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Cell adhesion induced using surface modification with cell-penetrating peptide-conjugated PEG-lipid: A new cell glue for 3D cell-based structures Yuji Teramura, Sana Asif, Kristina N Ekdahl, Elisabet Gustafson, and Bo Nilsson ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14584 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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ACS Applied Materials & Interfaces

Cell adhesion induced using surface modification with cell-penetrating peptide-conjugated PEG-lipid: A new cell glue for 3D cell-based structures

Yuji Teramura1,2* Sana Asif2, Kristina N. Ekdahl2,3, Elisabet Gustafson4, and Bo Nilsson2

1

Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo,

113-8656, Japan 2

Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Dag

Hammarskjölds väg 20, SE-751 85, Uppsala, Sweden 3

Linnæus Center of Biomaterials Chemistry, Linnæus University, SE-391 82 Kalmar, Sweden

4

Department of Women's and Children's Health, Uppsala University Hospital, SE-751 85,

Uppsala, Sweden

Keywords: Cell surface modification/ poly(ethylene glycol)-conjugated phospholipid (PEG– lipid)/Cell-penetrating peptide (CPP) / Cell adhesion / 3D structure

ACS Paragon Plus Environment

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[Abstract] We synthesized a novel material, cell-penetrating peptide-conjugated poly(ethylene glycol)-lipid (CPP-PEG-lipid), that can induce the adhesion of floating cells. Firm cell adhesion with spreading could be induced by cell surface modification with the CPP-PEG-lipids. Cell adhesion was induced by CPPs, but not by any other cationic short peptides we tested. Here, we demonstrated adherence using the floating cell line CCRF-CEM as well as primary human T cells, B cells, erythrocytes, and hepatocytes. As compared to cells grown in suspension, adherent cells were more rapidly induced to attach to substrates with the cell-surface modification. The critical factor for attachment was localization of CPPs at the cell membrane by PEG-lipids with PEG>20 kDa. These cationic CPPs on PEG chains were able to interact with substrate surfaces such as polystyrene surfaces (PS), glass surfaces, and PS microfibers that are negatively charged, inducing firm cell adhesion and cell spreading. Also, as opposed to normal cationic peptides that interact strongly with cell membranes, CPPs were less interactive with the cell surfaces because of their cell-penetrating property, making them more available for adhering cells to the substrate surface. No effects on cell viability or cell proliferation were observed after the induction of cell adhesion. With this technique, cells could be easily immobilized onto PS microfibers, an important step in fabricating 3D cell-based structures. Cells immobilized onto 3D PS microfibers were alive, and human hepatocytes showed normal production of urea and albumin on the microfibers. This method is novel in inducing firm cell adhesion via a one-step treatment.

[Introduction]


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The ultimate goal of regenerative therapy is the transplantation of functional tissues and organs to replace those lost as the result of pathology or tissue damage, using stem cells such as embryonic stem cells and induced pluripotent stem cells. Since tissues and organs are complicated 3D structures, 3D scaffolds are clearly required to properly orient living functional cells of different types. Recently, decellularized organs and tissue have been proposed as promising 3D scaffolds

1-3

. For example, decellularized kidney has shown preservation of the

tissue architecture of the extracellular matrix without nuclei or cellular components from donors 1

, and ureteral function has also been successfully recovered after reorganization with external

epithelial cells and endothelial cells in a rat model. These reports are very promising with regard to the use of stem cells to organize functional 3D organs and tissues. Although the kidney cells were infused into the artery and ureter under controlled pressure, it was not easy to control the cell density or the location of the cell attachment to the 3D scaffolds. Also, the cell adhesion process reqiured overnight incubation, according to their protocol. Probably the 3D scaffolds offer an environment for cell adhesion that differs from that of conventional 2D culture. Ideally, infused cells are immediately attached to the designated area of 3D scaffolds, significantly shortening the period until the transplantation into the patient, which is critical for saving recipients before their conditions become more serious. Therefore, the induction and control of cell attachment, not only to 2D substrate surfaces but also to 3D scaffolds, is of great importance. Thus far, cationic polymers, proteins, and antibodies detecting membrane proteins have been used to induce the initial cell attachment to the substrate surface

4-5

. However, the

interaction of cells with cationic polymers results in high cytotoxicity, and the antibody availability is limited. In addition, adherent cells can be patterned using extracellular matrices, such as fibronectin and RGD peptides, because specific interactions with integrins are involved


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in cell adhesion

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. However, this approach is limited by the fact that floating cells do not

express integrins for cell adhesion. However, biological signal transduction can be activated through the immobilization of these molecules. Another approach is the use of cell-surface engineering with single-stranded DNA (ssDNA) and immobilization of the complementary ssDNA

8-9

. Floating cells are patterned at specified areas by this method, but chemical

modification of both cells and substrates with ssDNA is necessary, and the availability of substrates with the chemical modification is limited. Yamazoe et al. have also found a small molecule that promotes cell adhesion and growth

10

; approximately 60% of the floating cells

were attached to the dish when this molecule was present in the culture medium. The interaction with heparan sulfate resulted in enhancement of cell adhesion and cell growth through signaling pathway activation. However, the efficiency of the cell adhesion was still low, and floating cells seemed to be attached to the surface but not firmly adherent. In addition, the presence of unattached molecules in excess can be harmful to organ and tissues. Thus, no materials have been yet been found to induce firm cell adhesion of both adherent and floating cells with high efficiency. In the present study, we examined the ability of various cationic short peptides, including cell-penetrating peptides (CPP)11-12, to induce cell adhesion by conjugating them to PEG-lipids, which are used for cell-surface modification (Fig. 1A). Fibronectin-derived peptide that mediates cell adhesion and cell membrane-derived peptides

13-15

were also used as neutral peptides. PEG-

lipid derivatives are incorporated into the lipid bilayer membranes of cells via hydrophobic interactions16, and the conjugated peptides can be immobilized onto the cell membrane via PEG chains (Fig. 1B). Here we used floating cells, (i.e., CCRF-CEM cells) as well as primary human T cells and B cells, erythrocytes, human hepatocytes, and the adherent cell lines L929 and


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HEK293 cells for our experiments. Finally, we examined cell attachment onto poly(styrene) (PS) microfiber-based 3D scaffolds to assess the effects of the surface modification with CPP-PEGlipids and examine the possibility of fabricating 3D cell-based structures using our modification.

[Experimental sections] Materials. 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) and three polyethylene glycol

(PEG)

moieties,

α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxy

carboxy)] (Mal-PEG(40k)-NHS, MW 40,000 Da), α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxy carboxy)] (Mal-PEG(20k)NHS, MW 20,000 Da), and α-3-[(3-maleimido-1-oxopropyl)aminopropyl-ω-(succinimidyloxy

carboxy)]

(Mal-PEG(5k)-

NHS, MW 5,000 Da) were purchased from NOF Corporation (Tokyo, Japan). Succinimidyl-[(Nmaleimidopropionamido)-tetracosaethylene glycol] ester (Mal-PEG(1k)-NHS, MW 1,000 Da) was purchased from Thermo Scientific (Rockford, IL, USA). The protein desalting spin column and Nunc Low Cell Binding dishes were purchased from Thermo Scientific (Waltham, MA, USA). Diethyl ether, dichloromethane, chloroform, and triethylamine were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Dulbecco’s phosphate-buffered saline (PBS; pH 7.4), RPMI 1640 medium, DMEM, fetal bovine serum (FBS), penicillin, streptomycin, EDTA (0.5 M), AlexaFluor 594-phalloidin, 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), and trypan blue solution were purchased from Invitrogen Co. (Carlsbad, CA, USA). Glass-bottom culture dishes were purchased from MatTek Co. (Ashland, MA, USA). All synthetic peptides


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except FITC-labeled glycine-cysteine (FITC-GC), heparinase I from Flavobacterium heparinum, bovine serum albumin (BSA), and chloroform-d were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). FITC-labeled glycine-cysteine (FITC-GC) was synthesized by BEX Co., Ltd (Tokyo, Japan). Alexa Fluor488 streptavidin (Alexa488-streptavidin), Celltracker Green, and Celltracker Orange were purchased from Life Technologies Co. (Carlsbad, CA, USA). Paraformaldehyde 4% phosphate buffer solution was purchased from Wako Pure Chemical (Osaka, Japan). Triton X-100 was purchased from Amersham Biosciences (Piscataway, NJ, USA). Tissue culture polystyrene (TCPS) dishes were purchased from IWAKI (Tokyo, Japan). Anti-CD4 antibody was purchased from Spring Bioscience (Pleasanton, CA, USA). Anti-CD8 antibody was purchased from GenTex, Inc. (Irvine, CA, USA).

Cell culture. The CCRF-CEM cell line established from acute lymphoblastic leukemia T cells was obtained from the Health Science Research Resources Bank (Tokyo, Japan). Suspended CCRFCEM cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin at 37°C in 5% CO2 and 95% air. Mouse fibroblasts (L929) were purchased from the cell bank of Riken BRC (Ibaraki, Japan). L929 cells were cultured in DMEM with 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin. Human peripheral blood CD4+ T cells (primary human T cells) and human peripheral blood CD19+ B cells (primary human B cells) were purchased from Cell Applications, Inc. (San Diego, CA, USA). Primary human T cells and B cells were cultured in culture medium according to the company’s instructions. Human embryonic kidney 293 cells (HEK293) were obtained from the


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JCRB Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan) and cultured in DMEM with 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin. Whole blood was taken from the author (YT) using a puncture needle (Sanwa Kagaku Kenkyusho, Co., Ltd, Nagoya, Japan) and collected in PBS solution containing 10 mM EDTA. After washing with PBS solution containing 10 mM EDTA by centrifugation (180g, 5 min) at room temperature (RT), the erythrocytes were used immediately. T cells, B cells, and erythrocytes were maintained at 37°C in 5% CO2 and 95% air.

Synthesis of peptide-PEG-lipids. Synthesis of Mal-PEG (1k, 5k, 20k, 40k)-lipids Mal-PEG-lipids with different-length PEG chains (1 KDa, 5 kDa, 20 kDa, and 40 kDa) were synthesized based on our previous reports8-9, 16. Four different sizes of Mal–PEG(1k, 5k, 20k, 40k)–NHS (50, 180, 174, and 100 mg, respectively) were combined with DPPE (33, 22, 6.0, and 1.7 mg, respectively), triethylamine (50 µL), and dichloromethane (4 mL). These solutions were stirred for 4 days at RT. Precipitation with diethyl ether yielded Mal–PEG(1k, 5k, 20k, 40k)–DPPE as powders (58 mg, 70% yield; 180 mg, 89% yield; 160 mg, 88% yield; and 92 mg, 90%

yield).

The

YGRKKRRQRRRC; YGRKKRRQRRRC;

following

peptides

FITC-Tat, R4,

were

conjugated

to

FITC-YGRKKRRQRRRC;

CRRRR;

R8,

CRRRRRRRR;

K8,

Mal-PEG-lipids biotin-Tat, CKKKKKKKK;

(Tat, biotinRA7,

CWGGRARARARARARARA; R12, CRRRRRRRRRRRR; R16, CRRRRRRRRRRRRRRRR; HIV-1 Rev, TRQARRNRRRRWRERQRC; HTLV-II Rex, TRRQRTRRARRNRC; FITC-RGD, FITC-RGDSGGGC;

FITC-RDG,

RDGSGGGC;

FITC-REDV,

CGGGREDVY;

GPI,


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CGSSKSPSKKKKKKPGD; and FITC-GPI, CGSSKSPSKKKKKKPGD-FITC)

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11-15

; a cysteine

residue was added to the N- or C-terminus of all peptides to allow conjugation to Mal-PEG-lipid via a thiol-maleimide reaction. All synthetic peptides were used without any purification. The Mal-PEG(40k)-lipids (5.0, 4.0, 5.0, 3.5, 4.0, 4.2, 3.0, 3.4, 3.5, 3.3, 3.6, 4.4, 4.6, 3.0, 2.1 and 2.5 mg) were mixed with Tat, FITC-Tat, biotin-Tat, R4, R8, K8, RA7, R12, R16, HIV-1 Rev, HTLV-II Rex, FITC-RGD, FITC-RDG, FITC-REDV, GPI and FITC-GPI (0.2, 0.2, 0.2, 0.064, 0.13, 0.12, 0.15, 0.17, 0.22, 0.21, 0.17, 0.12, 0.12, 0.10, 0.13 and 0.13 mg, respectively) and PBS (500, 400, 500, 350, 400, 420, 300, 340, 350, 330, 360, 440, 460, 300, 210 and 250 µL, respectively). After thorough mixing, the solutions were incubated at RT for 24 h and then purified on spin columns equilibrated with PBS to obtain the peptide-PEG-lipids. The FITClabeled peptides were used for flow cytometric analysis and fluorescence observation. FITClabeled control peptide (FITC-GC) was used as a control. Mal-PEG(40k)-DPPE (20 mg) was mixed with FITC-GC (0.5 mg) in PBS (1.0 mL) and incubated at 40°C for 24 h. The mixture was purified in a spin column equilibrated with PBS. Mal-PEG(20k)-lipids (1.8, 3.0, 3.0, 2.4, 3.0, 1.5, 1.5, 1.5, and 1.5 mg) were mixed with Tat, R4, R8, K8, RA7, R12, R16, HIV-1 Rev, and HTLV-II Rex (0.18, 0.11, 0.20, 0.14, 0.29, 0.15, 0.19, 0.19, and 0.14 mg, respectively) and PBS (180, 300, 300, 480, 300, 150, 150, 150, and 150 µL, respectively). After thorough mixing, the solutions were incubated at RT for 24 h and then purified on spin columns equilibrated with PBS. Mal-PEG(5k)-lipids (3.7, 3.0, 4.8, 2.5, and 1.2 mg) were mixed with Tat, FITC-Tat, R8, K8, and RA7 (1.0, 1.0, 1.0, 0.5, and 0.41 mg, respectively) and PBS (370, 300, 480, 250, and 120 µL, respectively). After thorough mixing, the solutions were incubated at RT for 24 h and then purified on spin columns equilibrated with PBS.


8

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Mal-PEG(1k)-lipids (1.0, 1.0, and 1.0 mg) were mixed with Tat, R8, and K8 (1.0, 0.8, and 0.5 mg, respectively), PBS (100, 100, and 100 µL, respectively), and methanol (100, 100, and 100 µL, respectively). After thorough mixing, the solutions were incubated at RT for 24 h and then purified on spin columns equilibrated with PBS to obtain the peptide-PEG-lipids.

Cell-surface modification with peptide-PEG-lipids. CCRF-CEM cells (5 × 105 cells) were collected by centrifugation (180g, 3 min, RT) and washed twice with PBS prior to use in the experiments. After the supernatant was removed, 50 µL of peptide–PEG(1k, 5k, 20k, 40k)–lipid (1 mg/mL in PBS) was added to the cell pellet and incubated for 30 min at RT with gentle agitation. After washing with PBS by centrifugation (180g, 3 min, RT), the cells were resuspended in culture medium and then cultured on a TCPS dish. CCRF-CEM cells were also treated with FITC-labeled peptide–PEG–lipids using the same methods. Concentrations of 1, 10, and 100 µg/mL and 1 mg/mL Tat-PEG(40k)-lipid were used for the surface modification of CCRF-CEM cells. As a control experiment, 0.5 mg/mL FITCTat solution was added to the CCRF-CEM cell pellet after centrifugation (180g, 3 min, RT) and incubated for 30 min at RT. After washing with PBS by centrifugation (180g, 3 min, RT), the cells were resuspended in culture medium and then cultured on a TCPS dish. CCRF-CEM cells were labeled with Celltracker Green and Celltracker Orange following the manufacturer’s instructions. The cells were washed twice with PBS using centrifugation (180g, 3 min, RT). After the green and orange-labeled cells were treated with Tat-PEG(40k)lipid, the mixed cells were cultured in culture medium on a glass-bottom dish for observation by confocal laser scanning microscopy (LSM510, Carl Zeiss Microscopy Co., Ltd., Jena Germany).


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Erythrocytes, primary human T cells (2×105 cells), and B cells (2×105 cells) were also treated with peptide-PEG-lipids or FITC-Tat-PEG-lipid using the same methods and cultured in culture medium.

Analysis of Tat-PEG-lipids on CCRF-CEM cells. In order to detect Tat-PEG-lipid on the cell surface after the adhesion of the CCRF-CEM cells, biotin-Tat-PEG-lipid was used for cell-surface modification. CCRF-CEM cells (1×105 cells) were collected by centrifugation (180g, 3 min, RT) and washed twice with PBS. After the supernatant was removed, 100 µL of biotin-Tat-PEG(40k)–lipid (1 mg/mL in PBS) was added to the cell pellet, followed by a 30-min incubation at RT with gentle agitation. After washing with PBS by centrifugation (180g, 3 min, RT), half of the cells were resuspended in culture medium and then cultured on a glass-bottom dish. The rest of the cells were mixed with Alexa488streptavidin (50 µg/mL in PBS) and incubated for 10 min at RT. After washing with PBS, the cells were inspected by confocal laser-scanning microscope. In addition, Alexa488-streptavidin (50 µg/mL in PBS) was added to the CCRF-CEM cells that adhered to the dish 1 h after culturing in culture medium on a glass-bottom dish. After a 10-min incubation at RT, the dish was washed with PBS and observed with a confocal laser-scanning microscope. As a control experiment, CCRF-CEM cells were incubated with Alexa 488-streptavidin (50 µg/mL in PBS) for 10 min at RT and then washed with PBS and inspected with the confocal laser-scanning microscope. For quantitative analysis of peptide–PEG(40k)–lipids on the cell surface, FITC-labeled peptides were used for cell-surface modification. CCRF-CEM cells (5×105 cells) were treated


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with FITC-Tat-PEG(40k)-lipid, FITC-RGD-PEG(40k)-lipid, FITC-RDG-PEG(40k)-lipid, FITCREDV-PEG(40k)-lipid, FITC-GPI-PEG(40k)-lipid, or FITC-control-PEG(40k)-lipid using the method described above. The treated cells were cultured on a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-coated dish (Nunc Low Cell Binding dishes). PMPC is a wellknown biocompatible polymer that can completely suppress protein adsorption and cell adhesion. The cells were then analyzed by flow cytometry (EPICS XL; Beckman Coulter, Inc., Brea, CA, USA) and confocal laser scanning microscopy.

Analysis of adherent Tat-PEG-lipid-modified CCRF-CEM cells. After CCRF-CEM cells, T cells, and B cells were treated with peptide-PEG(1k, 5k, 20k, 40k)-lipids, the cells were cultured on TCPS dishes in culture medium. The adhesion ratios of CCRF-CEM cells, T cells, and B cells were calculated through observation with a phase-contrast microscope (BX60; Olympus Co., Ltd., Tokyo, Japan). In order to count live CCRF-CEM cells, the floating cells were collected from the culture dish after 2 days, and the number of live cells was calculated by trypan blue exclusion. CCRF-CEM cells were treated with 1mg/mL TatPEG(1k, 5k, 20k, 40k)-lipid using the methods described above and non-treated cells were also used as a control group. The calculated cell number was normalized to the cell number on day 0. The cell viability was also examined by MTT assay according to the manufacturer’s instructions (EMD Millipore Co., Temecula, CA, USA). After the CCRF-CEM cells and L929 cells were treated with Tat-PEG(40k)-lipid, R8-PEG(40k)-lipid, and HTLV-II Rex-PEG(40k)-lipid, they were seeded into wells and cultured in medium. MTT assays were performed on these cells over time and compared to the control cells without any treatment.


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To compare the adhesion ratios under various culture conditions, Tat-PEG(40k)-lipidtreated CCRF-CEM cells were cultured in serum-free medium or culture medium supplemented with 10 mM EDTA in TCPS dishes. In addition, CCRF-CEM cells were pre-treated with heparinase I before treatment with Tat-PEG(40k)-lipid. After the cells were incubated in 1 U/mL heparinase I solution at 37°C for 1 h, they were treated with Tat-PEG(40k)-lipid using the same methods. The treated cells were cultured on TCPS dishes in culture medium. Anti-CD4 or antiCD8 antibody was also used to pre-treat CCRF-CEM cells at a 1:50 dilution in PBS for 30 min at RT, and then these cells were treated with Tat-PEG(40k)-lipid using the same methods. To stain actin filaments in adherent CCRF-CEM cells, the cells were fixed in 4% formaldehyde solution (in PBS) at RT for 30 min, permeabilized with 0.2% Triton X-100 solution (in PBS) for 15 min at 4°C, and blocked with 1% BSA solution (in PBS) at RT for 60 min. The cells were then stained with phalloidin at 1:40 dilution and DAPI at 1:10,000 dilution for 20 min at 4°C. After being washed with PBS, the cells were inspected by confocal laser-scanning microscopy.

Inhibition of induced cell adhesion. A solution of Tat–PEG(40k)–lipid was added to a TCPS dish and incubated for 30 min at RT. The dish was washed with PBS just before use. CCRF-CEM cells (5 × 105 cells) were treated with Tat–PEG(40k)–lipid (50 µL, 1 mg/mL in PBS) and incubated for 30 min at RT with gentle agitation. After washing with PBS by centrifugation (180g, 3 min, RT), the cells were resuspended in culture medium and then cultured on the Tat–PEG(40k)–lipid-treated TCPS


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dishes. The same experiments were performed using K8-PEG(40k)-lipid and R8-PEG(40k)-lipid, under the same experimental conditions.

Kinetic analysis of the adsorption of cationic short peptides onto the PS surface by QCMD. QCM-D measurements were performed with a Q-senseE4 (Biolin Scientific AB, Stockholm, Sweden) 17. The oscillator frequency (∆f) decreases when any substance is adsorbed onto the sensor surface. Thus, the amount of adsorbed material on a given surface can be measured on the basis of the change in the oscillator frequency. The gold QCM-D sensor chips were coated with polystyrene (PS) solution (10 mg/mL, in toluene) with a spin coater (KYOWARIKEN, Co. Ltd, Tokyo, Japan) and treated with O2 plasma for 3 min (300 W, 100 mL/min gas flow, PR500; Yamato Scientific Co., Ltd., Tokyo, Japan) before QCM-D measurements. The sensor chip was first exposed to PBS (pH 7.4), until a stable baseline signal was achieved. Then, a solution of one of the various peptides (0 to 100 µg/mL, in PBS) was injected, followed by a wash with PBS.

Cell attachment to 3D PS microfibers. PS microfibers were fabricated by electrospinning (Sprayer ES-1000, Fuence Co., Ltd. Saitama, Japan). Polystyrene pellets (Mw = 9.0×105) were dissolved in a mixed solvent of tetrahydrofuran and N, N’-dimethylformamide (1:1, by volume) with Triton-X (0.5 wt%). The PS solution (20wt%) was placed in a syringe fitted with a needle, and the feeding rate of the PS


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solution was 0.9 mL/h. The microfiber was collected onto a rotating drum (at 600 rpm) covered with aluminum mesh (wire diameter = 0.10 mm, aperture = 0.154 mm, aperture ratio= 36.8%) as a microfiber support substrate for 20 min. The PS solution was sprayed at an applied voltage of 20 kV while the collector was grounded. The distance between the collector and the syringe was 100 mm. The PS microfiber fabric pieces (~1 mm x 1 mm x 50 µm) were immersed in ethanol for 1 h and then washed with PBS just before use for cell experiments. CCRF-CEM cells, L929 cells, and human hepatocytes were treated with Tat-PEG(40k)-lipid and then seeded onto PS microfiber fabric pieces for 15 min at RT. After being washed with the culture medium, the cells were observed by confocal laser-scanning microscopy. Human hepatocytes were cryopreserved and purchased from 3H Biomedical (no 3H-023), and they were prepared as previously reported 18

. The hepatocyte viability was assessed by the trypan blue exclusion method. Cells with a post-

thaw viability exceeding 70% were used for further experiments. For urea assays, the collected supernatants were analysed with a urea assay kit (Sigma-Aldrich Chemical Co.) using colorimetric analysis at 520 nm.

Statistical analysis. Results are presented as means ± SD. Data plotting and statistical analysis (Fig. 2B, 4D) were performed using Prism version 6 for Macintosh software (Graphpad, San Diego, CA, USA). A p-value < 0.05 was considered significant.

[Results]


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Influence of Tat-PEG-lipids and PEG chain length on cell adhesion All the peptides that were conjugated to PEG-lipids are listed in Table 1. Each peptide was conjugated to the PEG-lipids with 1 kDa, 5 kDa, 20 kDa, or 40 kDa PEG and used for cellsurface modification. The listed short peptides are classified into two groups: one of positively charged peptides, including R4, RA7, GPI, CPPs such as Tat, and R8; and the other of neutral peptides such as RGD, RDG, and REDV. First, we examined the cell-surface modification of the Tat peptide, a representative CPP, using PEG-lipids with different PEG chains (Fig. 1C). FITCTat was detected inside the CCRF-CEM cells immediately after its addition to the cell suspension, and it then rapidly disappeared from the cells because of its high membrane permeability (Fig. 1C)19. In contrast, FITC-Tat-PEG-lipids with 5-kDa or 40-kDa PEG (FITCTat-PEG(5k, 40k)-lipids) were detected on the cell membrane but not inside cells, indicating that the Tat peptide with its high cell permeability can be localized to the cell membrane by hydrophobically anchoring it with PEG-lipids. Interestingly, FITC-Tat-PEG(40k)-lipid-treated CCRF-CEM cells started to adhere and firmly spread onto the culture dishes (tissue culture polystyrene (TCPS) dishes) within 10 min, whereas FITC-Tat-PEG(5k)-lipid-treated cells did not adhere, though some cells aggregated and became weakly tethered to the surface. FITC-TatPEG(40k)-lipids were still detected on the cell surface even at 3 or 24 h after initial cell adhesion (Fig. 1C).


15


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Page 16 of 39

A CPP-PEG-lipid CPP

PEG: 20k, 40k

m=14 (DPPE)

B CPP (Tat, R8, etc) CPP-PEG-lipid

Hydrophobic interac on

PEG (20k, 40k) lipid

Cell membrane

Cell surface modifica on with CPP-PEG-lipid Induced cell adhesion

Adherent cells (L929) Floa ng cells (CCRF-CEM, T cell, B cell, erythrocyte, hepatocyte)

3D PS microfibers

C

0hr

3hr

24hr

FITC-Tat

FITC-Tat-PEG(5k)-lipid

FITC-Tat-PEG(40k)-lipid


16

Page 17 of 39

D control

1k

20k

40k

G

100

100 1mg/mL

Adhered cell ra o (%)

80 60 40 20

100µg/mL

80

10µg/mL

60

1µg/mL

40 20 0

40 k

5k

1k

20 k

co nt

ro

l

0

40k

0

10 20 30 40 Culture me (hr)

50

Molecular weight of PEG in Tat-PEG-lipid

3.5 24h 3 2.5 2 1.5 1 0.5 0

0d a Co y nt ro Ta l t-1 k Ta t-5 Ta k t-2 0 Ta k t-4 0k

F

5k

Normalized cell number ra o in medium (-)

E

Adhesion ra o at 6hr (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 days culture

Figure 1. Influence of cell-surface modification with CPP-conjugated PEG-lipids on cell adhesion. (A) Chemical structure of amphiphilic CPP-PEG-lipids. Here CPP with 20- and 40-kDa PEG are used. (B) Schematic representation of cell-surface modification with CPP-PEG-lipids by hydrophobic interaction and the induction of firm cell adhesion to 3D PS microfibers. (C) Confocal laser-scanning microscopy of CCRF-CEM cells after treatment with FITC-Tat (top), FITC-Tat-PEG(5k)-lipids (middle), and FITC-TatPEG(40k)-lipids (bottom). Scale bars: 10 µm. (D) Influence of the molecular size of the PEG chain on cell adhesion of CCRF-CEM cells modified with CPP-PEG-lipids. Phase


17


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Page 18 of 39

contrast images of CCRF-CEM cells 3 h after the cells were modified with Tat-PEGlipids with 1 kDa, 5 kDa, 20 kDa, or 40 kDa PEG. The control is non-modified CCRFCEM cells. For Tat-PEG(40k)-lipids, green and red fluorescently labeled cells were prepared with Celltracker Green and Orange, and then the mixed cells were treated with Tat-PEG(40k)-lipids for confocal laser-scanning microscope observation on glass-bottom dishes. Scale bars: 50 µm (phase contrast microscopy) and 10 µm (confocal laserscanning microscopy). (E) Quantitative analysis of the adhesion ratios of CCRF-CEM cells treated with Tat-PEG(1k, 5k, 20k, 40k)-lipids after 6 h of culture. The control is non-modified CCRF-CEM cells. (F) Dependence of the adhesion ratio on the TatPEG(40k)-lipid concentration in Tat-PEG(40k)-lipid-treated CCRF-CEM cells. Cells were treated with Tat-PEG(40k)-lipids at 1, 10, or 100 µg/mL or 1 mg/mL and cultured on TCPS in culture medium. (G) Cell growth ratios for CCRF-CEM cells treated with Tat-PEG(1k, 5k, 20k, 40k)-lipids after 2 days of culture, as compared to non-modified control CCRF-CEM cells. Inset: Phase contrast image of CCRF-CEM cells that were cultured on TCPS, 24 h after treatment with Tat-PEG(40k)-lipid. Error bars indicate standard deviation; n = 3. Scale bars: 50 µm.

To further examine the cell adhesion of CCRF-CEM cells on TCPS, we used Tat-PEG-lipids with differently sized PEG chains (1 kDa, 5 kDa, 20 kDa, 40 kDa) for cell-surface modification (Fig. 1D). When the cells were treated with Tat-PEG(20k, 40k)-lipids, the cells spread and adhered firmly (adhesion ratios, 97% and 95%, respectively, at 6 h; Fig. 1E). Similar results were obtained on a glass surface. When cells that had been fluorescently labeled with green or red were treated with TatPEG(40k)-lipids, all the adherent cells were red or green, indicating a lack of cell fusion. Intercellular interactions were expected to occur because Tat is a CPP, but no such interactions were seen between cells, and the only cell adhesion was induced by localized Tat-PEG(40k)lipids. Although some cells were immobilized to the surface when treated with Tat-PEG(5k)lipids, clear cell adhesion was not observed (7% at 6 h; Fig. 1E). In the case of Tat-PEG(1k)lipids, the cells were in a suspended state similar to that of control non-modified CCRF-CEM cells (0% adherent at 6 h; Fig. 1E). In the case of PEG-lipid without any peptides, all the cells


18

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were found in suspension. These results indicate that the induced cell adhesion depends mainly on the length of the PEG chain used as a spacer in the Tat peptide on the PEG-lipid, and this length is a critical factor. Thus, the localization of Tat at the cell membrane by PEG-lipids with PEG>20 kDa is important for the induction of cell adhesion. Most Tat-PEG(20k, 40k)-lipid-treated cells adhered to a dish within 10 min (97% and 99%, respectively), and the number of adherent cells remained constant up to 6 h (Fig. 1F, Fig. S1). At 24 h and 48 h, the adhesion ratios decreased to 52% and 19%, respectively, and the rest of the cells were in suspension. The number of live floating cells was calculated at 2 days, since it was not possible to collect the adherent cells, even by pipetting with trypsin. The total number of floating cells collected was comparable for the control non-modified CCRF-CEM cells and the Tat-PEG(20k, 40k)-lipid-treated cells (Fig. 1G). Considering that 19% of the cells adhered at 48 h, the growth rate of the adherent cells was hardly influenced. We also performed MTT assays to evaluate the cytotoxicity of our modification for CCRF-CEM and L929 cells (Fig. S2) and found that there was no cytotoxicity in either the floating or the adherent cell lines. Our results indicate that the modified, adherent cells can proliferate and detach from the dish surface during cell division. Tat-PEG(1k)-lipid-treated cells were seen to be floating (non-adherent), and their cell growth rate was unaffected by the surface modification. However, the treatment of cells with Tat-PEG(5k)-lipids caused high cytotoxicity, and some cells died immediately. Therefore, the number of cells at 48 h was still lower than at time 0. When the cells were treated with various concentrations of the Tat-PEG(40k)-lipid to change the incorporation ratio of the Tat peptide on the cell surface, the adhesion ratio was dependent on the concentration (Fig. 1F), indicating that cell adhesion was induced by the Tat peptide anchored to the cell membrane by PEG(40k)-lipids.


19


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Page 20 of 39

Table 1. Peptide sequences and binding constants kon

Cell adhesion inductionb

Cell membrane permeability12, 20

Sequence

[M-1s-1]a

Tat

YGRKKRRQRRRC

4.9×103

+++

+++

R4

CRRRR

1.7×103

−

−

R8

CRRRRRRRR

5.8×102

+++

+++

K8

CKKKKKKKK

9.3×102

+++

+++

RA7

CWGGRARARARARARARA

1.1×103

+

−

R12

CRRRRRRRRRRRR

1.0×103

++

+++

R16

CRRRRRRRRRRRRRRRR

1.9×103

++

+++

HIV-1 Rev

TRQARRNRRRRWRERQRC

4.0×103

+++

+++

HTLV-II Rex

TRRQRTRRARRNRC

3.2×103

+++

+++

RGD

RGDSGGGC

4.5×100

−

−

RDG

RDGSGGGC

7.1×100

−

−

REDV

CGGGREDVY

1.1×101

−

−

GPI

CGSSKSPSKKKKKKPGD

5.5×103

−

−

Peptide

a

kon was calculated from binding assays on a polystyrene (PS)-coated sensor surface using QCMD measurements. koff was assumed to be zero, since no detachment was observed. b

Cell adhesion was determined from observation of the floating cell line, CCRF-CEM, which was treated with peptide-PEG(40k)-lipids, on TCPS dishes.

Effect of the cell-penetrating property of CPP on PEG-lipids and cell adhesion Next, various peptides (Table 1) were conjugated to PEG-lipids to examine the cell adhesion of CCRF-CEM cells (Fig. 2A, Fig. S3). Cell adhesion onto TCPS dishes was induced, with changes in cell shape, only when the CPPs were conjugated to PEG(20k, 40k)-lipids such as Tat, R8 K8, R12, R16, HIV-1 Rex, and HTLV-II Rex. Although R4, RA7, and GPI are also


20

Page 21 of 39

positively charged, they did not induce cell adhesion. When neutral peptides such as RGD, RDG, and REDV were used, no cell adhesion occurred. In order to examine the interaction between these peptides and the PS surface, which is a mimic of TCPS, we used QCM-D to analyze the kinetics of the interaction (Fig. 2B, Table 1). QCM-D measurements showed the rapid binding of all the positively charged peptides to the PScoated sensor surface (Table 1), and they showed more than a ~102-fold higher kon than for other peptides such as RGD, RDG, and REDV, indicating that all the positively charged peptides could strongly interact with the TCPS surface. However, only CPP, of all the positively charged peptides, was able to induce firm cell adhesion. The exact same results were obtained for CPPPEG(20k)-lipids (Fig. S4, S5). We found that the cell-penetrating property of the peptide is important for inducing cell adhesion when the PEG chain of the PEG-lipids is 20 kDa or 40 kDa. In fact, even though R8 was conjugated to PEG(1k,5k)-lipids, the treated cells did not adhere firmly; the same result was observed for Tat-PEG(1k,5k)-lipids (Fig. S6, S7). A 100 R4

80

60

HTLV-II Rex

40

20

I GP

V RE D

RD G

RG D

R1 6 HI V1 Re HT v LV -II Re x

R1 2

RA 7

K8

R8

R4

t

0 Ta

Adhesion ra o of CCRF-CEM at 6hr (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pep des in CPP-PEG(40k)-lipid


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39

C 0hr

6hr

0hr

6hr

0hr

2day

Human T cells

Human B cells

Human erythrocyte


22

Page 23 of 39

D 100 Adhesion ra o at 6hr (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 Human T cells

40

Human B cells 20 0 R4

R8

R12

R16

Tat

HIV-1 HTLVII Rev Rex

Pep des on PEG(40k)-lipid

Figure 2. Screening of peptide-PEG(40k)-lipids for the ability to induce cell adhesion of CCRF-CEM cells. (A) Influence of the peptides conjugated to PEG(40k)-lipid on firm cell adhesion: the ratio of firmly adherent CCRF-CEM cells to non-adherent cells (adhesion ratio) at 6 h. Inset: Phase-contrast image of CCRF-CEM cells treated with R4PEG(40k)-lipid (top) and HTLV-II Rex-PEG(40k)-lipid at 6 h. Error bars indicate standard deviation; n = 3. Scale bars: 50 µm. (B) Kinetic analysis of the binding of the Tat peptide to the PS surface as analyzed by QCM-D. The gold sensor surface was coated with PS and treated with oxygen plasma before the QCM-D measurements. A solution of Tat peptide (in PBS) was injected, followed by a PBS wash. The binding constants of all peptides were calculated from the QCM-D measurement, for which the dissociation was assumed to be zero (see Table 1). (C) Induction of cell adhesion in primary human T cells (top), B cells (middle), and human erythrocytes (bottom) by surface modification with Tat-PEG(40k)-lipids. These images were observed at 0 h, 6 h, and 2 days, respectively, by phase contrast microscopy. Insets: Primary human T cells and B cells were treated with FITC-Tat-PEG(40k)-lipids and observed immediately by confocal laser-scanning microscopy. Scale bars: 20 µm. (D) Adhesion ratios of primary T cells and B cells that were treated with CPP-PEG(40k)-lipids and cultured at 6 h. Error bars indicate standard deviation; n = 3.

We also examined other floating cells, primary human T cells and B cells and human erythrocytes with Tat-PEG(40k)-lipids (Fig. 2C). When T and B cells were treated with FITCTat-PEG(40k)-lipids, fluorescence was clearly observed on the cell surface of both kinds of cell (insets, Fig. 2C), indicating that Tat could localize to the cell surface as in CCRF-CEM cells. The


23


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Page 24 of 39

treated T cells and B cells immediately adhered to TCPS dishes, and firm cell adhesion was observed as in the CCRF-CEM cells (Fig. 2C, top and middle). These treated cells did not spread as much as did the adherent CCRF-CEM cells induced by Tat-PEG(40k)-lipids; also, the cell adhesion was actually induced over 2 days. Other CPP-PEG(40k)-lipids were then examined with the T and B cells. These cells were treated with various CPP-PEG(40k)-lipids using the same methods, and the adhesion ratios were calculated (Fig. 2D). We found that the CPP-PEG(40k)-lipids could induce cell adhesion in both the cell line and the floating primary cells. Similar results were obtained for human erythrocytes: Tat-PEG(40k)-lipid-treated erythrocytes adhered to TCPS dishes; stable adhesion was induced over 4 days without any lysis of the erythrocytes (Fig. 2C, bottom). No lysis or damage to the erythrocytes indicates that there was no cytotoxicity produced by the cell-surface modification with Tat-PEG(40k)-lipids, since erythrocytes are very sensitive to any chemical modification as well as to physical and mechanical stress. When adherent cell lines such as HEK293 cells were treated with Tat-PEG(40k)-lipids, the initial cell adhesion was more rapid than in non-modified cells, indicating that the Tat-PEG(40k)-lipids promoted cell adhesion and that the subsequent cell adhesion process was not influenced by the cell-surface modification (Fig. S8). Therefore, CPPPEG-lipids are also useful for inducing the initial cell adhesion of adherent cell lines.

Conditions for induction of cell adhesion by CPP-PEG-lipids In order to examine the induced cell adhesion by CPP-PEG(40k)-lipids, we performed further analyses (Fig. 3). When Tat-PEG(40k)-lipid-treated CCRF-CEM cells were cultured in serum-free medium, the same cell adhesion was observed, although the ratio was a little lower


24

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than under normal conditions (Fig. 3A, E), indicating that extracellular matrix molecules such as fibronectin are not involved in the induced cell adhesion. In addition, firm cell adhesion was clearly observed when the treated cells were cultured in EDTA-supplemented culture medium, indicating that the interaction of cell adhesion molecules such as integrins and cadherins is also not involved in the induced cell adhesion (Fig. 3B, E). Since negatively charged cell-surface glycans such as heparan sulfate are reported to be important for cell adhesion along with integrins

21

, we used heparinase to degrade the cell-surface glycans on CCRF-CEM cells. After

the cells were pre-treated with heparinase, they were treated with Tat-PEG(40k)-lipids using the usual methods. Firm cell adhesion of the treated cells was observed, indicating a lack of interaction between the Tat peptide and heparan sulfate (Fig. 3C, E). Specific blocking with antiCD4 and anti-CD8 antibodies did not inhibit the induced cell adhesion, indicating the Tat peptide does not interact with these receptors (Fig. 3E). We also examined the influence of the substrate surface on the induced cell adhesion by using cationic surface and anti-protein repelling surfaces: For this purpose we used a selfassembled monolayer (SAM) of alkanethiols carrying an amine group (NH2-SAM) as the cationic surface

22

. Since the alkanethiols chemisorb onto a gold surface, it was possible to

fabricate the positively charged surface. When Tat-PEG(40k)-lipid-treated CCRF-CEM cells were cultured on the NH2-SAM surface, the treated cells did not adhere strongly, but were only weakly immobilized, and the cells gradually detached from the surface with time (Fig. 3E). We also used poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-coated dishes as a proteinrepelling surface. PMPC is a well-known polymer that can suppress protein adsorption and cell adhesion

23-24

. We expected that the Tat peptide would not interact with the PMPC-coated


25


surface, and, in fact, no cell adhesion occurred between the Tat-PEG(40k)-lipid-treated cells and PMPC-coated dishes (Fig. 3E, Fig. 5B).

A

C

B

D Culture dish pretreated with Tat-PEG-lipid

Heparinase

+ EDTA

No serum

E

100

Adhesion ra o at 6hr (%)

80 60 40 20

NH 2SA TT a M a tre t--PPE at EGGed li-p su lipdirfa d ce

PM PC

CD 8

CD 4

TA pa rin as e

ED

He

se ru m

0

No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 39

Figure 3. Influence of culture conditions on the cell adhesion of Tat-PEG(40k)-lipidtreated CCRF-CEM cells and analysis of adherent cells. Adhesion behavior of TatPEG(40k)-lipid-modified CCRF-CEM cells (A) in the absence of serum at 6 h and (B) in the presence of 10 mM EDTA at 3 h. (C) Effect of heparan sulfate on the cell adhesion of Tat-PEG(40k)-lipid-modified CCRF-CEM cells. CCRF-CEM cells were treated with TatPEG(40k)-lipid after pre-treatment with heparinase. (D) Inhibition of induced cell adhesion. A PS culture dish was incubated with Tat-PEG(40k)-lipids before the addition of CCRF-CEM cells treated with Tat-PEG(40k)-lipids. (E) Adhesion ratios of TatPEG(40k)-lipid-modified CCRF-CEM cells after 6 h of culture on TCPS dishes under various culture conditions, including CD4 and CD8 treatment and culture on a proteinrepelling surface (PMPC-coated dish) and a cationic NH2-SAM surface. Scale bars: (A) 20, (B) 20, (C) 50, and (D) 40 µm.


26

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In an inhibition study, we pretreated the TCPS surface with Tat-PEG(40k)-lipids, and then cultured Tat-PEG(40k)-lipid-treated CCRF-CEM cells on the treated surface. The treated CCRFCEM cells did not adhere to the surface (Fig. 3D). Since the TCPS surface was already occupied with pretreated Tat-PEG(40k)-lipids, the Tat peptides on the cell surface could not interact with the surface and not induce cell adhesion. The same results were seen on R8-PEG(40k)-lipids and K8-PEG(40k)-lipids. These results suggest that only direct interaction between a positively charged CPP and a negatively charged substrate surface is essential to inducing cell adhesion.

Cell attachment to a microfiber-based 3D scaffold Since the surface modification with CPP-PEG-lipid was actually able to induce a firm and rapid cell adhesion, we tried to attach cells to a PS microfiber-based 3D scaffold as a model of decellularized organs and tissue (Fig. 4). When floating CCRF-CEM cells were treated with Tat-PEG(40k)-lipids and added to microfiber fabric pieces, they immediately attached to the microfibers. In contrast, hardly any cells were seen on the microfibers incubated with control non-treated cells (Fig. 4A); almost all the cells failed to attach to the PS microfibers and just passed through the space between them. Similar results were observed for adherent L929 cells and human hepatocytes (Fig. 4B, C). The treated L929 cells and hepatocytes immediately attached to the microfibers, whereas fewer cells were seen to attach in the case of the non-treated control cells. After 2 days in culture, the L929 cells had spread well and grown on the microfibers. In the case of the hepatocytes, the immobilized hepatocytes on the microfibers produced and secreted urea into culture medium in the normal way, indicating that the immobilized hepatocytes were alive and functional (Fig. 4D).


27


A

Treated CCRF-CEM

B

Treated L929 0 day

Control CCRF-CEM

C

Treated hepatocyte

D ***

800

Control L929 0 day

Control hepatocyte

2 day

Urea (ng/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 39

2 day

600 400 200 0

Control hepatocyte

Treated hepatocyte

Figure 4. Cell attachment to 3D PS microfibers induced by CPP-PEG-lipids. Cell attachment of (A) CCRF-CEM cells, (B) L929 mouse fibroblast cells, and (C) human hepatocytes treated with Tat-PEG-lipid onto PS microfibers. As a control, non-treated CCRF-CEM cells, L929 cells, and human hepatocytes were used. Cells were observed by confocal laser-scanning microscopy. Those cells were labeled with Celltracker Green. Scale bars: 20 µm. (D) Urea assay from human hepatocytes on PS microfibers. After human hepatocytes were immobilized onto PS microfiber fabric, they were incubated in culture medium for 3 h at 37oC. Error bars indicate standard deviation; n = 4. Significant differences (***=p20kDa (> ca.10 nm), CPP could “reach” over the membrane proteins and


32

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gain access to the substrate surface. On the other hand, shorter PEG chains (1 kDa, 5 kDa) were not long enough, so the CPPs were surrounded by higher membrane proteins. Furthermore, another important consideration is that CPPs have both cationic and cell-penetrating properties. CPPs attached to PEG-lipids can freely move inside and outside across the cell membrane. However, once the negatively charged surface was exposed to the CPPs, the CPPs moved toward the surface and rapidly formed electrostatic bonds with the surface, which induced firm cell adhesion. On the other hand, other cationic peptides such as R4, RA7 and GPI, which also showed a rapid binding property like that of the CPPs, did not induce cell adhesion. Presumably, these cationic peptides are strongly interactive with the negatively charged cell membrane, and all the peptides were already immobilized onto the cell surface. Therefore, these cationic peptides were not available for interaction with the substrate surface because the peptides were surrounded by other membrane proteins. CPPs are also cationic; however, they were less interactive with cell membrane than were other cationic peptides. With this technique, it was easy to immobilize cells onto 3D microfibers immediately, since cell adhesion was quickly induced by CPP-PEG-lipids. Once the treated cells contacted the microfibers, the cells attached to them even though the contact area was small; this behavior was different from the behavior we observed for control non-treated cells. The treated cells continued to adhere to the microfibers because normal cell adhesion by the integrins gradually emerged before the effect of the CPP-PEG-lipids had totally dissipated. During 3D cell culture, some cells detached and fell off the microfibers. We could see some cell attachment in non-treated L929 cells, but almost all the cells detached from the microfibers, indicating that the 3D microfiber cell environment may be different from 2D culture conditions. Therefore, the enhancement of


33


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Page 34 of 39

initial attachment with the CPP-PEG-lipids should improve the recellularization of organs and tissues.

[Conclusion] We found that CPP-PEG-lipids with 20-kDa or 40-kDa PEG could induce firm adhesion of floating cells without cytotoxicity, as well as rapid adhesion of adherent cells. The induced cell adhesion resulted from the strong interaction between CPP and the negatively charged substrate surface. Therefore, our CPP-PEG-lipids provide a new molecular design for inducing cell adhesion. The induction of cell adhesion with these materials will be useful for fabricating 3D cell-based structures using decellularized organs and tissue.

AUTHOR INFORMATION Corresponding author* Yuji TERAMURA Tel: +81(0)3-5841-1174 E-mail: [email protected]

Author Contributions


34

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources Bilateral Joint Research Project (Japan-Sweden) of the Japan Society for the Promotion of Science (JSPS) and STINT, a Grant-in-Aid for Young Scientists (A) (No. 26702017), as well as a Grant-in-Aid for Scientific Research on Innovative Areas (No. 26106709) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.

[Acknowledgement] We thank Dr. Deborah McClellan for editorial assistance. This research was supported in part by a Bilateral Joint Research Project (Japan-Sweden) of the Japan Society for the Promotion of Science (JSPS) and STINT, a Grant-in-Aid for Young Scientists (A) (No. 26702017), as well as a Grant-in-Aid for Scientific Research on Innovative Areas (No. 26106709) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.

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D. A. Perfusion-Decellularized Matrix: Using Nature's Platform to Engineer a Bioartificial Heart. Nat Med 2008, 14, 213-221. (3)

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Table of Contents Graphic and Synopsis

CPP-PEG-lipid CPP

cells Induced cell adhesion Surface modifica on with CPP-PEG-lipid

3D PS microfibers

Membrane protein

Cell membrane PEG: 20k, 40k Ca onic short pep de - - -+++ - - - - - - +++ ------3D fiber


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Cell Adhesion Induced Using Surface Modification ... - ACS Publications

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