Development of a novel drug delivery system consisting of an antitumor agent tocopheryl succinate

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Development of a novel drug delivery system consisting of an antitumor agent tocopheryl succinate Susumu Hama a, 1, Satoru Utsumi a, 1, Yuki Fukuda a, Kayoko Nakayama a, Yuriko Okamura a, Hiroyuki Tsuchiya a, Kenji Fukuzawa b, Hedeyoshi Harashima c, Kentaro Kogure a,⁎ a b c

Department of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Faculty of Pharmacy, Yasuda Women's University, 6-13-1, Yasuhigashi, Asaminami-Ku, Hiroshima 731-0153, Japan Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan

a r t i c l e

i n f o

Article history: Received 18 February 2012 Accepted 15 May 2012 Available online 23 May 2012 Keywords: Tocopheryl succinate Nanovesicles Tumor suppression

a b s t r a c t We have developed a novel drug delivery system (DDS) using an antitumor agent, α-tocopheryl succinate (TS). TS has attracted attention as a unique anti-cancer drug for its ability to induce apoptosis in various cancer cells. Furthermore, TS itself readily forms nanovesicles (TS-NVs) and is a prospective tool for use as an antitumor DDS. However, TS-NVs are unstable for encapsulating drugs and passive targeting delivery to tumor tissue via enhanced permeation and retention effect. Therefore, to improve the stability of vesicles, we developed a novel nanovesicle consisting of TS and egg phosphatidylcholine (TS-EPC-NVs). The stability of vesicles of TS-EPC-NVs was significantly higher than that of TS-NVs. As a result, the in vivo antitumor activity of TS-EPC-NVs was more potent than that of TS-NVs. The enhanced antitumor activity of TS-EPC-NVs was found to be due to its effective intratumoral distribution. Moreover, the in vitro anticancer efficiency of TS-EPC-NVs increased seven-fold. We suggest that the improvement is due to homogenous cellular uptake and enhanced cytosolic delivery of the nanovesicles via alteration of intracellular trafficking. Furthermore, TS-EPC-NVs encapsulating siRNA showed significant knockdown efficiency. In summary, TS-EPC-NVs represent a novel and attractive drug delivery system. The system shows antitumor activity of the encapsulated drug and the carrier itself. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In current antitumor therapy, it is well accepted that liposomes can be used to encapsulate an anticancer drug and that this approach is ideal for solid tumors. For example, liposomes encapsulating a single drug, such as Doxil, have been clinically used. However, solid tumors contain cancer cells varying in their resistance to anticancer drugs. Thus, the efficacy of liposomes encapsulating a single drug is inadequate and improvements are necessary to eradicate the tumor. In past clinical research, combination therapy using several drugs, each of which has a different mechanism, has been shown to enhance the therapeutic effect compared to monotherapy [1–3]. Therefore, to enhance the availability of combination therapy, drug carriers for multidrug therapy have been developed [4–7]. Among these approaches, it is known that the use of liposomes has benefits. Since liposomes can encapsulate several drugs at a suitable ratio and are passively delivered to tumor tissue by enhanced permeation and retention (EPR) effect, not only the synergistic effect is promising, but also side effects are diminished. In previous reports, liposomes ⁎ Corresponding author. Tel.: + 81 75 595 4663; fax: +81 75 595 4762. E-mail address: [email protected] (K. Kogure). 1 Susumu Hama and Satoru Utsumi equally contribute to this study. 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2012.05.031

encapsulating hydrophobic antitumor drugs, such as paclitaxel, have been extensively studied to improve their aqueous dispersion and to enhance their capacity to encapsulate hydrophilic drugs or functional nucleic acids into the aqueous compartment [8]. In designing a drug delivery system for a hydrophobic drug, it is important that the drug-containing liposomes have both an effective bio‐distribution and bioavailability. However, drug-containing liposomes tend to vitiate the EPR effect due to the high affinity for serum proteins and their destabilization [9,10]. In general, to avoid these undesirable interactions and poor bio‐distribution, polyethylene glycol (PEG) has been added to the surface of liposomes. Although PEG-modified liposomes are effectively delivered to tumor tissues via the EPR effect, the liposomes retain intrinsic problems, such as low cellular uptake and accelerated blood clearance [11,12]. Consequently, it is desirable to develop a drug carrier with minimal interactions with biogenic substances yet one which remains in the circulation for prolonged periods. Ideally, such a carrier would have high cellular affinity and lack PEG. Based on previous reports, anionic substances, including liposomes, are known to interact to a lesser extent with biogenic substances than do cationic drugs and carriers [13]. Thus, liposomes consisting of anionic compounds would be expected to be ideal drug carriers for multidrug therapy and provide alternatives to PEGmodified liposomes.

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Recently, α-tocopheryl succinate (TS), a succinic acid ester of αtocopherol (α-T), was suggested to have promise as a material for nanoparticles with antitumor activity. TS possesses various antitumor effects, such as specific induction of apoptosis for various cancer cells and prevention of tumor angiogenesis [14–17]. Furthermore, there is no cumulative toxicity with TS as distinct from many anti-cancer drugs because TS is hydrolyzed to α-T, silencing apoptotic activity [18]. However, clinical application of TS has been restricted due to its poor solubility. In previous reports, high density lipoprotein (HDL) -linked and PEG-linked TS have been developed [19,20]. Although these compounds exhibited higher antitumor activity than the TS molecule in vivo, they did not control TS delivery to tumor tissues nor offer co-delivery of other drugs. We have focused on the distinct property of TS, i.e., its ability to be dispersed in water as vesicles due to its amphipathic structure [21]. Previously, we prepared nanovesicles consisting solely of TS (TS-NVs) and examined their anticancer effects. We found that intravenous administration of TS-NVs significantly slowed tumor growth, indicating that TS-NVs have potential to act as a drug carrier with anticancer effects [22]. However, because TS-NVs have low vesicle stability in the presence of divalent cations, acidic pH, and serum [23], TS-NVs are not suitable to encapsulate other drugs. Therefore, to put TS-NVs into practice for actual tumor therapy, increasing the stability of TS-NVs is necessary. In this study, to improve the stability of TS-NVs, we developed novel liposomes for drug delivery consisting of TS and egg phosphatidylcholine (EPC) which can form a stable lamellar structure (TS-EPCNVs) [24]. We compared the antitumor effects of TS-EPC-NVs with TS-NVs. Furthermore, we studied the mechanism of the antitumor effect of TS-EPC-NVs. In addition, since it was reported that TS enhances doxorubicin (DOX)-induced apoptosis via promotion of DOX influx and suppression of DOX efflux [25], we examined preparation of TSEPC-NVs encapsulating siRNA, as an alternative anticancer drug, and its functionality as a drug delivery system.

20 min in a bath-type sonicator (NEY). TS-EPC-NVs were prepared as TS-NVs except the thin film contained TS (50 mM) and EPC (32 mM). To control the particle size, TS-NVs and TS-EPC-NVs were passed through a Mini-extruder (Avanti Polar Lipids) using a polycarbonate membrane (0.1 μm pore size). Particle size and surface potential of the nanovesicles dispersed in PBS (−) were measured by dynamic light scattering and laser doppler electrophoresis, respectively, with a Zetasizer nano (Malvern Ins. Ltd.). To assess the membrane fluidity, the degree of fluorescence polarization of the nanovesicles containing 0.002 mol% DPH was measured by BEACON2000 (AMZ System Science).

2.3. Confocal laser scanning microscopic observations of TS-NVs and TS-EPC-NVs B16-F1 cells were cultured on 0.002% poly-L-lysine- (PLL) coated glass bottom dishes at a density of 1 × 105 cells/dish for 24 h in DMEM containing 10% fetal bovine serum (FBS). After washing with PBS (−), cells were treated with TS-NVs or TS-EPC-NVs (25 μM TS) containing 1 mol% Rh-PE for 1 h in serum-free DMEM. To describe the subcellular location of nanovesicles, endosomes/lysosomes (end/lys) and nuclei (nuc), cells were stained with LysoTracker Green DND-26 and Hoechst33342, respectively, before acquiring the images. A series of images were obtained by confocal laser scanning microscopy (CLSM) (LSM510META, Carl Zeiss Co. Ltd., Jena, Germany) equipped with an oil-immersion objective lens (Plan-Apochromat 63/NA1.4). To investigate the intracellular trafficking of nanovesicles during inhibition of the endocytotic pathway, the cells were pre-incubated in serumfree DMEM containing 0.4 M sucrose at 4 °C for 30 min, followed by treatment with Rh-PE-labeled nanovesicles at 4 °C for 30 min. After washing with PBS (−) with heparin, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min. The cells were mounted with VECTASHIELD with DAPI and were observed by CLSM, as described above.

2. Materials and methods 2.4. Cell viability assay by Wst-1 2.1. Materials and animals α-Tocopheryl succinate (TS), 1,6-diphenyl-1,3,5-hexatriene (DPH) and Hoechst33342 were purchased from Sigma Aldrich (St. Louis, MO, U.S.A.). Egg phosphatidylcholine was obtained from NOF CORPORATION (Tokyo, Japan). 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) were purchased from Avanti Polar Lipid (Alabaster, AL, U.S.A.). Premix WST-1 Cell Proliferation Assay System was obtained from TAKARA BIO INC (Kyoto, Japan). Caspase-Glo 3/7 assay and luciferase assay system were purchased from Promega. LysoTracker Green DND-26 and Cell Tracker CM-DiI were obtained from Invitrogen (Carlsbad, CA, U.S.A.). B16-F1 cells, a mouse melanoma cell line, were obtained from DS Pharma Biomedical Co., Ltd (Osaka, Japan). Stearylated octaarginine (STR-R8) and anti-luciferase siRNA (21-mer, 5′-GCGCUGCUGGUGCCAACCCTT-3′, 5′-GGGUUGGCACCAGCAGCGCTT-3′) were synthesized by KURABO (Osaka, Japan) and Invitrogen (Carlsbad, CA, U.S.A.), respectively. Male Hos:HR-1 hairless mice were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). All mice were maintained and used in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee, Kyoto Pharmaceutical University (Kyoto, Japan).

B16-F1 cells were seeded on 96-well Cell BIND Plates (Corning) at 3 × 103 cells/well. After incubation at 37 °C for 24 h, TS dissolved in ethanol (TS-solution) or nanovesicles (TS-NVs or TS-EPC-NVs) was added at 12.5, 25, and 50 μM (TS concentration) for 48 h. The live cells were quantified with the Wst-1 assay. Cell viability was estimated by dividing the absorbance of 440 nm from the TS-treated sample by that of the non-treated group. To evaluate the involvement of the endocytotic pathway, the cells were incubated with TS containing 5 μM chloroquine at 37 °C for 24 h, followed by Wst-1 assay as described above.

2.5. Detection of apoptotic cells by Annexin-V and caspase 3/7 activity Annexin-V-positive cells were detected with the Annexin-V FLUOS Staining kit according to the manufacturer's instructions. Annexin-V fluorescein was measured with a FACS Caliber flow cytometer equipped with Cell Quest software (BD Biosciences). The caspase activity of TStreated cells was determined with a Caspase-Glo 3/7 Assay kit according to the manufacturer's instructions. The chemiluminescence was measured by PLATE manager Infinite M200 (TECAN), and the data are shown as relative light units (RLU).

2.2. Preparation of TS-NVs and TS-EPC-NVs

2.6. Hemolysis assay

TS-NVs were prepared as described previously [22]. To form a thin film containing TS, TS dissolved in ethanol (50 mM) was dried with nitrogen gas on the bottom of a glass tube. The thin film was hydrated in PBS (−) containing 40 mM NaOH for 10 min, followed by sonication for

Suspended rat erythrocytes were continuously mixed with TSsolution, TS-NVs, or TS-EPC-NVs (50 μM TS concentration) at 37 °C for 3 h. After centrifugation at 800 ×g for 5 min, the absorbance at 540 nm in the provided supernatant was measured by PLATE manager.

2.7. Quantification of cellular uptake of nanovesicles containing TS by HPLC and flow cytometry Intracellular amounts of TS were assayed by HPLC for cells treated with 50 μM TS for 3 h, as reported previously [22]. The samples were subjected to HPLC (Waters) using a column of Wakosil 5C18 (150 × 4.6 mm, Wako Pure Chemical Industries, Ltd., Osaka, Japan) monitored at 286 nm corresponding to λmax of TS. The solvent system used was methanol/water (99:1, v/v). Cell-associated nanovesicles were quantified by flow cytometry. B16-F1 cells were treated with DOTAP/EPC (1:1) liposomes, TS-NVs, or TS-EPC-NVs containing 1 mol% Rh-PE for 30 min. After washing twice with PBS(−) containing 2% FBS, the resuspended cells were subjected to flow cytometry.

2.8. Animal experiments The B16-F1 cell-bearing mice were prepared according to our previous report [22]. The B16-F1 cell suspension (1 × 10 6 cells) was mixed with ECM Gel (Sigma) at a ratio of 5:1 (v/v), and then cells were inoculated under the skin of male hairless mice (Hos:HR-1 strain) that were six to eight weeks old (Day 0). From 4 to 16 days after tumor inoculation, TS-NVs, TS-EPC-NVs (10 μmol TS) or saline was injected intravenously five times every three days. Tumor volume was determined according to the formula, Tvol = length × width 2 × 0.5. To examine the hepatic toxicity in TS-EPC-NVs-administered mice on Day 29, glutamate pyruvate transaminase (GPT) in the plasma was assessed with the Transaminase CIxI-test (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To examine the intratumoral distribution of nanoparticles, DiIlabeled TS-NVs and TS-EPC-NVs were administered intravenously to melanoma-bearing mice five days after tumor inoculation. At 24 h after administration, the isolated tumor tissues were embedded into O.C.T compound, and tissue sections (16 μm) were prepared with a LEICA CM 1100 (Leica). Intratumoral nanovesicles were observed by CLSM as described above.

2.9. Preparation of siRNA-encapsulated TS-EPC-NVs siRNA-encapsulated TS-EPC-NVs were prepared according to previous reports with minor modification [26,27]. Anti-Luc siRNA was condensed with STR-R8 at a nitrogen:phosphate (N/P) ratio of 3.0. A lipid film consisting of TS and EPC (5:3.2) was hydrated in the condensed siRNA solution containing 0.8 mM NaOH for 10 min, followed by sonication with a bath-type sonicator for 2 min. The final lipid concentration of TS-EPC-NV was 0.55 mM.

2.10. Transfection assay B16-F1 cells stably expressing luciferase (B16-F1-luc) were prepared according to the general protocol of neomycin selection. B16F1-luc cells were seeded at a density of 1 × 10 4 cells/well in 24 wells. After incubation for 24 h, the cells were treated with siRNA (20 pmol)-encapsulated TS-EPC-NVs in serum-free DMEM (250 μL/ well) for 3 h, followed by the addition of DMEM containing 10% FBS (1 mL/well) and additional incubation for 21 h. To examine the luciferase activity, the transfected cells were lysed with Reporter Lysis Buffer (Promega) according to the manufacturer's instructions. The cell lysates were reacted with luciferase substrate (Promega), and relative right units (RLU) were measured by Luminescenser-PSN (ATTO). The total protein concentration was determined with a BCA Protein Assay Kit (Thermo scientific). Luciferase activity is shown as RLU/mg protein.

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2.11. Statistical analysis Statistical significance was determined using One-way ANOVA, followed by Tukey's Honestly Significant Difference test. P values b 0.05 were considered to be significant. 3. Results 3.1. Physicochemical properties of TS-NVs and TS-EPC-NVs We prepared TS-EPC-NVs by mixing TS and EPC (1.7:1 mol/mol). The physicochemical properties of TS-NVs and TS-EPC-NVs are summarized in Table 1. The average diameters of both vesicles were below 100 nm after sizing with an extruder. The ζ-potentials of both nanovesicles were significantly negative. TS-EPC-NVs, in which TS was diluted with EPC, showed tempered ζ-potential compared with TS-NVs. Moreover, TS-EPC-NVs showed lower fluorescence polarization degree than TS-NVs, indicating that TS-EPC-NVs have higher membrane fluidity compared with TS-NVs. These data suggest that TS-EPC-NVs were successfully constructed. We next evaluated the variation of particles size in the presence of acidic pH, divalent cations, and serum after incubation for 24 h. At acidic pH, the diameters of both vesicles were almost constant below 200 nm. Furthermore, we additionally investigated the particles size of TS-NVs and TS-EPC-NVs in acidic buffer immediately after preparation of NVs. As a result, both NVs showed constant particles size despite pH changes (Supplemental Fig. 1). These data suggest that both nanovesicles were stable at acidic pH (Fig. 1A). In response to divalent cations or serum, the diameters of TS-NVs were significantly increased by aggregation of the vesicles, but TS-EPC-NVs remained almost constant (Fig. 1B, C). The results suggest that TS-EPC-NVs showed higher stability in the presence of divalent cations, and serum compared with TS-NVs. 3.2. Antitumor effects of TS-NVs and TS-EPC-NVs on B16-F1 cell-bearing mice To determine the influence of increased vesicle stability on the tumor inhibitory effect, we compared the in vivo antitumor effects of TS-EPC-NVs and TS-NVs (administered intravenously) on the growth of mouse melanoma. TS-EPC-NVs were more effective at preventing tumor growth than TS-NVs (Fig. 2A, B). The percentages of growth inhibition by TS-EPC-NVs and TS-NVs were 79% and 70%, respectively. In the TS-EPC-NVs injected mice, tumor growth was significantly suppressed 10 days after tumor inoculation. Moreover, the antitumor effect of TS-EPC-NVs was observed earlier than that of TS-NVs (Fig. 2A). Furthermore, tumor regression was observed in two of six mice injected with TS-EPC-NVs. Intravenous administration of either TS-EPC-NVs or TS-NVs failed to cause weight loss or hepatotoxicity (Table 2). These results suggested that TS-EPC-NVs showed potent tumor growth inhibition without remarkable toxicity. To clarify the reason why TS-EPC-NVs was more efficacious than TS-NVs, we next compared the intratumoral distribution of both nanovesicles using mouse melanoma-bearing mice. DiI-labeled TS-EPC-NVs showed a broader and more homogenous distribution in the tumor tissue compared to TS-NVs (Fig. 2C). It is suggested that the effective

Table 1 Physiochemical characteristics of TS-NVs and TS-EPC-NVs.

TS-NVs TS-EPC-NVs

Size (nm)

ζ-potential (mV)

Fluorescence polarization degree

94.5 ± 10.3 88.1 ± 7.7

− 49.0 ± 3.6 − 37.6 ± 4.6

207.2 ± 28.7 174.4 ± 17.9

The data are expressed as the mean ± SD values from at least three different preparations.

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A

A

B 80

300 200 100 0 6.0

6.5

7.0

pH

Relative tumor volume

Diameter (nm)

400

Diameter (nm)

TS-EPC-NVs

40 20 mm

20 mm

*

20

* 0

400

TS-NVs

; Cont. ; TS-NVs ; TS-EPC-NVs

60

B **

0

4

* * **

7 10 13 16

*

20

Days

300

C

TS-NVs

TS-EPC-NVs

200 100 0 0

3

Divalent cations (mM )

C *

300 200 100 0

0

50 µm

50 µm

Fig. 2. Suppression of tumor growth by TS-NVs and TS-EPC-NVs in B16-F1 cell-bearing mice.TS-NVs and TS-EPC-NVs were injected into melanoma-bearing mice via tail vein root five times every three days, as indicated by arrows in the graph. Tumor volume was measured, and data are shown as the ratio to the tumor volume in each mouse four days after tumor inoculation (A). Values and bars represent the means and SD, respectively. *P b 0.05 and **Pb 0.01 versus control mice. (B) Typical images were captured 20 days after tumor inoculation. (C) Comparison of the intratumoral distributions of TS-NVs and TS-EPC-NVs. Red and blue signals show nanovesicles and nuclei, respectively. Data are shown as typical images in three individual experiments.

400

Diameter (nm)

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846

50

effectively and the mechanism for cytotoxicity of TS-EPC-NVs may differ from that of TS-NVs.

FBS (%) Fig. 1. Variation of particles size of TS-NVs and TS-EPC-NVs in the presence of acidic pH, divalent cations, or serum.TS-NVs and TS-EPC-NVs were incubated in acidic buffer (A), or PBS (−) containing divalent cations (B) or FBS (C) at 37 °C for 24 h. Particle diameters were measured using Zeta Sizer Nano. White and black columns indicate TS-NVs and TS-EPC-NVs, respectively. Values represent the means of three individual experiments. Bars represent SD. *P b 0.05 and **P b 0.01.

intratumoral distribution of TS-EPC-NVs may contribute to the potent antitumor effect by TS-EPC-NVs. 3.3. Effect of TS-NVs and TS-EPC-NVs on cultured cells We next examined the effects of TS-EPC-NVs on in vitro cancer cell growth. All TS formulations prevented cell proliferation in dose- and time-dependent manners, but nanovesicles containing TS exhibited greater cell growth inhibition than did TS-solution (Fig. 3A, B). When the cells were treated with 25 or 50 μM TS, TS-EPC-NVs showed an almost two-fold greater inhibitory effect than did TSNVs (Fig. 3A). Interestingly, TS-NVs reduced cell viability initially (treatment for 12 h) compared to TS-solution and TS-EPC-NVs, but at later stages (treatment for 36 and 48 h), TS-EPC-NVs showed significant cytotoxicity (Fig. 3B). Furthermore, morphological analysis demonstrated that TS-EPC-NVs-treated cells showed a homogenous rounded shape without cellular membrane injury, differing from TSNVs (Fig. 3C). These results suggest that TS-EPC-NVs‐induced apoptosis

3.4. In vitro cytotoxicity of TS-NVs and TS-EPC-NVs We next characterized the cytotoxicity of nanovesicles containing TS. It was previously demonstrated that TS-solution induces apoptosis [14,15]. We initially asked whether our nanovesicles also induced apoptosis when assessed by the Annexin‐V-staining method. We found that the fraction of Annexin‐V-positive cells increased following treatment with TS-NVs and TS-EPC-NVs (Fig. 4A, B), indicating that these nanovesicles induced apoptosis, like TS‐solution. 2 h after treatment with TS-NVs or TS-EPC-NVs, the proportions of apoptosispositive cells were approximately 75% and 20%, respectively, and after 6 h, the percentages of apoptotic cells were approximately 23% and 50%, respectively. These results suggest that TS-NVs‐induced apoptosis earlier than did TS-EPC-NVs. Then, to examine apoptosis via a pathway undetectable by Annexin‐V staining, we assessed caspase 3/7 activities induced by TS‐nanovesicles. After 24 h of treatment, TS-EPC-NVs exhibited almost three- and nine-fold higher

Table 2 Glutamic-pyruvate transaminase (GPT) activities.

GPT (IU/L)

Cont.

TS-NVs

TS-EPC-NVs

28 ± 11.4

26 ± 2.0

25 ± 8.2

A

Cont.

** Counts

** 50

20

50 12.5 25

TS-solution

50 12.5 25

TS-NVs

TS-solution TS-NVs TS-EPC-NVs

Viability (%)

100

*

** **

50

*

** * **

14 12 10 8 6 4 2 0

0

12

24

10 3

TS-solution

10

0 10 0

10 4

10 1

10 2

10 3

10 4

AnnexinV (fluorescence intensity)

TS-EPC-NVs

Caspase3/7 activity (X105RLU)

** **

10 2

20

AnnexinV (fluorescence intensity)

50 (µM)

C B

10 1

TS-NVs TS-EPC-NVs

10

0 12.5 25

Cont.

TS-NVs

0 10 0

30

TS-EPC-NVs TS-solution

36

D **

**

**

Hemolysis (% of complete hemolysis)

Viability (%)

100

B

30

**

Counts

A

847

60

**

**

**

40

20

0

48

Time (h)

C Cont.

TS-solution

Fig. 4. Cytotoxicity of TS-NVs and TS-EPC-NVs in B16-F1 cells. Cells were treated with TS-solution, TS-NVs, or TS-EPC-NVs for 2 h (A), 6 h (B), or 24 h (C) and apoptotic cells were assessed by flow cytometry using the Annexin-V method (A, B) or the Caspase-Glo 3/7 assay (C). (D) TS-solution was mixed with erythrocytes for 3hr, and hemolysis was measured by absorbance at 540 nm. Values represent the means of three individual experiments. Bars represent SD. **P b 0.01.

TS-EPC-NVs can induce apoptosis without biomembrane injury, unlike TS-NVs and TS-solution.

TS-NVs

TS-EPC-NVs

Fig. 3. Suppression of B16-F1 cell viability by TS-NVs and TS-EPC-NVs. (A) B16-F1 cells were treated with TS dissolved in ethanol (denoted as TS-solution), TS-NVs, or TS-EPCNVs for 48 h at the indicated TS concentrations. (B) The cells were treated with TSsolution, TS-NVs, or TS-EPC-NVs (50 μM) for the indicated times. Cell viability was assessed by WST-1, and is shown as the ratio to non-treated cells. Values represent the means of three individual experiments. Bars represent SD. *P b 0.05 and **P b 0.01. (C) Morphological images of cells which were treated with 50 μM TS for 48 h. Scale bar indicates 100 μm.

activity than did TS-NVs and TS-solution, respectively (Fig. 4C). These results indicate that TS-EPC-NVs were more potent at inducing apoptosis than were TS-NVs or TS-solution. Furthermore, since it was previously reported that TS possesses a surfactant-like property [28,29], the direct influence of the nanovesicles on biological membranes was examined. We found that TS dissolved in DMSO (TSsolution) and TS-NVs caused erythrocyte hemolysis, whereas TSEPC-NVs showed no such effect (Fig. 4D). These results suggest that

3.5. Cellular uptake of TS-NVs and TS-EPC-NVs To clarify why TS-EPC-NVs induces apoptosis effectively, we examined the intracellular amounts of TS. Uptake efficiency of TS molecules was analyzed by HPLC. The amount of TS in cells treated with TS-NVs was almost the same as that with TS-solution, whereas the amount of TS in TS-EPC-NVs-treated cells was approximately 40% lower than in TS-NVs-treated cells (Fig. 5A). These results suggest that TS-EPC-NVs‐ induced apoptosis strongly, in spite of the low amounts of TS in cells treated with TS-EPC-NVs. Actually, the relative activity of TS-EPC-NVs was approximately five-fold higher than that of TS-NVs. Then, to clarify why TS-EPC-NVs possessed high relative activity, we examined the amount of nanovesicles taken up per cell by flow cytometric analysis. Total cellular uptake of nanovesicles was almost the same following treatments with TS-NVs and TS-EPC-NVs (Fig. 5B). Surprisingly, although cellular uptake of anionic nanoparticles is generally ineffective [30], negatively charged TS-nanovesicles showed almost comparable cellular uptake to conventional cationic liposomes. Interestingly, the variation in the amount of TS-EPC-NVs taken up per cell was smaller than that observed with TS-NVs (Fig. 5B). 3.6. Intracellular fate of TS-NV and TS-EPC-NV We next examined whether TS-EPC-NVs were taken up in the cells as vesicles using fluorescently labeled nanovesicles. When fluorescent nanovesicles are taken up as vesicles, nanovesicles are observed as

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Uptake of TS (%)

A

A TS-NVs

**

TS-EPC-NVs

**

100

50

40 µm

40 µm

0

B B

15

Cationic-Lip

TS-NVs

TS-EPC-NVs

Cationic-Lip +sucrose

TS-NVs +sucrose

TS-EPC-NVs +sucrose

cationic lip. TS-EPC-NVs

10

5

0 100

TS-NVs

101

102

103

104

cellular uptake (fluorescence intensity) Fig. 5. Cellular uptake of TS-NVs and TS-EPC-NVs. (A) Intracellular TS was assayed by HPLC in cells treated with 50 μM TS for 3 h. Data are shown as the percent of applied TS. Values represent the means of three individual experiments. Bars represent SD. **Pb 0.01. (B) Cell-associated TS-nanovesicles were quantified by flow cytometry after 30 min TS treatment. Typical histograms are shown from three individual experiments.

clusters in the cells. Comparing the CLSM images of TS-EPC-NVs with those of TS-NVs, in cells treated with TS-EPC-NVs for 1 h, many intracellular red clusters were recognized (Fig. 6A). Furthermore, some TS-EPCNVs were colocalized with endosomes/lysosomes, suggesting that TSEPC-NVs were taken up as vesicles through an endocytotic pathway. To confirm the uptake pathway of TS-EPC-NVs, we examined the intracellular localization of TS-EPC-NVs during inhibition of the endocytotic pathway by treatment with sucrose, an endocytosis inhibitor, at 4 °C. DOTAP/EPC liposomes and TS-EPC-NVs were observed in the cytosol as clusters under conventional culture conditions (Fig. 6B). Cellular uptake of TS-EPC-NVs was inhibited by the addition of sucrose, suggesting that TS-EPC-NVs were taken up through the classical endocytotic pathway. Previously, it was reported that TS-induced apoptosis is triggered by interaction with mitochondrial or cytosolic proteins [31–33]. That is, endosomal escape of TS-EPC-NVs is likely necessary for the induction of apoptosis. Therefore, we examined the effect of chloroquine, which destabilizes endosomes, on TS-EPC-NVs-induced cytotoxicity. Initially, we confirmed that neither TS-EPC-NVs (25 μM) nor chloroquine (5 μM) affected cellular viability 24 h after treatment (Fig. 6C, inserted panel). As shown in Fig. 6C, TS-EPC-NVs-induced cell death was accelerated by co-treatment with chloroquine, whereas TS-solution and TSNVs were not affected. These results suggest that TS-EPC-NVs were delivered to target proteins following endosomal escape. To obtain further evidence for cytosolic delivery of TS-EPC-NVs, siRNA was encapsulated into TS-EPC-NVs as reported previously [26,27], and the transfection efficiency was examined. When the siRNA was condensed with a polycation, the average diameter of the polyplex was approximately 100 nm, and the ζ-potential was positive. By encapsulating the polyplex

C 100

Cont. Chlor.

50 0

Viability (%)

Counts

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0 1 5 10 20 Chlor. (µM)

**

80 60 40 20 0

Fig. 6. Intracellular fate of TS-NVs and TS-EPC-NVs. (A) CLSM images of TS-NVs and TSEPC-NVs in B16-F1 cells. TS-NVs and TS-EPC-NVs (25 μM) were added to B16-F1 cells. After the 1 h incubation, the intracellular location of nanovesicles was observed by CLSM. Red, green, and blue signals indicate nanovesicles, end/lys, and nuc, respectively. (B) The intracellular locations of TS-NVs and TS-EPC-NVs during inhibition of the endocytotic pathway. Cells were treated at 37 °C with nanovesicles containing TS for 30 min, or at 4 °C in medium containing 0.4 M sucrose. (C) Cell viability following treatment of cells with TS‐nanovesicles (25 μM) and 5 μM chloroquine (chlor) for 24 h. Inserted panel shows cell viability in the medium containing only chloroquine. Data are shown as the ratio to non-treated cells. Values represent the means of three individual experiments. Bars represent SD. **P b 0.01.

within the TS-EPC membrane, the average diameter was approximately 200 nm, and the ζ-potentials were −20 ± 1.4 mV. The increased diameter and reversed charge of the particle suggested that siRNA was encapsulated into TS-EPC-NVs as reported previously [26,27]. On the

other hand, siRNA condensed with polycation could not be encapsulated into TS-NVs, probably owing to lower membrane stability of TS-NVs than that of TS-EPC-NVs in the condition. As shown in Fig. 7, in B16-F1 cells expressing luciferase stably, TS-EPC-NVs encapsulating anti-luciferase siRNA showed approximately 45% knockdown efficiency. These results suggested that TS-EPC-NVs were delivered to the cytosol.

4. Discussion In the present study, we developed a novel drug delivery system consisting of TS with antitumor activity. TS has been considered an ideal anti-cancer agent and is easily vesiculated [22], suggesting that nanovesicles consisting of TS would be useful as a drug carrier which would possess antitumor activity. However, nanovesicles consisting of only TS (TS-NVs) have technical limitations due to their collapse or conversion from its lamellar structures into hexagonal II structure in response to divalent cations, acidic pH, or serum [23]. Thus, TS-NVs lack the stability required for a drug carrier. Therefore, to improve the stability of the nanovesicles, we constructed a novel nanovesicle consisting of TS and egg phosphatidyl choline (EPC) (TS-EPC-NVs). Because these nanovesicles containing EPC form a stable lamellar structure [24], we examined the antitumor effect of TS-EPC-NVs and compared it with TS-NVs. As shown Fig. 1, TS-EPC-NVs showed higher stability in the presence of divalent cations and serum than did TS-NVs. Since TS is a weakly acidic compound, TS-NVs could not be prepared under neutral or acidic pH conditions. That is, the charged moiety of TS is important to form vesicles. In the presence of divalent cations or protons, it is thought that neutralization of the negative charge of TS results in the destabilization of the TS-NVs membrane. In particular, the diameter of TS-NVs increased in the presence of divalent cations compared with acidic pH. It is suggested that the interaction of TS molecules with divalent cations, such as Ca 2+ and Mg 2+, on the TS-NVs membrane, results in increased hydrophobicity of TS followed by aggregation or structural change of TS-NVs [21,34–36]. By contrast, the diameter of TS-EPC-NVs was nearly constant with or without divalent cations. These different responses to divalent cations are thought to have relevance to the degree of assembly of TS molecules. Because TS molecules are diluted into the membrane in TS-EPC-NVs, it is thought that TS-EPC-NVs might interact comparatively weakly with divalent cations compared to TS-NVs. Furthermore, EPC form a stable

25

luciferase activity (X107 RLU/mg protein)

** 20

** 15 10 5 0 Cont.

Luc.

LFN2000

Cont.

Luc.

TS-EPC -NVs

Fig. 7. Transfection efficiency of siRNA-encapsulated TS-EPC-NVs.Specific knockdown efficiency was evaluated using anti-luciferase siRNA-encapsulated TS-EPC-NVs (20 pmol/10,000 cells) in stable luciferase-expressing B16-F1 cells. Positive controls for transfection applied Lipofectamine2000 (LFN2000). Luciferase activities were measured 48 h after transfection in lysates of transfected cells. Data are shown as relative light units (RLU)/ mg protein in these cells. Values and bars represent the means and SD, respectively. **P b 0.01.

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lamellar structure [24], indicating that TS-EPC-NVs could maintain a stable vesicular confirmation even in the presence of divalent cations. TS-EPC-NVs showed a more potent antitumor effect than did TSNVs (Fig. 2). This result could be explained by the differences of bio-distribution, such as tumor accumulation and intratumoral distribution, and cytotoxicity of the nanovesicles. In general, the tumor accumulation of nanovesicles is achieved by passive targeting based on EPR effects. EPR effects depend strongly on particle size. A previous study indicated that PEG-liposomes, having a particle diameter below 200 nm, were effectively distributed to tumor tissue in mice bearing B16 cells [37]. As shown in Fig. 1, TS-EPC-NVs formed stable nanovesicles, and the vesicle diameter was maintained virtually below 200 nm under in vivo condition. Actually, we quantitatively compared TS-EPC-NVs and PEG-modified particles in their tumor accumulation. Tumor accumulation of TS-EPC-NVs was only half that of PEG-modified particles, suggesting that TS-EPC-NVs exhibited sufficient tumor accumulation (data not shown). Furthermore, it is known that the size of nanoparticles exerts an influence on intratumoral penetration [37]. In this study, TS-EPC-NVs showed more homogenous distribution and cellular uptake in tumor tissue compared with TS-NVs (Fig. 2C). The difference of intratumoral distribution between TS-EPC-NVs and TS-NVs also might be involved in the constant vesicle size of TS-EPC-NVs in tumor tissue. Additionally, it was previously reported that liposomes, having high membrane fluidity, show effective penetration into tissues [38,39]. As described in Table 1, the fluorescence polarization of TS-EPC-NVs was lower than that of TS-NVs. This result suggests that TS-EPC-NVs, which were diluted with EPC (a flexible phospholipid [40]) had higher membrane fluidity than did TS-NVs. Hence, it is possible that extensive intratumoral delivery of TS-EPC-NVs is due to flexibility of vesicles as well as conservation of vesicle size in tumor tissue. Thus, increased vesicle stability of TS-EPC-NVs might have improved intratumoral delivery, resulting in the enhanced antitumor effect. Furthermore, as shown in Figs. 3 and 4, TS-EPC-NVs exhibited significantly higher cytotoxicity compared with TS-NVs. To clarify the differences in toxicity, we focused on the mechanism of cell death. TS-NVs and TS-solution induced not only an increase of Annexin‐V-positive cells at early time phases (Figs. 3B, 4A), but also membrane instability (Figs. 3C, 4D). Since it was previously reported that TS has detergent-like activity [28,29], we speculate that the disruption of the plasma membrane is involved in the induction of cell death by TS-NVs and TS-solution. On the contrary, TS-EPC-NVs‐ induced potent caspase 3/7 activity without membrane disruption. These results might be caused by differences in the cellular uptake pattern based on the stability of the vesicles. Particle size has a large influence on cellular uptake of nanoparticles [41,42]. As shown in Fig. 5B, TS-EPC-NVs, which are stable nanovesicles, showed homogenous cellular uptake among cells. Furthermore, TS-EPC-NVs were observed as vesicles inside cells (Fig. 6A). Hence, TS-EPC-NVs were uniformly taken up as vesicles, leading to the enhanced cytotoxicity. To better understand the mechanism of TS-EPC-NVs-induced cytotoxicity, we examined the intracellular trafficking of TS-EPC-NVs. As shown in Fig. 6A and B, TS-EPC-NVs were observed in the cytosol, and sucrose, an endocytosis inhibitor, prevented cellular uptake of TS-EPC-NVs. In addition, chloroquine, which induces endosomal disruption, enhanced cell death induced by TS-EPC-NVs (Fig. 6C). Accordingly, TS-EPC-NVs taken up via the endocytotic pathway were effectively delivered to the cytosol, suggesting that enhanced cytotoxicity by TS-EPC-NVs is due to the increased amount of TS-EPCNVs present as vesicles in the cytosol. As shown Fig. 6B, the image of cellular uptake of TS-NVs seemed to be inhibited by the addition of sucrose at 4 °C like TS-EPC-NVs. As shown in Fig. 6B, TS-NVs were observed at inside of cells as diffused red signals under conventional culture conditions, on the other hand, the red signals derived from TS-NVs could not be observed in the presence of sucrose at 4 °C. However, chroloquine did not enhance TS-NVs-induced cytotoxicity.

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Therefore, TS-NVs taken up partly by endocytosis might not be responsible for TS-NVs cytotoxicity. Although the detailed mechanical analysis regarding the difference of cytotoxicity between TS-NVs and TS-EPC-NVs are ongoing, we consider that it might be related with internalization mechanism and intracellular trafficking. As shown in Fig. 6B, TS-EPC-NVs, not TS-NVs, were observed as red dots inside cells. Furthermore, the cytotoxic effect of TS-EPC-NVs was enhanced by co-presence of chroloquine (Fig. 6C). Therefore, it is suggested that TS-EPC-NVs released from endosomes might induce apoptosis via activating apoptosis-inducing mechanism at the cytoplasmic region. On the other hand, chroloquine did not affect TSNVs-induced cytotoxicity (Fig. 6C). Therefore, TS-NVs taken up partly by endocytosis might not be responsible for TS-NVs cytotoxicity. These results suggested that TS-NVs, which are essential for cytotoxicity, were taken up by the energy-independent manner, such as the distribution of disassembled TS-NVs into membranes, although TSEPC-NVs internalized via endocytosis showed significant cytotoxicity. To confirm the cytosolic delivery of TS-EPC-NVs, we encapsulated a siRNA into TS-EPC-NVs and examined the transfection activity. As shown in Fig. 7, TS-EPC-NVs showed markedly high transfection efficiency. In general, to exert its inhibitory activity, siRNA must be transfected by a liposome-based carrier for effective delivery to the cytosol. That is, intracellular trafficking of siRNA, such as cellular uptake and endosomal escape, determine the transfection efficiency [43]. Although TS-EPC-NVs showed sufficient cellular uptake, the amount of uptake of TS-EPC-NVs was slightly lower than that of cationic liposomes (Fig. 5B). In addition, TS-EPC-NVs were taken up by endocytosis (Fig. 6). Considering the differing uptake efficiencies between TS-EPC-NVs and cationic liposomes, the high transfection efficiency of TS-EPC-NVs could be responsible for potent endosomal escape. To facilitate endosomal escape of the drug carrier, cholesteryl hemisuccinate (CHEMS), which is a similar succinic ester of TS, has been used for pH-sensitive liposomes and gene carriers [44,45]. Given the mechanism of endosomal escape of CHEMS, it is important that the bilayer membrane is converted into a hexagonal II phase from a lamellar one, followed by membrane destabilization and fusion [46,47]. Hence, TS-EPC-NVs could possess a mechanism of endosomal escape similar to CHEMS. The efficient endosomal escape of TS-EPC-NVs was confirmed by knock down of mRNA via TS vesicles encapsulating siRNA. This result also indicates that TS-EPCNVs are useful carriers for various drugs, such as anticancer agents and siRNAs. TS-EPC-NVs are novel drug carriers that have anticancer activity and effectively deliver their contents to a tumor without showing undesirable toxicity. Because TS enhances doxorubicin (DOX)-induced apoptosis by increasing the intracellular level of DOX [25], TS-EPCNVs could be used to encapsulate other anticancer drugs, such as DOX, and would therefore represent a rational drug delivery system for cancer therapy. Furthermore, because TS-EPC-NVs can deliver an encapsulated drug effectively to cytoplasm in the cell, we believe that TS-EPC-NVs would be an appropriate tool for delivering an antitumor nucleic acid agent.

5. Conclusions In the present study, we developed nanovesicles consisting of TS and EPC as a co-delivery system for anticancer drugs. TS-EPC-NVs, as stable vesicles, showed more potent antitumor effects than TS-NVs in cultured cells and in tumor-bearing mice. The enhanced antitumor effects of TSEPC-NVs were attributed to the effective intratumoral distribution, cellular uptake, and delivery to the cytosol, all of which were due to increased vesicle stability. Collectively, TS-EPC-NVs are attractive drug carriers that show both antitumor activity of the encapsulated drug and the carrier itself. Supplementary materials related to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2012.05.031.

Acknowledgments This work was supported in part by the Japan Society for the Promotion of Science and by the Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research.

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