Multi-functionalization of Cells with a Self-assembling Molecule to

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Multi-functionalization of Cells with a Selfassembling Molecule to Enhance Cell Engraftment Ippei Takashima, Kosuke Kusamori, Hayase Hakariya, Megumi Takashima, Thi Hue Vu, Yuya Mizukami, Naotaka Noda, yukiya takayama, Yousuke Katsuda, Shin-ichi Sato, Yoshinobu Takakura, Makiya Nishikawa, and Motonari Uesugi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00109 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on March 3, 2019

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Multi-functionalization of Cells with a Self-assembling Molecule to Enhance Cell Engraftment Ippei Takashima,†Kosuke Kusamori,‡Hayase Hakariya,†Megumi Takashima,†Thi Hue Vu,† Yuya Mizukami,∥Naotaka Noda,† Yukiya Takayama,‡Yousuke Katsuda,†Shin-ichi Sato,† Yoshinobu Takakura,∥Makiya Nishikawa,‡,∥,* and Motonari Uesugi†,⊥,§,* †

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

‡

Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan.

∥

Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

⊥

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Uji, Kyoto 611-0011, Japan

§

School of Pharmacy, Fudan University, Shanghai 201203, China

KEYWORDS Cell-based therapy, cell-surface modification, self-assembly.

ABSTRACT: Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using selfassembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-Tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pre-treated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.

■ INTRODUCTION In regenerative medicine, many researchers have invested in the potential of cell-based therapy where transplanted cells are used to recover the lost function of a damaged organ.1 Recently, the development of induced-pluripotent stem cells (iPSC) has revolutionized the field of cell-based therapy by creating a new process to prepare the cells which circumvents major ethical and practical issues.2 Cell-based therapy holds great promise for the treatment of diseases such as diabetes, neurodegenerative diseases, and cardiovascular diseases;1-3 yet, some problems remain unresolved. A majority of the suspended cells quickly die after injection into the host, making only a small percentage of the cells viable for engraftment and necessitating an enormous number of cells to achieve the desired therapeutic effect.4-7 To avoid cell death and enhance cell engraftment, various approaches have been developed. Hydrogels, natural polymers and biocompatible synthetic polymers have been employed to enhance cell survival by providing protection from harsh biological conditions.8-12 However, hydrogels have little effect on apoptosis inhibition because the biomaterial itself does not induce cell-survival signals, and harmful biological factors can contaminate the hydrogels.12, 13 Growth factors have also significantly contributed to cell-based therapy by

enhancing cell-survival signals, angiogenesis, and so on.5, 14, 15 One shortfall of using growth factors in cell-based therapy is their short half-lives resulting from rapid dissociation under in vivo conditions.16, 17 The combination of hydrogels and growth factors has also been used in in vivo experiments because the former enhance the retention time of growth factors in the target tissue while the latter counteract apoptosis induced by pro-apoptotic molecules which gradually infiltrate the hydrogel.12-14 These reports informed us that a combination of various factors is necessary to improve cell engraftment for application to in vivo conditions, and we sought to contribute by developing other agents for enhancement of cell engraftment. One possible method might be synthetic modifications of the cellular surface with functional molecules, such as antibodies, growth factors, enzymes, bioactive peptides, riboenzymes, metal-organic materials including catalytic complexes and metal-organic frameworks (MOFs), and synthetic drugs. Many cell-surface modifications have been developed, including metabolic labeling of cellsurface glycoproteins with synthetic sugars,18-21 liganddirected labeling of cell-surface proteins,22-26 and membrane labeling with lipid-anchored molecules.27-30 However, these modifications have never been employed toward cell engraftment.


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In the present study, we report a synthetic cell-surface modification using a synthetic self-assembling molecule to increase the cell survival of treated cells. In the second step, we applied this system to modify the cellular surface with an unorthodox function: cell-invasion activity. This strategy aims to, first, assist cell survival and, second, to facilitate penetration and engraftment to the target tissue. Imparting these two characteristics, cell survival and cell invasion, to cells is a promising strategy to increase the effective number of cells injected and improve the engraftment rate. ■ RESULTS AND DISCUSSION Design of adh2.0. We previously developed a small molecule, adhesamine (adh),31, 32 that enhances cell attachment and growth by self-assembling in the presence of the haparan sulfate of syndecan, which is a key protein in cell attachment, cell growth, and cell survival signals.33, 34 Adhesamine was originally discovered as a compound that promotes cell adhesion to the surface of culture plates and enhance cell growth. However, adhesamine is not potent enough to inhibit anoikis of suspended, floating cells. One approach to improving its activity was to enhance its self-assembling property. We conjugated adhesamine to a series of peptides containing phenylalanine residues, a wellknown self-assembly unit.35, 36 A total of nine adhesamine-peptide conjugates were synthesized to find the optimal number of phenylalanine residues and linker length (Scheme 1).

Scheme 1. Structures of adhesamine and adhesamine derivatives conjugated with each peptide domain X.

We first examined the affinities of the adhesamine derivatives to heparin by isothermal titration calorimetry (ITC) (Table 1 and Fig. S1). The adhesamine molecule conjugated to the SFF peptide (adh2.0) shows the highest affinity to heparin, which is 3.5-fold greater than that of adhesamine (Table 1). Peptide SFF itself has no detectable affinity to heparin (Fig. S1B). Adh2.0 maintains the high selectivity of adhesamine to heparin with no detectable affinity to chondroitin sulfate A (Fig. S1D). These data indicate that the conjugated FF peptide

Table 1. Summary of the affinity to heparin of adhesamine and its derivatives. app

Name

Ka 6 -1 (x10 M )

app

⊿H

app

⊿G

app

⊿S

(kcal/mol/deg)

adh

10

-47

-9.7

-0.1

SFF

nd

nd

nd

nd

adh2.0

35

-52

-10.5

-0.1

1

4

-34

-9.2

-0.1

2

18

-45

-10.2

-0.1

3

26

-46

-10.4

-0.1

4

35

-52

-10.4

-0.1

5

29

-102

-10.4

-0.3

6

21

-54

-10.2

-0.1

7

31

-52

-10.4

-0.1

8

32

-48

-10.4

-0.1

[a] nd: not detectable. [b] Raw data is shown in Fig. S1.

Figure 1. Cell viability of NIH3T3 cells after incubation under an anoikis condition: serum-free DMEM, 0.5% (w/v) methylcellulose, 1% (v/v) DMSO in ultralow attachment 96-well plate for 3 days with each adhesamine derivative (100 μM). The cell viability was measured by WST-8 assay and normalized to the cells treated with adh2.0. Error bar is the standard deviation (n = 3).

augments adhesamine’s binding to heparin without compromising the selectivity. We also investigated the ability of the adhesaminepeptide conjugates to inhibit anoikis, a form of detachment-induced apoptosis that is likely a major cause of cell death in suspended cells used for cell-based therapy. NIH3T3 cells, an anchorage-dependent mouse embryo fibroblast cell line often used in anoikis studies, were treated with each conjugate at 100 µM (Fig. 1). A round-bottom, ultralow-attachment plate and methylcellulose were used to disrupt cell-substrate interactions and cell-cell interactions,37 and the cells were strained twice to ensure a single cell suspension. After a 72 hour incubation under anoikis conditions, the viability of the cells was quantified by measuring NADPH dehydrogenase activity (WST-8 assay). The cells treated with adh2.0 retained the highest cell viability after a 3 day incubation (Fig. 1).



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Figure 2. Characterization of adh2.0 aggregates. (A) Distribution of particle diameters calculated by DLS analyses and (B) cryo-TEM image of the adh2.0-heparin complex in serum-free DMEM with 100 μM adh2.0 and 1 μM heparin. (C) Visualization of the cellular surface with or without 100 μM adh2.0 treatment by FE-SEM. The panels on the right show the magnified images of the selected area. The cell surface dot structures are marked by the yellow arrows. (D, F) Immunostaining of SDC4 (D, λex = 488 nm, λem = 500-550 nm) and PKCα (F, λex = 568 m, λem = BP617/73) after incubation with 100 μM adhesamine (adh) or 100 μM adh2.0 in serum-free DMEM. (E) Quantification of the average number of particles per 1 cell in Fig. 2D categorized by size (n = 5 cell images, calculated by ImageJ), * Statistical differences were evaluated by one-way analysis of variance (ANOVA), followed by the Dunnett’s test for multiple comparisons (p 3x107 M-1) leads to the enhancement in cell viability. The anoikis-inhibitory activity of adh2.0 was also compared with those of other viability-promoting molecules (Fig. S4). Under the anoikis conditions that we used, alginate and chitosan, hydrogel-forming polysaccharides, failed to show high anoikis-inhibitory activity at comparable weight concentrations to that of adh2.0. The activity of 100 µM adh2.0 was also higher than that of 10 ng/mL fibroblast growth factor 2 (FGF2), a concentration often used in cell culture. The self-assembly structure of adhesamine on heparan sulfate. We expect that the FF peptide is positioned in close proximity to the pyrimidine of adhesamine and thus forms the robust self-assembly structure through ππ stacking between aromatic groups. To gain insights into this aggregate’s intermolecular interactions, we examined NOESY spectra of adh2.0 in solution with heparin (Fig. S5). The results suggest that the pyrimidine moiety of adh2.0 is adjacent to not only a neighboring pyrimidine but also the phenyl groups (Fig. S5C, D). In addition, the CD spectrum of adh2.0 showed a decrease in absorbance at 220 nm and increase around 200 nm when in complex with heparin, suggesting the formation of a β turn-like structure in the SFF peptide domain (Fig. S6). These results indicate that the formation of the βturn-like structure positions pyrimidine and phenyl groups close to each other. We further studied the self-assembly structure by dynamic light scattering (DLS) and electron microscopy (EM). The DLS measurements disclosed the formation of submicron particles (~600 nm diameter) of adh2.0 bound to heparin (Fig. 2A, S7), whereas the combination of adh2.0 and 4 μM chondroitin sulfate A showed no particle formation (data not shown). Using cryo-TEM we further observed aggregation of adh2.0 (100 μM) and heparin (1 μM) in aqueous solution (Fig. 2B) and formation of submicron particles which were absent in samples with adh2.0 (300 μM) or heparin (3 μM) alone (Fig. S8A, B). We also observed these adh2.0 complexes on the cellular surface using field emission scanning

electron microscopy (FE-SEM) which revealed many particles on the cell (Fig. 2C). The formation of the particles was significantly inhibited by the treatment of heparinase II, which cleaves heparan sulfate polysaccharides (Fig. S8C). Together, these results indicate that adh2.0 interacts with cell-surface heparan sulfate to coat the cellular surface with aggregate particles. Intracellular signaling for cell survival induced by adh2.0. Our previous studies indicate that adhesamine exerts its biological activity by inducing clustering of syndecan-4 (SDC4),31, 32 a heparan sulfate-modified protein that is known to be involved in cell attachment. Immunostaining with an anti-SDC4 antibody showed that treatment with adh2.0 induced SDC4 clustering in a similar fashion to adhesamine (Fig. 2D). The adh2.0treated sample showed greater numbers and sizes (> 0.4 μm2) of SDC4 clusters compared to those observed in the adhesamine-treated sample (Fig. 2E), suggesting that adh2.0 more potently induces SDC4 clustering compared to adhesamine. Oligomerization and clustering of SDC4 are known to activate PKCα through re-arrangement of PKCα binding molecules, such as phosphatidylserine and diacylglycerol33, 38-40.33, 38-40 Immunostaining with an antiPKCα antibody showed that adh2.0 treatment rapidly induces PKCα movement from the cytosol to the membrane (Fig. 2F, S9). Temporally, SDC4 clustering precedes PKCα relocation, implying that the former stimulates the latter (Fig. S10). MAPK activity is known to be regulated by PKCα.41-45 To identify the specific signaling pathway for adh2.0induced cell survival, we monitored MAPK phosphorylation levels after treatment with adh2.0 in a western blot time-course experiment. Treatment with adh2.0 resulted in gradual amplification of ERK phosphorylation (induction of cell survival signals), but inhibited JNK phosphorylation (induction of apoptotic signals) compared to controls (Fig. 2G, H).46 The transient ERK phosphorylation was similar to that induced by growth factors.47, 48 In fact, ERK phosphorylation is regulated in a similar manner for cells cultured under standard culture conditions (incubation with 10% (v/v) FBS on standard plastic culturing plates, referred to as SC). On the other hand, we found no significant changes in the phosphorylation levels of three other cell-growth related kinases, p38 MAPK46, Akt49, and FAK50 (Fig. S11). We additionally examined the inhibition of ERK signaling using UO126,51 a selective inhibitor of MEK, which is an upstream kinase of ERK. We also used UCN01,52 a selective PKCα inhibitor, and triciribine,53 an Akt inhibitor. UO126 and UCN01 decreased the cell viability produced by adh2.0 treatment (Fig. S12). On the other hand, triciribine exhibited much less inhibition of cell viability.



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migration and viability.55 These considerations inspired us to functionalize adh2.0 with MMP2 to increase its engraftment-reinforcing properties. We considered the Halo-Tag56 most appropriate for our purposes compared to other methods, such as the SNAPtag or CLIP-tag,57, 58 in terms of the tag size and the reaction rate. The Halo-Tag system is composed of two components: a haloalkane dehalogenase mutant and a synthetic ligand containing a reactive alkylchloride linker. A panel of adh2.0-alkylchloride conjugates were synthesized for the purpose of finding the alkylchloride conjugate that best maintains cell viability and heparin binding. The adhesamine derivative conjugated with two SFFK peptides, one to each aldehyde of adhesamine (Fig. S13A, molecule 9), exhibited limited cell-viability enhancement (Fig. S13B). In contrast, the derivative conjugated to only one SFFK peptide (Fig. S13D, molecule 10) retained the cell-viability enhancement properties (Fig. S13E), making 10 the clear choice for further modification with an alkylchloride substituent. Coupling of 10 with an alkylchloride substituent gave adh3.0 (Fig. 3A) which maintains high affinity to heparin and cell viability enhancement (Fig. S14). The other alkylchloride conjugates (molecules 11 and 12) showed negligible cell viability enhancement (Fig. S15), suggesting that the activity is sensitive to the location of modification. Figure 3. Cellular surface modification with adh3.0 and eGFP-H. (A) Structure of adh3.0. (B) The structural images of eGFP-H and conceptual illustration of the cell-surface modifications with eGFP. (C) Confocal images of cell surface modified with eGFP. Cells were incubated with eGFP-H, adh3.0, mixture of adh3.0 and eGFP-H, or mixture of adh2.0 and eGFP-H in serum-free DMEM ([adh2.0/adh3.0] = 50 μM, [eGFP-H] = 7.5 μM). “Merged” images are the stacked images of bright field, Hoechst 33342 (λex = 405 nm, λem = 417-477 nm) and eGFP (λex = 488 nm, λem = 500-550 nm).

These data indicate that adh2.0 treatment activates cell survival by regulating MAPK signals (ERK and JNK) and not by Akt signaling, or FAK signaling induced by attachment of cells to the culture plate. Design of adh3.0. We have found that adh2.0 selfassembles upon binding to heparan sulfate, forming submicron particles on the cellular surface and increasing cell viability. We envisioned that additional functionalization of this architecture might enhance cell transplantation. It has been reported that non-invasive NIH3T3 cells acquired invasion activity after transfection with Tgat (trio-related transforming gene), and overexpression of active MMP2.54 MMP2 degrades the extracellular matrix, especially type I and IV collagen, and clears the way for cell migration. Moreover, the decomposed ECM releases growth factors, attachment proteins, and angiogenesis factors which enhance cell

Cell-surface modification of transplanted cells. As a proof of concept, we used an eGFP-Halo fusion protein (eGFP-H), together with adh3.0, to modify the cellsurface with eGFP (Fig. 3B). The design, purification process, and activities (fluorescence of eGFP/Halo-tag reactivity) of the recombinant eGFP-H are shown in Fig. S16. Observation under a confocal microscope showed that treatment of NIH3T3 cells with adh3.0 (50 μM) and eGFP-H (7.5 μM) led to the formation of bright fluorescent particles distributed over the cellular surface (Fig. 3C for 2D images, Fig. S17A for Z-stack images, and Fig. S17B for flow cytometry). Treatment with either adh3.0 or eGFP-H alone, or with the combination of adh2.0 and eGFP-H failed to produce detectable fluorescence signals (Fig. 3C). Immunostaining confirmed that the observed eGFP fluorescence signals overlapped with the SDC4 clusters induced by adh3.0 treatment (Fig. S17C). Additionally, we examined the effect of the ratio of eGFP-H to adh3.0 (50 μM) on the cell-surface modification. The results show that 7.5 μM eGFP-H is optimal, and we used this ratio for further studies (Fig. S18). We then applied the adh3.0/Halo-Tag system to modify the cellular surface with MMP2. The design, purification process, and activities (enzymatic activity of MMP2/Halo-tag reactivity) of MMP2-Halo fusion protein (MMP2-H) are shown in Fig. S19. Immunostaining with an anti-MMP2 antibody demonstrated that the


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enhanced cell invasion activities while treatment with adh3.0 and MMP2-H separately did not (Fig. 4B, C, S20C). Similarly, treatment with adh2.0 and MMP2-H did not reinforce cell invasion activity, indicating that conjugation through the Halo-Tag technology is required for the observed increase in invasive activity. Finding that the invasive activity was inhibited by marimastat (200 nM), a well-known inhibitor of MMP2 and MMP9,59 links the invasion activity to MMP2 action. These data suggest that the covalent conjugate of adh3.0 and MMP2-H decorates the cellular surface through self-assembly to enhance both cell viability and invasion activity.

Figure 4. Cellular surface modification with adh3.0 and MMP2H. (A) The structural images of MMP2-H and conceptual illustration of the cell-surface modifications with MMP2. (B) Microscopy (4x) of the invasive cells stained with crystal violet in the invasion assay using Cytoselect CBA-110. Insets show the entire well. (C) The invasive cells were quantified with an invasion assay ([adh2.0/adh3.0] = 50 μM, [marimastat] = 0.2 μM, [MMP2-H] = 7.5 μM, n = 3). After staining with crystal violet, the invasive cells were lysed and the absorbance at 570 nm was measured. * Statistical differences were evaluated by one-way analysis of variance (ANOVA), followed by the Dunnett’s test for multiple comparisons (p

Multi-functionalization of Cells with a Self-assembling Molecule to

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