One-step encapsulation of siRNA between lipid-layers of multi-layer polycation liposomes by lipoplex freeze-thawing

    One-step encapsulation of siRNA between lipid-layers of multi-layer polycation liposomes by lipoplex freeze-thawing Hiroyuki Koide, Ayaka Okamoto, Hiroki Tsuchida, Hidenori Ando, Saki Ariizumi, Chiaki Kiyokawa, Masahiro Hashimoto, Tomohiro Asai, Takehisa Dewa, Naoto Oku PII: DOI: Reference:

S0168-3659(16)30026-8 doi: 10.1016/j.jconrel.2016.01.032 COREL 8088

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

24 September 2015 5 January 2016 18 January 2016

Please cite this article as: Hiroyuki Koide, Ayaka Okamoto, Hiroki Tsuchida, Hidenori Ando, Saki Ariizumi, Chiaki Kiyokawa, Masahiro Hashimoto, Tomohiro Asai, Takehisa Dewa, Naoto Oku, One-step encapsulation of siRNA between lipid-layers of multi-layer polycation liposomes by lipoplex freeze-thawing, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.01.032

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One-step encapsulation of siRNA between lipidlayers of multi-layer polycation liposomes by lipoplex freeze-thawing

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Hiroyuki Koide,† Ayaka Okamoto,†Hiroki Tsuchida,† Hidenori Ando,† Saki Ariizumi,† Chiaki Kiyokawa,† Masahiro Hashimoto,† Tomohiro Asai,† Takehisa Dewa‡ and Naoto Oku†*

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† Department of Medical Biochemistry, Graduate division of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526 Japan;

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Corresponding Author

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‡Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555 Japan

Naoto Oku, PhD

Department of Medical Biochemistry, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526 Japan TEL: +81-54-264-5701

FAX: +81-54-264-5705

e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCL) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5 cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72 h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a “packed multi-layer” structure after the freezethawing of “single-layer” PCL and siRNA complex, suggesting that siRNA exists between the lipid layer working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freezethawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for “one-step” encapsulation of siRNA into liposomes by freeze-thawing.

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KEYWORDS: small-interfering RNA; polycation liposomes; freeze-thaw; siRNA encapsulation

ABBREVIATIONS

BCA, bicinchoninic acid; DAPI, 2-amidino-diphenyl-indole; DCP-DETA, dicetyl phosphatediethylenetriamine; DEPC, diethylpyrocarbonate; DOPE, dioleoylphosphatidylethanolamine; EPR, enhanced permeability and retention; FITC, fluorescein isothiocyanate; lipoplex, liposome and siRNA complex; N/P, nitrogen/phosphorus ratio; PCL, polycationic liposomes; PEG, polyethylene glycol; siRNA, small-interfering RNA; TEM, transmission electron microscope.

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ACCEPTED MANUSCRIPT 1. Introduction

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Small interfering RNA (siRNA), a nucleic acid having RNA interference activity, is a short double-stranded RNA composed of 21-23 base pairs. The siRNA interacts with and cleaves sequence-specific mRNA [1-3]. siRNA has been widely used for investigating specific protein functions in the field of scientific research. Because of its fewer side effects and its potential therapeutic effects, siRNA is expected to be a therapeutic agent for intractable diseases such as cancer. Since siRNA is degraded in the bloodstream by RNases and has little transfection efficiency owing to its low permeation through the plasma membrane [4, 5], cationic liposomes, polycations and lipidic nanoparticles have been used as a siRNA delivery vector to overcome these problems [5-8]. The major approach for the delivery is the attachment of the siRNA on the lipidic vector surface to make a lipoplex. However, the cationic groups of these siRNA vectors tend to induce cytotoxic effect. For the purpose of siRNA delivery, we previously developed dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) for preparing polycation liposomes (PCLs), and found that siRNA complexed with these PCLs induced a remarkable gene-silencing effect in vitro and in vivo [9-11]. Moreover, the PCLs showed fewer cytotoxic effects than the conventional cationic liposomes, although it is preferred that the cationic charges in the PCL should be decreased as much as possible.

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It is well known that attaching siRNA onto the cationic liposome or PCL surface through electrostatic interaction is the major method for siRNA holding and delivery [7, 9]. The method does not require any special techniques or machines, as lipoplexes can be prepared by simply incubating siRNA and the vector for several minutes. Although siRNA in the PCL-complex induces significant knockdown of target mRNA in vitro, binding affinity of siRNA to PCLs is not very strong. In fact, siRNA tends to detach from the PCL surface after polyethylene glycol (PEG)-modification [9]. Therefore, an additional interaction between siRNA and PCL, such as hydrophobic interaction by conjugation of cholesterol to the siRNA, should be required [9, 1214]. The other approach is the encapsulation of siRNA inside a liposome, lipidic nanoparticle or like vector [15, 16]. The most important advantage of this approach is that siRNA degradation in the bloodstream is prevented. Lipidic vectors encapsulating siRNA are suitable for manipulating surface charge and probe-modification for active targeting, as well as for protecting against degradation by plasma RNases. Therefore, this approach is a quite useful one for in vivo siRNA delivery. However, additional technologies or organic solvents are required for preparation of siRNA-encapsulated lipidic nanoparticles [17-20]. Therefore, an easier method for encapsulating siRNA, such as a one-step encapsulation method, is still awaited for efficient siRNA delivery. The liposome is a closed vesicle composed of a lipid bilayer membrane. The liposome can encapsulate hydrophilic materials in its interior water phase and hydrophobic materials in the

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lipid membrane and, therefore, is widely used as a drug delivery tool. In fact, Doxil®, which is a doxorubicin (Dox)-encapsulated polyethylene glycol (PEG)-modified liposome, has been used clinically [21, 22]. The Doxil® formulation reduces side effects and enhances the anticancer activity of Dox through enhanced permeability and retention (EPR) effect [23, 24]. In general, a multi-layer structure is formed after hydration of a lipid film [25]. Since the multi-layer liposome is large heterogeneous in size, with a small volume of water phase per liposomal lipids, the single-layer liposome was developed by “freeze-thawing” of the multi-layer liposome [26]. The freeze-thawing method has also been used for the encapsulation of small hydrophilic molecules into the aqueous pocket of the liposome [27]. It is known that freeze-thawing of single-layer liposomes with calcein increases the encapsulation ratio of calcein into the “single-layer” liposome [28]. However, there has been no previous study on the influence of freeze-thawing on liposomes with siRNA.

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In the present study, we investigated the effect of freeze-thawing on PCL-based lipoplexes and found that freeze-thawing of single-layer PCLs and siRNA complex formed multi-layer structures, suggesting that siRNA was effectively encapsulated between the lipid layers of these multi-layer PCLs. The freeze-thawed lipoplexes protected siRNA from RNases and enhanced the knockdown efficiency of target mRNA in vitro. In addition, the accumulation of siRNA in tumor was increased by systemic administration of PEGylated freeze-thawed lipoplexes compared with the PEGylated conventional ones. These results suggest that this freeze-thawing strategy can offer a novel approach to develop a “one-step” preparation of siRNA-encapsulated PCLs or cationic liposomes.

2. Materials and Methods 2.1. Materials Cholesterol, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were kindly donated by Nippon Fine Chemical Co. (Takasago, Hyogo, Japan). Dicetylphosphate-tetraethylenepentamine (DCP-TEPA) was synthesized as described previously [9]. siRNAs for Luc2 was purchased from Hokkaido System Science Co. (Hokkaido, Japan). The nucleotide sequences of siRNA with a 2nucleotide overhang (underline) were 5’-GGCUACGUCCAGGAGCGCACC-3’ (sense) and 5’UGCGCUCCUGGACGUAGCCUU-3’ (antisense). Fluorescein isothiocyanate was conjugated with siRNA for preparing FITC-labeled siRNA. Alexa750 was also conjugated with siRNA at the 3'-end of the anti-sense strand for the in vivo study. All other reagents were of analytical grade. 2.2. Synthesis of DCP-DETA Dicetyl phosphate-diethylenetriamine (DCP-DETA) was synthesized by using a procedure analogous to that reported previously [29, 30]. Dimerized dicetyl phosphate anhydride (216 mg, 0.2 mmol) in anhydrous chloroform (0.5 mL) was added to a solution of

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diethylenetriamine (62 mg, 0.6 mmol) in 0.5 mL of anhydrous pyridine. The reaction mixture was stirred under nitrogen atmosphere for 4 h at room temperature. After removal of the solvent under reduced pressure, the residue was suspended in distilled water and then filtered to remove any unreacted diethylenetriamine. The residue was subsequently subjected to column chromatography using aminated silica gel (Chromatorex NH, Fuji Silisia Chemical LTD, Aichi, Japan) with an eluting solution of CHCl3 and subsequent CHCl3/MeOH (39/1, v/v), giving 73 mg (57%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 0.88 (t, 6H), 1.25 (s, 52H), 1.66 (m, 4H), 1.80 (br s, 4H), 2.68–2.83 (m, 5H), 2.99 (br m, 2H), 3.25 (br m, 1H), 3.98 (bm, 4H); MALDITOF-MS for (C36H79N3O3P)+ calcd 632.99, found 632.53.

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2.3. Preparation of lipoplexes Dioleoylphosphatidylethanolamine (DOPE), cholesterol, and dicetyl phosphatediethylenetriamine (DCP-DETA; 1:1:1 as a molar ratio) were dissolved in t-butyl alcohol and freeze-dried. PCLs were produced by hydration of the lipid mixture with DEPC-treated RNasefree water. Liposomes were sized by extrusion 10 times through a polycarbonate membrane filter having 100-nm pores (Nucleopore, Maidstone, UK). Liposomes and siRNA were mixed and incubated for 20 min at room temperature to form liposome/siRNA complexes. To prepare freeze-thawed lipoplex, the complex was frozen in liquid nitrogen and thawed in a water bath at 43 °C with vortexing. The particle size and ζ-potential of the complexes diluted with 0.5 mM PBS (pH=7.4) were measured by using a Zetasizer Nano ZS (Malvern, Worcs, UK). For the in vivo study, lipoplexes were decorated with polyethylene glycol (PEG) by incubating them with DSPE-PEG6000 (10 mol% to total lipids) at 40°C for 10 min. Dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dicetylphosphatetetraethylenepentamine (DCP-TEPA) liposomes (DOPE : cholesterol : DPPC, DOTAP or DCPTEPA=1:1:1) were also prepared by same procedure. 2.4. Electrophoretic assay The amount of siRNA that was not or only loosely attached to lipoplexes was checked by performng15% polyacrylamide gel electrophoresis, where the siRNA in stable complexes did not enter the gel. The gel was stained for 30 min in GelRed, and siRNA was detected by using a LAS-3000 mini system (Fuji Film, Tokyo, Japan). 2.5. Stability of siRNA in the absence and presence of serum Naked siRNA or lipoplexes were incubated in 90% fetal bovine serum (FBS; Sigma– Aldrich, St. Louis, MO) at 37°C for 72 h. Then, the siRNA was extracted from the serum by using TRIzol reagent and subjected to 15% polyacrylamide gel electrophoresis for detecting intact siRNA, as described above. 2.6. LC-MS measurement of siRNA in conventional and freeze-thawed lipoplexes

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The HPLC-IT-MS system consisted of a NANOSPACE SI-2 high-performance liquid chromatography (SHISEIDO, Tokyo, Japan) and a LCQ Fleet TM ion-trap mass spectrometer (Thermo Scientific™, MA, USA). An Asahipak ODP-50 2D column (5 m, 250 mm×2.0 mm i.d., Shodex) was used as the analytical column, and was maintained at 60 °C. The injection volume was 10 µL. The mobile phase, consisting of solvent A (0.01M triethylamine in acetic acid buffer [pH 6.0]) and solvent B (acetonitrile), was delivered at the flow rate of 0.2 mL/min. The gradient elution was as follows: B%=2-100 (0-15 min). The scan mode was used from m/z 1639.20-1644.20 (antisense strand), 1676.62-1681.62 (sense strand). The wavelength of detection was set at 260 nm.

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2.7. Transmission electron microscopy (TEM) Conventional liposomes (PCLs), lipoplexex or freeze-thawed lipoplexes (0.5 mM as total lipid concentration) in a volume of 5 μL were placed on a grid (Nisshin EM, Tokyo, Japan) and dried-out by a stream of warm air for 3 times. Then, each sample was negatively stained with 10 μL of 1 w/v% ammonium molybdate for 1 min, and imaged with an HT7700 TEM System (Hitachi High-Technologies, Tokyo, Japan). The images were recorded with a CCD camera at 1024 × 1024 pixels (Advanced Microscopy Techniques, Woburn, MA, USA).

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2.8. Cell cultures B16F10 murine melanoma cells were obtained from ATCC (Manassas, VA); and B16F10-Luc2 Bioware® Ultra Cell Line, a luciferase-expressing cell line stably transfected with the firefly luciferase gene (Luc2), were purchased from Caliper Life Sciences (Hopkinton, MA). Both types of cells were cultured in D-MEM/Ham’s F-12 containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 in the air. 2.9. Confocal microscopy B16F10 seeded onto MAS-coated micro slide glasses (Matsunami glass, Osaka, Japan) at a density of 1.0  ×  104 cells/well were transfected with FITC-labeled siRNA (FITC-siRNA) complexed with DiI-C18-labeled liposome (FITC-siRNA concentration was 20 pmol/0.2  ml/well). Twenty-four hours after transfection of each sample, the cells were washed 3 times with PBS (pH 7.4) containing heparin (30 units/ml) and then 3 times alone, and thereafter fixed with 4% paraformaldehyde. Nuclei were stained with 4,6-diamino-2-phenylindole (DAPI; 10  µg/ml; Invitrogen). Next, localization of FITC-labeled siRNA and DiI-C18-labeled liposomes in the B16F10 cells was observed under an LSM510 META confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). 2.10. siRNA uptake B16F10 cells were seeded onto 24-well plates (BD Bioscience, San Jose, CA) at the density of 1.0  ×  104 cells/well. Naked FITC-siRNA (100 nM, Hokkaido System Science Co.) or FITC-labeled siRNA formulated in conventional or freeze-thawed lipoplexes were added onto

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the cells. Six, 12 or 24 h after the transfection, the cells were washed with PBS and lysed with 1 w/v% n-octyl-β-d-glucoside (Dojindo, Kumamoto, Japan) containing the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/mL leupeptin, 2 μg/mL aprotinin, and 2 μg/mL pepstatin A (all from Sigma–Aldrich). The fluorescence intensity of FITC was determined with a Tecan Infinite M200 microplate reader (Salzburg, Austria) operated according to the manufacturer’s instructions (ex. 485 nm, em. 535 nm). Total protein content measured with a BCA Protein Assay Reagent Kit (PIERCE Biotechnology, Rockford, IL) according to the manufacturer’s instructions.

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2.11. Knockdown efficiency B16F10-Luc2 cells (1 × 104 cells/0.2 mL/well) were seeded onto 96-well Luminunc white plates (Nalge Nunc International, Napervilla, IL) and precultured overnight. For transfection with siRNA, the medium was changed to a fresh one containing 10% FBS but no antibiotics. The cells were then treated for 24 h with 50 pmol siLuc2 formulated in lipoplexes or lipofectamine2000 at a final siRNA concentration of 50 nM. After these complexes had been removed, the cells were cultured for an additional 24 h. Then, 100 μL ONE-Glo Reagent (Promega, Madison, WI) was applied to each well for determining relative luminescence units (RLUs) and cell viability was measured by using a Cell Titer-Fluor Cell Viability Assay kit (Promega) in accordance with the manufacturer's instructions.

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2.12. Experimental animals Five-week-old BALB/c male mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The animals were cared for according to the Animal Facility Guidelines of the University of Shizuoka. All animal experiments were approved by the Animal and Ethics Review Committee of the University of Shizuoka. 2.13. siRNA biodistribution Colon26 NL-17 cells (1 × 106 cells/mouse) were implanted subcutaneously into the posterior flank of 5-week-old BALB/c male mice. The biodistribution study was performed at day 20 after tumor implantation. Size-matched Colon26 NL-17-bearing mice were injected with naked Alexa750-conjugated siRNA, PEGylated conventional lipoplex or PEGylated freezethawed lipoplex via a tail vein. Then, biodistribution of Alexa750-conjugated siRNA was measured with an in vivo imaging system (Xenogen IVIS Lumina System) coupled to Living Image software for data acquisition (Xenogen Corp., Alameda, CA, USA). Forty-eight hours after the injection, these mice were sacrificed under deep anesthesia for the collection of blood. Then, after the mice had been bled from a carotid artery, their heart, lungs, liver, spleen, kidneys and tumor were removed and measured for fluorescence activity. 2.14. Statistics

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3. Results and discussion 3.1. Preparation and characterization of freeze-thawed lipoplexes

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Dicetyl phosphate-diethylenetriamine (DCP-DETA) was newly synthesized. PCLs composed of DCP-DETA, dioleoylphosphatidylethanolamine (DOPE), and cholesterol (DCPDETA : DOPE : cholesterol = 1 : 1 : 1 as a molar ratio) and sized for 100-nm particles were prepared as described in Materials and Methods. The particle sizes and ζ-potentials of conventional lipoplexes and freeze-thawed ones prepared at various N/P ratios are summarized in Table 1.

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We firstly validated the influence of freeze-thawing on the siRNA-holding capacity to the PCL by electrophoretic assay (Fig. 1a,b). In this assay, free siRNA and loosely bound siRNA could be detected. The siRNA band was clearly observed at N/P = 5 and was slightly thinner at N/P = 10 in the conventional lipoplex group, suggesting that the siRNA was not strongly attached to the PCL surface at an N/P ratio of less than 10, where the electrostatic interaction was not strong enough to hold the siRNA. In the case of the conventional lipoplex, about half of the DCP-DETA would be expected to exist on the outer layer of PCL; and only the primary amine (pK=6.2) but not the secondary amine (pK=8.9) was mainly protonated [31], since electrophoresis was conducted at pH 8.3. Therefore, an N/P ratio of 5 means roughly 1.25/1 as the cation/anion ratio. Consequently, it was possible that loosely bound siRNA could be detached from the PCL in the electrophoretic buffer. In contrast, the siRNA band at the N/P ratios of 5 and 10 after the lipoplex had been freeze-thawed 3 times became thin, suggesting that the siRNA had been protected from detachment, namely, encapsulated into the PCL. Importantly, the siRNA was not degraded by the freeze-thawing, as examined by LC-MS (Supplementary Fig. 1a,b). Interestingly, the band of siRNA was dramatically thinner after the 3 cycles of freeze-thawing at the N/P ratio of 5 (Fig. 1b). These results suggest that repeating freeze-thawing of the lipoplex increased the siRNA-encapsulating capacity to the PCL. In addition, more than 3 cycles of freeze-thawing would be required for significant increasing of the siRNA encapsulation. The increase of siRNA-encapsulating capacity was also observed in 1,2-dioleoyl-3trimethylammonium-propane (DOTAP)- or dicetylphosphate-tetraethylenepentaamine (DCPTEPA)-containing liposomes (Supplementary Fig. 2a,b), but not in dipalmitoylphosphatidylcholine (DPPC)-containing liposomes with neutral surface charge (Supplementary Fig. 2c). Therefore, it is suggested that, in general, cationic liposomes can increase siRNA-encapsulating capacity by the freeze-thawing. Next, to confirm the stability of the siRNA in the presence of serum, we incubated both types of lipoplex for 72 h at 37 °C in 90% serum that had not been heat inactivated. Then, un-degraded siRNA was extracted by TRIzol® and detected by performing the electrophoretic assay (Fig. 1c). None of siRNA bands

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were observed in conventional lipoplex groups, even when the N/P ratio was raised to 10. In contrast, siRNA bands were observed in all groups of freeze-thawed lipoplex. This result indicates that siRNA in the conventional lipoplex was degraded by RNases in the serum but that the degradation was inhibited by freeze-thawing of the lipoplex, suggesting that protection from serum RNase was due to encapsulation of siRNA in the PCL by the freeze-thawing.

Figure 1. Influence of freeze-thawing of lipoplex on siRNA holding capacity to lipoplex. (a) After the preparation of conventional or 3-times freeze-thawed lipoplexes, siRNA which was not attached to the lipoplex was separated by electrophoresis in a 15% acrylamide gel and stained with GelRed. (b) After 0, 1, 3 or 5 cycles of freeze-thawing of the lipoplexes, siRNA that was not attached to the lipoplex was separated by electrophoresis and stained with GelRed. (c) Naked siRNA, conventional, and 3-times freeze-thawed lipoplexes (N/P= 1, 3, 5 or 10) were incubated in 90% FBS for 72 h at 37 °C. After extraction of siRNA from the samples, undegraded siRNA was detected. Untreated siRNA was used as a control.

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3.2. Formation of multilayer lipoplexes through freeze-thawing

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The freeze-thawing of lipoplex increased siRNA-holding, or -encapsulating, capacity to the PCL. Therefore, we hypothesized that this freeze-thawing had changed the lipid-layer structure of the PCL. To confirm this hypothesis, we examined the lipid layer of the conventional and freeze-thawed lipoplexes by a negatively stained transmission electron microscopy (TEM, Fig. 2a-h). Several lipid layers, 5 layers were observed after hydration of the freeze-dried lipid mixture (Fig. 2a,e). This multi-layered structure was reduced to a single-layer one after freezethawing (Fig. 2b,f). The single-layer structure did not change after the addition of siRNA to make the lipoplex (Fig. 2c,g). Surprisingly, when this lipoplex was freeze-thawed 3 times, several lipid layers, 4 in the showing image, were observed (Fig. 2d,h). Fig. 2i-l shows a schematic diagram of the structural changes during preparation of the freeze-thawed lipoplex. In general, water molecules bind to the hydrophilic head of liposomal lipids in aqueous solution. However, dehydration occurs during the freezing step, with breakdown of the lipid bilayer. Then, the bilayers are reconstructed by a fusion process during rehydration, namely, thawing. Therefore, repeating the freeze-thawing process decreases the number of lipid layers. However, our data showed that freeze-thawing of a single-layer PCL and siRNA complex increased the number of lipid layers, unlike the case of freeze-thawing of the liposome alone. A possible explanation for this phenomenon is that the siRNA on the PCL surface strongly interacted with cationic lipids of the surrounding PCL by electrostatic interaction in the freezing step, which in turn triggers the formation of an additional lipid layer on the lipoplex. Therefore, repeating the freeze-thawing of lipoplex would increase the number of lipid layers, with siRNA acting as a binder.

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Figure 2. Influence of freeze-thawing of lipoplexes on their structure. (a-h) TEM image of (a,e) PCL after hydration (a,e), (b,f) freeze-thawed PCL (b,f), (c,g) conventional lipoplex (c,g) or (d,h) freeze-thawed lipoplex (d,h). (e-h) Higher magnification images. (i-l) Schematic presentation of PCL or lipoplex structures: multi-layered PCL (i), single-layered PCL after hydration (freeze-thawing) of multi-layered PCL (j), lipoplex (k), freeze-thawed lipoplex (l). (e-h) Arrowheads indicate lipid-layers. (a-d) Bar; 200 nm. (e-h) Bar; 50 nm.

3.3. Cellular uptake and knockdown effect of siRNA in freeze-thawed lipoplex Next, the cellular uptake of siRNA in conventional and freeze-thawed lipoplexes into B16F10 mouse melanoma cells was examined (Fig. 3a). Conventional and freeze-thawed lipoplexes were prepared with fluorescein isothiocyanate (FITC)-conjugated siRNA and then added to the cells. At 24 h after the transfection, the fluorescence intensity in the cells was measured. Fluorescence intensity of siRNA in the cells increased in an N/P ratio-dependent manner for both conventional and freeze-thawed lipoplex. However, the fluorescence intensity of the freeze-thawed lipoplex-treated cells was significantly higher than that of the conventional lipoplex-treated cells at each N/P ratio.

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Since siRNA release from the lipoplex in the cytosol is important for its knockdown effect on target mRNA, siRNA and PCL localization in the cells were observed by confocal laser-scanning microscopy at 24 h after transfection by using FITC-conjugated siRNA and DiIC18-labeled PCL (Fig. 3b). Most FITC-fluorescence, i.e., siRNA, and DiI-fluorescence, namely, PCL, were separately localized in the cells in both conventional and freeze-thawed lipoplexes, indicating that most of the siRNA had been released from the PCLs in the cells. Since PCLs are known to be taken up by cells through endocytosis, this observation indicates that siRNA was released into the cytosol from endosomes. Interestingly, fluorescence-labeled siRNA and PCL were separately localized in the cells treated with the freeze-thawed lipoplexes. These results suggest that siRNA could be delivered by freeze-thawed lipoplexes into the cytosol and the freeze-thawed lipoplexes have potential to induce knockdown of target mRNA similar to conventional lipoplexes. To evaluate the knockdown efficacy and cytotoxicity of the freezethawed lipoplex, we transfected luciferase-expressing B16F10 (B16F10-Luc2) cells with conventional or freeze-thawed lipoplexes at the selected N/P ratios shown in Fig. 3c,d. Since siRNA for luciferase (siLuc2) was used in the present study, knockdown efficiency was easily assessed in terms of luciferase activity. Although the knockdown effect of the conventional and freeze-thawed lipoplexes was N/P ratio dependent for both, the effect of the freeze-thawed lipoplex was higher than that of the conventional lipoplex at all N/P ratios tested (Fig. 3c). Surprisingly, although the cytotoxicity of the conventional lipoplexes was increased by raising the N/P ratio, that of freeze-thawed lipoplex was not, even when the N/P ratio was elevated to 10 (Fig. 3d). In the case of conventional lipoplexes, elevation of the N/P ratio would increase the number of free cationic groups on the PCL surface; and these free cationic groups might cause significant cytotoxicity. In contrast, in the case of the freeze-thawed lipoplex, these cationic groups would reside inside of the lipoplex by binding with siRNA between the bilayers during freeze-thawing, and so the number of free cationic groups on the surface of the freeze-thawed lipoplex should be decreased. Therefore, the cytotoxicity of the freeze-thawed lipoplex would be significantly decreased. Alternatively, the topological distribution of DCP-DETA between the outer bilayer and inner bilayers might have changed during reconstitution of PCL by freezethawing, and DCP-DETA would be more condensed in the inner bilayers due to binding by siRNA. In fact, the zeta-potential of the freeze-thawed lipoplex was lower than that of the conventional lipoplex (Table 1). This evidence is consistent with the possible reasons for the decreased cytotoxicity of freeze-thawed lipoplexes mentioned above, although further precise study is needed to clarify the mechanism responsible for the decrease in the positive charge on the surface of the freeze-thawed lipoplex. Our next question was whether there would be an optimum number of freeze-thawing cycles for efficient knockdown. The maximum knockdown effect was observed with lipoplexes that had been freeze-thawed 3 times. Less or more than 3 cycles of freeze-thawing slightly decreased the knockdown efficacy (Supplementary Fig. 3).

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Figure 3. Cellular uptake and knockdown effect of freeze-thawed lipoplexes. (a) siRNA uptake into the B16F10 cells at selected N/P ratios. Twenty-four hours after addition of conventional (blue bar) or freeze-thawed lipoplexes (red bar) to the cells, fluorescence intensity of FITC-siRNA in the cells was measured. (***P < 0.001 vs. conventional lipoplex) (b) Localization of siRNA and PCL in B16F10 cells. B16F10 cells were transfected with conventional or freeze-thawed lipoplexes composed of FITC-siRNA and DiI-C18-labeled PCL. Twenty-four hours after the incubation, the transfected cells were fixed with 4% paraformaldehyde. Nuclei were stained with DAPI (blue). Localization of siRNA (green) and PCL (red) was observed by confocal laser-scanning microscopy. Scale bars  =  10  µm. (c) Knockdown effect of conventional (blue bar) or freeze-thawed lipoplexes (red bar). B16F10Luc2 cells were transfected with conventional or freeze-thawed lipoplexes. After 48 hours of additional incubation, luciferase activities were measured and normalized by the total protein content. (d) The protein content of living cells was measured by use of the BCA protein assay. (***P < 0.001, **P < 0.01, *P < 0.05 vs. conventional lipoplex). C; control (PBS), L; lipofectamine® 2000

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3.4. siRNA delivery using freeze-thawed lipoplexes in vivo Finally, to deliver siRNA to tumor in situ, we modified conventional and freeze-thawed lipoplex with polyethylene glycol 6000 (10% as total lipid). In addition, Alexa750-conjugated siRNA was used for measurement of siRNA biodistribution in vivo. The size and ζ-potential of PEGylated conventional and freeze-thawed lipoplexes were 198.0 nm, -10.9 mV (PEGylated conventional lipoplex) and 261.9 nm, -4.84 mV (PEGylated freeze-thawed lipoplex), respectively. We previously reported that siRNA detaches from the PCL after PEGylation [9]. To confirm whether siRNA remained stable or not after PEGylation of the PCL, the amount of siRNA detached from the lipoplex after the PEGylation was assessed by use of the electrophoretic assay. As shown in Fig. 4a, the band of detached siRNA was detected in the lane that contained the PEGylated conventional lipoplex, consistent with previous results [9]; however, the detectable band was thin in that containing the PEGylated freeze-thawed lipoplex. This result indicates that only a little amount of siRNA, which might be bound to the outer surface of PCL, was detached from the freeze-thawed lipoplex, supporting the idea that a large amount siRNA had been efficiently encapsulated into it. To determine the biodistribution of siRNA in tumor-implanted mice, colon26 NL-17 carcinoma cells were subcutaneously implanted into the mice. Twenty days after tumor implantation, PEGylated conventional or freeze-thawed lipoplexes, as well as naked siRNA, were intravenously injected into the mice. The biodistribution of siRNA in the PEGylated conventional lipoplexes was very similar to that of naked siRNA (Fig. 4b). Although in vivo near infrared imaging is not quantitative and incomparable among tissues because the fluorescence intensity is strongly affected by the depth from the surface of the body [32], most fluorescence is detected in the bladder after the intravenous injection, suggesting that siRNA was present in its free form in the bloodstream after injection of PEGylated conventional lipoplexes. In addition, ex vivo imaging showed that siRNA accumulated in the liver, spleen, and kidneys, but not in the tumor, at 48 h after the injection (Fig. 4c). In contrast, the biodistribution of siRNA-fluorescence in the PEGylated freeze-thawed lipoplexes was different from that of the naked siRNA or PEGylated conventional lipoplex. Strong signals were observed in the whole body up to 3 h post injection, suggesting that much siRNA was circulating in the bloodstream. At 48 h after the injection, fluorescence signal was observed in the tumor tissue, indicating that siRNA had accumulated in it. Ex vivo imaging showed that siRNA delivered with PEGylated freeze-thawed lipoplexes had not only accumulated in the liver, spleen, and kidneys but also in the tumor at 48 h after the intravenous injection. These results demonstrate that the conventional lipoplex induced efficient knockdown in vitro; however, the most of siRNA were detached from the PCL after the PEGylation. On the other hand, siRNA formulated in freeze-thawed lipoplex showed the characteristic of long-term circulation, except that bound to the outer surface of the lipoplex, and accumulated in the tumor, suggesting that encapsulated siRNA by the freeze-thawing of lipoplex stably holds siRNA during circulation and may induces efficient knockdown in vivo after systemic administration.

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Figure 4. siRNA delivery using freeze-thawed lipoplexes in vivo (a) After the PEGylation of conventional or freeze-thawed lipoplexes, siRNA detached from the lipoplexes was separated by electrophoresis in 15% acrylamide gel and stained with GelRed. (b) siRNA biodistribution in tumor-implanted mice. Tumor-bearing mice were intravenously injected with Alexa750-conjugated naked siRNA or Alexa750-conjugated siRNA in PEGylated conventional or freeze-thawed lipoplexes. Then, siRNA biodistribution was measured by IVIS at several times. (c) Ex vivo images. Forty-eight hours after the injection, these mice were sacrificed and measured for fluorescence intensity of Alexa750-conjugated siRNA. H, Lu, Li, Sp, K, and T indicate heart, lungs, liver, spleen, kidney, and tumor, respectively.

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4. Conclusions In the present study, we evaluated the influence of freeze-thawing of polycation liposomes bearing siRNA. The siRNA-holding capacity to the PCLs was increased, and siRNA degradation by RNases was inhibited by freeze-thawing of the lipoplexes. We found that the freeze-thawing of single-layer PCLs and siRNA complex formed a multi-layer structure, suggesting that the siRNA was present between the lipid layers of the multi-layered PCLs. Although the knockdown effect of the conventional lipoplex increased in an N/P ratio-dependent fashion, cytotoxicity was also N/P ratio-dependently enhanced. However, the knockdown effect of the freeze-thawed lipoplexes was N/P ratio-dependently increased without any evidence of cytotoxicity. In addition, PEGylated freeze-thawed lipoplexes delivered siRNA to tumors in mice after their intravenous injection in vivo. Although there are a number of siRNA encapsulation methods, the freeze-thawing method offers the potential for a novel approach for “one-step” encapsulation of siRNA in liposomes.

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Table 1. Size, polydispersity index (PDI), and zeta-potential of conventional and freezethawed lipoplexes at several N/P ratios.

Particle size (nm)

PDI

ζ-Potential (mV)

PCL

152 ± 10

0.16 ± 0.04

26.0 ± 4.5

N/P = 1

179 ± 8

0.24 ± 0.04

N/P = 3

180 ± 5

0.23 ± 0.06

-33.4 ± 2.7

N/P = 5 N/P = 10

188 ± 10 608± 33

0.23 ± 0.04 0.12 ± 0.06

-8.1 ± 8.3 +17.1 ± 5.0

Freeze-thawed

Particle size (nm)

PDI

ζ-Potential (mV)

PCL

524 ± 39

N/P = 1

298 ± 27

N/P = 3

319 ± 24

N/P = 5

295 ± 12

N/P = 10

468 ± 42

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Conventional

0.28 ± 0.05

+34.4 ± 4.8

0.28 ± 0.03

-40.3 ± 2.5

0.26 ± 0.02

-36.9 ± 3.8

0.27 ± 0.03

-20.0 ± 2.5

0.36 ± 0.03

-8.8 ± 2.0

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-32.2 ± 8.9

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Supporting Information. Information on LC-MS data and materials and methods, such as synthesis of DCP-DETA and preparation of freeze-thawed lipoplexes.

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Conflict of Interest: The authors declare no competing financial interest.

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Acknowledgement

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This research was also supported by a grant-in-Aid for scientific research from the Japan Society for the Promotion of Science (JSPS) and by The Kurata Memorial Hitachi Science and Technology Foundation.

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[9] T. Asai, S. Matsushita, E. Kenjo, T. Tsuzuku, N. Yonenaga, H. Koide, K. Hatanaka, T. Dewa, M. Nango, N. Maeda, H. Kikuchi, N. Oku, Dicetyl phosphate-tetraethylenepentaminebased liposomes for systemic siRNA delivery. Bioconjug Chem 22 (2011) 429-435. [10] H. Koide, T. Asai, H. Kato, N. Yonenaga, M. Yokota, H. Ando, T. Dewa, M. Nango, N. Maeda, N. Oku, Susceptibility of PTEN-positive metastatic tumors to small interfering RNA targeting the mammalian target of rapamycin. Nanomedicine 11 (2015) 185-194. [11] H. Koide, T. Asai, K. Furuya, T. Tsuzuku, H. Kato, T. Dewa, M. Nango, N. Maeda, N. Oku, Inhibition of Akt (ser473) phosphorylation and rapamycin-resistant cell growth by knockdown of mammalian target of rapamycin with small interfering RNA in vascular endothelial growth factor receptor-1-targeting vector. Biol Pharm Bull 34 (2011) 602-608. [12] V.V. Ambardekar, H.Y. Han, M.L. Varney, S.V. Vinogradov, R.K. Singh, J.A. Vetro, The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes. Biomaterials 32 (2011) 1404-1411. [13] C. Wolfrum, S. Shi, K.N. Jayaprakash, M. Jayaraman, G. Wang, R.K. Pandey, K.G. Rajeev, T. Nakayama, K. Charrise, E.M. Ndungo, T. Zimmermann, V. Koteliansky, M. Manoharan, M. Stoffel, Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25 (2007) 1149-1157. [14] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8 (2009) 129-138. [15] Q. Lin, J. Chen, Z. Zhang, G. Zheng, Lipid-based nanoparticles in the systemic delivery of siRNA. Nanomedicine (Lond) 9 (2014) 105-120. [16] H. Hatakeyama, H. Akita, E. Ito, Y. Hayashi, M. Oishi, Y. Nagasaki, R. Danev, K. Nagayama, N. Kaji, H. Kikuchi, Y. Baba, H. Harashima, Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. Biomaterials 32 (2011) 4306-4316. [17] C.M. McCrudden, H.O. McCarthy, Current status of gene therapy for breast cancer: progress and challenges. Appl Clin Genet 7 (2014) 209-220. [18] A. Akinc, M. Goldberg, J. Qin, J.R. Dorkin, C. Gamba-Vitalo, M. Maier, K.N. Jayaprakash, M. Jayaraman, K.G. Rajeev, M. Manoharan, V. Koteliansky, I. Rohl, E.S. Leshchiner, R. Langer, D.G. Anderson, Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol Ther 17 (2009) 872-879. [19] D.V. Morrissey, J.A. Lockridge, L. Shaw, K. Blanchard, K. Jensen, W. Breen, K. Hartsough, L. Machemer, S. Radka, V. Jadhav, N. Vaish, S. Zinnen, C. Vargeese, K. Bowman, C.S. Shaffer, L.B. Jeffs, A. Judge, I. MacLachlan, B. Polisky, Potent and persistent in vivo antiHBV activity of chemically modified siRNAs. Nat Biotechnol 23 (2005) 1002-1007. [20] K. Kusumoto, H. Akita, S. Santiwarangkool, H. Harashima, Advantages of ethanol dilution method for preparing GALA-modified liposomal siRNA carriers on the in vivo gene knockdown efficiency in pulmonary endothelium. Int J Pharm 473 (2014) 144-147.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Influence of freeze-thawing of lipoplex on siRNA holding capacity to lipoplex.

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(a) After the preparation of conventional or 3-times freeze-thawed lipoplexes, siRNA which was not attached to the lipoplex was separated by electrophoresis in a 15% acrylamide gel and stained with GelRed. (b) After 0, 1, 3 or 5 cycles of freeze-thawing of the lipoplexes, siRNA that was not attached to the lipoplex was separated by electrophoresis and stained with GelRed. (c) Naked siRNA, conventional, and 3-times freeze-thawed lipoplexes (N/P= 1, 3, 5 or 10) were incubated in 90% FBS for 72 h at 37 °C. After extraction of siRNA from the samples, undegraded siRNA was detected. Untreated siRNA was used as a control.

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Figure 2. Influence of freeze-thawing of lipoplexes on their structure.

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(a-h) TEM image of (a,e) PCL after hydration (a,e), (b,f) freeze-thawed PCL (b,f), (c,g) conventional lipoplex (c,g) or (d,h) freeze-thawed lipoplex (d,h). (e-h) Higher magnification images. (i-l) Schematic presentation of PCL or lipoplex structures: multi-layered PCL (i), single-layered PCL after hydration (freeze-thawing) of multi-layered PCL (j), lipoplex (k), freeze-thawed lipoplex (l). (e-h) Arrowheads indicate lipid-layers. (a-d) Bar; 200 nm. (e-h) Bar; 50 nm.

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Figure 3. Cellular uptake and knockdown effect of freeze-thawed lipoplexes.

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(a) siRNA uptake into the B16F10 cells at selected N/P ratios. Twenty-four hours after addition of conventional (blue bar) or freeze-thawed lipoplexes (red bar) to the cells, fluorescence intensity of FITC-siRNA in the cells was measured. (***P < 0.001 vs. conventional lipoplex) (b) Localization of siRNA and PCL in B16F10 cells. B16F10 cells were transfected with conventional or freeze-thawed lipoplexes composed of FITC-siRNA and DiI-C18-labeled PCL. Twenty-four hours after the incubation, the transfected cells were fixed with 4% paraformaldehyde. Nuclei were stained with DAPI (blue). Localization of siRNA (green) and PCL (red) was observed by confocal laser-scanning microscopy. Scale bars  =  10  µm. (c) Knockdown effect of conventional (blue bar) or freeze-thawed lipoplexes (red bar). B16F10Luc2 cells were transfected with conventional or freeze-thawed lipoplexes. After 48 hours of additional incubation, luciferase activities were measured and normalized by the total protein content. (d) The protein content of living cells was measured by use of the BCA protein assay. (***P < 0.001, **P < 0.01, *P < 0.05 vs. conventional lipoplex). C; control (PBS), L; lipofectamine® 2000 Figure 4. siRNA delivery using freeze-thawed lipoplexes in vivo (a) After the PEGylation of conventional or freeze-thawed lipoplexes, siRNA detached from the lipoplexes was separated by electrophoresis in 15% acrylamide gel and stained with GelRed. (b) siRNA biodistribution in tumor-implanted mice. Tumor-bearing mice were intravenously injected with Alexa750-conjugated naked siRNA or Alexa750-conjugated siRNA in PEGylated

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conventional or freeze-thawed lipoplexes. Then, siRNA biodistribution was measured by IVIS at several times. (c) Ex vivo images. Forty-eight hours after the injection, these mice were sacrificed and measured for fluorescence intensity of Alexa750-conjugated siRNA. H, Lu, Li, Sp, K, and T indicate heart, lungs, liver, spleen, kidney, and tumor, respectively.

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Figure 3

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Figure 4

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Table 1. Size, polydispersity index (PDI), and zeta-potential of conventional and freezethawed lipoplexes at several N/P ratios.

Particle size (nm)

PDI

ζ-Potential (mV)

PCL

152 ± 10

0.16 ± 0.04

26.0 ± 4.5

N/P = 1

179 ± 8

0.24 ± 0.04

N/P = 3

180 ± 5

0.23 ± 0.06

-33.4 ± 2.7

N/P = 5 N/P = 10

188 ± 10 608± 33

0.23 ± 0.04 0.12 ± 0.06

-8.1 ± 8.3 +17.1 ± 5.0

Freeze-thawed

Particle size (nm)

PDI

ζ-Potential (mV)

PCL

524 ± 39

N/P = 1

298 ± 27

N/P = 3

319 ± 24

N/P = 5

295 ± 12

N/P = 10

468 ± 42

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Conventional

0.28 ± 0.05

+34.4 ± 4.8

0.28 ± 0.03

-40.3 ± 2.5

0.26 ± 0.02

-36.9 ± 3.8

0.27 ± 0.03

-20.0 ± 2.5

0.36 ± 0.03

-8.8 ± 2.0

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-32.2 ± 8.9

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Graphical Abstract

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