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Efficient siRNA delivery by lipid nanoparticles modified with a non-standard macrocyclic peptide for EpCAM-targeting Yu Sakurai, Wataru Mizumura, Manami Murata, Tomoya Hada, Shoshiro Yamamoto, Kenichiro Ito, Kazuhiro Iwasaki, Takayuki Katoh, Yuki Goto, Asako Takagi, Michinori Kohara, Hiroaki Suga, and Hideyoshi Harashima Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00362 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Molecular Pharmaceutics

Original article Title

Efficient siRNA delivery by lipid nanoparticles modified with a non-standard macrocyclic peptide for EpCAM-targeting Yu Sakurai1,§, Wataru Mizumura1,§, Manami Murata1, Tomoya Hada1, Shoshiro Yamamoto1, Kenichiro Ito2, Kazuhiro Iwasaki3, Takayuki Katoh2, Yuki Goto2, Asako Takagi4, Michinori Kohara4, Hiroaki Suga2, Hideyoshi Harashima1

1 Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan. 2 Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan. 3 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-8656, Japan. 4 Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan §

These authors equally contributed to this manuscript

*Corresponding author Correspondence should be addressed to Hideyoshi Harashima ([email protected]) Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. E-mail: [email protected] TEL: +81-11-706-3919 FAX: +81-11-706-4879

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Abstract The development of a specific, effective method for the delivery of therapeutics including small molecules and

nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been

developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because

cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibit the cellular uptake of

LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would

clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use

in an innovative nano-drug delivery system. Herein, we assessed the effect of non-standard macrocyclic peptides

against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a Random non-standard

Peptides Integrated Discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A

liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting

MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in

EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for

delivering therapeutics was comparable with that of non-modified MEND. In additional, when systemically

injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg

without no obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to

identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in

developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.


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Keywords cancer; siRNA delivery; liposome; non-standard macrocyclic peptide; active targeting



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1. Introduction The use of lipid nanoparticles (LNPs) is currently expanding for use in the delivery of therapeutic agents to target cells.1 It is especially noteworthy that nucleic acids are a popular cargo because LNPs prevent nucleic acids

from degradation and clearance from the body by encapsulating the nucleic acids within lipid envelops, which are susceptible to enzymatic degradation in the circulation and are readily excreted by the glomerular route.2 For

example, small interfering RNA (siRNA), a promising molecule for treating currently un-curable diseases has

become a subject of interest because siRNA can suppress an expression of any genes in sequence-dependent

manner. Actually, the pH-sensitive LNP containing cationic lipid MC3 is under phase II clinical trials for patients with heredity amyloidosis caused by transthyretin proteins (ATTR).3 In addition, LNPs would be applicable for use in the field of genome editing as a vector of Cas9 mRNA and small guide RNA.4, 5 However, the range of

applications of LNP is now restricted to the treatment of hepatological diseases, since they are taken up by the liver.6, 7

LNPs selectively accumulate in hepatocytes because the apolipoprotein (Apo) E that is bound to the surface of

LNP is recognized by the low density lipoprotein receptor (LDLR). It has been previously reported that various

types of LNPs are internalized into hepatocytes via the ApoE-LDLR pathway,

8-11

which results in almost all of

LNPs being accumulated in the liver tissue.11, 12 Therefore, targeting other cells in other organs by LNPs, the

development of an active targeting system that enables LNPs to recognize a specific cell via a ligand molecule will

be needed.

For this purpose, although a number of peptides with a sequence that allows for binding to a receptor have been


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reported, only a few active targeting systems have reached the stage of clinical trials, except for very high affinity ligands, such as tri-GalNAc, a ligand against the asialoglycoprotein receptor (KD ~3 nM),13 transferrin and an antibody against a cancer-specific antigen.14 We recently developed a novel method for identifying non-standard

macrocyclic peptides with a high affinity to a target protein. The developed system is referred to as a Random non-standard Peptides Integrated Discovery (RaPID) system.15,

16

The RaPID system consists of a ribosomal

RNA-amino acid conjugated with a Flexible in vitro translation (FIT) system and a diverse library of random amino

acid sequences with non-standard amino acids including N-methylation, non-basic side chains and cyclic structures

linked to mRNA via a puromycin linker. We previously identified a peptide sequence with a high affinity (Epi-1, KD 1.7 nM) for the epithelial cell adhesion molecule (EpCAM),17 a type 1 transmembrane glycoprotein that is expressed in some types of cancer such as hepatocellular carcinomas and colorectal cancer.18, 19 EpCAM has been found to be involved in the progression of cancer, such as by cell migration and proliferation.20, 21 These properties

suggest that EpCAM would be a useful target for active targeting system.

We recently developed a liposome-type siRNA delivery system, a multi functional envelop-type nano-device (MEND) containing a pH-sensitive cationic lipid YSK05.22, 23 YSK05 was designed to lead to membrane fusion

specifically at an acidic pH, which allows the MEND to efficiently escape from endosome/lysosome and release its siRNA cargo to the cytosol.24 In addition to pH-responsive fusion, a MEND containing YSK05 (YSK-MEND) is

highly bio-compatible in the bloodstream because YSK05 has no negative/positive charge at physiological pH. In

previous reports, YSK-MEND was found to successfully deliver siRNA to liver tissue via ApoE-LDLR at a low dose (ED50 ~0.06 mg/kg).10, 25 The YSK-MEND succeeded in gene silencing in tumor tissue at a relatively high



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dose (ED50 ~1.5 mg/kg),26, 27 at least in part, because ApoE-LDLR-mediated uptake was not sufficient to achieve efficient knockdown in the tumor tissue.

We report herein on the construction of a EpCAM-targeting MEND (ET-MEND) by combining

EpCAM-targeting peptide Epi-1 and LNPs for achieving effective knockdown in cancer cells. The ET-MEND

efficiently delivered siRNA and cytotoxic reagents under both in vitro and in vivo conditions. The concept of

combining LNPs and non-standard macrocyclic peptides would be useful for delivering therapeutics to a wide

variety of cells.


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2. Experimental Section 2.1 Materials

Cholesterol, Dulbecco’s modified Eagle’s medium (DMEM), monooleate polyethylene glycol2,000 (PEG) and TRI Reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-distearoyl-sn-glycerophosphocholine

(DSPC),

PEG-1,2-dimyristoyl-sn-glycerol

(PEG-DMG),

PEG-1,2-distearoyl-sn-glycerol

(PEG-DSG)

and

N-hydroxysuccinimide-PEG-distearoyl-sn-glycerophosphoethanolamine (NHS-PEG-DSPE) were purchased from

the NOF CORPORATION (Tokyo, Japan). The High-Capacity RNA-to-cDNA kit, LysoTracker Green DND-26

and Pierce BCA Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Amicon

Ultra-15

was

purchased

from

MERCK

1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine

MILLIPORE

perchlorate

(DiI)

(Darmstadt,

and

Germany).

1,1'-dioctadecyl-3,3,3',3'-

tetramethylindodicarbocyanine (DiD) were purchased from PromoKine (Heidelberg, Germany). All of the

non-labeled siRNA and Cy5-labeled siRNA were obtained from Hokkaido System Science (Sapporo, Japan).

Sequences of the siRNA were as follows; anti-polo-like kinase 1 siRNA (siPLK1), sense: AGA uCA CCC uCC

UuA AAu AUU, antisense: UAU UUA AgG AGG GUG AuC UUU (upper case: RNA, lower case: 2’-OMe-RNA).

Anti-luciferase siRNA (siGL4), sense: CCG UCG UCU UCG UGA GCA AdTdT, antisense: UUG CUC ACG AAU

ACG ACG GdTdT. Primer pairs used in this study were as follows. PLK1, forward: CTC CTT GAT GAA GAA

GAT CAC C, reverse: GAA GAA GTT GAT CTG CAC GC. GAPDH, forward: CCT CTG ACT TCA ACA GCG

AC, reverse: CGT TGT CAT ACC AGG AAA TGA G. The Cytotoxicity LDH Assay Kit-WST and 1 mg/mL

solution of Hoechst33342 were purchased from DOJINDO (Kumamoto, Japan). THUNDERBIRD SYBR qPCR



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Mix was obtained from TOYOBO (Osaka, Japan). [3H]-cholesteryl hexadecyl ether (CHE), Hionic Fluor, Soluene-350 were obtained from PerkinElmer (Waltham, MA, USA). Phosphate buffered saline without Mg2+ and Ca2+ (PBS (−)) and Transaminase CII-test wako were obtained from Wako Pure Chemical Industries (Osaka, Japan).

PEG-monooleyl ether and doxorubicin (DOX) were purchased from Tokyo Chemical Industry (Tokyo, Japan). All

other reagents used were of analytical grade.

2.2 Preparation of the EpCAM-targeting peptide and peptide-PEG conjugate

Epi-1 D

was

identified

and

synthesized

as

previously

reported.17

The

sequence

of

Epi-1

is

WRPTRYRLLPWWICGSGSGSK (Cys and the acetyl group of the N-terminus was linked via a thioether bond,

the superscript “D” denotes the next amino acid is the D- enantiomer). A proposed mechanism for cyclization

within Epi-1 is shown in Figure S1A. To display Epi-1 on the surface of MEND, Epi-1 was conjugated to NHS-PEG-DSPE, which contained an activated carboxylic acid NHS in the head group of the PEG, via amide bond.

Epi-1 and NHS-PEG-DSPE were incubated in PBS (−) for 48 h at room temperature. The mixture was then added

to a dialysis membrane Spectra/Por6 (MWCO 3,500 Da), and dialyzed three times against double distilled water

(DDW). Finally, the dialysate was freeze-dried to give a white powder (Pep-PEG-DSPE). The chemical structure of

Pep-PEG-DSPE is shown in Figure S1A. The synthesis was confirmed by matrix assisted laser desorption

ionization – time of flight mass spectrometry (Figure S1B).

2.3 Preparation of MENDs


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The MENDs were prepared as previously described.24, 28 Briefly, 1,500 nmol of YSK05, 1,500 nmol of chol and

45 nmol of PEG-DMG were dissolved in 400 µL of tertiary butanol (t-BuOH) and 40-160 µg of siRNA in 2 mM

citrate buffer (pH 4.0) was then added dropwise to the lipid mixture. The siRNA/lipid mixture was diluted with

PBS (−), and then was subjected to ultrafiltration with an Amicon Ultra-15 twice to remove t-BuOH and

un-encapsulated siRNA. The result was a “bare” MEND that had essentially no prolonged circulation time, most of which was accumulated in the liver after systemic injection,25, 29 because DMG was not sufficiently hydrophobic to permit the PEG-lipid to be retained on the surface of the LNP.30 To allow MEND to accumulate in the tumor tissue,

it was post-modified with an additional 3 mol% of hydrophobic PEG-DSG at 3 mol%. The MENDs were incubated

with the PEG-DSG solution in 7.5% ethanol (EtOH) for 30 min at 60°C. For preparation of the ET-MEND, the

MEND was post-modified with 3-X mol% of PEG-DSG and X mol% of PEG-DSPE following the same procedure.

The particle size distribution and zeta-potential of the MENDs were determined with a Zetasizer Nano ZS (Malvern

Instruments Ltd., Malvern, UK). The siRNA encapsulation efficiency and recovery ratio were determined by means of a RiboGreen assay, as previously described.24, 31 To label the MEND a lipophilic dye DiI or DiD, DiI or DiD was

added to the lipid solution. For labeling the siRNA, approximately 10 µg of Cy5-labeled siRNA was added to the

siRNA solution instead of non-labeled siRNA.

2.4 Preparation of doxorubicin-loaded liposomes

To a glass tube containing 750 nmol of DSPC in CHCl3, 750 nmol of chol in CHCl3 was added, and the CHCl3 was then removed by a stream of N2 gas to form a thin lipid layer. To prepare liposomes (LPs), 1.5 mL of an



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ammonium sulfate buffer (300 mM, pH 4.0) was added to the thin lipid layer, followed by sonicating the glass tube

for 15 min at 12 watts with an Astrason 3000 sonicator (Misonix, Farmingdale, NY, USA). The LPs were then

subjected to ultrafiltration through an Amicon Ultra-15 to alter the external water phase. The resulting material was

recovered with 500 µL of PBS (−). A 100 µL aliquot of this liposome solution was then mixed with 5 mol% of

PEG-DSPE (PEG-LP) or 3 mol% of PEG-DSPE and 2 mol% of Pep-PEG-DSPE (ET-LP) in 10% EtOH for 30 min

at 60°C. Additionally, since Epi-1 is moderately hydrophobic, the LP was prone to form aggregates during

post-modification. Therefore, to prevent this aggregation, 10 mol% of PEG-monooleyl ether was added to the

solution. The mixture was then subjected to ultrafiltration using an Amicon Ultra-15 to remove EtOH. The resulting

material was mixed with DOX at a drug/lipid ratio 1/10 (weight ratio) for 30 min at 60°C. Finally, the mixture was

subjected to ultrafiltration with an Amicon Ultra-15 to remove free DOX. The size and zeta-potential of the

particles was determined with a Zetasizer Nano ZS. When the DOX concentration in the LP solution was

determined by fluorescent intensity (excitation 480 nm, emission 650 nm) with a Infinite M200 plate reader (Tecan,

Männedorf, Switzerland), the DOX recovery ratio was usually nearly 100%.

2.5 Cell culture and in vitro cellular uptake

All cells were cultured in culture media supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and

100 mg/mL of streptomycin in a humidified 5% CO2 chamber. DMEM (1,000 mg/L glucose) was used for HCT116, Huh-7, HT-1080, HEK293T, A549 and HeLa, and DMEM (4,500 mg/L glucose) was used for Hep3B. For uptake experiments, 1.0×105 or 2.0×105 cells were seeded onto 6-well plates for 24 h before the experiment. Cells were


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incubated with MENDs at approximately 20 nM (siRNA concentration) for 2 h. The fluorescent intensity of each

MEND added to cells was adjusted to the same amount by a fluorometer prior to the treatment. Cells were

trypsinized after washing with PBS (−), and the dispersed cells were then centrifuged for 3 min at 4°C (500 G) to

form a cell pellet. The pellet was washed FACS buffer (0.5% bovine serum albumin, 0.1% sodium azide in PBS

(−)). Cells were suspended in 1 mL of FACS buffer after repeating this wash procedure twice. The suspension was

then subjected to flow cytometer analysis with a FACSCalibur (BD Falcon, San Jose, CA, USA). The data were

analyzed by the Cell Quest software (BD Falcon). DiD and Cy5-siRNA were used for detection in Figures 1 and

figure 3, respectively.

2.6 Observation of intracellular fate and intratumoral distribution of MENDs

Three days before the experiment, 10,000 HCT116 cells were seeded onto a glass-base dish. Cells were treated

with DiI-labeled MENDs for 2 h and then incubated with 10 µg/mL Hoechst33342 and 5 µM LysoTracker Green

DND-26 for 30 min to stain the nuclei and endo/lysosomes after washing with PBS (−). Cells were observed with a

con-focal laser scanning microscopy (CLSM) system Nikon A1R (Nikon, Tokyo, Japan) at 37°C under a 5% CO2 atmosphere. In the case of the intratumoral distribution study, the 400 µm thick slices were obtained from the

excised tumor with a microslicer DTK-1000 (Dosaka-em, Kyoto, Japan). The slices were then immersed in PBS (−)

containing 10 µg/mL Hoechst33342 and 10 µg/mL Alexa488-anti-mouse CD31 (Biolegend, #102514) for 30 min.

The slices were finally observed with a CLSM Nikon A1R system.



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2.7 Evaluation of knockdown efficiency

Cells were dissolved by treatment with the TRI Reagent 24 h after siRNA transfection. For in vivo experiments,

approximately 10 mg of tissue was homogenized in 1 mL of TRI Reagent with a PreCellys24 (Bertin Technologies,

Montigny-le-Bretonneux, France). The following procedure was the same. Total RNA was extracted following the

manufacturer’s instructions. Then, 1.0 µg of total RNA was reverse-transcribed using a High-Capacity

RNA-to-cDNA kit. The synthesized cDNA solution was diluted to an appropriate concentration, and was subjected

to quantitative PCR with a LightCycler 480 II (Roche Diagnostics, Basel, Switzerland) following the previously reported protocol.27 The level of mRNA expression was calculated by the ∆∆Ct method.

2.8 Animal experiment

ICR mice (4-week-old, male) and BALBc/AJcl nu/nu (4-week-old, male) were purchased from Japan SLC

(Shizuoka, Japan) and Japan CLEA (Shizuoka, Japan), respectively. For manipulating tumor-bearing mice, the mice were inoculated in the right flank with 1.0×106 Huh-7 cells in 70 µL of PBS (−)). Tumor volume was calculated a following equation; a×b2/2 (a: major axis, b:minor axis). When the tumor volume reached >150 mm3, animal

experiments were carried out. All of the animal experiments were reviewed and approved by Hokkaido University

Animal Committee.

2.9 Pharmacokinetics analysis


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For the long-term evaluation of blood concentration, fluorescently labeled MENDs were used. At the indicated

times, 12 µL of blood was collected from the tail vein after the DiD-labeled MENDs had been injected. The

collected blood was immediately dissolved by 228 µL of 1% sodium dodecyl sulfate (SDS). The blood

concentration of the MENDs were determined from a standard curve, prepared by mixing known-amounts of

MENDs with blood obtained from non-treated mice. Concerning accumulation in normal organs, the MENDs were labeled with a lipid marker [3H]-CHE by adding a [3H]-CHE EtOH solution to a lipid solution during the preparation procedure. Plasma, blood, liver, spleen, kidney,

lung, heart, colon, jejunum, muscle and subcutaneous fat were excised 6 and 24 h after an injection of 3,000,000

dpm of the radio isotopically (RI)-labeled MENDs. Prior to the separation of plasma, 50 µL of blood was mixed

with 1 mL of Soluene-350. The color of the solvent was quenched by 3 additions of 100 µL of hydrogen peroxide.

A 10 mL aliquot of Hionic Fluor liquid scintillation cocktail was then added to the quenched blood. Plasma was

separated from the remaining blood by centrifugation (1,000 G, 4°C, 15 min), 50 µL of plasma was then added to

10 mL of Hionic Fluor without any additional treatment. Other organs were first liquefied by incubating them in 2

mL of Soluene-350 for 12 h at 55°C. A 10 mL volume of Hionic Fluor was added to the dissolved organs. All of the

samples in Hionic Fluor were kept in 4°C refrigerator to reduce the occurrence of non-specific chemiluminescence

and the RI activity was then measured with an ALOKA 6100 scintillation counter (Hitachi-Aloka Medical, Tokyo,

Japan).

2.10 Toxicological analysis



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To assess liver toxicity caused by the systemically injected MENDs, aspartate transaminase (AST) and alanine

transaminase (ALT) levels were measured by a Wako Transaminase CII-test kit. In addition, lactate dehydrogenase

(LDH) was measured using a Cytotoxicity LDH Assay Kit-WST for evaluating somatic toxicity. Serum was

separated from the blood by centrifugation (1,000 G, 15 min, 4°C). Blood was collected at 1 and 24 h

post-administration from the tail vein.

2.11 Statistical analysis

Student’s t-test was carried out for pairwise comparison of two groups. For comparison among three or more

groups, non-repeated analysis of variance (nrANOVA), followed by Bonferroni test or SNK test. P value < 0.05

was regarded as being a statistically significant difference.


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3 Results 3.1 EpCAM-dependent cellular uptake of EpCAM targeting-MEND

EpCAM positive and negative cells were used to assess the enhancing effect of the EpCAM targeting peptide

Epi-1 on the cellular uptake of LNP. The results for the characterization of the PEG-MEND and ET-MEND are

shown in Table 1. HCT116, Huh-7 and Hep3B were all found to express high levels of EpCAM (Figure S3). On the other hand, the EpCAM expression for the HT-1080, HEK293T, A549 and HeLa cells was negligible in all

cases (Figure S3). Epi-1 was then conjugated to the head of PEG-DSPE to anchor it to the surface of the LNP (Pep-PEG-DSPE). To

determine the optimum amount of Epi-1 conjugates, cellular uptake was measured after the cells were treated with

the MEND that had been post-modified with X% of Pep-PEG-DSPE and 3-X% of PEG-DSG (Figure 1A). When more than 1.0 mol% of Pep-PEG-DSPE was incorporated into MENDs, they were liable to spontaneously

aggregate. Therefore, we assessed the enhancement effect of Epi-1 ranging from 0 – 1.0 mol%. Although cellular

uptake was increased with increasing Epi-1, 1.0% modification also enhanced the internalization, even in EpCAM

negative cell lines (HT-1080, HEK293T, A549 and HeLa). On the other hand, as the scrambled sequence of Epi-1

failed to improve the cellular uptake of the MENDs, the 3 dimensional structure of Epi-1, not just hydrophobicity

or other non-specific binding, appears to be important for the recognition of EpCAM. The enhancement effect of

Epi-1 is summarized in Figure 1B. The cellular uptake of the 0.5 mol% peptide-modified MEND was 5-7 fold

higher than that for the non-modified MEND, while no enhancement was observed in case of EpCAM negative cell

lines. Regarding 1.0 mol% modification, cellular uptake was enhanced by 15-25 fold as the result of modification



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with Epi-1 conjugates. However, even in the case of EpCAM negative cell lines, Epi-1 enhanced the cellular uptake

of MENDs in a nonspecific manner. We therefore conclude that a 0.5% modified MEND (EpCAM-targeting

MEND; ET-MEND) was the optimum particle for use.

Figure 1 Increase in cellular uptake of the ET-MEND. A) Cellular uptake of EpCAM-positive (HCT116, Huh-7) and EpCAM-negative (HeLa, A549, HEK293T, HT-1080) cell lines. When the amount of Epi-1 was varied from 0% to 1.0%, the intracellular fluorescence intensity was observed 2 h after the addition of

DiD-modified MENDs. Scr indicates that the peptide has the same amino acids with a scrambled sequence.


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ANOVA was performed for statistical analysis, followed by Bonferroni test (vs. 0%). *:P

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