WO2016034730A1 - Silk protein structures - Google Patents

Abstract

The present invention provides a capsule having a shell of material that comprises an assembly of a silk protein, such as an assembly that is a non-aggregated assembly of the silk protein, such as an assembly where the a-helix, β-sheet (native) and random coil content is at least 55%. Also provided are methods for preparing the capsule, which comprises the step of contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a protein, thereby to form a capsule shell at the boundary of the discrete region, wherein the first and second phases are immiscible.

Description

SILK PROTEIN STRUCTURES

Related Application

The present case claims the benefit and priority of GB 1415679.8 filed on 04 September 2014 (04/09/2014), the contents of which are hereby incorporated by reference in their entirety.

Field of the Invention

This invention relates to capsules, particularly microcapsules, based on a network of an assembled silk protein, and methods for the preparation of such capsules, and their use in methods of delivering encapsulated components. Also provided are fibres having a sheath that is an assembled silk protein.

Background

Silk has attracted significant attention over the years owing to its extraordinary mechanical properties which include excellent elasticity, strength, biodegradability and biocompatibility (Rising et al. Zoolog. Sci. 22, 273-281 (2005); Toshiki et al. Nature Biotechnology 18, 81 -84 (2000); Allmeling et al. Journal of Cellular and Molecular Medicine 10, 770-777 (2006); Wang et al. Bio materials 27, 6064-6082 (2006)). B. mori silkworm silk has been widely used in biomedical structural materials including sutures for micro surgery scaffolds for tissue engineering, and bulk silk emulsions for drug delivery (Pritchard et al. Expert Opinion on Drug Delivery 8, 797-81 1 (201 1 ); Lammel et al. Biomaterials 32, 2233-2240 (201 1 ); Vepari et al. Progress in Polymer Science 32, 991-1007 (2007); Gosline et al. The Journal of Experimental Biology 202, 3295-3303 (1999); Vollrath et al. Nature W O, 541-548 (2001 ); Nicholson et al. Biopolymers 33, 847-861 (1993); Link et al. Angewandte Chemie

International Edition 45, 2556-2560 (2006)). The mechanical properties of native silk are unsurpassed (Ha et al. Biomacromolecules 6, 1722-1731 (2005)). Due to the high viscosity of native silk and its aggregation propensity (Liu et al. J. Biosci. Bioeng. 108, 496-500 (2009)), artificial silk-based materials have, to date, mainly been synthesised from

reconstituted silk which is easier to process and more stable in solution (Teule et al. Nat. Protocols 4, 341 (2009); Gosline et al. Journal of Experimental Biology 202, 3295-3303 (1999); Tao et al. Advanced Materials 24, 1067-1072 (2012); Holland et al. Advanced Materials 24, 105-109 (2012); Wang et al. Advanced Functional Materials 22, 435-441 (2012)). This is a fundamental limitation to the use of native silk in materials science.

The properties of silk biomaterials are determined not only by the intrinsic properties of the native protein (Vepari et al. Progress in Polymer Science 32, 991 -1007 (2007); Gosline et al. The Journal of Experimental Biology 202, 3295-3303 (1999)), but are also fundamentally dependent the spinning processes that lead to the transition of the soluble form of native silk to a random coil structure to the solid fibrous form which consists of ordered hydrogen bond- rich β-sheet aggregates (Vollrath et al. Nature 410, 541-548 (2001 )). Silk spinning relies on a critical shear rate for inducing crystallisation in aqueous solution and the production of uniaxially aligned fibrous structures (Nicholson et al. Biopolymers 33, 847-861 (1993)).

Vepari and Kaplan discuss the use of silk proteins to form hydrogel materials. Here, silkworm proteins are permitted to form β-sheet aggregates from bulk silk protein solutions, for example in response to pH changes, changes in calcium ion concentration and changes in temperature. The gelation of the silk protein is apparently a gross transformation of the bulk silk protein solution.

Summary of the Invention

In a general aspect the present invention provides capsules having a shell of material that comprises an assembly of a silk protein and a fibre having a sheath that comprises an assembly of a silk protein. The assembly of a protein is obtained or obtainable by the self- assembly of one or more silk proteins.

The capsules and fibres of the present invention may be regarded as microgels, such as hydromicrogels. These structures are easy to synthesise, biodegradable and non-toxic, and are suitable for encapsulation and releasing components, such as small molecules.

Advantageously, the capsules and methods described herein can make use of a natural (non-recombinant) silk protein.

Surprisingly, the inventors have found that capsules, such as substantially spherical capsules, may be prepared where the assembly of the silk protein is not a β-sheet aggregate. Thus the assembly does not contain a substantial fibril content. Furthermore, the inventors have found that silk protein that is contained within a capsule remains in a non-aggregate form. The silk from the capsule may be released in this form.

In other structures, such as fibres, the silk protein may be provided as an aggregate of the silk protein, for example where there is a β-sheet aggregate form. The β-sheet aggregate form is present as substantial portion of the total silk protein content in structures such as a silk protein cylinder and a silk protein fibre.

The silk capsules and fibres may advantageously be used to encapsulate a component and release that component at a desired location. For example, the component may be a non-aggregated silk protein, as noted above.

Accordingly in a first aspect of the invention there is provided a capsule having a shell of material that comprises an assembly of a silk protein, such as a non-aggregate assembly of a silk protein. Here, the assembly of the silk protein is not a β-sheet aggregate. The shell is obtained or obtainable from the self-assembly of the silk protein.

In one embodiment, the capsule is substantially spherical. In one embodiment, the shell of the capsule is formed at the fluid boundary of a dispersed droplet in a continuous fluid phase. In one embodiment, each capsule has an average size of at most 10 μηη in the largest cross section, such as the diameter.

In one embodiment, the capsule is substantially cylindrical. In one embodiment, the shell of the capsule is formed at the fluid boundary of a dispersed slug in a continuous fluid phase. In one embodiment, the assembly of the silk protein is a β-sheet aggregate.

In a second aspect of the invention there is provided a fibre having a sheath of material that comprises an aggregate of a silk protein, such as an aggregated assembly of a silk protein. The sheath is obtained or obtainable from the self-assembly of the silk protein. In one embodiment, the sheath of the fibre is formed at the fluid boundary of a fluid flow within in an immiscible continuous fluid phase. In one embodiment, the assembly of the silk protein is a β-sheet aggregate.

In a third aspect of the invention there is provided a capsule or a fibre holding a component, wherein the capsule is a capsule of the first aspect of the invention and the fibre is a fibre of the second aspect of the invention.

The ability to hold and protect a component and release that component as required makes the spheres and fibres useful delivery vehicles for those components. In particular embodiments, the inventors have found that a silk protein may itself be encapsulated, and surprisingly, the silk protein may be encapsulated in a non-aggregated form.

In a fourth aspect of the invention there is provided a method for the preparation of a capsule having a shell that comprises an assembly of a silk protein, wherein the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a silk protein, thereby to form a capsule shell at the boundary of the discrete region, wherein the first and second phases are immiscible.

Typically the protein is provided in a second phase that is an aqueous phase. The first flow may be an oil phase, such as a fluorinated oil phase.

In one embodiment, the second phase comprises a component for encapsulation, and step (i) provides a capsule having a shell encapsulating the component. In one embodiment, the method further comprises the subsequent step of (ii) heating the droplet.

In one embodiment, the method further comprises the subsequent step of (iii) collecting the outflow from the channel, thereby to obtain a droplet, which has a capsule. This step may be performed before or after the heating step.

In a fifth aspect of the invention there is provided a method for the preparation of a fibre having a sheath that comprises an aggregation of a silk protein, wherein the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a fluid flow of the second phase between flows of the first phase, wherein the second phase comprises a silk protein suitable for forming an aggregation of a silk protein, thereby to form a fibre sheath shell at the fluid boundaries of the second phase, wherein the first and second phases are immiscible.

Typically the silk protein is provided in a second phase that is an aqueous phase. The first flow may be an oil phase, such as a fluorinated oil phase.

In a further aspect there is provided a method of delivering a component to a location, the method comprising the steps of:

(i) providing a capsule having a shell encapsulating a component or a fibre having a sheath encapsulating a component, according to the third aspect of the invention;

(ii) delivering the capsule or fibre to a target location; and

(iii) releasing the component from the shell of the capsule or the sheath of the fibre.

The present case allows the capsule and fibre to protect an encapsulated component, and release that component as and when required. This component may be a protein, such as a silk protein.

In another embodiment, the protein making up the shell or sheath may itself be released at a target location.

Thus, in a further aspect of the invention there is provided a method of delivering a protein to a target location, the method comprising the steps of:

(i) providing a capsule having a shell or a fibre having a sheath, wherein the shell comprises an assembly of a silk protein and the sheath comprises an aggregation of a silk protein, as described in the first and second aspects of the invention;

(ii) delivering the capsule or fibre a target location;

(iii) disrupting the shell or sheath, thereby to release the protein.

In one embodiment, a protein released from the shell or sheath is not in an aggregation, and optionally is also not part of an assembly. Description of the Figures

Figure 1 is (a) a series of light microscopy images of the B. mori silk protein structures synthesised in a single T-junction device for use according to an embodiment of the invention where the upper left image shows a single T-junction fluidic device with an image of the T-junction shown at the upper right. The remaining light microscopy images show the different silk structures that may be prepared using the methods described herein using B. mori silk protein such as (i) sphere, (ii) cylinder, (iii) short fibre, (iv) thin fibre, and (v) thick fibre. The scale bars are 10 μηη; (b) a series of schematic representations of the different silk structures, showing, from left to right, sphere, cylinder, short fibre, thin fibre, and thick fibre; and (c) a schematic presentation of the silk droplets structures generated as a function of the B. mori silk protein concentration (mg/mL) with the change in the aqueous:oil flow rate ratio in the T-junction fluidic device.

Figure 2 is a series of images of B. mori silk protein structures prepared by the methods described herein, where (a) to (e) are light microscopy (left) and AFM images (right and upper right) and (f) to (k) are 3D reconstruction of the confocal images, where the structures shown are (a) and (f) spherical, (b) and (g) cylindrical, (c) short fibre, (d) and (h) thin fibre, (e) and (i) thick fibre, (j) double shell sphere, and (k) double shell cylinder. The z-stack central cut image is shown in the right corner of the confocal images. For light and confocal microscopy images the Scale Bars is 5 μηη. For the inset AFM images the Scale Bar is 300 nm

Figure 3 is (a) the FTIR spectra of the native silk, silk sphere, sausage, short, thin and thick fibres structures, showing the change in intensity (au) with change in wavenumber (cm^"1). The AFM images of native and aggregative silk are shown on the top of FTIR spectra; (b) a pair of charts showing the structural changes to the B. mori silk protein upon its conversion into the variety of structures; and (c) shows the results of an AFM nanoindentation study of populations of silk capsules in air and liquid, showing the percentage population of capsules with specified DMT modulus values (GPa). The upper chart in Figure (b) shows the percentage population of, from left to right, a-helix, β-sheet (native), β-sheet (aggregate), random coil, and β-sheet (antiparallel) structure in native silk, sphere, cylinder (sausage), short fiber, thin fiber, thick fiber and aggregate. The lower chart in Figure (b) shows the percentage population of ordered and disordered B mori silk protein in these structures. The population calculations were based on Amide I and II vibrational changes in FTIR spectra.

Figure 4 is (a) a scheme shows the formation of a spherical silk capsule at a T-junction fluidic device according to an embodiment of the invention; (b) a series of images of B. mori silk, silk capsules, released silk and aggregated silk; (c) a series of Cryo-SEM micrographs showing the morphology of B. mori silk protein, silk capsules, released and aggregated silk; The Scale Bars are 100 μηη; (d) a graph showing the change in the amount of silk protein released (ng) over time (h) from a B. mori silk capsule by fast-freezing (liquid nitrogen), centrifugation (700 rpm, 3 min) and silk release to aqueous media, where the inset is a magnification of the initial 6 minute lease profile; (e) a graph showing change in the amount of aggregated B. mori silk protein as a percentage of the total B. mori silk protein amount over time (h) after the release of the silk protein release from the a B. mori silk capsule; and (f) is a chart (left) showing amount of native silk protein fraction detected for a native silk protein preparation and a capsule (at time 0 and after 1 month), and an aggregate after heating and a chart (right) showing the efficiency of silk release from a capsule according to an embodiment of the invention, using a liquid nitrogen snap-freezing method, gentle centrifugation and continuous washing of the silk capsules.

Figure 5 is (a) a series of images showing vials holding, from left to right, a mixture of aqueous B. mori silk protein and oil, aqueous B. mori silk protein and oil with RBBR dye, B. mori silk protein capsules holding the dye, and dye released from B. mori silk protein capsules; (b) a graph showing the percentage quantity of glucose (left), tetracycline (centre) and RBBR dye (right) encapsulated into a B. mori silk protein structure; and (c) is a series of graphs showing amount of glucose (top), tetracycline (centre) and RBBR dye (bottom) released from various silk protein structures in aqueous media over time (min.) as a percentage of the total amount encapsulated within that structure at time 0, where the structures are from left to right, capsule (sphere), cylinder (sausage), short fibre, thin fibre, and thick fibre. In the glucose release graph the lines are, from top to bottom at time 4,000 min., thin fibre, short fibre, cylinder, capsule, and thick fibre; in the tetracycline release graph the lines are, from top to bottom at time 3,000 min, thin fibre, short fibre, cylinder, capsule, and thick fibre; and in the RBBR dye release, from top to bottom at time 3,000 min, thin fibre, short fibre, cylinder, and capsule (these three roughly together), and thick fibre.

Figure 6 is a series of graphs showing the percentage caspase activity after exposure of various B. mori silk protein structures to SHY5 human cells. Figure 6 (a) shows the preliminary results, whilst, Figure 6 (b) shows the results from repeat experiments.

Figure 7 is (a) a series of light microscopy images of the silk structures synthesised in a double T-junction device for use according to an embodiment of the invention where the upper left image shows a double T-junction fluidic device with an image of the double T-junction shown at the upper right. The remaining light microscopy images show the different silk structures that may be prepared using the methods described herein using B. mori silk protein such as (a) double shell sphere, (b) half sphere, (c) disk, (d) fused double spheres, (e) fused multiple spheres, (f) fused sphere and sausage, (g) double shell sausage, (h) joined multiple spheres, (i) double shell sort fibre and (j) double shell thick fibre; (b) a series of light microscopy images of silk (i) double shell spherical, (vii) double shell cylinder and (x) double shell thick fiber structures at three time points T1 , T2 and T3 during the flow method. Scale Bars are 20 μηη; and (c) is a graph showing the change in viscosity (Pa.s) with change in B. mori silk protein concentration (mg/mL). Figure 8 is (a) a fluorescent spectra, showing the excitation and emission maxima of native and aggregative B. mori silk protein. For the silk protein in aggregative state the excitation maxima was detected at 315 nm and emission at 425 nm; (b) the FTIR spectra of the native silk, double shell silk sphere, double shell sausage and double shell thin fibre structures; and (c) a graph showing the percentage amount of ordered and disorder silk protein in four different B. mori silk protein structures.

Figure 9 is (a) a series of FTIR spectra indicating structural changes in silk protein (NSF) exposed to shaking; (b) a series of FTIR spectra indicating structural changes in

encapsulated silk protein (NSF) exposed to shaking; (c) a series of FTIR spectra indicating structural changes in silk (NSF) exposed to elevated temperatures; (d) a series of FTIR spectra indicating structural changes in encapsulated silk (NSF) exposed to elevated temperatures; (e) a series of FTIR spectra indicating structural changes in silk (NSF) exposed to phosphate buffer solution; and (f) a series of FTIR spectra indicating structural changes in encapsulated silk (NSF) exposed to phosphate buffer solution. The FTIR spectra show change in intensity (a.u.) with change in wavenumber (cm^"1). The chart insertions in each FTIR graph show the % of native silk component in each tested solution.

Figure 10 is (a) binding activity measurements for NbSyn87 mixed with silk protein (NSF); (b) binding activity measurements for NbSyn87 released from silk capsules; and (c) size exclusion of scFvC4 nanobodies, scFvC4 nanobodies mixed with silk (NSF) dope and scFvC4 nanobodies released from silk (NSF) capsules. The first absorbance maxima is attributed to the silk (NSF) content, while the second is attributed to nanobodies. The binding activity measurements show change in Ru with change in time (s). The size exclusion measurements shown change in absorbance as measured at 280 nm (mAU) with change in elution volume (Ve) (ml_).

Figure 1 1 is (a) schematic representation of the encapsulation and release of antibody domains by silk structures; (b) encapsulation efficiency studies for the C4scFv single chain Fv domain in spheres, cylinders, short, thin and thick fibres; (c) release kinetics for C4scFv from different silk structures; (d) biacore sensorgrams (SPR) of the binding of NbSyn86 to immobilised osynuclein: (i) a control sample of monomeric NbSyn86, (ii) NbSyn86 after encapsulation and release treatment in the absence of silk (NSF), (iii) NbSyn86 released from gelled silk structures (which contain 2% of aggregated silk (NSF)), (iv) NbSyn86 released from gelled silk structures which contain 60% of aggregated silk; and (e) a graph of the equilibrium binding values for the different released NbSyn86 samples (from Fig. 1 1 (d) versus the initial (pre-encapsulation) concentration of NbSyn86. The data were fitted to a 1 :1 bimolecular binding model to estimate the affinity constant of NbSyn86 for osynuclein, and were also used to estimate the loss of activity between samples. Detailed Description of the Invention

The present inventors have established that capsules and fibres may be prepared having a shell that is obtainable from the assembly of a silk protein. The capsules and fibres are formed using fluidic generation techniques, amongst others, and make use of the ability of a silk protein to assemble, either as an aggregate or another assembly. The ability of a silk protein to form a discrete shell or sheath is surprising given the previously reported behaviour of silk proteins, which often form amorphous assemblies.

A capsule or a fibre is obtainable through the use of fluidic preparation techniques. These techniques are particularly beneficial in that it generates droplets, for example, having a very low distribution of sizes, which results in capsules having a very low distribution in sizes. Moreover, the methods of the invention allow close control over the formation of the product capsule and fibre. Simple changes in the fluidic preparation technique, such as changes in flow rates and silk protein concentration, may be used to control the size and nature of the product structure obtained, the size of the pores in the shell, and the thickness of the shell, amongst others.

The techniques described herein are suitable for preparing substantially spherical capsules where the capsule shell comprises an assembly of a silk protein. In one embodiment, the assembly is not an aggregate of the silk protein. Thus, the shell does not have a major β- sheet aggregate component. Such structures are formed by selection of particular flow ratios in the fluidic preparation step. High shear rates at the junction of a fluidic device, which is linked to the relative flow rate ratios of the fluids meeting at the junction, is associated with the formation of capsules having non-aggregated forms of the silk protein. The ability to form a structure where the silk protein is in a non-aggregate form is entirely unexpected.

The fibres may have a sheath that comprises a β-sheet aggregate of the silk protein. These are useful alternatives to the capsules where the silk protein is not in aggregate form.

The capsules and fibres of the invention are shown to be robust, and are capable of withstanding temperatures of at least 95°C. The capsules also believed to maintain their integrity at reduced pressure.

The highly viscous nature of silk protein, such as NSF, and its tendency to aggregate presents a significant challenge for the storage and processing of this potentially highly functional material. The present work demonstrates that a multiphase flow of silk in microfluidic systems can be used to overcome these limitations through the flexible processing of silk into a wide range of structures, such as capsules and fibres, with different physical and chemical properties. Silk structures provide a practical solution for the long term storage and control of silk dope, with an increase in stable storage time of several orders of magnitude relative to

conventional storage under bulk conditions.

The capsules and fibres of the invention are suitable for encapsulating a component. Using the fluidic droplet preparation techniques described herein, a capsule shell or fibre sheath may be constructed in the presence of a component to be encapsulated. Thus, in one procedure the shell or sheath may be formed and the component encapsulated.

Advantageously therefore, the capsule or fibre may be constructed without the need for a later passive diffusion step after the capsule construction. Furthermore, the method of encapsulation allows high rates of incorporation of the material into the capsule or fibre, and material waste is therefore minimised. The encapsulated component may be released with ease using techniques such as freezing, centrifugation and washing.

The capsules and fibres, for example spherical capsules, are suitable for holding silk protein in a non-assembled, such as a non-aggregated form.

In addition, silk structures such as capsules and fibres can be used to encapsulate other components, such as functional antibodies, in a way that offers significant protection against their aggregation and loss of function. Protein molecules, including antibodies, are increasingly used in therapeutic applications, but often possess a high tendency to undergo unwanted aggregation processes and lose function. The ability of the silk structures to control and curtail this behaviour is therefore of considerable significance for the long term storage of proteins in functional states.

The capsules of the invention may be referred to as microgels, for example hydromicrogels, where they are provided holding and within a fluid, such as water.

Although hydrogels of silk protein have been previously described, these hydrogels are based on the β-aggregation of a silk protein (see Vepari and Kaplan). The hydrogels do not have a discrete structure, and the silk is clearly not a non-aggregated assembly, such as described herein. An assembly of the present case may be regarded as having a hydrogel character in view of the formation of an extensive network of silk protein in an aqueous environment (such as boundary of a dispersed second phase in the methods described herein).

The present application describes a general approach for generating capsules and fibres that are composed of silk proteins, such as entirely composed of silk proteins. The compartmentalisation of the precursor silk proteins in capsule or fibre form may be achieved through the use of droplet microfluidics, and formation of the capsule shell or fibre sheath is mediated through the assembly of precursor silk proteins, for example into networks of entangled amyloid fibrils. This approach is a convenient route to generate capsules and fibres having an assembly of proteins only at the shell or the sheath. The methods of the invention allow for the encapsulation of a variety of both hydrophilic and hydrophobic small molecules into the capsules and fibres.

Silk protein capsules and fibres are non-toxic to human cell lines. The capsules and fibres are effective at encapsulating drug molecules, and they may be used for the local release of those drugs, which provides for an enhanced pharmacological action, as exemplified through the use of two common antibiotics.

Due to their biocompatibility, self-assembling dynamic nature and effective delivery characteristics, the capsules of the invention represent a promising new class of structured protein materials.

The methods described herein provide a general strategy for generating microgels from silk protein, such as native B. mori silk. The highly viscous nature of silk, and its tendency to aggregate over time presents a significant challenge for the storage and processing of this potentially highly functional material. However, the work in the present case shows that silk protein processing using a multiphase flow overcomes these major limitations and allows flexible processing of native silk into a wide range of micron scale structures with different physical and chemical properties.

These materials offer great promise not only for the encapsulation and release of small molecules, but also offer a practical solution for the long term storage and controlled of native silk over with an increase of over two orders of magnitude relative to conventional storage under bulk conditions. Due to these special characteristics and in combination with the unique biocompatibility of silk based structures, silk microgels represent an attractive new class of material with tunable properties.

WO 2007/141 131 describes the preparation of protein assemblies based on spider silk protein.

Although WO 2007/141 131 refers to the preparation of fibres and spheres, there is no evidence to show that such structures could be prepared by the methods described.

WO 2007/141 131 only shows that threads may be prepared, and there is no suggestion that these could hold a component. Furthermore, the methods of WO 2007/141 131 rely on the use of laminar aqueous flows to form the thread structure. Thus, an aqueous silk-containing stream is brought into contact with aqueous ionic streams at a junction. The thread forms in the aqueous silk-containing stream, which is bounded by the aqueous ionic streams. Thus, WO 2007/141 131 makes use of water in water emulsions to form silk-based structures. Certain aspects and embodiments of the present invention may also be distinguished over WO 2007/141 131. For example, in certain embodiments, the capsule and fibres of the present case make use of a natural silk protein, such as reconstituted natural silk protein. In contrast, the work in WO 2007/141 131 is limited to spider silk recombinant protein.

The capsules and the fibres of the present case are also suitable for holding components within the shell or sheath. The structures described in WO 2007/141 131 are not said to be suitable for holding components, and there is no description of a capsule shell of fibre sheath.

The aggregation methods in WO 2007/141 131 are based on the use of an ionic aqueous stream. The work in WO 2007/141 131 also appears to describe a complete aggregation of the silk protein, whereas the methods of the present case allow for an assembly that is not an aggregation, and different structures with different surface characteristics may be prepared.

The present inventors have shown that the formation of capsules, such as spherical capsules, and fibres may be formed at the boundary of an aqueous phase and a

non-aqueous phase, such as an oil phase. Here, the shear forces are large and changes to those shear forces (by changes to the flow rate ratio of the non-aqueous flow to the aqueous flow at the junction) may be used to alter the structure of the product formed, and may also be used to change the nature of the silk protein assembly, for example to favour the formation of a non-aggregate assembly of silk protein, or to favour the formation of an aggregate assembly of silk protein where the fibrils are aligned perpendicular to, or linear to, the longitudinal axis of the structure.

As shown in the comparative examples in the present case, the inventors have studied the behaviour of silk in water in water emulsions. In contrast to the work in WO 2007/141 131 , the present inventors have found that ordered silk structures cannot be prepared from such fluid mixtures. The water in water emulsions were certainly not suitable for the formation of silk capsules.

WO 2014/012099 and WO 2014/012105 describe the encapsulation of compounds in silk particles. Both documents discuss increasing the beta-sheet content in silk fibronin in order to make the silk particles water insoluble. Apparently that this can also alter the rate of release of an encapsulated molecule and/or alter the rate of degradation of the silk matrix. The beta-sheet content of silk fibronin can vary from an amount of about 20% to about 75%. However, there is no discussion of the relative composition of the native and aggregated forms of beta sheets in the silk fibronin and how this relates to the behaviour of silk proteins in forming aggregated or non-aggregated assemblies of silk proteins. WO 2014/012099 also includes experimental results relating to the composition of a silk coating layer on the outside of a silk particle (see Figure 29 of WO 2014/012099, for example). The composition is broken down into the percentage of beta-sheets, random coil and alpha helix, amongst others, as measured by FTIR. However, the absorption band indicated for beta-sheets (1616-1637 cm^-1) does not distinguish between the native and aggregated forms of beta-sheets.

Hermanson et al. and EP 1757276 describe capsules, bags and balloons made from engineered spider silk C16. Hermanson et al. say that during formation of a capsule, C16 underwent a conformational change from a structure that was mainly random coil, to one that was beta-sheet rich. However, there is no detailed discussion about the silk structure. Indeed, neither document distinguishes between the native and aggregated forms of beta sheets or indicate percentage composition of alpha helix, beta-sheet (native) and random coil of the product capsules, bags and balloons.

The invention is now described in more detail with reference to the each relevant feature of the capsule and the fibre.

Capsules and Fibres

A capsule of the invention comprises a shell. The shell is a network that is formed from the assembly of a silk protein. The shell defines an internal space, which is suitable for holding a component. Thus, in one embodiment, the capsules of the invention extend to those capsules encapsulating a component within the shell. The shell may form a barrier limiting or preventing the release the component encapsulated within.

A fibre of the invention comprises a sheath. The sheath is a network that is formed from the assembly of a silk protein. The sheath defines an internal space, which is suitable for holding a component. Thus, in one embodiment, the fibres of the invention extends to those fibres encapsulating a component within the sheath. The sheath may form a barrier limiting or preventing the release of material encapsulated within.

In one embodiment, the internal space defined by the shell or sheath may be regarded as a hollow space which is substantially free of silk protein in an assembled form. Thus, an assembly of proteins is a part of the shell or sheath only. These are to be distinguished from particles and non-hollow fibres, where there is no substantial internal space.

In one embodiment, a capsule or a sheath holds within the internal space of the shell silk protein in non-assembled (or monomeric) form. For example, a substantially spherical capsule may hold silk protein in non-assembled form.

In one embodiment, a fibre has an internal network that is an assembly of the silk protein. This assembly may form a mesh of material that extends across the internal space defined by the sheath. In this embodiment, the internal space is nevertheless suitable for holding a component within.

The capsules of the invention do not have an internal network of an assembled silk protein.

The component may be releasable from the capsule, through pores that are present in the shell. In some embodiments, the pores are sufficiently small to prevent the component from being released. Thus, the assembly of proteins making up the shell may be at least partly disassembled thereby permitting release of material from within the shell. Similarly, a component may be releasable from a fibre through pores in the sheath.

In one embodiment, a capsule or a fibre holds water within the internal space of the shell. The water may be an aqueous solution comprising the protein. As noted above, in some embodiments, the shell holds a silk protein in non-assembled form.

An encapsulated material may be provided in addition to water and the silk protein that are for use in the assembly of the shell or sheath.

In one embodiment, a shell or sheath holds a non-aqueous phase within. An encapsulated material may also be provided within this non-aqueous phase. This embodiment is less preferred as the preparation of an oil dispersion in water may not yield capsules and fibres of the invention. The inventors have noted that when an aqueous continuous phase is used, and that phase contains the protein, a fibril network is formed through the aqueous phase, and the formation of discrete shells and sheaths at the phase boundaries may not result. For this reason, it is preferred that the second phase is an aqueous phase, and the silk protein is provided in this aqueous second phase, optionally together with a component to be encapsulated.

Where a capsule or fibre is said to encapsulate a component, it is understood that this encapsulated component may be present within the internal space defined by the shell or the sheath. In one embodiment, the encapsulant is also present, at least partially, within the pores of the shell.

The presence of a component within the internal space may be determined using suitable analytical techniques which are capable of distinguishing the shell material and the encapsulant. As described herein, silk protein capsules may be analysed by UV/vis spectroscopy, FTIR spectroscopy, and atomic force microscopy.

In one embodiment, each of the silk protein and the component (where that component is not a silk protein) may have a detectable label or suitable functionality that is independently detectable (orthogonal) to the label or functionality of the other. In one embodiment, each of the silk protein and the component has an orthogonal fluorescent label. For example, one has a rhodamine label and the other has a fluorescein label. Laser scanning confocal microscopy techniques may be used to independently detect the fluorescence of each label, thereby locating each of the silk protein and the encapsulant. Where the component signals are located at the same point as the signals from the silk protein, it is understood that the component resides within a pore of the shell.

Other labels, such as Nile Red dye may be used to label the assembly of the silk protein.

The general shape of the shell or the sheath, and therefore the shape of the capsule or the fibre, is not particularly limited. In practice however, the shape of the capsule may be dictated by its method of preparation. In the preparation methods described herein, the capsule and the fibre may be prepared using fluidic formation techniques.

Typically, a capsule shell is formed at the boundary of a discrete (or discontinuous) phase in a continuous phase. For example, one phase may be an aqueous phase (typically the second phase), and the other may be a water immiscible phase (typically the first phase). The discrete region may be a droplet, having a substantially spherical shape. The shell formed is therefore also substantially spherical.

Adaptations to the fluidic conditions allow for the formation of discrete regions that are not spherical. For example, the discrete region may take the form of a slug (or cylinder).

In certain embodiments, a capsule may be obtained where the shell has a substantially spherical shape. In this embodiment, the assembly of a silk protein is not an aggregation.

A fibre sheath may be formed at the boundary of a second phase held between two immiscible first phases. For example, one phase may be an aqueous phase (typically the second phase), and the other may be a water immiscible phase (typically the first phase). The second phase may be a discontinuous phase in a continuous first phase. Thus the second phase has a discrete size, and the fibre sheath that is formed at the boundary has a discrete length. Alternatively, the second phase may be a continuous phase within continuous first phases. Thus, the second phase may be a fluid flow held between two fluid flows of the first phase. Here, there is no discrete size along the flow direction. The fibre that is formed at the boundary has no discrete size along its length.

A capsule or fibre may be subjected to a drying step, which reduces the amount of solvent (for example, water) in and around the structure. As a result of this step, the structure may shrink in size. At first the structure maintains a substantially spherical, cylindrical or fibrous shape. After further drying, the structure may partially or fully collapse in on itself. The structural integrity is maintained and the shell or fibre simply distorts to accommodate changes in the internal volume. Thus, the capsules and fibre of the invention include those structures where the shell is, for example, an at least partially collapsed sphere, cylinder or fibre.

Given the formation of a shell or a sheath at a fluid boundary (for example, at the fluid boundary of a droplet dispersed in an immiscible continuous phase), references to the dimensions of the fluid encompassed by that boundary (such as a droplet) may also be taken as references to the dimensions of the capsule or fibre.

The inventors have established that capsules and fibres that have been shrunk, for example by desolvation, may subsequently be returned to their original substantially spherical shape, by, for example, resolvating the capsule.

In some embodiments, a formed capsule is not dehydrated after formation.

The size and shape of a capsule may therefore be assumed from the size and shape of the discrete region that is formed during the flow preparation methods described herein. The size and shape of a fibre may be assumed from the size and shape of the second fluid flow between flows of the first phase.

Alternatively, the shape of a capsule or a fibre may be determined by simple observation of the formed capsule using microscopy, such as light microscopy, scanning electron microscopy or confocal microscopy. Where the shell material comprises a label, the detection of the label through the shell will reveal the capsule shape. For example, where the label is a fluorescent label, laser scanning confocal microscopy may be used to locate the shell material and its shape.

A capsule generally refers to a substantially spherical capsule. Such are formed from substantially spherical droplets.

A fibre generally refers to an elongate structure, having a discernable length that is greater than the width of that fibre.

The size of a capsule is not particularly limited. In one embodiment, the capsule is a microcapsule and/or a nanocapsule.

In one embodiment, each capsule has an average size of at least 0.1 , 0.2, 0.5, 0.8, 1 , 5 or 6 μΠΊ in the largest cross section.

In one embodiment, each capsule has an average size of at most 200, 100, 75, 50, 20, 10 or 8 μΠΊ in in the largest cross section.

In one embodiment, the capsule size is in a range where the minimum and maximum sizes are selected from the embodiments above. For example, the capsule size is in range from 0.8 to 100 μηη, such as 5 to 10 μηη in the largest cross section. Average size refers to the numerical average of measured diameters for a sample of capsules. Typically, at least 5 capsules in the sample are measured. A cross section measurement is taken from the outmost edges of the shell.

In one embodiment, the fibre is a microfibre and/or a nanofibre. A fibre has a length and a width.

In one embodiment, a fibre has an average length of at least 3, 4, 5 or 6 μηη.

In one embodiment, a fibre has an average length of at most 1 ,000, 100, 75, 50, 20, or 10 μΠΊ.

In one embodiment, the width of a fibre in its largest cross section is at least 0.1 , 0.2, 0.5 or 1.0 μΠΊ.

In one embodiment, the width of a fibre in its largest cross section is at most 100, 50, 25, 20, 10, 5, or 2.5 μηη.

The cross-section of a capsule, or the length or width of a fibre, may be determined using simple microscopic analysis of the formed structure. For example, formed capsules and fibres may be placed on a microscope slide and the capsules analysed. Alternatively, the capsule or fibre size may be measured during the preparation process, for example as the capsules and fibres are formed in a channel of a fluidic device (i.e. in line).

The measurement of the cross section may also be achieved using techniques related to the detection of a detectable label that is associated with the silk protein in the assembly, or functionality inherent in the silk protein itself. The silk protein may have a fluorescent label which may be detected by laser scanning confocal microscopy techniques or the shell material may be labelled with a label that is bound to the shell after capsule formation. The presence of multiple labels within and around the capsule shell allows the cross-sectional shape to be determined, and the largest cross-section measured. Alternatively the methods of analysis may make use of inherent functionality present in the silk protein or in the silk protein aggregate (such as a-helix and β-sheet structure).

In the preparation method described herein a capsule is prepared using a fluidic droplet generation technique. The capsule shell is formed in a droplet or a slug, which is created in a channel of a fluidic droplet generating device, at the boundary of, for example, an aqueous phase of the droplet or slug with a continuous water-immiscible phase. The size and shape of the capsule is therefore substantially the same as that of the droplet or the slug. Similarly, a fibre sheath is formed at the boundary of, for example, an aqueous flow between water- immiscible flows, and the size and shape of the fibre is substantially the same as that of the aqueous flow between the water-immiscible phase.

The shell or sheath thickness may be at most 10 μηη, at most 5 μηη, at most 2 μηη, at most 1 μηη, or at most 0.5 μηη. Shell and sheath thickness may be measured in the same way as the shell and sheath cross sections, as described above, measuring from the outer edge of the shell or sheath to the internal edge of the shell or sheath.

The present inventors have established that the capsules and fibres of the invention may be prepared with a low size distribution. This is particularly advantageous, as a large number of capsules may be prepared, each with predictable physical and chemical characteristics.

In one embodiment, a capsule or fibre has a relative standard deviation (RSD) of at most 1.5%, at most 2%, at most 3%, at most 4%, at most 5%, at most 7%, or at most 10%.

As previously noted, the shell or sheath has pores. The pores are gaps in the assembly of silk protein.

In one embodiment, the pores may be of a size to permit the passage of material therethrough. For example, components encapsulated within may pass through the pores of to be released from the capsule or fibre. Conversely, the pores may be of sufficient size to allow components to pass into the shell or fibre internal space, and thereby become encapsulated. Such may be referred to as a passive diffusion encapsulation step. Such a technique may be used to provide a structure having an encapsulant within. As described herein, the present inventors have provided alternative methods for the encapsulation of material in the shell and sheath preparation steps. Such methods allow for a more efficient loading of material, as the material is entirely encapsulated within the shell or sheath.

In one embodiment, the pores may be of a size that is too small to permit passage of material therethrough. For example, encapsulated components may be prevented from passing through the pores of the shell, and therefore cannot be released from the capsule or the fibre. Such material may be released from, for example, disrupting the assembly of a silk protein. Disruption of the shell or sheath in this way creates larger pores through which material may pass.

The size of a pore may be gauged experimentally using a range of encapsulated

components each having a different cross-section, such as a different diameter. The cross- section may be known or may be predicted based on an understanding of the likely configuration of the component. The pore size may be determined based on which components are released from the capsule and which are not.

A capsule or fibre comprising an encapsulated component may be prepared using the methods described herein. Once the capsule or fibre (with encapsulant) is prepared, the capsule or fibre and its aqueous surroundings may be analysed for loss of material from within the shell out to the external phase. The encapsulated compounds may have an analytical label to aid detection. Suitable labels include fluorescent labels which are detectable using standard fluorescence microscopy techniques.

In one embodiment, the pore size is at most 20, at most 15, at most 10, at most 5, at most 1 or at most 0.5 μηη.

In one embodiment, the pore size is at most 500, at most 300 nm, at most 200, at most 100, at most 50, or at most 20 nm.

In one embodiment, the pore size is at least 0.5, at least 1 , or at least 5 nm.

In one embodiment, the pore size is in a range where the minimum and maximum pore sizes are selected from the embodiments above. For example, the pore size is in range 1 to 20 nm.

The present inventors have found that the pore size of a formed product, such as a capsule, may be increased by washing the capsule with fluid, such as an aqueous fluid, for an extended period, for example continuous washing for at least 30 min, at least 60 min, or at least 90 min. The continuous washing is also associated with a disintegration of the assembly

As expected, the pore size is influenced by the amount of silk protein present in the fluid phases used during the capsule and fibre preparation. Increasing the amount of protein present (for example increasing the concentration of protein) is believed to increase the density of material in the assembly, thereby reducing the size of the pores in the formed assembly. It is noted also that increases in the silk protein concentration are also associated with changes in the shape and structure of the assembly. At higher silk protein

concentrations the formation of fibres is favoured and the formation of aggregates of the silk protein are favoured. Capsules, such as spherical capsules, and non-aggregates of the silk protein are favoured at lower silk protein concentrations.

Where an encapsulant of a relatively small size is to be encapsulated, the capsule may be prepared with pores of relatively small diameter, thereby to limit or prevent loss of the encapsulant out of the shell. Where a relatively large encapsulant is to be encapsulated, the pore size may be larger.

A capsule shell or a fibre sheath may comprise one or more layers of material, where each layer is an assembly of a silk protein. The layers may be formed from different silk proteins, thereby to provide distinct layers within the capsule shell. Preferably, the layers are obtainable from the same silk protein. Neighbouring layers in a capsule shell may be connected through the interaction of β-sheet structures, for example, between protein molecules in each layer.

The shell and the sheath may be viewed as a mesh extending in three dimensions. This mesh if the assembly of a silk protein. Although the assembly may have a depth of material, such as a thickness described herein, it is understood that the formation of the shell and the sheath will nevertheless provide an internal space in which a component may reside. Thus, the present invention is not intended to encompass particles having no internal space.

Alternatively the capsule shell or fibre sheath may comprise a plurality of concentric layers of network material that are not interlinked. For example there is no assembly between layers of a multi-layered capsule or fibre. In any such embodiment, the reference to size refers to the cross section of the outermost structure.

In a multi-layered structure one or more capsules may be held within a fibre, and one or more fibres may be held in a capsule. One or more capsules may be held in a capsule, and one or more fibres may be held in a fibre. One or each capsule or fibre may hold an encapsulant.

In such a nested capsule of fibre there may be an internal space between the inside wall (of the shell or sheath) of the outer structure and the outer shell wall of the inner structure, where the structure may be a capsule or fibre. This space may be suitable for holding a component, such as a component described herein. The inner structure may itself hold a component, either instead of or in combination with the outer structure holding a component. Where the inner and the out structures hold a component, these components may be the same or different.

Where a component is held in the internal space between the inside wall of the outer structure and the outer wall of the inner structure, that component may be a hydrophobic component. Methods for the preparation of nested capsules make use of water in oil in water (for example) droplets, where the oil phase is ultimately incorporated as a fluid within the space between capsule shells formed at the droplet boundaries.

In other embodiments this internal space may simply hold a fluid, such as the oil used in the preparation methods described herein. This may be the case where the inner structure is only very slightly smaller than the outer structure, and there is insufficient space to hold a component between the walls of the inner and outer structures.

As discussed above, the shell or sheath may include detectable labels or detectable functionalities which are present or associated with the silk protein.

A detectable functionality is functionality of the assembly of a silk protein having a characteristic that is detectable over and above the characteristics that are present in other components of the capsule, or even other functionalities of the same component. The detectable functionality may refer to a particular chemical group that gives rise to a unique signal in, for example, IR, UV-VIS, NMR or Raman analysis. The functionality may be a radioactive element. Typically a part of the assembly of a silk protein or the encapsulant is provided with a detectable label, as the introduction of a chosen label allows the use of techniques that are most appropriate for the property that is to be measured.

The assembly of a silk protein is stable and may be stored without loss of the assembly structure. The integrity of the assembly therefore allows the capsule to be used as a storage vessel for an encapsulant. The capsules and fibres of the invention are thermally stable and the shell is known to maintain its integrity at least up to 95°C.

The capsules of the invention have a long shelf life. The present inventors have confirmed that structural integrity is maintained for at least 10 months.

In one embodiment, a capsule has a shell that is or consists of an assembly of a silk protein. In one embodiment, a fibre has a sheath that is or consists of an assembly of a silk protein.

Silk Protein

The capsules and fibres of the present case have shells and sheaths that comprise an assembly of a silk protein. A reference to a silk protein may be a reference to silk, such as naturally occurring silk, which typically comprises a variety of different silk proteins. The amounts and identity of each protein are dependent upon the source of the silk, for example from moth or spider.

Variant and derivative forms of naturally occurring silk are also suitable for use in the present invention. The silk protein may a recombinant silk protein.

Silk proteins are particularly suitable for use in the present case owing to their propensity to assemble, such as aggregate, for example to form fibrils. Thus, a silk protein may be present as an aggregation, for example in a cylindrical capsule or a fibre.

The present inventors have found that silk fibroin, and particularly, native silk fibroin, may be used to prepare capsules and sheaths using fluidic techniques.

In the worked examples of the present case native silk protein freshly extracted from B. mori silkworm gland is used. Native silk protein may be referred to as native silk fibroin (NSF).

As explained above, the present inventors have also established methods for the

preparation of a capsule where the silk protein is present in non-aggregate form. The ability to assemble a silk protein in this way is surprising and allows the capsule to be used to deliver components, such as the silk protein itself, to desired locations.

In one embodiment, a silk protein is a protein from a silk obtained or obtainable from a silkworm or a spider. In one embodiment, the silk protein is a silk protein from a silk from an organism that is a member of a species selected from the group consisting of Bombyx, Nephila, Araneus, Argiope, Latrodectus, Leucauge, Plectreurys, and Kukulcania.

The silk protein may be a silkworm silk protein.

The silk protein may be a silkworm silk protein obtained or obtainable from Bombyx mori. The silk protein may be a natural (non-recombinant) silk protein.

The silk protein may be a spider silk protein, such as a spider silk protein obtained or obtainable from an orb weaver spider, such as Nephila clavipes or Nephila edulis.

In one embodiment, the silk protein is a silk fibroin, such as an unspun silk fibroin.

A silk protein may refer to fibroin optionally together with sericin. For example, silkworm silk comprises fibroin, which may be a dimer including light chain and heavy chain components, optionally together with sericin, which may forma coating on the fibroin (see Vapri et al.). Fibroin may be separated from sericins by treatment with alkaline solution.

The silk protein may be associated with a glycoprotein, for example P25.

In one embodiment, a silk protein is a fibroin, substantially free of sericin.

A silk protein may refer to spidroin proteins 1 or 2 (Spidroin I or II). Spidroin proteins are believed to be free of sericin.

A silk protein may refer to a reconstituted silk protein. Thus, for example, a silk fiber is obtained from a silk worm or a spider, and the silk protein molecules making up the fiber, such as fibroin, are extracted, and prepared as an aqueous silk solution. A method for the preparation of an aqueous silk solution is summarized in Vapri et al. (see Figure 1 and accompanying text).

A silk protein may refer to native silk.

A silk protein may be glycosylated, for example at most 1 %, at most 5% or at most 10 % glycosylated. For example, the major protein component of spider silk has about 5% glycosylation (see Vollrath et al.).

A reference to a silk protein includes a reference to modified proteins, such as those having an analytical label. Where a label is provided, that label does not interfere with the protein's ability to form aggregates.

In the methods of preparation described herein, a silk protein is permitted to self-assemble thereby to form an aggregate of that protein as a capsule shell or a fibre sheath. In these methods the reference to a silk protein is typically a reference to that protein not in an assembly with another protein. Thus, a capsule shell or a fibre sheath consists essentially of the silk protein.

A silk protein may be provided in its native or functioning state, and it may be denatured during the process of preparation to allow for the formation of the assembly. The denaturing step may be required where the native state of the protein is not associated with the formation of aggregates, such as the formation of amyloid structures.

In one embodiment, the silk protein is a silkworm protein obtained or obtainable from Bombyx mori or a variant thereof, wherein the variant is a polypeptide having at least about 50% identity to a silk protein obtainable from Bombyx mori. The variant may be a polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to a silk protein obtainable from Bombyx mori.

Variants which are polypeptides comprising an amino acid sequence at least 50% identical to a silk protein obtainable from Bombyx mori may comprise one or more amino acid additions, substitutions and/or deletions relative to the amino acid sequence of the silk protein obtainable from Bombyx mori. Variants may comprise one or several amino acid additions, substitutions and/or deletions relative to the amino acid sequence of a silk protein obtainable from Bombyx mori. Variants may comprise 1 - 150, 1 - 100, 1 - 50, 1 - 20 or 1 - 10 amino acid additions, substitutions and/or deletions relative to the amino acid sequence of a silk protein obtainable from Bombyx mori.

Amino acid sequence identity and similarity and nucleic acid sequence identity may be measured using standard bioinformatics software tools, such as the freely available

EMBOSS, or BLAST, software tools. Default parameters are generally used. For example EMBOSS Needle pairwise sequence alignment can be used to determine amino acid sequence identity. EMBOSS Needle pairwise sequence alignment, which uses the

Needleman-Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)), can be used to determine amino acid sequence similarity, for example using default parameters and using a BLOSUM scoring matrix such as the BLOSUM62 scoring matrix. Default parameters may be used with a gap creation penalty = 12 and gap extension penalty = 4.

Assembly

The assembly is a network of silk protein molecules held together by non-covalent bonding. The assembly may be a non-aggregated assembly, for example in the shell of a capsule, or an aggregated assembly, for example in a sheath of a fibre. In certain embodiments, the assembly is a collection of a silk protein in a non-aggregated form. Such an assembly has a very low content of β-sheet aggregate form. Thus the assembly does not contain a substantial fibril content.

Without wishing to be bound by theory, the silk protein is believed to form an assembly through non-specific hydrogen bond interactions between protein molecules. The proteins are also believed to interact via the side chain groups of Tyr and Trp amino acids. Whilst it is noted that an aggregation is based on hydrogen bonding between β-sheets in different protein molecules, the non-aggregated assembly does not contain a large amount (if any) of this type of interaction. This is apparent from the FTIR analysis of the assembly, for example, which is described below.

The level of β-sheet aggregate form may be determined experimentally, for example by FTIR measurements. For example, as a silk protein under goes an aggregation a change may be observed in the intensity of a peak in the FTIR spectrum. For example, by monitoring changes to the amide I (between 1600 and 1700 cm^-1) and II (1510 and

1580 cm^-1) absorption bands.

As described herein for the B. mori silk protein, increased β-sheet aggregation between protein molecules is associated with a shift in an absorption peak from ca. 1630 cm^"1 to ca. 1620 cm^-1, and a change in the intensity of that absorption peak. Such an assembly also has a very low content of β-sheet (turn) antiparallel aggregate. An increase in the β-sheet (turn) antiparallel aggregate content is seen on aggregation and is associated with a decrease in the intensity of an absorption peak centred at ca. 1700 cm^-1 in the FTIR spectrum. The assembly has a high content of random coil. A decrease in the random coil content is seen on aggregation and is associated with an increase in the intensity of an absorption peak centred at ca. 1650 cm^-1 in the FTIR spectrum.

An assembly where the silk protein in a non-aggregated form may be said to have a disordered state. Thus, the a-helix, β-sheet (native) and random coil content of a non- aggregated form is greater than the β-sheet (aggregate) and β-sheet (antiparallel aggregate) content of the assembly. For example, the a-helix, β-sheet (native) and random coil content is at least 55%, at least 65%, at least 75%, at least 85%, at least 95% of the total content of the assembly.

An assembly where the silk protein in an aggregated form may be said to have an ordered state. Thus, the a-helix, β-sheet (native) and random coil content of a non-aggregated form is less than the β-sheet (aggregate) and β-sheet (antiparallel aggregate) content of the assembly. For example, β-sheet (aggregate) and β-sheet (antiparallel aggregate) content is at least 55%, at least 65%, at least 75%, at least 85%, at least 95% of the total content of the assembly. The % values given above are based on the intensity of the a-helix and β-sheet peaks in the FTIR spectrum (see, for example, Figures 3 and 8 in the present case). For comparative measurements between silk free, assembled and aggregated forms, the silk protein concentration is identical.

Changes in the assembly of the silk protein may also be monitored by fluorescent spectroscopy. Changes in the blue region of the excitation/emission spectrum are associated with changes in the β-sheet aggregate content of an assembly. For example, excitation maximum at 315 nm and emission maximum at 425 nm may be used. An increase in intensity at these maxima is associated with an increase in the β-sheet aggregate content.

The present inventors have also found that a silk protein may form an aggregate of the silk protein. In alternative aspects of the invention there is provided a capsule having a shell of material that is an aggregation of a silk protein. Also provided is a fibre having a sheath of material that is an aggregation of a silk protein.

Preferably, a capsule of the invention is a capsule having a shell of material that is a non- aggregate assembly of a silk protein. In the methods of the invention the capsule is generally formed with a non-aggregate assembly of a silk protein. Thus, the native β-sheet content is high and the aggregate β-sheet content is low, as described above.

A reference to a capsule having a shell that is an assembly of a silk protein may also be construed as a reference to a capsule having a shell that a non-aggregate of a silk protein.

A fibre of the invention may be a fibre having a sheath of material that is an aggregation of a silk protein. In the methods of the invention the capsule is generally formed with an aggregation of a silk protein. Thus, the native β-sheet content is not high and the aggregate β-sheet content is not low.

In a shell or sheath where the silk is an aggregation, the aggregate β-sheet content may be 40% or more, 50% or more, or 60 % or more, as a fraction of the total content aggregate, native and anti-parallel β-sheet, random coil, and a-helix content of the shell or sheath.

In certain embodiments, the assembly is a collection of a silk protein in an aggregated from. Such an assembly has a very high content of β-sheet aggregate form. Thus the assembly has a substantial fibril content.

The presence (or not) of fibrils within a capsule shell or fibre sheath may be confirmed by study of the structure surfaces by AFM. The aggregation of the protein molecules may occur spontaneously under the conditions of the fibre formation step.

The assembly may be regarded as a cross-linked polymer network.

The assembly may be fibrous silk protein aggregates. The structure of the assembly may be referred to as an amyloid structure.

The assembly, and therefore the capsule shell, may be insoluble, for example in water.

A silk protein present within an assembly is typically present in a misfolded state.

In one embodiment, the silk protein possess a beta sheet within its secondary structure.

Typically, the silk protein molecules in an aggregation are bound together in an assembly by interaction of beta sheet structures between neighbouring silk protein molecules ("cross-beta sheet"). Thus the quaternary structure includes an arrangement of silk proteins interacting through the beta sheet.

In one embodiment, the beta sheet structures are formed during the assembly process.

The assembly may include a plurality of silk protein fibrils that are cross-linked.

The assembly may be an aggregate having fibrils of the silk protein. The fibrils may be present in the shell of a cylindrical capsule, or may be present in the sheath of a fibre. The fibrils may be orientated along an axis of a fibre. A cylinder or a fibre has a longitudinal axis which is the axis along the length of the cylinder or the fibre. Generally this axis corresponds to the length of the structure in the flow channel along the direction of flow. In one embodiment the fibrils are parallel to the longitudinal axis. This arrangement may be present in the shell of the cylinder. In one embodiment the fibrils are aligned substantially

perpendicular to the longitudinal axis. This arrangement of fibrils may be present in the sheath of a fibre.

An arrangement of fibrils parallel to the cylinder longitudinal axis is associated with lower concentrations of silk protein and higher shear rates in the flow methods of preparation.

In one embodiment, a fibre has a sheath that is an aggregation of a silk protein, where the aggregation comprises fibrils of the assembled silk protein, and the silk fibrils are aligned in the sheath along the longitudinal axis of the fibre. Such an arrangement is preferred where the fibre is relatively short, for example where the length of the fibre is no more than 15 times, such as no more than 5 or 10, the cross section of the fibre.

In another embodiment, the silk fibrils are aligned in the sheath along the latitudinal axis of the fibre. Such an arrangement is preferred where the fibre is relatively short, for example where the length of the fibre is more than 15 times, such as more than 20 time, the cross section of the fibre. The inventors have found that a non-aggregate assembly of a silk protein may be converted to an aggregate assembly of a silk protein. For example, heat, changes in pH and changes in hydration levels may be used to promote the formation of, for example, β-sheet aggregates.

In one embodiment, a shell or sheath may comprise an assembly that is a mixture of silk protein in non-aggregate form and aggregate form.

Where a capsule or fibre holds a silk protein within, the conditions for the generation of the aggregate assembly of a silk protein may also lead to the assembly, such as the aggregate assembly, of the silk protein within the shell or sheath. This assembly may then at least partially occupy the internal space of the capsule or fibre. In one embodiment, the assembly of the silk protein within the capsule or fibre may yield a particle or non-hollow fibre.

Young's Modulus

A capsule or a fibre may have a Young's modulus with certain limits. The modulus may be a DMT (Derjaguin-Muller-Toporov) modulus.

The Young's modulus may be determined at room temperature, such as 20°C, using AFM nanoindentation methods, such as described herein.

In one embodiment, a capsule or a fibre has a Young's modulus of at least 0.5, at least 1 .0, at least 1.5, or at least 2.0 GPa.

In one embodiment, a capsule or a fibre has a Young's modulus of at most 6.0, at most 7.0, at most 8.0, or at most 10.0 GPa.

The modulus may refer to the number average modulus of a collection of capsules or fibres. The modulus of the capsule or fibre may be measured in air or liquid (such as water).

Encapsulant

A capsule or fibre of the invention may be used to encapsulate a component (the encapsulant). In one embodiment there is provided a capsule or a fibre comprising an encapsulant. The capsule or fibre is suitable for storing a component, and this component may be later released as required at a chosen location.

It is understood that a reference to an encapsulated component is not a reference to a solvent molecule. For example, the encapsulated component is not water or is not an oil or an organic solvent. The encapsulant is therefore a component of the capsule that is provided in addition to solvent that may be present within the shell. Otherwise, the component is not particularly limited.

In one embodiment, an encapsulated component is a silk protein for use in the preparation of the capsule shell or the fibre sheath. This protein is not in an assembly with the silk proteins that make up the shell or the sheath. An encapsulated silk protein may be present in monomeric (non-assembled, such as non-aggregate) form.

In the methods of the invention the capsule shell and the fibre sheath are prepared from a fluid containing a silk protein. Not all the silk protein may assemble to form the shell or sheath. Some of the protein may be retained within the shell or sheath. In one embodiment, the silk protein is retained within the shell or sheath in a non-assembled form.

A silk protein may be contained within the shell or sheath, and may be contained in addition to another encapsulant. Thus, a further encapsulant may be a component of the capsule or fibre that is provided in addition to the encapsulated silk protein.

The capsules and fibres of the invention may be used to encapsulate a wide range of components.

In one embodiment, the encapsulated component has a molecular weight of at least 100, at least 200, at least 300, at least 1 ,000, at least 5,000 (1 k), at least 10,000 (10k), at least 50,000 (50k), at least 100,000 (100k) or at least 200,000 (200k).

In one embodiment, the encapsulant is a therapeutic compound.

In one embodiment, the encapsulant is a biological molecule, such as a polynucleotide (for example DNA and RNA), a polypeptide (such as a protein) or a polysaccharide.

In one embodiment, the encapsulant is a polymeric molecule, including biological polymers such as those polymers mentioned above.

In one embodiment, the encapsulant is a virus, antibody, microorganism, or hormone.

In one embodiment, the encapsulant is an antibody.

Where the encapsulant is a protein, that protein differs from the protein that makes up the shell of the capsule. Thus, the protein may not be a silk protein, or may be a different silk protein.

The size of the capsule is selected so as to accommodate the size of the encapsulant.

Thus, the internal diameter (the distance from innermost wall to innermost wall) is greater than the greatest dimension of the encapsulant. In one embodiment, the encapsulant has a detectable label. The detectable label may be used to quantify and/or locate the encapsulant. The label may be used to determine the amount of encapsulant contained with the capsule.

In one embodiment, the detectable label is a luminescent label. In one embodiment, the detectable label is a fluorescent label or a phosphorescent label.

In one embodiment, the detectable label is a visible label.

In one embodiment, the fluorescent label is a rhodamine or fluorescein label.

Methods for the Preparation of Capsules and Fibres

In one aspect of the invention there is provided a method for the preparation of a capsule having a shell that comprises an assembly of a silk protein, wherein the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a dispersion of discrete regions, such as droplets, of the second phase in the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a silk protein, thereby to form a capsule shell at the boundary of the discrete region, wherein the first and second phases are immiscible.

Also provided is a method of preparing a fibre having a shell that comprises an assembly of a silk protein, wherein the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a fluid flow of the second phase between flows of the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a silk protein, thereby to form a fibre sheath shell at the fluid boundaries of the second phase, wherein the first and second phases are immiscible.

In both methods the silk protein is typically provided in a second phase that is an aqueous phase. Where the protein is provided in a first phase that is an aqueous phase there is a tendency for the protein to assemble as fibrils throughout the aqueous phase. Thus an assembly of the silk protein such as to form a discrete shell at the boundary of the first aqueous phase and the second phase may not be observed.

In the methods of the invention a dispersion of the second phase is created within the continuous first phase. In one embodiment, the second phase is an aqueous phase and the other phase is a water immiscible phase, for example an oil phase.

In one embodiment, the second phase comprises a component for encapsulation, and the step (i) provides a capsule having a shell encapsulating the component.

In one embodiment, the method further comprises the subsequent step of (ii) collecting the outflow from the channel, thereby to obtain a droplet, which has a capsule. In one embodiment, the flow of the second phase is brought into contact with the flow of the first phase substantially perpendicular to the first phase. In this embodiment, the channel structure may be a T-junction geometry. The path of the channel may follow the path of the flow of the first phase, in which case the second flow will be substantially perpendicular to the resulting combined flow in the channel. Alternatively, the path of the channel may follow the path of the flow of the second phase, in which case the first phase flow will be

substantially perpendicular to the resulting combined flow in the channel.

Methods utilising a T-junction geometry provide discrete regions, typically droplets, of the aqueous phase in the oil phase as a result of induced shear forces within the two phase system.

In one embodiment, an additional flow of the first phase is provided. The first phase flows are brought into contact with each side of the second phase flow in a channel, and the flow of phases is then passed through a region of the channel of reduced cross-section (an orifice) thereby to generate a discrete region, preferably a droplet, of the second phase in the channel. Such methods, which have an inner second phase flow and two outer first phase flows, are referred to as flow-focussing configurations.

Methods using flow-focussing techniques provide discrete regions, typically droplets, of the second phase in the first phase as a result of the outer first phase applying pressure and viscous stresses to the inner second phase, thereby generating a narrow flow of that phase. This narrowed flow then separates into discrete regions, typically droplets, at the orifice or soon after the combined flow has passed through the orifice.

In one embodiment, the discrete region is a droplet, such as a substantially spherical droplet. In one embodiment, the discrete region is a slug (or a cylinder).

After the discrete region is formed in the channel, the discrete region may be passed along the channel to a collection area. The residence time of the discrete region in the channel is not particularly limited. In one embodiment, the residency time is sufficient to allow the shell to form.

As the discrete region is passed along the channel it may be subjected to a mixing stage whereby the components of the discrete region are more evenly distributed around that discrete region. In one embodiment, the channel comprises a winding region. The winding region may take the form of a substantially sigmoid path through which the discrete region is passed.

In one embodiment, the second phase further comprises a component for encapsulation, and the step (i) provides a capsule encapsulating the component. Discrete regions of second phase are generated in the channel as the immiscible first phase shears off the second phase. The frequency of shearing is dependent on the flow rate ratio of the two phases. The inventors have also established that the flow ratio may be used to control the shape of the formed discrete regions and therefore the shape of the resulting capsule. Typically, spherical discrete regions are favoured where the flow rate of the first flow is higher than the flow rate of the second flow.

In one embodiment, the ratio of flow rates for the aqueous to the oil phase is 1 :X, where X is 1 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, or 10 or more.

In one embodiment, the ratio of flow rates for the aqueous to the oil phase is 1 :X, where X is at most 100, at most, 50, at most 20, or at most 15.

The inventors have also found that the concentration of the silk protein in the second phase may be used to control the shape of the formed discrete regions and therefore the shape of the resulting capsule. Typically, spherical discrete regions are favoured where the concentration of the silk protein in the second phase is relatively low.

In one embodiment, the concentration of the silk protein in the second phase is at least 0.01 , at least 0.05, at least 0.1 , at least 0.5, or at least 1 .0 mg/mL.

In one embodiment, the concentration of the silk protein in the second phase is at most 50, at most 25, at most 15, at most 10, at most 8, or at most 7 mg/mL.

In one embodiment, the concentration of the protein in the first or second phase is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the concentration of the concentration of the protein in the first or second phase is in the range 1 to 8 mg/mL.

In one embodiment, the concentration of the protein in the second phase is at least 0.05, at least 0.1 , at least 0.2, at least 0.3, at least 0.5, at least 1 .0, at least 5.0 or at least 10 μΜ. In one embodiment, the concentration of the protein in the first or second phase is at most 500, at most 200, at most 100, at most 75, at most 50 μΜ.

In one embodiment, the concentration of the protein in the first or second phase is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the concentration of the concentration of the protein in the first or second phase is in the range 1 to 50 μΜ.

As shown herein, the inventors have established that changes in the silk protein

concentration may be used to control the shape and morphology of the capsule or fibre. Typically, a non-aggregated assembly of a silk protein is favoured where the concentration of the silk protein in the second phase is low, and an aggregated assembly of a silk protein is favoured where the concentration of the silk protein in the second phase is high. Where it is intended to prepare a non-aggregated assembly of a silk protein, the concentration of the silk protein in the second phase may be at most 15, at most 10, at most 8, at most 7, at most 5, at most 3, or at most 2 mg/mL.

Where it is intended to prepare a non-aggregated assembly of a silk protein, the

concentration of the silk protein in the second phase may be at most 25, at most 20, at most 10, or at most 5 μΜ.

Where it is intended to prepare an aggregated assembly of a silk protein, the concentration of the silk protein in the second phase may be at least 3, at least 5, at least 7, at least 10, or at least 15 mg/mL.

Where it is intended to prepare a non-aggregated assembly of a silk protein, the

concentration of the silk protein in the second phase may be at least 5, at least 10, at least 20, at least 25 μΜ.

Changes in silk protein concentration and/or changes in flow rate ratios may be used to control capsule and fibre shape and morphology.

In one embodiment, the flow rate is selected so as to provide a set number of droplets per unit time (for example, droplets per second).

The droplets may be prepared at a rate of at most 10,000, at most, 5,000, at most 1 ,000 or at most 500 Hz.

The droplets may be prepared at a rate of at least 1 , at least 10, at least 50, at least 100, or at least 200 Hz.

In one embodiment, the droplets may be prepared at a rate that is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the rate is in range 100 to 500 Hz.

In one embodiment, a method may comprise the subsequent step of (ii) at least partially drying the capsule or fibre. The drying step at least partially removes solvent (which may be water or organic solvent) from the capsule or fibre and may be referred to as desolvation.

There are no particular limitations placed on the method for drying the capsules and fibres. In one embodiment, the capsules obtained may simply be allowed to stand at ambient conditions, and the solvent permitted to evaporate.

In one embodiment, a method optionally comprises a washing step, whereby an obtained capsule or fibre is washed with a solvent. The purpose of the washing step may be to remove surfactant (where used) or any other component used in the shell- or sheath-forming step. In one embodiment, a method comprises the drying the capsule or fibre and subsequently resolvating the capsule or fibre. The resolvation step may be performed minutes, hours, days, weeks or months after step (ii) is complete.

In one embodiment, a reference to a size of a droplet is also a direct reference to a size of a capsule. The droplet is a droplet formed in a channel of a fluidic device or a droplet that is collected from the channel of such a device. The size refers to a droplet that has not been subjected to a drying step.

A capsule formed directly after preparation is substantially spherical. Desolvation of the capsule may result in the collapse of the capsule as the spherical edge becomes distorted. The shell material appears to fold in a random manner.

In the preparation method described herein, a droplet is formed and the shell of a capsule forms at the interface of the droplet. The formed droplet may be subjected to a desolvation step, thereby resulting in the shrinkage of the capsule shell. In one embodiment, the size of the capsule refers to the size of a capsule that has been subjected to a dehydration step.

The flow rate of the first phase and/or the second phase may be varied to allow preparation of droplets, and therefore capsules, of a desired size. As the flow rate of the first phase is increased relative to the second phase, the average size of the droplet decreases, and the formed capsule size decreases also.

Typically, the flow rate of the first phase is at least 1.5, 2, 3, 4, 5 or 10 times greater than that of the second phase, as noted above.

In one embodiment, the flow rates of the first and the second phases are selected so as to provide droplets having a desired average diameter.

The average particle size may be determined from measurements of a sample of droplets collected from the flow channel using simple microscopy techniques.

In one embodiment, the each droplet is a microdroplet.

In one embodiment, the each droplet is a nanodroplet.

In one embodiment, the average size of the droplet is at least 0.1 , 0.2, 0.5, 0.7, 1 , 5 or 6 μηη in the largest cross section.

In one embodiment, the average size of the droplet at most 100, 75, 50, 20, 10 or 8 μηη in in the largest cross section.

In one embodiment, the average size of the droplet is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the average size is in the range 5 to 10 μηη. The droplet formed from the fluidic preparation has a narrow size distribution. This may be gauged empirically by observation of the packing of collected droplets. A hexagonal close packing arrangement of collected droplets is indicative of a low monodipsersity value (see, for example, L.J. De Cock et al. Angew. Chem. Int. Ed. 2010, 49, 6954).

The concentration of the silk protein as used in the second phase may be altered. Changes in concentration of the silk protein may alter the physical and chemical properties of the shell material subsequently formed. In one embodiment, the concentration of silk protein may be altered in order to increase the thickness of the shell and/or to decrease the number and/or size of pores in the capsule shell. Representative silk protein concentrations are described above.

In one embodiment, step (i) is performed at ambient temperature, such as a temperature in the range 15 to 25°C.

In one embodiment, step (i) is performed at about 5, 10, 15, 20, 25, or greater than 25°C. Apparatus

The preferred methods of the present invention call for a flow of a second phase and a flow of a first phase, which is immiscible with the second phase, to be brought together in a channel, for example to generate a dispersion of the second phase in the first phase.

Methods for the generation of a flow of a first phase and a second phase are well known in the art.

In one embodiment, each flow may be generated from a syringe under the control a programmable syringe pump. Each syringe is loaded with an appropriate aqueous solution or water-immiscible phase.

In the method of the invention, droplets and fibres may be collected only when the flows are at the required flow rate.

The channel in which the second phase and first phase flows are contacted is not particularly limited.

In one embodiment, the channel is a microfluidic channel.

In one embodiment, the channel has a largest cross-section of at most 1 ,000, at most 500, at most 200, at most 100 or at most 50 μηη.

In one embodiment, the channel has a largest cross-section of at least 0.1 , at least 1 , at least 10 or at least 20 μηη.

The channel may be provided in an appropriate substrate. The substrate is one that will not react with the components of the complexable composition.

The substrate may be a PDMS-based substrate. The preparation of substrates for use in fluidic flow techniques are well known to those of skill in the art. Examples in the art include the preparation described by Yang et al. (Yang et al. Lab Chip 2009, 9, 961 ), which is incorporated herein.

Second Phase

The second phase is immiscible with the first phase. The second phase may be referred to as a dispersed phase, particularly once it has contacted the first phase and is separated into discrete regions, such as droplets.

In one embodiment, the second phase is an aqueous phase. Therefore, the first phase is water immiscible.

Typically a silk protein is provided in an aqueous phase. When the silk protein is provided in the second phase, a capsule may be formed having a shell that is an assembly of silk protein and the capsule is optionally provided with a network of material within the shell that is an assembly of the silk protein.

In one embodiment, the flow rate of the second phase is at most 1 ,000, at most 500, at most 250, or at most 100 μΐ/h.

In one embodiment, the flow rate of the second phase is at least 0.05, at least 0.1 , at least 0.5, at least 1 , at least 5, at least 10, or at least 50 μΙ_/|-ι.

In one embodiment, the flow rate of the second phase is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the flow rate of the second phase in the range 0.1 to 500 μί/h, such as 0.1 to 100 μΙ_/|-ι.

The flow rate of the second phase refers to the flow rate of that phase before the phase is contacted with the first phase.

First Phase

The first phase is immiscible with the second phase. The first phase may be referred to as a continuous or carrier phase. Typically, the first phase is a water immiscible phase.

In one embodiment, the flow rate of the first phase is at most 1 ,000, at most 500, or at most 250 μΙ_/ϊι.

In one embodiment, the flow rate of the first phase is at least 10, at least 50, or at least 100 μΙ_/ϊι.

In one embodiment, the flow rate of the first phase is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the flow rate of the first phase in the range 100 to 250 μΙ_/Ι"ΐ.

The flow rate of the first phase refers to the flow rate of that phase before the phase is contacted with the second phase. Where a flow focusing technique is used to develop discrete regions of a second phase, the flow rates of the two first phases may be the same.

The first phase may additionally comprise a surfactant. The surfactant is provided in the first phase in order to stabilise the macroemulsion that is formed in the fluidic preparation methods. The step of forming the discrete region (such as a droplet) may require the presence of a surfactant. Furthermore, the presence of a surfactant is useful in limiting or preventing the coalescence of the droplets collected.

The surfactant chosen is not particularly limited, and encompasses any surfactant that is capable of promoting and/or stabilising the formation of discrete regions, such as droplets, of the second phase in the first phase.

Suitable surfactants for use in the present invention include those described by Holtze et al. Lab Chip 2008, 8, 1632. Typically such surfactants comprise an oligomeric perfluorinated polyether (PFPE) linked to a polyethyleneglycol. Such surfactants are especially useful for stabilising water-in-fluorocarbon oil emulsions.

The surfactant is present at most 0.1 %, at most 0.2%, at most 0.5%, at most 0.75%, at most 1 %, at most 2%, at most 5% w/w to the total phase.

The surfactant is present at least 0.05% or at least 0.07% w/w to the total phase.

In one embodiment, the first phase has a solubility in the second phase of at most 50, at most 20, at most 10, or at most 5 ppmw.

In one embodiment, second phase has a solubility in the first phase of at most 50, at most 20, at most 10, or at most 5 ppmw.

Aqueous Phase

The present invention calls for the use of an aqueous phase either as the dispersed phase in the methods of the invention. Methods for the preparation of suitable aqueous solutions comprising the protein will be apparent to those of skill in the art.

Typically the aqueous phase is at substantially neutral pH.

The pH of the aqueous phase may be at least 5, at least 6 or at least 6.5.

The pH of the aqueous phase may be at most 7.5, at most 8 or at most 9.0.

In some embodiments the aqueous phase may additionally comprise one or more additives, such as salts. However, it is noted that certain additives, such as salts, may induce the aggregation of a silk protein. As such, the nature and concentration of the additive must be carefully controlled. In the flow methods of the present case it is a trivial task to changes the additive concentration in the aqueous flow, and to determine whether a formed capsule or fibre has an assembly of a silk protein or an aggregation.

Water Immiscible Phase

The present invention calls for the use of a phase that is immiscible with water. That phase may be an oil-based phase (oil phase) or an organic solvent-based phase (organic phase), or a combination of the two.

In one embodiment, the water immiscible phase is a liquid phase.

In one embodiment, the water immiscible phase is not itself an aqueous phase that is immiscible with the water phase. Although it is possible to use water phases as the first and second phases, this is not preferred as the structures that are obtainable in such as system do not exhibit the desired assembly properties, such as a non-aggregated assemblies in a capsule of the invention.

The oil phase has as a principal component an oil. The oil is a liquid at ambient

temperature.

The oil is inert. That is, it does not react with the protein, or any other component used to form a capsule of the invention. The oil does not react with the shell.

In one embodiment, the oil is a hydrocarbon-based oil.

In one embodiment, the oil is a mineral oil.

In one embodiment, the oil is a fluorinated hydrocarbon oil.

In one embodiment, the oil is a perfluorinated oil. An example of a perfluorinated for use in the invention is FC-40 (Fluoroinert as available from 3M).

In one embodiment, the oil is a silicone oil.

In one embodiment, the water immiscible phase has as a principal component an organic solvent. For example, the organic solvent is selected from chloroform and octane.

Capsule with Encapsulant

The methods of the invention are suitable for the incorporation of a component into a capsule or a fibre. The capsule or fibre produced therefore comprises an encapsulated material (an encapsulant).

In a further aspect of the invention there is provided a method for the preparation of a capsule having a shell that comprises an assembly of a silk protein, wherein the capsule holds a component, the method comprises the step of: (i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprise a silk protein suitable for forming an assembly of a silk protein, and the second phase further comprises a component for encapsulation, thereby to form a capsule shell at the boundary of the discrete region, wherein the capsule holds the component and the first and second phases are immiscible.

Also provided is a method of preparing a fibre having a shell that comprises an assembly of a silk protein, wherein the fibre holds a component, the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a fluid flow of the second phase between flows of the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a silk protein, and the second phase further comprises a component for encapsulation, thereby to form a fibre sheath shell at the fluid boundaries of the second phase, wherein the fibre holds the component and the first and second phases are immiscible.

The methods of the invention are particularly attractive as they allow substantially all the component present in the second phase flow to be encapsulated within the capsule shell or the fibre sheath. The formation of the assembly of the protein occurs at the interface with the first phase. Substantially all the component, therefore, is encapsulated within the formed shell or the formed sheath. The present method therefore provides an efficient method for the incorporation of component into a capsule or fibre.

In one embodiment, the method is a method for the preparation of a capsule or fibre encapsulating a plurality of components. In this embodiment, the aqueous phase comprises at least a first component to be encapsulated and a second component to be encapsulated. The plurality of components may be provided in separate sub-flows that are contacted prior to contact with the first phase or at substantially the same time as the second phases are brought into contact with the flow of the first phase

In one embodiment, the concentration of a component to be encapsulated in the second phase is at least 0.01 , at least 0.02, at least 0.05, at least 0.1 , at least 0.2, at least 0.3, at least 0.5, at least 1 .0, or at least 5.0 μΜ.

In one embodiment, the concentration of a component to be encapsulated in the second phase is at most 500, at most 200, at most 100, at most 75, at most 50, or at most 10 μΜ. In one embodiment, the concentration of a component to be encapsulated in the second phase is in a range where the minimum and maximum rates are selected from the embodiments above. For example, the concentration of a component to be encapsulated in the second phase is in the range 0.02 to 50 μΜ. In one embodiment, the concentration of the component to be encapsulated refers to the concentration in the second phase after any sub-flows, where present, have been brought together.

The concentration of the component in the second phase may also represent the

concentration of the component held within the capsule.

The present invention provides a capsule that is obtained or obtainable from any of the methods described herein. The capsule may comprise an encapsulated component, which may also be prepared using the methods described herein.

Analysis of Capsule and Fibre

In the sections above, the analysis of the shell or sheath material, shape, and dimensions are described. For example, a capsule may be analysed by simple bright field microscopy to determine the shape of the capsule shell. The images obtained may also be used to determine the cross-section, typically the diameter, of the capsule shell.

The capsule shell and fibre sheath may also be analysed for shape, cross-section and its thickness using scanning electron microscopy and confocal microscopy.

Use of Capsules and Fibres

The capsules and fibres described herein are suitable for use as encapsulants for material. This material may be stored within the capsule or fibre and released as required.

The release characteristics of the capsule and fibre may be altered by changing the shell or sheath structure, for example by increasing the silk protein concentration or by changes to the arrangement of silk proteins within the shell or sheath. Such changes can be achieved by altering the silk protein concentration and altering the flow rates in the methods of preparation, as described herein.

In one embodiment there is provided a capsule of the invention comprising an encapsulated component. In another embodiment, there is provide a fibre of the invention comprising an encapsulated component.

In a further aspect there is provided a method of delivering a component to a location, the method comprising the steps of:

(i) providing a capsule having a shell encapsulating a component or a fibre having a sheath encapsulating a component, as described herein;

(ii) delivering the capsule or fibre to a target location;

(iii) releasing the component from the shell or sheath. In other aspects of the invention the capsule of the invention may be used to deliver a silk protein, which protein may be an encapsulated component and optionally may also be a protein that makes up the shell or sheath to a target location. As shown in the worked examples, the self-assembly of a protein may be disrupted, thereby leading to the dispersion of a silk protein from the capsule or the fibre. In this way, the material making up the capsule may be released.

Thus, in a further aspect of the invention there is provided a method of delivering a protein to a target location, the method comprising the steps of:

(i) providing a capsule having a shell or a fibre having a sheath, wherein the shell and the sheath are an assembly of a silk protein, as described herein;

(ii) delivering the capsule or fibre a target location;

(iii) disrupting the shell or sheath, thereby to release the silk protein.

In one embodiment, a released silk protein is not in an aggregation, and optionally is also not part of an assembly. The inventors have found that the silk protein may be stored long term (for example, for one month or more) in non-aggregated form, and may subsequently be released in non-aggregated form too, as and when it is required.

In one embodiment, the capsule or sheath holds a silk protein, such as a silk protein in a non-aggregated form

In one embodiment, the location is in vivo.

In one embodiment, the location is ex vivo.

In one embodiment the release of the encapsulated component is in response to an external stimulus.

In one embodiment the release of the encapsulated component is in response to a change in the local conditions.

In on embodiment, the change in local conditions may be a change in pH, a change in temperature, a change in oxidation level, change in concentration, or the appearance of a reactive chemical entity.

In the worked examples, the inventors have shown that freezing, centrifugation and washing may all be used to disrupt the assembly of a silk protein, thereby to release the silk protein and any encapsulated component.

Freezing of a capsule or fibre may refer to use of liquid nitrogen freezing techniques, such as snap freezing techniques.

Centrifugation may refer to ultracentrifugation where the capsule or fibre experiences acceleration of at least 1 ,000 G, at least 10,000 G, at least 50,000 G, at least, 100,000 G, or at least 500,000 G, for example with a ultracentrifuge. Here, the disruption of the assembly of a silk protein is believed to be due to increased hydrostatic pressure during the centrifugation. It is noted that during the centrifugation some of the silk protein may form an aggregation as a response to the shear forces that are encountered during the process. Nevertheless, the majority of the silk protein release is in free form, and is not in an aggregation.

In a washing release, a capsule or fibre is washed with a fluid, either continuously or in stage, such as with an aqueous fluid, to bring about a disintegration of the assembly of a silk protein.

The extent of release of a silk protein may be determined by UV absorption measurements of the sample.

In a further aspect of the invention there is provided a method for preparing a capsule, the method comprising the steps of:

(i) providing a capsule having a shell that is a non-aggregate assembly of a silk protein, as describe herein;

(ii) converting the non-aggregate assembly of the silk protein to an aggregate assembly of the silk protein.

Similarly, there is provided a method for preparing a fibre, the method comprising the steps of:

(i) providing a fibre having a sheath that is a non-aggregate assembly of a silk protein, as describe herein;

(ii) converting the non-aggregate assembly of the silk protein to an aggregate assembly of the silk protein.

In both methods, step (ii) may include heating the capsule or fibre, or altering the pH of an aqueous mixture containing the capsule or fibre, or dehydrating an aqueous mixture containing the capsule or fibre.

Other Preferences

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Experimental

Silk-Based Capsules Materials

The following materials were used for silk capsule preparation: native silk protein (NSF) freshly extracted from the B. mori silkworm gland, fluorinert FC-70 (Sigma) and

/V,A/-£>/s-(n-propyl) polyethylene oxide-£>/s(2-trifluoromethyl polyperfluoroethylene oxide) amide surfactant. For encapsulation and release kinetics studies aqueous solutions of 0.01 % of Remazol Brilliant BlueR (RBBR) dye (Sigma-Aldrich) were used.

Microfluidics

Single and double T-junction droplet maker PDMS (polydimethylsiloxane, 50,000 Mw) chips were fabricated by sol-gel methods according to an established protocol (see Qin et al. Nature Protocols 2010, 5, 491 ). The synthesis of the silk capsules was performed on a microfluidic system having 20 μηη wide channels. Small quantities of immiscible liquid reagents, 1 mL of aqueous native silk protein and 1 mL of fluorinert oil (containing 2% w/v of /V,A/-£>/s-(n-propyl)polyethylene oxide-bis-(2-trifluoromethyl polyperfluoroethylene oxide) amide surfactant), were contacted at the T-junction of the microfluidic channels under hydrodynamic pumping.

The initial concentrations of aqueous (pH = 7) silk protein varied from 1 mg/mL to 10 mg/mL. Each aqueous sample pumped through the holding component (e.g., a piece of capillary) and mixed with a second immiscible liquid (fluorinert oil) on the T-junction of the microfluidic device. The single shape silk protein capsules were generated with use of

A/,A/-bis(n-propyl)polyethylene oxidebis(2-trifluoromethyl polyperfluoroethylene oxide) amide surfactant (2% w/v) in order to stabilise obtained shape and avoid premature protein aggregation. For silk shapes formation on double T-junction device the aqueous silk and oil solutions were mixed on the first T-junction device to form initial shape and then passed through the second T-junction with aqueous silk as a continuous phase. After the formation step, the products were washed with aqueous (pH = 7) DDW (double- distilled water) in order to remove the surfactant and unreacted protein residues.

For encapsulation studies the RBBR dye (0.01 % w/v), tetracycline and glucose (Sigma- Aldrich) components was dissolved in aqueous protein media. Glucose was quantified using a commercial glucose assay kit (Sigma-Aldrich).

Silk microgel structures were synthesised using microfluidic droplet maker device by forming micron sized droplets on a T-junction of aqueous oil phases. The creation of the five basic structures (sphere, sausage, short, thin and thick fibres) was achieved by passing aqueous silk solution through continuous oil phase, where the viscosity of the silk solution at the phase boundary determined the final shape of the resulted silk droplets.

The variety of the silk structures was expanded (doubled) by introducing a second T-junction onto the microfluidic droplet maker device (see Figs. 7 (a) and 7 (b)).

The conversion of the native silk protein into β-sheet aggregates was followed by changes to the fluorescent properties of the silk portions. Blue excitation (315 nm) and emission (425 nm) maxima were selected for the detection of β-sheet aggregates and the fluorescent spectra is presented in Fig. 8 (a).

The maximum emission intensity of the intrinsic blue fluorescence signal for the silk aggregates was in the range of 425 nm to 450 nm, with a small variation attributed to the biological diversity of the 'donor' silkworms. This characteristic spectral shift allowed aggregated silk to be detected and localised spatially through confocal microscopy.

Capsule Preparation and Use

Microfluidic techniques were used to apply a controlled shear to induce the transition of mainly random coil native silk into assembled silk microstructures, such as aggregated β-sheet rich microstructures, formed as a monodisperse microemulsion. Non-aggregated forms of assembled silk were also formed, as described below.

Silk structures, including capsules, were synthesised at a T-junction in a microfluidic device (Fig. 1 (a) and 1 (b)) by contacting flows of an fluorinated oil phase either side of an aqueous flow containing native silk in a microchannel (as described above). The aqueous flow was dispersed as highly monodisperse discrete regions, such as droplets, in an oil continuous phase. The shear experienced by the silk during droplet formation step lead to the formation of gellified material as the native silk converted to its assembled form, such as an aggregated or non-aggregated from, at the aqueous/oil interface where the shear was greatest. The conversion of the silk protein (NSF) into β-sheet aggregates was followed by changes in its fluorescent properties. Blue excitation (315 nm) and emission (425 nm) maxima were detected for β-sheet aggregates and their fluorescent spectra are presented herein.

It was found that by controlling viscosity of the silk solution through changes in the shear rates, a variety of different silk aggregate structures could be formed, including spheres, cylinders and variable length and fully continuous fibres. The range of structures could be further increased by introducing a second T-junction in tandem with the first T-junction on the same fluid flow chip. In this manner, the silk structures produced in the first junction could be wrapped by an aqueous silk solution to form double shell structures or deformed to generate more intricate shapes (see Figure 7 (a)).

It was found that the final shape of the silk protein assembly was determined by the concentration and viscosity of the precursor silk protein solution and the relative aqueous silk protein:oil flow rates (Fig. 1 (d)). The viscosity of aqueous native solution increased linearly with the increase in the protein concentration (see Figure 7 (c)). For high concentrations, for example above 5 mg/mL and with correspondingly high viscosity values, elongated aggregates were the predominant form. By contrast, when the silk concentration was lowered, for example to 2 mg/mL, spherical microdroplets were formed. This observation suggests a dominant effect of surface tension at low concentration and of shear forces at high concentration. In agreement with this idea, the change in ratio of the inner to outer phase, controlling the applied shear, governed the final shape of the silk aggregates in the viscosity dominated regime at high silk concentration (see Figs. 1 (c) and 1 (d)).

The efficiency of conversion silk protein into a silk aggregate was studied by UV

spectroscopy (see below) through determination of the concentration of the unconverted silk remaining in solution after isolation of the aggregate phase. For all aggregate structures, the observed conversion efficiency was above 87%, highlighting the efficient nature of the processing approach.

The dependence of the morphology of the silk aggregates on the different droplets shapes was studied by light microscopy, atomic force microscopy (Figs. 2 (a)-(e)) and confocal microscopy (Figs. 2 (f)-(k)). Fibrillated morphology was observed on the surfaces of the cylindrical and fiber structures (See AFM image of the shapes' surfaces on the right corner of each light microscopy image in Figs. 2 (a)-(e)). The surface of the spherical shape of silk microgels exhibited smooth morphology (Fig. 2 (a)) with no evidence of surface fibrillation. By contrast, aligned silk fibrils were observed by AFM on the surface of the elongated microgels. For cylindrical silk microgels, the nanofibrils are oriented parallel to the long axis of the microgel particle, while for fibre-like structures, the component nanofibrils align perpendicularly to the fibril axis. The difference in silk fibrils alignment rises from the difference in mechanism of microgel formation. It is proposed that the cylindrical shapes are formed through the shear induced elongation of droplets, while the continuous fibrils formed at high shear rates gel at the nozzle and are then undergo buckling while being pushed through the channel.

The shear stress for the silk structure formation inside the microfluidic channels was calculated by multiplying the viscosity of the aqueous NSF solution and the shear rate (γ = v/h). The shear rate for the fluids in the microdroplet device was defined by maximum flow velocity per area (τ = γ/Α). The results are shown in Table 1.

Table 1 - Shear Stress

The transformation of native silk from its random coil structure to β-sheet aggregates was followed through the changes in its fluorescent spectra that accompany this transition and are characterised by the increase of the fluorescent signal in the blue range of the fluorescent spectra with excitation maxima at 315 nm and emission at 425 nm (see

Fig. 8 (a)). This characteristic spectral shift allows aggregated silk to be detected and localised spatially through confocal microscopy. The results in Fig. 2 (f)-(k) reveal an accumulation of aggregated silk on the outside of the particles where the shear during formation is largest. For double layered particles, both interfaces display a shell of aggregated silk (Figs. 2 (j) and (k)).

The structural changes of silk protein upon conversion into microgel shapes was studied by Fourier transform infrared spectroscopy (FTIR). The infrared spectrum, in particular the characteristic amide I and II bands are highly sensitive to the secondary structure of the protein. Native silk protein has predominantly a disordered random coil secondary structure (Ha et al. Biomacromolecules 6, 1722-1731 (2005)) (Figs. 3 (a) and 3 (b)) with a small β- sheet and a-helix content, while the aggregated state is β-sheet rich. We found that the differences in the morphologies of the microgel particles were reflected in differences in the relative abundance of β-sheet secondary structure relative to random coil. Indeed, the spherical shapes exhibited the lowest value of β-sheet content (ca. 40% of native β-sheet content), while the fibre shape had the highest (ca. 80% of β-sheet aggregates). These results are in good agreement with AFM surface morphology observations (Figs. 2 (a)-€) that indicate pronounced aggregation on the surface of the elongated forms of the particles. These differences can be rationalised by considering the process of silk microgel formation. The spherical particles are formed under conditions of low shear, and therefore only the surface of droplets denatures and gels. The inside of such structures remains in the native form and is encapsulated by a thin shell of aggregated silk as shown in Figs. 3 (a) and 3 (b). With increasing shear rates, the gelation becomes more extensive, which results in the full conversion of the native silk into the aggregated form in the case of the elongated morphologies, Figs. 3 (a) and 3 (b).

In addition, the Young's modulus of the NSF micron-scale capsules was measured by means of AFM nanoindentation and peak force quantitative nanomechanical mapping (PFQNM), both in air and in liquid. 3D topographic AFM images of a spherical micron-scale capsule in air and in liquid were recorded (not shown) and the corresponding height profiles were measured (data not shown). The average Young's modulus of the shell was measured to be 4.6 GPa in air and 3.8 GPa in liquid, respectively (see Fig. 3(c)).

Microfluidic processing allows native silk to be transformed into capsules with a degree of aggregation that can be tuned to form either shells containing native silk or fully aggregated microgels. We now exploit this factor to design an approach for the long-term encapsulation of aggregation-prone native silk for protein storage and subsequent controllable release. A macroscopic volume of silk spherical microgels was prepared, see Fig. 4 (a), which consist of a thin shell with a thickness of 1-2 μηη as shown in Fig. 4 (a), with the rest of the volume of the particle consisting of native silk encapsulated by the shell. Under bulk conditions, native silk is only stable for a few hours, but we found that within the microcapsules, the silk protein retained its native properties over incubation times of several months. Furthermore, after up to two months storage, the native silk can be released from the shells through rupturing the outer shell by means of low temperature or increased hydrostatic pressure from

centrifugation, Figs. 4 (b)-€.

The release was monitored by measuring the concentration of soluble protein through its UV absorption, Fig. 4 (d). The results reveal that 98% of the silk can be recovered from the capsules by liquid nitrogen snap freezing. Release by ultracentrifugation allowed 80% of the protein to be recovered, with approximately 20% being lost due to aggregation during the release as a consequence of the mechanical shear encountered during the centrifugation process. The structure of the native silk stored for 2 months was shown to be

indistinguishable from free native silk by FTIR. Moreover, the stored silk retained its tendency to aggregate. Indeed, the high level of protection against aggregation offered by encapsulating silk in our microcapsules is highlighted by the fact that the silk released from such structures is susceptible for reconversion to microgel shells and further storage in a process which could be repeated at least 5 times with the same starting material.

In order to probe further the stabilising effect conferred on the silk in solution within the structures, the structural changes of the soluble protein in bulk solution and in the gelled structure were monitored as a function of environmental parameters such as mechanical shear, temperature and ionic strength.

To this effect, silk (NSF) solutions in both bulk and encapsulated forms were exposed to mechanical stress by continuous shaking, and then followed the conformational changes by FTIR spectroscopy (Fig. 9(a) and (b)). Observations show almost complete transformation of the NSF into β-sheet rich aggregates within only 25 s (Fig. 9(a)) under bulk conditions, while silk (NSF) encapsulated inside structures retained its native structure (Fig. 9(b)) following the same treatment for at least 12 h of continuous shaking. Both systems were then exposed to elevated temperatures and high ionic strength. The results are shown in Fig. 9(c) to (f), and demonstrate that soluble silk (NSF) formed fibrils rapidly (within a few seconds) with increases in temperature or ionic strength, while encapsulated silk remained unaffected for up to 12 h.

Taken together, these data demonstrate that the silk capsules protect encapsulated silk from a range of factors promoting aggregation. In addition, the compartmentalisation, achieved by means of encapsulation intrinsically lowers the rate of aggregation processes.

In addition to their capacity to act as long term storage micro-containers protecting native silk against aggregation, we investigated the potential of silk microgels to serve as a small molecule carriers, a possibility that is particularly interesting for drug delivery in view of the outstanding biocompatibility of silk. For probing the encapsulation and release of small molecules, we focused on Remazol Brilliant BlueR dye 30 (RBBR), tetracycline antibiotic and glucose encapsulation and release under physiological conditions. The loading efficiency and release kinetics of the RBBR and tetracycline molecules was studied by following the changes in their characteristic UV, while glucose was quantified using a glucose detection kit (see below).

Fig. 5 (a) shows the preparation of a silk protein capsule holding RBBR dye, and its subsequent release from the capsule.

The loading efficiency studies for performed systematically for the different morphologies are summarised in Fig. 5 (b). For all types of shapes, the loading efficiency exceeded 88% demonstrating effective encapsulation and storage. The release kinetics could be altered by changes to the morphology of the microgels. Thick fibrous structures exhibited the slowest release rate for RBBR, tetracycline and glucose, and thin fibre structures possessed the fastest release rate, while spherical, cylindrical and short fibre structures displayed intermediate release kinetics. The results shown in in Fig. 5 (b) reveal marked differences in the rate of release of the small molecules. RBBR exhibited the slowest release rate for the all silk structures and was not fully released even after one week, a results originating from the interaction of dye molecules with the silk protein. Behaviour similar to RBBR was observed for glucose. By contrast, hydrophobic tetracycline antibiotic molecule reached its maximum release rate after 48 hours.

Further encapsulation and release experiments were performed in phosphate buffer media (PBS). The results show almost no differences in the small molecule release rates in the buffer solution (data not shown).

The ability of silk (NSF) capsules to provide long-term storage for silk (NSF) itself suggested their potential use for the stabilisation of other sensitive protein species against aggregation. Antibodies provide an important example of protein structures possess a high propensity to aggregate, a factor which can limit significantly their efficacy and shelf-life for medical purposes. The outstanding bio-medical compatibility of silk should be of great value as a potential pharmaceutical carrier, and thus the encapsulation, stabilisation and release of several active antibody species was examined, including a single-chain Fv binding domain specific for the protein huntingtin, C4scFv25, and two single chain Fv domains specific for osynuclein, Nb-Syn86 and NbSyn8726.

First, the encapsulation and release efficiencies were tested using single chain Fv domains that were labelled with AlexaFluor647 (see details below, and schematic in Fig. 1 1 (a)). Very high loading efficiencies were achieved (> 95%, Fig. 1 1 (b)) in all cases as well as efficient and rapid release kinetics (see Fig. 1 1 (c)), without any loss of binding activity or solubility (see Fig. 10).

Next, the effect of the silk (NSF) structures on the stability of the domains was investigated. NbSyn86 was chosen for this study, which has previously been shown to have relatively low thermal stability in bulk solution (Fig. 1 1 (d)) and a high propensity to self-aggregate resulting in a significant reduction of its binding activity.

Experimental results showed that the gelled structures themselves did not affect the stability of the antibody domain, as the binding activity of NbSyn86 before and after encapsulation and release was measured to be identical (Fig. 1 1 (d) and (e)). Moreover, the micron-scale capsules demonstrated their capability of enhancing dramatically the stability of the single chain Fv domain (see Fig. 1 1 (d)). Indeed, in the absence of the silk (NSF) structures, NbSyn86 rapidly aggregated when heated to 65°C, while identical heat treatment of the domain encapsulated by NSF led to no measurable effect on the activity of antibody binding.

Moreover, the 2% of the soluble silk protein (NSF) itself had been transformed into silk (NSF) aggregates when NbSyn86 loaded structures were heated to 65°C, which confirm the capability of soluble as well as aggregated silk to inhibit aggregation of antibody domains. Thus, the silk structures offer full protection against aggregation of NbSyn86 even under condition of prolonged heating that lead to 60% aggregation of the NSF in the structures. These results suggest a route towards the development of very effective stabilisation systems for storage of highly sensitive functional macromolecular species.

It was also found that the silk structures have an outstanding ability to encapsulate, store and release in controlled manner small molecules such as glucose and the antibiotic tetracycline under physiological conditions (see below).

Finally, the biocompatibility of the silk structures with human cells was studied. To this end, a caspase toxicity assay were performed to evaluate the viability of the human

neuroblastoma SHY5 cells (see above). The results, shown in Fig. 6, demonstrate that all types of silk structures were non-toxic to the human cell lines, which confirms the

biocompatibility of the structures for biomedical applications.

For all types of shapes, the loading efficiency exceeded 88% demonstrating effective encapsulation and storage. The release kinetics could be tuned by controlling the morphology of the microgels. The results show that the thick fibrous structures exhibited the slowest release rate and thin fibre shape possessed the fastest release rate, while spherical, cylindrical and short fibre structures displayed intermediate release kinetics.

Thus, silk structures were synthesised using microfluidic droplet maker device by forming micron sized droplets on a T-junction of aqueous oil phases. The creation of the five basic structures (spherical capsule, and sausage, short, thin and thick fibres) was achieved by passing aqueous silk solution through continuous oil phase, where the viscosity of the silk solution and the silk protein concentration determined the final structure of the resulted silk droplets. The variety of the silk structures was expanded (doubled) by introducing a second T-junction on microfluidic droplet maker device. This allowed for the preparation of nested capsule and fibres.

The conversion of the native silk protein into -sheet aggregates followed by changes in its fluorescent properties. Blue excitation (315 nm) and emission (425 nm) maxima were detected for-sheet aggregates and its fluorescent spectra is presented in Fig. 8 (a).

Confocal and Light Microscopy

For light and confocal fluorescent microscopy, samples were deposited as the aqueous dispersions, without further purification, onto a glass slide. The silk structures were analysed by confocal microscopy (Laser Scan Confocal, Zeiss Microscope) using following UV 405 nm at 25 mW (for violet excitations) laser. Due to the native fluorescent signal emitted from aggregated silk protein, the silk structures were analysed by confocal microscopy without colouring pre-treatment (i.e. without labelling). The emission maxima, in the blue region of the fluorescence spectrum, originated from the aggregated component of the capsules, while for single shell structures the aggregated silk content was detected at the interface of each micron-scale capsule. The double shell structures exhibited blue emission from the internal as well as the external shells of the silk structures. 3D images were reconstructed using the Imaris image analysis program (on average, 412 z-stack slices per each protein shell).

Atomic Force Microscopy

For atomic force microscopy (AFM) silk protein structures were cross-linked with 4% of paraformaldehyde solution (20 min. incubation at room temperature), in order to keep silk shapes structure intact, and then rinsed with increased concentration of aqueous ethanol solutions (20%, 50%, 70% and 100%. The structures were deposited on mica slide and dried at ambient conditions. The protein structures were then analysed and characterised by AFM microscope, H-02-0067 NanoWizard II (JPK Instruments).

AFM Nanoindentation Studies

PF-QNM measurements were performed by using a MultiMode VIII Scanning Probe

Microscope (Bruker, USA) operated in intermittent mode either under ambient conditions or in a liquid environment at a scan rate of 1 Hz. The microscope was covered with an acoustic hood to minimise vibrational noise. The AFM cantilevers were calibrated on defined samples (Bruker, USA) covering the following ranges of Young's moduli: from 100 MPa to 2 GPa (for low-density polyethylene) and from 1 to 20 GPa (for polystyrene). The analysis of the Derjaguin-Mueller-Toporov (DMT) modulus was performed using Nanoscope Analysis software. Measurements were made at room temperature (ca. 20°C).

Silk Release Studies

The release of native silk from the inner content of the spherical shape silk structures was achieved by a fast-freeze method (liquid nitrogen), gentle centrifugation (700 rpm, 3 min) or incubation of the silk structures in aqueous media. The degree of aggregation for the released silk was measured by FTIR spectroscopy, following the change in the β-sheet vibration band.

Efficiency, Loading Capacity and Release Kinetics Measurements

The efficiency of conversion native silk protein into the silk structures using microfluidic technique was studied by calculating the percentage of protein participating in gel droplet formation. After the preparation of the silk structures was accomplished and the resulting gels were washed, in order to remove unreacted protein, and the concentration of unreacted protein was measured by UV absorption by using NanoDrop 2000 UV spectrophotometer (Thermo Scientific). A bicinchoninic acid (BCA) protein detection kit (Thermo Scientific) was also used, following UV absorption of the dye at 562 nm. In no case did the difference between the two approaches exceed 3%.

In addition, the loading efficiency and release profiles of the C4scFv antibody domain from silk (NSF) structures were probed using an AlexaFluor647 labelled domain. The loaded structures were washed with PBS at intervals of time from 10 min to 30 d, and the solutes after each washing were analysed by UV and fluorescence spectroscopy.

For studies of encapsulation and release kinetics, aqueous solutions of 0.01 % of Remazol Brilliant BlueR (RBBR) dye, tetracycline antibiotic and glucose solution (Sigma-Aldrich, UK) were used. For encapsulation studies the RBBR dye (0.01 % w/v), tetracycline and glucose (Sigma-Aldrich, UK) components was dissolved in aqueous protein media (NSF dissolved in water). Glucose was quantified using a glucose assay kit (Sigma-Aldrich, UK).

The loading capacity and release kinetics for silk structures was studied by UV spectroscopy following the change in UV absorption maxima at 592 nm for RBBR, 360 nm for tetracycline and 540 nm for glucose (using a glucose detection kit according to the manufacture's protocol: see the manufacture protocol for glucose detection kit in Sigma-Aldrich). For encapsulation and release efficiency studies more than 10 repeated experiments were performed. In order to study the release kinetics of silk structures, the loaded samples were washed with doubly distilled water (DDW) (pH = 7) within following intervals of time: 10 min., 30 min., 1 h., 3 h., 7 h., 12 h., 24 h., 48 h., 3 d., 7 d., 10 d. Each washing solution was than analysed by UV-spectroscopy and exact concentration of the released dye was detected.

FTIR Structural Analysis

The structural analysis of the silk structures was performed using FTIR-Equinox 55 spectrometer (Bruker). The washed samples (washed with DDW), without further pre-treatment, were loaded to the FTIR holder and analysed by subtracting water reference. The atmospheric compensation was subtracted from original FTIR spectra and secondary derivative with 25 smoothing points was applied for further analysis. The small differences in FTIR spectra of NSF are due to the extraction of NSF from different worms; these difference do not impact on the interpretation of results.

Fluorescence Spectroscopy Analysis.

The fluorescence spectra of native and aggregated NSF in bulk and in gels was monitored by fluorescence spectroscopy using a Cary Eclipse fluorescence spectrophotometer. The samples were pre-scanned to calculate the excitation and emission maximum. The emission maxima were determined by exciting samples at wavelengths varying from 300 nm to 415 nm with intervals of 5 nm each scan. The excitation maximum was detected by measuring spectrum at fixed emission wavelengths varying from 400 nm to 515 nm with an interval of 5 nm for each scan.

Cell Viability Assays

SH-SY5Y human neuroblastoma cells were incubated in a 96-well plate with 100 μΙ_ of following silk shapes for 24 h at 37°C: 1 ) native silk; 2) silk capsules (spheres); 3) silk microgels with cylindrical shape; 4) short silk fibres; 5) thin silk fibres; 6) thick silk fibres. After 24 h in culture in Opti-MEM Reduced Serum Medium (Gibco) cell viability was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), and a plate reader (Fluostar Optima, BMG Labtech).

Expression, Purification and Labelling of Antibody Fragments

The antibody fragment, NbSyn87, was previously isolated through phage display selection following the immunization of a lama with the A53T variant of human a-Syn, and the expression and purification of NbSyn87 was performed according the protocol described in the same paper (see Guilliams et al.).

To obtain Alexa-Fluor 647 labelled NbSyn87, a solution of 70 nmol of NbSyn87 was mixed with 1 .5 equivalent of Alexa Fluor 647-succinimidyl ester (Life Technologies, Paisley, UK) in 100 mM sodium carbonate buffer (1 mL, pH 9.0). The reaction mixture was then incubated in the dark and at room temperature (RT) for 5 hours. After the reaction, the free dye was separated from the labelled protein using a PD10 desalting column, containing Sephadex 25 resin (8 mL) (GE-Healthcare, Little Chalfont, UK). The labelling yield and stoichiometry were determined by absorbance spectrophotometry.

The nanobody, NbSyn86, was obtained from the same phage-display library that yielded NbSyn87 and though identical selection strategies as described previously30. NbSyn86 was also expressed and purified in an identical way to that described for NbSyn87. The cloning expression and purification of the C4 scFv protein are described elsewhere (De Genst et al; in preparation). Labeling of C4 scFv with Alexa-fluor 647 was obtained in an identical way to that described for NbSyn87.

Analytical Gel Filtration Experiments

To evaluate the conformational integrity of scFvC4 upon release after encapsulation by capsules, size exclusion experiments were performed using a Superdex 200 (10/30) high resolution gel filtration column (GE Heathcare). Samples of scFvC4 of 100 μί volume were injected onto the column using an Akta basic instrument (GE Healthcare). Elution profiles were recorded for one column volume by measuring the absorbance at 280 nm. Activity Measurements of NbSyn86 and NbSyn87 using SPR

The NbSyn87 binding activity was measured using surface plasmon resonance using a Biacore 2000 (GE Healthcare) instrument. One flow-cell of a CM5 sensor chip (GE

Healthcare) containing 250 RU of immobilised osynuclein and a blank control flow-cell, were prepared using EDC/NHS amine coupling chemistry according to the manufacturer's recommendations.

A concentration range of NbSyn87, corresponding to initial concentrations ranging from 0 nM to 100 nM was then prepared in HBS-EP running buffer pH 7.4 (GE Healthcare, UK) for each sample containing NbSyn87 and subsequently injected for 2 min onto the sensor chip at a flow rate of 30 μΙ_/Γηίη to follow the association of the binding reactions. The

dissociation of the nanobody in the running buffer at a flow-rate of 30 μΙ_/Γηίη was followed for 10 min immediately after injection.

The kinetic curves were fitted to a binding model that accounts for mass-transport effects, implemented in the BIA evaluation software (GE Healthcare, UK). The binding

measurements involving NbSyn86 were performed using a Biacore 3000 instrument and a CM5 sensor chip that was coated with 150 RU of osynuclein using amine coupling chemistry. The preceding flow-cell served again as a blank reference surface. From all samples used in the encapsulation experiments that contained NbSyn86, a series of concentrations were prepared ranging from 0 nM to 200 nM. Kinetic traces were recorded as described above for the NbSyn87 samples. The kinetics of the interaction were, however, too fast to be measured accurately. Therefore the equilibrium binding levels were measured and plotted against the initial concentration of NbSyn86. Fitting of these data to a Langmuir binding model yielded an estimate of the Kd of the interaction for each sample.

Comparative Example

In a comparative, example an attempt was made to prepare silk structures in a T-junction microfluidic device, such as described above, using a water in water microemulsion. This preparative technique is based on the work described in WO 2007/141 131 , which suggests that a variety of silk structures may be prepared in this way.

Thus, a flow of an aqueous silk solution was contacted with a flow of water at the T-junction of a microfluidic device, where the flow of water was used as the continuous phase. The formation of silk droplets, such as silk capsules, was not observed.

In a further experiment concentrated HCI was added to the continuous aqueous flow (to pH 2). Native silk has a reduced solubility in acidic solutions. As before, the flow of the aqueous silk solution was contacted with a flow of the now acidified water at the T-junction of a microfluidic device. No droplets were seen to form. Silk aggregation was observed.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

Allmeling et al. Journal of Cellular and Molecular Medicine 10, 770-777 (2006)

Anna et al. Applied Physics Letters 2003, 82, 364

Cole Cancer Chemother. Pharmacol. 1986, 17, 259

EP 1757276

Gosline et al. Journal of Experimental Biology 202, 3295-3303 (1999)

Gosline et al. The Journal of Experimental Biology 202, 3295-3303 (1999)

Gosline et al. The Journal of Experimental Biology 202, 3295-3303 (1999)

Guilliams et al. J. Mol. Biol. 425, 2397-241 1 (2013)

Ha et al. Biomacromolecules 6, 1722-1731 (2005)

Hermanson et al. Advanced Materials, 18, 1810 - 1815 (2007)

Holland et al. Advanced Materials 24, 105-109 (2012)

Holtze et al. Lab Chip 200S, 8, 1632

Lammel et al. Biomaterials 32, 2233 - 2240 (201 1 )

Link et al. Angewandte Chemie International Edition 45, 2556-2560 (2006)

Liu et al. J. Biosci. Bioeng. 108, 496-500 (2009)

Nicholson et al. Biopolymers 33, 847-861 (1993)

Nicholson et al. Biopolymers 33, 847-861 (1993)

Pritchard et al. Expert Opinion on Drug Delivery 8, 797-81 1 (201 1 )

Qin et al. Nature Protocols 2010, 5, 491

Rising et al. Zoolog. Sci. 22, 273-281 (2005)

Tao et al. Advanced Materials 24, 1067-1072 (2012)

Teule et al. Nat. Protocols 4, 341 (2009)

Toshiki et al. Nature Biotechnology 18, 81-84 (2000)

Vepari et al. Progress in Polymer Science 32, 991 - 1007 (2007)

Vepari et al. Progress in Polymer Science 32, 991-1007 (2007)

Vollrath et al. Nature O, 541-548 (2001 )

Vollrath et al. Nature O, 541-548 (2001 )

Wang et al. Advanced Functional Materials 22, 435-441 (2012)

Wang et al. Biomaterials 27, 6064 - 6082 (2006)

WO 2014/012099

WO 2014/012105

Claims

Claims:

1. A capsule having a shell of material that comprises an assembly of a silk protein.

2. The capsule of claim 1 , wherein the assembly is a non-aggregated assembly of the silk protein, such as an assembly where the a-helix, β-sheet (native) and random coil content is at least 55%.

3. The capsule of claim 1 or claim 2, wherein the capsule is substantially spherical.

4. The capsule of any one of the preceding claims, wherein the capsule has an average size of at most 10 μηη in the largest cross section.

5. The capsule of any one of the preceding claims, wherein the capsule holds a component.

6. The capsule of claim 5, wherein the component is a non-aggregated silk protein.

7. A fibre having a sheath of material that comprises an assembly of a silk protein.

8. The fibre of claim 7, wherein the assembly is an aggregated assembly of the silk protein, such a β-sheet aggregation, for example such as an assembly where the a-helix, β-sheet (native) and random coil content is less than 45%.

9. The fibre of claim 7 or claim 8, wherein the fibre holds a component.

10. The capsule according to any one of claims 1 to 6, or a fibre according to any one of claims 7 to 9, wherein the silk protein is non-recombinant silk protein, such as a native silk fibroin.

1 1 . The capsule according to any one of claims 1 to 6, or a fibre according to any one of claims 7 to 9, wherein the silk protein is a reconstituted silk protein.

12. The capsule according to any one of claims 1 to 6, or a fibre according to any one of claims 7 to 9, wherein the silk protein is obtained or obtainable from a silkworm or a spider.

13. The capsule or fibre according to claim 12, wherein the silk protein is obtained or obtainable from an organism that is a member of a species selected from the group consisting of Bombyx, Nephila, Araneus, Argiope, Latrodectus, Leucauge, Plectreurys, and Kukulcania.

14. The capsule or fibre according to claim 13, wherein the silk protein is obtained or obtainable from an organism that is a Bombyx species.

15. The capsule or fibre according to claim 14, wherein the silk protein is obtained or obtainable from Bombyx mori.

16. The capsule or fibre according to claim 13, wherein the silk protein is obtained or obtainable from an organism that is a Nephila species.

17. The capsule or fibre according to claim 16, wherein the silk protein is obtained or obtainable from Nephila clavipes or Nephila edulis.

18. The capsule according to any one of claims 1 to 6, or a fibre accruing to any one of claims 7 to 9, wherein the silk protein is substantially free of sericin.

19. A method for the preparation of a capsule having a shell that comprises an assembly of a silk protein, wherein the method comprises the step of:

(i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a protein, thereby to form a capsule shell at the boundary of the discrete region, wherein the first and second phases are immiscible.

20. The method of claim 19, wherein the discrete region is a substantially spherical droplet.

21 . The method of claim 19 or claim 20, wherein the assembly of a silk protein is a non-aggregated assembly of the silk protein, such as an assembly where the a-helix, β-sheet (native) and random coil content is at least 55%.

22. The method of any one of claims 19 to 21 , wherein the flow rate of the first phase is greater than the flow rate of the second phase, such as at least 1.5, 2, 3, 4, 5, or 10 times greater than the flow rate of the second phase.

23. The method of any one of claims 19 to 22, wherein the concentration of the silk protein in the second phase is at most 10 μΜ or at most 5 mg/mL.

24. The method of any one of claims 19 to 23, wherein the second phase is an aqueous phase.

25. A method for the preparation of a fibre having a sheath that comprises an assembly of a silk protein, wherein the method comprises the step of: (i) contacting a flow of a first phase and a flow of a second phase in a channel, thereby to generate in the channel a fluid flow of the second phase between flows of the first phase, wherein the second phase comprises a silk protein suitable for forming an assembly of a silk protein, thereby to form a fibre sheath shell at the fluid boundaries of the second phase, wherein the first and second phases are immiscible.

26. The method of claim 25, wherein the assembly of a silk protein is an aggregated assembly of the silk protein, such as such a β-sheet aggregation, for example such as an assembly where the a-helix, β-sheet (native) and random coil content is less than 45%.

27. The method of any one of claim 25 or claim 26, wherein the flow rate of the first phase is at most 5 times greater than the flow rate of the second phase.

28. The method of any one of claims 25 to 27, wherein the concentration of the silk protein in the second phase is at least 10 μΜ or at least 5 mg/mL.

29. The method of any one of claims 25 to 28, wherein the second phase is an aqueous phase.

30. A method of delivering a component to a location, the method comprising the steps of:

(i) providing a capsule having a shell encapsulating a component according to claims 5 to 6, or a fibre having a sheath encapsulating a component according to claim 9;

(ii) delivering the capsule to a target location; and

(iii) releasing the component from the shell or sheath.

31 . The method of claim 30, wherein the component is a silk protein that is not in an assembly.

32. The method of claim 30 or claim 31 , wherein the component is released on freezing, centrifugation or washing.