High-Density Protein Loading on Hierarchically Porous Layered Double Hydroxide Composites with a Rational Mesostructure

Hierarchically porous biocompatible Mg-Al-Cl type LDH composites containing aluminum hydroxide (Alhy) have been prepared using a phase-separation process. The sol-gel synthesis allows for the hierarchical pores of the LDH-Alhy composites to be tuned, leading to a high specific solid surface area per unit volume available for high molecular weight protein adsorptions. A linear relationship between effective surface area, S EFF , and loading capacity of a model protein, bovine serum albumin (BSA) is established following successful control of the structure of the LDH-Alhy composite. The threshold of mean pore diameter, D pm , above which BSA is effectively adsorbed on the surface of LDH-Alhy composites, is deduced as 20 nm. In particular, LDH-Alhy composite aerogels obtained via supercritical drying exhibits extremely high capacity for protein loading (996 mg/g) due to a large mean mesopore diameter (> 30 nm). The protein loading on LDH-Alhy is >14 times that of a reference LDH material (70 mg/g) prepared via a standard procedure. Importantly, BSA molecules pre-adsorbed on porous composites were successfully released on soaking in ionic solutions (HPO 42− and Cl − aq.). The superior capability of the biocompatible LDH materials for loading, encapsulation, and releasing large quantity of proteins was clearly demonstrated.


Introduction
Protein immobilization on solid surfaces is of relevance to a wide range of research areas with potential applications in biotechnology and physiology. 1 The activity of immobilized proteins is an important consideration which affects inorganic/bio interfacial properties, such as antifouling and antibacterial properties, 2 and hemo-/bio-compatibilities. 3 Various solids have been studied as supports for proteins, 1 including layered double hydroxides (LDHs), which are promising candidates due to their outstanding biocompatibility and an ability to limit denaturation of immobilized proteins. 4 The active conformation of proteins is retained on twodimensionally flat and highly hydrophilic surfaces of LDHs, 5 to avoid denaturation which otherwise takes place on curved inorganic surfaces. 6,7 As a result, heme proteins, which usually denature on inorganic solids, can be immobilized on LDH surfaces without losing their inherent activity. Immobilized heme proteins are currently used as bio-electrodes with high sensitivity. 8,9 Synthesizing LDHs with meso/macropores is highly promising to achieve high capacity loading of protein. The rational design of the porous structure in nm to sub-μm scale is especially important because micropores (< 2 nm) and relatively small mesopores, that typically adsorb ion/small molecules, cannot accommodate large protein molecules (~tens of nm in size). The surface area accessible by proteins (defined here as effective surface area, S EFF ) strongly depends on the pore diameter, D p , of porous LDHs. To date, LDHs of micron-and submicron scale structures have been reported as particles, 10,11 sheets, 12 plates with grooves, 13 however, increasing S EFF is challenging and hard to achieve with these materials. This is because LDH crystals as building-blocks used to assemble materials are relatively large, typically in sub-μm. 13,14 Limiting the dimensions of LDH crystals to nanoscale, assembling them into 3D porous solids, and optimizing their meso and macroporous structures remains a big challenge when attempting to maximize S EFF and thereby protein loading. Recently, we have reported the preparation of monolithic LDHs with hierarchical pores. 15 The hierarchical pores of macro (1 μm) and meso (8 nm) formed spontaneously via a facile sol-gel reaction. It was also reported that target oxyanions (CrO 4 2− , SO 4 2− , MoO 4 2− , etc) and small molecules diffuse rapidly through the macropores and adsorb on a large surface derived from the mesopores. 16 However, the development of LDHs with tens-nm-pores which are optimized to maximize S EFF through structural hierarchy is still needed in order to exploit the applications of biocompatible solid supports with a high capacity for protein loading.
We here prepare biocompatible composites of LDH and aluminum hydroxide (Alhy) with two levels of hierarchical tunable pores in the range of tens-nm and a few-μm. Dependence of the mean mesopore diameter D pm on the synthesis parameters were investigated to tune the hierarchical porosity of the LDH-Alhy. Then, the adsorption of a large protein, bovine serum albumin (BSA) on the LDH-Alhy surfaces of various S EFF was conducted to establish a relationship between S EFF and loading capacity (Fig. 1a). D pm could be tuned from 12 nm to 53 nm, leading to a wide range of S EFF for adsorption of BSA molecules. Particular interest was focused on aerogels obtained via supercritical drying 17  as "X-LDH-40" and "X-LDH-120", respectively. Supercritical drying was conducted with supercritical CO 2 (80 °C and 14.0 MPa), yielding aerogels which are labeled as "A-LDH" in this following. the Barrett-Joyner-Halenda (BJH) method. Mean pore diameter, D pm , and total pore volume, V p (cumulative pore volume calculated from pores with D p < 183 nm) were estimated from the distribution curves. The BJH method was also applied to assess partial specific surface area (S (>xnm) ) derived from pores larger than a cut-off value. For example, S (>5nm) corresponds to a specific surface area obtained by integrating surfaces of pores with D P > 5 nm. The Brunauer-Emmett-Teller (BET) method was also applied to estimate specific surface area, S BET .
Synchrotron X-ray micro-computed tomography (μ-CT) was employed to non-destructively obtain three dimensional (3D) images of the drying process of the monoliths. High resolution synchrotron-based X-ray tomographic image was obtained from the Diamond-Manchester branchline I13-2 at Diamond Light Source. 19 The samples were kept at 300 K on the beamline using an in house environmental stage. 20 A polychromatic filtered parallel-beam setup was used with a 0.81 μm effective pixel size and ∼2 μm spatial resolution. Over the 180° rotation, 3600 projections were collected at 0.05 s exposure time and tomographically reconstructed into a 3D volume using software developed at Diamond Light Source. 21 (1), , where C s (mg/g) is the amount of BSA adsorbed by LDH, C i (mg/mL) and C eq (mg/mL) are initial and equilibrium concentrations of BSA in the solution, V (mL) is the volume of the solution, and m (g) is the mass of LDH. Freundlich equation (eq. (2)) was used as isotherm model for BSA adsorption on the sample solids.
, where K f (mg/g) is Freundlich constant and n f is the adsorption intensity.

Synthesis of hierarchically porous LDH-Alhy with tunable porosities
The hierarchically porous LDH-Alhy composites were prepared via hydrolysis and condensation reactions of metal salts by alkalinization in the presence of propylene oxide (PO) according to our previously published method. 15  Simultaneously, the mesopores (in nm range) were formed within the gel skeleton (Fig. 1a). The mesopore size, D p , and mesopores volume, V p , which expectedly influence the S EFF and the adsorption capacity of proteins, varied as a function of W PEO (the amount of PEO additive) and drying conditions. Alhy which forms porous networks together with LDH crystals display hydroxylated surface that are biocompatible with proteins, as confirmed by their use as adjuvant in some vaccines. 26 As a result, the LDH-Alhy composites offer extensive solid surface used for protein loading.
The LDH-Alhy composites produced here and Ref-LDH possess very different microstructures (Fig. 2).  Table 1). The formation of smaller mesopores with increasing W PEO is caused by decrease of the solvent which is to be mesopores after drying in LDH-Alhy-rich solid phase, leading to a more packed LDH and Alhy crystallites. 27 The characteristics of mesopores also depends on the drying conditions. A-LDHs possess the largest D pm and V p among the three sets of LDH-Alhy composites because the shrinkage is minimized by applying the supercritical drying. 17 To get better insight on the shrinkage upon drying process of the monolithic LDHs, X-ray μ-CT was performed while the gels were left to dry at 300 K (Fig. 3). Figure 3 shows 3D images of a small volume of the same gel before and after drying. It shows that shrinkage was isotropic, leading to a volume shrinkage of 85% and a linear shrinkage of 46%. The relatively large D pm and V p of X-LDH-120 compared to X-LDH-40 is due to smaller degree of shrinkage on drying. The faster drying at 120 °C forms cracks in the monolithic specimen and releases stress generated at the drying front, retarding isotropic shrinkage and leading to larger mesopores. 28 It should be emphasized again that X-LDH-40, X-LDH-120, and A-LDHs possess the identical chemical composition and the crystallinity, and differences among these samples are only porosity in nm and μm scales. In summary, D pm of LDH-Alhy composites were successfully tuned between 12 nm and 53 nm by W PEO value and drying condition.

Effect of pore structure on protein adsorption
The LDH-Alhy composites with various D pm and V p were assessed as bio-supports with a high capacity for protein loading. As an adsorbate, BSA was used, which is a large multi-domain protein with a hydrodynamic radius of 3.6 nm and 3D size of 5×7×7 nm 3 . 29 Serum albumin, a major soluble constituent of the plasma proteins, has many physiological functions. 30 Moreover, BSA has been used as a model protein to investigate reactions of physiological disorders, such as diabetes, 31 and its sustained-release 32 , immobilization 7 , and bio-probes are of interest. 33 Aluminum hydroxide and LDH, have been individually used in the form of crystalline platelets as adsorbents for BSA molecules. 34,35,36 Preliminary results (not shown) of BSA adsorption using previously-reported hierarchically porous LDHs 15 (prepared by ambient drying without the solvothermal treatment) lead to negligible amount of protein adsorption. Where, mesopore size was too small to accommodate protein molecules, and only macropores were available for protein adsorption; surface area derived from macropores 37 is less than 10 m 2 /g.  Fig. 4b and c, respectively. Due to the lack of realistic adsorption models for fitting the interactions between solid surfaces and proteins, many studies have reported the use of Freundlich model as an approximative tool to evaluate and compare various solid/protein systems. 38 Freundlich adsorption isotherms for X-LDH-40, X-LDH-120, and A-LDH are represented in Fig. S3. The plots can be linearly fitted by equation (2) except for the case of X-LDH-40 at W PEO =0.02 g whose microstructure is highly inhomogeneous because of structure deformation during drying. K f value increases in the order of X-LDH-40 < X-LDH-120 < A-LDH as summarized in Table 2 (Tables S1 and S2). Indeed, while surface of Ref-LDH with a higher zeta potential (+42 mV) should promote higher BSA adsorption than LDH/Alhy composite (+37 mV for X-LDH-40), the reverse is observed; BSA has an isoelectric point of pI = 4.7 39 and is negatively-charged in the present adsorption condition (pH= ~7). Figure 5 shows plots of K f values of LDH-Alhy composites against S (>xnm) that is a specific surface area obtained by integrating surface areas derived from pores of D p > x nm; for example, S (>5nm) is a sum of surface areas derived from pores with a diameter of D p > 5 nm. The LDH-Alhy composites prepared in the present study have tunable pore characteristics and S (>xnm) was controllable to a large extent. Figure 5 shows  (Fig. 4a).
Similar size effect of mesopores on BSA adsorption was qualitatively reported on mesoporous silica, where SBA-15 with the pore diameter of 24 nm showed much better BSA adsorption than those with 3.8 and 7.7 nm in diameters. 40 The present results give a very systematic and quantitative evidence for a threshold in pore diameter for effective adsorption of BSA molecules to mesoporous materials. The approach demonstrated here will be a promising platform to maximize S EFF for respective proteins with different molecular sizes.

Ability for encapsulation and releasing of immobilized proteins
The capability of encapsulating immobilized protein and their sustained-release are also important feature for bio-adsorbents. A previous study reported that BSA desorption from Zn-Al LDH took place by the addition of competitive anionic adsorbates. 41 Herein, HPO 4 2− and Cl − ions were used as competitive ions to release pre-adsorbed BSA molecules. Table 3 summarizes the results of protein desorption from A-LDH and Com-LDH. 52% of BSA desorbed in 72 h from A-LDH in the case of using HPO 4 2− as competitive anion, which is comparable to that from the Com-LDH (49%). This result confirms that the protein desorption as well as the adsorption takes place with a large capacity for the present LDH-Alhy composites. Cl − ions did not induce the desorption of BSA both for Com-LDH and A-LDH, which is due to the lower affinity of Cl − and LDH surface, and smaller minus charge compared to HPO 4 2− . While the LDH-Alhy composites (X-LDH and A-LDH) have a weight ratio of LDH : Alhy = 1 : 1, the desorption percentage by Cl − of these composites is almost same as pure LDH (~2%) (       Tables Table 1. Mesopore characteristics of LDH-Alhy composites.