the nanoparticles,

2

cells were maintained at 37 °C by a cage incubator (Okolab) and images were directly recorded

3

every 5 s for 1,200 s with an EM-CCD camera (iXon Ultra) and projected with the Andor IQ3

4

software. Images were acquired using a lens 60x Plan Apo lambda 1.4 NA oil. Acquisitions

5

were performed using an excitation filter QUAD 561 nm and an emission filter 607/36 nm on

6

an external wheel. Exposition time was set to 0.2 sec with a laser power of 1%. Multi-position

7

acquiring was performed in a z-scan mode (11 z-scans with a step size of 0.5 µm). All images

8

were adjusted with the same brightness and contrast parameters with the ImageJ software.

9

Thereafter, with the ImageJ software as well as, the overall fluorescence intensity (given as

10

grey value) of the whole image in each z-step positions was acquired after background

11

subtraction (radius of 10 pixels). For the graphical representation, variations in fluorescence

12

intensity were normalized between the values at the beginning (t 0 s= 0% = auto-fluorescence

13

of the cell) and at the end of the measurement (t 1,200 s = 100%). Two to three independent

14

experiments were performed with up to seven analyzed positions per dish including each 11 z-

15

scans.

16 17 18 19

ACKNOWLEDGEMENTS

20

We thank Sylvain De Rossi (Montpellier RIO imaging microscopy platform) for technical

21

advices and Coralie Genevois (Bordeaux) for cell lines generation. This work was supported

22

by the Fondation pour la Recherche Médicale (DBS 20140930769), the Labex TRAIL

23

TARGLIN (ANR-10-LABX-57) and the Centre National de la Recherche Scientifique.

24 25 26

30


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1

ASSOCIATED CONTENT

2

Supporting Information:

3

Supporting Information is available free of charge on the ACS Publications website at DOI:

4

Supplementary Material:

5

This document contains the CD spectra of the amphipathic peptide, the evaluation of the heparin

6

effect on the siRNA-loaded nanoparticle destabilization by gel shift assay, the DLS size

7

distributions of WRAP1:siRNA and WRAP5:siRNA (R = 20 with 500 nM siRNA) expressed

8

in percentage of intensity, results on the efficiency of WRAP:siRNA nanoparticles in the

9

presence of serum and confocal microscopy images.

10 11

AUTHOR INFORMATION

12

Corresponding Author

13

* [email protected]

14 15

Author Contributions

16

The manuscript was written through contributions of all authors. All authors have given

17

approval to the final version of the manuscript.

18 19

CONFLICT OF INTEREST

20

The author declare no conflict of interest

21 22

REFERENCES

23 24 25 26 27 28

(1) (2)

de Fougerolles, A.; Vornlocher, H.-P.; Maraganore, J.; Lieberman, J. Interfering with Disease: A Progress Report on siRNA-Based Therapeutics. Nat. Rev. Drug Discov. 2007, 6 (6), 443–453. https://doi.org/10.1038/nrd2310. Ozcan, G.; Ozpolat, B.; Coleman, R. L.; Sood, A. K.; Lopez-Berestein, G. Preclinical and Clinical Development of siRNA-Based Therapeutics. Adv. Drug Deliv. Rev. 2015, 87, 108–119. https://doi.org/10.1016/j.addr.2015.01.007.

31



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(3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

(13)

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TABLE OF CONTENTS

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New peptide-based nanoparticles to rapidly and efficiently “Wrap’n Roll" siRNA into cells

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Authors: Karidia Konate, Marion Dussot, Gudrun Aldrian, Anaïs Vaissière, Véronique Viguier,

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Isabel Ferreiro Neira, Franck Couillaud, Eric Vivès, Prisca Boisguerin, Sébastien Deshayes*

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Keyword: siRNA delivery, cell penetrating peptides, nanoparticle, gene knock-down, cancer

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We designed new short tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP)

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as new delivery tool for siRNA delivery. Our results reveal that siRNA-loaded WRAP

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nanoparticles (> 100 nm) have significant potential for a fast, safe and efficient transfer of

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siRNA for diverse applications.

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Peptide-Based Nanoparticles to Rapidly and Efficiently âWrap 'n Roll

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