SOLVENT-FREE LIQUID FROM REGENERATED SILK FIBROIN: A WRITEABLE AND SHAPEABLE MATERIAL

Abstract

The present invention provides a solvent-free viscoelastic RSF liquid by modifying the RSF surface with polyethylenimine and polyethylene glycol-based polymer surfactant, which surrounds each RSF molecule by forming a dual shell and thereby minimizes the inter RSF interactions and thus prevents formation of β-sheet aggregation. The engineering of RSF surface with PEI and PS significantly improved the conformational stability and storage time of RSF liquid compared to native RSF as silk I conformation of RSF liquid remained intact for more than 8 months.

Claims

1. A composite for retaining the silk-I coiled conformation in RSF silk fibroin comprising RSF silk fibroin with combination of oppositely charged polymeric species.

2. The composite as claimed in claim 1, wherein cationic polymeric species is polyethyleneimine (PEI).

3. The composite as claimed in claim 1, wherein the anionic polymeric species is poly (ethylene glycol-based polymer surfactant (PS).

4. The composite as claimed in claim 3, wherein poly (ethylene glycol-based polymer surfactant (PS) are selected from ##STR00002##

5. The poly (ethylene glycol-based polymer surfactant (PS) as claimed in claim 4, wherein R is C12-C14, y is 12 for PS-3, 30 for PS-4 and 32 for PS-5 and n is 10-12.

6. A viscoeastic RSF silk liquid comprising 6 wt % of RSF silk fibroin and combination of oppositely charged polymeric species, wherein the oppositely charged polymeric species are 15 wt % of polyethyleneimine (PEI) and 77 wt % of poly (ethylene glycol-based polymer surfactant (PS).

7. The viscoelastic RSF silk liquid as claimed in claim 6 prevents the interchain RSF interactions via the formation of a dual coronal shell around the RSF random coil chain.

8. The viscoelastic RSF silk liquid as claimed in claim 6 behaves like a liquid at above 45° C.

9. The viscoelastic RSF silk liquid as claimed in claim 6 behaves like a soft solid at 25° C.

10. The viscoelastic RSF silk liquid as claimed in claim 6 retains its silk I (random coil/helix rich) conformation for more than 8 months.

11. The viscoelastic RSF silk liquid as claimed in claim 6, on treatment with glutaraldehyde vapor at 50° C. results in the structural transition from silk I (random coil/helix rich) to silk II (β-sheet rich).

12. A process for preparation of RSF silk liquid comprising the steps of a. coupling of an aqueous solution of regenerated silk fibroin (RSF) with branched PEI polymer in presence of carbodiimide, at pH 6.5 and 4° C. to produce a positively charged cationized RSF (cRSF) solution, wherein PEI consists of 1°, 2° and 3° amines and is protonated at pH 6.5, and wherein the mole excess ratio for RSF:PEI:carbodiimide is in ratio of 1:30:50.

13. The process as claimed in claim 12 further comprising steps b. removing excess PEI and carbodiimide by extensive dialysis before mixing PEG based polymer surfactants (PS), c. adding PEG based polymer surfactant followed by freeze drying at temperature at −60° C. to produce freeze-dried PS-cRSF. d. heating the freeze-dried PS-cRSF of step (c) at 50° C. to produce a RSF-polymer biconjugate based viscous liquid-like material.

14. The process as claimed in claim 12, wherein the process is solvent free.

Description

BRIEF DESCRIPTION OF FIGURES

[0035] FIG. 1 illustrates anionic polymeric species (PS)

[0036] FIG. 2 (a): Temperature sweep rheology experiment was performed at 1 rad s.sup.−1 and 0.1% shear strain. The data indicated elastic solid like behavior from temperature 25 to 44° C. as storage modulus (G′) was higher than loss modulus (G″) where as shows liquid like nature above 44° C. suggesting viscoelastic nature and dual behavior with temperature.

[0037] FIG. 2 (b): Viscosity was measured with respect to applied shear rate which show decrease in viscosity from 500 Pa.Math.s (shear rate-0.1 s.sup.−1) to 250 Pa.Math.s at 10 s-1 shear rate suggesting the shear thinning nature of RSF liquid at 50° C.

[0038] FIG. 3 illustrates FIG. 3 (a): Schematic of RSF liquid used as an ink for writing further to be crosslinked with glutaraldehyde vapor at 50° C. for 24 h;

[0039] FIG. 3 (b): A cuboidal shaped soft solid slab (RSF@SS) of RSF liquid casted a PDMS template;

[0040] FIG. 3 (c): Temperature sweep rheology of RSF@SS, GC-RSF@SS, PS, and GC-PS. Closed and open symbols show the storage (E′) and loss (E″) moduli, respectively

[0041] FIG. 4 illustrates step wise process for preparation of RSF silk liquid.

[0042] FIG. 5 (a): ATR FTIR of native RSF silk fibroin (nRSF) showed shift in amide I band shifted from 1646 to 1623 cm.sup.−1 indicating conformational transformation from silk I to silk II within 1 month.

[0043] FIG. 5 (b): ATR FTIR of RSF silk showed retention of silk I conformation over 8 months.

[0044] FIG. 6 illustrates FIG. 6 (a): The lyophilized PS-cRSF was heated to 50° C. to achieve RSF Liquid;

[0045] FIG. 6 (b): DSC profile of PS and RSF liquid were recorded from −60° C. to 60° C., and the rate of heating was kept 10° C. min-1. PS and RSF liquid showed endothermic melting transition temperature at ˜39° C. and ˜45° C. and exothermic crystallization temperature at ˜8° C. and ˜11° C.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be omitted for clarity and conciseness.

[0047] It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component surface” includes reference to one or more of such surfaces.

[0048] The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. It will be further understood that the terms “comprises” and/or “comprising” used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof. Also, expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0049] The present invention describes and discloses RSF silk liquid and a method for preparation thereof and to provide RSF silk fibroin, which retains its Silk-I conformation. Further described herein is a system which prevents transformation of silk-I conformation into silk-II conformation.

[0050] In the present invention, the method for preparation of RSF silk liquid comprises the steps of: [0051] a. coupling of an aqueous solution of regenerated silk fibroin (RSF) with cationic polymeric species in presence of carbodiimide, at about pH 6.5 and about 4° C. to produce a positively charged cationized RSF (cRSF) solution, [0052] b. removing excess of cationic polymeric species and carbodiimide by extensive dialysis before mixing anionic polymeric species, [0053] c. adding anionic polymeric species followed by freeze drying at temperature at −60° C. to produce freeze-dried PS-cRSF. [0054] d. heating the freeze-dried PS-cRSF of step (c) at about 50° C. to produce a RSF-polymer bioconjugate based viscous liquid-like material.

[0055] As disclosed above, the process for preparation of viscoelastic RSF silk liquid is completely free of solvent and which thus retains the native conformation of silk-I.

[0056] The heating of PS-cRSF results in melting of PS chains and formation of a RSF-polymer biconjugate based viscous liquid-like material at about 50° C., and provides the viscoelastic RSF silk liquid of the present invention.

[0057] The cationic polymeric species can be polyethyleneimine (PEI) and the anionic polymeric species can be poly (ethylene glycol-based polymer surfactant (PS). The oppositely charged polymers are liquid at room temperature, and therefore the polymers and RSF silk fibroin forms a composite, which is viscoelastic in nature.

[0058] Therefore, the method for preparation of RSF silk liquid comprises the steps of: [0059] a. coupling of an aqueous solution of regenerated silk fibroin (RSF) with branched polyethyleneimine (PEI) polymer in presence of carbodiimide, at about pH 6.5 and about 4° C. to produce a positively charged cationized RSF (cRSF) solution, [0060] wherein PEI consists of 1°, 2° and 3° amines and is protonated at pH 6.5, and [0061] wherein the molar excess ratio of acidic amino acid residues of RSF:PEI:carbodiimide is in ratio of about 1:30:50, [0062] b. removing excess PEI and carbodiimide by extensive dialysis before mixing PEG based polymer surfactants (PS), [0063] c. adding PEG based polymer surfactant (PS) followed by freeze drying at temperature at −60° C. to produce freeze-dried PS-cRSF. [0064] d. heating the freeze-dried PS-cRSF of step (c) at about 50° C. to produce a RSF-polymer biconjugate based viscous liquid-like material.

[0065] PEG based polymer surfactants of the composite is selected from

##STR00001## [0066] wherein R is C12-C14 and y is 12 for PS-3, 30 for PS-4 and 32 for PS-5 and n is 10-12. (FIG. 1).

[0067] It has been found that the viscoelastic RSF silk liquid obtained by the aforesaid method retains silk in silk-I conformation. RSF silk liquid displays dual characteristic behavior as function of temperature as it shows soft solid like nature at room temperature and liquid like at 50° C. (FIG. 2a) and also shows shear thinning (FIG. 2b; viscosity decreases with shear and temperature) behavior with respect to temperature and applied shear which enables its utilization in writing/printing and shaping applications (FIGS. 3a and 3b) at different temperatures.

[0068] In another embodiment, the present invention describes and discloses a composite system based on bioconjugation and protein surface engineering of RSF silk fibroin, which prevents transformation of silk-I conformation of RSF fibroin into silk-II conformation, and retain the native original conformation. Said system for retaining the silk-I conformation of RSF fibroin, which comprises providing a composite of RSF fibroin with combination of oppositely charged polymeric species. RSF silk liquid is a composite system comprises of RSF silk fibroin, polyethylenimine and a PEG based polymer surfactant (FIG. 4).

[0069] In an embodiment of the present invention there is provided a viscoelastic RSF silk liquid, which retains the native conformation of RSF silk fibroin. Said viscoelastic RSF silk liquid comprises RSF silk fibroin (˜6 wt %) and combination of oppositely charged polymeric species, wherein the oppositely charged polymeric species are polyethyleneimine (PEI ˜15 wt %) and poly (ethylene glycol-based polymer surfactant (PS ˜77 wt %).

[0070] The viscoelastic RSF silk liquid prevents the interchain RSF interactions via the formation of a dual coronal shell around the RSF random coil chain. It has RSF in the predominant random coil conformation, and can be used for applications of injection molding, compression molding, and casting into different shapes.

[0071] Further, the viscoelastic RSF silk liquid shows phase transition; behaves like a liquid above 45° C. and acts as a soft solid at 25° C. This phase transition is mainly attributed to the interaction between polymer surfactant chains.

[0072] Rheological experiments reveal that RSF liquid exhibits temperature dependent viscoelastic and shear thinning behavior. Such solid-liquid transition property between 45-50° C. of RSF viscoelastic material offers applications, e.g., as an ink for different writing, and as a shapeable material (rectangular, dog-bone, or circular disk shapes).

[0073] The inventors of the present invention, while analyzing various characteristics of this newly formed RSF silk liquid observed that the RSF silk liquid retains its silk I (random coil/helix rich) conformation over at least 8 months however RSF silk fibroin solution with same concentration (6 wt %) shows silk I to silk II transformation within 30 days (FIG. 5).

[0074] The present invention further provides that RSF silk liquid on treatment with glutaraldehyde vapor at 50° C. results in the structural transition from silk I (random coil/helix rich) to silk II (β-sheet rich). This allows tuneability in mechanical properties of the materials fabricated using RSF Liquid, thereby providing a route to prepare library of different products for desired biomaterial applications.

[0075] Further embodiments of the present invention will be explained by examples.

Example 1: Preparation of Viscoelastic RSF Silk Liquid

[0076] 100 mL of 1 mg/mL native RSF (nRSF; Mw=179 kDa) aqueous solution was modified with a branched PEI (Mw=800 Da) using a carbodiimide activated coupling reaction at pH 6.5 and 4° C. The molar ratio of acidic amino acid residues of RSF:PEI:carbodiimide was taken as 1:30:50 for effective coupling. Excess PEI and carbodiimide was removed by extensive dialysis. This modification resulted in a positively charged cationized RSF (cRSF) solution (FIG. 4).

[0077] In the last step, the freeze-dried PS-cRSF (FIG. 6a-b) was heated to 50° C., which resulted in melting of PS chains and formation of a RSF-polymer bioconjugate based viscous liquid-like material at 50° C., designated from now on as RSF liquid.

[0078] The secondary structure studies using a combination of ATR-FTIR and circular dichroism indicate that the native-like confirmation of RSF (i.e. silk I) is intact and remains unchanged during the surface engineering process and even in RSF liquid.

[0079] This liquid flows under gravity at temperatures above 45-50° C. DSC profile of PS and RSF liquid were recorded from −60° C. to 60° C., and the rate of heating was kept 10° C. min-1 (FIG. 6c). PS and RSF liquid showed endothermic melting transition temperature at ˜39° C. and ˜45° C. and exothermic crystallization temperature at ˜8° C. and ˜11° C.

Example 2: Applications of RSF Silk Liquid in Writing Application and Shaping of Materials

[0080] (i) Various types of patterns are written using RSF Liquid (FIG. 3a). [0081] (ii) RSF liquid at 50° C., was subjected to shaping using PDMS templates having rectangular/dog-bone/circular disc geometries as shown in FIG. 3b. A cuboidal slab of RSF soft solid (RSF@SS) was produced on cooling to room temperature. This was further crosslinked using a similar glutaraldehyde vapour method. To understand the effect of crosslinking, temperature sweep (20-35° C.) rheology was performed on RSF@SS, and glutaraldehyde crosslinked RSF soft solid (GC-RSF@SS). Further, control experiments were performed on PS, and GC-PS. Crosslinking led to an enhancement of modulus by a factor of 2 for the RSF@SS (FIG. 3c). Lastly, RSF liquid can also be subjected to applications with high strain rate requirements for e.g. in compression molding.

ADVANTAGE OF THE INVENTION

[0082] This new class of concentrated RSF liquid helps in preventing the intermolecular interactions between the RSF chains. The RSF liquid retains its silk I (random coil/helix rich) conformation over at least over 8 months. The solid-liquid transition property between 45-50° C. of RSF viscoelastic material offers applications, e.g., as an ink for different writing, and as a shapeable material (rectangular, dog-bone, or circular disk shapes). Further, RSF silk liquid on treatment with glutaraldehyde vapor at 50° C. resulted the structural transition from silk I (random coil/helix rich) to silk II (β-sheet rich), which allows tuneability in mechanical properties of the materials fabricated using RSF Liquid, thereby providing a route to prepare library of different products for desired biomaterial applications.