Synthesis and Advanced Characterization of Polymer–Protein Core–Shell Nanoparticles

: Enzyme immobilization techniques are widely researched due to their wide range of applications. Polymer–protein core–shell nanoparticles (CSNPs) have emerged as a promising technique for enzyme/protein immobilization via a self-assembly process. Based on the desired application, different sizes and distribution of the polymer–protein CSNPs may be required. This work systematically studies the assembly process of poly(4-vinyl pyridine) and bovine serum albumin CSNPs. Average particle size was controlled by varying the concentrations of each reagent. Particle size and size distributions were monitored by dynamic light scattering, ultra-small-angle X-ray scattering, small-angle X-ray scattering and transmission electron microscopy. Results showed a wide range of CSNPs could be assembled ranging from an average radius as small as 52.3 nm, to particles above 1 µ m by adjusting reagent concentrations. In situ X-ray scattering techniques monitored particle assembly as a function of time showing the initial particle growth followed by a decrease in particle size as they reach equilibrium. The results outline a general strategy that can be applied to other CSNP systems to better control particle size and distribution for various applications.


Introduction
The self-assembly of functional bio-active materials is a promising field of research which has applications related multiple scientific disciplines including chemistry, medicine, bioengineering, and materials science. Self-assembled materials arise from multiple noncovalent intermolecular interactions. The often dynamic nature of these materials allow for the design of materials with self-healing, cytocompatibility, and biodegradable properties. Recent work has described the preparation of polymer-protein core-shell nanoparticles (CNSPs) [1][2][3][4][5]. These materials are prepared via a stepwise self-assembly process resulting a polymer core covered by a protein "corona" shell. The assembly process occurs via a mechanism similar to Pickering emulsions, in which proteins displace water at the interface of the polymer to reduce the interfacial energy [6][7][8][9]. The biomolecules on the surface of the polymer core are stabilized via hydrogen bonding interactions. This weak bonding interaction results in the formation of the protein corona which yields no loss in protein functionality [1,[10][11][12][13]]. This entropically driven, self-assembly process also allows for the size of the particles to be controlled. Synthesis can also be completed with a wide variety of biomolecules which open the door for applications related to general enzyme stabilization, drug delivery, imaging, and catalysis. Although much research has been done related to the composition and applications of the polymer protein CSNPs, there has not been a thorough study on the particle size tunability and assembly process.
Dynamic light scattering (DLS) has shown to be a powerful technique for determining the size and distribution of colloidal systems [14][15][16][17][18]. DLS is based on the Brownian motion of dispersed particles and measures fluctuations in the intensity of scattered light caused by constructive and destructive interference [19]. DLS instruments determine particle size and distribution based on a few factors including sample temperature, viscosity, and refractive index [20]. Accurate size determinations also require samples to be a clear, infinitely dilute solution, per the Stokes-Einstein equation. Overly concentrated solutions lead to multiple scattering and particle-particle interactions which produce an artificially lower particle size measurement [21]. DLS has shown to be an extremely useful technique for characterizing colloidal systems but suffers from inherent limitations. The synthesized polymer-protein CSNPs in this work were well characterized via DLS, but the technique lacks the ability to study particle formation during the assembly process as it requires a high-concentration solution.
X-ray scattering techniques such as small-angle (SAXS) and ultra-small-angle X-ray scattering (USAXS) offer distinct advantages over traditional light scattering techniques. SAXS is a technique which measures scattered X-rays as a result of variations in electron density within a sample [22,23]. The 2D detector measures scattered X-rays at low angles. The 2D detector image is integrated to yield I(Q) vs. Q, where Q = (4π/λ)(sin θ) for scattering angle 2θ (λ = wavelength of the X-ray). SAXS data can provide structural information related to size, shape, surface structure, distribution, and relative distance to other objects for features from 1 nm to up to 100 nm [22][23][24][25][26][27]. In contrast to DLS, X-raybased techniques such as SAXS do not have limitations based on the concentration, color, fluorescence, or temperature of the sample. USAXS extends the 100 nm size limitation of SAXS by utilizing Bonse-Hart double crystal to collect discernible data at even lower scattering angles, allowing measurements of features up to 1 µm in size [28,29].
This work utilizes advanced characterization techniques such as SAXS and USAXS to investigate the assembly process of polymer-protein CSNPs. Poly(4-vinylpyridine) (P4VP) was used for the polymer while bovine serum albumin (BSA) served as the protein shell. DLS and transmission electron microscopy (TEM) were used to study the effect reagent concentration has on particle size including demonstrating the upper and lower limits for which assembly occurs. Particle size and distribution play an especially important role related to specific applications. For example, for biomedical and drug delivery applications the accepted particle size range is ≤100 nm [30,31]. While for other processes, such as use as an industrial catalyst, larger particle sizes may be more desirable for easier separation from the product stream.

BSA-P4VP Core-Shell Nanoparticle Synthesis
All CSNP unless noted otherwise were prepared in a 3.7 mL glass vial by adding 0.12 mL of P4VP in MeOH to 0.5 mL of BSA in an aqueous buffer solution. A series of identical reactions were completed with varied reagent concentrations. Based on the described solution volumes, reactions involving reagents of equal concentrations results in a polymer-to-protein mass ratio of 0.24. Previous work has discussed the ability to control the size of polymer-protein CSNPs by altering the polymer solution volume and, therefore, the polymer-protein mass ratio [1,2]. To further investigate the limits of the polymerprotein self-assembly method, BSA and P4VP solutions were prepared at concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 10.0, and 15.0 mg/mL. Reactions were run for all possible reagent concentration combinations. Each reaction underwent the evaporation purification process which involves letting the solution stir in open air at 25 • C for 48 h. This allows the methanol from the P4VP solution to evaporate, resulting in the CSNPs in the aqueous buffer solution. For the other purification method, dialysis, the solution is transferred to a dialysis tube after the initial thirty-minute reaction time. The dialysis tube is then suspended in a 1.0 L solution of the HEPES/NaCl buffer solution while stirring. The 1.0 L solution is replaced with a fresh solution after 2, 4, 8, and 12 h. The key difference with the dialysis method is that the porous dialysis tubing allows excess, unreacted protein to be removed from the reaction solution.

Particle Size Characterization of CSNPs
The particle size and distribution of the P4VP-BSA CSNPs was investigated with DLS and TEM. DLS measurements were collected on each of CSNP reactions. Figure 1a shows an overview of the size distribution based on the BSA and P4VP concentrations. The plot shows that the smallest particles, with a radius of 52.3 nm, are obtained when the protein concentration is the highest (15 mg/mL) and the P4VP concentration is the lowest (0.5 mg/mL). The average particle size increases as the P4VP concentration increases and vice versa decreases as the BSA concentration is lowered. This is consistent with previous work which demonstrates that particle size can be controlled as the polymer-protein mass ratio is adjusted [1,12,32]. A table containing the average particle radii can be seen in Table  S1. For reactions with the standard 0.24 polymer-to-protein mass ratio, the data shows that particle size decreases as the concentration of both reagents increases. For simplification purposes, reactions will be described using a P X B Y format, where "P" represents P4VP, "B" represents BSA, and "X" and "Y" signify their respective concentrations (mg/mL). When comparing the particle size as the concentration of each reagent is adjusted, it becomes evident the amount of protein available in the solution has a much larger effect on particle size than the polymer. For example, for reactions P 0.5 B 15 and P 15 B 15 , CSNPs are formed with an average radius of 52.3 and 117.5 nm, respectively. The increase from 0.5 to 15 mg/mL P4VP results in a 224.7% increase in particle size. When comparing to reactions P 0.5 B 1 and P 15 B 1 , the average CSNP radius increases from 309.6 to 4107.1 nm, a 1326.6% increase. This increase even more pronounced for reactions of P >2 B 0.5 where the resulting particle formed reached the upper limit of detection for the DLS instrument and could not be accurately measured. Figure 1b shows the DLS lognormal size distributions for three different CSNP reactions. For the three reactions plotted, P 0.5 B 2 , P 2 B 2 , and P 15 B 2 , an obvious increase in particle distribution is seen as the P4VP concentration is increased. For all BSA-P4VP reactions, an increase in average particle size is also associated with the increase in particle distribution. These results show how the average CSNP size and distribution can be controlled by varying reagent concentration and easily monitored via DLS.

Particle Size Characterization of CSNPs
The particle size and distribution of the P4VP-BSA CSNPs was investigated with DLS and TEM. DLS measurements were collected on each of CSNP reactions. Figure 1a shows an overview of the size distribution based on the BSA and P4VP concentrations. The plot shows that the smallest particles, with a radius of 52.3 nm, are obtained when the protein concentration is the highest (15 mg/mL) and the P4VP concentration is the lowest (0.5 mg/mL). The average particle size increases as the P4VP concentration increases and vice versa decreases as the BSA concentration is lowered. This is consistent with previous work which demonstrates that particle size can be controlled as the polymer-protein mass ratio is adjusted [1,12,32]. A table containing the average particle radii can be seen in Table S1. For reactions with the standard 0.24 polymer-to-protein mass ratio, the data shows that particle size decreases as the concentration of both reagents increases. For simplification purposes, reactions will be described using a PXBY format, where "P" represents P4VP, "B" represents BSA, and "X" and "Y" signify their respective concentrations (mg/mL). When comparing the particle size as the concentration of each reagent is adjusted, it becomes evident the amount of protein available in the solution has a much larger effect on particle size than the polymer. For example, for reactions P0.5B15 and P15B15, CSNPs are formed with an average radius of 52.3 and 117.5 nm, respectively. The increase from 0.5 to 15 mg/mL P4VP results in a 224.7% increase in particle size. When comparing to reactions P0.5B1 and P15B1, the average CSNP radius increases from 309.6 to 4107.1 nm, a 1326.6% increase. This increase even more pronounced for reactions of P>2B0.5 where the resulting particle formed reached the upper limit of detection for the DLS instrument and could not be accurately measured. Figure 1b shows the DLS lognormal size distributions for three different CSNP reactions. For the three reactions plotted, P0.5B2, P2B2, and P15B2, an obvious increase in particle distribution is seen as the P4VP concentration is increased. For all BSA-P4VP reactions, an increase in average particle size is also associated with the increase in particle distribution. These results show how the average CSNP size and distribution can be controlled by varying reagent concentration and easily monitored via DLS.

In-Situ CSNP Characterization via USAXS
In situ USAXS and SAXS were used to investigate the polymer-protein CSNP assembly process. A reaction vial was setup on the synchrotron X-ray beamline with a 12 V peristaltic pump providing constant flow through a complete circuit which moved the solution to the X-ray path and back to the reaction vial. An illustration of the flow cell and beamline setup can be seen in Figure 2a. The detecting components for both SAXS and USAXS experiments are mounted on high precision, micro-stepping motors which allows

In-Situ CSNP Characterization via USAXS
In situ USAXS and SAXS were used to investigate the polymer-protein CSNP assembly process. A reaction vial was setup on the synchrotron X-ray beamline with a 12 V peristaltic pump providing constant flow through a complete circuit which moved the solution to the X-ray path and back to the reaction vial. An illustration of the flow cell and beamline setup can be seen in Figure 2a. The detecting components for both SAXS and USAXS experiments are mounted on high precision, micro-stepping motors which allows them to be quickly moved in and out of the beam path without needing recalibration. This allows for sequential SAXS and USAXS scans with downtime on the order of seconds. For each in situ USAXS/SAXS experiment, reagents were combined in an open-air beaker following procedures described in the experimental section. Initially, reactions of P 2 B 2 were measured but the relatively low concentration lacked scattering intensity and was unable to yield an acceptable signal to noise ratio. After incrementally increasing reagent concentrations, a P 10 B 10 reaction yielded acceptable scattering intensity, as seen in the combined USAXS and SAXS scattering profiles seen in Figure 2b. The CSNP reaction was measured for 180 min, after which no structural changes could be observed. The USAXS/SAXS data was combined, processed, and fit using the Nika and Irena packages as part of the Igor Pro software [33,34]. Each scan shows to distinct features at different q ranges. The low q range (0.0003-0.002) feature is generated by the CSNPs while the high q (0.02-0.4) feature is characteristic of free, unbonded BSA in the solution [35,36]. Each of these scattering features were fit using lognormal size distribution models. Figure 2c shows a plot of the lognormal size distribution of the CSNPs as a function of time. The first scan, collected one minute after the reaction, shows particles with a lognormal mean radius of 236.1 nm. After twenty minutes the CSNPs begin to stabilize and are no longer increasing in size. Over the next 180 min the particles slowly start to reduce their average size as distribution stays the same. After 200 min, the particles stabilized resulting in a lognormal mean radius of 176.7 nm. The data shows that the bulk of CSNP assembly occurs within the first twenty minutes, however the slow reduction in particle size after the initial formation suggests that particle stabilization continues to occur for up to five hours. The average radius calculated from the USAXS data is consistent with DLS measurements, which returned an average radius of 182.2 nm for these particles. them to be quickly moved in and out of the beam path without needing recalibration. This allows for sequential SAXS and USAXS scans with downtime on the order of seconds. For each in situ USAXS/SAXS experiment, reagents were combined in an open-air beaker following procedures described in the experimental section. Initially, reactions of P2B2 were measured but the relatively low concentration lacked scattering intensity and was unable to yield an acceptable signal to noise ratio. After incrementally increasing reagent concentrations, a P10B10 reaction yielded acceptable scattering intensity, as seen in the combined USAXS and SAXS scattering profiles seen in Figure 2b. The CSNP reaction was measured for 180 min, after which no structural changes could be observed. The USAXS/SAXS data was combined, processed, and fit using the Nika and Irena packages as part of the Igor Pro software [33,34]. Each scan shows to distinct features at different q ranges. The low q range (0.0003-0.002) feature is generated by the CSNPs while the high q (0.02-0.4) feature is characteristic of free, unbonded BSA in the solution [35,36]. Each of these scattering features were fit using lognormal size distribution models. Figure 2c shows a plot of the lognormal size distribution of the CSNPs as a function of time. The first scan, collected one minute after the reaction, shows particles with a lognormal mean radius of 236.1 nm. After twenty minutes the CSNPs begin to stabilize and are no longer increasing in size. Over the next 180 min the particles slowly start to reduce their average size as distribution stays the same. After 200 min, the particles stabilized resulting in a lognormal mean radius of 176.7 nm. The data shows that the bulk of CSNP assembly occurs within the first twenty minutes, however the slow reduction in particle size after the initial formation suggests that particle stabilization continues to occur for up to five hours. The average radius calculated from the USAXS data is consistent with DLS measurements, which returned an average radius of 182.2 nm for these particles.

Transmission Electron Microscopy (TEM) Characterization
DLS and USAXS provided valuable information related to particle size and distribution of the CSNP systems. To further characterize the CSNPs and analyze particle features such as shape and morphology, TEM images were obtained. Figure 3 shows TEM images from a series of P4VP-BSA reactions. The particles seen in the figure show a gradual increase in diameter as the polymer-protein mass ratio is increased. The TEM measured particle size radius in Figure 3a,c-f are 168, 200, 300, 361, and 440 nm, respectively. The high-magnification images demonstrate the uniform spherical shape and smooth surface. It is worth noting that as particle size increases towards the upper limit, there is no identifiable degeneration of the spherical shape or surface smoothness. Figure 3b also shows a low magnification image of a CSNP reaction. Based on particle size distributions determined via DLS and USAXS data fitting, the range of particle sizes seen in Figure 3b are well within the expected range. The results seen in the TEM images provide useful insight that cannot be obtained by scattering techniques such as DLS and USAXS. These results also help confirm the particle sizes evaluated by these techniques.

Transmission Electron Microscopy (TEM) Characterization
DLS and USAXS provided valuable information related to particle size and distribution of the CSNP systems. To further characterize the CSNPs and analyze particle features such as shape and morphology, TEM images were obtained. Figure 3 shows TEM images from a series of P4VP-BSA reactions. The particles seen in the figure show a gradual increase in diameter as the polymer-protein mass ratio is increased. The TEM measured particle size radius in Figure 3a,c-f are 168, 200, 300, 361, and 440 nm, respectively. The high-magnification images demonstrate the uniform spherical shape and smooth surface. It is worth noting that as particle size increases towards the upper limit, there is no identifiable degeneration of the spherical shape or surface smoothness. Figure 3b also shows a low magnification image of a CSNP reaction. Based on particle size distributions determined via DLS and USAXS data fitting, the range of particle sizes seen in Figure 3b are well within the expected range. The results seen in the TEM images provide useful insight that cannot be obtained by scattering techniques such as DLS and USAXS. These results also help confirm the particle sizes evaluated by these techniques.

BSA-P4VP Core-Shell Nanoparticle Synthesis
For a typical reaction, P4VP (M W 60,000) in MeOH (2.0 mg/mL, 0.12 mL) was added in 40 µL increments to a 3.7 mL glass vial containing 0.5 mL of a BSA solution (2.0 mg/mL) in a 10 mM HEPES, 125 mM NaCl buffer. The solution was constantly stirred during the mixing of reagents. The vial was then sealed for thirty minutes before undergoing either dialysis or evaporation purification methods.

DLS Characterization
DLS measurements were performed on a Nanobrook Omni by Brookhaven Instruments (Holtsville, NY, USA) with an incident laser with a wavelength of 640 nm and a detection angle of 90 • . For a typical P 2 B 2 measurement, the CSNP solution (7.0 µL) was diluted to 2.0 mL with DI H 2 O. The resulting solution was then transferred to a standard plastic cuvette. Each DLS measurement is the average of five consecutive five-minute scans. Sample dilutions were adjusted accordingly as the reagent concentrations were increased or decreased to ensure the dilute limit required for DLS was met.

USAXS/SAXS Characterization
USAXS and SAXS measurements were performed at the 9-ID-C beamline at the Advanced Photon Source at Argonne National Laboratory (Lemont, IL, USA) [37]. The X-ray energy was 24 keV with exposure times of 120 s and 20 s for USAXS and SAXS measurements, respectively. The flow cell in the X-ray path was constantly cycles with the CSNP solution using a 12 V peristaltic pump with a flow rate of 3.0 mL/min. The solution was stirred at 300 RPM using a magnetic stir bar during the entirety of the experiment. Data processing and fitting was done using the Irena [33] software suite by J. Ilavsky and P. R. Jemian (Lemont, IL, USA). The Irena program operates as a module of the Igor Pro software package (Wavemetrics, Portland, OR, USA).

TEM Characterization
FE-TEM was performed using a JEOL JEM2100F microscope operated at 200 kV (JEOL, Peabody, MA, USA). Images were collected in both high and low magnification modes. Samples were prepared on 400 mesh carbon-coated copper grids (Ted Pella INC., Redding, CA, USA). For each sample, a grid was submerged in a 100 µL droplet containing a 50:50 mixture of sample solution and ultra-pure DI H 2 O. Grids were left in the droplet for ten minutes before being transferred to another 100 µL droplet of ultra-pure DI H 2 O to rinse. After ten minutes, the grids were removed and allowed to dry.

Conclusions
In summary, both in situ and ex situ characterization techniques were used to monitor the assembly process of polymer-protein CSNPs. To the best of our knowledge, this work describes the first ever monitoring of polymer-protein CSNP assembly via USAXS. The in situ USAXS/SAXS experimental setup was able to monitor the evolution of CSNP particle size and distribution for the entirety of the assembly process. DLS and TEM were also used to outline a series of reactions which provides the foundation for the synthesis of P4VP-BSA CSNPs ranging from~100 nm to 1.2 µm in diameter. Going forward, the ability to control particle size by altering the polymer-protein mass ratio can be applied to research looking to utilize other polymer/protein combinations. The in situ USAXS experiment also demonstrated the gradual particle size changes as a function of time. In future work, we plan to experiment with stopping the assembly process prior to completion to investigate its effect on particle size and distribution.