Efficient siRNA Delivery by Lipid Nanoparticles Modified with a

Aug 8, 2017 - The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor ti...
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Efficient siRNA Delivery by Lipid Nanoparticles Modified with a Nonstandard 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*,† †

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

S Supporting Information *

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 inhibits 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 nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard 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 were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any 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. KEYWORDS: cancer, siRNA delivery, liposome, nonstandard macrocyclic peptide, active targeting (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

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 uncurable diseases, has become a subject of interest because siRNA can suppress an expression of any genes in a 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 © XXXX American Chemical Society

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May 3, 2017 June 27, 2017 August 8, 2017 August 8, 2017 DOI: 10.1021/acs.molpharmaceut.7b00362 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

(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 MILLIPORE (Darmstadt, Germany). 1,1′-Dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) were purchased from PromoKine (Heidelberg, Germany). All of the nonlabeled 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). Antiluciferase 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 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). PEGmonooleyl 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 was identified and synthesized as previously reported. 17 The sequence of Epi-1 is D 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 Denantiomer). 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 headgroup 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 3500 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−timeof-flight mass spectrometry (Figure S1B). 2.3. Preparation of MENDs. 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 unencapsulated 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

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 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 cancerspecific antigen.14 We recently developed a novel method for identifying nonstandard macrocyclic peptides with a high affinity to a target protein. The developed system is referred to as a random nonstandard peptides integrated discovery (RaPID) system.15,16 The RaPID system consists of a rRNAamino acid conjugated with a Flexible in vitro translation (FIT) system and a diverse library of random amino acid sequences with nonstandard amino acids including N-methylation, nonbasic 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 an active targeting system. We recently developed a liposome-type siRNA delivery system, a multifunctional envelop-type nanodevice (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 biocompatible 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 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 nonstandard macrocyclic peptides would be useful for delivering therapeutics to a wide variety of cells.

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-hydroxysuccinimidePEG-distearoyl-sn-glycerophosphoethanolamine (NHS-PEGDSPE) were purchased from the NOF CORPORATION B

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Molecular Pharmaceutics PEG-lipid to be retained on the surface of the LNP.30 To allow MEND to accumulate in the tumor tissue, it was postmodified 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 postmodified 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 nonlabeled 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 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 W with an Astrason 3000 sonicator (Misonix, Farmingdale, NY, USA). The LPs were then subjected to ultrafiltration through an Amicon Ultra15 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-PEGDSPE (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 postmodification. Therefore, to prevent this aggregation, 10 mol % of PEGmonooleyl 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 zetapotential 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 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.

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. ANOVA was performed for statistical analysis, followed by Bonferroni test (vs 0%). *:P < 0.05, **P < 0.01. (B) Summary of cellular uptake. Fold increase of cellular uptake of ETMEND against nonmodified MEND was shown. ANOVA was carried out, followed by SNK test. *:P < 0.05, **:P < 0.01 against HCT-116, † :P < 0.05, ††:P < 0.01 against Huh-7. Data represents mean ± SD.

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 C

DOI: 10.1021/acs.molpharmaceut.7b00362 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Characterization of the siRNA MENDs PEG-MEND ET-MEND

Diameter (mm)

PdI

Zeta-potential (mV)

siRNA encapsulation effciency

siRNA recovery ratio

83 ± 6 92 ± 10

0.15 ± 0.05 0.24 ± 0.01

−6 ± 3 −3 ± 1

91 ± 2% 88 ± 2%

86 ± 6% 77 ± 8%

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-antimouse CD31 (Biolegend, #102514) for 30 min. The slices were finally observed with a CLSM Nikon A1R system. 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 HighCapacity 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 ΔΔCt 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. 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 nontreated 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 nonspecific chemiluminescence and the RI activity was then measured with an ALOKA 6100 scintillation counter (Hitachi-Aloka Medical, Tokyo, Japan). 2.10. Toxicological analysis. 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 postadministration 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, nonrepeated analysis of variance (nrANOVA), followed by Bonferroni test or SNK test. P value < 0.05 was regarded as being a statistically significant difference.

3. RESULTS 3.1. EpCAM-dependent cellular uptake of EpCAMtargeting 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 postmodified with X% of Pep-PEGDSPE 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 nonspecific 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 nonmodified MEND, while no enhancement was observed in the case of EpCAM negative cell lines. Regarding 1.0 mol % modification, cellular uptake was enhanced by 15−25-fold as the result of modification 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 (EpCAMtargeting MEND; ET-MEND) was the optimum particle for use. D

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Molecular Pharmaceutics The characterizations of the MEND that was nonmodified (PEG-MEND) and the ET-MEND are listed in Table 1. To verify that the ET-MEND was taken up by cells, and not simply bound, cells that were treated with the fluorescently labeled PEG-MEND and the ET-MEND were observed by CLSM. In the case of the PEG-MEND, no fluorescent signals were detected at all in HCT116 cells (Figure 2). On the other hand,

Figure 3. Enhancement effect of EpCAM-targeting Epi-1 on the therapeutic effect of its cargo. EpCAM-positive HCT116 and EpCAMnegative HT-1080 were incubated with free DOX, Dox-loaded LNPs with/without EpCAM-targeting peptide Epi-1 for 2 h. The viability of the two cell lines was determined by the amount of protein at 46 h after the removal of DOX. **:P < 0.01 (Student’s t test, vs LP). Data represents mean ± SD.

3.0-fold higher than that of PEG-LP. In contrast, the EC50 for ET-LP was 8.6-fold higher than that of PEG-LP in the case of HCT116. We next examined the impact of Epi-1 on the efficiency of siRNA delivery. To assess the enhancement of siRNA internalization, EpCAM positive HCT116 cells were treated with Cy5-labeled siRNA encapsulated in PEG- and ETMENDs. The ET-MEND delivered siRNA 27-fold more efficiently than the PEG-MEND (Figure 4A, B). We next evaluated the gene silencing effect of the ET-MEND. In EpCAM positive Hep3B and HCT116 cells, the ET-MEND inhibited the target mRNA more robustly than the PEGMEND (Figure 4C). In contrast to positive cells, the gene silencing abilities of both PEG- and ET-MENDs were comparable in the case of EpCAM negative HT-1080 cells. Accordingly, EpCAM-targeting peptide Epi-1 enhanced the pharmacological effect of LNPs 3.3. In vivo evaluation of ET-MEND. To target tumor tissue, LNPs should have a prolonged circulation time because the level of tumor accumulation was reported to be proportional to the area under the curve (AUC).33,34 Therefore, the pharmacokinetics properties of both MENDs were investigated. When mice were administered with the PEGand ET-MENDs via the tail vein, the blood concentration of the PEG-MEND was higher than that of ET-MEND (Figure 5A). The calculated AUC (0−24 h) of the PEG- and ETMEND was 183%ID·h/mL blood and 79%ID·h/mL blood, respectively. The accumulation of each of the MENDs in other organs (liver, spleen, kidney, lung, heart, colon, jejunum, muscle, and subcutaneous fat) was also measured using the lipophilic RI marker [ 3 H]-CHE (Figure 5B and C). Significantly higher amounts of the ET-MEND accumulated in the liver and kidney than the PEG-MEND at both 6 and 24 h postinjection. Although the ET-MEND tended to be delivered to the colon, jejunum, lung, and kidney, the EpCAM expression level was much higher in other organs (Figure S4), and no obvious increases in accumulation were observed. This might be because the ET-MEND was not able to approach the EpCAM that is expressed on epithelial cells distant from the vasculature in normal organs due to the presence of a rigid endothelial barrier.35 These results suggest that the ET-MEND could be used to target EpCAM-positive cancer cells owing to moderately high blood concentration and the absence of recognition of EpCAM in normal organs. To assess the enhancement effect of EpCAM-targeting peptide Epi-1 on the efficiency of siRNA delivery to tumor

Figure 2. Cellular uptake of the ET-MEND in EpCAM positive cells. Cellular uptake of EpCAM-positive HCT116 with CLSM. Cells were observed 2 h after the addition of DiI-labeled MENDs at 20 nM (siRNA concentration). Blue, green, and red dots mean nuclei (Hoechst33342), endo/lysosome (LysoTracker Green DND-26), and MENDs (DiI), repectively. Scale bars are 10 μm.

a substantial amount of the ET-MEND was internalized into HCT116 cells. Accordingly, the use of the Epi-1 conjugates Pep-PEG-DSPE significantly improved the cellular uptake of the MENDs, especially in the case of the EpCAM positive cell lines. In addition, we compared the ET-MEND with PEGMEND with regard to the intracellular fate of siRNA. As a result, a larger amount of was siRNA encapsulated in ETMEND localized in the cells than for the PEG-MEND (Figure S3). 3.2. Enhancement of pharmacological effect by EpCAM-targeting peptide. We then evaluated the ability of the Epi-1-modified LNP to enhance the delivery of its cargo. To simplify the effect of Epi-1 modification, doxorubicin was selected as a cargo, because the cytotoxic effect caused by doxorubicin (DOX) would dominantly affect the cellular uptake of LNP, while the delivery efficiency of siRNA depends, not only on cellular uptake, but also on endosomal escape. For simplicity, DOX was used as the therapeutic agent. In this experiment, the LNP was composed of the conventional lipid DSPC and chol with 5 mol % of PEG-DSPE (PEG-LP), 3 mol % of PEG-DSPE, and 2 mol % of Pep-PEG-DSPE (ET-LP). Characterization data for these two LPs are shown in Table 2. Table 2. Characterization of DOX-Loaded LNPs PEG-LP ET-LP

Diameter (nm)

PdI

Zeta-potential (mV)

111 ± 3 121 ± 12

0.15 ± 0.03 0.15 ± 0.01

−15 ± 2 −9 ± 1

In the case of EpCAM positive HCT116, the cytotoxic effect of ET-LP was superior to that of PEG-LP. At 100 μM DOX, the cell killing effect of ET-LP was significantly higher than that of PEG-LP (Figure 3). On the other hand, the cytotoxic effect of ET-LP was equal to that of PEG-LP. The half maximal effective concentration (EC50) was calculated by fitting Hill’s 4parameter equation.32 As shown in Table 3, in the case of the EpCAM negative HT-1080, the EC50 of ET-LP was only E

DOI: 10.1021/acs.molpharmaceut.7b00362 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 3. EC50 for the Cell Killing Effect by DOX-Loaded LNPs HCT116 EC50

HT-1080

Free DOX

PEG-LP

Pep-LP

Free DOX

PEG-LP

Pep-LP

1.1 μM

63 μM

7.3 μM

0.64 μM

20 μM

6.7 μM

Figure 6. in vivo siRNA delivery by ET-MEND. (A) Intratumoral distribution of PEG- and ET-MEND. Tumor raw sections were observed with a Nikon A1R system 24 h after the injection. Green and red pixels indicate vessels and MENDs, respectively. Scale bars are 100 μm. (B) Gene silencing effect of MENDs. When Hep3B-bearing mice were treated with 0.5 mg/kg of siRNA encapsulated in MENDs, mRNA expression was determined by qRT-PCR. **:P < 0.01 (Student’s t test, vs PEG-MEND). In the left graph, Scr. peptide means the randomized sequence with the same amino acid as Epi-1. Data represents mean ± SD.

Figure 4. Improvement in siRNA delivery by modifying LNP with Epi-1. (A) Uptake of siRNA encapsulated in LNPs. Cellular uptake of Cy5-siRNA loaded in LNPs was determined by flow cytometry 2 h after the addition. Light histogram showing representative data from three independent experiments. Black, orange, and red lines indicate nontreatment (N.T.), PEG-MEND, and ET-MEND, respectively. The graph indicates the fluorescent intensity of cells treated with PEG- or ET-MEND. **:P < 0.01 (Student’s t test, vs PEG-MEND). Data represents mean ± SD.

However, the accumulation of the PEG-MEND appeared to be slightly higher than that of the ET-MEND, possibly due to the short circulation time of ET-MEND. This assumption was supported by a flow cytometry analysis (Figure S5). Although the gene silencing effect of the ET-MEND was expected to be weak due to its low level of accumulation, the ET-MEND was significantly superior to the PEG-MEND in the gene silencing despite the low accumulation (Figure 6B). To confirm whether

tissue, the distribution and the knockdown effect of the ETMEND when it was intravenously administered into Hep3Bbearing mice were examined. Concerning intratumoral distribution, both the PEG-MEND and ET-MEND were observed to be present in the tumor tissue (Figure 6A).

Figure 5. Pharmacokinetic properties of the ET-MEND. (A) Blood concentration profile of PEG- and ET-MEND. Blood was collected via the tail vein 0.016, 0.16, 0.5, 1, 3, and 24 h after the injection of the fluorescent MENDs. Blood concentration was determined by the fluorescent intensity from samples of collected blood. (B) Pharmacokinetics evaluation of PEG- and ET-MEND. Plasma, blood, liver, spleen, kidney, lung, heart, colon, jejunum, muscle, and subcutaneous fat were collected 6 and 24 h after the administration of the RI [3H]-CHE labeled MENDs. The RI activities of the lysates were determined with a liquid scintillation counter. Data represents the mean ± SD. F

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Molecular Pharmaceutics the gene silencing effect by the ET-MEND is mediated by EpCAM, we performed in vivo knockdown experiments with human hepatocellular carcinoma Huh-7 cells, which express EpCAM less highly than Hep3B (Figure S6A). The findings indicate that the ET-MEND showed no gene silencing effect (Figure S6B). Taken together, the EpCAM-targeting peptide Epi-1 appears to have some potential for enhancing the pharmacological effect of LNPs. 3.4. Toxicological analysis. Finally, we tested the nonspecific toxicity of the ET-MEND. Since the ET-MEND accumulates nonspecifically in the liver and spleen (Figure 5B), it is possible that the ET-MEND could cause adverse damage in an organ, specifically the liver, in which most of the systemically injected ET-MEND accumulates. AST and ALT levels in serum were measured as an index of hepatological toxicity when healthy ICR mice were administered with the PEG-MEND and ET-MEND at a dose of 2.0 mg/kg. As a result, the AST and ALT activities were slightly increased by the injection of the ET-MEND at 1 h postinjection, but not significantly (Figures 7A, B). Additionally, when LDH was also measured as an

hydrophilicity) is responsible would be beneficial and would clearly advance the progress of nanoparticle-mediated medicine. The enhancement in cellular uptake by a nonstandard macrocyclic peptide Epi-1 was very selective both in vitro (Figure 1) and in vivo (Figure 6). In the EpCAM-low expressing Huh-7 xenograft, the ET-MEND failed to show a gene silencing effect (Figure S6). In addition, the ET-MEND did not become distributed in the colon and jejunum in spite of the fact that a high level of EpCAM mRNA was detected (Figures 5 and S4). As EpCAM was expressed in epithelial celljunctions, not the vascular lumen,40 ET-MEND, after systemic injection, would not likely reach an area adjacent to EpCAM in the jejunum and colon. On the other hand, in the case of neoplasia tissue, the spatial structure of epithelial cells, stroma, and vasculature was disrupted. Therefore, the ET-MEND was able to access EpCAM in the tumor tissue. Accordingly, EpCAM would be expected to be an attractive target for ligandmediated active targeting. The findings reported herein indicate that the use of a nonstandard macrocyclic peptide can be very useful for creating a selective, efficient active targeting system. As the RaPID system allowed us to obtain a specific ligand with a very high affinity, it became possible to target various cells/tissues. If a breakthrough technology that would permit nanoparticles to pass through the vessel wall in normal tissue, which has a tight junction between endothelial cells, could be developed, it would be possible to deliver nanoparticles to any cells/tissues of interest.

Figure 7. Toxicological analysis of ET-MEND. The activity of enzymes in serum was determined at 1 and 24 h after the injection of 2.0 mg/kg each MENDs. Statistical analysis was carried out by nonrepeated ANOVA. If the P value was over 0.05, the difference was regarded as not significant (N.S.). Data represents the mean ± SD.

5. CONCLUSIONS In this study, we constructed epithelial cell adhesion molecule (EpCAM)-targeting lipid nanoparticles (ET-MEND) by modifying a MEND with a nonstandard macrocyclic peptide Epi-1 that had a high affinity for EpCAM, which was previously identified using a random nonstandard peptides integrated discovery (RaPID) system. The ET-MEND delivered therapeutics (siRNA and doxorubicin) in a highly selective manner, and the improvement in cellular uptake by Epi-1 modification was very high (∼100-fold). The ET-MEND also inhibited the expression of the target gene more strongly after systemic injection than the PEG-MEND. As nonstandard macrocyclic peptides could be designed toward target proteins of interest, the combination of a MEND and the RaPID system has the potential for use in the treatment of a wide range of diseases.

indicator of somatic damage, neither the PEG-MEND nor the ET-MEND caused an increase of LDH in comparison with N.T. (Figures 7C). These moderate, transient elevations in AST and LDH can be attributed to the increased accumulation of ET-MEND in the liver. Judging from toxicological data at 24 h postinjection.

4. DISCUSSION In this study, we report on the development of a combination of lipid nanoparticles (LNPs) and a nonstandard macrocyclic peptide Epi-1 for use in a new class of cancer therapy. Although the affinity of Epi-1 to EpCAM (KD ∼ 1.7 nM) is slightly higher than that of a typical ligand such as folic acid 3 nM,36 transferrin 5.1 nM,37 cyclic RGD peptide 5.05 nM,38 Epi1 greatly enhanced the cellular uptake of LNPs (∼30-fold). We previously reported that a transferrin modification of ∼10-fold enhanced the cellular uptake of LNPs and that a cyclic RGD modification of ∼5-fold enhanced that of LNPs.22,39 The difference between the nonstandard macrocyclic peptide Epi1 and other typical ligands regarding their ability to enhance the uptake of LNPs cannot be attributed to the slight difference in their KD values. The underlying principle responsible for this is currently unclear, since there is little information regarding what factors in ligands and receptors affect the capacity to potentiate the cellular uptake of nanoparticles. Further studies directed at elucidating which factor (receptor, number, endocytosis pathway, recycling rate, ligand, KD, kon, koff,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00362. Structural information regarding Epi-1 and Pep-PEGDSPE, expression of EpCAM in each cell line, intracellular fate of siRNA, expression level of murine EpCAM in normal organs, distribution of intravenously administered PEG and ET-MEND, and silencing effect in Huh-7 cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Address: Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. Telephone: +8111-706-3919. Fax: +81-11-706-4879. G

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Molecular Pharmaceutics ORCID

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Yu Sakurai: 0000-0001-6584-8350 Author Contributions ⊥

Y.S. and W.M. contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Dr. Milton S. Feather for appropriately modifying the manuscript. This study was supported, in part, by research grants (Research on Development of New Drugs, Health and Labour Sciences Research Grant, and Initiative for Accelerating Regulatory Science in Innovative Drug, Medical Device, and Regenerative Medicine) from the Japan Ministry of Health, Labour and Welfare (MHLW), Grant Number PH44280004, and Platform Project for Supporting in Drug Discovery and Life Science Research and Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency For Medical Research And Development (AMED) (to H.S.).

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