Development of a Microscale Red Blood Cell-Shaped Pectin-Oligochitosan Hydrogel System Using an Electrospray-Vibration Method: Preparation and Characterization

Introduction

Hydrogel microcapsule systems have been widely applied in the biological fields including drug delivery, cell immobilization/encapsulation, and tissue engineering (1, 2). Pectin is one of the most commonly used macromolecules for hydrogel capsule fabrication. Pectin is a naturally occurring biopolymer derived from citrus fruit and apple peels, composed of galacturonic acid with carboxyl groups, and shows an excellent biodegradable and biocompatible nature (3-4-5). Pectin is stable under low pH conditions and resists proteases and amylases; however, it can be easily digested by colonic enzymes such as pectinase. This makes pectin an ideal drug carrier for controlled drug delivery to the lower gastrointestinal (GI) tract. Moreover, pectin has also shown great potential for tissue engineering applications, such as bone tissue engineering (5, 6). Depending on the degree of methyl esterification, pectin can be classified as low methyl (LM) pectin and high methyl (HM) pectin. LM pectin has been shown to form hydrogels in the presence of divalent cations, such as Ca²⁺ and Ba²⁺, at a lower critical concentration than that of HM pectin (4, 7-8-9). Our previous study (10) discovered that oligochitosan, functioning as a non-ionic cross-linker, could also gel the pectin to form red blood cell-shaped hydrogel capsules.

The red blood cell-like shape offers significant advantages over the conventional spherical shape, toward biological applications. In particular, the biconcave shape provides a favorable surface area-to-volume ratio and allows marked deformations compared with spheres (11, 12). With the larger surface area-to-volume ratio, it is well known that the diffusion rate across the capsule membrane needs to be adequate to support the encapsulated cell growth, which is critical for both cell encapsulation and tissue engineering applications (13, 14). Currently, the inorganic red blood cell-like capsules have been used for drug delivery and biomedical imaging (12, 15).

Low molecular weight chitosan, especially oligochitosan, is becoming a significant material for a variety of biological applications - mainly owing to its improved solubility, biocompatibility, and mechanical properties (16-17-18). It is well studied that divalent cations may cause an immune response and is potentially toxic in applications such as cellular encapsulation (10, 19-20-21-22-23). Use of oligochitosan in the formation of hydrogels increases its biocompatibility with the elimination of the divalent cations.

There are several ways to fabricate hydrogel spheres/capsules at the microscale, which include air-jet encapsulation, electrostatic spray, laminar jet breakup, and microfluidic channel/nozzle methods (13). Among these methods, the electrostatic spray method is appealing due to its ease of operation, high efficiency, and allowance for a sterile preparation environment (13, 24), especially the combination of the electrostatic spray method with a vibration technique provides an alternative way for microsphere/capsule fabrication, which allows for the fabrication operation to be conducted at a relatively low voltage conditions. The preparation of macroscale pectin-oligochitosan microcapsules using the simple extrusion process was reported in our previous study (10). The microscale pectin-oligochitosan capsules show other potential applications for multiple biological applications, such as creating artificial red blood cells. Thus, the goal of this work is to explore the possibility of fabricating microscale pectin-oligochitosan hydrogel capsules by using the vibration/electrostatic spray method and to further characterize them, especially those characteristics related to their potential biological applications: controlled bioactive agent delivery, three-dimensional (3D) cell culture, regenerative medicine and tissue engineering, as well as creating artificial red blood cells/blood substitutes. In particular, the designed pectin-oligochitosan microcapsule system exhibited a dual-responsiveness, responsive to both high pH and colonic enzymes. To our knowledge, this is the first study to test the encapsulation of cells in this microscale microcapsule system.

Materials and Methods

Preparation of biconcave hydrogel microcapsules

Pectin and oligochitosan solutions were freshly prepared. In brief, 3% (w/v) pectin solution (dissolved in deionized [DI] water by gentle stirring) was extruded into the 5% (w/v) oligochitosan solution (dissolved in DI water by gentle stirring; used as the gelation bath) under constant stir (at a speed of 300 rpm) at room temperature by using an encapsulator device (B-390, BÜCHI® Labortechnik AG). The hydrogel microcapsules formed immediately once the micro-scale droplets reached the gelation solution. The device was used to generate the micro-scale-sized droplets (electrospray-vibration method), which is based on the principle that a laminar flowing liquid jet breaks up into equally sized droplets by superimposed vibration, while the electrostatic repulsion forces (produced by the imposed electrical field) disperse the droplets into a gelation bath (Fig. 1). The following parameter settings were used: voltage of 2000 V; vibration frequency of 2000 Hz; air pressure of 600 mbar; flow rate of 0.2 mL/s; and nozzle diameter of 150 µm. After approximately 5 min, the suspension was set still, allowing the microcapsules to sink to the bottom, then the microcapsules were collected by removing the supernatant. The collected hydrogel microcapsules were stored at 4°C for further analysis.

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Fig. 1

A schematic illustration of the procedure/mechanism for preparing micro-scale pectin/oligochitosan hydrogel capsules. Arrows indicate the concave surface of the capsules. Scar bar: 1000 µm.

Results and discussion

Morphological and chemical analysis of the hydrogel microcapsules

A typical image of the hydrogel microcapsule is shown in Figure 2A. The biconcave microcapsule morphology is easily noticeable and the size is uniform (diameter: 354 ± 6.3 µm; thickness: 101 ± 22.0 µm). To further examine the hydrogel morphology, a high definition scanning electron microscopy (SEM) image of the pectin/oligochitosan hydrogel microcapsules was captured and is shown in Figure 2B. The core-shell structure has been confirmed and published in a previous study (10). As shown in the SEM image, folds could be noticed on the microcapsule surface. This is most likely caused by the collapse of the capsule shell during the drying process, when preparing the samples for SEM imaging. Fourier Transform Infrared Spectroscopy (FTIR) was used to monitor the interaction between the pectin and oligochitosan during the hydrogel microcapsule formation (Fig. 3). As expected, these interactions are demonstrated through the changes in the spectra, occurring in the 1800-1400 cm⁻¹ range. In the microcapsule spectrum, the pectin's ionized carboxylic acid (COO⁻) peak at 1580 cm⁻¹ is much weaker in intensity and can barely be resolved as a shoulder to the amide I peak at 1602 cm⁻¹. The ester carbonyl group (COO-R) peak intensity, on the contrary, significantly increased as compared to that in the pure LM pectin spectrum, while its position shifted from 1722 cm⁻¹ to 1741 cm⁻¹. These changes can be interpreted as an increase in the number of COO-R species at the expense of the ionized carboxylic acid groups as a result of their interaction with the oligochitosan amine groups (27, 28). This is further supported by the shift of the amide I peak from 1592 cm⁻¹ in pure oligochitosan to 1602 cm⁻¹ in the microcapsule spectrum (28). These changes indicate the formation of LM pectin-oligochitosan complexes in the hydrogel capsules.

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Fig. 2

Optical (A) and SEM (B) images of micro-scale pectin oligochitosan hydrogels. The arrows indicate the concave erythrocyte shape.

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Fig. 3

FTIR spectra of LM pectin, oligochitosan, and micro-scale pectin/oligochitosan hydrogels.

pH/enzyme responsiveness of the hydrogel microcapsules

To test the capability of the microcapsules for controlled drug delivery applications, the responsiveness of the hydrogel capsules to pH (synthetic body fluids) and enzyme (pectinase) was studied. The capsules remained stable within the DI water and artificial gastric fluid, while they were found to swell (remained biconcave disc-shaped before breaking) and degrade (gel erosion) in artificial intestinal fluid over time (Fig. 4). High pH conditions caused the deprotonation of the amine groups (from NH₃⁺ to NH₂) of oligochitosan molecules (pKa ∼6.5), which in turn degraded the pectin/oligochitosan cross-linking (polyelectrolyte complex) formed by the ionic interactions (29-30-31). This hydrogel microcapsule system could be used as a novel drug carrier for lower gastrointestinal (GI) tract delivery, which requires protection of drug from degradation and release in the stomach and increased drug release as the drug carrier passes down the intestinal tract, owing to an increased pH. Erosion of the polymeric gel would be considered a key mechanism when using this system for oral drug delivery to the GI tract. In addition, the pectin has multiple regions (methylesterified carboxyl groups) on it, which allow for hydrophobic molecules to interact while still maintaining the hydrophilic attributes, and this fact makes it an excellent carrier of hydrophobic drugs (9, 30, 32-33-34). Colonic microbial enzymes degrade the polysaccharide, which further facilitate the drug release in the lower GI tract (32, 33). Pectin-oligochitosan was confirmed to be sensitive to the pectinase, even at a low concentrations tested during this study (5.7 U/mL), while, at the same time, the pectin was not degraded by gastric (pepsin) or intestinal (trypsin) enzymes.

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Fig. 4

Responsiveness of the micro-scale pectin/oligochitosan hydrogel capsules. Hydrogel micro capsules were incubated in different media: (A) and (B): DI water; (C) and (D): Artificial gastric fluid; (E) and (F): pectinase solution; and (G) and (H): artificial intestinal fluid. Left panel: 0 min, and right panel: 45 min after incubation. Scar bar: 500 µm.

Cellular encapsulation

The feasibility of encapsulating and culturing bacteria within the hydrogel microcapsule system was investigated. The cells proliferated well within the microcapsule and formed cell clusters/aggregates/colonies from single cells (Fig. 5). These results indicate that the encapsulation process was not adverse to the cells, and the nutrients were able to diffuse through the hydrogel membrane to the encapsulated cells. To enhance the cell adhesion and growth, especially for mammalian cells, the Arg-Gly-Asp peptide (RGD)-modified pectin could be used (35).

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Fig. 5

Images show the encapsulated E. coli cells at different time points within the micro-scale pectin oligochitosan hydrogels: (A) 0 h and (B) 24 h after encapsulation. Scar bar: 500 µm.

Conclusion

The micro-scale pectin-oligochitosan hydrogel capsule can be prepared by using the vibration/electrostatic spray method. The hydrogel microcapsule exhibits a biconcave disc (red blood-cell) shape. Compared with the spherical shape, the biconcave disc shape provides a larger surface area-to-volume ratio (i.e. advantage of rapid biotransport of molecules), ideal for 3D cell culture. It will provide a means to overcome the insufficient biotransport of nutrients/waste, which is a major cause for failure of 3D cell culture (13). Moreover, this hydrogel capsule system can be used to develop artificial cells and/or blood substitutes by encapsulating hemoglobin into the microcapsules; this is an ongoing project for the team. With further studies of the responsiveness of pectin-oliogochitosan microcapsules, promising applications of this system in drug delivery could be foreseen, especially the delivery of bio-agents, such as probiotics, to the lower GI tract (36, 37).