Tumor Microenvironment-Sensitive Liposomes Penetrate Tumor

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Tumor microenvironment-sensitive liposomes penetrate tumor tissue via attenuated interaction of the extracellular matrix and tumor cells, and accompanying actin depolymerization Satoko Suzuki, Shoko Itakura, Ryo Matsui, Kayoko Nakayama, Takayuki Nishi, Akinori Nishimoto, Susumu Hama, and Kentaro Kogure Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01688 • Publication Date (Web): 05 Jan 2017 Downloaded from http://pubs.acs.org on January 6, 2017

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Tumor microenvironment-sensitive liposomes penetrate tumor tissue via attenuated interaction of the extracellular matrix and tumor cells, and accompanying actin depolymerization Satoko Suzuki‡, Shoko Itakura‡, Ryo Matsui, Kayoko Nakayama, Takayuki Nishi, Akinori Nishimoto, Susumu Hama*, Kentaro Kogure†. Department of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.

KEYWORDS: tumor microenvironment, tumor penetration, charge-invertible nanoparticles, actin depolymerization

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ABSTRACT

Delivery of anti-cancer drugs into tumor cores comprised of malignant cancer cells can result in potent therapeutic effects. However, conventional nanoparticle-based drug delivery systems used for cancer therapy often exhibit inefficient tumor-penetrating properties, thus suggesting the need to improve the functional design of such systems. Herein, we focus on the interactions between cancer cells and the extracellular matrix (ECM), and demonstrate that liposomes modified with slightly acidic pH-sensitive peptide (SAPSp-lipo) can penetrate in vivo tumor tissue and in vitro spheroids comprised of cancer cells and ECM. We previously reported SAPSp-lipo, tumor microenvironment-sensitive liposomes, are effectively delivered to tumor tissue (Hama S, et al. (2015) J Control Release 206: 67-74.). Compared with conventional liposomes, SAPSp-lipo could be delivered to deeper regions within both spheroids and tumor tissues. Furthermore, tumor penetration was found to be promoted at regions where actin depolymerization was induced by SAPSp-lipo, and inhibited by the polymerization of actin. In addition, SAPSp-lipo attenuated the interaction between cancer cells and ECM, contributing to the penetration of SAPSp-lipo. These results suggest that SAPSp-lipo penetrates tumors via the interspace route and is accompanied by actin depolymerization. Taken together, SAPSp-lipo demonstrates potential as a novel tumor-penetrable drug carrier for induction of therapeutic effects against malignant cells that comprise tumor cores.


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INTRODUCTION Multi-functional nanoparticles used as drug delivery systems (DDSs) can achieve anti-cancer therapeutic effects via targeted delivery of anti-cancer drugs to tumor tissues. Although nanoparticles with prolonged blood circulation are passively distributed to tumor tissues via the enhanced permeation and retention (EPR) effect,1 improvement of the post-extravasation process, which involves intratumoral diffusion and penetration, is necessary. Such improvements can facilitate the effective uptake of nanoparticles by target cells, and subsequent induction of potent anti-cancer effects against cells located within the tumor core, far from the tumor blood vessels.2,3 The sizes of nanoparticles that can be used to achieve effective tumor-specific delivery are limited by the need to avoid renal filtration (cutoff size < 8 nm), yet permit extravasation in normal tissue via the narrow spaces (5-12 nm) of normal blood vessels.4,5 Therefore, larger sizes hamper the effective diffusion and permeation necessary for delivery to the tumor core.3 Overcoming this size handicap of anti-cancer DDSs requires improvements that rely upon an understanding of the limiting factors of their intratumoral diffusion and permeation, such as high interstitial fluid pressure (IFP) and the stiff extracellular matrix (ECM).6,7 The ECM network is a barrier for the transport of macromolecules, as it is composed of charged matrix polymers, such as hyaluronan and collagen, which prevent transport of highly-charged macromolecules due to electrostatic interactions with these matrix polymers.8,9 Thus, control of these electrostatic interactions may lead to the development of anti-cancer DDSs that exhibit effective intratumoral distribution and penetration. It has previously been reported that homogenous distribution of nanoparticles within tumors can be achieved if the zeta-potential of the nanoparticles falls within the range of -20 mV to +10 mV.9 However, mild negatively-charged nanoparticles and polyethylene glycol (PEG)-modified


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nanoparticles, which are designed to avoid interactions with matrix polymers, are difficult to deliver to the tumor core, owing to low tumor retention via their wash out by IFP.6 On the other hand, antibody-modified nanoparticles and nanoparticles with high positive charges often accumulate in peripheral tumor blood vessels due to interactions with target cells and matrix polymers, resulting in limited delivery to the tumor core.10,11 As a rational strategy to overcome these obstacles, it is suggested that conversion of the mild negative charge of nanoparticles to positive or neutral can facilitate efficient diffusion and penetration, and ultimately retention, following delivery to the tumor core. It is known that the pH at the tumor core is slightly lower than that at peripheral tumor blood vessels.12 Therefore, nanoparticles whose surface charges are inverted from negative to either positive or neutral in response to slightly acidic pH, are expected to show potential as anti-cancer DDSs capable of efficient delivery to high-grade malignant cancer cells located within the tumor core.13,14 We previously developed tumor microenvironment-sensitive liposomes of which liposomes were modified with slightly acidic pH-sensitive peptide comprised of histidine (His) and glutamic acid (Glu) residues (SAPSp-lipo).15 SAPSp-lipo is negatively-charged at pH 7.4, which allows it to avoid interactions with biogenic substances, whereas the surface charge of SAPSplipo is inverted from negative to positive in response to a slightly acidic pH environment (e.g. pH 6.5), resulting in cytoplasmic delivery of the encapsulated drugs to target cancer cells at slightly acidic pH, a condition that is relatively consistent with the core region of tumors. Herein, we demonstrate that SAPSp-lipo penetrates and diffuses into both tumor tissue and spheroids comprised of cancer cells and ECM. The mechanism of penetration of SAPSp-lipo is determined to occur via the interspace route, which includes the region between cancer cells and the ECM.


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EXPERIMENTAL SECTION

Materials and animals. B16-F1 cells, a mouse melanoma cell line, were obtained from DS Pharma Biomedical Co., Ltd (Osaka, Japan). Egg phosphatidylcholine (EPC) and N-(carbonylmethoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEGDSPE) were obtained from NOF Corporation (Tokyo, Japan). 1,2-dioleoyl-3trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N(lissamine rhodamine B sulfonyl) (Rh-PE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol, dihexadecyl phosphate and ECM gel were purchased from Sigma Aldrich (St. Louis, MO, USA). Growth factor-reduced BD Matrigel Matrix was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Goat anti-integrin β1/CD29 antibody and recombinant mouse epidermal growth factor were purchased from R&D systems (Minneapolis, MN, USA). Rabbit anti-collagen I polyclonal antibody and rhodamine phalloidin were purchased from Abcam (Cambridge, UK) and Cytoskeleton, Inc. (Denver, CO, USA), respectively. 3,3'dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC18(3)) and Alexa Fluor 594 chicken anti-rabbit IgG were obtained from Invitrogen (Carlsbad, CA, USA). Stearylated slightly acidic pH-sensitive peptide (SAPSp) (stearylGGGGHGAHEHAGHEHAAGEHHAHE-NH2) was synthesized by Scrum, Inc. (Tokyo, Japan). Male Hos:HR-1 hairless mice (8 weeks) were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal


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Experiments of the Kyoto Pharmaceutical University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Preparation of liposomes. Liposomes were prepared by a simple hydration method according to our previous report.15 Briefly, to prepare cationic liposomes (Cationic-lipo), EPC and DOTAP dissolved in ethanol were mixed at a molecular ratio of 7.6:1 and the lipid mixture was dried using nitrogen gas. The resultant lipid film was then hydrated by addition of PBS (-) (total lipid concentration: 10 mM), followed by sonication in a bath-type sonicator (NEY). The lipid composition of cationic liposomes (Cationic-lipo) was EPC/DOTAP (7.6/1 mol/mol). Liposomes were modified with SAPSp (SAPSp-lipo) by incubation with stearylated SAPSp (5 mol%) for 30 min. The lipid compositions of anionic liposomes (Anionic-lipo) and PEGylated liposomes (PEG-lipo) used in this study were EPC/DCP (9/1 mol/mol) and EPC/cholesterol/ PEG-DSPE (1.85/1/0.15 mol/mol/mol), respectively, and were prepared as described above. The particle sizes and surface charges of the liposomes were measured using a Zetasizer Nano (Malvern Ins. Ltd.). Spheroid penetration of liposomes. To prepare spheroids containing ECM, B16-F1 cells were suspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% BD Matrigel Matrix, and then cultured on a 96-well NanoCulture Plate (SCIVAX Life Sciences, Inc. Kawasaki, Japan) at 37°C and 5% CO2 under humidified conditions. Following incubation for 96 h, stable spheroids were transferred to a 0.002% poly-L-lysine (PLL)-coated 35 mm glassbottom dish in DMEM containing 10% FBS, and incubated for 24 h. After washing with PBS(-), spheroids were treated with Rh-PE-labeled SAPSp-lipo, Cationic-lipo and PEG-lipo (lipid concentration: 0.81 mM) in medium prepared at pH 7.4 and 6.5 for 1 h, and the nuclei were then stained by hoechst33342. After washing with DMEM, the distribution of the liposomes within


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the spheroids was observed by confocal laser scanning microscope (Nikon A1; Nikon Co Ltd, Tokyo, Japan). Actin-staining of cells and tumor sections. B16-F1 cells were seeded on 0.002% PLL-coated 8 well Lab Tek II chamber slides at a density of 3 × 104 cells/well. After washing with PBS(-), the cells were treated with SAPSp-lipo, Cationic-lipo, Anionic-lipo and PEG-lipo (lipid concentration: 0.81 mM) in medium prepared at pH 7.4 for 1 h. To evaluate the relationship between liposomal penetration and cytoskeletal changes in the tumor tissue, mice bearing B16F1 cells were prepared according to a previous report.16 Briefly, a cell suspension (3.5 × 106 cells) was mixed with ECM matrigel at a ratio of 5:1 on ice. The mixture was inoculated at 4 sites under the skin of the back of a hairless mouse (Hos: HR-1, 8 weeks). SP-DiO-labeled PEGlipo and SAPSp-lipo were administered into the center of the tumors at a dose of 50 µl/mouse (the amount of lipid was 4.1 × 10-7 mol/mouse) with or without EGF (10 ng/mouse), 8 days after inoculation (mean tumor volume: 244 mm3). Tumor volume (Tvol) was determined from caliper measurements according to the formula, Tvol = length × (width)2 × 0.5. Tumor tissues were isolated 5 h after administration and were embedded in OCT compound, and tissue sections (14 µm) were prepared using a LEICA CM 1100 instrument (Leica). Immediately after tumor isolation, all mice were euthanized by the deep inhalation anesthesia of isoflurane for relief of their suffering. From the results of monitoring of body weight, unhealthy mice were not observed until the experimental endpoint. Humane end point for the euthanasia of mice used in this study was set by over 20% weight loss, prior to the experimental endpoint. Three mice were used for actin staining of the tumor sections. The cells and tissue sections were fixed in phosphatebuffered saline (PBS) containing 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and permeabilized in PBS containing 1% Triton X-100 for 10 min at room


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temperature. The cells were then incubated with rhodamine phalloidin to stain the cellular actin, in accordance with the manufacturer's protocol. After washing, cells were mounted in VECTASHIELD with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Actin polymerization within the treated cells was evaluated by CLSM observation. To determine the degree of actin polymerization in a tumor section and cells, the fluorescence intensity of F-actin per an image (1024 × 1024 pixels) was estimated using the Columbus software (PerkinElmer, Waltham, MA, USA). Additionally, the fluorescence intensities of liposomes and F-actin were measured at a certain distance from the centers of fluorescent regions, indicating liposomal accumulation within the tumor, and the changes in these fluorescence intensities in four different directions/liposomal region were analyzed using Zeiss LSM 510 META software. Immunofluorescent staining of integrin β1 and collagen I. SP-DiO-labeled PEG-lipo and SAPSp-lipo were administered into the center of the tumors at a dose of 50 µl/mouse (the amount of lipid was 4.5 × 10-7 mol/mouse). Three mice were used for immunofluorescent staining of integrin β1 and collagen I. Tissue sections were prepared from the tumor isolated 5 h after administration, and were subsequently fixed and permeabilized as described above. After washing with PBS (-) containing 0.05% tween-20 (PBS-T), tissue sections were blocked in Protein Block solution (DAKO Cytomation, Inc. California, USA), and incubated with rabbit anti-collagen I antibodies diluted 1:50 with PBS (-) containing 0.5% FBS or goat anti-integrin β1/CD29 antibodies labeled with Alexa 350 (according to our previous report17) at 15 µg/ml in PBS (-) containing 0.5% FBS overnight at room temperature in a moist chamber. To detect collagen I, Alexa 594-conjugated chicken anti-rabbit antibodies diluted 1:1000 were used as secondary antibodies with 1 h incubation at 37°C. After washing with PBS-T, tissue sections were mounted in PBS (-) containing 80% glycerol and observed by CLSM (Nikon A1; Nikon Co


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Ltd, Tokyo, Japan). The fluorescence intensities of collagen I and integrin β1 per image, and at certain distances, were measured using the Columbus and NIS-Elements software, respectively, as described above. Statistical analysis. Statistical significance was determined by ANOVA and Student’s t-test. P values

Tumor Microenvironment-Sensitive Liposomes Penetrate Tumor

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