Building cell-containing multilayered phospholipid polymer hydrogels for controlling diffusion of bioactive reagent

Cytocompatible and multilayered phospholipid polymer hydrogels containing living cells and a specific bioactive reagent were used to determine the role of diffusion of a bioactive reagent in regulating cell-fate. Within the multilayered hydrogels prepared by layer-by-layer assembling process, a bioactive reagent reservoir layer and a cell-laden layer were separated with a finely formed dual-crosslinked hydrogel multilayer that was composed of 2-methacryloyloxyethyl phosphorylcholine polymer and photo-reactive poly(vinyl alcohol). The dual-crosslinked hydrogel was less permeable than the single-crosslinked hydrogel and thus served as a diffusion-controlling barrier. We demonstrated the use of this multilayered hydrogel by subjecting the human cervical cancer HeLa cell-line to paclitaxel. We found that cell viability was regulated by the thickness of the diffusion-controlling barrier and bioactive reagent concentration. Increase in barrier thickness and decrease in bioactive reagent concentration decreased apoptosis of HeLa cells. Our results suggest that the gradient of a bioactive reagent formed was due to the diffusion-controlling barriers, which explains the observed diffusion-dependent cell behavior. This study provides insight into the regulation of bioactive reagent diffusion in micrometer scale hydrogels and the associated diffusion-dependent effects on cell behavior. This finding can contribute in the designing of a platform for studying diffusion-dependent cell behavior for tissue engineering and regenerative medicine applications.


Introduction
2][3] All together mimicked some key features of the native extracelluar matrix (ECM) and facilitated cell spreading, migration and differentiation. 4,57][8] Mimicry of these gradients in hydrogel could be particularly important in functional tissue engineering approaches, which aim to use combinations of cells, materials, and bioactive reagents to recreate natural tissue structure and function. 10][11][12][13][14] Several approaches such as photolithographic technology 10,11 , soft lithographic process 12 , microfluidic technology 13,14 , have been used to spatially control the diffusional properties of hydrogel.While these new approaches represent great progress, each one has its specific drawbacks, such as complicated manipulations or lengthy constructions.
Layer-by-layer (LbL) assembly technology, which can mimic highly organized and stratified architectures of biological tissues, can be useful for the integration of specific reagents into biomaterials.6][17] It is an economical, easily applicable, and versatile tool for the controlled fabrication of surface coatings or tissue scaffolds.
A variety of bioactive reagents, such as proteins, DNA, RNA, and nanoparticles, Page 4 of 24 RSC Advances

RSC Advances Accepted Manuscript
[13] However, until recently, stable incorporate cell into single layers of the LbL assembly was a challenge, with the exception of a cell spraying LbL procedure 18 .For the first time, we have reported a spinning-assisted LbL procedure that allows precise spatial control of multiple cell types in 3D without cellular damage. 19This method has been previously used to encapsulate tumor cells and stroma cells to study distance-dependent cell-cell interactions. 20en a water-soluble phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)] (PMBV) and poly(vinyl alcohol) (PVA) were mixed together, based on the specific reaction between the VPBA unit in PMBV and the diol group in PVA a hydrogel (PMBV/PVA) was spontaneously formed at room temperature.This hydrogel formation occurs under mild reaction conditions and can effectively encapsulate of biological components, such as bacteria 21 , living cells 22,23 , and bioactive reagents 24,25 .
In this study, photo-reactive PVA (Azido unit pendant water-soluble photopolymer, AWP) 26 was used to prepare a dual-crosslinked hydrogel (PMBV/AWP) to increase the cross-linking degree of the hydrogel.Thus, this hydrogel plays the role of a diffusion-controlling barrier, by which the diffusion rate of the bioactive reagents contained in the hydrogel can be controlled as a function of thickness of the barrier layer.We used the anticancer bioactive reagent, paclitaxel (PTX), as a model bioactive reagent.Although PTX is extremely hydrophobic, it can be dissolved in a PMBV aqueous solution with high efficiency due to the presence of hydrophobic domains in aqueous medium. 24,25Therefore, PTX can be loaded in situ into the PMBV/PVA hydrogel to prepare a reservoir layer of the bioactive reagent.

RSC Advances Accepted Manuscript
What's more, we developed a spinning-assisted LbL method for building a sandwich multilayered hydrogel platform, that is (PMBV/PVA as a PTX-loaded layer)-(PMBV/AWP as a diffusion-controlling barrier)-(PMBV/PVA as a cell-incorporated layer). 19,20  aim of this study was to investigate the diffusion of a bioactive reagent in sandwich structured multilayered hydrogels, and to determine the effect of the thickness of the diffusion-controlling barrier and the concentration of the bioactive reagent on cell behavior.The proposed methodology may contribute to the technological advancement of tissue engineering by offering the possibility to regulate the biomaterial composition and control different biological effects during tissue reconstruction.
Fluorescence-labeled PTX (Oregon-green 488-labeled) was purchased from Invitrogen (Carlsbad, CA, USA).AWP was purchased from Tokyo Gosei Co. Ltd., Japan.Other organic reagents and solvents were commercially available reagents of extra-pure grade and were used without further purification.
PMBV was synthesized by a conventional free radical polymerization of corresponding monomers, as previously described 19,20 .The mole fractions of MPC,
Fluorescence-labeled PMBV, poly(MPC-co-BMA-co-VPBA-co-MTR) (PMBV-R) was synthesized by radical polymerization 19 .The chemical structures of PMBV and AWP are shown in Figure 1a.The dual-crosslinking reaction between PMBV and or AWP is shown in Figure 1b.

Cell culture procedure
We used human cervical cancer HeLa cells that showed a stable expression of fluorescent ubiquitination-based cell cycle indicator (Fucci) fusion proteins.The Fucci fusion proteins were used as cell cycle sensors to perform quantitative analysis of cell activity kinetics. 28HeLa-Fucci cells were purchased from Riken Cell Bank (Ibaraki, Japan).They were cultured with DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum at 37°C in a 5.0 vol% CO 2 humidified atmosphere.

Solubilization of PTX with the polymer solutions
PTX was dissolved in a PMBV solution. 24Briefly, PTX (1.0 mg/mL) was dissolved in 99.5 wt% ethanol, and PMBV was dissolved in PBS with polymer concentrations of 50 mg/mL.Then a PTX-loaded PMBV solution was prepared by vortexing a mixture of the PTX and PMBV solutions at a ratio of PMBV/PTX = 90/10, and then removing the ethanol under reduced pressure.The PTX that was not dissolved was removed by filtration.The final PTX concentration in the PMBV solution was 75 µM (data not shown).

RSC Advances Accepted Manuscript
The PMBV(-R) solution (5.0 wt%) was prepared in PBS.The AWP solution (2.0 wt%) was prepared using a diluted stock solution (6.0 wt%) comprised of a mixture of 99.5 wt% ethanol and PBS at a ratio of PBS/ethanol = 30/10.The PMBV/AWP multilayer hydrogel was prepared by a spinning assisted LbL procedure (Figure 2) similar to our previously report 15,16 .Briefly, the AWP solution was manually spread on a 35 mm plastic culture dish to form a thin membrane.AWP-coated dishes were then air-dried in a fume hood at room temperature.Then they were irradiated for 1.0 min with UV light (50 mW/cm 2 ) from a UV Spot Cure (SP-7, Ushio Inc., Yokohama, Japan).Then the PMBV solution was deposited onto the AWP coated surface.After For the sample preparation for FT-IR reflection absorption spectroscopy (RAS), the dual-crosslinked multilayered hydrogels were applied onto Au spattered glass substrates and irradiated with UV light for 0 or 3.0 min.After drying overnight in a desiccator, the membranes were measured by FT-IR RAS (FT-IR 6300/Jasco).

Incorporation of a bioactive reagent and cells into multilayered hydrogels
The following procedure is shown in Figure 2. HeLa-Fucci cells were dispersed in 5.0

Evaluation of the cell proliferation-cycle distribution and cell viability
The numbers, positions, and fluorescence of HeLa-Fucci cells were measured using a BIOREVO BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan) at 12 h and 48 h.For quantitative analysis of fluorescent cells, at least 300 cells in five independent fields were analyzed.

RSC Advances Accepted Manuscript
Cells designated as apoptotic were those that displayed the characteristic morphological features of apoptosis, including cell volume shrinkage, chromatin condensation, and the presence of membrane-bound apoptotic bodies.According to this apoptotic cell morphology, the percentage of apoptotic cells was calculated.

Evaluation of bioactive reagent diff ff ff ffusivity in dual-crosslinked PMBV/AWP multilayer hydrogels
For the evaluation of bioactive reagent diffusion in the PMBV/AWP hydrogels, the diffusion coefficient of PTX-F was determined by fluorescence correlation spectroscopy (FCS) measurements (CLSM, Carl Zeiss) as previously described. 29The

Statistical analysis of the data
Student's t-test was carried out to determine whether the observed differences were statistically significant (p < 0.05).

Fabrication of multifunctional hydrogel layers
The LbL assembling procedure was applied for preparation of multilayered hydrogels composed of PMBV and PVA.We successfully controlled the thickness of the multilayered hydrogels by changes in both the polymer concentration and number of layers. 19 this study, we embedded PTX in a PMBV/PVA layer, and then separated this layer with a cell-containing PMBV/PVA layer by PMBV/AWP layers.These multilayered hydrogels are novel because: 1) their micrometer size allows for the incorporation of both bioactive reagents and cells in their architecture, which would

RSC Advances Accepted Manuscript
relatively high viscosity. 19 confirm the photoreaction in the dual-crosslinked hydrogel membranes, dried membranes were measured by FT-IR RAS (see Figure S1 in the Supporting Information).A specific peak of the azide group was detected at 2123 cm −1 before UV irradiation.This peak was eliminated after UV irradiation, indicating a photochemical-crosslinking of the azide group.

Bioactive reagent diffusivity within multilayered hydrogels
To understand the permeability of dual-crosslinked hydrogels, we measured the diffusion coefficient of PTX-F across different multilayered hydrogels.As shown in Figure 5a, the diffusion coefficient of PTX-F across the PMBV/AWP hydrogel (photoirradiation for 3.0 min) was much lower than the single-crosslinked PMBV/AWP hydrogel (no irradiation) or PMBV/PVA.A probable reason for this phenomenon is that dual-crosslinked PMBV/AWP has the highest crosslinking degree, which prevents the diffusion of PTX-F.Therefore, PMBV/AWP could work as a diffusion-controlling barrier and generate unique spatial gradients of PTX-F.As shown in Figure 5b, PTX-F was encapsulated into a reservoir, and was allowed to gradually diffuse through the diffusion-controlling barrier.Over the course of 48 h, green fluorescent PTX-F gradually emanated, providing a spatial gradient field over a micrometer-size scale (Figure 5b and c).The evolution of the PTX-F intensity gradients demonstrated that the gradient gradually decreased with time and reached equilibrium after 48 h (Figure 5c).
One point that should be emphasized here is the constant permeability of PMBV/PVA(or AWP) hydrogel under cell culture conditions.As mentioned above, PMBV/PVA hydrogel is chemical crosslinked hydrogel.The boronic acid/diol complex can be dissociated by sugar solutions, such as sorbitol, which has stronger Page 12 of 24 RSC Advances

RSC Advances Accepted Manuscript
affinity to boronic acids than that of diol group, and thus degrade PMBV/PVA hydrogel. 23,30,31 Tere are no matrix metalloproteinases (MMP) sensitive segments in PMBV/PVA hydrogel, so that PMBV/PVA hydrogel cannot be destroyed by MMP secreted by cells.Therefore, it is impossible for cells to degrade the hydrogels and subsequently enhance the permeability of the hydrogel.][32]

Effects of PTX on cell proliferation-cycle distribution
Next, we demonstrated the applicability of our approach to control PTX diffusion within the hydrogel layers by changes in the thickness of the diffusion-controlling barrier.First, we determined the effects of PTX on cell proliferation-cycle distribution in HeLa-Fucci cells at 12 h after stimulation.Fucci allows determination of the dynamics of live-cell proliferation during a cell cycle; G1 and S/G2/M phase cells expressing Fucci emit red and green fluorescence, respectively. 28At 12 h, significant G2/M arrest was observed (Figure 6a and d).Besides, confirming the previous report 33 , we also observed that abnormal red fluorescence appeared in addition to the original green fluorescence at the M phase.Cells collapsed several hours after the appearance of the abnormal red fluorescence, suggesting that the emergence of the abnormal red fluorescence is an early indicator of mitotic catastrophe.As shown in Figure 6d, the percentage of (G2/M phase cells + abnormal cells) in the four groups in descending order were as follows: control group ≈ thin barrier group > intermediate barrier group > thick barrier group.No significant differences were observed between the control group and the thin barrier group.This shows that one can tune the biological activity by altering the number of PMBV/AWP layers composing the barrier.In order to further verify this barrier thickness dependent phenomenon, the Page 13 of 24 RSC Advances

RSC Advances Accepted Manuscript
cell viability of the above four groups was also characterized. Figure 6b and c show the cell viability after 48 h of bioactive reagent stimulation.Indeed, there were significant differences among them.While one PMBV/AWP layer does not show a significant barrier effect compared with the control group, five layers efficiently hinder the effects of PTX.A barrier composed of three PMBV/AWP layers partially hinders the diffusion of PTX, leading to a cellular viability between that of one and five PMBV/AWP layers.In addition, we found a dose effect: higher embedded-PTX concentrations lead to higher apoptosis of HeLa-Fucci cells (see Figure S2 in the Supporting Information).
These results can be explained based on the following mechanism: PTX can diffuse through a diffusion-controlling barrier and into the cell-containing gel and can come in contact with the cells.However, the amount of PTX in the cell-containing hydrogel is dependent on the thickness of the diffusion-controlling barrier and initial concentration of the loaded bioactive reagent.With an increasing number of barrier layers, the diffusion of PTX became limited and induced a weaker stimulation effect to HeLa-Fucci cells.
This strategy will now be extended to include the incorporation of customized multi-agents and cell-containing systems to mimic diffusion-controlled cell behavior in vivo.We envision that this system could serve as a powerful tool in tissue engineering and cell biology to address basic questions related to spatiotemporal signaling of biomolecules, such as morphogens.For example, the recapitulation of morphogen gradients within 3D structures and an examination of their influence on the fate of developing pluripotent stem cells, is possible. 34,35 e 14 of 24 RSC Advances

Conclusion
In this study, we have engineered sandwich structured multilayered hydrogels where a cell-containing gel layer was separated from the bioactive reagent reservoir layer by dual-crosslinked hydrogels.The multilayered hydrogels could be micropatterned three-dimensionally by a single mechanism or dual mechanisms to spatially control hydrogel permeability on the micrometer scale and permit examination of diffusion of bioactive reagent on encapsulated cell behavior.By manipulating thickness of dual-crosslinked hydrogel, which worked as a diffusion-controlling barrier, the barrier effects were tunable.Thus, we have shown that PTX embedded in the reservoir layer decreased apoptosis in HeLa cells as the barrier thickness increased and the PTX concentration decreased.Overall, the permeability of hydrogel system was introduced a design parameter for regulating cellular behaviors, and this multilayered hydrogel system provides a new platform for studying diffusion-dependent cell behavior for tissue engineering and regenerative medicine applications.

RSC Advances Accepted Manuscript
1.0 min, to allow the boronic acid-diol interaction, the dish was rotated at 2000 rpm to remove the excess PMBV solution and form a layer of PMBV/AWP membrane.Next, the AWP solution and PMBV solutions were alternately deposited to form a (PMBV/AWP)-(PMBV/AWP) n hydrogel membrane, where the value of n (n = 1, 3, 5) indicates the number of PMBV/AWP layers.Finally, the multilayered hydrogel membranes were air-dried, irradiated with UV light (50 mW/cm 2 ) for 3.0 min and submerged in PBS for swelling.With the help of PMBV-R to indicate PMBV distribution in the multilayered hydrogels, the structure and thickness of the multilayered hydrogels were characterized using a Zeiss LSM 510 confocal laser-scanning microscope (CLSM; Carl Zeiss, Oberkochen, Germany).
wt% PMBV solution, and a 5.0 wt% PMBV solution containing PTX (750 nM, 375 nM, and 188 nM) was also prepared.Briefly, the 5.0 wt% PVA solution was dropped on an irradiated PMBV/AWP layer and spun at 2000 rpm followed by the deposition of 50 µL PMBV solution containing PTX.Then the PTX loaded layer was alternately deposited with AWP solution and PMBV solution to form a (PMBV/AWP)-(PMBV/PTX/PVA)-(PMBV/AWP) n multilayer hydrogel, where the value of n = 1, 3, and 5. Next, 4.0 µL PMBV/HeLa-Fucci cell suspensions were spotted on the top of the growing multilayered hydrogel.The final structure was denoted as a (PMBV/AWP)-(PMBV/PTX/PVA)-(PMBV/AWP) n -(PMBV/HeLa-Fucci cell/PVA)-(PMBV/PVA) multilayer hydrogel.Finally, 2.0 mL DMEM was added to each dish and all the samples were incubated at 37°C in a 5.0 vol% CO 2 humidified atmosphere for 48 h.We used four groups to evaluate the cell response as a function of diffusion-controlling barrier thickness, which are shown in PTX-F/PBS solution (200 nM, 2.0 mL) was added onto multilayered PMBV/AWP (0 min irradiation), multilayered PMBV/AWP (3.0 min irradiation), and multilayered PMBV/PVA, which were built on 35 mm (glass 27ϕ) glass base dishes (Iwaki) and stored at 37°C for 24 h to equilibrate the samples.Then the diffusion coefficients of PTX-F were determined at eight positions within the multilayered hydrogels to get an average value.The experiments were repeated three times.In order to observe the diffusion process of PTX-F within multilayered hydrogel, a (PMBV-R/AWP)-(PMBV/2.0 µM PTX-F/PVA)-(PMBV-R/AWP) 3 -(PMBV-R/PVA) (PMBV/PVA) multilayer hydrogel was prepared.Cross-section fluorescence images of the multilayered hydrogel were taken at 0.5, 12, and 48 h under the same settings using CLSM (Carl Zeiss).
not be easily achieved by other LbL technologies, and 2) separating the bioactive reagent loaded layer and the cell-containing layer with different thicknesses of PMBV/AWP layers in a multilayered hydrogel allows for fine-tuned accessibility of bioactive reagents into the cells.As shown in Figure 3, confocal microscopy imaging shows a multilayered hydrogel consisting of fluorescent-labeled PMBV, fluorescent-labeled PTX, and HeLa-Fucci cells.In the vertical cross-section, a sandwich structure of bioactive reagent loaded layer, diffusion-controlling barrier, and cell-incorporated layer was visualized.As shown in Figure 4a and b, the thickness of dual-crosslinked PMBV/AWP could be finely tuned by varying the number of gel layers.The HeLa-Fucci cell-laden PMBV/PVA layer was on top of the dual-crosslinked PMBV/AWP layers.The thickness of one layer of 5.0%-PMBV/5.0%-PVAwas about 15-20 µm, which was enough to hold cells.Our previous research proved that the concentration of PVA was very important to form a cell-containing layer, because it could provide enough thickness and mechanical property to hold the cells due to its Page 11 of 24 RSC Advances

Figure 2 .Figure 3 .
Figure 2. Fabrication of multilayered hydrogel with encapsulation of PTX and cells.

Table 1
Experimental groups to study HeLa-Fucci cells associated with PTX stimulation under different conditions.Number of hydrogel layer between PTX-loaded layer and HeLa-Fucci cell laden layer b.Number of PMBV/AWP hydrogel layer (diffusion-controlling barrier) c.Number of PMBV/PVA hydrogel layer