Half-Life Modeling of Basic Fibroblast Growth Factor and its Media Concentration from Growth Factor-Eluting Polyelectrolyte Multilayers

: Growth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 days and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH=4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH=5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH=4 and pH=5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH=4 assembly condition had higher cells counts, while the PEM assembled at pH=5 and PEM with no FGF2 increased cell counts and spreading compared to exogenous FGF2 supplementation and the FGF2-free controls. At 4 days, the cell count in the FGF2-eluting PEM assembled at was higher than both the 8 ng/mL exogenous condition and the controls. Cell spreading was highest in the PEM-only control followed by the FGF2-eluting PEM assembled at pH=5, indicating that the PEM and FGF2 may have a synergistic effect on cell attachment and proliferation. These results show that FGF2 introduced from a GF-eluting PEM is capable of moderately increasing the number of viable cells early during culture. It has been established that presentation other GFs from the surface directly can provide more bioavailable GF. This work is an early sign this also However, observed benefits 2 days cell and have 2-day


Introduction
The controlled release of biologically relevant molecules is of great interest in the biomedical field, where exogenous methods of supplementation can be costly and risk contamination of products meant to directly treat patients. 1 Polyelectrolytes have been employed as core components of many controlled-release systems due to their versatility. 2 From the same starting materials, polyelectrolytes can form a number of different structures, including polyelectrolyte multilayer (PEM) coatings, polyelectrolyte complexes (PECs), complex coacervates, or microcapsules. [3][4][5][6][7][8][9][10][11][12] The structure and properties of the resulting material depend on factors including the assembly method, polyelectrolyte concentrations, pH, and temperature. 3,[13][14][15] For this reason, polyelectrolytes have been used for a range of cell scaffolds and surface coatings capable of releasing specific nanoparticles, pharmaceuticals, and proteins. [7][8][9][10][11]16 PEMs specifically have been used extensively to control the release of growth factors (GFs) such as basic fibroblast growth factor (FGF2), bone morphogenetic protein (BMP-2), and transforming growth factor beta 1 (TGF-β1). 3,6,17,18 FGF2 is especially important for the growth of both fibroblasts and mesenchymal stem cells and is linked to improved proliferation and decreased cell death rate, and can also prevent differentiation to undesirable phenotypes. 3,[19][20][21][22][23] PEMs are well-suited to the controlled release of GFs due to their ability to maintain GF bioactivity and release GFs over periods ranging from weeks to months. 3,6,17 Furthermore, when PEMs without GFs are used alone to modify culture surface properties, they can improve cell proliferation compared to common culture substrates and implanted device materials such as tissue culture plastic (TCP) and titanium. 3,6,17 The ability to simultaneously control surface properties and GF release rates, coupled with the ease of processing, make PEMs excellent materials for improving cell outcomes both in vitro and in vivo when paired with GFs.
While numerous publications report that GF-eluting PEMs greatly improve cell outcomes, many do not quantify the actual concentrations of GF released into the system. 6,18,24,25 Methods for measuring GF concentration include enzyme-linked immunosorbent assay (ELISA), fluorescent labeling, and western blot. [3][4][5]17,[26][27][28] However, all of these methods have limitations. Fluorescent labeling requires modification of the starting protein, potentially changing the properties of or damaging the GF of interest. 29 Western blot requires much higher concentrations than the 0.1-100 ng/mL optimal dosage of GFs such as FGF2 and the number of conditions that can be characterized simultaneously is limited by gel and well sizes. 19,[30][31][32] ELISA may detect non-bioactive protein that possesses the necessary binding groups, and may not detect bioactive forms of the protein if they cannot bind with ELISA antibodies. 33 For both methods, these limitations are compounded by the fact that GFs degrade over time under physiological conditions. At a specific time point, the concentration detected will inevitably be lower than the true amount released due to the loss of GF bioactivity. Despite the impact of protein degradation on measurement accuracy, there is almost no research on the half-lives of even the most common growth factors. In the literature, there are references to FGF2 half-life, but there is only one experimental investigation that quantifies the half-life value. Shiba et al. used western blot and estimated a half-life ranging from 4.7 to 13.7 hours depending on the presence of polyphosphate media supplements. 30 Han et al. measured FGF2 degradation in phosphate buffered saline (PBS) using ELISA, but no further analysis of the half-life was performed in their work. 11 Due to the differences in the methodology and concentration readings, generating an accurate model of FGF2 degradation using ELISAderived results necessitates determining its ELISA-detectable half-life.
In this work, we developed a working model of growth factor release from PEMs and subsequent degradation by obtaining an ELISA-detectable half-life of FGF2 and applying it to a new dataset. Half-life values were obtained by measuring concentrations of FGF2 dissolved in both PBS and Dulbecco's modified Eagle medium (DMEM) over a period of 4 days. PEMs consisting of poly-L-histidine (PLH) and poly(methacrylic acid) (PMAA) were prepared at a range of assembly pH values in order to obtain a new data set for GF release. Combining these results provided an ELISA-detectable concentration profile of FGF2 from the GF-eluting PEMs. The ability of the PEMs to improve cell proliferation and attachment was determined using CRL-2352 fibroblasts.

PEM Half Life
The ELISA-detectable half-life for FGF2 was obtained to more accurately predict FGF2 concentrations. We specify "ELISA-detectable" to differentiate the values from other methods that may result in different readings. Figure 1 shows FGF2 concentration curves obtained over 4 days and the same data on a natural logarithmic scale. The initial time point taken 1 hour after plating shows a lower value than the expected starting concentration, likely due to a combination of a small amount of initial adsorption, including during mixing and transfer of aliquots to vials, and losses during freezing. Nonetheless, the results showed a gradual decrease in ELISA-detectable FGF2 concentration over the course of the 4-day study, resulting in an over 10-   This is likely because the folding/unfolding reaction does not reach equilibrium during this time.
The FGF2 used for these experiments was reconstituted from a lyophilized state, diluted, then heated to the experimental temperature, so it seems reasonable that equilibration would not be achieved immediately. The result is a fast degradation rate in the first few hours of the experiment.
To account for the non-equilibrium conditions during initial plating, only data from 8 h to Average half-life values were determined for each condition and are given in Table 1 30 The data in the current work agrees better with the activity loss studied via ELISA presented in Han et al. 11 This difference in reported values is not necessarily due to the differences in western blot and ELISA, rather the reported western blot values are for a 24 hr study, which would result in a lower half-life if the experiment was performed while in a non-equilibrium state.

pH Dependence of PMAA/PLH PEMs
In order to both explore the effects of assembly pH for PEMs on tissue culture plastic, and to obtain a data set for modeling purposes, a release study was performed on PEMs assembled at pH=4, pH=5, pH=6, pH=7, and pH=8. Figure 2 shows the results of this study. Assembling the PEM at pH=4 results in the highest FGF2 concentration after 7 days at 4.71±1.13 ng/mL, which is statistically significantly higher than PEMs assembled at pH=5, pH=7, and pH=8 (p<0.05). FGF2 release from PEMs assembled at pH=5 (2.3±0.44 ng/mL) and above were not statistically significantly different. pH=8, the addition of PMAA results in rapid stripping followed by adsorption resulting in a mass slightly below or equal to the initial value. At pH=7, the QCM-D results show that there is minimal adsorption of the PMAA layer and PEM formation is primarily driven by PLH. At pH=8, the addition of PMAA results in rapid stripping followed by adsorption resulting in a mass slightly below or equal to the initial value. For PMAA/PLH PEMs, it is likely that the range between pH=5 and pH=6 are where the polyelectrolytes have the highest charge density, resulting in a driving force favoring adsorption.
At even the very first layer, we see increased PLH adsorption, which is then balanced by a greater mass of PMAA adsorbed in the second layer. This is repeated throughout the adsorption process, resulting in a doubled mass when comparing the pH=6 PEM to the pH=4 PEM.
Similarly, the collapse of PEM mass in the intermediate pH range occurs as the ionization of both individual polyelectrolytes decreases. 35 For the PMAA/PLH polyelectrolyte, this leads to instability in the PEM resulting in stripping. During PEM assembly, adsorption and stripping directly compete. 36 On an individual layer level, it can be seen that when PMAA is introduced at pH=7 and pH=8, stripping overtakes adsorption, resulting in loss of polyelectrolyte mass before more is adsorbed in that same layer. As PEMs are dynamic, rearrange during formation, and do not typically form discrete layers, loss in mass likely correlates to a simultaneous loss in FGF2.
By comparing the QCM-D results to the FGF2 release curves, some conclusions can be drawn. Thin and unstable PMAA/PLH PEMs release lower amounts of FGF2 due to stripping of portions of the PEM mass during adsorption. This can be seen clearly from the pH=7 and pH=8 conditions. Increasing PEM mass is correlated to lower FGF2 release, but may not be the cause.
A likely explanation is that the PEMs at pH=5 and pH=6 conditions are less able to maintain FGF2 bioactivity compared to the pH=4 condition. Differences in chain arrangement, charge, and moisture uptake at the higher pH values can all result in decreased FGF2 bioactivity.
Comparing the remaining amount of FGF2 adsorbed to the substrate after the release study provides further insight on the differences of the different pH conditions. Remaining FGF2 surface concentrations were not statistically significantly different for the pH=4, pH=5, and pH=6 samples (6.7±2.31 ng/cm 2 , 6.6±2.95 ng/cm 2 , 4.2±0.51 ng/cm 2 respectively). However, the amount of remaining FGF2 for the pH=7 and pH=8 conditions are lower, at 3.1±1.30 ng/cm 2 and 0.6±0.22 ng/cm 2 , respectively. This reinforces the idea that less FGF2 was adsorbed in the pH=7 and pH=8 PEMs, but not at the lower assembly pH values. However, as the amounts of FGF2 released were statistically similar throughout the pH=5 to pH=8 range, it is clear that the release kinetics and arrangement of the polyelectrolyte chains within the PEM differ for each assembly pH condition.
PEM stability is likely the greatest contributing factor to the GF release results. Previous work on titanium-coated substrates showed that mass loss when exposed to PBS was minimized for a PEM formed at pH=4, compared to those formed at pH values closer to that of PBS (pH=7.4). 17 In this system, polyelectrolyte choice plays an important role and the most stable PMAA/PLH PEM forms at pH=4 where the positive charge of the polyamino acid, PLH, is greatest among the tested conditions. This increased stability results is a coating that is most able to maintain and release ELISA-detectable FGF2.  Figure 4a shows this methodology applied to the data obtained at the tested pH values compared to exogenous FGF2 supplementation. Graphs of specific curves to reach the final modeled release can be seen in the Supporting Information. The exogenous supplementation data seen in Figure 4b was obtained by applying Equation 3 by itself.  increase rapidly over the course of the first day, but gradually decline over the next 3 days. Figure   4b shows the FGF2 concentration curve for an exogenous condition where FGF2 is supplemented initially and after 2 days without total media replacement. For the exogenous condition to have the same amount of FGF2 after 4 days as the pH=4 condition, a concentration between 8-16 ng/mL of FGF2 would be required.  PEM-only and uncoated surfaces served as controls.   The results at 4 days do not follow the trends seen at 2 days. At 4 days, all FGF2-positive conditions show statistically significant increases to cell count compared to the PEM-only and uncoated controls. This is reasonable, as proliferation signaling is a primary function of FGF2. 39,40 Furthermore, the FGF2-eluting PEM at pH=4 has a statistically significantly higher cell count than the 8 ng exogenous condition, indicating that the FGF2-eluting PEM may be superior to exogenous delivery at certain concentrations. However, for both GF-eluting PEMs and exogenous FGF2, control of concentration is extremely important.

Concentration Modeling
Interestingly, the order of relative cell sizes differs greatly at 4 days compared to 2 days.  Care must be taken in modifying substrate propertieswhile improved fibroblast spreading is correlated with improved attachment, it may also be indicative of a more myofibroblastic, chondrogenic, or osteoblastic character. 39,41 However, this concern can be addressed by supplementing FGF2 exogenously, or releasing it over time when culturing cells on a PEM. FGF2 is known to allow fibroblasts to more effectively maintain their phenotype. Ideally, the PEM improves attachment by increasing the surface charge, while the FGF2 prevents undesired differentiation and improves proliferation. This could have significant implications for cell expansion in bioreactor systems, as the shear forces risk the causing detachment or undesired differentiation of cells. 28,42 From a proliferation standpoint, the (PMAA/PLH)5 PEM and uncoated polystyrene have statistically similar cell counts, which is consistent with our previous results. 3 However, confocal imaging and staining highlight that the extent of cell spreading is affected by the presence of a PEM. While exogenous addition of FGF2 to media is known to improve fibroblast proliferation, 3,39,40 this work provides a better understanding of the optimal concentration ranges under which FGF2 should be supplemented. Furthermore, FGF2-eluting PEMs can increase fibroblast growth rates, likely due to improvements in early attachment and growth.

Conclusions
In this work, an ELISA-detectable half-life value was obtained for FGF2. This half-life was then used to model actual FGF2 concentrations in cell culture systems. The effects of assembly pH on FGF2 release was studied and serves as a representative system for the developed model.
The highest FGF2 release rate was seen from PEMs assembled at pH=4, while PEMs assembled at pH=5 to pH=8 showed approximately equal FGF2 release rates. Based on QCM-D results, the differences in release profile can be attributed to the ability of the PEM at this pH to better maintain growth factor bioactivity, and not related to PEM mass directly. The model showed that the pH=4 condition could maintain FGF2 concentrations between 2 ng/mL and 6 ng/mL after 8 hours of culture time in a cell culture system with a 1.9 cm 2 surface area and 600 μL of cell culture media.
The PEM assembled at pH=5 could maintain a concentration between 0.5 ng/mL and 2.5 ng/mL.
Fibroblasts were cultured at these same conditions to determine the effects of the FGF2eluting PEMs on cell attachment and proliferation. After 2 days, the FGF2-eluting PEMs had increased cell counts and spreading compared to exogenous FGF2 supplementation and the FGF2free controls. At 4 days, the cell count in the FGF2-eluting PEM assembled at pH=4 was higher than both the 8 ng/mL exogenous condition and the controls. Cell spreading was highest in the PEM-only control followed by the FGF2-eluting PEM assembled at pH=5, indicating that the PEM and FGF2 may have a synergistic effect on cell attachment and proliferation.
These results show that FGF2 introduced from a GF-eluting PEM is capable of moderately increasing the number of viable cells early during culture. It has been established that presentation of other GFs from the surface directly can provide more bioavailable GF. This work is an early sign that this also applies to FGF2 released from a PEM. However, the observed benefits are

Materials
Plasma-treated polystyrene petri dishes were purchased from Corning. Poly

FGF2 Half-Life Determination
FGF2's half-life was determined in both PBS and DMEM to understand its degradation in a simulated cell culture environment. No additives such as fetal bovine serum were included, as they vary in concentration depending on the culture system. Before the study, 5 wells in a 6-well polystyrene cell culture plate were blocked with 2% BSA overnight at 37 °C to limit FGF2 adsorption during the experiment. After blocking, the plates were washed with 37 °C PBS twice after which 4 mL of PBS or DMEM with the appropriate FGF2 concentration were added to the wells. At the appropriate time point, a 100 μL aliquot was taken from each of the wells without replacement and stored frozen until the ELISA study. The first time point was after a 1-hour incubation period at 37 °C to limit the effects of adsorption to the well walls on the results. To account for non-equilibrium conditions, half-life results were obtained by fitting the data from 8 hrs to 96 hrs to a first order rate law.

Substrate Preparation and PEM Coating.
Polyelectrolytes were prepared at a concentration of 1 mg/mL and Tris buffer was prepared at 0.05M. Solution pH values were adjusted using 1M HCl and 1M NaOH. Before coating, the substrates were cut into 1 cm 2 squares and cleaned with deionized water, ethanol, and deionized water again. Following the cleaning, 50 µg/mL FGF2 in pH=7.6 Tris buffer was pipetted onto one side of the substrate and allowed to adsorb for 15 minutes, followed by three washes in deionized water for 1 minute each. Using a dip coater (6 Position Compact SILAR Coating System, MTI), the substrates were immersed into 1 mg/mL PMAA for 15 minutes at the appropriate pH followed by two 90-second wash steps with water at the same pH. Another 15-minute adsorption step was performed for 1 mg/mL PLH, followed by two more wash steps. These two steps were repeated five times to form 5 bilayers, resulting in a FGF2-(PMAA/PLH)5 PEM. The samples were dried using compressed nitrogen gas and stored at 4 °C overnight.

FGF2 Release Study
To determine FGF2 release rates, the coated substrates were immersed in 1 mL of PBS in individual scintillation vials and incubated at 37 °C. At the specified time points, PBS was collected and stored frozen at -30 °C and 1 mL of fresh PBS was added to the vial. After collecting the aliquots at the 7-day time point, substrates were rinsed for 30 seconds in 0.5 mL of 0.1M HCl followed by a second rinse with 0.5 mL of 0.1M NaOH. The HCl and NaOH aliquots were combined, pH adjusted to pH=7.4 and stored at -30 °C. 4 ABTS sandwich ELISA kits (PeproTech) were used to quantify FGF2 release and ELISA was performed in accordance to supplier instructions. Aliquots from the release studies were thawed and returned to room temperature immediately prior to their use. Absorbance readings of the developed ELISA plates were obtained using a Molecular Devices SpectraMax M2 plate reader at a reading wavelength of 405 nm with a reference wavelength of 650 nm.

Quartz Crystal Microbalance with Dissipation Monitoring
QCM-D was used to monitor PEM assembly at different pH values using a Q-Sense E4 (Biolin Scientific) system with polystyrene-coated sensors to mimic the experimental substrate. 1 mg/mL polyelectrolyte solutions were fed at a 50 µL/min flow rate for each layer followed by a 10-minute wash step using water of the appropriate pH. An initial PLH first layer was used in place of FGF2 as both are positively charged below pH=7. Frequency and dissipation were obtained at odd overtones from the 3rd to 13th representing the harmonic resonances of the quartz crystal. The 3rd overtone was chosen to calculate the PEM mass as it best represents the bulk character of the film. 43 The Sauerbrey equation for rigid films was used to determine the mass of the assembled PEM based on the raw frequency data (Equation 1): where m is the calculated adsorbed mass, f is the resonant frequency, n is the overtone number, and C is the sensitivity constant, which is 17.7 ng/(cm 2 Hz) for these 5 MHz QCM-D sensors. Use of the Sauerbrey equation is appropriate if the films are rigid, characterized by a stable ratio of dissipation change to frequency change between overtones and is generally applicable for any PEM system under 40 nm in thickness. 44

Cell Culture
Individual 4-well plates were coated manually under the same assembly conditions as described above. In place of dip coating, the solutions were deposited using a multi-pipettor and were aspirated after the appropriate time interval. 200 µL of 50 µg/mL FGF2 was pipetted to the bottom of the well, followed by 300 µL of DI water, then by 300 µL of polyelectrolyte solution and pH-adjusted wash water for each step, forming the full PEM. GF-eluting PEMs were prepared at pH=4 and pH=5. A PEM-only condition and uncoated surfaces were used as negative controls.
Two conditions with 8 ng and 4 ng of exogenous FGF2 added at t=0 days and t=2 days were used as positive controls. The exogenous conditions had a PEM-only coating to serve as a direct comparison to the FGF2-eluting surfaces. The coated plates were sterilized under UV light for 10 minutes before starting culture.
Fibroblasts were initially expanded in complete growth medium (IMDM containing 10% FBS, 1% L-glutamate, and 1% penicillin streptomycin). Cells at passage 4 were seeded at a density of 5,000 cells/cm 2 (9,500 cells/well) in individual 4-well plates for each condition and cultured in 600 µL of IMDM containing 1% FBS for 2 and 4 days at 37 ºC and 5% CO2. At the specified time points, the 4-well plates were removed from the incubator. The media was aspirated and the cells were washed twice using 1X PBS with Mg 2+ and Ca 2+ . The cells were fixed in 200 µL of 4% paraformaldehyde in PBS for 10 minutes, after which two more rinses in PBS were performed.
The fixed cells were stored at 8 °C until staining.
To permeabilize the cells, 0.1% Triton X-100 in PBS was added to the culture wells for 5 minutes followed by two washes in PBS again. Cells were incubated in a 1 wt.% BSA solution in PBS for 30 minutes to prevent non-specific binding of the fluorescent stains. After blocking, the wash step was repeated. The cell nuclei were stained using 300 µL of Hoechst solution diluted 1:3,000 in PBS for 10 minutes under a foil cover. Phalloidin coupled with Alexa Fluor 488 was diluted to 2.5 vol.% before staining. 300 µL of solution was added per well and the stain was developed for 20 minutes under foil followed by two final washes with PBS.
The cells were imaged using a Leica SP8 Confocal Microscope. Cell counts based on the nuclei count and surface area values were obtained using ImageJ. 45

Statistical Analysis
Statistical analysis was performed using a single factor analysis of variance ( U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein.

Author Contribution Statement
ID and AMP conceived and planned the experiments. ID carried out the experiments, took lead in the analysis of results, and wrote the initial draft of the manuscript. ID and AMP edited together to create the final version of the manuscript. AMP supervised the project.

Competing Interests Statement
The authors declare no competing interests.