Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite

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Biodegradable Neuro-compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection and Delivers Anti-Alzheimer drug Anindyasundar Adak, Gaurav Das, Surajit Barman, Saswat Mohapatra, Debmalya Bhunia, Batakrishna Jana, and Surajit Ghosh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12114 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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ACS Applied Materials & Interfaces

Biodegradable Neuro-compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection and Delivers AntiAlzheimer Drug Anindyasundar Adak,1# Gaurav Das, 1# Surajit Barman,1 Saswat Mohapatra,2, Debmalya Bhunia,1 Batakrishna Jana,1 Surajit Ghosh*1,2 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India. # These two authors contributed equally CORRESPONDING AUTHOR INFORMATION: Fax: +91-33-2473-5197/0284; Tel: +91-33-2499-5872; E-mail: [email protected] KEYWORDS: Peptide hydrogel, Microtubule, 2D and 3D Neuron cell culture, Neuroprotection, Neurite outgrowth, Drug Delivery

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ABSTRACT: A novel neuro-compatible peptide based hydrogel has been designed and developed, which contains microtubule stabilizing and neuroprotective short peptide. This hydrogel shows strong three dimensional cross-linked fibrillary networks, which can capture water molecules. Interestingly, this hydrogel serves as excellent biocompatible soft-material for 2D and 3D (neuro-sphere) neuron cell culture and provides stability of key cytoskeleton filaments such as microtubule and actin. Remarkably, it was observed that this hydrogel slowly enzymatically degrades and releases neuroprotective peptide, which promotes neurite outgrowth of neuron cell as well as exhibits excellent neuroprotection against anti-NGF induced toxicity in neuron cells. Further, it can encapsulate anti-Alzheimer and anti-cancer hydrophobic drug curcumin, releases slowly and inhibit significantly the growth of 3D spheroid of neuron cancer cells. Thus, this novel neuroprotective hydrogel can be used for both neuronal cell transplantations for repairing brain damages as well as delivery vehicle for neuroprotective agents, anti-Alzheimer and anti-cancer molecules. INTRODUCTION Human brain is inexplicable machinery comprising of networks of millions of neurons that interconnect and maintain its function.1 Any damages due to neurodegenerative diseases, stroke and various injuries in brain or central nervous system (CNS) cause neuron cell death, which lead to severe complications in psychological, motor and cognitive functions.2-6 The major concern with this complex machinery is its limited regenerative capabilities.7 Due to the complexity of the brain, repairing the damages is a challenging task.8 Currently, neuron cell transplantation-based approach has shown greatest promise among various approaches for repairing and regenerating the damage of neural tissues.9 However, transplanted cells often suffer from poor in vivo survival rates and fail to show clinical success10-14 due to lack of highly


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biocompatible neuron cell transplantable scaffold. To address this issue, various soft biocompatible neuron cell transplantable scaffolds have been developed, which keeps neuron cell healthy but clinical success of these scaffolds are poor.15-17 Thus, further development for the fabrication of highly biocompatible scaffold is crucial for successful neuronal cell transplantation and regeneration. Peptide self-assembly plays important role in fabrication of potential 3D biomaterials scaffolds for neuron cell culture in physiological condition.18, 19 Interesting example of peptide self-assembling scaffold reveals the concept of alternating amino acid sequences positive charge (Arg), hydrophobic (Ala) and anionic (Asp), which facilitates the self-assembly process to form a stable β-sheet structure18, 20, 21 followed by formation of hydrogel containing dense fibrillary networks, which mimics the extracellular matrix (ECM)18,

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and served as

excellent scaffold for neural stem cells culture22-26, axonal regeneration, growth of blood vessel inside the injured brain.27,

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In this manuscript, we have designed a novel lipopeptide, which

contain a hydrophobic long chain, microtubule stabilizing peptide and hydrophilic amino acids (Lys/K). Amphiphilic character of designed molecules facilitates self-assembly process.29-36 Thus, hydrophobic long chain i.e. palmitic acid (PA) has been incorporated at the N-terminal of this peptide which enhanced the hydrophobic character. The rationale behind the incorporation of microtubule stabilizing peptide is due to the fact that, in Alzheimer’s disease (AD) microtubule associated protein tau gets hyper-phosphorylated, which causes disassociation of tau from microtubule lattice and results severe microtubule disruption and loss of axonal transportation.37-40 Apart from AD, other neuronal damages are also associated with microtubule or cytoskeleton disruption and release of serine proteases41. Reports showed that a small octapeptide (NAPVSIPQ) stabilizes microtubule by reducing the hyper phosphorylation of tau, binds strongly with microtubule lattice and promotes neurite outgrowth42-49. Next, we introduced


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triple lysine residues at the C-terminal position of this peptide to promote the amphiphilicity and generate cell adhesion as it was known that lysine units help in cell adhesion because of its positive charge. Combination of the above described concept resulted in a new molecule, which is palmitic acid (PA) attached NAPVSIPQKKK peptide (abbreviated as PA-NK) (Figure 1a). Due to its unique amphiphilic character it spontaneously self-assembled to form β-sheet structure, which results in interconnected fibrillary network structure and entraps large number of water molecules to form a well-defined hydrogel. This hydrogel is highly biocompatible, which serves as an excellent platform for 2D neuron cell adherence, 3D neuron cell growth (neuro-sphere) and keeps microtubule and actin stable. It slowly enzymatically degrades to release neuroprotective peptide NAPVSIPQK, which promotes neurite outgrowth and exhibits significant neuroprotection against anti-NGF induced toxicity in neuron cells. Further, it can encapsulate and release anti-Alzheimer and anti-cancer drug (curcumin) slowly and inhibit the growth of 3D spheroid of neuron cancer cell. EXPERIMENTAL SECTION Materials Chemicals. Fmoc-Rink Amide AM resin and Fmoc-amino acids, HBTU were purchased from Novabiochem. Methanol, Dimethylsulfoxide (DMSO), Pipyridine, Diisopropylethylamine (DIPEA), Pyridine, Ether, and Trifluoroacetic acid were purchased from Spectrochem. Phenol, Dichloromethane (DCM), Ethanedithiol (EDT), Hydrogen peroxide (30% solution), Acetone, Dichloromethane and N, N′-Dimethylformamide (DMF) were purchased from Merck. TritonX100 was purchased from SRL. N, N′- Diisopropylcarbodiimide (DIC), 5(6)-Carboxyfluorescence (FITC), 5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole dihydrochloride


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(DAPI), curcumin, Proteinase K enzyme, NGF (Nerve growth factor), anti-NGF, dulbecco’s modified eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Horse serum, MES, trypsin-EDTA solution, DMSO for cell culture and formaldehyde solution (molecular biology grade) were purchased from Sigma Aldrich. Propidium iodide and Calcein AM from Thermo Fisher. Penicillin-Streptomycin, neutravidin, Alexafluor568-carboxylic acid succinimidyl ester and fetal bovine serum (FBS) were purchased from Invitrogen. Guanosine-5’[(α,β)-methyleno]triphosphate, Sodium salt (GMP-CPP) was purchased from Jena Bioscience. Anti-alpha Tubulin (EP1332Y) antibody and Goat polyclonal anti-Rabbit IgG H&L (Cy3.5 ®) were purchased from Abcam. For purification, we used Simadzu HPLC system with Symmetry C-18 (Waters) semi preparative reverse phase column. Pure product was lyophilized in Vertis 4K freeze drier after column purification. HPLC grade water and Acetonitrile were purchased from J. T. Baker. All the chemicals were used without further purification. Cell Culture Cell culture: Neuro-2a cells (mouse neuroblastoma cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heal inactivated Fetal Bovine Serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO2 atmosphere at 37 ºC. Rat pheochromocytoma cells (PC12) were cultured in RPMI supplemented with 10% heat inactivated horse serum (HS) and 5% heat inactivated FBS. The cells were then induced into neuronal cells by addition of NGF (100 ng/mL) to the medium supplemented with 1% HS for 5 days. Peptide Amphiphile (PA-NK) Synthesis


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NK (NH2-NAPVSIPQKKK-CONH2) was synthesized by solid phase peptide synthesis method in CEM-LIBERTY1 automated microwave peptide synthesizer using Rink Amide AM resin. Deprotection of Fmoc group is carried out by 20% v/v piperidine in DMF. HBTU (5 equiv.) and DIPEA (10 equiv.) were used as coupling agent. After the synthesis of peptide, palmitic acid group was attached at the N-terminal of peptide using HBTU and DIPEA in DMF for 6 hr. After that palmitic acid attached peptide (abbreviated as PA-NK) was cleaved from the resin with a mixture of TFA, EDT, H2O, phenol 94:2.0:2.0:2.0 (v/v/v/v) at room temperature for 2 h and precipitated in cold ether. PA-NK was lyophilized and purified by reverse phase HPLC (Simadzu) and characterized by MALDI Mass spectroscopy. Preparation of Peptide Hydrogel PA-NK (20 mg) was dissolved in (1 mL) phosphate-buffered saline (10 mM) to get 2 wt% solution at pH 7.4 and sonicated for few minutes to get homogeneous mixture. Then the resulting mixture is heated gently for 5-10 minutes. After that short-wavelength ultraviolet light has been passed through it for sterilization and cell culture purpose. After that the solution is kept at room temperature and within an hour well defined homogeneous hydrogel was obtained, which is confirmed by inverting the vials. Characterization of the PA-NK Hydrogel Fourier Transforms Infrared Spectroscopy FT-IR analysis of the PA-NK hydrogel (2 wt%) was carried out on a Perkin-Elmer Spectrum 100 FT-IR spectrometer using KBr pellets by placing hydrogel sample in a CaF2 cell. FT-IR spectrum was recorded in Perkin-Elmer Spectrum 100 series spectrometer using LiTaO3 detector. Background correction was accomplished each time to remove errors.


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Atomic Force Microscopy (AFM) Study The morphology of the hydrogel was studied using a Nanosurf C3000 Controller Atomic Force Microscopy. The samples were prepared by depositing 0.2 wt% of dilute gel sample onto a clean microscopic glass slide. Then, the glass slide was dried under vacuum at room temperature for overnight. The experiment was carried out under Nanosurf C3000 Controller Atomic Force Microscopy in dynamic mode. Transmission Electron Microscopy (TEM) Study For further confirmation about the morphology of the hydrogel TEM was performed. A small diluted gel sample was prepared from 1 wt% hydrogel. This diluted sample was deposited on the carbon coated TEM grid (Quantifoil, Jena, Germany). The excess sample solution was removed by blotting paper followed by addition of 2% uranyl acetate. After that the grids was washed with Milli-Q water. The electron microscopy was performed at an accelerating voltage of 120 kV. Rheology Study For Dynamic frequency sweep experiment, AR 2000, 2 wt% PA-NK hydrogel was prepared and the experiment carried out in TA Instrument, USA rheometer with a cone-plate (diameter 40 mm and angle 4º). Determination of the Biocompatibility of the Compounds by the MTT Assay Biocompatibility of the hydrogel was checked by MTT assay in Neuro-2a cells. Around 5000 cells were seeded per well in a 96-well plate (total medium volume of 100 µL). After 24 hours, post-seeding, the medium was replaced with solutions of different concentration of hydrogel


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compounds to each well (five wells for each concentration). The MTT assays were performed after 48 hr. Cells without treatment was used as the control. In vitro Microtubule Stabilization Two solutions were prepared to carry out this experiment. In first one, 2.5 µL tubulin, 0.5 µL Alexa 568 tubulin and 20 µL BRB80 buffer were mixed (Mixture 1). For the second one, 10 µL of mixture 1, 5 µL GMP-CPP and 1 µL MgCl2 and 34 µL BRB80 were mixed together to prepare mixture 2. Then, mixture 2 was incubated at 37ºC for 2.5 hours. After that the solution was centrifuged (Eppendorf centrifuge) at 12000 rpm for 8 minutes and a pallet was obtained, which was dissolved in 20 µL BRB80 buffer and kept at 37ºC incubation for the formation of microtubule. Next, 0.5 wt% hydrogel solution was mixed with the microtubule in BRB80 buffer and incubated at 37ºC. A microtubule solution mixed with palmitic acid (PA) and another one containing only microtubule solution alone were incubated at 37 ºC (Both were considered as control). After that, time dependent confocal fluorescence microscopic studies were performed to observe the in vitro microtubule stabilization. In vitro Proteolytic Degradation by Proteinase K For in vitro proteolytic degradation study, first PA-NK compound was dissolved in HEPES (100 mM) buffer to obtain 1 mM solution. Then, Proteinase K enzyme was mixed with PA-NK solution in HEPES buffer (3.2 units/ mL). After that small fractions were analyzed time to time by MALDI-TOF mass spectroscopy. Neurite Outgrowth Experiment by PA-NK Hydrogel in Neuro-2a Cells


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For neurite outgrowth experiment, first PA-NK hydrogel was coated at the bottom surface of the round shaped cover glass dish by 500 µL 2 wt % PA-NK hydrogel. After that, Neuro-2a cells were placed on the dish to adhere and kept in incubation for culture at 37ºC for 48 hours for 1st experiment and 7 days for 2nd one. Cells were washed with PBS buffer and treated with 0.2% (v/v) triton X in PBS in order to permeable the cells and again washed with PBS buffer. Then, cells were treated with rabbit monoclonal anti-alpha tubulin IgG (Abcam) primary antibody followed by secondary antibody (Goat pab to Rb IgG (Cy3.5s) to visualize microtubule network, Alexa 488 phalloidin to visualize actin filaments, DAPI to visualize nucleus in confocal fluorescence microscope (Olympus equipped with Andor iXON3 897 EMCCD). Quantitative Analysis of Neurite Outgrowth Length Consequently, cells were analyzed under microscope (Olympus) at DIC mode. Cells with extracellular process extending longer than two cell bodies were considered as neurite-bearing cells. Evaluation of neurite length was carried out using analysis and calculation mode of CellSens software. Live/dead Cell Assay First, N2a cells were seeded upon PA-NK (2 wt%) coated cell culture dish. After the attachment of the cells on the surface of PA-NK hydrogel (for 24 hours), cells were treated with a solution containing calcein-AM (2 µM) and propidium iodide (PI) (4 µM) for 30 minutes. After that, cells were washed with PBS buffer and excess dye was removed. Next, confocal microscopic images of the cells were observed using Olympus microscope equipped with Andor iXON3 897 EMCCD.


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Neuroprotection Study PC12 cells were differentiated for 5 days in the medium with NGF. Cell viability was assessed by MTT reduction. Briefly, the fifth day differentiated cells were seeded into 96 well plates (1×104 cells/mL) and cultured for 24 hrs. The cells were then treated with anti-NGF along with different concentrations of PA-NK hydrogel (5 µM, 10 µM and 20 µM) for 20 h. For MTT assay, 50 μL of 5 mg/mL MTT solution dissolved in PBS was added to each well and incubated for 4 h at 37°C. After 4h MTT solution was removed and formazan was solubilized using 1:1 DMSO-methanol. Color development was measured using microplate ELISA reader (Thermo; Multiskan™ GO Microplate Spectrophotometer) at 550 nm of wavelength. After that microscopic images were taken in Olympus microscope equipped with Andor iXON3 897 EMCCD. 3D Cell Culture of Neuro-2a Cells inside the Hydrogel For 3D cell culture at first Neuro-2a spheroid cells were grown up to a certain volume. After that the spheroids were transferred to two culture dish, one containing 2 wt% (w/v) PA-NK hydrogel solutions with the addition of serum and for another there was no serum. Both the dishes were kept in the 37ºC to form hydrogel. Confocal microscope was performed each day to collect images in DIC mode of the spheroids. In vitro Drug Release The curcumin loaded peptide hydrogel was prepared in a glass vial for the in vitro drug release experiment. Next, 1 mL of phosphate buffer solution (PBS, pH 7.4) was added into the top of the hydrogel. At a predetermined time interval, the total volume of PBS was removed and 1 mL fresh PBS was added. Time dependent drug release from hydrogel was performed in triplicate


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and the experiments were carried out at physiological temperature (37 °C) for 1 week. The amount of curcumin released from the hydrogel was measured using a Fluorescence spectrophotometer (PTI Technology, QM 40) at 420 nm excitation wavelength. The cumulative drug release was calculated as: Cumulative amount released (%) [(F – F0)/Ff]×100, where, F0 = Maximum value of the control. Ff = Maximum value of the final reading. Comparative Cytotoxicity Study Cytotoxicity of free curcumin and curcumin loaded PA-NK hydrogel in Neuro-2a cancer cells was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction. The cells were seeded at a density of 10000 cells per well in a 96-well plate one day prior to the treatment. Then Neuro-2a cells were treated with different concentration (6.125 µM, 12.5 µM, 25 µM, 50 µM) curcumin loaded PA-NK in DMEM medium containing 10% FBS for 24 h and compared with free curcumin. Following the termination of experiment, cells were washed and promptly assayed for viability using MTT. Results were expressed as percent viability = [(A550 (treated cells)-background) / (A550 (untreated cells)-background)] x 100

Growth Inhibition Analysis of Neuro-Sphere/3D Tumor Spheroidal Cultures The Multicellular Tumor Spheroidal culture of Neuro-2a cells were performed using nonadhesive culture system. In brief, neuro-sphere of Neuro-2a cells has been generated using liquid overlay or non-adherent method. In brief, 2D monolayer cells were detached, transferred into the 35 mm dish, previously coated with 2 wt% PA-NK hydrogel and incubated at 37oC till neurosphere/3D spheroid formation. Morphological structures of neuro-sphere/spheroid were captured in bright field using inverted Olympus microscope equipped with EMCCD camera and


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represented as day one. Then the neuro-sphere/spheroids were divided in groups such as without treatment group as control and different treatment groups such as, only curcumin (25 µM), curcumin-hydrogel (25 µM). After that neuro-sphere/spheroid morphology was assessed up to day 9 using inverted Olympus microscope. Volume of the sphere was analyzed using following formula. Volume = 0.5 × Length (major axis) × Width2 (minor axis). Tubulin: Tubulin was extracted from goat brain. The purification of tubulin from goat brain and it’s labelling with Alexa fluorophore were performed as described in the literature.50 RESULTS AND DISCUSSION The lipopeptide or palmitic acid (PA) attached peptide (PA-NK) (Fig. 1a) was synthesized by Fmoc solid phase peptide synthesis procedure, purified by reverse phase HPLC (Fig. S1) and lyophilized to obtain pure PA-NK (Fig. S2). Next, we prepared the 1 wt% PA-NK hydrogel in phosphate-buffered saline (PBS) (Fig. 1b and S3, detailed in experimental section), where intermolecular hydrogen bonding facilitates to form β-sheet like structure, which further selfassembles to form dense fibers like network.51-54 To understand the detailed mechanistic insights of the self-assembly process of the PA-NK hydrogel, first we performed FT-IR experiment. From the FT-IR experiment, we found that amide-I peak of the PA-NK hydrogel at 1627 cm-1 (Figure S4), which corresponds to β-sheet structure.55-57 These changes in secondary structure clearly indicate that the PA-NK molecules form hydrogel through the formation of β-sheet structure. Next, we carried out the Transmission electron microscopy (TEM) and Atomic force microscopy (AFM) to analyse the interior of the PA-NK hydrogel. TEM and AFM studies revealed that formation of three dimensional cross-linked dense fibril networks because of strong inter-fiber interactions (Figure 1c, 1d, S5). The width of the fibers is around 20 nm and above 1


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µm long. These three dimensional networks are capable of capturing water or drug molecules. In addition, we also observed that hydrogel was homogeneous in nature. Next, to check the mechanical strength of the hydrogel, rheology experiment was carried out with 1% (w/v) PA-NK hydrogel. From a frequency sweep experiment (at a constant strain of 0.1%), it was observed that PA-NK hydrogel have greater storage modulus (G') than loss modulus (G") and a significant difference between them, suggesting that PA-NK hydrogel acting like proper hydrogel (Fig. 1e). G' and G" values were weakly dependent on angular frequency over the range (1-100 rad/s) studied. Our key objective was to develop highly biocompatible hydrogel which will be extremely useful for neuronal cell transplantation. With that aim, we have checked the biocompatibility and cytotoxicity of our hydrogel. For this purpose we have chosen Neuro-2a cells.58 MTT assay was performed using up to 1 mM (0.14 wt%) of PA-NK and it revealed that the PA-NK hydrogel is non-cytotoxic and biocompatible in nature as we observed that cell viability of Neuro-2a cells after treatment with 1 mM of PA-NK (0.14 wt%) hydrogel was almost 100% (Figure 1f). This study clearly revealed that PA-NK hydrogel will be suitable for cell culture and tissue engineering purpose. Next, we have studied whether the PA-NK hydrogel was able to keep the neuron cell morphology healthy or not. For that purpose, we have cultured the Neuro-2a cells in PA-NK hydrogel and checked its morphology under microscope and found the cell retained its healthy morphology (Figure 1g) as comparison to control i.e. without PA-NK hydrogel (Figure S6). Microtubule is the key cytoskeleton of cell, which is crucial for the cell proliferation, cell division and cell migration. Therefore, fabrication of hydrogel is important, which can keep


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microtubule in healthy condition. For that purpose, Alexa568 labelled Guanosine-5’-[(α,β)methyleno]triphosphate (GMP-CPP) microtubule seeds were incubated with hydrogel at 37ºC for 8 hours. Time dependent fluorescence microscopy study has been performed to characterize the stability of the GMP-CPP microtubule seeds. Fluorescence microscopic image reveals that Alexa568 labelled GMP-CPP microtubule seeds were stable inside the hydrogel and they are forming bundle as we observed intense fluorescent signals from seeds (Figure 2b). Further, two control experiments have been performed, one in absence of hydrogel and another in presence of only palmitic acid solution, which showed thin seeds with occasional depolymerized microtubules (Figure 2a, S7). Next, we explored whether the hydrogel has a role in intracellular microtubule stabilization or not. For that purpose, we have cultured Neuro-2a cells onto the PA-NK hydrogel coated coverslip for 6 hours followed by treatment with primary antibody Anti-alpha Tubulin (1: 300), secondary antibody (Goat pab to Rb IgG (Cy3.5s); 1: 500) and 1 mg mL-1 of Hoechst. Fluorescence microscopic image revealed that microtubules were healthier when cells were cultured onto the hydrogel compared to absence of hydrogel (Fig. 2c, 2d). This result confirmed that our newly designed PA-NK hydrogel provides healthy and biocompatible support for neuron cell culture and maintain healthy cell morphology. Next, we tried to understand the mechanism of the microtubule stabilization. It has been described before that octapeptide NAPVSIPQ (NQ) peptide stabilizes intracellular microtubules and promotes neurite outgrowth of neuron cells.49 Further it is also known that damaged neuron cells and neuronal cancer cells secrete different proteinases.59, 60 Therefore, we have checked whether PA-NK hydrogel degrades in presence of proteinase and releases neuroprotective octapeptide NAPVSIPQ or not. Here, we used proteinase K, which have broad specificity towards proteins and peptides and preferentially cleave at


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aromatic and aliphatic amino acids residues.61 PA-NK (1 mM) was dissolved in HEPES buffer and incubated with Proteinase K (3 units/mL) at 37ºC and small fractions were analysed by MALDI-TOF Mass with different time interval in order to know the proteolytic cleavage site. Mass spectrum reveals the presence of the neuroprotective nonapeptide NAPVSIPQK (NQK) (Figure S8-S11) and the intensity of this fragment in MALDI-TOF Mass spectroscopy gradually increases with incubation time (from 24 to 48 hours). Mass of the PA-NK molecule was 100% initially but after 48 hour the mass became 10% (Figure 3). So, from this in vitro proteolytic degradation study by Proteinase K, it is confirmed that the PA-NK hydrogel slowly degrades (after 12 hours) and producing NAPVSIPQK (NQK) peptide sequence, which may promote neurite outgrowth as well as neuroprotection. This result is extremely encouraging as biodegradability of neuronal cell transplantation scaffold is essential for successful transplantation process. Moreover, our main focus of this hydrogel development was to transplant neuron cells into the damaged area of brain, which in general contains considerable amount of secreted proteinases. Since the PA-NK hydrogel shows healthy environment for the culture of neuron cells and releases neuronal growth factor like peptide through slow proteolitically degradation mechanism, we are curious to know whether this hydrogel can facilitate the neurite outgrowth or not. As we know neuronal growth factor promotes neurite outgrowth, which is the key process for neuron cell differentiation and migration.62 We have cultured Neuro-2a cells for 48 hours upon PA-NK coated coverslip. Cells were stained with primary antibody Anti-alpha Tubulin (1: 300), secondary antibody (Goat pab to Rb IgG (Cy3.5s) for microtubule filaments and Alexa 488 phalloidin for actin filaments. Fluorescence microscopic images reveal small, tangled ray like neurite outgrowth (Figure 4) in case of both microtubules stained and actin stained images.


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These images clearly revealed that our hydrogel promotes neurite outgrowth in absence of any external growth factor. These results confirmed that slow enzymatic degradation of PA-NK releases microtubule stabilizing neuroprotective peptide, which promoted neurite outgrowth.49 To the best of our knowledge this is the first report of a hydrogel, which biodegrades and releases neuronal growth factor peptide, unlike earlier report, which described that hydrogel entrapped neuronal growth factors help neurite outgrowth.63 Further to confirm the fact that PANK hydrogel holds proper condition for the cell survival and promotes neurite outgrowth, we have cultured the Neuro2a cells inside the PA-NK hydrogel for 7 days and stained the actin filaments of the cells with Alexa 488 phalloidin and nucleus was stained with DAPI. From microscopic image, strong fibre like interconnected neurite outgrowth was found of Neuro-2a cells (both in DIC and merged images; Figure 5a-d). We observed that the neurite extension of Neuro-2a cell significantly promoted after 7 days culture in PA-NK hydrogel (2 wt.%) and the length was increased around 50% compared to control (N2a cells without PA-NK gel after 7 days culture) (Figure 5e and S12). This experiment revealed that cells were healthy as we observed that dense Neuro-2a cells homogeneously distributed and formed network like structure because of release of neuroprotective nonapeptide NAPVSIPQK (NQK) from PA-NK hydrogel by proteolytic degradation. To further confirm that the PA-NK hydrogel maintains healthy environment towards neuron cells, we have performed live/dead cell assay. N2a cells were seeded on a PA-NK hydrogel (2 wt%) coated tissue culture plate and kept in incubation for 24 hours. In another standard tissue culture plate N2a cells are seeded for 24 hours and considered as control. After live/dead cell assay, microscopic images were captured using confocal fluorescence microscope (Figure S13). Confocal fluorescence microscopic images revealed excellent attachment and growth of N2a cells on the PA-NK hydrogel (the green cells are viable


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and red cells are dead). Therefore, from above experiments, it is confirmed that PA-NK hydrogel is non-cytotoxic, facilitates neurite outgrowth of neuron cells by proteolytic degradation and able to provide healthy environment for the neuron cells. Inspired from the above facts, we are eager to know whether our PA-NK hydrogel is able to show neuroprotection against anti-nerve growth factor (anti-NGF) induced cytotoxicity since NGF deprivation consequences overproduction of Aβ and death of neuron cells.64-66 For that purpose we have chosen PC12 cells since it expresses nerve growth factor (NGF) receptors.47 PC12 cells were differentiated for 5 days in the medium with NGF. At this point when the cells had already acquired neural morphology, they were treated with anti-NGF for 20 hours along with different concentration of PA-NK hydrogel (Figure 6a). Microscopic images revealed that PA-NK hydrogel inhibited the induced toxic effect by anti-NGF whereas anti-NGF alone caused severe cell death of PC12 cells (Figure 6a). MTT assay also indicated that the PA-NK treated at different concentrations (5 µM, 10 µM, 20 µM) was able to ameliorate the neural morphology and health of the PC12 cells against anti-NGF induced toxicity (Figure 6b) and PA-NK hydrogel at 20µM concentration exhibited around 94% neuroprotection. This result suggested that the PANK hydrogel delivered significant neuroprotection to the neuron cells. The 2D monolayer cells fail to mimic the complex environment of brain tissue, because it lacks cell to cell, cell to extracellular matrix interactions.67 Therefore, it is essential to develop a 3D system using PA-NK hydrogel, which can help to understand the proper interaction of cell to ECM interactions. Here, first we have grown the Neuro-sphere (spheroid) of Neuro2a cells and transferred into the PA-NK hydrogel followed by monitoring of the spheroids up to 20 days in two parallel batches. In the first batch, the spheroids cells were cultured with serum and in the second batch spheroids cells were cultured without serum. Microscopic images were captured in


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bright field mode each day till the end of the experiment. From Figure 7a, it is clear that the spheroid cells were viable both in presence and absence of serum. Further the nuclei of the cells was stained with DAPI to confirm whether the cells were actually viable or not, and it is confirmed that all the cells were viable and healthy. The most interesting thing is that the cells were viable even without the serum up to 20 days, which confirms that the cells have acquired both necessary fluids and extra cellular matrix like proper environment for survival from PA-NK hydrogel. After observing interesting results of PA-NK hydrogel, we became interested whether PA-NK hydrogel can entrap hydrophobic drug molecules or not as we know that delivery of hydrophobic molecules into the neuron cells is a challenging task. Here, we choose curcumin as a hydrophobic drug because it targets microtubule68 act as anti-Alzheimer, anti-cancer and neuroprotecting agent.69 Interestingly, we found that PA-NK hydrogel can efficiently entrap curcumin molecules within 2 wt% (w/v) hydrogel. Next, we checked whether, the PA-NK hydrogel can release the curcumin or not. We added equal volume of PBS buffer on the curcumin loaded hydrogel. Interestingly, it was found that curcumin is slowly diffused from hydrogel to PBS solution (Figure 7b). The release of curcumin was further monitored using fluorescence spectrophotometer in a time dependent manner and monitored up to 72 hr. Graph represent percent (%) of drug release with time (Figure 7c and S14), which reveals that curcumin is released from the hydrogel up to 72 hour. Further, we observed that morphology of the hydrogel was unchanged (Figure 7b). Therefore, release of incorporated curcumin from the hydrogel was due to diffusion and dependent on the interaction between the drug and the hydrogel network. This experiment confirmed that PA-NK hydrogel can be a useful prototype for the purpose of slow drug release. Next, we tried to understand whether the drug loaded


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hydrogel can release and deliver the hydrophobic drug or not. For that purpose, we performed MTT assay and compared the cell viability of control curcumin and curcumin loaded PA-NK hydrogel. Neuro-2a cells are treated with different concentrations (50 µM to 1µM) of curcumin loaded hydrogel and curcumin solution alone (as control, Fig. 8a). We have found that curcumin loaded hydrogel shows better killing efficiency against Neuro2a cells (~15%) (Figure 8a) compared to control. Finally, we have enquired whether our hydrogel is capable of delivering hydrophobic anticancer drugs curcumin into the 3D spheroid cells or not. Since we know that 2D monolayer cells lacks appropriate tumour environment,70 for that reason, we have analysed the effect of the curcumin loaded hydrogel on tumor mimicking 3D spheroid (neuro-sphere) system. Neuro-2a spheroids were developed and treated with curcumin (200 µM) loaded hydrogel. Microscopic images from day 1 to 9 (Figure 8b, 8c) revealed that there was significant shrinkage of spheroids volume for the curcumin loaded PA-NK hydrogel compared to control or free curcumin. This result reveals that our PA-NK hydrogel can deliver the hydrophobic drugs and shows significant anticancer effect not only in 2D monolayer cells but also in 3D spheroid cells. This result is also extremely encouraging that our novel peptide based hydrogel can be used as anticancer drug delivery at the site of brain tumour. CONCLUSIONS In summary, we report the design of a novel biodegradable neuro-compatible peptide based hydrogel. This hydrogel shows strong three dimensional cross-linked network structure, which is capable of entrapping large amount of water molecules. Interestingly, this hydrogel serves as highly biocompatible platform for 2D and 3D neuron cell culture. It slowly enzymatically


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degrades and releases neuroprotective peptide NAPVSIPQK, which promotes neurite outgrowth as well as displays excellent neuroprotection against Anti-NGF induced toxicity of neuron cells. This hydrogel can promotes neurite outgrowth and provide neuroprotection to PC12 neuron cells in absence of externally added neuronal growth factor (NGF). Moreover, it can encapsulate hydrophobic drugs, slowly release the hydrophobic drug into the Neuro-2a cells and significantly inhibit the growth of 3D spheroid of neuron cells. Finally, we envision that our novel biodegradable hydrogel will serve as an excellent scaffold for neuron cell transplantation in damaged area of brain as well as delivery vehicle for neuroprotective agents, anti-Alzheimer and anti-cancer molecules. ASSOCIATED CONTENT Supporting Information This

Supporting

Information

are

available

free

of

charge

via

the

internet

at

http://pubs.acs.org.Characterisation of PA-NK by High Performance Liquid Chromatography (HPLC) and Matrix-assisted Laser Desorption/Ionization (MALDI) mass spectrometry, Schematic diagram descripting the mechanism of PA-NK hydrogel formation, FT-IR spectra of PA-NK hydrogel, AFM study, Control images of N2a cells, In vitro Alexa 568 labelled GMPCPP microtubule seeds formation study in presence of palmitic acid solution (PA), Detailed study of proteinase K experiment, Control study of neurite outgrowth study, Live/dead cell assay, Curcumin release study and its release kinetics. AUTHOR INFORMATION Corresponding Author


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E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Authors wish to thank Prof. A. K. Nandi and his student Mr. Sujoy Das for rheology study, Dr. Jayati Sengupta and Mr. Chiranjit Biswas for Cryo-EM, NCCS-Pune for cell lines. AA, SB and SM thank UGC, GD thanks ICMR, DB thank DST and BJ thanks CSIR for their fellowships. SG kindly acknowledges DST (EMR/2015/002230) and CSIR-Network project (BSC-0115) for financial assistance. ABBREVIATIONS NAPVSIPQ, NQ. NAPVSIPQK, NQK. Palmitic acid, PA. Palmitic acid attached NAPVSIPQKKK peptide, PA-NK. Curcumin, CUR. Neuro-2a, N2a. REFERENCES (1) Nowakowski, R. S. Stable Neuron Numbers from Cradle to Grave. Proc. Natl. Acad. Sci. 2006, 103, 12219-12220. (2) Wikström, L. Activation of Endogenous Stem Cells in the Brain-A Novel Approach to Central Nervous System Disease. Drug Discovery. 2009, 6, 33-35.


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(64) Matrone, C.; Di Luzio, A.; Meli, G.; D’Aguanno, S.; Severini, C.; Ciotti, M. T.; Cattaneo, A.; Calissano, P. Activation of the Amyloidogenic Route by NGF Deprivation Induces Apoptotic Death in PC12 cells. J. Alzheimer’s Dis. 2008, 13, 81-96. (65) Xu, Z.; Maroney, A. C.; Dobrzanski, P.; Kukekov, N. V.; Greene, L. A. The MLK Family Mediates c-Jun N-terminal Kinase Activation in Neuronal Apoptosis. Mol. Cell. Biol. 2001, 21, 4713−4724. (66) Rajasekhar, K.; Madhu C.; Govindaraju, T. Natural Tripeptide-Based Inhibitor of Multifaceted Amyloid β Toxicity. ACS Chem. Neurosci. 2016, 7, 1300-1310. (67) Antoni, D.; Burckel, H.; Josset, E.; Noel, G. Three-Dimensional Cell Culture: A Breakthrough In Vivo. Int. J. Mol. Sci. 2015, 16, 5517-5527. (68) Gupta, K. K.; Bharne, S. S.; Rathinasamy, K.; Naik, N. R., Panda, D. Dietary Antioxidant Curcumin Inhibits Microtubule Assembly through Tubulin Binding. FEBS J. 2006, 273, 53205332. (69) Ono, K.; Hasegawa, K.; Naiki, H.; Yamada, M.; Curcumin has Potent Anti-amyloidogenic Effects for Alzheimer's Beta-amyloid Fibrils In Vitro. J. Neurosci. Res. 2004, 75, 742-750. (70) Hirschhaeuser, F.; Menne, H.; Dittfeld, C.; West, J.; Mueller-Klieser, W.; KunzSchughart, L. A. Multicellular Tumor Spheroids: An Underestimated Tool is Catching up again. J. Biotechnol. 2010, 148, 3-15.


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Figure 1. (a) Chemical structure of palmitic acid (PA) conjugated with NK peptide (PA-NK), containing microtubule (MT) stabilizing and neuroprotective residues. (b) Snapshot of inverted glass vial shows PA-NK hydrogel with 1 wt% compound in PBS buffer. (c) TEM image of PANK hydrogel reveals interconnected fibril network structure. (d) AFM image of PA-NK hydrogel reveals densely packed fibril structure. (e) Dynamic frequency sweep study of PA-NK hydrogel (2 wt%) at 1% strain, G' and G" refer to storage and loss modulus respectively. (f) Cell viability study reveals that the PA-NK hydrogel is non-cytotoxic in nature. (g) Microscopic image in DIC mode of Neuro-2a cell in PA-NK hydrogel reveals the cells are healthy in nature. Scale bar corresponds to 20 µm.


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Figure 2. (a) Fluorescence microscopic image of Alexa 568 labelled GMP-CPP microtubule seeds on glass side reveals thin densely packed microtubules. (b) Fluorescence microscopic image reveals Alexa 568 labelled GMP-CPP microtubule seeds inside the PA-NK hydrogel are healthy and stable. (c) Fluorescence microscopic image reveals the microtubule network of Neuro-2a cell in cell culture dish in absence of hydrogel after 6 hour incubation. (d) Fluorescence microscopic image reveals the microtubule network of Neuro-2a cell in presence of PA-NK hydrogel after 6 hour incubation. Scale bars correspond to the 20 µm for a, b and 20 µm for c,d, respectively.


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Figure 3. The generation of neuroprotective peptide NQK through the time dependent proteolytic degradation of PA-NK hydrogel.


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Figure 4. Neurite outgrowth of Neuro-2a cells after 48 hours culture: (a) Microtubule-stained fluorescence microscopic image reveals hyper-branched morphology of Neuro-2a cells. Inset shows the hyper-branched morphology of Neuro-2a cells at other position. (b) Actin-stained fluorescence microscopic image reveals hyper-branched morphology of Neuro-2a cells. Inset shows the hyper-branched morphology of Neuro-2a cells at other position. (c) Microtubulestained fluorescence microscopic image reveals hyper-branched morphology of Neuro-2a cells. (d) Actin-stained fluorescence microscopic image reveals hyper-branched morphology of Neuro2a cells. Scale bars correspond to 20 µm.


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Figure 5. Neuro-2a (N2a) cell culture inside the hydrogel for 7 days. (a) DIC, (b) DAPI stained Nucleus, (c) Actin stained by Alexa 488 phalloidin, (d) merged image. Images indicate neurite outgrowth as we can observe long filaments like growth from neuron cell after culture in PA-NK hydrogel. Average length of neurite in PA-NK hydrogel compare to control N2a cells calculated by Cellsens software (e). Scale bars correspond to the 100 µm.


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Figure 6. Neuroprotection Assay: Microscopic images and MTT assay represent higher survival of PC12 cells treated with anti-NGF in the presence of PA-NK hydrogel (a) and (b) respectively. Results indicate excellent neuroprotection against anti-NGF toxicity in neuron cells. Scale bar corresponds to 50 µm.


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Figure 7. (a) Neuro-2a spheroid (Neuro-sphere) culture in PA-NK hydrogel up to 20 days with serum and without serum, nuclei stained with DAPI (b) Snapshots of glass vial shows curcumin releases from curcumin loaded PA-NK hydrogel up to 72 hours. (c) Curcumin release kinetics.


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Figure 8. (a) Comparative cell viability assay between control curcumin and curcumin loaded PA-NK hydrogel in Neuro-2a cells, (b) Inhibition of Neuro-2a spheroid growth upto 9 days : line graph representing inhibition of Neuro-2a spheroid growth upto 9 days. (c) DIC images of spheroid growth inhibition with curcumin loaded hydrogel compared to free curcumin.


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Graphical Content:


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Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite

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