In-situ, light-guided axon growth on biomaterials via photoactivatable

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Biological and Medical Applications of Materials and Interfaces

In-situ, light-guided axon growth on biomaterials via photoactivatable Laminin peptidomimetic IK(HANBP)VAV Aleeza Farrukh, Shifang Zhao, Julieta I. I. Paez, Atria Kavyanifar, Marcelo J Salierno, Adolfo Cavalie, and Aranzazu del Campo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15517 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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ACS Applied Materials & Interfaces

In-situ, Light-Guided Axon Growth on Biomaterials via

Photoactivatable

Laminin

Peptidomimetic

IK(HANBP)VAV Aleeza Farrukh,(1,2) Shifang Zhao,(1,3) Julieta I. Paez,(1) Atria Kavyanifar,(4) Marcelo Salierno,(4) Adolfo Cavalié,(5) Aránzazu del Campo*(1,3) (1) INM – Leibniz Institute for New Materials, Campus D2 2, 66123, Saarbrücken, Germany (2) Max Planck Graduate Center, Forum Universitatis 2, Building 1111, 55122 Mainz, Germany (3) Saarland University, Chemistry Department, 66123 Saarbrücken, Germany (4) Institute of Physiological Chemistry, University Medical Center Johannes Gutenberg University, Hanns-Dieter-Hüsch-Weg 19, D-55128 Mainz, Germany (5) Experimental and Clinical Pharmacology and Toxicology, Saarland University, 66421 Homburg

KEYWORDS: photo-triggered cell adhesion, laminin peptidomimetics, directional neuronal growth, IKVAV

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ABSTRACT: The ability to guide the growth of neurites is relevant for reconstructing neural networks and for nerve tissue regeneration. Here, a biofunctional hydrogel that allows light-based directional control of axon growth in-situ is presented. The gel is covalently modified with a photoactivatable derivative of the short laminin peptidomimetic IKVAV. This adhesive peptide contains

the

photoremovable

group

2-(4'-amino-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol

(HANBP) on the Lys rest that inhibits its activity. The modified peptide is highly soluble in water and can be simply conjugated to –COOH containing hydrogels via its terminal -NH2 group. Light exposure allows presentation of the IKVAV adhesive motif on a soft hydrogel at desired concentration, and at defined position and time point. The photoactivated gel supports neurite outgrowth in embryonic neural progenitor cells (eNPCs) culture and allows site-selective guidance of neurites extension. In-situ exposure of cell cultures using a scanning laser allows outgrowth of neurites in desired pathways.


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INTRODUCTION Directional migration and positioning of neural cells is an important process during development of neural tissue that has to be reconstructed during nerve repair or when building neural networks for technical applications.1-2 Most reported approaches to guide neurons use topographical features (channels, fibers) on the substrate to spatially confine elongating neurites and favor their growth along predefined directions.3-4 Recently, the light beam from a scanning laser has been also been used to guide neurite growth on substrates. A near-IR laser (800 nm) spot placed in front of a nerve’s leading edge enhanced the growth of the dendrites following the beam focus.5 Likewise, an IR laser (1455 nm) has triggered the elongation of dendrites and axons by 10 μm in 1 min.6 The mechanism by which neurons follow light stimulus in these two examples remains to be elucidated, though it is generally accepted that neurite growth follows gradients of forces or temperature caused by illumination. A unique advantage of light to guide neurite growth is the possibility of defining the neuronal pathway in situ, i.e. in the presence of cells, and with precise and flexible temporal and spatial resolution. We recently reported a strategy for light-guided directional growth of neurons on biomaterials modified with photoactivatable cell adhesive peptides.7-8 In this case, guidance is provided by molecular recognition between photoactivated ligands at the material surface and complementary receptors on the cell membrane, and not by physical forces as in the examples mentioned before. The phototriggerable laminin peptidomimetic CSIK(HANBP)VAVSADR [IK-12(HANBP)], containing the neuron adhesive IKVAV sequence and the photoremovable protecting group 2-(4'amino-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol, HANBP, was used for this purpose.9-14 Laminin is an abundant protein in the neural niche, with a demonstrated fundamental role in neuronal migration, differentiation and neurite outgrowth.15-16 The IKVAV sequence in particular is present


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at the globular region of the α-chain in Laminin-1 protein and interacts with integrin receptors α3β1, α4β1 and α6β1.17 The photoremovable protecting group 2-(4'-amino-4-nitro-[1,1'-biphenyl]3-yl)propan-1-ol, HANBP, introduced at the Lys rest of CSIKVAVSADR caused temporal inhibition of the bioactivity of the peptide.8 HANBP chromophore was selected on the basis of its high water solubility due to its polar substituents, and the possibility of photolysis at wavelengths up to the visible rante (λmax = 490 nm).18-19 This o-nitrobiphenyl donor–acceptor based chromophore shows higher photolysis efficiency than frequently used o-nitrobenzyl chromophores (NVOC or DMNPB), and it is reported to show appreciable two-photon cross-section, which could be relevant for future application in 3D cell cultures.8,

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Hydrogels modified with

CSIK(HANBP)VAVSADR supported patterned growth and migration of neuronal cells along preirradiated patterns. However, IK-12(HANBP) showed some limitations in the applications: it has poor water solubility (1 µm as measured by light scattering. The IKVAV pentapeptide formed the largest ones (>1 µm). The introduction of charged flanking amino acids (CE4IK, CE2IK, CIKC and IK-19), polar amino acids (CSIK) or pegylated chains ((EG)2-IK) to the sequence proved insufficient to avoid aggregation. These results demonstrate that IKVAV sequence forms very stable aggregates in water, and these aggregates are present in all reported works with this peptide and its longer derivatives.26-27 The introduction of the HANBP group at the amine group of the Lys seems to be the only effective way to disturb β-sheet formation21 and improve solubility. Table 1. Characterization of aggregate formation in aqueous solutions of different IKVAV containing peptide sequences by light scattering. Peptide Sequence

Abbreviation

Net Charge Aggregate (pH 7.0)

size*

IKVAV28

IKVAV

1

> 1 µm

(EG2)-IKVAV29

(EG2)-IK

1

370 nm

CSIKVAV30

CSIK

0.9

1 µm

CEEGIKVAV27

CE2IK

-1.1

260 nm

CEEEEGIKVAV27

CE4IK

-3.1

120 nm

CCRRIKVAVWLC31

CIKC

2.8

> 1 µm

CSRARKQAASIKVAVSADR 14

IK-19

3.9

400 nm

IK(HANBP)VAV

IK(HANBP)VAV

1

-

* Measured by DLS. Measurement conditions: 0.2 mM peptide solution in water at room temperature.


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The photochemical properties of IK(HANBP)VAV were then analyzed. Light exposure of IK(HANBP)VAV solution for increasing times at 420 nm (LUMOS 42, Atlas Photonics, 1.2 mWcm-2) lead to a drop of absorbance at its max = 490 nm and the appearance of a broad maximum around 400 nm (Figure 1C). Analytic HPLC analysis of the irradiated solution showed the disappearance of the IK(HANBP)VAV signal and the parallel appearance of IKVAV to a yield of 83% after 1 h of irradiation (Figure S1 A-B). These results show that IK(HANBP)VAV has excellent photosensitivity and photochemical yield for in-situ application in biological experimentation. Interestingly, the photolytic properties of IK(HANBP) are better than those of the previously reported longer peptide CSIK(HANBP)VAVSADR at the same irradiation conditions (61 % in 1h).7 It is important to note that we did not fully exploit the possibilities of the chromophore in this study, and the photochemical yield of this chromophore at max = 490 nm are expected even better. In order to test the bioactivity of IK(HANBP)VAV, cell experiments with neuronal cells were performed on soft hydrogels (2 kPa). For this purpose, poly(acrylamide-co-acrylic acid) P(AAmAA) hydrogels were used.32 The gels were first activated with EDC/NHS chemistry and then incubated with a 0.2 mM solution of IK(HANBP)VAV in PBS (pH 7.4). In order to corroborate the coupling, UV spectra of the gels were measured after modification. The appearance of an absorbance band at 490 nm, which remained stable after long term washing with different solvents, confirmed covalent binding of the N-terminal -NH2 group of the peptide to the activated COOH groups of the hydrogel. Upon light exposure (420 nm, 0.34 mWcm-2) and washing, the absorbance decreased, evidencing the photocleavage of the chromophore from the gel (Figure 1D). A 86% cleavage was obtained after 8 minutes of irradiation in contrast to slow photoloysis (29% in 8 minutes) of IK-12 (HANBP). Note that photocleavage of HANBP chromophore occurs at much


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lower irradiation dose than photolysis of commonly used o-nitrobenzyl chromophore in biology studies (~7.2% for NVOC and 6.3% for DMNPB at 8 minutes irradiation with same lamp at same wavelength).7 Neural progenitor cells (eNPCs) isolated from cortex of mouse embryo (E 14.5) were used to test the bioactivity of IK(HANBP)VAV modified gels. Initial experiments to find out appropriate ligand densities to support neuronal culture were performed with the widely used IK-19 peptidomimetic. Gels were incubated with peptide concentrations between 0.01 and 1mM. Gels incubated with 0.2 mM concentration of IK-19 supported neuronal survival and development of neuronal morphological parameters, i.e. multipolar neurites, extensive neurite outgrowth and branching and significant axon extension (Figure 2). This concentration was then used for all other experiments in order to allow comparison of the performance of the different peptides. Comparable levels of maturation were also observed on gels coated with LN or PL/LN (Figure 2). Neurons cultured on gels functionalized with IKVAV derivatives from Table 1 showed low viability and did not develop neuronal phenotypes (Figure 2A). The aggregation of the peptides in the incubation solution, as reported in Table 1, is expected to hinder reaction of the peptides with the gel and hamper accessibility of the membrane receptors for cell attachment.


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Figure 2. Immunofluorescence images of representative neural progenitor cells (E 14.5) on 2 kPa P(AAm-AA) hydrogels functionalized with IKVAV-derived peptidomimetic sequences and LN controls (A), and quantification of neuronal morphological parameters: length of axon, neuronal polarity, number of branches and cell survival (B), 24 h after cell seeding. DCX stains cell body, SMI marks axonal filament and nucleus is stained by DAPI. The significance of data obtained for the different samples was analysed by Tukey-test (mean ± SD, ANOVA, ** p

In-situ, light-guided axon growth on biomaterials via photoactivatable

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