Phospholipid Polymer Hydrogel Research Articles

Phospholipid polymer hydrogels with rapid dissociation for reversible cell immobilization.

A reversible and cytocompatible cell immobilization polymer matrix with a rapid dissociation rate was prepared using a zwitterionic phospholipid polymer bearing phenylboronic acid and poly(vinyl alcohol) (PVA). A reversible and spontaneously forming phospholipid polymer hydrogel is reported for use as a cell immobilization matrix which caused no invasive damage to the cells. To improve the possibility of applying the hydrogels as a reversible cell immobilization matrix, the stimuli-responsive dissociation rate of polymer hydrogels was designed to have a more rapid rate to ease the recovery of the immobilized cells. In this study, a phospholipid polymer containing 3-methacrylamide phenylboronic acid (MAPBA) as the phenylboronic acid unit was synthesized. The water-soluble phospholipid polymer (PMB-MAPBA) can spontaneously form polymer hydrogels after mixing with PVA solution under normal pressure, room temperature, and neutral pH conditions. Also, the dissociation of the hydrogels after the addition of D-sorbitol completely occurred within 10 minutes. The cells were easily immobilized on the hydrogels during the preparation process. Also, the recovery ratio of the immobilized cells was improved due to the rapid dissociation of the hydrogels. The reversible and spontaneously formed phospholipid polymer hydrogels are promising for use as soft materials for platforms for cell engineering.

Phospholipid Polymer Hydrogel Matrices with Dually Immobilized Cytokines for Accelerating Secretion of the Extracellular Matrix by Encapsulated Cells.

Construction of 3D tissues by various types of cells with specific characteristics is an important and fundamental technology in tissue reconstruction medicine and animal-free diagnosis system. To do so, an excellent extracellular matrix (ECM) is needed for encapsulation of cells and maintaining cell activity. Spontaneously forming hydrogel matrix is used by complexation between two water-soluble polymers, 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups and poly(vinyl alcohol). Two cytokines for cell proliferation are immobilized in the hydrogel matrix to control the activities of the encapsulated cells. The cytokine-immobilized hydrogel matrix can encapsulate both L929 fibroblasts and normal human dermal fibroblasts under mild condition. The physical properties of the hydrogel matrix can follow the proliferation process of the encapsulated cells. The encapsulated cells secrete ECM in the polymer hydrogel networks upon 3D culturing for 7 days. Consequently, the tissue-mimicking ECM hybrid hydrogels are fabricated successfully.

Hybridization of a phospholipid polymer hydrogel with a natural extracellular matrix using active cell immobilization.

Three-dimensional tissue organization is still an obstacle in the field of tissue engineering, which generally involves cell immobilization, proliferation, and organization. As an artificial extracellular matrix (ECM) for providing a suitable environment of cells to construct tissues, combination of cytocompatible polymer hydrogels and natural ECM produced by the immobilized cells was considered. In this research, we designed a spontaneously forming hydrogel system between two water-soluble polymers for the immobilization of cells. These polymers were poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid-co-N-succinimidyloxycarbonyl tetra(ethylene glycol)methacrylate) (PMBVS) and poly(vinyl alcohol) (PVA) to form a PMBVS/PVA hydrogel in a cell culture medium under mild conditions. Basic fibroblast growth factor (bFGF) was conjugated with PMBVS (PMBV-bFGF). To enhance the growth of the immobilized cells, mouse fibroblast L929 cells were immobilized in the PMBVS/PVA hydrogel and the PMBV-bFGF/PVA hydrogel, and their proliferation and secretion of the ECM under stimulation with bFGF was observed. The ECM infiltrated and replaced the hydrogel, resulting in the formation of a hybrid hydrogel with the ECM and laden cells.

リン脂質ポリマーハイドロゲルを用いたon-chip細胞保存

近年，各分野において細胞の利用は急速に拡大している。これにともない，従来の細胞保存に必要とされる精密機器や専門的知識へ依存しない，また過酷な環境でも細胞が安定に保存できる手法の確立が熱望されている。そこで，筆者は，2-メタクリロイルオキシエチルホスホリルコリン（MPC）ポリマーハイドロゲルとマイクロチップ技術を融合することで，無灌流培養条件下のon-chip細胞保存法，および室温・冷蔵温度での非培養環境下のon-chip細胞保存法を開発してきた。本稿では，MPCポリマーハイドロゲルを用いたこれらの細胞のon-chip保存法について解説する。

Novel tissue-binding phospholipid polymer hydrogels for mucosal tissue regeneration

Novel tissue-binding phospholipid polymer hydrogels for mucosal tissue regeneration

Bioinspired Phospholipid Polymer Hydrogel System for Cellular Engineering

SummaryThe properties of the microenvironment surrounding cells are important for the control of cell functions. The polymeric cellular environment has great potential in this regard because environmental properties can be manipulated. We propose a spontaneously forming phospholipid polymer hydrogel system composed of 2 kinds of pre‐polymers to control cellular functions via hydrogel physical properties. These pre‐polymers are cytocompatible poly(2‐methacryloyloxyethyl phosphorylcholine‐co‐n‐butyl methacrylate‐co‐p‐vinylphenylboronic acid) (PMBV) and water‐soluble poly(vinyl alcohol) (PVA). The p‐vinylphenylboronic acid units in PMBV can react with the hydroxy groups of PVA in aqueous medium and form cross‐linkages. This spontaneously formed PMBV/PVA hydrogel can be dissociated again via the addition of sugar compounds. To alter the physical properties, we simply change the concentration or mixing ratio of the pre‐polymers. The storage modulus of the PMBV/PVA hydrogel matrix was controlled from 0.3 kPa to 2.5 kPa, which corresponds to very soft natural tissue. Cells can be encapsulated in the hydrogel. When the storage modulus of the PMBV/PVA hydrogel was above 1.0 kPa, the proliferation of encapsulated cells was suppressed and provided uniform cells in G1 phase in cell cycle progression. High G1 phase fraction (>90%) may lead to excellent differentiation efficiency, which results in great insight for stem cell engineering. The PMBV/PVA system is expected to become a key material in cellular engineering for regulating cellular function without undesirable biological events.

Efficient differentiation of stem cells encapsulated in a cytocompatible phospholipid polymer hydrogel with tunable physical properties

A large number of lineage-committed progenitor cells are required for advanced regenerative medicine based on cell engineering. Due to their ability to differentiate into multiple cells lines, multipotent stem cells have emerged as a vital source for generating transplantable cells for use in regenerative medicine. Increment in differentiation efficiency of the mesenchymal stem cell was obtained by using hydrogel to adjust the proliferation cycle of encapsulated cells to signal sensitive phase. Three dimensional (3-D) polymer networks composed of poly(2-methacyloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)) (PMBV) and poly(vinyl alcohol) (PVA) were prepared as a hydrogel. The proliferation of cells encapsulated in the PMBV/PVA hydrogel was highly sensitive to the storage modulus (G′) of the hydrogel. That is, when the G′ value of the hydrogel was higher than 1.0 kPa, the cell proliferation was ceased and the proliferation cycle of cells was converged to G1 phase, whereas when the G′ value was below 1.0 kPa, cell proliferation proceeded. By changing the G′ value of hydrogels under encapsulation the cells, proliferation cycle of encapsulated mesenchymal stem cells was regulated to G1 phase and thus signal sensitivity were increased. 3-D polymer networks as hydrogels with tunable physical properties can be effectively used to control proliferation and lineage-restricted differentiation of stem cells.

Cytocompatible and spontaneously forming phospholipid polymer hydrogels

Given the importance of regulating living cell functions in three-dimensional (3D) environments, we have designed a cytocompatible and spontaneously forming polymer hydrogel matrix. Water-soluble phospholipid polymers bearing a phenylboronic acid unit, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), and poly(vinyl alcohol) (PVA), are candidate systems for preparing the hydrogel matrices. Aqueous solutions of PMBV and PVA can be used to form hydrogels based on reversible complexation between the phenylboronic acid groups in PMBV and the diol groups in PVA, even under cell culture conditions. Uniform cell encapsulation is easily achieved with hydrogel formation, and cells survived well in the hydrogel. By applying a spinning-assisted layer-by-layer (LbL) process, multilayered PMBV/PVA hydrogels containing living cells can be assembled. This multilayered hydrogel, which mimics the stratified structure of in vivo tissues, allows the layer-specific encapsulation of cells and temporary storage of bioactive molecules. Distance-dependent cell–cell interactions are investigated using the multilayered hydrogel where two cell-laden layers are separated by a finely controlled multilayered hydrogel. In addition, dual-crosslinked multilayered hydrogels are also assembled by alternative depositions of PMBV and photoreactive-PVA solutions, followed by photoirradiation. The dual-crosslinked hydrogel has a lower diffusivity of bioactive molecules than that of single-crosslinked hydrogel and therefore acts as a diffusion-controlling barrier. We demonstrate the utility of this dual-crosslinked hydrogel by examining ways to regulate the diffusion of bioactive molecules in the hydrogel and investigating the diffusion-dependent effects on cell behavior. In conclusion, these hydrogel matrices can provide insights into the regulation of cell behavior in 3D matrices. In turn, our results may contribute to the future design of 3D cell culture systems and tissue regenerated medicine based on cell engineering.

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

We developed multilayered phospholipid polymer hydrogels containing living cells and a specific bioactive reagent for the regulation of cell-fate.

Redox-active cytocompatible phospholipid polymer hydrogels for three-dimensional electrical control of encapsulated living cells

Three-dimensional encapsulation of a living human cervical cancer HeLa cell in a redox-active cytocompatible phospholipid polymer hydrogel and the regulation of cell functions with simple bioelectrical stimulation were investigated. A redox-active and cytocompatible polymer based on the water-soluble phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)-co-vinylferrocene (VFc)) (PMBVF) was synthesized by conventional radical polymerization method. A HeLa-Fucci cell, a human cervical cancer HeLa cell stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were cultured and suspended into the medium, in which a PMBVF polymer were pre-dissolved with desired concentration. Poly(vinyl alcohol) (PVA) solution was added to this PMBVF/HeLa-Fucci medium solution with gently mixing at room temperature, in which case, a reversible PMBVF/PVA hydrogel with encapsulated HeLa-Fucci cells was obtained. The morphologies of the PMBVF/PVA hydrogel and the three-dimensional encapsulated HeLa-Fucci cells were confirmed with microscopy. Cyclic voltammograms results indicated that PMBVF/PVA hydrogel still had excellent electrochemical properties even in the presence of living HeLa-Fucci cells. A constant voltage (0.4 V vs. Ag/AgCl) was applied to the extracellular environment of encapsulated cells as electrical stimulation and the cell cycle progression was calculated base on the analysis of fluorescent images. Compared with that without electrical stimulation, a delay of the cell cycle progression could be observed between 8 h and 18 h while the cells were encapsulated in the hydrogel with electrical stimulation. The investigation of cell functions based on a three-dimensional electrical stimulation with a redox-active cytocompatible phospholipid polymer hydrogel will promote the development of electrochemical therapy of cancer and tumor.

Multilayered phospholipid polymer hydrogels for releasing cell growth factors

Polymer multilayered hydrogels were prepared on a titanium alloy (Ti) substrate using a layer-by-layer (LBL) process to load a cell growth factor. Two water-soluble polymers were used to fabricate the multilayered hydrogels, a phospholipid polymer with both N, N-dimethylaminoethyl methacrylate (DMAEMA) units and 4-vinylphenylboronic acid (VPBA) units [poly(MPC-co-DMAEMA-co-VPBA) (PMDV)], and the polysaccharide alginate (ALG). PMDV interacted with ALG through a selective reaction between the VPBA units in PMDV and the hydroxyl groups in ALG and through electrostatic interactions between the DMAEMA units in PMDA and the anionic carboxyl groups in ALG. First, the Ti substrate was covered with photoreactive poly vinyl alcohol, and then the Ti alloy was alternately immersed in the respective polymer solutions to form the PMDV/ALG multilayered hydrogels. In this multilayered hydrogel, vascular endothelial growth factor (VEGF) was introduced in different layers during the LbL process under mild conditions. Release of VEGF from the multilayered hydrogels was dependent on the location; however, release continued for 2 weeks. Endothelial cells adhered to the hydrogel and proliferated, and these corresponded to the VEGF release profile from the hydrogel. We concluded that multilayered hydrogels composed of PMDV and ALG could be loaded with cell growth factors that have high activity and can control cell functions. Therefore, this system provides a cell function controllable substrate based on the controlled release of biologically active proteins.

Cytocompatible and reversible phospholipid polymer hydrogels for encapsulation to provide unified quality cells

As the field of regenerative medicine based on stem cell engineering starts to play the important role in the new generation of bioengineering, the cells are starting to be treated as one of the materials to be controlled and optimized rather than those to be observed. Temporal and spatial encapsulation of the cells by the hydrogels are getting an attention as a novel way to handle cells in the three dimensional condition. In this study, cytocompatible and reversible phospholipid polymer hydrogels were prepared by mixing the aqueous solutions of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The murine pluripotent stem cell line C3H10T1/2 is known to suppress its proliferation when encapsulated in the PMBV/PVA hydrogel with storage modulus of 1.2 kPa. The cell cycle of the encapsulated cells were unified to G1 phase in cell proliferation cycle after 1 day of encapsulation. The differentiation of C3H10T1/2 to osteogenic cells by bone morphogenetic protein 2 (BMP-2) was evaluated by polymerase chain reaction method after induction of BMP-2 signal for 3 days. The cells encapsulated in the PMBV/PVA hydrogel showed 1.7-hold increase of early-stage osteoblast gene expression with well-defined quality will make substantial contribution to the field of regenerative medicine.

Quantitating distance-dependent, indirect cell–cell interactions with a multilayered phospholipid polymer hydrogel

Multilayered polymer hydrogels containing living cells were assembled for assessing the distance-dependent effects of soluble factors secreted by stroma cells on tumor cell cycle progression in vitro. A layer of tumor cells and a layer of stroma cells were separated with finely controlled spacing in a multilayered sandwich composed of a 2-methacryloyloxyethyl phosphorylcholine polymer and poly(vinyl alcohol) hydrogel. We demonstrated the utility of this tool for investigating intercellular communication between human cervical cancer HeLa cells and supportive stromal L929 fibroblast cells in co-culture. Time-lapse microscopic analyses of HeLa cells showed short distances (15 μm) between tumor cells and stroma cells induced a continuous increase in the percentage of HeLa cells in the S/G2/M phases of the cell cycle, while longer distances (70 μm) between the cells caused a slower increase followed by a sharp increase in the percentage of cells in S/G2/M phases. One possible explanation is gradient formation in the diffusion-dominant multilayer hydrogels by water-soluble factors such as those inducing growth, differentiation, and proliferation. This study provides insights into the potential effects of diffusion of soluble factors and related distance-dependent effects on cell behavior, which may contribute to the design of future co-culture systems.

The use of the mechanical microenvironment of phospholipid polymer hydrogels to control cell behavior

We considered that properties of the microenvironment surrounding cells are important for the control of the cell functions. Cytocompatible polymer hydrogels are good candidates to study such microenvironment. Here, we prepared spontaneously forming hydrogels composed of two polymer systems, namely poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The PMBV/PVA hydrogels could be reversibly dissociated by the addition of d-sorbitol. The storage modulus was measured for evaluating the mechanical properties of the PMBV/PVA hydrogels. The storage modulus could be controlled in the range 0.30–2.5 kPa by changing the cross-linking density of the hydrogels. After pluripotent stem cells were encapsulated within the PMBV/PVA hydrogels during the preparation of the hydrogel under normal cell-culturing conditions, the proliferation rate and the cell cycle of the encapsulated cells were observed. Cells lived for more than three days in every PMBV/PVA hydrogel. However, the proliferation significantly depended on the storage modulus of the hydrogels. Although the cell cycle of the initial cells was heterogenous, it developed uniformity toward the G1 phase when the cells were encapsulated within the PMBV/PVA hydrogel with a storage modulus of 1.1 kPa for three days. That is, the mechanical properties of the PMBV/PVA environment influenced the biological functions of the cells encapsulated in the hydrogels. From these results, we conclude that PMBV/PVA hydrogels are useful for adjusting cell cycles and proliferation, thus providing uniform cells for applications in the field of cell engineering.

A simple procedure for the preparation of precise spatial multicellular phospholipid polymer hydrogels

A precise spatial multicellular polymer hydrogel matrix was prepared by successive assembly of cell-laden hydrogel layers alternated by hydrogel layers without cells based on the spontaneous hydrogel formation between 2 aqueous polymer solutions. The polymers used were a water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA). Each cell-laden layer was deposited as a cell-laden PMBV solution on a PMBV/PVA precursor film. PMBV/PVA multilayer hydrogel was stacked on the top of a cell-laden layer by sequential coating with spinning of the PMBV and PVA solutions. This process allowed the formation of the PMBV/PVA multilayer hydrogel with finely controlled thickness. Finally, we succeeded in cell patterning by using a multilayer hydrogel matrix, forming a sandwich of 2 cell-laden layers separated by a PMBV/PVA multilayer hydrogel. The cells remained alive during the spinning process and maintained their metabolism for at least 24h. This precise spatial multicellular PMBV/PVA hydrogel can be used to examine interactions between many different cells and construct customized microenvironments for multicellular co-cultures.

Phospholipid polymer hydrogel microsphere modulates the cell cycle profile of encapsulated cells

Recently, the understanding of the relationship between cell functions and the physical properties around cells has gathered much attention in the fields of cell engineering and physical biology. In this report, we describe the modulation of the cell cycle in cytocompatible hydrogel microspheres with varied physical properties. We prepared hydrogel microspheres composed of cytocompatible pre-polymers: poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate-co-4-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The diffusion coefficients of proteins varied with both the protein size and concentration of the pre-polymer used to construct the PMBV–PVA hydrogel microspheres. The elastic moduli of the PMBV–PVA hydrogels were in the range of 24–367 Pa. We monitored the cell cycle of single cells encapsulated in single PMBV–PVA hydrogel microspheres in order to eliminate the influence of cell-to-cell interactions. Using a fluorescent ubiquitination-based cell cycle indicator, we found that the cell cycle progression of single HeLa cells was delayed within PMBV–PVA hydrogel microspheres with a higher stiffness. In contrast, a significant delay of the cell cycle was not observed within hydrogel microspheres with a lower stiffness. Although there was a difference in diffusivity between both types of hydrogel microspheres, control experiments showed that this range of diffusivity did not affect cell cycle progression. Thus, we conclude that cell cycle progression is regulated by the difference in stiffness around the cell. We believe that the PMBV–PVA hydrogel microsphere offers a useful cellular environment for the control of cell function by regulating a wide variety of stimulatory factors around cell.

The effect of the encapsulation of bacteria in redox phospholipid polymer hydrogels on electron transfer efficiency in living cell-based devices

Development of living cell-based devices holds great promise in many biomedical and industrial applications. To increase our understanding of the process, we investigated the biological and electrochemical properties of a redox phospholipid polymer hydrogel containing an electron-generating bacteria (Shewanella oneidensis MR-1). A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMBVF), was our choice for incorporation into a hydrogel matrix that promotes encapsulation of bacteria and acts as an electron transfer mediator. This hydrogel formed spontaneously and encapsulated Shewanella in three-dimensional structures. Visual analysis showed that the encapsulated Shewanella maintained viability and metabolic activity even after long-term storage. Cyclic voltammetry measurement indicated that the PMBVF/poly(vinyl alcohol) (PMBVF/PVA) hydrogel had stable and high electron transfer efficiency. Amperometric measurement showed that the hydrogel could maintain the electron transfer efficiency even when Shewanella was encapsulated. Thus, the PMBVF/PVA hydrogel not only provides a mild environment for long-term bacterial survival but also maintains electron transfer efficiency from the bacteria to the electrode. We conclude that hydrogel/bacteria hybrid biomaterials, such as PMBVF/PVA/Shewanella, may find application in the fabrication of living cell-based devices.

Encapsulation of <i>shewanella</i> in the redox phospholipid polymer hydrogel for microbial fuel cell fabrication

In this research, encapsulation of Shewanella oneidensis MR-1 in the phospholipid polymer hydrogel and electrochemical property of the hydrogel with immobilized shewanella was investigated. Cytocompatible and electron transfer mediated polymer based on the water-soluble phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)-co-vinylferrocene (VFc)) (PMBVF) was synthesized by conventional radical polymerization method. Shewanella which have the ability to generate and transfer extracellular electron without any external mediator, have significant potential on the fabrication of microbial fuel cell. The water-soluble PMBVF was dissolved in the cell culture medium containing shewanella cells. Poly(vinyl alcohol) (PVA) solution was added to the PMBVF solution with gently mixing, in which case, a reversible PMBVF/PVA hydrogel with encapsulated shewanella was obtained. The viability of the encapsulated shewanella was calculated by the fluorescence microscopy. The electrochemical property of the PMBVF/PVA hydrogel containing shewanella was measured by cyclic voltammograms (CV) and amperometric i-t curve. PMBVF/PVA hydrogel can provide mild environment for the shewanella and maintain the cellular function for longtime. The generated current was higher than that cultured with normal medium.

Spherical Phospholipid Polymer Hydrogels for Cell Encapsulation Prepared with a Flow-Focusing Microfluidic Channel Device

To prepare spherical polymer hydrogels, we used a flow-focusing microfluidic channel device for mixing aqueous solutions of two water-soluble polymers. Continuous encapsulation of cells in the hydrogels was also examined. The polymers were bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenyl boronic acid groups (PMBV) and poly(vinyl alcohol) (PVA), which spontaneously form a hydrogel in aqueous medium via specific molecular complexation upon mixing, even when they were in cell culture medium. The microfluidic device was prepared with polydimethylsiloxan, and the surface of the channel was treated with fluoroalkyl compound to prevent sticking of the polymers on the surface. The microfluidic channel process could control the diameter of the spherical hydrogels in the range of 30-90 μm and generated highly monodispersed diameter spherical hydrogels. We found that the polymer distribution in the hydrogel was influenced by the PVA concentration and that the hydrogel could be dissociated by the addition of d-sorbitol to the suspension. The single cells could be encapsulated and remain viable in the hydrogels. The localized distribution of polymers in the hydrogel may provide an environment for modulating cell function. It is concluded that the spontaneous hydrogel formation between PMBV and PVA in the flow-focusing microfluidic channel device is applicable for continuous preparation of a spherical hydrogel-encapsulating living cell.

Regulation of cell proliferation by multi-layered phospholipid polymer hydrogel coatings through controlled release of paclitaxel

We fabricated multi-layered hydrogels on titanium alloy (Ti) surfaces by applying alternating layers of a water-soluble phospholipid polymer (PMBV) and polyvinyl alcohol (PVA). This was accomplished by a layer-by-layer (LbL) process that is based on the formation of reversible covalent bonds between the boronic acid subunits in the PMBV and the hydroxyl groups in the PVA. When placed in an aqueous medium, PMBV acquires a polymeric aggregate structure with hydrophobic domains that can effectively solubilize hydrophobic molecules such as the anticancer drug paclitaxel (PTX) used in this study. The PTX-containing PMBV layer acted as a reservoir in the multi-layered hydrogels. To obtain diverse release profiles, the PTX was loaded in either the top layer (top-type) or the bottom layer (bottom-type) of the hydrogels; additional layers of PMBV and PVA, without PTX, functioned as a diffusion-barrier. In cell culture experiments, top-type hydrogels demonstrated excessive suppression of human epidermal carcinoma A431 cell proliferation over 5 days due to the initial high concentration of released PTX. However, bottom-type hydrogels were able to maintain a constant cell number profile. The release of PTX from multi-layered hydrogels was governed by both diffusion through the diffusion-barrier and dissociation of the hydrogel through an exchange reaction of phenylboronic acid subunits with the low-molecular weight d-glucose in the cell culture medium. In the cell culture experiments, the cell cycle was arrested in S and G2/M phases, as expected following PTX-mediated growth inhibition; control hydrogels did not demonstrate any appreciable cell cycle arrest. We concluded that cell proliferation could be controlled by the concentration of PTX released from the multi-layered hydrogels prepared through the LbL process. This system when used to solubilize bioactive agents at an appropriate layer within the hydrogel has potential for localized and surface-mediated delivery of bioactive molecules from biomedical devices.