FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
PATENTS RULES, 2006
COMPLETE SPECIFICATION
(SECTION 10; RULE 13) TITLE:
SELF-ASSEMBLED AMYLOID HYDROGEL FOR NEURAL TISSUE ENGINEERING
IITB-MONASH RESEARCH ACADEMY
IIT BOMBAY, POWAI, MUMBAI 400 076
INVENTORS:
1. SHUBHADEEP DAS
2. DR. SAMIR K. MAJI
3. DR. JOHN FORSYTHE
4. DR. DAVID FINKELSTEIN

FIELD OF THE INVENTION
The invention relates to hydrogels, especially peptide based amyloid hydrogels for mesenchymal stem cell culture and differentiation, preferably towards neurons. The invention also relates to a method for designing and developing such peptide based amyloid hydrogels. The invention further relates to 3D culture of stem cells in this hydrogels and transplantation of cells with this hydrogel for repair and restoration of neurons in the brain.
BACKGROUND OF THE INVENTION
Molecular self-assembly of protein and peptides is a popular and attractive strategy to construct variety of nanostructures and supramolecular hydrogels for various nanotechnology and biotechnologieal applications ,
Although, controlling the protein secondary structure for the modulation of protein self-assembly and their corresponding supramolecular structure is shown to be feasible, modulating mesocopic property is much easier to control using small peptides,
Previously, various small peptides based hydrogels were developed either for drug delivery or tissue engineering applications. Until now, no peptide based amyloid hydrogels have been used for mesenchymal stem cell culture and differentiation, e.g. towards neurons. Peptide hydrogels are a class of physical hydrogels where the component peptides self-assemble non-covalently to form an ordered nanostructure. Subsequently, a plurality of these nanostructures gather to form a supramolecular network.
Recently, with the advent of protein engineering, relatively simple peptide based hydrogels have gained increasing attention in biomaterial development. The major advantage of employing proteins/peptides in hydrogels is that their properties could be precisely controlled to achieve the desired functionality. Peptide hydrogels also score over others in biocompatibility, ease of synthesis and non-toxic degraded products, not necessarily found in polymeric gels. The network of nanofibers formed by the peptides, mimics the natural extracellular matrix (ECM) better than microfiber based polymer scaffolds.
[1] T. C Holmes, Trends Bioteohnol 2002, 20, 16.
To date, many peptide-based hydrogels have found applications in effective and fast recovery
post glaucoma surgery in eyes[2], regeneration of myocardium with neonatal cardiomyocyctes,

as hemostatic agents in post-surgery iatrogenic injury and imparting rapid vascularization to damaged tissues.
Gazit and coworkers developed a series of peptide based hydrogel based on Phe-Phe dipeptides corresponding to Aβ[3]. Fmoc-Phe-Phe peptides assume tubular amyloid like structures similar to the molecular dimensions of amyloid fibrils obtained from full length proteins. Cells grown on these hydrogeis showed viability similar to that cultured on poly-L-lysine coated surfaces. Further this hydrogel and its derivatives were used in different purposes like drug-delivery, bio-sensing etc.
With advancement of knowledge and insights on self-assembly of proteins, attention has been devoted to design and study minimalistic fragments that can form amyloids. Fragments from A-beta, 1APP, human calcitonin etc. are reported to form amyloid fibrils in-vitro like the full-length protein.
Zhang et at discovered earlier that amphipathic peptides self-assemble into nanoflbrils which entrap solvents to give hydrogeis. The nano-fibrils consist of beta sheet structures and had a diameter of 10-20 nm. These peptides were named as EAK 16-11 peptides.[4] Replacement of Glu with Arg and Lys with Asp residues gave rise to the RAD16 peptides that readily self-assembled into hydrogeis under physiological conditions.[5] These fibrils indeed exhibit red/green birefringence on binding with Congo Red.
Schneider et al [6]designed peptide hydrogeis where the peptides folds to form j3 hairpin to template intermolecular self-assembly. They developed H2N-VKVKVKVK-VDPPT-KVKVKVKV-NH2 (MAXI) peptide, which self-assembles into a bilayer β-sheet fibril at pH 9 or increased ionic strength. They also substituted different amino acids to study sequence dependent self-assembly and gelation kinetics. It was found that in general, introduction of polar residues in the hydrophobic face was destructive to varying degrees on the self-assembly process and hence hydrogel formation.
[2]X.-Da. Xu, L. Liang, C.-S. Chen, B. Lu, N.-l. Wang, F.-G. Jiang, X.-Z. Zhang, R.-X. Zhuo,
ACS applied materials & interfaces 2010,2,2663,
[3] E. G. Metial Reches, Current Nanoscience 2006,2, 105. 141 S. Zhang, T. C. Holmes, C. M. DiPersio, R. O. Hynes, X. Su, A, Rich, Biomaterials 1995,16, 1385. [5]H. Yokoi, T. Kinoshita, S. G. Zhang, Proa Natl, Acad. Set. U. S. A, 2005, 102, 8414. [6]J. P, Schneider, D. J. Pochan, B. Ozbas, ... American Chemical.., 2002, Amyloids are self-assembled protein/peptides aggregates associated with diseases such as
Alzheimer's, and Parkinson's. In these diseases, proteins/peptides misfold into aggregated

amyloid structure, which kills the neuronal cells. More than two dozen human diseases exist, which are directly or indirectly associated with amyloids aggregates. Recent studies however showed that soluble oligomers are much cytotoxic compared to matured fibrils, However, many reports suggest that amyloids also do native biological functions for the host, which support the host organism lor tl leir survival rather making diseases. Presence of non-pathogenic amyloids has been reported both in eukaryotes and prokaryotes, In humans, milanin biosynthesis is templated by amyloid fibril Pmel, Moreover, pituitary gland stores hormones in secretory granules as amyloids.
Apart from their functions (either diseases causation and or native functions), amyloid possess superior materials property due to their highly orderered and stable structure, their stiffness comparable to steel, and the possibility of their modulation of physiochemical property. Due to the superior quality of their material property, a series of hydrogels based of self-recognition motif of amyloidogenic protein α-syn has been designed. Although development of amyloid based biomaterials may look counterintuitive due to presence of toxic amyloids and their involvement in a dozen of human diseases, evolutionary conserved sequences in functional amyloids as well as proof that synthetic amyloids are not always toxic Supports the usage of amyloid as a material. Recently it has also been shown that amyloid nanofibrils do mimic the features of extracellular matrix proteins and can support cell attachment without any cell adhesion motifs like ROD.
Usage of peptide based scaffold in various tissue engineering application.
In neural tissue engineering scaffolds containing the laminin epitope 1KYAV were employed. The nanofibrous system promoted generation of axons in a mouse model with spinal cord injury. RADA 16-1 hydrogels have been shown to fill lesions formed by sciatic axotomy and facilitate ceil migration. Regeneration was also observed when RADA16-1 peptide hydrogel was implanted in damaged cortex. Peptide based scaffolds showed considerable promise in treatment of Duchenne's muscular dystrophy (DMD). Here, a 3D scaffold based on Fmoc π-β system has been enzymatically triggered to form hydrogel and deployed in vivo to present the missing protein laminin at proper site. The therapeutic benefit of this scaffold was tested in a zebra fish model. In bone tissue engineering, hybrid scaffolds were used for cell transplantation. The standard practice of using hydroxyapatite coating to promote osteoinduction in orthopaedic implants faces the problem of bio functionality and vascularization. Self-assembled peptide scaffolds were used to bio- functionalize inert titanium foam implants] to spatially confine hydroxyapatite crystals. Peptide scaffolds were also utilized in repair of cartilage.

Neural tissue engineering strategies
Neurons in brain undergo limited self-repair after damage or trauma is inflicted necessitating tissue engineering approaches to restore functional tissue. Common treatments for neurodegenerative diseases, like Parkinson's and Alzheimer's, suppress the symptoms and cannot cure the disease to the roots. Cell transplantation holds good promise as a therapy, but the hostile environment of the damaged tissue makes it difficult to promote regeneration. Earlier direct transplantation of cells was done to replace the damaged neurons with functional ones and restore normal dopamine levels in substantia niagra pars striatum. Direct transplantation of fetal midbrain tissue in has shown great promise in pre-clinical studies, but failed to fulfill expectations in clinical trials,
The major problem was unregulated release of dopamine from dendritic spines. This was mainly due to inappropriate connections with target cells in the striatum. These limitations were mainly attributed to poor cell viability in the graft with DA neurons representing approximately only 0.3% of the total implanted cells.
Also, the heterogeneity of cell population in the graft needs to be addressed. Hydrogel based tissue engineering scaffolds are capable of improving the situation by providing permissive environment for cell survival in the implant.
Low graft survival and migration of the transplanted cells are the two major bottlenecks in stem cell based therapies. Transplantation of neural precursor cells has demonstrated the power to repair host tissue, but unfortunately most of the cells die when directly injected in the brain ' ' Recently it was also shown that injectable hydrogel scaffolds provides additional physical strength enhancing survival of neural progenitor cells within stroke cavity.
Earlier studies assessing fate of stem ceils to repair tissue in-vivo met with limited success primarily due to low engraftment and transdifferentiation when transplanted in diseased or injured tissue. The major reason being the hostile microenvironment of the diseased tissue that subdues regeneration,

US 2009 0175785 discloses novel peptide-based hydrogels, composed of short aromatic peptides (e.g., homodipeptides of aromatic amino acid residues) are disclosed. The hydrogels
are characterized by remarkable rigidity and biocornpatibilily. Further disclosed are uses of
these hydrogels in applications such as tissue engineering, drug delivery, cosmetics, implantation, packaging and the like. Further disclosed are processes and kits for preparing
these hydrogels.
US 2011 0137328 discloses materials and methods for effective nerve repair with a hydrogel and optional adhesive. In a preferred embodiment, the invention provides nerve repair methods comprising the use of a fibrin glue and a polyethylene glycol (PEG) hydrogel to coapt severed nerve stumps or nerve grafts,
US 2014 0357837 discloses provides novel peptides that can be used to form hydrogels. The peptides are short (preferably 30 amino acid residues or less) and include hydrophilic and hydrophobic segments joined by a turning segment. The hydrogels are formed by altering the pH of a solution of these peptides to an acidic level, or by introducing a source of ions into a solution of these peptides. The resulting hydrogels are shear thinning gels that have high storage moduli and high rates of recovery after destruction. They find use in medical applications, including tissue engineering.
US 2014 0357823 discloses polymers, especially polymers useful as hydrogels, and to use of hydrogels for repair or restoration of tissue. In particular, the polymers and hydrogels of the present invention can be used for the repair or restoration of cartilage, especially articular cartilage. The polymers comprise at least a monomer for binding water, a monomer for imparting mechanical properties and a monomer for binding to an extracellular protein. The hydrogels comprise a polymer comprising at least a monomer for binding water and a monomer for binding to an extracellular protein. Crosslinking polymers by binding of said extra-cellular matrix protein forms hydrogels.
US 2015 0025005 discloses a glucose binding amphophilic peptide hydrogel insulin delivery system that is responsive to glucose concentrations under physiological conditions is provided, Insulin is encapsulated in a glucose binding hydrogel, made from self-assembling amphophilic peptides including a hydrophobic domain including a beta sheet forming region coupled to a charged hydrophilic domain modified to contain a glucose binding segment. The formulations are designed to release insulin as a function of blood glucose level, maintaining the patients' blood glucose level in an optimum range and avoiding both hyper- and hypoglycemia.

WO 2014 104981 discloses an amphiphilic linear peptide and/or peptoid as well as a hydrogel that includes the amphiphilic linear peptide/peptoid. The amphiphilic linear peptide/peptoid is capable of self-assembling into three-dimensional macromolecular nanofibrous networks, which entrap water, and forming a hydrogel. These peptides/peptoids include short amphiphilic sequences with a hydrophobic portion of aliphatic amino acids and at least one acidic, neutral, or basic polar amino acid. The amphiphilic linear peptide/peptoid is build-up of non-repetitive aliphatic amino acids, which may be in the L- or D-form. A plurality of such peptides/peptoids assembles to supramolecular helical fibers and forms peptide hydrogels after assembly. A corresponding hydrogel is formed in aqueous solutions at physiological pH and is thus useful for inter alia cell culture, tissue engineering, tissue regeneration, wound healing and release of bioactive moieties (including cells, nucleic acids, antimicrobials, micro-/nanoparticles, cosmetic agents and small molecule therapeutics), as well as for providing mechanical support for damaged or missing tissues. Such hydrogels can also be formed in situ, wherein the gelation process occurs within the body following the injection of a peptide solution. Such hydrogels, which are rigid, biocompatible and entrap up to 99.9% of water, are also well suited for applications utilizing electronic devices.
US 2014 0302144 discloses a pharmaceutical formulation for sustained delivery of a therapeutic agent, preferably a protein, polypeptide, an antibody or an antibody fragment, comprising one or more gel forming peptides wherein the formulation exhibits sustained delivery for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks or more. In one embodiment, the invention relates to a formulation comprising self-assembling peptides that undergo sol-gel transition in the presence of an electrolyte solution such as biological fluids and salts. The formulation can provide sustained release of antibody and antibody fragments, in particular, IgG. Antibody diffusivities can be decreased with increasing hydrogel nanofiber density, providing a means to control the release kinetics,
CHALLENGES AND GAPS IN UNDERSTANDING
1. Developing amyloid based hydrogels for promoting stem cell differentiation towards neurons.
2. Developing suitable cell transplantation vehicle for brain.
3. A scaffold that will contain the cells at the target site and provide a conducive microenvironment that will help in achieving its differentiation.

SUMMARY OF THE INVENTION
In accordance with the present invention, there are provided self-assembled, injectable amyloid, peptide-based hydrogeis produced from alpha synuclein (α-Syn) useful for neural tissue engineering in transplantation of stem cells in brain with minimally invasive surgery, said hydrogeis comprising cross-β sheet rich amyloid assembled into nano-fibrous meshwork to mimic natural extracellular matrix (ECM) having various application such as, but not limited to, excellent cell attachment and differentiation capabilities for neuronal lineage from human mesenchymal stem cells (hMSCs).
In one embodiment, the said hydrogeis comprise short peptide gelators developed from beta sheet rich region of 140-amino acid a-Syn and by altering the amino acids for dissolving them into an aqueous phosphate buffer at physiological pH,
In another embodiment, the said hydrogeis include short peptides with 5 amino acids developed from a shorter sequence within the 69-78 region and N-terminus of the said peptides protected with Fmoc group for enhancing intermolecular stacking interaction to favor the self-assembly
process.
In still another embodiment, the said short-peptides self-assemble into nano-fibrils with diameter of ~40 nm and height ~10 nm.
In yet another embodiment, the said hydrogeis include self-assembled nanofibrils or nano-fibrous meshwork having a cross-β organisation and said meshwork is used for culturing human mesenchymal stem cells (hMSCs) on said hydrogeis due to their non-toxicity to cells; said nano-fibrous meshwork mimicking natural extracellular matrix (ECM).
In a further embodiment, the said hydrogeis include anti-parallel orientation of the β-sheet structure in amyloids.
In a still further embodiment, the hydrogeis are shear thinning hydrogeis forming liquid while being injected, but reforming into hydrogel in-vivo for cell-delivery with minimally invasive surgery.
In a yet further embodiment, the said hydrogeis have higher stiffness by adding salts such as
CaCl2.

In one more embodiment, the said hydrogels are thixotropic in nature and suitable for their application in 3D cell-culture.
In an additional embodiment, the said hydrogels have predefined hydrophobic or n stacking interaction to support self-assembly under suitable conditions.
In another additional embodiment, the storage modulus (G') of said hydrogels is in the range of 20 to 30 Pa and the storage modulus thereof can be controlled by changing the salt from a monovalent to divalent salt, e.g. from NaCI to CaCl2.
In still another additional embodiment, the bulk modulus of said hydrogels can be controlled by varying the peptide and salt concentration therein.
In yet another additional embodiment, the said hydrogels have higher spread area and lower circularity than the substrate on seeding the bone marrow derived human mesenchymal stem cells (hMSCs) thereon, which impart a distinct neuron-like morphology to said hydrogels.
In a further additional embodiment, the said hydrogels facilitate cell attachment and assume neuronal morphology with substantially extended neurites to achieve distinct differentiation of human mesenchymal stem cells (hMSCs).
In a still further additional embodiment, the said hydrogels have moderate astrocyte response for therapeutic application for transplantation of human mesenchymal stem cells (hMSCs) in brain with minimally invasive surgery.
In a yet further additional embodiment, the said hydrogels have distinct differentiation towards neurons for transplantation of human mesenchymal stem cells (hMSCs) into the brain by easy loading and delivery at a desired site via a minimally invasive approach.
In one more additional embodiment, the said hydrogels self-assemble and form physical hydrogels at sites with components not linked by chemical bonds to modulate these hydrogels by changing the peptide structure and by adding salts to yield the targeted physical and bulk properties of the hydrogel.
In accordance with the present invention, a method is provided for producing self-assembled, injectable amyloid, peptide-based hydrogels from amyloidogenie proteins like alpha synuclein (a-Syn protein), the said method comprises the steps of:

• measuring β-sheet propensities of α-Syn protein having numerous amyloidogenic regions with varying amyloidgenicity using TANGO software;
• selecting one of the said amyloidogenic regions and altering the amino acid sequences for obtaining the targeted hydrophobic or π stacking interaction for supporting self-assembly under suitable conditions;
• developing short peptides with 5 amino acids from a shorter sequence within the 69-78 region thereof;
• protecting the N-terminus of said peptides with Fmoc group for further favoring the self-assembly process; and
• dissolving hydrogels A2, A3 and A5 in physiological buffer condition (20 mM phosphate buffer, pH 7.4) in 6mg/ml concentration after repeated heat cool cycles and in presence of 150 mM NaCl for forming self-sustaining hydrogels.
In an embodiment of the method, NaCl salt is replaced by CaCl2 in gelation of hydrogel A5 to make assembly with more stiff hydrogels with storage modulus (G') increased from 20 Pa to 90 Pa.
In another embodiment of the method, different residues are introduced in the core sequence, i.e. Fmoc-VTAVA with the side chain ionization thereof, varying according to the pH of the peptide dissolving buffer to develop pH responsive hydrogels based on said injectable amyloid
hydrogels.
In still another embodiment of the method, A4 is produced by substituting threonine (Thr) with Lysine (Lys) and hydrogels A6 and A7 are produced by replacing Thr with histidine (His) in hydrogels Al and A3, which formed hydrogels on pH trigger and this gel formation is assisted by enhanced stacking interaction between the imidazole side chain to alleviate the salt requirement.
In yet another embodiment of the method, the said amyloid inspired peptide hydrogels are developed based on amyloidogenic segment of α-Syn protein to trigger gel formation by different stimulus like heating/cooling or change in pH; said peptides reflect cross-β sheet rich amyloid and assemble into a nano-flbrous meshwork mimicking the natural extracellular matrix (ECM) excellent for ceil attachment and differentiation capabilities for neuronal lineage from human mesenchymal stem cells (hMSCs).

In a further embodiment of the method, the said amyloid hydrogels are shear thinning hydrogels forming liquid during injection thereof and reform into hydrogel in-vivo to facilitate transplantation of human mesenchymal stem cells (hMSCs) in brain with minimally invasive surgery.
In a still further embodiment of the method, the stem cell delivery to specific regions of brain is used as a prototypical system to demonstrate utilization of said injectable amyloid hydrogels for promoting cell survival and neuronal differentiation of human mesenchymal stem cells (hMSCs) in vivo.
DESCRIPTION OF THE INVENTION
Amyloid inspired short peptide gelators were obtained from alpha-synuclein protein. These peptides are developed from beta sheet rich region of alpha-synuclein and suitably modified by altering the amino acids such that it gets dissolved in aqueous buffer.
All the hydrogels developed are at physiological pH in phosphate buffer.
The sequence is selected and synthesized from the beta aggregation rich region. Then it is dissolved in phosphate buffer at physiological pH to make the hydrogel.
The self-assembled nanofibrils show a cross-beta organization and bind to amyloid specific dyes like thiofiavin-T and Congo red. Human cells could be cultured on these hydrogels, as they are not toxic to cells.
Moreover, additional cell adhesive motifs are not required, as the nanofibrils themselves mimic extracellular matrix.
Given the stiffness of the hydrogel as predicted from rheological experiments, they promote neuronal differentiation of mesenchymal stem cells.
The hydrogels developed in accordance with the present invention are thixotropic in nature and hence can also be suitably used for 3D cell culture.
On implanting in adult rat brains, this hydrogel is well tolerated by immune system of rat brain,

Another advantage of sheer thinning gels is that they serve as perfect candidates for ceil delivery with minimally invasive surgery.
After cells are transplanted in mice brain at the striatum and substantia niagra, the hydrogel enhanced the survival of the transplanted cells and was also able to contain them at the
transplant site,
On assessing the fate of the transplanted mesenchymal stem cells from brain sections, it was found that these are driven towards neurons.
α-Syn is a 140-amino acid protein (Fig. 1A) having numerous amyloidogenic regions with varying amyloidgenicity and high propensity for P-sheet formation; for e.g. regions 15-19, 36-41 and 69-78. The p-sheet propensities were measured using the software TANGO (Fig 1B). One of these regions was selected and then the amino acid sequences were altered such that the resulting peptide would have sufficient hydrophobic or π stacking interaction to support self-assembly under suitable conditions. Short peptides with 5 amino acids were developed from a shorter sequence within the 69-78 region. The N-terminus of the peptides was protected with Fmoc group, which is known to enhance intermolecular stacking interaction, further favoring the self-assembly process.
When peptides were dissolved in physiological buffer condition (20 mM phosphate buffer, pH 7.4), the peptides A2, A3 and A5 formed self-sustaining hydrogels (Fig. ID) in 6mg/ml concentration after repeated heat cool cycles and in presence of 150 mM NaCl. Addition of salts is essential to form hydrogels. Explanation of this salt effect could be attributed to charge shielding of component peptides during the self assembly process. This is further supported by the fact that replacing NaCl with CaCl2 in gelation of A5 hydrogel makes the assembly better, making stiffer hydrogels, reflected in increase of its modulus from 20 Pa to 90 Pa. In an attempt to develop pH responsive hydrogels based on this system, different residues were introduced in the core sequence i.e. Fmoc-VTAVA, the side chain ionization of which varies according to the pH of the buffer in which the peptide is dissolved, Thr with Lys in Al was substituted to produce A4. Thr was replaced with His in A1 and A3 to produce A6 and A7 respectively (Fig. IC). A4 dissolved in 20mM phosphate buffer above pH 10.4 but unfortunately did not form under any of the tested conditions. A6 and A7 formed hydrogels on pH trigger (Fig. 1D). The gel formation could be further assisted by the enhanced stacking interaction between the imidazole side chain of His, which might be alleviating the requirement of salts. Morphology of assembled state

To study the rneshwork responsible for solvent entrapment and gelation, field emission gun scanning electron microscopy (FEG-SEM) (Fig. 5) and atomic force microscopy (AFM) of dried hydrogels (Figure 1F & 6) were performed. Both the techniques revealed that the peptides self-assemble into nano-fibril with diameter ~40 nm and height ~10 nm.
Structural analysis of peptides in assembled state
1. FTIR (Fourier Transform Infra-Red spectra)
Fourier transform infra-red (FTIR) spectroscopy was employed to determine the secondary structures of the peptides in the assembled state. FTIR spectra from 1800 cm-1 to 1500 cm-1 were recorded to analyze the amide-I and amide-II bands/regions to acquire an idea of the secondary structure of the peptides in the material (Fig 1H & 16). Classic signatures of p-sheet structures are noted in the spectra obtained from the hydrogels, p-sheet structures are reported to exhibit absorptions at 1628 cm-1 to 1635 cm-1 and 1521 cm-1 to 1525 cm-1 in the amide I and amide II regions respectively. If the protein under investigation contains antiparallel β-sheet structures, a peak can be observed at a high frequency (above 1670 cm-1). This is due to transition dipole coupling in the p-sheet component[8].
The exact position of this peak is difficult to determine, but is reported to be usually at a frequency 50 cm-1 to 70 cm-1 higher than the main β-sheet component. All the spectra obtained from hydrogels invariably show an additional peak at ~ 1690 cm-1. This is probably due to the anti-parallel orientation of the β-sheet structure as present in amyloids.
2. Amyloid dye binding assay
Thioflavin-T (ThT) is a fluorescent dye widely used to ascertain amyloidgenicity of proteins/peptides as it specifically binds to the P-sheet region of the amyloid fibrils and not to monomeric form of the proteins/peptides. ThT upon binding to amyloids yields high fluorescence at 482 nm when excited at 450nm compared to the excitation and emission of the free dye at 385 nm and 445 nm. This dye does not show fluorescence on binding to monomeric proteins and hence the dye binding could be attributed to the aggregated state of the peptides. ThT binding assay was done with A2, A3, A5, A6 and A7. The fluorescence yield obtained at 480nm reveals that these peptide hydrogels under study are amyloidgenic in nature (Fig. 7). As a control, ThT was added to buffer only and its fluorescence was measured at 480nm. Congo

red (CR) tike ThT is also used routinely for detection of β-sheet structures in proteins. UV absorption study shows an increase in the intensity of CR absorption upon incubating with the hydrogels (Fig, 1G).
Storage modulus of A 5 andA6
Oscillatory rheology is used to measure the visco-elastic property of these hydrogels. Out of the storage modulus (G') and loss modulus (G") of the hydrogels, G' is plotted against frequency obtained at constant amplitude depicts the storage modulus of the material at the plateau region (Fig. 1E). The storage modulus (G') of hydrogel A5 is 20.21 ± 3.59 and A6 is that of 27.48 ± 5.1 Pa. The bulk modulus of these hydrogels can be altered by varying the peptide and salt concentration. Change from a monovalent to divalent salt, like from NaCl to CaCl3 also changes the storage modulus of the hydrogel.
In vitro toxicity assay of hydrogels using SHSY5Y
Since the present class of gels is composed of amyloid fibrils and certain amyloids are known to cause cellular toxicity, MTT assay with neuroblastoma cells using SHSY5Y was performed to evaluate the potential toxicity. These cells have a fibroblast like morphology until differentiated to neurons. Cells were seeded on 30µL hydrogel and incubated.
As controls, cells under identical conditions on polystyrene plates were incubated directly in presence and absence of buffer used for hydrogel. All these hydrogels are found to facilitate cell attachment (Fig. 9). However, the spreading of the cells was less on hydrogels compared to control sets. MTT assay was performed after 24 hours and cell viability for all hydrogels is more than 80% (Fig. 2C). SHSY5Y was further cultured on the hydrogel A5 for 48 hours to look at the differentiation potential of these hydrogels by monitoring their morphology (Fig. 2A) and neurite extension. Compared to the control, cells seeded on hydrogel assumed neuronal morphology with much extended neurites (Fig. 11)
Cross seeding capability of gel fibrils
Recently, it was suggested that α-Syn fibrils have capability to spread from one cell to another ceil and possess ability to recruit soluble α-Syn protein and transform them to insoluble PD like Lewy bodies (LBs) by accelerating fibrillation process (seeding ability). To check whether these peptide hydrogels derived from α-Syn protein possess any seeding capacity, in vitro wild type (wt) full-length α-Syn aggregation kinetics were performed in presence and absence of gel fibrils and wt α-Syn preformed fibril seeds. For seeding, 7 days incubated α-Syn fibril and A5

hydrogel were used. 300 µM soluble wt α-Syn protein was incubated in presence of 1 % and 2% (v/v) gel/fibril seeds in phosphate buffer (20 mM; pH 7.4) at 37°C with slight agitation. The ■fibril formation kinetics was monitored by ThT fluorescence. It was found that gel fibrils seed did not affect aggregation kinetics of WT α-Syn protein. However, pre-formed full-length a-Syn fibril seeds significantly accelerated α-Syn aggregation (as evident from the reduction in lag time of a-Syn aggregation (Fig. 8), The data suggested that α-Syn derived peptide hydrogels might not be capable of seeding full length α-Syn aggregation. Although, the fibrils constituting hydrogel A5 is not seeding the full length α-Syn, its degraded products over time may lead to the generation of fragments that may interact with α-Syn and modulate its aggregation state.
To evaluate this, aggregation kinetics experiments were performed using two different conditions for gel degradation in vitro. In one condition, phosphate buffer was placed on the top of gel surface (condition A) and incubated up to 21 days. To mimic possible degradation of the gel in vivo, the gel was further mixed with 3.8 µg/mL proteinase K, an enzyme most frequently used for non-specific degradation of proteins and amyloids 28,29 (condition B), and incubated for 21 days, After 7 and 21-day incubation, 2% v/v concentration of degraded gel products from both conditions A and B were added to WT α-Syn and aggregation was monitored via ThT fluorescence (Fig. 8B and 8C). At 7 days, no significant change in lag time was observed for α-Syn aggregation in different conditions (seeding with condition A with lag time 47 ± 2,6 h and condition B with lag time 47 ± 2.5 h and α-Syn alone with a lag time 49 ± 0.8 h) (Fig. 10). A similar trend was observed with the lag time of 57 ± 1,2 h for a-Syn alone at 21 days; for seeding with gel fragments from condition A, the lag time was 55 ± 2 h and for condition B the lag time was 58 ± 2 h (Fig 11). It was therefore concluded that neither gel fibril nor its degraded counterpart possess any seeding capacity for a-Syn fibrillation. However, WT a-Syn fibril seeds were able to almost eliminate the lag time for aggregation in all cases.
Human mesenchymal stem cell culture on A5 andA6 hydrogel
Bone marrow derived human mesenchymal stem cells (hMSCs) were seeded onto A5 hydrogel coated on a glass coversiip and incubated. The control cells were seeded only on glass coverslips. The cells incubated on the gels show different morphology from the control set from day 1 itself (Fig. 2B). The cells plated on glass coverslips are more spread, whereas the cells plated on the hydrogels are less spread and show distinct neuron like morphology (Fig, 14). Data analysis was done for two time points (Day 1 and Day 5) for spread area and circularity (Fig. 2D) of hMSCs cultured on A5 hydrogel, The mean cell spread area on A5 at day 1 is 2674 ± 959 µm2 as compared to control cells with 5963 ± 3639 µm2. On day 5, mean cell spread area on glass (control) and gel are 5785 ± 2025 µm2 and 4383 ± 2139 µm2 respectively. Circularity

of ceils cultured on gel is significantly less than those cultured on glass, as seen from the graph, However, the variation is not significant between cultures of day 1 and day 5. The cell spread area analysis shows that these stem cells assume elongated bipolar morphology right from the early stages of culture, which elongates further with time along with branching (Fig. 15). Identical trend is observed when hMSCs are cultured on A6 hydrogel (Fig. 13),
Transcript profiling of stem ceils cultured on A5
The expressions of neuron specific enolase (ENO), β-III tubulin (TUBB3), glial fibrillary acidic protein (GFAP), glutamate ionotropic receptor (GRIA3) genes were tracked in cells cultured for 5 days on hydrogel (A5) coated coverslips. The relative high expression ENO and TUBB3 (Fig. 2F) ascertains that the differentiation towards neuronal lineage has started. Low level GFAP expression denotes that cells are preferably directed towards neurons over astrocytes. Fold changes in the expression level of the above genes are described in (Table 1) The expressions of all genes are normalized relative to housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT),
Neuro-inflammatory response against AS
Post seven-day implantation, the hydrogel in the caudate putamen region of adult rat brain attracted both astrocytes and microglial cells. This is quite expected due to the introduction of a foreign material and the associated trauma of the implantation procedure. But the localizations of these cells were different. The recruited astrocytes were mainly confined to the material-tissue interface, whereas the microglial cells have infiltrated the hydrogel (Fig, 3 A & 3B). 21-days post-implantation (dpi), the acute inflammatory reaction had subsided and the numbers of astrocytes as well as microglia were drastically reduced (Fig. 3C & 3D), However, the presence of scaffold cannot be ascertained from the images obtained in case of 21-day brain sections (Fig. 8). It might be possible that the scaffold is removed by the invading microglia. Interestingly, the number of microglia and astrocytes are even less than the control set post 21 -dpi. The astrocyte and microglial response was quantified from the total fluorescent intensity of GFAP and Ibal respectively in the field under study (~500 µm from the center of implant). Seven days post implantation, the astrocyte response was higher in case of hydrogel as compared to the sham control. The astrocyte recruitment attenuated by twenty-one days and the number of GFAP positive cells decreased even less than the control set. An identical trend is observed in case of microglia as well (Fig. 20). Thus, it was found from the inflammatory response post-surgery that our amyloid based hydrogel could be potentially used for therapeutic purposes in transplanting cells into the brain,

Stem cell transplantation along with hydrogel A5
The efficacy of hydrogel was tested by transplanting growth factor primed green fluorescent protein tagged human mesenchymal stem cells (GFP-hMSCs) into two different areas of MPTP mice brain (Fig. 4A). Transplantation procedures were performed at two locations in the brain, the SN and the striatum. Since the microenvironment of these two regions vary, it was tried to find out as to which site is better for transplantation of exogenous cells. The rationale of using GFP tagged cells was to distinguish transplanted cells from the host cells. These stem cells could be clearly seen within our scaffold in a 3D culture (Fig. 2E & 8). Given the thixotropic nature of these gels, cells could be easily loaded and delivered at a desired site via a minimally invasive approach. In this study, the stem cells were primed with a growth factor cocktail of 250ng/ml SHU, 100ng/mL FGF-8, 50ng/mL FGF-2 in neurobasal media supplemented with 0.25% B27. The growth factor cocktail is reported to help hMSCs specifically drive to DA neurons. The cells were cultured for 5 days in the growth factors and then transplanted to substantia niagra and striatum of MPTP mice along with the hydrogel A5. A 23G needle with a suitable plunger fitted to a stereotactic frame was used to the deliver the payload.
After transplantation, the system was left undisturbed for 5 minutes, which assists gelation of the matrix, and then the needle was withdrawn, The brains were harvested after 7 days and sectioned to look into the implant. The hydrogel was able to contain the cells at the transplantation site as well as enhance their survival (Fig. 4B). There were marked differences in the area occupied by the cells at the transplant bed when this hydrogel is used as compared to the cell only control (Fig. 4C & 1.8). The brain sections for different types of neuronal cell markers was further immunostained to determine the fate of the implanted ceils (Figure 17 & 19), The cells transplanted at SN took positive stain for β-III tubulin, a neuronal marker, revealing that these cells are differentiating towards neurons, which is being explored further.
EXPERIMENTAL SECTION.
Thioflavln T binding assay: Amyloid formation of the peptides in the hydrogel was monitored by binding and fluorescence intensity changes of the (3-sheet sensitive dye, Thioflavin T (ThT). 10µl of ThT solution (stock 1mM) was added in 200µl of gel. The ThT fluorescence was measured immediately in 10mm path length quartz cuvette cell (Helima, Forest Hills, NY) on a spectrofluorimeter (Shimadzu-RF-530) with an excitation wavelength 450nm and emission spectra recorded between 460nm-500nm. The excitation and emission slit width was 3nm for ail the studies. ThT control was carried out with 10µl ThT dye solution (1mM) in 200µl of phosphate buffer (20mM, pH-7.4).

Congo Red binding assay: Congo red (CR) binding assay was done to confirm the findings
from ThT binding assay. CR stock solution was prepared by dissolving 25rng CR in 100 ml,
PBS containing 10% ethanol (stock-360 µM). 15µl of CR stock solution was mixed with 85µl liydrogel and incubated in dark for 5 min. UV absorption spectra was measured from 300 nm to 700 nm on a UV spectrophotometer (JASCO V-650). CR control experiment was done by incubating the dye solution with the buffer alone.
Scanning Electron Microscopy: To characterize the morphology of the peptides FEG-SEM was performed with all the hydrogels. Hydrogel samples were prepared for SEM by placing10 µl of hydrogel on a glass coverslip. The samples were allowed to air dry under vacuum and then washed twice with MilliQ water. The final dried samples were gold coated for 120 sec at 10 mAmp current before SEM was performed with JSM-7600F at 5 kV.
Scanning Probe Microscopy: Scanning probe microscopy (SPM) was performed for additional validation of morphology of peptide fibers. In brief 10µl gel sample was spotted on freshly cleaved mica surface followed by air drying. AFM imaging was done in tapping mode under a silicon nitride cantilever using Veeco Nanoscope IV multimode AFM. Minimum five different areas of three independent samples were scanned with a scan rate of 1.5 Hz.
Fourier Transform Infrared spectra: A thin film of hydrogel was allowed to dry upon freshly prepared KBr pellet and analyzed. Fine powder of KBr was made with a mortar and pestle and compressed in a hydraulic pressure pump with a pressure around 7 ton to make the pellet. The pellet was kept under IR lamp and 10 µl of gel sample were spotted on the pellet The hydrogels were briefly vortexed before spotting so as to utilize the shear thinning property of the hydrogels make them less viscous. Baseline correction was done using 20mM phosphate buffer, pH-7.4 spotted ad dried on a separate pellet. FTIR spectra were obtained using a Vertex 80 FTIR system, Bruker, Germany equipped with DTGS detector in the range of 1800-1500 cm-1 at a resolution of 4cm-l. using an average of 32 scans.
Cell culture: Neuroblastoma cell line SHSY-5Y, was used in the study for initial cell based experiments, Cells were cultured and maintained on T25 flasks in modified Dulbecco's modified Eagle's medium (DMEM; HiMedia, India) supplemented with 10% (v/v) fetal bovine serum FBS (Invitrogen, USA), 1 % (v/v) antibiotic at 37 °C and 5% CO2. Near confluent flasks of SHSY-5Y cells were rinsed with phosphate buffered saline (PBS) and incubated with trypsin-EDTA solution for 5 min and then re-suspended in culture medium (complete media). Media was changed every 3 days. Cell adhesion on hydrogels studied on 96 & 24 well-plates.

MTT assay: To check toxicity of hydrogels, SH-SY5Y was cultured on hydrogels (2D culture). 30 jtL of each gel was poured into 3 different wells in a 96 well-plate and sterilized under UV for 30 mins. Cells were seeded in the wells in 100 ul, medium (complete DMEM) at a cell density of-10,000 per well. Cells were seeded only in culture media and in media+buffer as controls. After 24 hours of incubation 10 µL of a 5 mg/ml MTT prepared in PBS was added to each well and the incubation was continued for 4 hrs. Finally, 100 µl of a solution containing 50% dimethylformamide (DMF) and 20% sodium dodecyl sulphate (SDS) (pH 4.7) was added and incubated for overnight. After incubation in a 5% CO2 humidified environment at 37°C, absorption values at 560 nm were determined with an automatic micro-titre plate reader (Thermo Fisher Scientific, USA). The background absorbance was also recorded at 690 nm and subtracted from the absorbance value of 560 nm.
Bone marrow derived mesenchymal stem cell culture on hydrogel: Coverslips (12 mm) were treated with 0.5M NaOH for 30 minutes and dried. These coverslips were then coated with (3-Aminopropyl) triethoxysilane (APES), Sigma, and incubated for 5 min at room temperature. The excess APES was removed via two washes with MilliQ water. The coverslips are then dried inside a laminar flow and treated with 0.5% gluteraldehyde for 30 minutes and dried again. Dry coverslips were used for casting hydrogels. 15 µl of hydrogel was drop cast on the coverslip and spread uniformly with the pipette tip. Hydrogel was prepared in the laminar flow and further sterilized under UV for 30 min after casting on coverslip. Coverslips with hydrogel was transferred into wells of a 24 well-plate (Nunc) and 5x103 hMSC was seeded on each. Initially a low volume (~ 50 µL) of cell solution was used to ensure that the cells remain on the hydrogel. Remaining complete media was added after 30 min incubation. Cells were cultured in knockout DMEM (Gibco) supplemented with Glutamax (Gibco), 10% FBS (HiMedia) and 0.25% antibiotic cocktail (HiMedia) at 37°C in a humified incubator with 5% C02, Full media change was given on every third day. The control set of cells were incubated only on glass coverslips under identical conditions. Human bone marrow derived mesenchymal stem cells was purchased from Stempeuties (Bangalore, India), and all experiments are performed with cells within passage number 4 to 7. Phase contrast images of cells on hydrogel and control were obtained every alternate day via Olympus IX-50 microscope at 10X resolution. Images were analyzed with ImageJ for obtaining cell spread area and circularity. Box whisker plots were obtained for cell circularity using OriginPro (v8).
Rheologicat measurements: Rheological measurements were obtained using an Anton Paar Rheometer using a parallel plate geometry of 25 mm diameter. 150 µL of pre-formed hydrogel was loaded between the plates for the study. The measurements were taken at 37°C in the dynamic oscillatory mode with constant amplitude of 0.05% with a gap size of 0.2 mm. The

frequency sweep was performed with angular frequency (ω) 100-0.1 1/s conducted for 15 mm. The linear visco-elastic region was determined from a preliminary strain sweep from 0,01% to
100% at a constant frequency.
RNA isolation and quantitative Real Time PCS: hMSCs were cultured at a density of 104 per well in a 24 well-plate. Two conditions were employed on the cells. One set was cultured on hydrogel and the control set on glass. Cells were pooled from 3 wells for each condition by trypsinisation before isolating RNA. Pooled cells were then mixed with TRlzol (Invitrogen) and passed through a 24G sterile needle 8-10 times to lyse them completely. This was followed by a chloroform extraction and subsequently RNA precipitation by isopropanol and nucleic acid carrier glycogen. Extracted RNA was quantified via nanodrop spectrophotometer, Total RNA was then reverse transcribed to cDNA with ProtoScript® First Strand cDNA Synthesis Kit (NEB) using random hexamers and Oligo(dT)20 primers according to manufacturer's protocol. Real Time PCR was performed using SYBR®Green mastermtx (Ambion) on lllumina Eco qPCR system. Pie-designed validated SYBR green primers were purchased from Sigma-Aldrich for the study,
Hydrogel implantation into rat brain: To assess the inflammatory response of the hydrogel A5, it was implanted inside adult male Wistar rats brain. Animals were housed 2 rats per cage, given free access to food and water, and kept on a 12/12 h light/dark cycle. Rats were administered with an intramuscular injection of a pre-drug mix consisting of 0.1 ml atropine, 0.2 ml xylazine diluted in 0,7 ml saline. Anaesthesia was induced with 3% isofluorane in oxygen with a constant flow rate 1.0 L/rnin, When unresponsive to toe pinch, the head was shaved and rats were placed into a stereotaxic instrument and skull was exposed. Bilateral craniotomies were performed at coordinates of anteroposterior +1.0 mm AP and lateral +2.5 mm ML (medial-lateral) from bregma. A 32mm 21G needle preloaded with the scaffold was loaded onto the stereotactic frame and the scaffold was implanted into brain to a depth of-7.0 mm DV (dorsal-ventral) (scaffold length ™ 5 mm). 20mM phosphate buffer was implanted as a control. For each rat, scaffold was implanted in the right hemisphere and control on the left. Experiments are set for two time points; 7 days and 21 days. After an antiseptic ointment was applied to the edges of the wound and sutured. Rats were fully recovered in warm cages prior to being returned to their home cages.
Immunohistochemistry: The rats were euthanized by cardiac perfusion under terminal anesthesia with 0.1 mL lethabarb and ice cold saline followed by 4% paraformaldehyde (PFA) solution. The brain after removal were furthered stored in 4% PFA for 24 hrs followed by immersion in 30% sucrose solution, until it sinks. 30 urn transverse sections were obtained

using a cryostat and one in every three sections were collected on a superfrost plus slide. Sections were then subjected to rabbit monoclonal primary antibodies against glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (IBM) and mouse
monoclonal antibody against non-phosphory fated neurofilament H (SMI-32). Alexa Fluor 488 and 568 secondary antibodies were used. 0,5% Thioflavm-S in MilliQ water was used to stain the amyloid components of the sections. Nucleus was counterstained with DAPI. Images were obtained in a Nikon Eclipse Ti-U fluorescent microscope at 10X resolution, Individual color channels are captured and merged in ImageJ
Quantification of total fluorescent intensity; Total fluorescent intensity (integrated pixel density) obtained from each channel was quantified with ImageJ. An area of ~500 µm was selected keeping the area of implant at the center for both hydrogef containing sections as well as sham. Background reduction was done taking signal intensity of five random fields in the region of interest (ROI). Corrected integrated density of the sections was plotted in KaliedaGraph software for comparative analysis.
Priming GFP-hMSC for differentiation: Enhanced Green fluorescent protein (e-GFP) expressing human bone marrow derived mesenchymal stem cells was purchased from Tulane Centre for stem cell research and regenerative medicine, Cells were cultured in medium consisting of αMEM supplemented with 16.5% FBS, 2mM L-glutamine, 100 U/mL penicillin and 100µg/mL streptomycin (all from Invitrogen, CA, USA). For expansion cells were plated at a density of 60 cells/cm2 in tissue culture flasks (Nunc, Thermo Scientific) and media was changed after 3 days. BM-hMSCs were passaged at 80% confluency using Tryple (Invitrogen), Differentiation cocktail for induction to dopaminergic neurons (DA neurons) was carried out on hMSCs (P6-P8) in neurobasal media supplemented with 0.25X B27, 250ng/mL SHH, 100 µg/mL FGF-8, 50ng/mL FGF-2. Cells were seeded in expansion media and cultured for 24 hours before addition of differentiation media. Cells were fixed with 4% PFA at different time points and immunostained for specific markers.
Implanting cells at striatum and SnPC: Implantation procedures were done 4-week post-MPTP administration. As pre-an esthetic, each animal was given intraperitonial injection containing 6 µg Atropine, 400 µg Xylazine and 50 µg Meloxicam in 0.2 mL saline. The animals were then placed in a nose cone with 3% isofiurane in oxygen at a flow rate of 1.5 L/min until no vigorous reflexes were observed. The animal was then transferred to a stereotactic frame with a supportive warming pad, The flow rate of the anesthesia was maintained at 0,5-1% isofiurane for maintenance of anesthesia. The scalp was parted with a incision and a burr hole was drilled at 2.7 mm anterior and 1,8 mm lateral to the bregma on either hemispheres, A 23G

needle pre-loaded with cells (and scaffold) was inserted at an angle 55° (from vertical) to a depth of 6mm from the cortical surface for implantation in SNpC. The same angle was used with a reduced depth, 3mm for implantation in the caudate putamen. A suitable plunger was used to deliver the contents of the needle into the target site. The arrangement was left undisturbed for 1-2 minutes to minimize backflow of the cells through the delivery vehicle. The wound was closed using cyanoacrylate (instant bonding glue). Post-surgery animals were transferred to a warming cage till they recovered consciousness.
Procedure for hading cells in 23G needle. Cells cultured in TC flasks with suitable media were taken off with Tryple express. Cells were then pelleted, counted and mixed with A5 hydrogel at a density of 50,000 cells per 10 µl of hydrogel. 10 µl of scaffold solution along with cells were loaded onto a 23G needle using a Pi 0 pipette.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1A is full-length sequence of α-Syn protein with gelator core sequence marked in red.
Figure 1B is a TANGO plot of the protein showing β-aggregation prone regions of α-Syn.
Figure 1C is a table containing peptide sequences which formed hydrogels A1-A7 and names assigned each of them.
Figure 1D shows an inversion test of all hydrogels. A4 did not form hydrogel.
Figure IE is a plot of G' v/s frequency showing stiffness of hydrogels.
Figure IF is an atomic force microscopy (AFM) image of dried hydrogels for studying the meshwork responsible for solvent entrapment and gelation.
Figure 1G shows a Congo red absorption plot of all hydrogels depicting amyloidogenic nature owing to increased absorption at 520 nm.
Figure 1H shows an FTIR scan plotted from 1500 to 1800 cm-1 depicting typical signature peaks for cross β-sheet structures of thermos-responsive amyloid hydrogels.
Figure 2A SH-SY5Y cultured for 72 hours on A5 hydrogel for 48 hours to look at the differentiation potential of these hydrogels by monitoring their morphology.

Figure 2B shows the morphology of hMSC when cultured on A5 and glass coverslips. Bone marrow derived human mesenchymal stem cells (hMSCs) were seeded onto A5 hydrogel coated on a glass coversiip and incubated.
Figure 2C is a cell viability plot with SH-SY5Y culture with MTT reduction assay performed after 24 hours.
Figure 2D shows the quantification of cell circularity of hMSC cultured on A5 compared to control glass.
Figure 2E shows a 3D culture of GFP-hMSC in hydrogel A5,
Figure 2F is the quantitative real time PCR showing up regulation of neuronal markers and down regulation of astrocyte marker GFAP. The graph is plotted after normalization of marker expression in control set (scale-100 µm),
Figure 3A shows the neuro-inflammatory response of astrocytes imrnunostained with anti-GFAP antibodies against hydrogel A5 (left panel) implanted and sham control (right panel).
Figure 3B shows the microglial cells imrnunostained with anti-Ibal antibodies for hydrogel A3 (left panel) implanted and sham control (right panel).
Figure 3C shows the quantification of the astrocyte population from imrnunostained brain sections 7 and 21 dpi.
Figure 3D shows the quantification of the microglia population from imrnunostained brain sections 7 and 21 dpi.
Figure 4A schematically shows the transplantation of primed hMSC,
Figure 4B schematically shows the implanted GFP-hMSC with hydrogel A5 (left) at caudate putamen after 7 days in-vivo,
Figure 4C is a box plot of area occupied by the surviving cells when transplanted with and without hydrogel A5.
Figure 5 shows field emission gun scanning electron microscopy (FEG-SEM) images of hydrogels A2, A3, A6 and A7 for studying the meshwork responsible for solvent entrapment and gelation.

Figure 6A shows an atomic force microscopy (AFM) image of A2, A3, A6 and A7 hydrogels (scale- 100nm) performed for studying the meshwork responsible for solvent entrapment and gelation-Figure 6B is an atomic force microscopy (AFM) high-resolution image of individual A5 hydrogel fibril and measurement of individual fibril height and diameter (scaIe-100 nm) performed for studying the meshwork responsible for solvent entrapment and gelation.
Figure 6C shows the atomic force microscopy (AFM) amplitude images of B series peptides, which did not form hydrogel (scale -500 nm) performed for studying the meshwork responsible for solvent entrapment and gelation.
Figure 7 shows Thioflavin-T(ThT) fluorescence of hydrogels under study to reveal that these peptide hydrogels under study are amyloidgenic in nature.
Figure 8A shows the aggregation of WT α-Syn was monitored by ThT fluorescence, α-Syn LMW was incubated at a concentration of 300 µM with 1% and 2% (v/v) pre-formed fibril seed and gel fibril seeds at 37 °C with slight agitation in 20 mM phosphate buffer, 0.01% sodium azide; pH 7.4.
Figure 8B shows the aggregation of WT a-Syn was monitored by ThT fluorescence. The aggregation kinetics monitored with 2% WT a-Syn seed along with 2% proteinaseK digested gel fibril (α-Syn + PK mix) and degraded fragments leaking out of gel (a~Syn+ PB) and WT α-Syn at day 7.
Figure 8C shows the aggregation of WT α-Syn was monitored by ThT fluorescence. The aggregation kinetics monitored with 2% WT α-Syn seed along with 2% proteinaseK digested gel fibril (α-Syn.+ PK mix) and degraded fragments leaking out of gel (α-Syn+ PB) and WT α-Syn at day 21.
Figure 9 shows the phase contrast images of SHSY5Y cells cultured for 24 hours on hydrogels and control, displays adhesion and spreading of neuroblastoma cell line SHSY5Y on all
hydrogels.
Figure 10 shows the Calcein-Am stained cells inside hydrogel A5 when cultured in 3D for 24 hours shows viable cells.
Figure 11 is a box plot of neurite length of SHSY5Y cells when cultured on hydrogel A5 and glass (control) for 48 hours to look at the differentiation potential of these hydrogels by monitoring their neurite extension.

Figure 12 shows Thioflavin S staining of rat brain sections with implanted A5 hydrogel. The amount of scaffold remaining after 7 and 21 days in-vivo could be seen in the figure (scale-150µm).
Figure 13 shows the mesenchymal stem cells cultured on A6 hydrogel and glass coverslips as control. This cell spread area analysis shows that these stem cells assume elongated bipolar morphology right from the early stages of culture, which elongates further with time along with branching.
Figure 14 shows F-actin staining of hMSCs adhered on A5 hydrogel and glass with fluorescent-labelled phalloidin displays the difference in actin fiber organization in hMSCs cultured on hydrogel A5 to that of glass,
Figure 15 shows the morphological differences of hMSCs at day 12 when cultured on A5 hydrogel compared to that of glass (control). Cells assume branched morphology from bipolar morphology at day 12.
Figure 16 shows the classic signatures of p-sheet structures are noted in the spectra obtained from the hydrogels exhibiting absorptions at 1628 cm-1 to 1635 cm-1 and 1521 ~1525 cm-1 in the amide I and amide II regions respectively.
Figure 17 shows the immunostained brain sections of MPTP mice. (A) Nestin staining of transplant bed at CpU (B) TH of CpU (C) Nestin staining of transplanted cells at substantia niagra (D) NFH staining of transplanted cells at substantia niagra (scale- 100 µm).
Figure 18 shows the implanted GFP-hMSC with hydrogel A5 (upper right) at substantia niagra of MPTP mice brain.
Figure 19 shows the immunostained sections of MPTP mice brain, Beta-3-tubulin stained cells in scaffold at substantia niagra. Scale-150 µm.
Figure 20 shows the immunostained sections of rat brains against astrocyte marker GFAP and microglia marker lbal,
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1A is full-length sequence of α-Syn protein with gelator core sequence marked in red.
Figure 1B is a TANGO plot of the protein showing β-aggregation prone regions of α-Syn. Position of core gelator sequence is marked along its peak.

Figure 1C is a table containing peptide sequences which formed hydrogels A1-A7 and names assigned each of them.
Figure 11) shows an inversion test of all hydrogels. A4 did not form hydrogel.
Figure IE is a plot of G' vs. frequency showing stiffness of hydrogels. Addition of CaC12 in¬creases the modulus making stiffer hydrogels. Here, oscillatory rheology is used to measure the visco-elastic property of these hydrogels. Here, G' is plotted against frequency obtained at constant amplitude depicts the storage modulus of the material at the plateau region.
Figure IF is an atomic force microscopy (AFM) image of dried hydrogels for studying the meshwork responsible for solvent entrapment and gelation. It shows the fibril networks of
hydrogel A5 (scale- 100nm). This technique reveals that the peptides self-assemble into nanofibril with diameter ~40 nm and height ~10 nm.
Figure 1G shows a Congo red absorption plot of all hydrogels depicting amyloidogenic nature owing to increased absorption at 520 nm. This UV absorption study shows an increase in the intensify of CR absorption upon incubating with the hydrogels.
Figure 1H shows an FT1R scan plotted from 1500 to 1800 cm-1 depicting typical signature peaks for cross β-sheet structures of thermos-responsive amyloid hydrogels.
Figure 2A SH-SY5Y cultured for 72 hours on A5 hydrogel for 48 hours to look at the differentiation potential of these hydrogels by monitoring their morphology. This shows elongated and branched morphology like neurons (arrows). The control sets with media only and media+buffer shows cells with spindle shaped morphology reflecting undifferentiated state.
Figure 2B shows the morphology of hMSC when cultured on AS and glass coverslips, Bone marrow derived human mesenchymal stem cells (hMSCs) were seeded onto A5 hydrogel coated on a glass coverslip and incubated. The control cells were seeded only on glass coverslips. The cells incubated on the gels show different morphology from the control set from day 1 itself. Cells after 5 days of culture on A5 show elongated bipolar morphology.
Figure 2C is a cell viability plot with SH-SY5Y culture with MTT reduction assay performed after 24 hours. Most scaffold support cells with more than 80% viability for all hydrogels. Ap40 and TritonX are used as negative controls.

Figure 2D shows the quantification of cell circularity of hMSC cultured on A5 compared to control glass. Data analysis was done for two time points (Day 1 and Day 5) for circularity of hMSCs cultured on A5 hydrogel, The circularity of cells cultured on gel is significantly less than those cultured on glass, as seen from the graph. However, the variation is not significant between cultures of day 1 and day 5.
Figure 2.E shows a 3D culture of GFP-hMSC in hydrogel A5, The efficacy of hydrogel was tested by transplanting growth factor primed green fluorescent protein tagged human mesenchymal stem cells (GFP-hMSCs) into two different areas of MPTP mice brain by performing transplantation procedures at two locations in the brain, the SN and the striatum. The rationale of using GFP tagged cells was to distinguish transplanted cells from the host cells. These stem cells could be clearly seen within our scaffold in a 3D culture. Due to the thixotropic nature of these gels, cells could be easily loaded and delivered at a desired site via a minimally invasive approach.
Figure 2F is the quantitative real time PCR showing up regulation of neuronal markers and down regulation of astrocyte marker GFAP. The graph is plotted after normalisation of marker expression in control set (scale-100 µm). The expressions of neuron specific enolase (ENO), p-111 tubulin (TUBB3), glial fibrillary acidic protein (GFAP), glutamate ionotropic receptor (GRIA3) genes were tracked in cells cultured for 5 days on hydrogel (A5) coated coverslips. The relative high expression ENO and TUBB3 ascertains that the differentiation towards neuronal lineage has started.
Figure 3A shows the neuro-inflammatory response of astrocytes immunostained with anti-GFAP antibodies against hydrogel A5 (left panel) implanted and sham control (right panel). At day 7 the astrocytes mostly exist at the scaffold tissue interface and surround the hydrogel, Blue channel represents cell nuclei stained with DAP1. The recruited astrocytes were mainly confined to the material-tissue interface,
Figure 3B shows the microglial cells immunostained with anti-Ibal antibodies for hydrogel A5 (left panel) implanted and sham control (right panel). At day 7 the microglia are mostly localized within the scaffold.
Figure 3C shows the quantification of the astrocyte population from immunostained brain sections 7 and 21 dpi. The plot represents total fluorescence intensity of GFAP stained slides after background correction (scale-150µm). 21.-days post-implantation (dpi), the acute inflammatory reaction had subsided and the numbers of astrocytes were drastically reduced,

Figure 3D shows the quantification of the microglia population from immunostained brain sections 7 and 21 dpi. The plot represents total fluorescence intensity of Ibal stained slides after background correction (scale-150 µm). 21-days post-implantation (dpi), the acute inflammatory reaction had subsided and the numbers of microglia were drastically reduced.
Figure 4A schematically shows the transplantation of primed hMSC. Cells were first plated and cultured in proliferation media for 24 hours. Subsequently they were primed with a differentiation media for 5 days and then transplanted with hydrogel A5 at SNPc of MPTP mice. Post 7 days in-vivo the brains were harvested and sectioned appropriately to reveal the graft. Schematic includes morphology data of cells at each stage during the study. This way, the efficacy of hydrogel was tested by transplanting growth factor primed green fluorescent protein tagged human mesenchymal stem cells (GFP-hMSCs) into two different areas of MPTP mice brain and transplantation procedures were performed at two locations in the brain, the SN and the striatum.
Figure 4B schematically shows the implanted GFP-hMSC with hydrogel A5 (left) at caudate putamen after 7 days in-vivo. Cells show better neuronal morphology than those implanted without hydrogel A5 (right). The hydrogel was able to contain the cells at the transplantation site as well as enhance their survival.
Figure 4C is a box plot of area occupied by the surviving cells when transplanted with and without hydrogel A5. Higher number of cells survives when transplanted with the hydrogel (scale-100 µm). There were marked differences in the area occupied by the cells at the transplant bed when this hydrogel is used as compared to the cell only control.
Figure 5 shows field emission gun scanning electron microscopy (FEG-SEM) images of hydrogels A2, A3, A6 and A7 for studying the meshwork responsible for solvent entrapment and gelation. It shows the nano-fibrillar network of peptide components in hydrogels (scale-100 nm). This technique also reveals that the peptides self-assemble into nano-fibrii with diameter ~40 nm and height ~10 nm.
Figure 6A shows an atomic force microscopy (AFM) image of A2, A3, A6 and A7 hydrogels (scale- 100nm) performed for studying the meshwork responsible for solvent entrapment and gelation. It shows the network of amyloid fibrils in hydrogels. This technique reveals that peptides self-assemble into nanofibril with diameter~40 nm and height ~10 nm.
Figure 6B is an atomic force microscopy (AFM) high-resolution image of individual A5 hydrogel fibril and measurement of individual fibril height and diameter (scale-100 nm)

performed for studying the meshwork responsible for solvent entrapment and gelation. This technique reveals that the peptides self-assemble into nanofibril with diameter ~40 nm and height~10 nm.
Figure 6C shows the atomic force microscopy (AFM) amplitude images of B series peptides, which did not form hydrogel (scale -500 nm) performed for studying the meshwork responsible for solvent entrapment and gelation. This technique reveals that the peptides self-assemble into nanofibril with diameter ~40 nm and height ~10 nm.
Figure 7 shows Thioflavin-T(ThT) fluorescence of hydrogels under study. ThT upon binding to amyloids yields high fluorescence at 482 nm when excited at 450 nm compared to the excitation and emission of the free dye at 385 nm and 445 nm respectively. This assay indicates presence of amyloid fibrils in the hydrogels. The fluorescence yield obtained at 480nm reveals that these peptide hydrogels under study are amyloidgenic in nature.
Figure 8A shows the aggregation of WT α-Syn was monitored by ThT fluorescence. a-Syn LM W was incubated at a concentration of 300 µM with 1 % and 2% (v/v) pre-formed fibril seed and gel fibril seeds at 37 °C with slight agitation in 20 inM phosphate buffer, 0.01% sodium azide; pH 7.4. As control, only seeds and WT a-Syn LMW was incubated under identical conditions. ThT fluorescence was measured at regular intervals at 480 nm and ThT fluorescence intensities were plotted against incubation time for each set.
Figure 8B shows the aggregation of WT α-Syn was monitored by ThT fluorescence. The aggregation kinetics monitored with 2% WT a-Syn seed along with 2% proteinaseK digested gel fibril (α-Syn+ PK mix) and degraded fragments leaking out of gel (α-Syn+ PB) and WT a-Syn at day 7.
Figure 8C shows the aggregation of WT a-Syn was monitored by ThT fluorescence. The aggregation kinetics monitored with 2% WT α-Syn seed along with 2% proteinaseK digested gel fibril (α-Syn+ PK mix) and degraded fragments leaking out of gel (a-Syn+ PB) and WT α-Syn at day 21. Identical lag time is seen for all samples except 2% WT a-Syn seed, The pre¬formed ■full-length α-Syn fibril seeds significantly accelerated α-Syn aggregation evident from the reduction in lag time of α-Syn aggregation.
Figure 9 shows the phase contrast images of SHSY5Y cells cultured for 24 hours on hydrogels and control, displays adhesion and spreading of neuroblastoma cell line SHSY5Y on all hydrogels. Cells were seeded on 30µL hydrogel and incubated. The control set contains cells cultured in TCPS along with 20 mM phosphate buffer at pH 7.4 in cell culture media. Compared

to Hie control cells, cultured on hydrogels show elongated morphology. Scale - 100 µm. As controls, cells under identical conditions on polystyrene plates were incubated directly in presence and absence of buffer used for hydrogel. All these hydrogels facilitate cell attachment.
Figure 10 shows the Calcein-Am stained cells inside hydrogel A5 when cultured in 3D for 24 hours shows viable cells. These stem cells could be clearly seen within our scaffold in a 3D culture.
Figure 11 is a box plot of neurite length of SHSY5Y cells when cultured on hydrogel A5 and glass (control) for 48 hours to look at the differentiation potential of these hydrogels by monitoring their neurite extension. As compared to the control, celts seeded on hydrogel assumed neuronal morphology with much extended neurites. This graph depicts that the cells cultured on hydrogel has longer neurite length than that of control.
Figure 12 shows Thioflavin S staining of rat brain sections with implanted A5 hydrogel. The amount of scaffold remaining after 7 and 21 days in-vivo could be seen in the figure (scale-150µm). Here, the presence of scaffold cannot be ascertained from the images obtained in case of 21-day brain sections. It might be possible to remove the scaffold by invading microglia,
Figure 13 shows the mesenchymal stem cells cultured on A6 hydrogel and glass coverslips as control. Stem cells exhibit difference in morphology when cultured on A6 hydrogel compared to glass. The cells cultured on hydrogel show more elongated and branched structure compared to the control. The trend is sim ilar to that of A5, Scale bar-100 µm. The cell spread area analysis shows that these stem cells assume elongated bipolar morphology right from the early stages of culture, which elongates further with time along with branching.
Figure 14 shows F-actin staining of hMSCs adhered on A5 hydrogel and glass with fluorescent-labelled phalloidin displays the difference in actin fiber organization in hMSCs cultured on hydrogel A5 to that of glass. Here, bone marrow derived human mesenchymal stem cells (hMSCs) were seeded onto A5 hydrogel coated on a glass coverslip and incubated. This type of actin organization is earlier reported in stem cells when cultured on soft substrate and they differentiate towards neuronal lineage (scale bar-100 nm).
Figure 15 shows the morphological differences of hMSCs at day 12 when cultured on A5 hydrogel compared to that of glass (control). Cells assume branched morphology from bipolar morphology at day 12. Control cells cultured on glass retains their fibroblast like morphology and also proliferated. Scale - 100 µm. The cell spread area analysis shows that these stem cells

assume elongated bipolar morphology right from the early stages of culture, which elongates further with time along with branching.
Figure 16 shows the classic signatures of β-sheet structures are noted in the spectra obtained from the hydrogels exhibiting absorptions at 1628 cm-1 to 1635 cm-1 and 1521 cm-1 to 1525 cm-1 in the amide I and amide II regions respectively,
Figure 17 shows the immunostained brain sections of MPTP mice, (A) Nestin staining of transplant bed at CpU(B) TH of CpU (C) Nestin staining of transplanted cells at substantia niagra (D) NFH staining of transplanted cells at substantia niagra. Scale- 100 µm. The brain sections for different types of neuronal cell markers were further immunostained to determine the fate of the implanted cells. The cells transplanted at SN took positive stain for β-III tubulin, a neuronal marker to reveal that these cells are differentiating towards neurons.
Figure 18 shows the implanted GFP-hMSC with hydrogel A5 (upper right) at substantia niagra of MPTP mice brain. Left panel shows cells implanted without scaffold. Box plot (lower) shows differences in area occupied by the implant. Vividly the cells survive better and occupy more area when implanted with the scaffold. Scale -100 µm. There were marked differences in the area occupied by the cells at the transplant bed when this hydrogel is used as compared to the cell only control,
Figure 19 shows the immunostained sections of MPTP mice brain. Beta-3-tubulin stained cells in scaffold at substantia niagra, Scale-150 µm. Here also, brain sections for different types of neuronal cell markers was further immunostained to determine the fate of implanted cells.
Figure 20 shows the immunostained sections of rat brains against astrocyte marker GFAP and microglia marker 1bal. Astrocytes immunostained with anti-GFAP antibodies for hydrogel A5 (upper left panel) implanted and sham control (upper right panel). Microglial cells immunostained with anti-lbal antibodies for hydrogel A5 (tower left panel) implanted and sham control (lower right panel) (scale -150 µm). 7-day post-implantation, the astrocyte response was higher in case of hydrogel as compared to the sham control. The astrocyte recruitment attenuated by twenty-one days and the number of GFAP positive cells decreased even less than the control set. An identical trend is observed in case of microglia as well. Thus, it was found from the inflammatory response post-surgery that amyloid based hydrogel developed in accordance with the present invention could be potentially used for therapeutic purposes in transplanting cells into the brain.

Mesenchymal stem cell culture on A5 hydrogel;

Relative values of gene expression of AS compared to glass
END 7.65 ± 0.36 ':.:■:■::' :■ ■' ".■'■'' ■ ■'■ ■ ■!"■■■.'
TUBB3 GFAP 0.69 ± 0.4

GRIA3 2.48 ± 0.39

Table I: values of relative gene expression when hMSCs are cultured on hydrogel A5 compared to that of glass.
β-[1] tubulin is primarily expressed in neurons and may be involved in neurogenesis and axon guidance and maintenance[9]. The appearance of NSE in neuron development is also a late event[10] and hence serves as useful index for tracking neural differentiation and maturation. Increment in the expression of these genes in cells cultured on hydrogels illustrates commitment of the seeded stem cells towards neurons. GRIA3 which is a glutamate ion channel receptor is predominantly present in the excitatory neuro receptors of mammalian brain and is activated in a variety of normal neurophysiologic processes,
Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", shall be understood to implies including a described element, integer or method step, or group of elements, integers or method steps, however, does not imply excluding any other element, integer or step, or group of elements, integers or method steps.
The exemplary embodiments described in this specification are intended merely to provide an understanding of various manners in which this embodiment may be used and to further enable the skilled person in the relevant art to practice this invention. The description provided herein is purely by way of example and illustration.
Although the embodiments presented in this disclosure have been described in terms of its preferred embodiments, the skilled person in the art would readily recognize that these embodiments can be applied with modifications possible within the spirit and scope of the present invention as described in this specification by making innumerable changes, variations, modifications, alterations and/or integrations in terms of materials and method used to

configure, manufacture and assemble various constituents, components, subassemblies and assemblies, in terms of their size, shapes, orientations and interrelationships without departing from the scope and spirit of the present invention.
While considerable emphasis has been placed on the specific features of the preferred embodiment described here, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiments without departing from the principles of the invention,
These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

WE CLAIM:
1. Hydrogels produced from alpha synuclein (α-Syn), said hydrogels comprise short peptide gelators developed from cross-p sheet rich of alpha synuclein (α-Syn) by altering the amino acids for dissolving them into an aqueous phosphate buffer at physiological pH.
2. Hydrogels as claimed in claim 1, wherein said short peptide with 5 amino acids are developed from a shorter sequence within the 69-78 region of the 140-amino acid a-Syn and N-terminus of the said peptides protected with Fmoc group for enhancing intermolecular stacking interaction.
3. Hydrogels as claimed in claim 2, wherein said short-peptides self-assemble into nano-fibrils with diameter of ~ 40 ran and height ~10 nm.
4. Hydrogels as claimed in claim 3, wherein said hydrogels include self-assembled nanofibrils or nano-fibrous meshwork having a cross-β organization and said meshwork is used for culturing human mesenchymal stem cells (hMSCs) on said hydrogels due to their non-toxicity to cells; said nano-fibrous meshwork mimicking natural extracellular matrix (ECM),
5. Hydrogels as claimed in claim 1, wherein said hydrogels include anti-parallel orientation of the p-sheet structure in amyloids.
6. Hydrogels as claimed in claim 1, wherein said hydrogels are shear thinning hydrogels forming liquid while being injected, but reforming into hydrogel in-vivo for cell-delivery with minimally invasive surgery.
7. Hydrogels as claimed in claim I, wherein said hydrogels have higher stiffness by adding salts such as CaCl2
8. Hydrogels as claimed in claim 1, wherein said hydrogels are thixotropic in nature and suitable for their application in 3D cell-culture,
9. Hydrogels as claimed in claim 1, wherein said hydrogels have predefined hydrophobic or π stacking interaction to support self-assembly under suitable conditions.

10. Hydrogels as claimed in claim 1, wherein the storage modulus (G') of said hydrogels
is in the range of 20 to 30 Pa and the storage modulus thereof can be controlled by changing
the salt from a monovalent to divalent salt, e.g. from NaCl to CaCl2.
11. Hydrogels as claimed in claim 1, wherein the bulk modulus of said hydrogels can be controlled by varying the peptide and salt concentration therein.
12. Hydrogels as claimed in claim 1, wherein said hydrogels have higher spread area and lower circularity than the substrate on seeding the bone marrow derived human mesenchymal stem cells (hMSCs) thereon, which impart a distinct neuron-like morphology to said hydrogels.
1.3. Hydrogels as claimed in claim 1, wherein said hydrogels facilitate cell attachment and assume neuronal morphology with substantially extended neurites to achieve distinct differentiation of human mesenchymal stem cells (hMSCs).
14. Hydrogels as claimed in claim 1, wherein said hydrogels have moderate astrocyte response for therapeutic application for transplantation of human mesenchymal stem cells (hMSCs) in brain with minimally invasive surgery,
15. Hydrogels as claimed in claim 1, wherein said hydrogels have distinct differentiation towards neurons for transplantation of human mesenchymal stem cells (hMSCs) into the brain by easy loading and delivery at a desired site via a minimally invasive approach.
16. Hydrogels as claimed in claim 1, wherein said hydrogels self-assemble and form physical hydrogels at sites with components not linked by chemical bonds to modulate these hydrogels by changing the peptide structure and by adding salts to yield the targeted physical and bulk properties of the hydrogel.
17. A method for producing hydrogels from amyloidogenic proteins like alpha synuclein (a-Syn), wherein the method comprises the steps of:

• measuring p-sheet propensities of a-Syn protein Slaving numerous amyloidogenic regions with varying amyloidgenicity;
• selecting one of the said amyloidogenic regions and altering the amino acid sequences for obtaining the targeted hydrophobic or π stacking interaction for supporting self-assembly under suitable conditions;

• developing short peptides with 5 amino acids from a shorter sequence within the 69-78 region of the 140-amino acid a-Syn;
• protecting the N-termintts of said peptides with Fmoc group; and
• dissolving hydrogels A2, A3 and A5 in physiological buffer condition (20 mM phosphate buffer, pH 7.4) in 6mg/ml concentration after repeated heat cool cycles and in presence of 150 mM NaCl to produce self-sustaining hydrogels.

18. Method as claimed in claim 17, wherein Nacl salt is replaced by CaCI2 in gelation of hydrogel A5 to make assembly with more stiff hydrogels with storage modulus (G') increased from 20 Pa to 90 Pa.
19. Method as claimed in claim 38, wherein introducing different residues in the core sequence, i.e. Fmoc-VTAVA with the side chain ionization thereof, varying according to the pH of the peptide dissolving buffer to develop pH responsive hydrogels based on said injectable amyloid hydrogels.
20. Method as claimed in claim 19, wherein A4 is produced by substituting threonine (Thr) with Lysine (Lys) and hydrogels A6 and A7 are produced by replacing Thr with histidine (His) in hydrogels A1 and A3, which formed hydrogels on pH trigger and this gel formation is assisted by enhanced stacking interaction between the imidazole side chain to alleviate the salt requirement.
21. Method as claimed in claim 20, wherein amyloid inspired peptide hydrogels are developed based on amyloidogenic segment of a-Syn protein to trigger gel formation by different stimulus like heating/cooling or change in pH; said peptides reflect cross-β sheet rich amyloid and assemble into a nano-fibrous meshwork mimicking the natural extracellular matrix (ECM) excellent for cell attachment and differentiation capabilities for neuronal lineage from human mesenchymal stem cells (hMSCs).
22. Method as claimed in claim 21, wherein said amyloid hydrogels are shear thinning hydrogels forming liquid during injection thereof and reforming into hydrogel in-vivo to facilitate transplantation of human mesenchymal stem cells (hMSCs) in brain with minimally invasive surgery,

23. Method as claimed in claim 22, wherein the stem cell delivery to specific regions of brain is used as a prototypical system to demonstrate utilization of injectable amyloid hydrogels for promoting cell survival and neuronal differentiation of human mesenchymal stem cells (hMSCs) in vivo.