Effect of the Aspect Ratio of Coiled-Coil Protein Carriers on Cellular

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Effect of the Aspect Ratio of Coiled-Coil Protein Carriers on Cellular Uptake Norihisa Nakayama, Sho Takaoka, Megumi Ota, Kentaro Takagaki, and Ken-Ichi Sano Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02616 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 4, 2018

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Effect of the Aspect Ratio of Coiled-Coil Protein Carriers on Cellular Uptake Norihisa Nakayama†, Sho Takaoka¶, Megumi Ota¶, Kentaro Takagaki¶, and Ken-Ichi Sano†, §, * † Graduate School of Environmental Symbiotic System Major, Nippon Institute of Technology, Miyashiro, Saitama 345-8501, Japan ¶ BioMimetics Sympathies Inc., Aomi, Koto-Ku, Tokyo 135-0064, Japan §Department of Applied Chemistry, Faculty of Fundamental Engineering, Nippon Institute of Technology, Miyashiro, Saitama 345-8501, Japan *Corresponding author. E-mail: [email protected]

KEYWORDS:

intracellular

delivery,

fibrous

protein,

reverse

biomimetics, -helical coiled-coil

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ABSTRACT

We

showed

cationic

previously

coiled-coil

that

a

rigid

artificial

and

protein

fibrous-structured

had

cell-penetrating

activity that was significantly greater when compared with a less-structured

cell-penetrating

peptide.

Nanomaterials

with

anisotropic structures often show aspect ratio-dependent unique physicochemical

properties,

as

well

as

cell-penetrating

activities. In this report, we have designed and demonstrated the cell-penetrating activity of a shorter cationic coiled-coil protein. An aspect ratio at 4.5:1 was found to be critical for ensuring

that

the

cell-penetrating cationic that

was

cationic

activity.

coiled-coil similar

to

coiled-coil At

protein a

an

protein

aspect

showed

ratio

showed of

3.5:1,

cell-penetrating

less-structured

short

strong the

activity

cationic

cell

penetrating peptide. Interestingly, at an aspect ratio 4:1, the cationic

coiled-coil

protein

exhibited

intermediate

cell-

penetrating activity. These findings should aid in the principle design of intracellular drug delivery carriers including coiledcoil artificial proteins, their derivatives and -helical cellpenetrating

peptides,

as

well

as

provide

a

framework

for

developing synthetic nanomaterials, such as metal nanorods and synthetic polymers.

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INTRODUCTION Asbestos

and

carbon

nanotubes,

in

which

having

rigid

and

anisotropic structure show enhanced cellular uptake ability and this is a factor complicit with their toxicity. toxicity,

which

is

termed

“fiber-toxicity”

1- 3

or

Despite the

“asbestosis”,

studies that develop carriers for cellular drug delivery systems (DDS) using fibrous materials have been extensively conducted.4-8 Such

interest

clearly

indicates

that

cellular

delivery

of

therapeutic molecules that cross the cell membrane is a critical subject in the development of DDS. Cellular delivery of pharmaceutics is also achieved with cellpenetrating peptides (CPPs).

9-11

The first CPP identified was the

TAT peptide derived from the human immunodeficiency virus (HIV) 12,13

TAT protein,

which has an amino acid composition that is

highly cationic. This physical feature spawned the production of an array of synthetic cationic CPPs.

14-17

CPPs are considered to

be less harmful to humans than other potential cell-penetrating materials such as carbon nanotubes because such peptides are biodegradable. By combining the advantages of fibrous materials and cationic CPPs, we created a coiled-coil protein carrier named CCPC 140, which

has

a

rigid

molecular surface.

18

and

fibrous-structure

with

a

cationic

CCPC 140 has a two-stranded parallel -

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Page 4 of 36

helical coiled-coil structure through its entire length and is modeled to be 20 nm in length with a diameter of 2 nm.

18

CCPC

140 was found to display better cell-penetrating activity when 14,

compared with previously reported CPPs.

18,

19

Here, typical

CPPs, such as octa-arginine R8, usually display cell-penetrating activity at several M. In contrast, CCPC 140 is active at the nanomolar

level.

18

Thus,

CCPC

140

appears

to

have

cell-

penetrating activity of at least 100-times greater than that of other CPPs. We also demonstrated that a CCPC 140 fused with green fluorescent protein (GFP) was delivered into cells 20-fold more efficiently than R8 fused with GFP.

20

We created CCPC 140 variants with isoelectric points (pI) lower (i.e.,

6.5

and

8.6)

than

the

pI

of

10.6

for

CCPC

140

to

investigate the origin of the superior cell-penetrating activity of

CCPC

140.

21

The

CCPC

140

pI

variants

were

effective

at

concentrations 100 times higher than that required for CCPC 140 cell-penetrating activity. showed

better

21

Nonetheless, these variants still

cell-penetrating

activity

than

other

cationic

CPPs. We also tried to evaluate the effect of the aspect ratio of CCPC 140 on cell-penetrating activity; however, this feature could not be addressed because thermal fluctuations were too large

for

structure.

short

CCPC molecules

to form a

stable

coiled-coil

18

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Anisotropic structured materials such as gold nano-rods have also gained increasing attention because of their superior cellpenetrating activity and mesoscopic properties.

22-25

Structural

anisotropy of gold nano-rods, such as their aspect ratio, has been suggested to affect their cell-penetrating activity. Recently,

it

is

reported

that

a

rod-shaped

DNA

26, 27

hierarchical

structure created by DNA origami also showed excellent cellpenetrating ability.

28

Challenges with controlling the synthesis

and quantitative analysis of these materials continue to hamper efforts to examine the effect of features, such as the aspect ratio, on their cell-penetrating activity. In this study, we designed and created novel CCPC variants to examine

the

activity.

The

effect newly

of

the

designed

aspect

ratio

artificial

on

cell-penetrating

proteins,

termed

LZ-

CCPCs, were confirmed to fold into tight -helical coiled-coil structures and the cell-penetrating activity of these LZ-CCPCs was examined.

EXPERIMENTAL DNA

Constructs

and

Expression

of

LZ-CCPC

Derivatives.

The

protein-coding DNA sequence of LZ-CCPC 140 was codon optimized for expression in Escherichia coli (E. coli) and synthesized by Eurofin genomics Inc. (Tokyo, Japan). The DNA sequences were designed to include NcoI and BamHI sites at the ends of the

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Page 6 of 36

coding sequence for subcloning into the same sites of the pET3d vector. Construction of a series of deletion LZ-CCPC variants was carried out using PCR with the primers presented in Table S1.

E.

coli

BL21(DE3)pLysS

cells

were

used

for

protein

expression. Transformed cells were cultured in modified LuriaBertani medium (1 wt% bacto-tryptone, 0.5 wt% yeast extract, 0.5 wt% NaCl) supplemented with 100 g/mL of carbenicillin at 37 °C. Induction of protein expression was achieved by adding isopropyl β-D-1-thiogalactopyranoside

to

the

culture

at

a

final

concentration of 0.2 M when the optical cell density at 600 nm reached 0.6–0.8. Cells were collected by centrifugation 3–5 h after induction, washed with 50 mM Tris-HCl, pH 8.0 and stored at −80 °C.

Purification

of

Recombinant

Proteins.

CCPC

140

and

its

derivatives were purified according to the procedure described in previous publications.

18, 20

A similar procedure was used for

purification of the LZ-CCPC derivatives.

18

In brief, expressed

cells were resuspended and incubated with 50 mM Tris-HCl, pH 8.0, with a protease inhibitor cocktail (EDTA-free, Roche) for 20 min at room temperature. Sodium deoxycholate was added to the sample at a final concentration of 0.1 mg/ml and the sample incubated for a further 20 min at room temperature. Cell debris

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were pelleted by centrifugation and washed twice in 50 mM TrisHCl, pH 8.0. Collected cell debris were suspended in 10 mM PIPES (piperazine–1,4–bis (2–ethanesulfonic acid))-NaOH, pH 7.0 and 2 M NaCl for 15 min to release target protein from cell debris. The

supernatant

was

collected

following

centrifugation

and

diluted with 10 mM PIPES-NaOH, pH 7.0 until a salt concentration of 0.3 M was reached. The diluted protein solution was purified by

anion

exchange

chromatography

using

SP-Sepharose

HP

(GE

material

was

healthcare). The

concentration

of

the

purified

protein

determined by the micro-biuret method.29

Circular obtained

Dichroism. on

a

JASCO

Circular J-820

dichroic

(CD)

spectropolarimeter

spectra using

a

were method

described previously.18 The relative helicity content of the CCPC and LZ-CCPC variants was evaluated with the following equation:

Relative helicity = [222nm]temp / [222nm]5 °C

In vitro Cell Penetration Assay. CCPC and LZ-CCPC variants were labeled with AlexaFluor 532. Two molar excess of AlexaFluor 532 succinimidyl solutions,

ester and

the

(Life

technologies)

reaction

incubated

was

added

for

2

to h

protein at

room

temperature. Unreacted dye was extensively removed by washing

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the

sample

with

PBS and using

Page 8 of 36

an

ultrafiltration Nanosep

3K

Omega device (Pall, Port Washington, NY). Fluorescently labeled proteins were diluted in PBS and added to cultured cells. We cultured three cell lines: A549 human lung adenocarcinoma epithelial cells, HeLa human cervical carcinoma cells and K562 human erythromyeloblastoid leukemia cells. A549 and HeLa cells were maintained in DMEM medium supplemented with 10% fetal calf serum (FCS) and K562 cells were maintained in RPMI 1640 medium supplemented

with

fluorescence

microscope

WRAYCAM-SR130M activated

10%

FCS.

Cells

Olympus

were

IX51

with

(Wraymer,

Osaka,

Japan)

sorting

(FACS)

Moxiflow

cell

observed

and

under

a

a

SCMOS

a

fluorescence-

instrument

camera

(ORFLO,

Ketchum, ID).

RESULTS and DISCUSSION In a previous report, we produced deletion variants of CCPC 140 with chain lengths of 55, 62, 69, 83 and 111 amino acids. Analysis showed

of

that

structures

CD

spectra

CCPC and

recorded

83 and

CCPC

exhibited

111

similar

on

these

adopted

deletion stable

cell-penetrating

when compared with that of CCPC 140.

18

18

variants

coiled-coil activities

In contrast, CCPC 69,

adopted only 56% -helical content at 37 °C because of thermal fluctuations and exhibited a 50% decrease in cell-penetrating activity

when

compared

with

that

of

CCPC

140.

18

Deletion

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constructs CCPC 55 and CCPC 62 did not form stable -helical coiled coil structures at 37 °C and showed further decreases in cell-penetration

18

activity.

Based on these observations,

we

concluded that the superior cell-penetrating activity of CCPCs is governed by structural rigidity and anisotropy. Unfortunately,

defining

the

minimum

length

of

CCPC

that

retained similar cell-penetrating activity as CCPC 140 was not possible because short CCPCs did not form a rigid -helical coiled-coil structure at 37 °C. The design of CCPC 140 was based on the structural frame of human skeletal muscle -tropomyosin. 18,

30

Tropomyosin

coiled-coil terminal

forms

structure

amino

acid

a

well-known

along

its

sequences

two-stranded

entire

of

length.

tropomyosin

-helical

31-33

from

The

N-

different

species are conserved and important both for molecular function and structure. sequence

of

34-36

the

Therefore, we did not change the amino acid

first

two

tropomyosin in CCPC 140.

18

periods

of

the

heptad

repeat

of

To generate the CCPC 140 sequence, 18

residues at the b, c and f positions of the heptad repeat of human

skeletal

muscle

-tropomyosin

were

exchanged

to

basic

amino acids. These amino acid exchanges did not disrupt the helical coiled-coil structure.

32, 33, 37, 38

However, the designed

CCPC sequences did not have sufficient structural stability when the length of these variants was shortened below 70 amino acids.

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18

Page 10 of 36

Hodges and co-workers demonstrated that the large hydrophobic

leucine residues at the a and d positions contribute more to the stability of the two stranded coiled-coil structure than the other amino acid residues, including other hydrophobic residues such as isoleucine, valine, phenylalanine, tyrosine and alanine. 39, 40

Greenfield and Hitchcock-DeGregori also demonstrated that

the first 40 residues of rabbit skeletal muscle -tropomyosin could

increase

stability

of

the

coiled-coil

structure

by

exchanging the amino acids to leucine at the a and d positions of the heptad repeat.

41

We designed CCPC variants, termed LZ-CCPCs, which formed an helical coiled-coil structure at 37 °C under physiological ionic conditions. Although there are design rules to improve dimeric stability,42

coiled-coil

however

in

this

study,

we

adopt

molecular design rules based on the knowledge from studies of tropomyosin. The amino acid sequence of LZ-CCPC is identical to CCPC 140 except the a and d positions of the third heptad repeat and onward were exchanged to leucine (Figure 1). Although the designed amino acid sequence of LZ-CCPC was codon optimized for production

of

recombinant variants,

the

species LZ-CCPC

successfully

proteins

in

was

obtained;

48,

not

LZ-CCPC

overexpressed

in

E.

E.

coli,

55

however,

and

coli

the

and

LZ-CCPC the

LZ-CCPC

shorter

62,

purified.

140

were

Thermal

melting profiles by measuring the circular dichroic signal at

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222 nm of these purified LZ-CCPC constructs in PBS was carried out to determine whether the exchange of amino acids at the a and d positions to leucine increased thermal stability (Figure 2). The thermal melting profiles revealed that LZ-CCPC 48, LZCCPC 55 and LZ-CCPC 62 have a relatively high -helical content that is comparable to that of CCPC 140 at 37 °C. The three LZCCPC variants maintained an -helical coiled-coil even at 90 °C. Far UV CD spectra profiles of LZ-CCPCs also provide an evidence for

maintaining

their

-helical

structure

(Figure

S1).

Consequently, an estimate of their exact helical content using the

previously

reported

method

could

not

be

obtained.

Nonetheless, the -helical content of the LZ-CCPC variants is sufficiently

high

to

ensure

structural

rigidity

and

cell-

penetrating activity, and comparable with that of cationic CCPC 140 deletion variants, such as CCPC 62 and CCPC 55 (Figure 2). We also calculated probability of dimeric coiled-coil formation of LZ-CCPC 62 by the Multicoil program (Figure S2).

43

In

designed sequence region of LZ-CCPC 62 (from Leu 15), the score of dimeric coiled-coil formation is nearly 1. We

then

examined

whether

the

deletion

variants

of

LZ-CCPC

showed superior cell-penetrating activity to that of CCPC 140. Both short CCPCs and LZ-CCPCs were fluorescently labeled with AlexaFluor 532. Labeled CCPCs and LZ-CCPCs were adjusted molar

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ratio

from

1

concentration

to and

two,

which

fluorophore

Page 12 of 36

was

confirmed

absorbance.

By

by

protein

fluorescently

labeling, CCPCs and LZ-CCPCs is a decrease in primary amines at the

surface

of molecule

the lysine side chain and/or amino-

terminus. Loss of primary amines of molecules can be attribution of decreasing in the cell-penetrating activity to lowering the pI.

The

labeled

CCPCs

and

LZ-CCPCs

were

added

to

HeLa

cell

cultures at 100 nM concentrations, and cellular uptake of the labeled CCPCs and LZ-CCPCs were examined using a fluorescent microscope (Figure 3). We were able to detect a fluorescence signal

from

cells

when

we

added

CCPC

140

at

a

final

concentration of 100 nM and after 3 h of administration (Figure 3A-C).

In

contrast,

CCPC

62

and

CCPC

55

showed

weak

cell-

penetrating activity (Figure 3D-I). These results are in good agreement with our previous report. showed

cell-penetrating

activity

18

In contrast, LZ-CCPC 62

equal

to

that

of

CCPC

140

(Figure 3J-L) and the cell-penetrating activity of LZ-CCPC 55 appeared to be lower than that of LZ-CCPC 62 (Figure 3M-O). We confirmed that the fluorescent signal came from inside of the cells using optical sectioning microscope (Figure S3). A further sequence

deletion,

LZ-CCPC

48,

showed

weak

cell-penetrating

activity that matched the activity of CCPC 55 and CCPC 62. We also

evaluated

the

cell-penetrating

activity

by

fluorescence

activated cell sorting (FACS) (Figure 4). Distribution profiles

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of both CCPC 140 and LZ-CCPC 62 showed similar results (Figure 4A & E), whereas the distribution profiles of CCPC 55, CCPC 62 and LZ-CCPC 48 showed a decrease in fluorescence intensity by almost an order of magnitude (Figure 4B, C, G). Interestingly, the distribution profile of LZ-CCPC 55 was broad and showed an intermediate cell-penetrating activity between the activities of LZ-CCPC 62 and LZ-CCPC 48 (Figure 4F). The cell strain dependency on the cell-penetrating activity of CCPCs

and

LZ-CCPCs

was

also

examined

using

A549

human

lung

adenocarcinoma epithelial cells at a concentration of 100 nM (Figures 5 & 6). As indicated in these figures, LZ-CCPC 62 was able to penetrate the plasma membrane as efficiently as CCPC 140, and LZ-CCPC 48 exhibited cell-penetrating activity as low as CCPC 62. LZ-CCPC 55 also showed intermediate cell-penetrating activity toward A549 cells, which is in good agreement with the experiments

using

distribution

HeLa

profiles

cells. using

We

K562

also

obtained

human

fluorescent

erythromyeloblastoid

leukemia cells at a concentration of 100 nM (Figure S4). The results

using

these

cells

were

in

good

agreement

with

the

experiments using HeLa and A549 cells. We also evaluated the short-term cytotoxicity (72 h) of CCPC 140 and LZ-CCPC 62 at 100 nM concentration (Figure S5). CCPC 140 did

not

exhibit

significant

short-term

cytotoxicity

among

in

HeLa, A549 and K562 cells under this experimental condition as

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18, 21

previous studies shown,

Page 14 of 36

and LZ-CCPC 62 also did not. The

results obtained using LZ-CCPCs have provided strong evidence that

molecular

anisotropy

is

important

for

enabling

cationic

coiled-coil proteins to pass through the plasma membrane. The molecular

length

and

diameter

of

designed

LZ-CCPCs

were

calculated from a corresponding length and diameter of crystal structure of tropomyosin.

31

LZ-CCPC 62 has an aspect ratio of

4.5:1 because it is modeled to be 9 nm in length and 2 nm in diameter. LZ-CCPC 55 and LZ-CCPC 48 have aspect ratios of 4:1 and 3.5:1, respectively. Our previous results examining CCPC 83, which

has

an

aspect

ratio

of

6:1,

showed

cell-penetrating

activity equal to that of CCPC 140 with an aspect ratio at 10:1 and that of LZ-CCPC 62. Thus, we conclude that an aspect ratio at 4.5:1 appears to be a critical threshold value for ensuring strong

cell-penetrating

activity

of

cationic

coiled-coil

proteins. In support of this concept, the cationic coiled-coil molecule LZ-CCPC 48 with an aspect ratio of 3.5:1 only shows cell-penetrating activity equal to that of unstructured short CCPC variants, CCPC 55 and 62, as well as short CPPs.

4, 18, 19

Interestingly, LZ-CCPC 55 with an aspect ratio 4:1 exhibited intermediate

cell-penetrating

activity.

Recombinant

proteins,

especially coiled-coil proteins, are relatively easy to design and

their

strictly

molecular

controlled.

length Because

and we

surface used

properties

recombinant

can

be

proteins

in

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Langmuir

this study, we were able to clarify the relationship between the molecular aspect ratio and cell-penetrating activity. The

coiled-coil

interaction motif,

motif 44, 45

is

an

abundant

protein-protein

and its use in molecular targeting tools

for cellular delivery has been studied extensively.

46-48

Some

CPPs can form an -helix under non-physiological conditions, 49

and

linked many

adopting

such

a

stable

secondary

structure

has

been

with cell-penetrating activity. Furthermore, there

studies

showing

an

improvement

in

the

14,

are

cell-penetrating

activity of cationic CPPs via the introduction of hydrophobic amino acids into their sequences. multimers

by

adopting

a

50-52

coiled-coil

These peptides may form structure,

which

likely

stabilizes their overall structure. We showed that the cellpenetrating activity of CPPs can be improved by incorporating a coiled-coil

motif

into

the

CPP

sequence.

In

addition

to

introducing a hydrophobic core sequence to CPPs to facilitate the formation of an intermolecular interface, i.e., coiled-coil, which

stabilized

the

structure,

the

formation

of

such

a

structural motif may also reduce cytotoxicity. In the latest report, Woolfson and co-workers demonstrates that the designed self-assembled coiled-coil peptide nanocages are able to control their

structure

provide

and

important

intracellular knowledge

in

delivery. the

53

principle

Our

findings

design

of

intracellular DDS carrier molecules not only for CCPCs, their

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Page 16 of 36

derivatives and -helical CPPs, but for nanomaterials such as 21, 22

metal nanorods and synthetic polymers.

Conclusion Anisotropic

structured

nanomaterials

have

gained

increasing

attention in the research field of DDS because of their unique mesoscopic But

little

properties is

known

and an

superior effect

cell-penetrating of

aspect

ratio

activity. on

cell-

penetrating activity. Our present study clearly proves that an aspect ratio at 4.5:1 appears to be a critical threshold value for ensuring superior cell-penetrating activity. Also, an aspect ratio of 3.5:1 only shows cell-penetrating activity equal to that of less structured CPPs. Our findings can provide important knowledge in the principle design of intracellular DDS carrier nanomaterials.

Figure captions Figure

1.

Amino

acid

sequence

comparison

of

the

-helical

coiled-coil proteins used in this study. The seven positions of the coiled-coil motif are described as a to g. Substituted amino acids are displayed in blue and red. Hu sk -Tm 140 is an abbreviation for human skeletal muscle -tropomyosin.

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Langmuir

Figure 2. Thermal unfolding profiles of CCPC 55, 62 and 140, and LZ-CCPC 48, 55 and 62.

Figure

3.

Analysis

of

the

cell-penetrating

activity

of

AlexaFluor 532 labeled CCPCs and LZ-CCPCs against HeLa cells. HeLa cells were incubated with 100 nM labeled CCPCs and LZ-CCPCs for 3 h. Panels A-C, D-F, G-I, J-L, M-O and P-R show the results for CCPC 140, CCPC 62, CCPC 55, LZ-CCPC 62, LZ-CCPC 55 and LZCCPC 48 administrated cells, respectively. A, D, G, J, M and P show phase-contrast images; B, E, H, K, N and Q show fluorescent images;

C,

F,

I,

L,

O,

and

R

show

the

merged

images.

All

fluorescence images were given pseudo-color and the levels were tuned by imaging software for visualization.

Figure

4. Quantitative

FACS

analysis

of

the

cell-penetrating

activity of AlexaFluor 532 labeled CCPCs and LZ-CCPCs against HeLa cells (A-G), and mean fluorescence intensities (H). HeLa cells were incubated without protein and with 100 nM labeled protein for 3 h incubation. A, B, C, E, F and G are CCPC 140, CCPC

62,

CCPC

55,

LZ-CCPC

62,

LZ-CCPC

55

and

LZ-CCPC

48

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administrated

cells,

respectively.

Page 18 of 36

In

D,

no-fluorescently

labeled protein was administrated.

Figure 5. Analysis of cell-penetrating activity of AlexaFluor 532 labeled CCPCs and LZ-CCPCs against A549 cells. A549 cells were incubated with 100 nM labeled CCPCs and LZ-CCPCs for 3 h. Panels A-C, D-F, G-I, J-L, M-O and P-R are CCPC 140, CCPC 62, CCPC 55, LZ-CCPC 62, LZ-CCPC 55 and LZ-CCPC 48 administrated cells, respectively. A, D, G, J, M and P show phase-contrast images; B, E, H, K, N and Q show fluorescent images; C, F, I, L, O and R show the merged images. All fluorescence images were given pseudo-color and the levels tuned by imaging software for visualization.

Figure

6. Quantitative

FACS

analysis

of

the

cell-penetrating

activity of AlexaFluor 532 labeled CCPCs and LZ-CCPCs against A549 cells. A, B, D, E and F are CCPC 140, CCPC 62, LZ-CCPC 62, LZ-CCPC 55 and LZ-CCPC 48 administrated cells, respectively. In C, no-fluorescently labeled protein was administrated.

ASSOCIATED CONTENT

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

of

oligonucleotides

used

in

this

study,

far

UV

CD

spectra of LZ-CCPCs, calculated dimerization probability of LZCCPC 62, optical sectioning images of AlexaFluor 532 labeled LZCCPC 62 administrated cells, and quantitative FACS analysis of the cell-penetrating activity of AlexaFluor 532 labeled CCPCs and LZ-CCPCs against K562 cells (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Phone: +81-480-33-7725 ORCID Norihisa Nakayama: 0000-0001-5498-4103 Sho Takaoka: 0000-0001-5042-9735 Megumi Ota: 0000-0002-6222-4830 Kentaro Takagaki: 0000-0001-8085-6201 Ken-Ichi Sano: 0000-0003-0715-5040

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Page 20 of 36

Notes Ken-Ichi

Sano,

Norihisa

Nakayama

and

Kentaro

Takagaki

were

inventors of LZ-CCPCs, which are patented (JP 2017-206464 A).

ACKNOWLEDGMENTS This work was partially supported by a JSPS KAKENHI to K-I. S. (16K01395), the Network Joint Research Center for Materials, and a special research grant from the Nippon Institute of Technology to K-I. S. We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

ABBREVIATIONS DDS,

drug

delivery

system;

CPPs,

cell-penetrating

peptides;

CCPC, coiled-coil protein carrier; CD, circular dichroism

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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