Long-term durability antibacterial microcapsules with plant-derived Chinese nutgall and their applications in wound dressing

2.1 Experimental materials

SA was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chitosan (deacetylation degree 92.36%) was purchased in Jinke Biochemical Co., Ltd (Zhejiang, China). Calcium chloride (CaCl₂), glacial acetic acid (CH₃COOH, 98% in concentration), methyl alcohol (CH₃OH), glutaraldehyde (25%) and sodium hydroxide (NaOH) were obtained from Lingfeng Chemical Reagent Co., Ltd (Shanghai, China), with analytical grade. CN extract was purchased from Huilin Bio-technology Co., Ltd (Shanxi, China).

2.2 Preparation of the CN - SA/ chitosan microcapsules

CN - SA/ chitosan microcapsules were fabricated by the orifice granulation method (19). Firstly, CN and SA - chitosan solutions were prepared as follows. Solution A: CN extract was dissolved into NaOH solution, which was produced by dissolving NaOH in distilled water. Solution B: NaOH was dissolved in CH₃COOH solution and the chitosan was added into the mixture. Secondly, the solution A was dropwise added into solution B, and the system (pH=5.5) was stirred for 30 min at 45°C. Thirdly, the glutaraldehyde was added to the reaction system to fix microcapsule shape. Finally, the suspension was dried in an electrothermal blowing dryer (Shanghai Yiheng Co., Ltd, China). The optimized parameters were attained by orthogonal experiment and shown in Table 1.

2.3 CN encapsulation efficiency and stability

The ultraviolet spectrum of CN in phosphate buffered saline (PBS, pH = 7.7) (20) was measured by UV- vis spectra photometer (TU-1901, Beijing Purkinje General Instrument Co., Ltd., Beijing, China). And the maximal absorption peak was at the wavelength of 256 nm (Figure S1). Accordingly, mass fraction – absorption intensity curve of CN was obtained (Figure S2). To calculate CN encapsulation efficiency, grinded microcapsules were dissolved in CH₃OH and dispersed by ultra-sonication. After filtration, the solution absorption intensity at 256 m wavelength was recorded and CN encapsulation efficiency was calculated as the ratio of the weight of CN in microcapsules to theoretical loading CN.

Besides, the storage stability of CN - SA/ chitosan microcapsules in -20°C was also studied. At a predetermined time, CN in microcapsules was dissolved in PBS (pH = 7.7) and its absorption intensity was measured.

2.4 Physical characterization of microcapsules

The morphology of the CN loaded and unloaded microcapsules was observed by scanning electron microscopy (SEM; Jeol Ltd., Japan) at an accelerating voltage of 10 kV. Image processing software ImageJ was applied to measure the diameter of at least 50 microcapsules from at least 10 images. Fourier transform infrared (FTIR) spectra of different samples was recorded by absorption mode at 2 cm^-1 intervals in the range of 4000–600 cm^-1 wave numbers, using a FT-IR spectrophotometer (Avatar380, USA). Wide-angle X-ray diffraction (WAXD) of samples with and without CN was recorded by an X-ray diffractometer (Rigaku D/MAX 2550/PC, Japan) operating at 40 kV and 200 mA, along with Cu Kα radiation.

2.5 In vitro release

The release characteristics of CN from antimicrobial microcapsules were measured in PBS (pH = 7.7) to simulate the CN release corresponding to the inflammation phase. Samples were placed in centrifuge tubes with the release medium at 37°C. At predetermined time points, 1 mL solution of each sample was extracted to measure the absorption intensity at 256 nm wavelength. Meanwhile, an equal amount of fresh PBS was supplemented to each centrifuge tube. And the accumulative release rate (%) was calculated as the ratio of released CN at each procedural time to the amount of loaded CN.

2.6 Antibacterial activity of microcapsules

Agar diffusion plate test was applied to evaluate antibacterial activity of microcapsules qualitatively according to ISO 20645:2004 (20). Typical bacteria Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) in wounds were selected as testing organisms (20) and their sensitivity was judged by the zone of inhibition. Microcapsule circle with the diameter of 20 mm was inoculated with approximately 1 mL of bacterial suspension ((1 × 10⁸ CFU/mL) per tryptic soya agar plate. The plates were incubated at 37°C for 24 h and then measured by a Vernier caliper to determine the zone of inhibition, which was calculated as following formula (1),

where H is the zone of inhibition (mm), D is the total diameter of the specimen and zone of inhibition (mm) and d is the diameter of the specimen (mm).

Oscillation method (GB/T 20944.3-2008, China Standards) was adopted to determine the antibacterial activity of microcapsules quantitatively. Inhibition rates (%) were measured by: (control bacterial suspension (CFU/mL) - sample bacterial suspension (CFU/mL))/ control bacterial suspension (CFU/mL).

2.7 Application of microcapsules onto wound dressings and their antibacterial properties

Cotton gauze is still a standard of care in the management of chronic wounds. It has reasonable gas permeability and effective exudates absorption (21). Here, we designed a composite wound dressing based on the cotton gauze and alginates. And antimicrobial microcapsules were applied on it (Figure 1). The outer cotton gauze layers helped exudates absorption, while the inner SA hydrogel provided a barrier for water vapor permeation. Antimicrobial microcapsules were fixed and incorporated into the SA hydrogel. Three layers were then laminated and antibacterial properties were evaluated according to agar diffusion plate test in ISO 20645:2004. For each sample, three specimens were tested.

Figure 1

Composite wounding dressing.

2.8 Statistical analysis

Data reported was the means and standard deviations, and the error bars in figures corresponded to standard deviations. t-tests were used to determine the statistical difference. The data in the figures were marked by (*) for p < 0.05.

3.1 CN encapsulation efficiency and stability

The encapsulation efficiency of CN is an important index for CN loaded microcapsules and is shown in Figure 2a. SA and chitosan provide essential basis in the shell formation of microcapsules. Insufficient SA or chitosan may lead to low shell strength, or even shell breakage under foreigner loads. However, excessive SA can increase viscosity of the Solution A and then decrease CN encapsulation efficiency. CaCl₂ works as a curing agent and its lack will also cause shell softness, while rising amount can lead to thicker shell and less CN encapsulated. For the four optimized microcapsules by orthogonal method, they had encapsulation efficiencies of 80.16%, 74.21%, 70.79% and 70.11% for A₁, B₁, C₁ and D₁, respectively.

Figure 2

(a) Encapsulation efficiency of microcapsules; (b) Stability of microcapsules; (c) SEM images and average diameters of microcapsules.

Absorption intensity of CN after storage under -20°C for different intervals is shown in Figure 2b. Compared with fluctuant intensity of CN extract, CN in microcapsules kept almost stable and unchanged. It indicated that microcapsules could protect CN from degradation and destruction due to electrostatic attraction between SA and chitosan.

3.2 Physical characterization of microcapsules

Microcapsules were successfully prepared by the orifice granulation method. After adding SA solution into chitosan solution, the interface became thicker as the aggregation caused by electrostatic attraction between the negative charges on SA and the positive charges of chitosan. And the above rigid shell was able to protect core materials. Figure 2c shows the images of microcapsules with good monodispersity, together with surface details. The surface of microcapsules with CN encapsulated is porous, which allows the release of CN to the surrounding medium. Cross-section image shows obvious layers of cores and shells. Microcapsules with core materials have more pores on the surface compared with microcapsules without core materials. Besides, the roughness of microcapsules increases with the decrease of encapsulation efficiency from A₁ to D₁ (22). Besides, mean size of CN - SA/ chitosan microcapsules are larger than microcapsules without CN included.

Encapsulation of CN is qualitatively confirmed by FTIR spectroscopy in Figure 3a. In the FTIR spectrum of the pure CN, the peaks at 3457 cm^-1, 3153 cm^-1, 1722 cm^-1 and 1617 cm^-1 are attributed to stretching vibration of O-H, C-H, C=O and -C=C-, respectively. Peaks at 1326 cm^-1, 1111 cm^-1, 1039 cm^-1 and 757 cm^-1 are due to bending vibration of -CH₃, C-O-C, C-N and ⌬, respectively. Compared with pure CN, the FTIR spectrum of the CN loaded microcapsules has peaks at 1326 cm^-1, 1111 cm^-1, 1039 cm^-1 and 757 cm^-1, which derive from the CN. Microcapsules without CN did not show any characteristic peaks of CN. According to the results of FTIR spectra analysis, CN loaded microcapsules were prepared successfully.

Figure 3

(a) FTIR spectroscopy of microcapsules; (b) XRD spectrum of microcapsules.

Figure 3b illustrates the changes in the intensity, height and weight of the reflection peak in CN extract and microcapsules. Sharp diffraction peak around 20° indicates high crystallinity and larger crystal size insideCN. Peaks around 20° in microcapsules confirmed the encapsulation of CN, while shorter ones indicated SA and chitosan in shells affected the original structure of CN. On one hand, SA and chitosan interacted with each other by electrostatic attraction. On the other hand, carboxyl of SA and amidogen of chitosan can bond with phenolic hydroxyl groups in CN, furtherly intervening with its crystal structures.

3.3 In vitro release

The release behaviors of CN from the microcapsules were investigated at pH = 7.7. Figure 4a gives a distinct picture of the release rate of CN. It was seen that the release of CN demonstrated a continuous and comparatively even process over the study period. The initial “burst release” was around 20% for B₁, C₁ and D₁, and 35% for A₁, which was very weak, during the first 12 h. It was primarily attributed to the protection of CN by shells. CN sustaining release reached the release proportions around 80% after 60 h. It is the basis of long-term durability of microcapsule antibacterial activity.

Figure 4

(a) CN release of microcapsules along with the time; (b) Microcapsule qualitative antibacterial activity; (c) Microcapsule quantitative antibacterial activity.

Acute wound healing process can be divided into four periods, among which inflammation phase is more likely to cause microorganism growth (23). In this period, the skin pH increases from 5-6 to 7 or more (24). In acid medium, the COO− groups in alginates transform into COOH groups. On the one hand, the hydrogen-bonding interaction among COOH groups is strengthened and the additional physical crosslinking is generated; on the other hand, the electrostatic repulsion among COO− group is restricted, and so the alginate shell tends to shrink. CN release from the core decreases (25). But partial chitosan degrades to defend microbes at the first stage. However, in inflammation stage, the skin pH increases in wounds and the ratio of COO− groups in polymer network increase. Consequently, the hydrogen-bonding interaction is broken and the electrostatic repulsion among negatively charged polymer chains increases, and so the polymer network tends to swell. Degraded chitosan also leads to more pores for CN release to kill the bacteria.

3.4 Microcapsule antibacterial activity

Antibacterial activity of microcapsules with CN loaded and unloaded against S. aureus and E. coli was investigated. The results (Figure 4b) revealed that CN-loaded microcapsules had strong antibacterial effects, with S. aureus inhibition zones of 7.67 mm and E. coli inhibition zones of 5.27 mm after incubation for 24 h. Microcapsules without CN encapsulation also illustrated antimicrobial property, with S. aureus inhibition zones of 3.25 mm and E. coli inhibition zones of 1.33 mm, which were the contributions of chitosan. In addition, it was observed that the antibacterial effect against E. coli was slightly weak compared to S. aureus. Quantitative evaluations (Figure 4c) also illustrated higher inhibition rates against S. aureus. Besides, antibacterial performance of microcapsules increased with the CN encapsulation rates, while microcapsules without CN performed similar inhibition rates. It confirmed the biocidal functions of CN. The inhibition rate could research 98.99% against S. aureus after 12 h and 100% after 24 h (Table S1) for microcapsule (A₁) loading concentration of 4.5 mg/mL. It remained 99.61% against E. coli after 6 h and kept 100% after 12 h (Table S2) for 5.0 mg/mL, demonstrating a long-term antibacterial performance.

So, at the early stage of wound healing, chitosan in microcapsule shells plays the role to defend bacteria and help hemostatic. At inflammation stage, alginate networks swell with the increase of skin pH and partial chitosan degrades with pores left. Thus, more CN in the core release to defend microbe.

3.5 Wound dressing antibacterial properties

Antimicrobial performance of composite wound dressings was investigated and shown in Figure 5. CN loaded dressings have S. aureus inhibition zones of 4.82 mm and E. coli inhibition zones of 4.11 mm, while dressings without CN showed S. aureus inhibition zones

Figure 5

Wound dressing antibacterial properties.

of 2.57 mm and E. coli inhibition zones of 2.15 mm. Because of indirect CN contact with bacteria, wound dressings have inferior antimicrobials compared with microcapsules. According to ISO 20645:2004, they still showed good antibacterial effect. Moreover, cotton gauze on the outer layer works as wicking of excessive exudation. Alginates in the middle can fix antibacterial microcapsules, as well as provide a barrier for water vapor permeation by forming hydrogels through ionic exchange. The whole system works cooperatively to help wound healing.