Modulating the release of proteins from a loaded carrier of alginate/gelatin porous spheres immersed in different solutions

Abstract

Background:

A biodegradable porous particle for the controlled biofactor delivery which assembly of pores in scaffolds can improve the permeation and diffusion of drugs or growth factors.

Objective:

Porous-spheres in millimeter scale were prepared by mixing sodium alginate and gelatin interpenetrating networks with cross-linkers; interconnected open pores were fabricated through solvent casting and particulate leaching.

Methods:

Morphological characteristics, degradation, and bovine serum albumin (BSA) release rates of the porous-spheres immersed in three different solutions, namely, deionized distilled water, simulated body fluid (SBF), and phosphate-buffered saline (PBS), were detected.

Results:

Porous-spheres with a large amount of gelatin exhibited an increase in water absorption rates without affecting scaffold strength and no cytotoxicity was elicited. Highly interconnected pores with a diameter of 100–200 µm were uniformly distributed in scaffolds. The weight loss in PBS was faster than that in other solutions; the highest release rate of BSA in SBF was observed for 2 h. The release rates also exhibited linear patterns from 2 h to 24 h in all of the groups.

Conclusions:

After 1 d of immersion in solutions, BSA release rates in scaffolds logarithmically decreased for 14 d. The degradation of porous-spheres also showed an inverse pattern.

Keywords

Sodium alginate gelatin porous-sphere solvent casting/particulate leaching cross-linker

1. Introduction

A temporary scaffold structures play an important role in cell seeding before implantation and following tissue reconstruction after implantation, as well as in tissue engineering and regenerative medicine. However, the effectiveness of scaffolds is limited by difficulty of nutrients and other essential molecules to diffuse through intricate porous networks of three-dimensional (3D) scaffolds [1,2]. Combined carriers used to attach biofactors into hydrogel scaffolds of polymer-based biodegradable porous-spheres can be used to improve this problem and can be suitable for medical and biomedical applications [3–6]. Biodegradable polymers, such as sodium alginate, gelatin, and chitosan, have been used in tissue engineering and regenerative medicine [2,7–9]. Gelation and cross-linking of sodium alginate are mainly achieved through the exchange of sodium ions from guluronic acids with divalent cations, such as Ca²⁺, Sr²⁺, or Ba²⁺ and the hardened network of calcium alginate is formed [10–12].

Gelatin extracted from animal skins is classified as denatured collagen with a triple helical superstructure of extended polypeptide chains [8,9]. Gelatin contains free carboxyl and amino groups on the backbone, and these groups cause gelatin to carry a positive charge in acidic solutions [9]. Gelatin also contains integrin binding sites for cell adhesion and differentiation; with these properties, gelatin is suitable for tissue engineering applications [13,14]. In gelation, many cross-linking agents have been used, such as glutaraldehyde, hexamethylene diisocyanate, carbodiimide, and acyl azides. Although these agents enhance mechanical properties of gelatin based scaffolds and also improve their water resistance [15–17], but the different agents would lead to the concerning problems associated to each reticulation method, such as cross-linking agent toxicity.

In this study, interconnected open pores in porous-spheres in millimeter scale were fabricated through solvent casting and particulate leaching [18,19] and following investigated the release protein of bovine serum albumin (BSA) porous-spheres prepared by mixing sodium alginate and gelatin interpenetrating networks by using cross-linkers.

2. Materials and methods

2.1. Materials

Alginic acid sodium salt (A2158), gelatin (Type B, G9382, molecular weight of 1,420,000 g/mole) extracted from bovine skin, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (E1769, EDAC) as one of the cross-linkers, and albumin from bovine serum (A9418, BSA) were purchased from Sigma-Aldrich, Poole, UK. Saccharose (Katayama Chemical Industries Co., Ltd.) as pore-forming agent with a size of 88 µm to 210 µm was ground by hand and sieved to control particle size. Anhydrous calcium chloride was used as another cross-linker (Panreac Quimica S.L.U).

2.2. Sample preparation

Porous-spheres were fabricated through solvent casting and particulate leaching; preparation procedures were based on previously described methods [18,19]. Colloidal suspension was prepared by mixing sodium alginate and gelatin at mass ratios of 1:2 and 1:4 with 10 ml of deionized distilled water (18.2 Ω d.d. water). The colloidal suspension was also mixed with saccharose particles (colloidal/particles = 1 ml/4 g) to form a particle-based colloid. The particle-based colloidal solution was added dropwise to the cross-linking solution; the solution was then mixed with 1% EDAC and 0.5% anhydrous calcium chloride to fabricate spherical porous-spheres at 4°C for 24 h. The colloidal porous-spheres were immersed in 1% EDAC solution at 4°C for 24 h to complete cross-linking. The colloidal porous-spheres were then immersed in d.d. water for 3 h at 26°C to leach the particles and dried in a vacuum through lyophilization for 3 d; in this process, pressure was reduced to <20 µHg (26 µbar).

2.3. Water absorption rate

Water absorption rate was calculated using the following equation: $\begin{matrix} Water absorption rate = (W_{w} - W_{d}) / W_{d} . \end{matrix}$

The water absorption rates of the porous-spheres were determined by immersing the porous-spheres in d.d. water at 37°C for 5, 10, 15, and 30 min and for 1, 2, and 24 h. After the residual solution was rinsed from the surface with a wiper, the weights of hydrated specimens ( $W_{w}$ ) were obtained immediately and compared with the weights of dried porous-spheres ( $W_{d}$ ). The weights of three duplicate specimens were measured at each time point ( $n = 3$ ).

2.4. Loading of porous-spheres with BSA

The porous-spheres were immersed in 0.1% BSA solution to carry out protein loading at 4°C for 24 h, frozen at −20°C for 2 h, and dried in a vacuum through lyophilization to fabricate porous-spheres with BSA.

2.5. Morphological observation

Dried and wetted porous-spheres were observed using an optical micro-scope (L803, Homa, Taiwan) at 37°C after 24 h of immersion; cross-sectional morphology was examined using a scanning electron micro-scope (SEM; S-3000 N, Hitachi, Japan) to investigate the pore distribution and morphological characteristics of porous-spheres.

2.6. Fourier transforms infrared spectroscopy (FTIR) analysis

The surface and internal structures of the porous-spheres were evaluated through attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR) (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA). Analysis was operated in a range of 650 cm⁻¹ to 4,000 cm⁻¹ to estimate functional groups.

2.7. Residual amount of amino groups and crosslinking index

Ninhydin (2,2-dihydroxy-1,3-indanedione) reagent (Sigma Aldrich, St Louis, MO) was used to investigate the residual amino groups of the BSA loaded alginate-gelatin porous-spheres before and after cross-linked with EDAC and calcium chloride. Ninhydin is a well-known reagent due to its ability to reacts with amines and amino acids for the colorimetric assay. The same composed contribution of alginic acid sodium salt and gelatin without crosslinking was measured as the control group. The groups without and with BSA loaded porous-spheres were dissolved in d.d. water at 37°C for 1 h. Those samples were placed in a boiling water bath for 10 min. After the sample solution cooling to room temperature, draw 200 µl sample solution mixed with 1.0 ml 95% ethanol. These samples were read using an enzyme-linked immunosorbent assay (ELISA) macro-plate reader (EZ Read 400, Biochrom, Cambridge, UK) and the optical density (OD) was measured at a wavelength of 570 nm. A standard curve derived from glycine (Sigma Aldrich, St Louis, MO). In addition to the residual amount of amino groups, we also calculated the different crosslinking index of the porous-spheres. $\begin{matrix} The crosslinking index = ({OD}_{control group} - {OD}_{experimental group}) / {OD}_{control group} \end{matrix}$

2.8. Degradation of porous-spheres

The degradation characteristics of the porous-spheres were investigated in d.d. water, Hank’s physiological solution of simulated body fluid (SBF), and phosphate-buffered saline (PBS). Macro-sphere samples were placed in Eppendorf tubes, and immersion solutions of d.d. water, SBF, and PBS were added. The immersed samples were maintained at 37°C in a water bath for 1 d. After the samples were immersed, the surface moisture of hydrated porous-spheres was removed with wipers; initial weights ( $W_{0}$ ) were recorded. The samples obtained from the immersion solution were rinsed daily; the residual solution from the surface was removed with wipers, and their weights ( $W_{1}$ ) from 2 d to 17 d ( $n = 3$ ) were recorded. The residual rate in immersion solutions of the porous-spheres was calculated using the following equation: $\begin{matrix} Residual rate = 100 % - [(W_{0} - W_{1}) / W_{0}] . \end{matrix}$

2.9. Release study

The release rates of the porous-spheres with BSA were investigated in d.d. water, SBF, and PBS. BSA-loaded porous-spheres weighing 0.01 g were suspended in Eppendorf tubes with 1 ml of immersion solution. The release rates of BSA for 1, 2, 4, and 8 h and for 1, 2, 4, 8, and 14 d in a water bath at 37°C were determined. Triplicate vials were used at each time point ( $n = 3$ ). The immersion solutions were withdrawn at each time interval; the amount of BSA released from porous-spheres was evaluated using a BCA™ protein assay kit (Prod#23225; Thermo Fisher Scientific, Rockford, IL, USA) at an optical density of a wavelength of 570 nm in an enzyme-linked immunosorbent assay (ELISA) macro-plate reader (EZ Read 400, Biochrom, Cambridge, UK).

2.10. Cell viability

In accordance with ISO 10993-5:2009, the cell viability of the porous-spheres was determined by using the extracts of the porous-spheres to culture of fibroblast cells (NIH3T3) ( $n = 6$ ). The cell line of NIH-3T3 was provided by the National Institute of Health (NIH) in Taiwan. NIH-3T3 was derived from newborn mouse fibroblasts and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Taiwan Ltd., MD) containing 10% bovine serum (BS) (Biological Industries, Haemek, Israel). The cells were cultured in a humidified atmosphere in a 5% CO₂ incubator. The culture medium was replaced every 2–3 days. The porous-spheres were sterilized by gamma-ray of 15 kGy (China Biotech Co., Taiwan). The extract was prepared at a ratio of 1:10 (1 g sample/10 ml medium), in which the porous-spheres were immersed in the culture medium for 24 h.

The cells were seeded in 96-well culture plate at a density of 1 × 10⁴ cells per well in DMEM culture medium and allowed to attach for overnight. The culture medium was removed and added the extract to culture for 24 h. After 24 h of culture, the cell viability of the porous-spheres on fibroblast cells (NIH-3T3) was determined by using a commercially available tetrazolium salt (XTT assay, Biological Industries, Israel). The extract was removed and added 50 µl of the XTT reaction solution (sodium30-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate and N-methyl dibenzo pyrazine methyl sulfate, 50:1) was added to the wells. After the plates were incubated for 4 h with XTT (37 1C and 5% CO₂ + 95% air), the optical density was obtained at a wavelength of 490 nm in an ELISA plate reader.

2.11. Statistical analysis

Water absorption rate was used T-test to evaluate statistical significance. One-way ANOVA and Tukey’s HSD was used to evaluate the statistical significance of the residual amount of amino groups, releasing amount of BSA and cell viability. The results were considered to be statistically significance level of $p < 0.05$ .

3. Results and discussion

3.1. Water absorption rate

The same patterns showed that a high gelatin content exhibited high adaptability to water absorption rate by varying the ratios between sodium alginate and gelatin at 1:2 and 1:4 (Fig. 1). At all of the time points, the water absorption ability of the porous-spheres with a high content of mixed gelatin was significantly greater than that of the porous-spheres with low gelatin content. After the porous-spheres immersed in the solution for 5 min, the porous-spheres became saturated with the solution; water absorption rate reached a maximum value and maintained a plateau level for 24 h.

Fig. 1.

Water absorption ability of porous-spheres prepared by mixing sodium alginate and gelatin at ratios of 1:2 and 1:4. Symbols * indicate each group after T-test statistical analysis are shown to be significantly different ( $p < 0.05$ ).

The water absorption rate of the porous-spheres is an important property of drug delivery systems because this parameter significantly affects drug release behavior. As alginate binds to calcium ions in a planar two-dimensional manner [20], excellent water uptake ability occurs; however, extended 3D cross-linking likely decreases water uptake [21]. Although the porous-spheres prepared by mixing sodium alginate and gelatin at a ratio of 1:2 contained more alginate in raw materials, the 3D spherical structure possibly limits water uptake. Carboxyl groups are partially ionized and electrostatic repulsion increases the degree of swelling under neutral conditions. Previous studies also showed that 100 g of gelatin contains approximately 100, 75, and 50 mEq of hydroxyl, carboxyl, and amino groups, respectively [22–24]. This finding shows that water permeation ability of porous-spheres depends on the amount of gelatin. Therefore, the porous-spheres prepared by mixing sodium alginate and gelatin at a ratio of 1:4 exhibited more efficient water absorption ability than those with a mixture of sodium alginate and gelatin at a ratio of 1:2. Sodium alginate-to-gelatin ratios of 1:4 were selective to the group subjected to subsequent measurements.

3.2. Morphological observation

The morphological characteristics of dried and wetted porous-spheres were investigated using digital photos; these characteristics were also observed through optical macro-scopy and SEM (Fig. 2). The porous-sphere preparation through a controlled process and the dried spherical particles showed the average diameter was around 2.17 mm and the standard deviation was 0.08 mm ( $n = 6$ ). After the porous-spheres were completely saturated with water, the diameter of the wetted state ranged from 2.5 mm to 3 mm; wetted porous-spheres showed no significant swelling with a more spherical shape than dried porous-spheres [Fig. 2(a) and (b)]. The phenomenon demonstrated a non-physical interlocking of networks among molecules. Chemical intermolecular bonding also occurs in scaffolds. Porous-spheres with highly interconnected pores are shown in Fig. 2(c). Many round pores were observed in porous-spheres with BSA. The wetted porous-spheres were dried again through lyophilization after BSA was loaded. Many pores in contact showed that the macro-sphere structure was strong after BSA loading processes, including immersion and lyophilization, were repeatedly performed. The cross-section images of porous-spheres without BSA (without BSA) and with BSA (with BSA) indicate that inner hollowed pores with diameters of 100 µm to 200 µm contained highly interconnected pores uniformly distributed in the porous-spheres (Fig. 2(d)). Solvent casting and particulate leaching were performed to develop porous-spheres in this study. The strategy was used to control particle size during particulate leaching to achieve desired homogeneous and highly interconnected pore sizes in 3D scaffold structures. This type of structure may be beneficial for drug diffusion because a highly porous structure can generate capillary forces; with these forces, fluids can penetrate porous-spheres to cause drugs to easily diffuse in water [25,26].

Fig. 2.

Images of porous-spheres. Digital photographs (a) in the dry state and (b) in the wet state. (c) Optical micro-scope images of microspheres without and with BSA. (d) SEM cross-section images of porous-spheres without and with BSA.

3.3. FTIR analysis of the porous-spheres

The FTIR spectra of alginate showed two characteristic absorption bands at 1,602 cm⁻¹ to 1,627 cm⁻¹ and 1,415 cm⁻¹ corresponding to the −COO group [27] and one absorption band at 1,031 cm⁻¹ corresponding to the C–O–C group (Fig. 3(a)). The absorption bands of gelatin were detected at 1,644, 1,552, and 1,241 cm⁻¹ corresponding to amide I (C=O and C–N), amide II, and amide III (main presentation of N–H bending vibration and C–N), respectively [28]. The wide absorption band at approximately 3,463 cm⁻¹ corresponds to the OH group [29].

Fig. 3.

(a) FTIR spectra of raw materials and porous-spheres without and with BSA. (b) ATR-FTIR spectra of external and internal bindings of porous-spheres without and with BSA.

Fig. 3.

(Continued.)

Fig. 4.

Residual amount of amino groups in alginate-gelatin before/after crosslinking and the porous-spheres loading with BSA. Symbols * indicate each group after one-way ANOVA statistical analysis are shown to be significantly different ( $p < 0.05$ ).

Spectra of the porous-spheres showed that the absorption bands of gelatin at 1,644 cm⁻¹ as amide I and the absorption bands of alginate at 1,031 cm⁻¹ corresponding to the C–O–C group shifted to lower wavenumber at 1,625 and 1,029 cm⁻¹, respectively (Fig. 3(a)). The absorption bands of gelatin at 3,463 cm⁻¹ corresponding to the OH group also shifted to a lower wavenumber at 3,291 cm⁻¹ because the stretching vibration of the N–H group bonded to the OH group; this result indicates an increase in hydrogen bonding [27,29]. All of these changes provided strong evidence of intermolecular interactions and good molecular compatibility between alginate and gelatin (Fig. 3(a) and (b)).

3.4. Residual amount of amino groups and crosslinking index

The residual amounts of amino groups in the porous-spheres composed of alginate-gelatin before and after crosslinked with crosslinking solution and furthermore the BSA loaded porous-spheres were shown in Fig. 4. The residual amount of amino groups in the porous-spheres before crosslinking (1.536 mM ± 0.028 mM) was significantly higher than the porous-spheres (0.367 mM ± 0.006 mM). The detection of amino groups in the BSA loaded macro-sphere (0.837 mM ± 0.012 mM) was significantly higher than in the macro-sphere without loading BSA. The measured crosslinking index in the group of porous-spheres after crosslinking was up to the value of 89.27% ± 0.47%. It showed the alginate-gelatin porous-spheres prepared by the crosslinking solution of EDAC and anhydrous calcium chloride could afford a high degree of polymerization while the integrity of the polymersomes was preserved.

3.5. Degradation of porous-spheres

The degradation characteristics of the porous-spheres were compared; our results showed that these characteristics differed in the groups immersed in different solutions of d.d. water, SBF, and PBS (Fig. 5). The residual rates of the porous-spheres exhibited linear patterns in different immersion solutions. The weight loss of the porous-spheres immersed in PBS was also faster than that of the porous-spheres immersed in SBF and d.d. water (Fig. 5).

Fig. 5.

Residual rates of porous-spheres immersed in d.d. water (blue), SBF (orange), and PBS (green).

Ionic compositions in solution greatly affect the strength and degradation behavior of porous-spheres on structures. In this study, 3D structures are initially obtained through complexion of alginate with calcium (Ca²⁺) ions [30,31]. The gel in gelatin is trapped and entangled in a structure as the mixture cools [32]. Therefore, the degradation of Ca²⁺-cross-linked alginate gel can occur when Ca²⁺ ions are removed [10]. This process can be accomplished by utilizing chelating agents, such as ethylene glycol-bis(β-aminoethyl ether)-N,N, $N^{'}$ , $N^{'}$ -tetraacetic acid, lactate, citrate, and phosphate, or high concentrations of ions, such as Na⁺ or Mg²⁺ [33]. If phosphate is used as a dissolution medium, the dissolution medium becomes turbid because Ca²⁺ dissociates from a polymer network and phosphate ions interact with Ca²⁺ to form calcium phosphate [10,33].

Compared with the SBF solution, PBS solution contained high PO₄ ³⁻ concentration even when Na⁺ concentration of PBS was consistent with that of SBF. Ca²⁺ ions in SBF inhibit the degradation occurring in alginate gel [10]; thus, the weight loss of the porous-spheres in PBS was faster than that of porous-spheres in SBF and d.d. water.

3.6. Release rate of the porous-spheres

Different immersion solutions of d.d. water, SBF, and PBS were used to investigate the release behavior of BSA in porous-spheres. The BSA releasing percentage in porous-spheres was showed similar pattern in different immersion solution. The BSA releasing percentage of in porous-spheres immersed in SBF was faster than in PBS from 8 to 24 h and both reached recorded level of 50% releasing. The releasing percentage of BSA loaded porous-spheres in SBF and PBS showed a consistent trend (Fig. 6(a)) after 1 d immersion. A high release amount was observed after the porous-spheres were immersed in solutions from 1 h to 2 h in all of the cases (Fig. 6(b)). The release amounts exhibited linear patterns and sustained release in all immersion solutions from 2 h to 24 h. The release amount in PBS was faster than that in other immersion solutions. Water absorption occurs because wetted porous-spheres likely absorb water through permeation in void regions of a polymer network until equilibrium is reached [34]. Therefore, protein release is induced when a polymer network undergoes relaxation because of osmotic pressure [35]. The increase in salt concentration is related to significant differences in osmotic pressure between inner and outer spheres because PBS contains ions; thus, the release of BSA from porous-spheres is accelerated. The BSA release amount of spheres in the PBS immersion solution was also higher than that in SBF and d.d. water immersion solutions in the early stage of BSA release (Fig. 6). After 1 d of immersion in solution, BSA release amounts logarithmically decreased from 2 d to 14 d; porous-spheres were consistently degraded.

Fig. 6.

(a) Release percentage and (b) releasing rates of porous-spheres with BSA immersed in d.d. water (blue), SBF (orange), and PBS (green). Symbols * indicate each group after one-way ANOVA statistical analysis are shown to be significantly different ( $p < 0.05$ ).

The fundamental principle for evaluation of the kinetics of protein or drug release was shown as the equation: $\begin{matrix} d M / d t = KS (C_{s} - C_{t}) \end{matrix}$ where, M is the mass transferred verusu time t; S is the surface of matrix formulation and $C_{s} - C_{t}$ is the driving force of concentration gradient.

On the basis of this principle, a wide range of mathematical models for depicting the diffusion of proteins in the hydrogel matrix have been fully developed over the past few years. However, the releasing rate of a protein through the physically crosslinked hydrogel can be affect by the crosslinked density, the volume fraction of water within the gel and the size of the protein. At the present study, the crosslinked density is the deciding factor for controlling the swelling behavior. Therefore, within the 1st h of initial release, the releasing rate of the three solutions is very slant, representing the release was very fast because the first paragraph is by the protein transport and the diffusion effect. The hydrogel-spheres immersed in the PBS and SBF solutions should be considered the ion concentration in the soaking environment and thus the release slowly reached equilibrium through osmotic pressure at the 2nd h. Subsequent through the ion exchange, the hydrogel-spheres produce different degrees of degradation, so it also affected the release rate.

3.7. Cell viability of the porous-spheres

After the cells were exposed to the extract for 24 h, 100% cell viability of the group in the medium with the reagent control was observed (Fig. 7(a)). After the cells were incubated for 24 h, the expressions of the extracted medium of the negative control group showed no changes in cell morphological characteristics, no cell lysis, and no reduction in cell growth. No changes in cell morphological characteristics were also observed in the experimental group incubated in the extract from the porous-spheres (Fig. 7(b)); cell viability showed that cell growth was slightly inhibited during proliferation (Fig. 7(a)). Quantitative and qualitative results further revealed that the extract from porous-spheres did not elicit cytotoxic effects.

Fig. 7.

Cell viability of porous-spheres: (a) quantitative and (b) qualitative results. Symbols * indicate each group after one-way ANOVA statistical analysis are shown to be significantly different ( $p < 0.05$ ).

4. Conclusions

Cross-linked sodium alginate and gelatin porous-spheres were fabricated through solvent casting and particulate leaching are a rapid and efficient strategy to prepare highly ordered uniform pores in porous-spheres. These porous-spheres also contain highly interconnected pores, exhibit enhanced water absorption ability, and yield appropriate degradation and protein release rates. Therefore, porous-spheres can be used as a carrier of specific proteins because these porous-spheres exhibit good molecular compatibility; these porous-spheres also elicit no cytotoxic effects and can be further embedded in other biomaterial matrixes as drug delivery systems.

Footnotes

Acknowledgements

The authors acknowledge the financial support provided by the Ministry of Science and Technology, Taiwan (Grant Nos. MOST 106-2622-E-035-002-CC2 and MOST 103-2221-E-035-099) and by the Southern Taiwan Medical Device Industry Cluster (CZ-02-02-02-105).

Conflict of interest

The authors declare that they have no conflict of interest.

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