Self-assembled coacervates of chitosan and an insect cuticle protein

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Self-assembled coacervates of chitosan and an insect cuticle protein containing a Rebers-Riddiford motif M. Coleman Vaclaw, Patricia A. Sprouse, Neal T. Dittmer, Saba Ghazvini, C. Russell Middaugh, Michael R Kanost, Stevin H. Gehrke, and Prajnaparamita Dhar Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01637 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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Self-assembled coacervates of chitosan and an insect cuticle protein containing a Rebers-Riddiford motif M. Coleman Vaclaw,† Patricia A. Sprouse,‡ Neal T. Dittmer,§ Saba Ghazvini,† C. Russell Middaugh,∥ Michael R. Kanost,§ Stevin H. Gehrke,†,‡ Prajnaparamita Dhar,*,†,‡ AUTHOR ADDRESS †

Bioengineering, University of Kansas 1530 W 15th St. Lawrence, KS 66045 USA

‡

Chemical and Petroleum Engineering, University of Kansas 1530 W 15th St. Lawrence, KS

66045 USA §

Department of Biochemistry and Molecular Biophysics, Kansas State University 141 Chalmers

Hall Manhattan, KS 66506 USA ∥Department

of Pharmaceutical Chemistry, 2095 Constant Ave. Lawrence, KS 66047 USA;

KEYWORDS Coacervates, Microrheology, Chitosan, Cuticle

ABSTRACT

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to

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bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micron-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif. Introduction Biomimetics, a relatively new material science approach switches focus from the design of synthetic materials and systems to the utilization of designs and structures already found and optimized in nature, to solve complex materials design problems. One particular subset of systems from which biomimetic inspiration can be drawn is the insect exoskeleton, formed from a material called cuticle. Insect cuticle may serve as a biomimetic template for materials due to its robust yet versatile mechanical properties. Insect cuticle is a composite material made of chitin nanofibers embedded in a matrix of protein and hydrophobic pigments. Different types of cuticle can have drastically different mechanical properties, even though they are composed of the same, relatively simple set of organic materials.1 It is generally accepted that differences in molecular interactions between the proteins and chitin are responsible for these large changes in mechanical properties.1 Therefore, understanding these protein-polysaccharide interactions and how they affect the mechanical


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properties of the materials they are found in is vital in biomaterials research and may lead to the development of novel biomaterials with enhanced properties.2-18 Cuticle is commonly divided into two separate categories based on cuticle composition, properties, and function. “Soft” cuticle mostly contains equal weight fractions of chitin and protein, 40-75% water and is a very flexible material.1,19,20 On the other hand, “hard” cuticle contains only 12% water while chitin makes up 15-50% weight fraction of dried cuticle which makes it stiff and contributes to the strength and rigidity of the insect exoskeleton.1,21 Chitin microfibrils found in cuticle consist of 18-21 chitin molecules that are arranged in various patterns but generally form fibers that are about 2.8 nm in diameter.21 The chitin nanofibers are secreted from crests of undulations on the epithelial cell membrane, while the protein matrix is secreted from the grooves between each crest.22 The chitin is therefore secreted into a protein matrix that offers many stabilizing interactions, lending the cuticle its increased strength. Chitosan, the partially deacetylated derivative of chitin, is another common component of insect cuticle and has already been used with success in a variety of biomedical applications.23-25 The amount of deacetylation of chitin in cuticle is variable and has been found to be between 10-20%.26 Insects have genes that encode chitin deacetylases and it has been hypothesized that one of the functions of these deacetylases may be to provide amino groups for cross-linking to proteins.26 The degree of deacetylation (DD) at which point chitin becomes chitosan is not clearly defined although a DD of 50-95% is typical.25 It is hypothesized that face stacking interactions between the aromatic moieties in the protein and the carbon rings of polysaccharides allow proteins to bind chitin/chitosan.27 Because of the presence of chitin and, to a lesser extent, chitosan in the cuticle, interactions between proteins and chitin are expected to be similar to those between protein and chitosan, although they may not be identical. Chitosan is more


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soluble than chitin in aqueous medium, which facilitates and simplifies many research applications in which it is used. Due to its solubility, and because of the relative ease in obtaining a purified form of it, chitosan was chosen for the present work. While several conserved families of insect cuticular protein genes have been identified,28,29 the most abundant family of cuticular protein genes in analyzed insect genomes contain a sequence known as the Rebers-Riddiford (RR) motif.28 The extended RR consensus sequence contains 68 residues and three distinct forms of the “extended sequence” have been identified: RR-1, RR-2, and RR-3.28 Proteins containing the RR-1 form are commonly found in “soft” cuticle whereas the RR-2 form is generally isolated in “hard” cuticle although this classification is based on limited data.30 The RR-3 form is less common and is based on only five sequences from postecdysial cuticle of arthropods.27,31 The RR consensus sequence has been hypothesized to be involved in chitin binding.27,30-34 However, there has not been sufficient direct experimental evidence to support this hypothesis. Binding of recombinant cuticular proteins to chitin beads has been shown experimentally, but the binding may not reflect in vivo binding mechanisms, either to chitin nanofibers, or chitosan.32,35,36 Therefore, the goal of the present study is to gain direct experimental evidence of chitosan binding to proteins containing the RR sequence. Specifically, we sought to test a hypothesis that RR binding of protein to chitosan would result in the formation of self-assembled structures, possibly coacervates, which would alter the solution viscosity. Our hypothesis is based on previous reports of cuticular proteins undergoing coacervate formation due changes in temperature, pH, and ionic strength.37 Further, observing changes in viscosity and other rheological properties of solutions undergoing coacervate formation isn’t without precedent. Recently, it was shown that coacervates formed from jumbo squid Dosidicus gigas histidine-rich


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proteins (DgHBPs) demonstrated relatively lower viscosity and shear-thinning behavior. It was proposed that this shear thinning behavior of the coacervates of DgHBP allows the proteins to more easily penetrate the beak’s chitin network and are partially responsible for the observed mechanical gradient found in squid beak.38 Therefore, we hypothesize that such coacervates may also play a vital role in insect cuticle, including changing the mechanical and rheological properties.39,40 Rheological experiments, while extremely useful in studying changes in solution viscosities, present several hurdles for studies involving recombinant proteins, since they often require large sample volumes which may be difficult to obtain if only limited quantities of a recombinant protein are available. Microrheology, a technique using micron or submicron sized particles as tracers to probe the solution viscosity, overcomes these drawbacks by observing the motion of probe particles in microliter-sized solutions. In particular, microrheology is advantageous as it can characterize local rheological properties in a solution and relate them to changes occurring in the microstructure of a sample of interest, such as protein folding and unfolding phenomena.41 This is also especially useful for suspension and coacervate systems where solution viscosity can be correlated to the formation and density of particulates.42 Microrheology techniques coupled with microscopy can simultaneously measure and visualize samples such that changes in rheological properties can be directly correlated to visible changes in a sample’s microstructure. In the present study, we investigate interactions of a full length RR-2 protein, CPR27, with chitosan. CPR27 is a 10 kDa RR-2 protein found in the rigid elytra (modified forewings) of the red floor beetle, Tribolium castaneum. This insect model was chosen because it is one of the most widely studied insects and many of the cuticle protein sequences have already been obtained.19,20,43-45 CPR27 was studied here because it is a major structural protein containing the


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RR-2 motif and is abundant in rigid cuticle but not in flexible cuticle.44 Furthermore, it has been shown that decreased expression of CPR27 by RNA interference led to elytra that were shorter, wrinkled, warped and less rigid than elytra of control beetles, suggesting that CPR27 plays an important role in the structural integrity of the cuticle.44 The second protein used in this study is CP30, a 19 kDa protein and the third most abundant protein in the elytra of T. castaneum.46 Although the localization of CP30 in cuticle is similar to that of CPR27, CP30 does not contain any form of the RR motif, thus serving as a negative control for our study. Further, its structural role in cuticle currently remains unknown. It has been shown that RNA interference of CP30 results in most insects failing to molt from pupa to adult. The insects that are able to successfully molt fail to fully expand their elytra.46 There is also evidence that CP30 cross-links with itself and putatively to CPR27 and therefore contributes to the strength of the cuticle.46 Interestingly, CP30 has a low complexity sequence with blocks of positively and negatively charged residues which may be involved in electrostatic interactions between the different cuticle components or even with itself.46 Therefore CP30 was used to rule out whether simple electrostatic interactions are responsible for the RR motif’s ability to bind chitin. The amino acid sequences for both proteins are shown in Figure 1.


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CPR27 Sequence QGGEGYGHHHLEEYIDYRARPRYHYDYNVHDHHTHDFHHQWEHRDGKEVKGEYSLIQPDGRRRTVEYRAGKHGADYRIKYE GHSHHGGIGSFGIGGN

CP30 Sequence VHASPHHEERRHEERRREEEKHHHHREGGEEGGRGREEEHHHHREEERKHHREEEERKHHHREEEERKHREEEERHHHREEE RKHHHREEEERHHHREREEERHHHHEGEEGGRGGGEEEGRGGEEHWGRGGEEEGGRGGGEEEEWGHGWGRREW Figure 1. CPR27 and CP30 amino acid sequences. The Rebers-Riddiford (RR) motif in CPR27 is highlighted in red. This motif is hypothesized to bind to chitin. CP30 does not contain an RR motif and is not expected to bind chitin and serves as the control for this study.

In this study an active microrheology technique was used to measure and detect RR protein induced changes in chitosan solution viscosity at several different protein and chitosan concentrations. This was combined with fluorescence microscopy and microflow imaging (MFI) to detect the formation of chitosan complexes induced by CPR27. Our results showed direct evidence of RR protein induced complexation of chitosan, leading to coacervate formation. Coacervate formation was also accompanied by significant lowering of the solution viscosity. We conclude that RR protein CPR27 binds to chitosan due to the presence of the RR chitinbinding motif, resulting in formation of coacervates, which may alter the solution viscosity. Such changes in solution viscosity may actually be responsible for gradients in mechanical properties in insect cuticles and may enable smoother penetration of proteins through the chitin-protein matrix similar to reported observations in the jumbo squid beak discussed above.38 Experimental Section Materials


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Two different chitosan variants were used in this study: a low molecular weight (2.7 kDa47) oligomer designated KITO1 was used for rheological measurements and another low molecular weight chitosan modified with an FITC label (KITO3) was used for fluorescent tracking of chitosan in solution. Both chitosan variants were obtained from Akina, Inc. (West Lafayette, Indiana). H-NMR and FTIR data from the manufacturer were used to calculate an approximate degree of deacetylation (%DD) and in both variants the %DD was greater than 90%. Rheological tests were performed by dissolving the chitosan and protein in 0.2 M sodium acetate buffer (pH5). To produce recombinant protein, the coding sequence (minus the signal peptide) for CPR27 was cloned into the expression vector pET-28 and used to transform the Escherichia coli strain Rosetta (DE3) (Novagen). Cultures were grown at 37 oC in LB medium supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenical. Protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Four to five hours post induction, bacterial cells were collected by centrifugation and stored at -20 oC. For protein purification, cell pellets were resuspended in 50 mM Tris (pH 8) and lysed on ice by sonication. Following centrifugation, the supernatant was discarded and the insoluble material was resuspended in 8 M urea by stirring overnight at 4 oC. The sample was centrifuged again and the supernatant was diluted 20-fold in 50 mM ammonium acetate (pH 5) with stirring for 16 to 24 h at 4 oC. Precipitated material was removed by centrifugation and the supernatant was loaded onto a HiPrep SP FF 16/10 cation exchange column (GE Healthcare). The column was washed with 50 mM ammonium acetate (pH 5) and eluted with a 50 mM to 2 M linear gradient of the same buffer. Fractions containing CPR27 were pooled and lyophilized. Spectra obtained using circular dichroism show that the CPR27 contains very little secondary structure. Expression and


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purification of CP30 were as described in the supplementary information of a previous publication.46 Stock solutions of protein were prepared from lyophilized protein by dissolution in sodium acetate/acetic acid buffer at twice the desired concentration. The stock solutions were then mixed with chitosan solutions in equal volumes to achieve the desired final concentration. Bulk Rheology Bulk rheology measurements were used to characterize the bulk viscosity and the viscoelasticity of the solution being examined and also to provide a calibration for the microrheology technique. These experiments were done using an AR2000 rheometer (TA Instruments, New Castle, DE) with a 40mm diameter 2o cone and plate geometry. Chitosan samples were allowed to dissolve overnight in sodium acetate buffer. First, shear stress sweeps ranging from 0.04 Pa to 4000 Pa were performed to ensure that measurements were taken outside of any shear thinning regions. Subsequent measurements were done at the lowest allowable shear stress to estimate the zero shear viscosity of the samples which was then compared with results obtained on the microrheology system. All experiments were performed at 23 ± 1 oC. It is important to note that, in order to measure the viscosity of bulk solution, conventional rheological methods could not be initially applied because it was unclear at which chitosan and protein concentrations an interaction resulting in a rheological effect could be identified. Such testing would require an unfeasible amount of protein if it were done with bulk methods. Therefore, an active microrheology technique, described in detail in the next section, was used. Active Magnetic Microrheology


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A custom-built active microrheology technique was used to obtain viscosity data using two sets of solenoids that create orthogonal magnetic fields as shown in Figure 2. Magnetic nanorods several microns in length and 300 nm in diameter were synthesized by electrochemical deposition of nickel into Anodyne disk filters as described elsewhere.48-50 Before starting an experiment the nanorods were sonicated for approximately 1 hour to break up any significant aggregates. Then nanorods in dilute concentrations were mixed with the samples of interest. Twenty μL of the sample of interest was loaded into a small well made from polydimethylsiloxane (PDMS) and placed between two glass slides. Initially the nanorods are allowed to align with one magnetic field at time t=0, at which point the first magnetic field is turned off and the other which is set at 90o to the original field is turned on. The change in magnetic field causes the nanorods to rotate from 0o to 90o with respect to the initial field, following the equations described below. The rotation of the nanorods was monitored by a CCD Andor Luca video camera attached to a Leica DM 2500M microscope. The videos were


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converted into a series of images and the angle of the nanorods with respect to time could be measured using NIH ImageJ software. For a purely viscous system the motion of the nanorods is described by first applying a balance of the magnetic torque and the viscous torque that are being applied to the nanorods in solution experience in the magnetic field:

Figure 2: Home-built active magnetic microrheology setup. A power supply and switch connected to two pairs of magnets form two magnetic fields that are approximately orthogonal. Magnetic nanorods turning in the sample are captured using a CCD camera connected to a Leica DM 2500M microscope.

𝜇𝑜 𝑚𝐻𝑠𝑖𝑛𝜃 = −𝑓𝑟 𝜂𝑙 3 𝑑𝜃/𝑑𝑡

(1)

where 𝜇𝑜 𝑚 is the magnetic moment, H is the magnitude of the magnetic field, θ is the angle of the rod with respect to the applied magnetic field, 𝑓𝑟 is the drag coefficient, η is the viscosity of the solution, and l is the length of the nanorod. The solution to the differential equation is given by:


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𝜃

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(2)

tan ( 2) = exp⁡(−𝑡/𝜏)

where τ = 𝑓𝑟 𝜂𝑙 3 /𝜇𝑜 𝑚𝐻 is the relaxation time. A plot of the angle of the nanorod versus time is generated and the data is fit to an exponential decay function from which the relaxation time is extracted. The viscosity of the solution is then calculated as: 𝜂 = 𝜏𝜇𝑜 𝑚𝐻/𝑓𝑟 𝑙 3

(3)

Equation 3 can be rewritten in terms of the magnetic field B (=𝜇𝑜 𝐻), and the magnetization M (=mV, where V is the volume of the nanorod). The solution viscosity is then calculated as: 𝜂 = 𝜏𝐵𝑀𝑉/𝑓𝑟 𝑙 3

(4)

The magnetic field was measured after each experiment using a Gauss meter. The magnetization was calibrated by performing a set of experiments on water and glycerol solutions whose viscosities are well documented. This was calculated to be about 1.2 * 105 A/m. The drag coefficient was calculated using the equation: 2𝑙

(5)

𝑓𝑟 = 𝜋/(3(ln ( 𝑑 ) − 0.8)

where l is the length of the nanorods and d is the diameter of the nanorods which was previously determined to be about 300 nm by transmission electron microscopy. Fluorescence images were taken with an upright fluorescence microscope (Leica DM 2500M). By switching between brightfield and fluorescence mode, the imaging was done immediately after microrheological measurements were recorded.


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Measurements were taken from 2.5 mg/mL to 300 mg/mL KITO1. This range of polyelectrolyte concentration was chosen because it spans the solubility of KITO1 in sodium acetate buffer of pH 5, which is the highest possible pH that allows for solubility of CPR27. Further, since large quantities of CPR27 are difficult to synthesize, initially two orders of magnitude lower constant concentration of CPR27 (3 mg/mL) was used to study KITO1/CPR27 interactions over a range of KITO1 concentrations. These concentrations were not affected significantly by the uptake of solvent into the PDMS as no nanorods were located outside of the well when observed under the microscope. Brightfield and Fluorescence Microscopy Brightfield and fluorescence microscopy were used to monitor the KITO1 and KITO3 solutions directly after rheological measurements in order to observe changes in microstructure and correlate them to changes in solution viscosity. An upright microscope (Leica DM 2500M) with an extra working distance lens was used at 40x magnification. An 89 North PhotoFluor II 200 W Metal Halide lamp with an excitation wavelength of 500 nm was used as the fluorescent light source and 358-461 nm filters were used to capture the emission. Images were taken with an Andor Luca CCD camera. FTIR To monitor the potential structural changes caused by chitosan and protein complexation, a realtime in situ monitoring of different concentrations of solution were made using an infrared spectrometer (Spectrum One, Fourier transform infrared spectrophotometer, Perkin-Elmer, Waltham, MA, USA) at a resolution of 4 cm-1. A fixed volume of 30 μL of chitosan, CPR27, and chitosan/CPR27 samples were put on an attenuated total reflectance (ATR) crystal (Perkin-


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Elmer, Waltham, MA, WSA). The ATR crystal was zinc selenide (ZnSe) with a transmission range between 650 and 4000 cm-1. Real-time IR spectra were continuously recorded for 64s. Three separate replications for each chitosan and CPR27 solution were obtained. Flow Microscopy Studies done on coacervates involving proteins containing an RR motif showed spherical droplets in solution.38 Therefore, to quantify the amount of coacervate particles formed and their relative concentration in solution, microflow imaging (MFI) was performed with a FlowCam VS Series (Fluid Imaging Technologies, Scarborough, ME) benchtop flow microscope. One set of experiments were performed at concentrations of 3, 30, 50, 100, and 150 mg/mL KITO1 with a constant 3 mg/mL of CPR27. The next set of experiments were performed at concentrations of 0.1, 1, 3, 5, and 10 mg/mL CPR27 with a constant 150 mg/mL of KITO1. These KITO1 concentrations were used due to high opacity where individual particles could not be distinguished. Control measurements were obtained by using samples of 150, 30, and 3 mg/mL KITO1 alone as well as 10, 1, and 0.1 mg/mL CPR27 alone. Each concentration was measured three times (n = 3). The prepared samples were passed through a 100 μm x 2 mm flow cell and observed with a 10x magnification lens. The software used to process images was capable of counting up to one million particles and sample volumes needed to reach this cap varied. Therefore results are presented as the average number of particles per frame. Samples were processed at a flow rate of 0.1 mL/min and a capture speed of 21 frames/s. The instrument was focused using NIST 10 μm Duke Standards (Thermo Fisher Scientific, Waltham, MA) prior to measurement. Particles were analyzed using the “Analyze Particles” function in ImageJ software. Initial measurements of KITO1-only solutions showed that undissolved chitosan microparticles and noise due to solution opacity were present. Therefore, the ImageJ function


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was set to locate particles that were greater than about 5 μm in diameter to filter out these particles. Statistics Data obtained from the microrheology experiments were analyzed using Origin Lab statistical software. Nanorod rotations were fit to a preset exponential decay function found in the software. Statistical analysis was done using a student’s t-test on the solution viscosities calculated from the turning of the nanorods with n >= 4 and α = 0.05. Results and Discussion Microrheology of KITO1 and CPR27 As a first step in this process, viscosity measurements were taken using KITO1 solutions over a range of concentrations from 2.5 to 300 mg/mL KITO1 (see supporting information). The results demonstrate that the viscosity of KITO1 increases with increasing polymer concentration, with almost complete agreement in the relative viscosity values obtained using the bulk rheometer and the microrheology technique. More importantly, the data shows two different regions where relative viscosity data are related to KITO1 concentration by simple power laws with differing exponents. This power-law dependence on polymer solution viscosity is in fact not surprising, as it is typically expected that the viscosity of a polyelectrolyte in low salt solutions should have a power law relationship with concentration with an exponent that is equal to 0.5 for semi-dilute un-entangled regions and 1.5 for semi-dilute entangled regions.51 These two regions, corresponding to a high or low polyelectrolyte concentration region, intersect at what is known as the critical entanglement concentration or overlap concentration. At low polyelectrolyte


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concentrations, the viscosity of the solutions has a lower dependence on concentration since the polymer chains interact minimally with each other. As the polymer concentration increases, this dependence remains the same until the overlap concentration is reached. At the overlap concentration, there are enough polymer chains in solution such that they overlap and constrain each other’s motion. As a result, the viscosity of the solution sharply increases with increasing

y = 8.92*10-6x1.54

y = 1.65*10-3x0.267 y = 1.07*10-5x1.30 y = 6.79*10-4x0.463

Figure 3: Microrheology data of KITO1 chitosan and KITO1 with CPR27. The data are divided into two regions that are defined by scaling laws set forth by Dobrynin et al where data follow a power law with an exponent of ~0.5 at low KITO1 concentrations (semi-dilute) and ~1.5 at high KITO1 concentrations (entangled). In the entangled region a large drop in viscosity upon addition of CPR27 is observed indicating interactions between KITO1 and CPR27. CPR27 concentration was constant at 3 mg/mL. Data were obtained at 23 oC.

concentration. Figure 3 shows that the power law exponents obtained using the microrheology system were 0.267 ± 0.076 and 1.54 ± 0.11 for low and high KITO concentrations, respectively, and an overlap concentration of 57.9 mg/mL was observed. In addition, the shear stress placed


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Figure 4: Viscosity dependence on CPR27 concentration. The red line marks the viscosity of 150 mg/mL of KITO1 without any protein. At lower protein concentrations no significant drop in viscosity is observed. As protein concentration increases reduction in the solution viscosity becomes more pronounced and significant.

on the sample in microrheology experiments was calculated to be 50-100 Pa. This corresponds to stresses that show little or no shear thinning in chitosan solutions on the bulk rheometer. Thus, in addition to the actual relative viscosity measurements, power law exponents in the semientangled regions and overlap concentrations show that the viscosity of KITO1 obtained on the microrheology system agree well with scaling theory for polyelectrolytes. Next, we measured changes to the viscosity of KITO1 as a result of interactions with CPR27 across the same KITO1 concentration range. Our results show that addition of CPR27 to different KITO1 concentrations led to a lowering of the viscosities of KITO1 solutions (Figure 3), especially at higher polyelectrolyte solution concentrations, in the entangled region. However, the dependence of KITO1 solution viscosity on solution concentrations, in the presence of CPR27, continued to demonstrate similar power law behavior as the KITO1


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solutions alone. The power law behavior observed in these results is the result of a polyelectrolyte’s ability to form entanglements with neighboring chains.51 A polymer’s ability to form entanglements is affected by its conformation in solution which is highly dependent on the concentration of polymer and salt. The fact that the results show KITO1 has similar power law behavior in the presence of CPR27 indicates that the presence of the protein molecules does not greatly influence KITO1’s conformation and ability to undergo entanglement. Furthermore, the addition of CPR27 to KITO1 solutions increases the critical entanglement concentration for KITO1. This suggests that as KITO1 aggregates with CPR27 it crashes out of solution, lowering the effective concentration of KITO1 in solution. Therefore, a higher polyelectrolyte concentration is needed to reach the critical entanglement level. The power law exponents for KITO1 with CPR27 data were 0.463 ± 0.028 for the semi-dilute unentangled region and 1.30 ± 0.06 for the semi-dilute entangled region. These changes in solution viscosity upon addition of CPR27 suggest interactions between the KITO1 and the CPR27. The observation that the reduction in viscosity occurs upon the addition of CPR27 to the system demonstrates that the protein perturbs the solution microstructure. It is still unclear how CPR27 accomplishes this. It may disrupts entanglements in the chitosan network or act as a site to which chitosan can bind. To investigate this problem, a similar set of experiments were performed at a constant KITO1 concentration with varying CPR27 concentrations. By varying the CPR27 concentration these experiments shed light as to the role CPR27 plays in the interaction with chitosan. Initially these studies were done with the KITO1 concentration held constant at 300 mg/mL, the concentration shown to have the largest drop in viscosity. The CPR27 concentration was varied in a range between 0.1 and 10 mg/mL (see supporting information). Unfortunately, the imaging technique used in this study (MFI) was unable to fully analyze solutions with 300


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mg/mL KITO1 due to their partial transparency. At this chitosan concentration the MFI analysis done in ImageJ was unable to properly threshold the resulting images to distinguish particles. To overcome this limitation tests were also done using 150 mg/mL KITO1 so that rheological results could be compared with MFI results while also still yielding similar viscosity changes observed in previous experiments. Figure 4 shows the viscosity plotted against CPR27 concentration at a constant 150 mg/mL KITO1 concentration. At low protein concentrations there was no significant change in the solution viscosity when compared with the samples containing only KITO1 (marked as the red line in Figure 4). As CPR27 concentrations increase the reduction in solution viscosity becomes more pronounced and significant with the highest protein concentrations yielding the largest drop in viscosity. There appears to be a critical protein concentration above which the complexation between chitosan and CPR27 is more dominant. These results suggest that CPR27 initiates some form of self-assembly in polyelectrolyte solutions of chitosan, but the nature of these interactions was unclear. If the interactions are simple and the Kd can be estimated as the midpoint in the viscosity change, values in the range of 300-400 μm can be estimated. Therefore, the reason for observed reduction in viscosity of these polyelectrolyte solutions was further explored and complemented using techniques focused on understanding microstructure changes in the KITO1 solutions. Microscopy of KITO1 and CPR27


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Our combined microrheology and microscopy technique allowed us to simultaneously detect changes in the solution microstructure. Figure 5 presents brightfield microscopy images of chitosan solutions in the absence (Figure 5A) and presence of CPR27 (3 mg/ml). Two important details are noted from these series of images: (i) A comparison of Figure 5A and 5B (250 mg/ml chitosan, with and without 3 mg/ml CPR27) shows that the introduction of protein induces the formation of clear aggregates (fluidic droplets) in an otherwise optically partially opaque solution and (ii) These same insoluble aggregates were not present upon the addition of CP30 to KITO1 solutions (Figure 5C). A

B

250 mg/mL KITO1

C

250 mg/mL KITO1 + 3 mg/mL CPR27

250 mg/mL KITO1 + 3 mg/mL CP30

Figure 5: Brightfield microscopy images of solutions of KITO1 and CPR27. KITO1 solutions show several impurities in the solution but no spherical particles. Micron-sized particles are observed upon addition of CPR27 and some examples are marked with red circles. The density of these particle is dependent on chitosan concentration and appear to be directly proportional with each other. No spherical particles were observed in solutions of KITO1 and CP30. Scale bar = 20 μm

To clearly characterize the dependence of aggregate formation and aggregate morphology on chitosan and protein concentrations we analyzed images produced by a series of Microflow Imaging (MFI) experiments. Preliminary experiments using KITO1-only solutions showed the presence of small undissolved chitosan particles. It was observed upon analysis of the resulting images that 95% of such KITO1 particles were smaller than 8 μm in diameter. This diameter was used as a cutoff when searching for particles in KITO1 + CPR27 solutions. The extent of aggregate formation was strongly correlated with both KITO1 and CPR27 concentrations (Figure 6). More aggregate particles appeared at higher KITO1 concentrations. This change in the


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number of aggregates was related to viscosity changes observed using microrheology. The mixtures with varying amounts of CPR27 show a similar, though markedly smaller, concentration dependence, with the exception of 10 mg/mL CPR27. At this concentration a significant spike in the number of particles in solution was observed. This is in agreement with previous microrheology data (Figure 4) showing the presence of a critical CPR27 concentration, about 3 to 5 mg/mL, above which viscosity changes occurred. Here the critical CPR27 concentration appears to be between 5 and 10 mg/mL suggesting that the critical concentration is near 5 mg/mL CPR27. Below this critical concentration the density of particles is much more dependent on chitosan than protein concentration. It is also important to note that even the lowest of protein concentrations yields higher particle counts than solutions with no protein, suggesting


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A)

3 mg/mL CPR27

B)

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150 mg/mL KITO1

C)

Figure 6: Results of aggregate counts and morphologies from MFI experiments. A) Number of particles for solutions with varying KITO1 concentrations. B) Number of particles for solutions with varying CPR27 concentrations. C) Average particle diameters for all concentrations tested.

that even small amounts of CPR7 cause some coacervate formation. Despite changes in both KITO1 and CPR27 concentration the average particle diameter remains relatively unchanged (Figure 6C) suggesting a stable (perhaps equilibrium) concentrate form. Nearly all the particles observed in these experiments were spherical except for larger protein aggregates that had a


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more extended conformation. The average diameter of the particles (9.58 ± 0.35 μm) was nearly the same at every concentration (Figure 6C). Therefore, we hypothesize that CPR27 serves as a nucleation site where chitosan can begin to aggregate, but the relative ratios of chitosan and protein is highly important in controlling the number of coacervate particles. The brightfield microscopy and MFI images showing the formation of insoluble aggregates reveal that the solution is similar to a colloidal suspension. Therefore, one would expect an increase in solution viscosity, particularly with an increase in the volume fraction of the aggregates. However, as seen in Figure 3, a decrease in viscosity was observed in the entangled region. This is surprising since the concentrations that yield the highest number of aggregates are also in the entangled region. We hypothesize that CPR27 induces complex coacervation in chitosan solutions, resulting in micron sized fluidic droplets at higher concentrations of chitosan. This is consistent with microrheology results, since the resulting aggregation lowers the effective chitosan concentration in solution, producing a decrease in viscosity. Further dynamic rheological testing of protein/chitosan solutions supports the idea that CPR27 induces complex coacervation. Dynamic results with KITO1 and CPR27 mixtures show that the storage modulus is much higher than that of KITO1-only solutions indicating an elastic component present in the protein-chitosan mixtures and absent in chitosan samples. Such elasticity is similar to that observed in coacervates formed from proteins in squid beak.38 This elastic component was also observed in microrheology image data where nanorods that were present amid dense packs of the observed fluidic droplets were unable to complete the 90 degree turn. It should be noted that only nanorods that were not in the immediate vicinity of these particles and therefore not interacting significantly with the particles themselves were used for microrheology analysis, to ensure that


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the changes in solution viscosity were due solely to molecular and structural changes in the solution as a whole, rather than changes in the solutions microenvironment. Microrheology and Microscopy of KITO3 and CPR27 While these observations clearly suggest that CPR27 initiates formation of complexes in KITO1 solutions, we also used a fluorescently labeled chitosan solution (KITO3, a FITC-labelled variant of chitosan), to further explore CPR27-chitosan interactions and the nature of the coacervates. Addition of CPR27 to this fluorescently tagged polyelectrolyte solution resulted in a drop in viscosity, similar to that observed for KITO1 solutions. Additionally, as seen with KITO1, KITO3 data were defined by power laws with exponents of 0.295 ± 0.102 and 2.42 ± 0.273 for the semi-dilute unentangled and the semi-dilute entangled regions, respectively, and an overlap concentration of 88.3 mg/mL. While these results show that the power law describing solution viscosity differs between KITO1 and KITO3, possibly due to the presence of the fluorescent label. Upon addition of CPR27 the power law exponents were shifted to 0.157 ± 0.0201 and 1.37 ± 0.0490 for the semi-dilute unentangled and semi-dilute entangled region, respectively, with an overlap concentration of 62.7 mg/mL. Interestingly, this power law exponent is comparable to that induced by CPR27 in KITO1 solutions, suggesting that the interactions between the different chitosan molecules (tagged vs. untagged) and CPR27 are similar. The drop in viscosity of the KITO3 solution is accompanied by formation of brightly fluorescent aggregates, (as shown in


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Figure 8 the particles are outlined with red circles), corresponding to aggregates of KITO3, providing further evidence that CPR27 induces aggregation of chitosan molecules.

-7 2.42

y = 1.01*10 x

-5 1.37

y = 1.36*10 x

y = 0.00204x

0.157

y = 0.0014x

0.295

Figure 7: Viscosity data for fluorescently tagged KITO3 and CPR27 solutions. Data follow similar power laws to those of KITO1. A higher overlap concentration may be due to large FITC groups being added to the chitosan. A significant drop in viscosity is observed again at high KITO3 concentrations indicating there is an interaction between KITO3 and CPR27. CPR27 concentration was held constant at 3 mg/mL. Data were obtained at 23 oC.

Figure 8: Fluorescence microscopic images of KITO3, CPR27, and CP30 solutions. Upon addition of CPR27 fluorescent aggregates, outlined with red circles, were observed indicating interaction of chitosan with CPR27. No aggregates were observed upon addition of CP30, a non-RR cuticular protein, suggesting that the RR motif participates in chitosan binding. Scale bar = 20 μm


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Coacervate formation unique to the RR motif Formation of chitosan nanoparticles and soluble and insoluble chitosan aggregates have previously been reported in several different studies.52,53 Often, the formation of these aggregates have been attributed to electrostatic interactions between the cationic chitosan and anionic colloidal particles or molecules resulting in the formation of intermolecular hydrated complexes that can ripen to form chitosan coacervates in solution. For example, a study on the coacervation of chitosan and gelatin showed that the complexation between the polysaccharide and protein resulted in phase-separated, heterogeneous solutions with polymer-rich and polymer-poor regions.52 At higher pH values, insoluble complexes began to form, which corresponded to a reduced viscosity52, similar to the results obtained in this study with chitosan and CPR27. Coacervates commonly form visible phase separations in solutions; however, chitosan and protein solutions are partially opaque and yellow in color such that visible phase separations were difficult to determine. To test whether the interactions between CPR27 and chitosan presented here are not simple electrostatic interactions, but can be attributed to specific RR-binding interactions, we compared the change in bulk viscosity of 300 mg/ml KITO3 solution due to the addition of another abundant cuticle protein, CP30, which does not contain the RR motif. CP30 consists of an interesting and unusual repeat structure of positively and negatively charged sequences. Therefore, if coacervation with chitosan is primarily the results of electrostatic interactions, which is true for many coacervate systems ,38,40,42 then CP30 should go through a similar selfassembly process, producing a detectable drop in viscosity and the presence of fluorescent particles similar to those found in Figure 8.The results, in contrast to CPR27, addition of CP30 showed no significant change in the viscosity of the chitosan solution (Figure 9). This lack of


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change in viscosity with the addition of CP30 was also accompanied by a lack of aggregate formation in KITO3 solutions, as evidenced by our fluorescence microscopy studies (supplementary information). CP30 contains the necessary charged residues to form electrostatic interactions with chitosan but lacks an RR motif, which suggests that the RR motif plays a role in the interaction of CPR27 with chitosan. 120

* 100

Relative Viscosity

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80

60

40

20

0 KITO3

KITO3 + CPR27

KITO3

KITO3 + CP30

Figure 9: Viscosity data of chitosan and chitosan/protein solutions. Solutions in all cases consisted of 300 mg/mL chitosan and 3mg/mL of the respective protein. Significant losses in solution viscosity were seen for KITO3/CPR27 mixtures, indicating complexation between the polysaccharide and the protein. The same losses were observed in KITO1/CPR27 mixtures. CP30, a protein without an RR motif, was used as a control. Solutions containing CP30 did not have a significant drop in viscosity. * denotes p

Self-assembled coacervates of chitosan and an insect cuticle protein

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