Systemic administration of siRNA with anti-HB-EGF antibody-modified

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Systemic administration of siRNA with anti-HB-EGF antibody-modified lipid nanoparticles for the treatment of triple-negative breast cancer Ayaka Okamoto, Tomohiro Asai, Yusuke Hirai, Kosuke Shimizu, Hiroyuki Koide, Tetsuo Minamino, and Naoto Oku Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01055 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Molecular Pharmaceutics

1

Systemic administration of siRNA with anti-HB-EGF antibody-modified lipid nanoparticles for

2

the treatment of triple-negative breast cancer

3 4

Authors:

5

Ayaka Okamoto

6

Minamino d, and Naoto Oku a,*

a,b

, Tomohiro Asai a, Yusuke Hirai a, Kosuke Shimizu

a,c

, Hiroyuki Koide a, Tetsuo

7 8

Affiliations:

9

a

Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka,

10

52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

11

b

12

c

13

Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1

14

Handayama, Higashi-ku, Hamamatsu city, Shizuoka, Japan 431-3192

15

d

16

Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793 Japan

Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, 102-0083, Japan

Department of Molecular Imaging, Institute for Medical Photonics Research, Preeminent Medical

Department of Cardiovascular Medicine, Graduate School of Medicine, Kagawa University, 1750-1

17 18

Footnote:

19

*Corresponding author. Tel: +81 54 264 5701, Fax: +81 54 264 5705.

20

E-mail address: [email protected]. (N. Oku)

21 22

Keywords

23

siRNA delivery, lipid nanoparticles, antibody, HB-EGF, triple-negative breast cancer

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1 ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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1 2

Abstract

3 4

Triple-negative breast cancer is one of intractable cancers that are not sensitive to the

5

treatment with existing molecular-targeted drugs. Recently, there has been much interest in RNA

6

interference-mediated treatment of triple-negative breast cancer. In the present study, we have

7

developed lipid nanoparticles encapsulating siRNA (LNP-siRNA) decorated with an Fab’ antibody

8

against heparin-binding EGF-like growth factor (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA

9

targeting polo-like kinase 1 (PLK1) was prepared and evaluated for its anticancer effect using

10

MDA-MB-231 human triple-negative breast cancer cells overexpressing HB-EGF on their cell

11

surface. Biodistribution data of radioisotope-labeled LNP and fluorescence-labeled siRNA indicated

12

that

13

carcinoma-bearing mice. Expression of PLK1 protein in the tumors was clearly suppressed after

14

intravenous injection of αHB-EGF LNP-siPLK1. In addition, tumor growth was significantly

15

inhibited by treatment with this formulation of siRNA and an antibody-modified carrier. These

16

findings indicate that αHB-EGF LNP is a promising carrier for the treatment of HB-EGF-expressing

17

cancers, including triple-negative breast cancer.

αHB-EGF

LNP effectively

delivered

siRNA to

18 19 20


tumor

tissue

in

MDA-MB-231

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1 2

1. Introduction

3 4 5

The clinical application of small interfering RNA (siRNA) has been attracting increasing

6

attention in recent years. Because of its high selectivity and diverse targetability [1, 2], siRNA has

7

received considerable attention as a therapeutic candidate for intractable diseases such as cancer.

8

Triple-negative breast cancer (TNBC) is known as a refractory cancer because it does not

9

express drug target genes, such as estrogen receptors (ER), progesterone receptors (PR), or human

10

epidermal growth factor receptor 2 (HER2) [3]. As TNBC accounts for about 15% of breast cancers

11

and tends to have high malignancy and poor prognosis [4], the development of a novel TNBC

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therapeutic strategy is urgently required, and many studies of RNA interference-based therapy with

13

siRNA have been reported. It is well known both that siRNA is likely to be eliminated from the

14

blood by rapid degradation and glomerular filtration through the kidneys, and that siRNA has

15

difficulty penetrating cell membranes [5]. Therefore, several studies have used siRNA-delivery

16

systems to treat TNBC, including cyclodextrin-grafted polyethylenimine (PEI) functionalized

17

mesoporous silica nanoparticles [6], siRNA conjugated to a diacyl lipid moiety [7], PEI substituted

18

with linoleic acid [8], chitosan-gold nanorods [9], cationic lipid assisted poly(ethylene

19

glycol)-b-poly(D,L-lactide)

20

(1-aminoethyl)iminobis[N-oleicylsteinyl-1-aminoethyl]propionamide]

21

nanoparticles [11]. These reports indicate that RNA interference by siRNA has potential as an

22

innovative therapeutic strategy for TNBC, if an appropriate carrier can be developed. While a variety

23

of technologies are available for the passive-targeting of siRNA to TNBC, until now there has been

24

no system for the active-targeting of specific tumors.

(PEG-PLA)

nanoparticles

[10], (ECO)-based

and lipid

25

Here, we have developed lipid nanoparticles for TNBC treatment that encapsulate siRNA

26

modified with an antibody targeting heparin-binding epidermal growth factor-like growth factor

27

(HB-EGF). HB-EGF is a ligand that binds to the EGF receptor (EGFR) and is related to various

28

physiological and pathological functions, such as heart development [12], perinatal distal lung

29

development [13], and wound healing [14]. In addition, HB-EGF is known to be highly expressed in

30

various cancers, including breast, ovarian, and gastric cancer [15]. HB-EGF is highly likely to be

31

involved in tumor progression by activating the signaling pathway for tumorigenesis [16], promoting

32

angiogenesis [17], and increasing tumor metastasis [18]. In TNBC patients, HB-EGF has been

33

reported to show the highest expression level among the EGFR ligands, which also include

34

amphiregulin, transforming growth factor-α (TGFα), and EGF [19]. It has been applied to the

35

targeting of particular tumors. For example, CRM197, which is a mutant of diphtheria toxin, has 3 ACS Paragon Plus Environment


1

been used as an HB-EGF inhibitor for the treatment of breast cancer [19, 20]. The C-terminal

2

receptor domain of the diphtheria toxin has been coated on poly(lactic-co-glycolic acid)

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nanoparticles to target HB-EGF-expressing glioblastoma [21]. These reports suggest that HB-EGF is

4

very likely to be useful as an address-molecule for tumor targeting.

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We have previously shown in vitro that anti-HB-EGF antibody-modified LNP-siRNA

6

(αHB-EGF LNP-siRNA) efficiently delivered siRNA to cancer cells overexpressing HB-EGF and

7

induced significant gene silencing [22]. While αHB-EGF LNP-siRNA appears to be suitable for

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RNAi-based therapy, little is known about its in vivo characteristics, such as its retentivity in blood

9

or RNAi activity in tumors. In this study, we developed the vector and evaluated its activity in vivo

10

using siRNA against polo-like kinase 1 (PLK1), which is related to cell viability through control of

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the cell cycle. After checking the effect of RNAi in vitro, we evaluated the qualities of αHB-EGF

12

LNP as a siRNA vector in vivo using a radioisotope-labeled lipid and fluorescent-labeled siRNA.

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αHB-EGF LNP was then loaded with siPLK1 for the treatment of MDA-MB-231 breast cancer, a

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type of TNBC cell that expresses HB-EGF on its cell surface. αHB-EGF LNP-siPLK1 was

15

administered to MDA-MB-231-bearing mice, and its utility as a candidate for TNBC treatment was

16

evaluated.

17 18 19


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2. Experimental Section

3 4 5

2.1 Materials

6 7

siRNAs against polo-like kinase 1 (siPLK1) and against luciferase 2 (siLuc2) were

8

purchased from Hokkaido System Science Co. (Hokkaido, Japan). Alexa750-conjugated siRNA

9

against green fluorescent protein (siGFP) was purchased from Japan Bio Services Co., Ltd. (Saitama,

10

Japan). The sequences of nucleotides with an overhang (2-nucleotide, underline) for siPLK1 were

11

5’-UAG AGG AUG AGG CGU GUU GTT-3’ (guide) and 5’-CAA CAC GCC UCA UCC UCU

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ATT-3’ (passenger), for siLuc2 were 5’-UUU GUA UUC AGC CCA UAG CTT-3’ (guide) and

13

5’-GCU AUG GGC UGA AUA CAA ATT-3’ (passenger), for siGFP were 5’-UGC GCU CCU GGA

14

CGU AGC CUU-3’ (guide) and 5’-GGC UAC GUC CAG GAG CGC ACC-3’ (passenger). siLuc2

15

was used as a control siRNA (siCont). In the in vivo experiment, 3’ end of the passenger strand of

16

siRNAs was modified with cholesterol. For near-infrared fluorescence imaging, 3’ end of the guide

17

strand of siGFP was modified with Alexa750. A palmitoyl conjugate of protamine-derived

18

13-amino-acid peptide (NH2-RRRRRRGGRRRRG(Lys[Palmitoyl])-CONH2, PP-13) was obtained

19

by custom synthesis from Operon Biotechnologies (Tokyo, Japan). Dimyristoylphosphoglycerol

20

(DMPG),

21

maleimide-conjugated DSPE-PEG5000 (DSPE-PEG-mal) were purchased from NOF Co. (Tokyo,

22

Japan). Dioleoylphosphatidylethanolamine (DOPE) and cholesterol were kindly gifted by Nippon

23

Fine Chemical Co. (Hyogo, Japan). Human HB-EGF-specific monoclonal antibody (clone 3E9) and

24

control mouse IgG (MGG-0500) were obtained from Medical & Biological Laboratories Co., Ltd.

25

(Nagoya, Japan) [23]. Primers of PLK1 and β-actin were obtained by custom synthesis from Rikaken

26

Co., Ltd. (Aichi, Japan). The primer sequences of PLK1 were 5’-CAC AGT GTC AAT GCC TCC

27

AA-3’ (forward) and 5’-TTG CTG ACC CAG AAG ATG G-3’ (reverse), and those of β-actin,

28

5’-CAT CCG TAA AGA CCT CTA TGC CAA C-3’ (forward) and 5’-ATG GAG CCA CCG ATC

29

CAC A-3’ (reverse). Anti-PLK1 rabbit polyclonal antibody and anti-β-actin rabbit polyclonal

30

antibody were purchased from Cell Signaling Technology (MA, USA) and Novus Biologicals (CO,

31

USA), respectively. Anti-rabbit IgG polyclonal antibody conjugated with horseradish peroxidase

32

(HRP) was purchased from GE Healthcare (Little Chalfont, UK).

distearoylphosphatidylethanolamine-polyethyleneglycol

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2.2 Preparation of anti-HB-EGF-Fab’-modified LNP-siRNA 5 ACS Paragon Plus Environment

(DSPE-PEG)

5000,

and


1 2

LNP-siRNA was prepared as described previously [24]. siRNA was mixed with PP-13

3

(1/16.8 as a molar ratio) to obtain cationic cores. LNP was prepared by wrapping the cores with lipid

4

bilayers containing DOPE, cholesterol and DMPG (6/5/2 as a molar ratio). To prepare the

5

[3H]-labeled and the fluorescence-labeled LNP-siRNA, [3H]cholesteryl hexadecyl ether and

6

3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), respectively, were added to the initial lipid

7

solution.

8

Fab’ antibody modification of LNP-siRNA was performed as described previously [22].

9

LNP-siRNA was incubated with DSPE-PEG (9.5 mol% to total lipid) and DSPE-PEG-mal (0.5

10

mol% to total lipid) at 37°C for 2 h. The Fab’ fragments of anti-HB-EGF antibody were added and

11

incubated at 4°C for 16 h. After ultracentrifugation (453,000 xg, 4°C, 15 min), LNP-siRNA modified

12

with anti-HB-EGF Fab’ (αHB-EGF LNP-siRNA) was re-suspended in RNase-free water.

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LNP-siRNA modified with Fab’ fragments of control mouse IgG (Control LNP-siRNA) was

14

prepared for control. The particle size and ζ-potential of the nanoparticles diluted with 10 mM

15

phosphate buffer (pH 7.4) were determined using a Zetasizer Nano ZS (Malvern, Worcs, UK). The

16

amount of Fab’ antibody modified on LNP-siRNA was measured by high-performance liquid

17

chromatography (Hitachi High-Technologies Corporation, Tokyo, Japan). Control LNP-siRNA and

18

αHB-EGF LNP-siRNA, respectively, were solubilized with 2% sodium dodecyl sulfate (SDS, Wako

19

Pure Chemical Industries, Ltd., Osaka, Japan), and subjected to a column of TSKgel G3000SWXL

20

(Tosoh, Tokyo, Japan). Mobile phase was composed of 0.1% SDS, 0.1 M NaH2PO4, 0.1 M Na2SO4.

21

pH of the mobile phase was adjusted to 6.7 with NaOH.

22 23 24

2.3 Cell culture

25 26

MDA-MB-231 human triple-negative breast cancer cells were obtained from ATCC

27

(Manassas, VA). The cells were cultured in RPMI-1640 medium (Wako Pure Chemical Industries,

28

Ltd., Osaka, Japan) containing 10% fetal bovine serum (FBS, AusGeneX, Oxenford, Australia),

29

100-units/mL penicillin G (MP Biomedicals, Irvine, CA), and 100-µg/mL streptomycin (MP

30

Biomedicals) in a humidified 5% CO2 incubator.

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2.4 Suppression of PLK1 mRNA expression

34 35

MDA-MB-231 cells were seeded onto a 6-well plate at a density of 1×105 cells / 2 mL and 6 ACS Paragon Plus Environment

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1

incubated overnight. The medium was changed to an antibiotic-free one containing FBS before

2

transfection. The cells were transfected with αHB-EGF LNP-siCont, Control LNP-siPLK1, or

3

αHB-EGF LNP-siPLK1 at a final concentration of 100 nM as siRNA, and then incubated for 24 h.

4

According to the manufacturer’s protocol, the total RNA of the cells was extracted with TRIzol LS

5

reagent (Thermo Fisher Scientific Inc., Kanagawa, Japan). One microgram of total RNA was applied

6

to the synthesis of complementary DNA with a First-Strand cDNA Synthesis Kit (GE Healthcare).

7

Real-time RT-PCR was performed in the presence of either human PLK1 or β-actin primers and

8

SYBR Premix Ex Taq II (Takara Bio, Shiga, Japan) using a Thermal Cycler Dice Real Time System

9

(Takara Bio). The conditions for PCR were as follows: 95°C for 30 sec (1 cycle), 95°C for 5 sec and

10

60°C for 30 sec (60 cycles).

11 12 13

2.5 Suppression of PLK1 protein expression

14 15

MDA-MB-231 cells were seeded onto a 6-well plate (5×104 cells / 2 mL) and pre-cultured

16

overnight. After a medium change to an antibiotic-free one containing FBS, the cells were

17

transfected with αHB-EGF LNP-siCont, Control LNP-siPLK1, or αHB-EGF LNP-siPLK1 (100 nM

18

as siRNA) for 24 h. After a medium change, the cells were cultured for additional 48 h. After the

19

cells had been washed with PBS, they were lysed with 0.1% SDS containing protease inhibitors (1

20

mM phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 2 µg/mL pepstatin

21

A) in 150 mM NaCl / 10 mM Tris-HCl (pH 7.5). The cell lysate was applied for Western blotting.

22 23 24

2.6 Western blotting

25 26

Protein concentration was measured by bicinchoninic acid (BCA) assay with a Pierce™

27

BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Cell lysates containing 10-µg protein were

28

subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore,

29

Billerica, MA). After the PVDF membrane had been blocked with 5% bovine serum albumin (BSA,

30

Sigma-Aldrich) in 0.1% Tween 20-containing Tris-HCl-buffered saline (TTBS, pH 7.4) for 1 h at

31

37°C, it was incubated with a primary antibody against PLK1 (1:1,000) or β-actin (1:5,000)

32

overnight at 4°C, and then with an HRP-conjugated secondary antibody (1:10,000) for 1 h at room

33

temperature. A chemiluminescent substrate (ECL-prime, GE Healthcare) was used to generate

34

chemiluminescence. The chemiluminescence signal was detected with a LAS-3000 mini system (Fuji

35

Film, Tokyo, Japan). 7 ACS Paragon Plus Environment


1 2 3

2.7 Growth inhibition assay

4 5

MDA-MB-231 cells were seeded onto a 96-well plate (Thermo Fisher Scientific Inc.) at a

6

density of 2×103 cells/well with RPMI-1640 medium and transfected with 20 µL of αHB-EGF

7

LNP-siCont, Control LNP-siPLK1, or αHB-EGF LNP-siPLK1 (100 nM; 20 pmol/200 µL as siRNA)

8

for 24 h. After a medium change, the cells were incubated for additional time periods as described

9

below. Cell viability was measured by WST-8 assay with a Cell Counting Kit-8 (Dojindo Laboratries,

10

Kumamoto, Japan) at 0, 1, 3, 5, and 7 days after transfection. In accordance with the manufacturer’s

11

protocol, WST-8 assay reagent (Cell Counting Kit-8 : medium = 1 : 9) was added after removing the

12

culture medium, and then the cells were incubated for 2 h at 37°C. To determine cell viability,

13

absorbance at 450 nm was measured. In cases in which the cells were cultured for more than 3 days,

14

the medium was changed at day 4.

15 16 17

2.8 Experimental animals

18 19

The animals were cared for according to the Animal Facility Guidelines of the University

20

of Shizuoka. All the animal experiments were conducted in compliance with the protocol that had

21

reviewed and approved by the Animal and Ethics Committee of the University of Shizuoka on April

22

1, 2016 (Approval No. 166198). Four-week-old BALB/c nu/nu female mice were purchased from

23

Japan SLC (Shizuoka, Japan). For preparation of tumor-bearing mice, MDA-MB-231 cells (1×107

24

cells/mouse) were subcutaneously injected into the left flank of BALB/c nu/nu mice. Each type of

25

LNP-siRNA was injected via a tail vein. Tumor volume was calculated by use of the following

26

formula: a × b2 × 0.4 (a, largest diameter; b, smallest diameter).

27 28 29

2.9 Biodistribution of αHB-EGF LNP-siRNA in mice

30 31

MDA-MB-231-carcinoma bearing mice were intravenously injected with [3H]-labeled

32

PEG LNP-siRNA, Control LNP-siRNA, or αHB-EGF LNP-siRNA (74 kBq / mouse). At 24 h after

33

injection, the mice were sacrificed under deep anesthesia with isoflurane (Wako Pure Chemical

34

Industries, Ltd.), and their blood was collected. The collected blood was centrifuged (3,000 rpm, 10

35

min, 4°C) to obtain plasma. Then, the liver, spleen, heart, lungs, kidneys, and tumor were removed 8 ACS Paragon Plus Environment

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1

and weighed. To lyse them, these excises were treated with Solvable (PerkinElmer, MA, USA). They

2

were then treated with hydrogen peroxide (Wako Pure Chemical Industries, Ltd.) for bleaching. After

3

incubation with Hionic-Fluor (PerkinElmer) overnight at room temperature, the radioactivity in the

4

plasma and in each organ was measured using a liquid scintillation counter (LSC-7400, Hitachi

5

Aloka Medical, Tokyo, Japan). The total amount in the plasma was determined based on the body

6

weight, where the plasma volume was calculated to be 4.27% of body weight.

7 8 9

2.10 Intratumoral distribution of αHB-EGF LNP-siRNA

10 11

DiO-labeled

Control

LNP-siRNA or

αHB-EGF

LNP-siRNA was

injected

to

12

MDA-MB-231 carcinoma-bearing mice via a tail vein. Twenty-four hours after injection, the mice

13

were injected with DyLight594®-conjugated Lycopersicon Esculentum (Tomato) Lectin (Vector

14

Laboratories, Inc., Burlingame, CA, USA) for staining vessels with blood circulation. Fifteen

15

minutes later, perfusion fixation of the organs was performed with 1% paraformaldehyde under deep

16

anesthesia with isoflurane, and the tumor was excised. The tumor was then embedded and frozen in

17

Tissue-Tek® O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan). Tumor sections (10-µm

18

thickness) were prepared, mounted on MAS-coated slides (Matsunami Glass, Osaka, Japan), fixed

19

with 1% paraformaldehyde, and blocked with 3% BSA in PBS. The nuclei were counterstained with

20

4’,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA, USA). Intratumoral

21

distribution of DiO-labeled LNP was observed using a confocal laser-scanning microscope (A1R+,

22

Nikon, Tokyo, Japan).

23 24 25

2.11 siRNA distribution in tumor-bearing mice

26 27

MDA-MB-231 carcinoma-bearing mice were injected with Alexa750-labeled Naked

28

siRNA, Alexa750-labeled siRNA formulated in Control LNP, or αHB-EGF LNP (10 µg / mouse as

29

siRNA) via a tail vein on the day when the tumor had reached a volume of approximately 300 mm3.

30

Biodistribution of Alexa750-labeled siRNA was then measured with an in vivo imaging system

31

(Xenogen IVIS Lumina System, Xenogen Corp., Alameda, CA, USA). Living Image software

32

(Xenogen Corp.) was used for data acquisition with 30 seconds exposure for each imaging point.

33

Twenty-four hours after the injection, perfusion fixation of the organs was performed with 1%

34

paraformaldehyde under deep anesthesia with isoflurane. The organs and tumor were excised, and

35

their fluorescence intensities were determined by IVIS. 9 ACS Paragon Plus Environment


1 2 3

2.12 Protein knockdown effect of αHB-EGF LNP-siPLK1 in tumor-bearing mice

4 5

MDA-MB-231 carcinoma-bearing mice were injected with αHB-EGF LNP-siCont,

6

Control LNP-siPLK1, or αHB-EGF LNP-siPLK1 via a tail vein on the day when the tumor had

7

reached a volume of approximately 250 mm3. Five days after treatment, the tumor was collected and

8

homogenized in Tissue-Protein Extraction Reagent (Thermo Fisher Scientific Inc.) containing

9

protease inhibitors using a Shakeman 2 vortex homogenizer (Biomedical Science, Tokyo, Japan) for

10

2 cycles of homogenization: 40 seconds of shaking, then 20 seconds of cooling on ice. The

11

homogenate was centrifuged 3 times (10,000 xg, 10 min, 4°C) in order to obtain the tumor protein

12

extraction. The protein concentration of the extraction was determined by BCA assay. Thirty

13

micrograms of the protein was applied to 10% SDS-PAGE. Expression of PLK1 and β-actin was

14

determined by Western blotting. Immunoblotting was performed with a primary antibody against

15

PLK1 (1:2,000) or β-actin (1:10,000) overnight at 4°C, and then with an HRP-conjugated secondary

16

antibody (1:10,000) for 1 h at room temperature.

17 18 19

2.13 Therapeutic experiment

20 21

MDA-MB-231 carcinoma-bearing mice were injected 4 times with samples (αHB-EGF

22

LNP-siCont, Control LNP-siPLK1, or αHB-EGF LNP-siPLK1) once a week (0.5 mg/kg as siRNA

23

dose per day). The tumor size and body weight change were monitored daily from one day before

24

sample injection. As an experimental control, PBS was injected instead of the LNP-siRNA samples.

25 26 27

2.14 Statistical analysis

28 29 30

Differences within a group were determined by analysis of variance (ANOVA) with the Tukey post-hoc test.

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1 2

3. Results

3

3.1 Characteristics of αHB-EGF LNP-siRNA

4 5

The physicochemical properties of each type of LNP-siRNA are shown in Table 1.

6

LNP-siRNA had a particle size of 129 ± 30 nm and a ζ-potential of -45 ± 7.1 mV in 10 mM

7

phosphate buffer (pH 7.4). On the other hand, αHB-EGF LNP-siRNA and Control LNP-siRNA both

8

had a particle size of around 160 nm and an almost neutral surface charge. The degree of

9

modification of anti-HB-EGF Fab’ antibody was about 130 µg Fab’ / 1 µmol lipid.

10 11

Table 1. Characteristics of LNP-siRNA, PEG LNP-siRNA, Control LNP-siRNA, and αHB-EGF

12

LNP-siRNA Size (d.nm)

PdI

ζ-Potential (mV)

Fab’ modification (µg/µmol lipid)

LNP-siRNA

129 ± 30

0.242 ± 0.045

-45 ± 7.1

-

PEG LNP-siRNA

103 ± 15

0.265 ± 0.008

-2.8 ± 1.2

-

Control LNP-siRNA

138 ± 17

0.247 ± 0.047

-8.3 ± 3.9

126 ± 18

αHB-EGF LNP-siRNA

167 ± 56

0.284 ± 0.084

-5.9 ± 2.6

129 ± 14

13 14 15

3.2 Gene silencing effect of αHB-EGF LNP-siRNA

16 17 18

Gene silencing activity of αHB-EGF LNP-siPLK1 against MDA-MB-231 cells was

19

determined. The relative amount of PLK1 mRNA on the cells was reduced by treatment with

20

αHB-EGF LNP encapsulating siPLK1 (Figure 1A). More than 80% of PLK1 mRNA expression was

21

suppressed by treatment with αHB-EGF LNP-siPLK1. Also, the amount of PLK1 mRNA in the

22

Control LNP-siPLK1-treated cells was slightly reduced (approximately 24% reduction). In addition,

23

PLK1 protein expression was clearly suppressed by treatment with αHB-EGF LNP-siPLK1 (Figure

24

1B). No silencing effects were observed in PLK1 mRNA or protein after treatment with αHB-EGF

25

LNP-siCont. Furthermore, cell growth was inhibited after treatment with αHB-EGF LNP-siPLK1

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(Figure 1C). At day 7, the αHB-EGF LNP-siPLK1-treated group showed about 45% inhibition

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compared with control (RNase-free water). On the other hand, Control LNP-siPLK1 and αHB-EGF



1

LNP-siCont had no effect on cell growth.

2


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Figure 1. Gene silencing by siRNA formulated in αHB-EGF LNP. (A) Reduction of PLK1 mRNA in MDA-MB-231 cells after the treatment with αHB-EGF LNP-siPLK1. The cells were transfected for 24 h with siPLK1 encapsulated in Control LNP or αHB-EGF LNP. The expression of PLK1 mRNA was determined by real-time RT-PCR. Data are shown as relative expression level of PLK1 mRNA to that in the control (vehicle: RNase-free water) with SD bars. Asterisks indicate significant differences (***P

Systemic administration of siRNA with anti-HB-EGF antibody-modified

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