(706g) Aqueous Fiber Spinning From a Renewable Protein Waste Material

Crystallin
proteins from animal eye lenses represent a renewable, environmentally
responsible biopolymer source for bio-derived fibers.  The crystallin protein
family is a diverse and highly conserved set of proteins that form the
macromolecular structure of the lenses of many vertebrates.  Despite
variability in lens anatomy across organisms, these proteins are consistently
organized in a supramolecular structure that is optically clear, preserving the
ability of the lens to refract light.  Due to their natural functions, these
proteins possess a range of interesting features which make them attractive for
the production of new biomaterials such as self-assembly and the formation of
stable, well ordered structures.  These structures include soluble protein,
nanofibrils, and amorphous aggregates, each derived from native conformations. 
Here, we describe the aqueous processing and spinning of crystallin proteins
generated from a waste material, namely fish eye lens, into durable fibers.  Crystallin
protein was isolated from North Atlantic Haddock (Melanogrammus
aeglefinus) eye lenses through a simple homogenization technique.  Intact lenses were
extracted and gently stirred at room temperature in an aqueous homogenization
buffer designed to minimize protein aggregation and stabilize intermediate
assembly states.  After 24 hrs, the fish lens homogenate (FLH) is clarified by
centrifugation and collected as an opaque suspension in which crystallin
protein is present in both amorphous and structured forms.  The FLH is
comprised of a series of related structural proteins, which are used for
subsequent investigations without further purification. 

Aqueous
spinning of fibers from the FLH entails a delicate balance of processing,
spinning and post-spinning variables.  The desired aqueous processing approach creates
a uniform, soluble protein solution removing insoluble aggregates.  The
processing approach must stabilize solution dynamics to facilitate proper ordering
and organization of the protein, at a molecular level.  Minimization or
suppression of aggregation during solution preparation and aging at relatively
high concentrations is required to generate a reproducible spin solution for
fiber spinning.  Toward designing suitable processing conditions, FLH was
dialyzed into biological buffers and concentrated through ultrafiltration. 
Several variables throughout the process were considered including buffer
composition, protein concentration and spin solution aging.  Dialysis buffer
composition was varied by supplementation with a number of amino acid
stabilizers (i.e. aggregation suppressors) at varying concentrations including
glycine, lysine and arginine. Buffer exchange was also conducted under varying temperature
and pH conditions.  While glycine and lysine enhanced protein solubility as
amino acid concentration increased, the influence on protein molecular assembly
was not sufficient since gelation occurred relatively rapidly during spin solution
aging.  However, supplementation with arginine improved protein solubilization
and stabilization, enhancing protein yields and allowing for the preparation of
concentrated spin solutions (~300-400 mg/ml).  Arginine resulted in reproducible,
stable solutions which, after sufficient aging, facilitated suitable protein
assembly states for subsequent formation of intact, durable fibers. 
Interestingly, the arginine supplemented buffer was basic in nature (pH = 10.5)
whereas glycine and lysine buffers were more acidic (pH 5 and 5.2
respectively).  The influence of pH relative to the presence of arginine on
protein solubilization and stabilization is still under investigation.  Optimal
processing temperature was determined as 4^oC. 

Spin
solutions of various concentrations and stages of supramolecular assembly were
spun into fibers through a patented aqueous spinning approach.  The spinning
technique was inspired by nature and, more specifically, the process by which a
spider spins native dragline silk.  The design of the spinneret is based on the
gradually tapered gland within the spider, which causes a shear-induced
transition of self-assembled silk protein into a fiber.  Our lab-scale
technique has previously been demonstrated in spinning recombinant spider silk
proteins into fibers with mechanical integrity equivalent to natural dragline
silk.  Spin solutions are loaded into the micro-spinneret, which has a volume
capacity of 500 ul although typical spin trials are 50-75 ul.  A syringe pump
applies a constant force at a constant rate (2-10 ul/min) to force the spin
dope through PEEK tubing with I.D. ranging from 0.15-0.25 mm into a coagulation
bath.  The fibers are allowed to coagulate within the bath and are collected
after removal from the coagulation bath and submersion into a water bath. 
Numerous spinning and post-spinning variables must be considered to optimize
fiber formation, durability and mechanical integrity from concentrated spin
dopes.  Here, this spinning technique was used to generate fibers from FLH
solutions containing arginine at various protein concentrations and stages of
spin solution aging, which correlate to various intermediate assembly states. 
Fiber spinning variables considered include coagulation bath composition and
spinneret I.D. while post-spinning variables include coagulation time and draw
ratio.  Coagulation tests were conducted to choose optimal coagulation
conditions for the various spin solutions.  Fibers up to 12 inches were
generated although typically fibers ranged from 2-3 inches in length.  Fibers
were of sufficient durability to withstand not only natural draw due to surface
tension while removing from the coagulation bath but also extension to twice
the original length prior to collection.  Drawn fibers were, on average, ~17 um
in diameter while undrawn fibers were ~28 um.  Light microscopy of the as-spun
fibers under white light was conducted to investigate surface and core defects
along the length of the fiber.  Molecular alignment along the fiber axis was analyzed
by investigating the birefringent nature of the single fibers under polarizing
light with a first order red plate at 530 nm.  In general, fibers exhibited
birefringence, which was indicative of some level of molecular order.  However,
the fibers did contain a variety of defects that limited subsequent mechanical
testing. 

Analysis
of a compilation of spin trials enabled the identification of integral
processing, spinning and post-spinning variables essential for formation of
fibers from crystallin proteins.  Spin solution aging is dependent on protein
concentration - higher concentration requires less aging to reach the
appropriate conditions to spin fibers.  Increasing draw decreased fiber
diameter, but molecular order was not enhanced at the current extensibility
(~2-fold).  Optimal coagulation time was determined as 15-20 min for formation
of the most durable fibers.  The information derived is being considered for
optimization of processing and spinning processes to produce fibers with enhanced
mechanical integrity.  Also under investigation is the use of
nano-reinforcements, specifically nanofibrils, to enhance fiber properties.  Crystallin
proteins readily form nanofibrils in solution, with branching and length being
controlled by processing conditions and temperature.  Nanofibrils occur by
subjecting the proteins to mildly destabilizing conditions that allow the
proteins to partially unfold and refold.  Recent advances in our lab enable the
control of nanofibril structure and morphology (e.g. length, bundling and
branching).  Nanofibrils of up to 10 um in length with varying degrees of
bundling and branching have been created and may serve as an additive that
could further facilitate formation of fibers with increasing mechanical
integrity from concentrated soluble protein spin solutions.  Alternatively,
fibers can be spun directly from solutions containing nanofibrils. 

Fibers
from crystallin proteins are a naturally-derived biomaterial that could be used
in composite or textile applications including new fiber reinforcing material
for (bio)composites or as a substitute for Nylon and polypropylene-based
textiles.  Furthermore, the fibers represent a chemically diverse (defined by
the amino acid side chains) platform for the future design of new multifunctional
materials for use in military and civilian applications.  This work represents
a significant advancement in the state-of-the-art regarding spinning of
bio-derived fibers from a renewable waste protein material and offers a basis
for spinning a range of biopolymers into useful materials.