The present invention is directed to a method of forming
multilayer silk protein films and a multilayer film obtained therefrom. The invention
is further directed to various materials, products and compositions containing said
multilayer film and to the use of said multilayer film in several applications.
Multilayer films are useful for a large variety of purposes
1-12. Applications could be said to fall into two general categories:
tailoring interactions of a surface with its environment and fabricating "devices"
with defined structural properties. The range of development areas includes coatings,
colloid stabilization, light-emitting or photovoltaic devices, electrode modification,
optical storage and magnetic films, high charge density batteries, biomaterials,
alteration of biocompatibility, enzyme immobilization, flocculation for water treatment
and paper making, functional membranes, separations, carriers, controlled release
devices, sensors, and nanoreactors. A key attribute of the preferred method of preparing
multilayer films and capsules is controlled vertical structuring on the nanometer
scale. A polypeptide multilayer film is defined as a multilayer film made of polypeptides.
In some instances another type of polymer is involved in the fabrication process,
for instance a chemically modified polypeptide 13, a non-biological organic
polyelectrolyte 14, or a polysaccharide15. A polypeptide film
might be deposited to confer specific bio-functionality on a surface that was otherwise
bio-inert or to convert a bioactive surface into one that is not adhesive to cells16-20.
Multilayer films of polypeptides are promising for the
development of applications which encompass some of the following desirable features:
anti-fouling, biocompatibility, biodegradability, specific bio-molecular sensitivity,
edibility, environmental benignity, thermal responsiveness, and stickiness or non-stickiness.
Silk proteins are ideally suited for such applications by virtue of their biochemical
nature, the control one can have over chemical structure in various approaches to
polymer synthesis, the ability to control formation of secondary structure, or the
availability of genomic data.
US 2005/0069950 A1
describes the fabrication of ultra thin multilayered films of polypeptides
on suitable surfaces by electrostatic layer-by-layer self assembly (ELBL). Further,
it describes a method for designing polypeptides for the nanofabrication of thin
films for applications in biomedicine and other fields. A novel method for identifying
sequence motifs of a defined length and net charge at neutral pH in amino acid sequence
information for use in ELBL and recording of a desired number of the motifs is provided.
claims the use of small, highly charged peptides in ELBL assembly.
Spider silks are protein polymers that display extraordinary
physical properties. Among the different types of spider silks, draglines are most
intensely studied. Dragline silks are utilized by orb weaving spiders to build frame
and radii of their nets and as lifelines that are permanently dragged behind. For
these purposes high tensile strength and elasticity are required. The combination
of such properties results in a toughness that is higher than that of most other
known materials. Dragline silks are generally composed of two major proteins whose
primary structures share a common repetitive architecture.
An orb web's capture spiral, in part composed of viscid
silk formed by the flagelliform gland, which is therefore named flagelliform silk,
is stretchy and can triple in length before breaking, but provides only half the
tensile strength of dragline silk.
Systems for the recombinant production of spider silk proteins
in E. coli have been developed earlier. As an example, it is referred
WO 2006/008163 A2
WO 2006/002827 A1
US provisional application No. 60/712,095
(unpublished). In the expression system described therein, single building
blocks (= modules) can be varied freely and can thus be adapted to the requirements
of the specific case. Modules of this type are disclosed also in Ref. 21.
However, up to now, there is no general method of forming
multilayer films from those silk proteins available. The methods which are known
in the state of the art can not be used in a general way to produce various multilayer
films made from silk proteins. As mentioned above, some publications rely on charged
surfaces in order to stabilize the multilayer films, but do not provide a universal
It is, therefore, an object of the present invention to
provide a method for producing multilayer silk protein films and to provide films
obtainable therefrom. It is a further object of the present invention to provide
various applications for those multilayer films.
These objects are achieved by the subject-matter of the
independent claims. Preferred embodiments are set forth in the dependent claims.
The inventors established for the first time a method for
forming multilayer films made from silk proteins of different origin. They showed
that multiple layers of silk proteins can be processed in order to form stable and
useful multilayer films. This finding is unexpected since from the state of the
art it could not be expected that stable multilayer films may be formed from silk
proteins without creating specific conditions as, for example, oppositely charged
According to a first aspect, the invention provides a method
of forming multilayer silk protein films comprising the steps of:
- a) providing one or more solutions of silk proteins dissolved or suspended in
a suitable solvent;
- b) forming said solution into a film;
- c) evaporating the solvent; and
- d) once or more repeating steps a) - c) in order to form a multilayer silk protein
It has been known for a certain time to cast films made
of silk proteins, in particular from spider silk proteins. Regarding the film casting
of spider silk proteins see ref. 22. However, forming multilayer films was not described
Apart from the fact that, surprisingly, multilayer films
can be produced by the method of the present invention, the advantage is provided
that tailored multilayer films can be produced. As an example, the thickness of
the films may be controlled by the concentration of the employed protein solution.
Furthermore, different silk proteins can be combined in the same or different layers
in order to achieve the desired characteristics. Additionally, the present approach
allows to combine the silk protein layers with other, for example, artificial polymers
in order to achieve various purposes (as they will be outlined below in detail).
In an embodiment, each single layer of the multilayer film
is formed from a silk protein solution comprising one or more types of silk proteins.
Which types of silks may be used in practicing the present invention will be explained
later. For example, the multilayer film is formed from layers comprising the same
(homogenous multilayer film) or different (heterogenous multilayer film) silk proteins.
As mentioned above, in a preferred embodiment, the multilayer
film comprises one or more layers made from silk proteins and one or more layers
comprising other proteinaceous or non-proteinaceous materials.
The non-proteinaceous material is preferably selected from
polystyrene, polyvinylchloride, poly(styrene sulfonate) (PSS), poly(allylamine hydrochloride)
(PAH), poly(acrylic acid) (PAA), and/or poly(diallyldimethylammoniumchloride) (PDADMAC).
The non-proteinaceous material may be used alone or in combination with other non-proteinaceous
materials and/or silk proteins and/or other proteinaceous materials.
Those other proteinaceous materials may be preferably selected
from collagens, elastin or keratin. Examples for those proteinaceous materials are
mussel byssus proteins, for example those being obtained from Mytilus sp.,
preferably from M. edulis, M. galloprovincialis, M. californians, or
MASCOLO and WAITE (1986) first identified chemical gradients
in byssus threads in Mytilus. After treatment of the threads with pepsin,
two pepsin-resistant collagen fragments, called ColP and ColD, having molecular
weights of 50 kDa and 60 kDa, respectively were identified. ColP can be found predominantly
in the proximal area and is hardly to be found in the distal area. In contrast,
the amount of ColD increases in the distal part to approximately 100% (LUCAS et
al., 2002; QIN & WAITE, 1995). In the byssus thread as well as in the mussel foot,
there is a further collagen-like protein which takes part in the construction of
the thread structure. This additional protein is called ColNG (NG = no gradient),
and is, in contrast to ColD and ColP, evenly distributed throughout the whole thread.
Its physiological function presumably is being an adapter between the two other
thread collagens (QIN & WAITE, 1998).
Keratins are a family of fibrous structural proteins which
are tough and insoluble, form the hard but nonmineralized structures found in reptiles,
birds and mammals. Keratins are also found in the gastrointestinal tracts of many
animals, including roundworms. There are various types of keratins. Silk fibroins
produced by insects and spiders are often classified as keratins.
Collagen is the main protein of connective tissue in animals
and the most abundant protein in mammals, making up about 40% of the total. It is
tough and inextensible, with great tensile strength, and is the main component of
cartilage, ligaments and tendons, and the main protein component of bone and teeth.
Collagen occurs in many places throughout the body, and occurs in different forms
known as types, which include Type I to Type XIII collagen, among others (there
are 27 types of collagen in total).
Elastin, is a protein in connective tissue that is elastic
and allows many tissues in the body to resume their shape after stretching or contracting.
Elastin helps skin to return to its original position when it is poked or pinched.
It is primarily composed of the amino acids glycine, valine, alanine and proline.
Elastin is made by linking many soluble tropoelastin protein molecules to make a
massive insoluble, durable cross-linked array.
However, the present invention is not limited to these
proteinaceous materials and many others may be used.
As a solvent, preferably a polar solvent is used. The polar
solvent preferably is selected from water, formic acid, hexafluoroisopropanol and/or
acetic acid. Water is most preferred due to its good availability and nontoxicity.
In this solvent, the silk proteins are solved or suspended. Furthermore, the solvent
has to be chosen from those substances which can easily be evaporated in order to
leave behind the solved proteins, thus forming an individual film layer.
A "solution" in the meaning of the present invention means
any liquid mixture that contains silk proteins and is amenable to film casting.
Those solutions may also contain, in addition to protein monomers, higher order
aggregates including, for example, dimers, trimers, and tetramers. The solutions
may include additives to enhance preservation, stability, or workability of the
A suspension herein is defined as a dispersion of solid
particles in a liquid. If the particles are ~100 nm in diameter, the suspension
In a preferred embodiment, the silk protein is selected
from insect silk proteins or spider silk proteins, preferably natural or recombinant
silk proteins, preferably silks from Insecta, Arachnida or analogues therefrom.
In particular preferred are the dragline and/or flagelliform
sequences from dragline or flagelliform proteins of orb-web spiders (Araneidae and
Spider silks in general are protein polymers that display
extraordinary physical properties, but there is only limited information on the
composition of the various silks produced by different spiders (see Scheibel, 2004).
Among the different types of spider silks, draglines from the golden orb weaver
Nephila clavipes and the garden cross spider Araneus diadematus are
most intensely studied. Dragline silks are generally composed of two major proteins
and it remains unclear whether additional proteins play a significant role in silk
assembly and the final silk structure. The two major protein components of draglines
from Araneus diadematus are ADF-3 and ADF-4 (Araneus Diadematus
It is noted that the term "spider silk protein" as used
herein does not only comprise all natural sequences but also all artificial or synthetic
sequences which were derived therefrom.
Accordingly, the spider silk sequences may be derived from
sequences which are termed "authentic" herein. This term means that the underlying
nucleic acid sequences are isolated from their natural environment without performing
substantial amendments in the sequence itself. The only modification, which is accepted
to occur, is where the authentic nucleic acid sequence is modified in order to adapt
said sequence to the expression in a host without changing the encoded amino acid
The authentic sequences are preferably derived from the
amino terminal non-repetitive region (flagelliform proteins) and/or the carboxy
terminal non-repetitive region (flagelliform and dragline proteins) of a naturally
occuring spider silk protein. Preferred examples of those proteins will be indicated
According to a further embodiment, the authentic sequences
are derived from the amino terminal non-repetitive region (flagelliform proteins)
and/or the carboxy terminal non-repetitive region (flagelliform and dragline proteins)
of a naturally occuring spider silk protein.
According to one preferred embodiment, the dragline protein
is wild type ADF-3, ADF-4, MaSp I, MaSp II and the flagelliform protein is FLAG.
The term ADF-3/-4 is used in the context of MaSp proteins produced by Araneus diadematus
(Araneus diadematus fibroin-3/-4). Both proteins, ADF-3 and -4 belong to the class
of MaSp II proteins (major ampullate spidroin II). It is explicitely referred to
, the contents of which are incorporated herein by reference.
Monomeric sequence modules have been developed which are
also forming a starting point of the present invention. These modules are derived
from the genes ADF3 and ADF4 of the spider Araneus diadematus as well as
the gene FLAG of the spider Nephila clavipes. Variations of the employed
sequences of ADF3 and ADF4 are publicly available (available under the accession
numbers U47855 and U47856). The first two genes (ADF3 and ADF4) are coding for proteins
which are forming the dragline thread of the spider, the third is coding for a protein
of the flagelliform silk. Based on the amino acid sequence of these proteins, several
modules were designed:
GPYGPGASAA AAAAGGYGPG SGQQ
(SEQ ID NO: 1)
GSSAAAAAAA ASGPGGYGPE NQGPSGPGGY GPGGP
(SEQ ID NO: 3)
(SEQ ID NO: 2)
GPGGAGGPYG PGGAGGPYGP GGAGGPY
(SEQ ID NO: 4)
GGTTIIEDLD ITIDGADGPI TISEELTI
(SEQ ID NO: 5)
(SEQ ID NO: 6)
GPGGAGPGGY GPGGSGPGGY GPGGSGPGGY
(SEQ ID NO: 7)
GPYGPGASAA AAAAGGYGPG CGQQ
(SEQ ID NO: 8)
GPYGPGASAA AAAAGGYGPG KGQQ
(SEQ ID NO: 9)
GSSAAAAAAA ASGPGGYGPE NQGPCGPGGY GPGGP
(SEQ ID NO: 10)
GSSAAAAAAA ASGPGGYGPE NQGPKGPGGY GPGGP
(SEQ ID NO: 11)
GSSAAAAAAA ASGPGGYGPK NQGPSGPGGY GPGGP
(SEQ ID NO: 12)
GSSAAAAAAA ASGPGGYGPK NQGPCGPGGY GPGGP
(SEQ ID NO: 13)
GGTTIIEDLD ITIDGADGPI TICEELTI
(SEQ ID NO: 14)
GGTTIIEDLD ITIDGADGPI TIKEELTI
(SEQ ID NO: 15)
(SEQ ID NO: 16)
(SEQ ID NO: 17)
GPGGAGPGGY GPGGSGPGGY GPGGCGPGGY
(SEQ ID NO: 18)
GPGGAGPGGY GPGGSGPGGY GPGGKGPGGY
(SEQ ID NO: 19)
From these amino acid modules, synthetic spider silk protein
constructs were assembled. These modules and the spider silk proteins derived therefrom
are among others forming the starting material in the present method of forming
The invention is further directed to the use of specific
peptide TAGs. These tags (for example Tag's as disclosed in SEQ ID NO: 20-27, below)
contain cysteine or lysine. The sequence of the TAG is so selected that an interaction
with the rest of the silk protein and an influence of the assembling behavior can
be precluded to the greatest possible extent.
The following Tag's were developed for preferred use in
the spider silk constructs:
(SEQ ID NO:20)
(SEQ ID NO:22)
(SEQ ID NO:23)
(SEQ ID NO: 24)
(SEQ ID NO: 25)
(SEQ ID NO: 26)
(SEQ ID NO: 27)
Preferred examples of synthetic silk proteins made from
these modules may be found in chapter examples and preferably are (AQ)24NR3
Examples of silk producing insects from which silk proteins
may be obtained are Bombyx mori, Antheraea mylitta (oriental moth that produces
brownish silk) among others. The latter is producing tussah silk. Tussah silk is
a brownish silk yarn or fabric made from wild silk cocoons of a brownish color.
These worms feed on leaves from various plants and trees such as oak, cherry, and
wild mulberry. Further examples of such insects are caddies flies (e.g.
Hydropsyche slossonae), moths (e.g. Galleria mellonella (wax moth),
Ephestria kuehniella (flour moth), Plodia interpunctella (indian meal
moth), or Hyalophora cecropia (silk moth)).
The layers of the multilayer film preferably comprise one
or more agents incorporated therein or located between two adjacent layers. Those
agents preferably are selected from salts, dyes, metals, chemicals and/or pharmaceutical
agents. In this regard, it is referred to Figures 9, 11 and 12 which show the principles
of such an incorporation. The substances to be incorporated may be solid, semi-solid
or liquid without limitation.
For example, the pharmaceutical agent may be selected from
the group consisting of analgetics; hypnotics and sedatives; drugs for the treatment
of psychiatric disorders such as depression and schizophrenia; anti-epileptics and
anticonvulsants; drugs for the treatment of Parkinson's and Huntington's disease,
aging and Alzheimer's disease; drugs aimed at the treatment of CNS trauma or stroke;
drugs for the treatment of addiction and drug abuse; chemotherapeutic agents for
parasitic infections and diseases caused by microbes; immunosuppressive agents and
anti-cancer drugs; hormones and hormone antagonists; antagonists for non-metallic
toxic agents; cytostatic agents for the treatment of cancer; diagnostic substances
for use in medicine; immunoactive and immunoreactive agents; antibiotics; antispasmodics;
antihistamines; antinauseants; relaxants; stimulants; cerebral dilators; psychotropics;
vascular dilators and constrictors; anti-hypertensives; drugs for migraine treatment;
hypnotics, hyperglycemic and hypoglycemic agents; anti-asthmatics; antiviral agents;
and mixtures thereof.
Further, multilayer films according to the invention may
be designed which are acting as drug delivery system topically or systemically.
A topical system may comprise a multilayer system, wherein a viscous liquid is incorporated
and which system is applied to the skin for a predefined time. During that time,
the liquid penetrates through one or more layers of said multilayer film, thus providing
a defined amount of said liquid to the skin surface. In this case, the multilayer
film may be regarded as TTS (transdermal therapeutic system). Pharmaceutical substances
as hormones or nicotine might be administered by that way.
As an alternative, solid substances might be incorporated
within and/or between one or more layers of said multilayer. After oral administration,
the particles will migrate through the layers or will be forced out by influx of
body fluids (omotic systems) or by slowly dissolving one or more of the outer layers
and subsequent (sustained) release of the respective substance.
In other applications, for example clothing, the multilayer
film may be specifically adapted to the required field of use, for example, might
have one or more windtight or waterresistant layers. Additionally, as an example,
silver might be incorporated into the layers in order to provide an antiseptic effect.
According to a further preferred embodiment, the silk proteins
are covalently functionalized before or after step 1b) as defined above. In this
connection, it is also referred to the accompanying examples, see Figures 7 and
The layers may also be further processed in order to achieve
additional characteristics. For example, since water insolubility is a prerequisite
for most applications of protein films (which are mostly water soluble), the films
may be processed in order to become water-insoluble. Suitable methods for this purpose
are treatment with potassium phosphate or methanol.
The silk protein solution for casting the respective layer
of the multilayer film is containing 0.1-20%, preferably 0.5-10%, more preferably
1-3% w/v of silk protein. It is important to note that the concentration of the
solution is crucial since it will determine the actual thickness of the film. Also
by this means, tailored multilayer films of layers having a predetermined thickness
can be produced adapted to the specific envisioned application.
The layers of the multilayer film of the present invention
may be formed by any available method, preferably by moulding, spincoating or casting
the solution onto a suitable support. The type of support is generally not restricted,
however, supports of polystyrene, glass or silane (or any other surface which is
resistant to the employed solvents) may be named as suitable supports.
In a second aspect, the present invention is directed to
a multilayer film obtainable by the method as defined above.
According to a third aspect, the invention provides a cosmetical
composition; a pharmaceutical or medical composition, preferably drug delivery system,
artificial cell, contact lens coating, sustained-release drug delivery system, artificial
skin graft; food composition; automotive part; aeronautic component; computer or
data storage device; building material; textile or membrane comprising the above
Furthermore, the present invention provides the use of
that multilayer film in medicine.
The present invention will be illustrated by the following
non-limiting Examples and the accompanying figures.
The figures are showing the following:
Film casting of spider silk proteins:
CD-spectra of synthetic silk proteins (AQ)24NR3 and C16
dissolved in 6 M guanidinium thiocyanate followed by dialysis against 5 mM potassium
phosphate pH 8.0 (straight line) or dissolved in HFIP (dotted line).
CD-spectra of protein films made from (AQ)24NR3 and C16.
Films were cast from a protein solution in HFIP directly on a plain quartz glass
and analyzed by CD-spectroscopy (dotted line). The film was subsequently processed
with 1 M potassium phosphate and reanalyzed. Due to inaccuracies in defining the
thickness of the films, &THgr;MRW could not be determined.
Modification of C16 films cast from a HFIP solution and processed
with potassium phosphate. (A) Efficient coupling of fluorescein (yellow colour)
only occurred when the carboxyl groups of C16 were activated (+) using
EDC. In contrast only little fluorescein bound to films without EDC activation (-).
(B) Activity of coupled &bgr;-galactosidase was monitored using X-Gal as substrate.
The occurrence of a blue precipitate indicated enzyme activity only on films that
had been activated with EDC (+), while non-activated films only showed residual
enzymatic activity (-).
Casting of multilayer silk films.
Casting of multilayer silk films with different functionalities.
Figure 6 Casting of multilayer silk films with different functionalities.
Figure 7 Chemical coupling of agents to silk proteins as shown for example
with EDC (N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide) induced coupling of an
Specific functionalization of silk surfaces. In multilayer films polarity can
be obtained by employing a multilayer silk film cast from different proteins.
Incorporation of agents in multilayer silk films.
Incorporation of agents in multilayer silk films. Differently colored chemicals
were added to the silk protein solution prior to casting as a proof of principle.
Incorporation of solid agents in a sandwich multilayer silk film.
Incorporation of fluid agents in a sandwich multilayer silk film.
In order to cast films the inventors previously employed
the two synthetic silk proteins, (AQ)24NR3 and C16, which
are derived from the dragline silk proteins ADF-3 and ADF-4 from the garden spider
Araneus diadematus. These proteins were chosen based on previous observations
that ADF-3 and ADF-4 as well as its derivatives display a markedly different solubility
and assembly behaviour. Measuring circular dichroism (CD) of (AQ)24NR3
and C16 solutions revealed a different influence of aqueous buffer and
HFIP on secondary structure. In aqueous solution both proteins displayed a CD-spectrum
with a single minimum at a wavelength below 200 nm which is indicative of a mainly
random coiled protein (Figure 1).
In contrast, the spectra of both proteins in HFIP displayed
one minimum at 201 - 202 nm and an additional minimum ((AQ)24NR3) or
shoulder (C16) at 220 nm which is indicative of an increased &agr;-helical
content (Figure 1). Such an effect of fluorinated alcohols on proteins and peptides
has been reported previously and has also been observed for silk fibroin and a synthetic
silk protein derived from the dragline silk protein MaSp1 from the spider
Films were cast from 200 µl HFIP solutions containing
2% w/v protein on a polystyrene surface (or on quartz glass for CD-measurements).
After evaporation of the solvent, (AQ)24NR3 and C16 both formed
transparent films that could easily be peeled off the surface. Assuming complete
evaporation of the solvent and the density of the protein film to be identical with
the reported value of 1.3 g/cm3 for spider dragline silk, the thickness
of the films was calculated to range from 0.5 to 1.5 µm. As-cast (freshly prepared)
films made of either protein dissolved upon contact with water. Since water insolubility
is a prerequisite for most applications of protein films, we searched for a processing
method in order to render films insoluble. Potassium phosphate is known to induce
aggregation and formation of chemically stable structures of the employed silk proteins.
Also methanol has been used to obtain insoluble silk morphologies. Accordingly,
processing (incubating) of as-cast films with 1 M potassium phosphate or methanol
resulted in the conversion of water-soluble films into water-insoluble ones.
To investigate the structural properties of the films,
the secondary structure of the underlying proteins was investigated by CD-spectroscopy.
As-cast films revealed a spectrum with two pronounced minima at 208 nm and 220 nm
indicative of an &agr;-helical content higher than that of soluble proteins. After
processing with 1 M potassium phosphate, films revealed spectra with a single minimum
at 218 nm, which is typical for a &bgr;-sheet rich protein structure (Figure 2).
Similar results were obtained after processing films with methanol (data not shown).
Thus, the transition from water-solubility to water-insolubility was paralleled
by a conversion of the protein's secondary structure from &agr;-helix to &bgr;-sheet.
To test their chemical stability, films were submerged
for 24 hours in 8 M urea, 6 M guanidinium hydrochloride and 6 M guanidinium thiocyanate.
As-cast films of both proteins as well as (AQ)24NR3 films processed with
potassium phosphate or methanol were soluble in all of these denaturants. In contrast,
C16 films processed with potassium phosphate or methanol could only be
dissolved in guanidinium thiocyanate. This remarkable chemical stability of C16
films is identical to that of natural dragline silk and to that of recombinantly
produced and assembled ADF-4. Previous studies could correlate assembly properties
and stabilities of assembled structures directly with the amino acid sequences of
the silk proteins. Thus, properties of spider silk films can directly be modified
by altering the primary structure of the silk protein via manipulation of the corresponding
silk gene (Figure 2).
Many applications of protein films require the presence
of specific functionalities on the film's surface. In order to demonstrate, that
the employed spider silk films can be modified with small organic molecules as well
as biological macromolecules such as proteins, the chromophor fluorescein and the
enzyme &bgr;-galactosidase were chemically coupled to C16 films processed
with potassium phosphate as a proof of principle. The coupling was achieved by activating
surface exposed carboxyl groups of C16 using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC). The films were further incubated with ethylenediamine leading to the formation
of an amide. The remaining free amino group of ethylenediamine was subsequently
coupled to fluoresceinisothiocyanate resulting in the efficient covalent linkage
of fluorescein via formation of a stable thiourea derivative (Figure 3). Similarly,
incubation of &bgr;-galactosidase with EDC-activated C16 films led
to the formation of amide bonds between carboxyl groups of C16 and primary
amines (e.g. from lysine residues) of &bgr;-galactosidase which were accessible
at the enzyme's surface. After repeated washing of such modified films, &bgr;-galactosidase
activity could be detected using 5-bromo-4-chloro-3-indolyl-&bgr;-D-galactopyranoside
(X-Gal) as a substrate (Figure 3).
The inventors could demonstrate that protein films can
be obtained from synthetic spider silk proteins. The films, which initially were
water soluble, can be processed with potassium phosphate or methanol leading to
water-insolubility, which is a major requirement for many applications. Comparison
of the chemical stabilities of films made from two different synthetic spider silk
proteins suggested that the properties of the films were based on the primary structure
of the proteins. Employing our previously established cloning strategy for spider
silk genes, it will be possible to generate silk proteins that form films displaying
specific properties. Since different functional molecules can be covalently attached
to the film's surface, a great variety of technical or medical applications can
therefore be approached.
The proteins (AQ)24NR3 and C16 can
be cast into several films starting from HFIP or formic acid solutions. However,
any other silk protein built upon our modules (sequence 1-27) as well as natural
insect and spider silk proteins can be employed. The protein solution is cast on
polystyrene, glass or silane (or any other surface which are resistant to the employed
solvents) and the solvent is completely evaporated afterwards. Films cast from hexafluoroisopropanol
solutions are water soluble. To achieve water-insolubility these films have to be
methanol, ethanol or potassium phosphate. Films cast from formic acid are insoluble
in water without processing.
The thickness of the films can be controlled by the concentration
of the employed protein solution (data not shown). Importantly, films can be cast
from solutions of a single protein (One-protein films) or of two proteins (Two-protein
The inventors analyzed whether films cast from solutions
containing two protein components (Two-protein films) revealed a different structure
or stability in comparison to One-protein films. (AQ)12 and C16
or (AQ)24NR3 and C16NR4 were dissolved
in HFIP (1% w/v each, 2% w/v in total). For sequence information, it is referred
, incorporated herein by reference.
Two-protein films were cast from (AQ)12/C16
(molar ratio 1 : 1) or (AQ)24NR3/C16NR4 (molar ratio 1 : 1.8)
mixtures. Remarkably, Two-protein films showed a combination of the properties of
the films cast from the single silk proteins. As-cast Two-protein films made of
(AQ)12/C16 have been soluble in all tested reagents. After
processing with methanol, these films became insoluble in water and urea, but soluble
in solutions of GdmC1 and GdmSCN, reflecting a chemical stability between that of
plain C16 or plain (AQ)12 films. As-cast Two-protein films
of (AQ)24NR3/C16NR4 could not be completely dissolved in water.
After water treatment, amorphous protein aggregates were remaining. The formation
of intermolecular disulphide bridges between the NR-regions could be excluded to
cause amorphous aggregation, since the behaviour of the films did not change in
the presence of a reducing agent such as &bgr;-mercaptoethanol (5% (v/v)). Processing
of the (AQ)24NR3/C16NR4 films with methanol led to chemical
stability in water and urea.
In the case of Two-protein films, the treatment with methanol
led to a chemical stability influenced by both proteins. Compared to conventional
synthetic polymers, where blending usually causes mixed properties, such finding
is astonishing for proteins. In general, the structure and interaction of proteins
is complex and depends on many factors. When mixed, two proteins, that do not interact,
usually remain their chemical stability found in the absence of the second protein.
In case they do interact, usually both proteins show a higher chemical
stability. In our case, both proteins seemed to interact, but the gained chemical
stability was between that of the single compounds.
Multilayered films can be obtained by casting further layers
on already existing films (Figure 4, 5, and 6). All layers of a multilayer film
can be made of spider silk, but film layers can additionally be made of other materials
such as insect silk, elastin, collagen, keratin, polystyrene, polyvinylchloride,
poly(styrene sulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(acrylic
acid) (PAA), poly(diallyldimethylammoniumchloride) (PDADMAC), etc. The thickness
of the silk films can be controlled by the protein concentration. Each layer can
contain a different silk protein (natural insect or spider silk, or recombinant
silk based on our modules sequence 1-27) with different chemical and physical properties.
Further, each film can contain differently modified silk proteins (the modification
can take place before film casting). Finally, each layer can be post-cast processed
with a desired functionality by chemically coupling an agent to the respective silk
protein (Figures 7 and 8).
Substances can be incorporated into films cast from recombinant
spider silk proteins by adding them before casting (Figure 9 and 10). Alternatively
the substance can be given on top of a silk film and another silk film can be cast
on it (Figures 11 and 12).
As a summary, the following objects can be achieved by the present invention:
Single silk film layers can either be covalently functionalized
before or after casting. Further, blends between silk and agents (such as salts,
dyes, metals, chemicals, drugs, etc.) can be prepared prior to casting. Casting
layer by layer generates multilayer films with different functionalities in each
layer. Such multilayer multifunctional silk protein films are entirely new. Additionally,
blending of different spider silk proteins, each functionalized differently, for
casting a single film is new. Thereby, each single film of a multilayer protein
film can provide different functionalities, yielding a complex three-dimensional
scaffold with defined spatial distributions of single functions. In case, the single
function can communicate, smart three-dimensional structures are the result. Finally,
silk films can be layered with other existing polymer films, creating mixed multilayer
films of different components.
Industrial applicability of the invention:
Multilayer, multifunctional scaffolds and structures are
a basis for a huge amount of innovative products for food science, waste disposal,
and the cosmetical, medical, pharmaceutical, automotive, aeronautic, etc. market.
The applications involve for example, device coatings (e.g. for advanced endothelial
cell attachments), drug delivery systems, artificial cells, contact lens coatings,
sustained-release drug delivery systems, biosensors, and functionally advanced materials
with various electrical (e.g. light-emitting diodes), magnetic, electrochromic,
and optical properties (e.g. enhancing the brightness of handheld computers and
computer screens, reducing the amount of interference with cellular signals, and
enabling automobiles to function more efficiently by reflecting infrared light (heat
rays) and thereby reducing the burden on air conditioners).
Multilayer silk films can take light that would normally
be absorbed and turn it into useful light, and increase brightness. In automotive
industry multilayer silk films can be employed that reflect solar infrared heat,
since the non-conductive films are completely clear, a property that could be useful
in architectural applications where increased daylight transmission is desirable.
Multilayer film that are comprised of hundreds of layers of transparent silk polymers
reflect due to optical interference effects. The wavelengths that are reflected
and transmitted change as a result of the angle at which they're held.
Multilayers films can been used to form thin nanoporous
and microporous membranes and can been exploited as nanoreactors for the synthesis
of metallic nanoparticles.
The hydrogen-bonded multilayer silk films can be employed
in applications such as micropatterning and drug delivery, where control of the
deconstruction rate of the hydrogen-bonded films is desirable. One approach to modulating
the deconstruction behavior of multilayered thin films is via structural design
of the films.
Another application would be enantiomeric separations.
Chirality is key to molecular recognition in biology. The pharmaceutical industry's
need for single enantiomer drugs has spurred the development of techniques for preparative-scale
separation of chiral molecules. Membrane-based separations have attracted much attention
due to operational simplicity and low cost.
Further applications of multilayer silk films are artificial
skin grafts. Major burn accidents involve extensive damage to the skin. Immediate
coverage is needed to limit loss of fluid and aid tissue repair and regeneration.
The structural and functional properties of an ideal skin substitute should closely
match autograft skin. Plasticity of the substitute preparation procedure and its
composition provide added value for coverage, minimizing rejection and activation
of the inflammatory response. Multilayer spider silk films could be useful for preparing
artificial skin grafts, as the method provides a simple means of producing films
without limit to size, shape, and composition and polypeptides are inherently biocompatible.
Nanofiltration (NF) is a pressure-driven membrane separation
process that is used for applications such as water softening, brackish water reclamation,
and dyesalt separations. Such applications do not require the high NaCl rejections
that are typical of reverse osmosis (RO) membranes, so NF occurs at significantly
lower pressures than RO and, hence, requires less energy. For this reason, the use
of NF is growing rapidly, but continued development of selective, high-flux membranes
that are stable and resist fouling should enhance the utility of this separation
technique. In both RO and NF, membranes consist of a selective skin layer on a highly
permeable support because the minimal thickness of the skin layer allows a reasonable
flux despite its dense nature. Typical procedures for creating such membrane structures
include phase inversion and formation of composite membranes by interfacial polymerization,
grafting, or film deposition on a preformed porous support. Composite membranes
are particularly attractive because they require only small amounts of the potentially
expensive skin material. Multilayer silk films provide a controlled method for forming
the skin layer of membranes for NF, gas-separation, and pervaporation.
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