Original Article Study on Encapsulation of Chlorine Dioxide in Gelatin Microsphere for Reducing Release Rate

Int J Clin Exp Med 2015;8(8):12404-12410 www.ijcem.com /ISSN:1940-5901/IJCEM0008189

Original Article Study on encapsulation of chlorine dioxide in gelatin microsphere for reducing release rate

Ying Ci1, Lin Wang1, Yanchuan Guo2, Ruixue Sun3, Xijie Wang2, Jinyou Li1

1Chinese Academy of Inspection and Quarantine, Beijing 100176, China; 2Technical Institute of Physics and Chemistry, CAS, Beijing 100190, China; 3Nan Yang Normal University, Nanyang 473061, Henan, China Received March 18, 2015; Accepted July 1, 2015; Epub August 15, 2015; Published August 30, 2015

Abstract: Objective: This study aims to explore the effects of encapsulation of chlorine dioxide in a hydrophilic biodegradable polymer gelatin to reduce its release rate. Methods: An emulsification-coacervation method was adopted. The characterizations of chlorine dioxide-gelatin microspheres were described. Using UV-vis spectrophotometer the

λmax of chlorine dioxide was observed at 358 nm. The particle size and distribution of chlorine oxide-gelatin microspheres was measured by a dynamic light scattering (DLS) method, the diameter was (1400~1900) nm. The entrapment of chlorine dioxide-gelatin microspheres was confirmed by IR. The surface morphology, size, and shape of chlorine dioxide-gelatin microspheres were analyzed using Scanning electron microscope (SEM). Results: It showed that the encapsulated microspheres size was around 2000 nm with uniform distribution. The percentage entrapment of chlorine dioxide in the encapsulated samples was about 80~85%. A slow release study of chlorine dioxide from the encapsulated biopolymer (gelatin) in air was also carried out, which showed continuous release up to ten days. Conclusions: It can be concluded that it is possible to make a slow release formulation of ClO2 by entrapped in a hydrophilic biodegradable polymer gelatin. ClO2-gelatin microspheres can stable release low concentration ClO2 gas over an extended period.

Keywords: Chlorine dioxide, gelatin, encapsulation, microsphere, release rate

Introduction sterilization, deodorization and purification; refrigerators or freezers sterilization and food Chlorine dioxide (ClO ) is a broad-spectrum 2 preservation; household anti-mildew and textile bactericide with many advantages of good bleaching. Therefore, it is important to develop antibacterial activity [1-5], such as wide appli- a sustained-release ClO -loaded solid agent, cation of pH, low toxicity to human body, and 2 with the capacity to stably release low concen- lower production of potential carcinogen [6, 7]. tration of ClO gas for a long-term. It is widely used in the fields of drinking water 2 treatment, industrial cooling water treatment, Natural hydropolymers are perhaps the best wastewater treatment [8, 9], air deodorization choice available as the encapsulating material. and sterilization [10], food antiseptic and pres- Gelatin is a nature polymer that is biodegrad- ervation [11, 12]. ClO is a yellow-green gas 2 able and biocompatible. Gelatin microspheres under standard condition, it is unstable in (GMs) have been known to display a variety of water and even explosive by inter-action with applications in different technological fields light [13]. When a large number of ClO2 is used in industry, it is often produced on-site or sta- including medicine, biology, chemical and elec- ble ClO solution with stabilizers [14] (such as tronic information industry because of their 2 diverse and excellent physical, chemical, bio- carbonate or borate). By mixing the stable ClO2 solution and activator (such as acid), the gas of logical and amphoteric properties [15]. In this study, ClO has been encapsulated using gela- ClO2 will be produced. However, this character- 2 istic makes it not suitable for small number of tin. ClO2-containing gelatin microspheres were applications, for instance, in treatment of water prepared by an emulsification-coacervation and wastewater in small factories; small or method, which provide gradual and controlled household water tank disinfection; indoor air release of ClO2 gas. Slow release formulation of ClO2

continuous stirring for 20 min. Then, this solution was slowly poured into 500 mL Soybean oil, and emul- sified by shear emulsifier (800 rpm) for 30 min. Span-80 was used as the emulsifier for the system. The whole reaction kept at 45°C. The solution was cooled with an ice bath for 40 min, and then was added 250 mL of acetone.

The ClO2-containing gelatin microspheres were sepa- rated by dehydrated 1 h, filtered under vacuum and dried at room temperature.

Release rate test

Figure 1. Comparison of UV-Vis spectrum of gelatin void and encapsulated ClO2. The measurement of ClO2 gas is made in the atmo-

Materials and methods sphere into which the ClO2 gas is generated. For example, if the generating mixture is exposed Materials to water vapor in air, the concentration of ClO2 gas in ppm will be measured based on the total The ClO2 was prepared by our laboratory. The atmosphere including the air and water vapor. Gelatin (food grade, Deionized) was obtained from Gelatin and Nano-micro Carbon material 20 gram samples were stored in sealed glass research group of Technical institute of physics jars. Every two hours, we took 10 mL gas by and chemistry, CAS. Soybean oil was purchased needle and dissolved the gas in 10 mL distilled from Beijing Essen Emerald Oil Co., Ltd, food water. We investigated the release rate of ClO2 grade. Span 80, Glutaraldehyde and Acetone based on the concentrations of ClO2 solutions were purchased from Beijing Chemical Reagent in different time at room temperature. The solu- Company, china. All the chemicals and reagents tions were analyzed by a UV-Vis spectropho- were of analytical grade. tometer.

Sample characterization Entrapment of ClO2

UV-Vis spectroscopy (Thermo, UV-Visible) and The entrapment rate of chloride dioxide in IR spectroscopy (PE, PerkinElmer spectrum encapsulated polymer material was deter- 100) were used as analytical tools for chemical mined by dissolving 20 g of dried microcap- analysis. Scanning electron microscope (SEM, sules in the distilled water. The concentration Model Hitachi 4300-S) was utilized for mor- of ClO2 generated can be calculated by measur- phology studied of the formed polymer micro- ing the absorbance in a UV-Vis spectrophotom- spheres. Particle size distribution was carried eter at 358 nm using the standard absorbance out by Dynamic Light Scattering (DLS, Malvern vs. concentration plot at 358 nm (Figure 2). S-90 series).

Entrapment rate (%) = (Amount of ClO2 entrap- Preparation of microspheres by emulsification- ped/initial amount of ClO2 taken) × 100 coacervation method Antibacterial test

ClO2-containing gelatin microspheres were prepared by an emulsification-coacervation meth- The slow released chlorine dioxide was continu- od [16, 17]. Deionized gelatin (10 g) was dis- ously diluted with sterilized nutrient broth in solved in 50 mL ClO2 aqueous solution under tube and up to 10 tubes. The 0.1 ml of bacteria

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experimental group (chlorine dioxide-gelatin microspheres).

Results

UV-Vis spectroscopy analysis

The maximum absorbance

of ClO2 dissolved in water was observed at 358 nm

wavelength (λmax), as show-

ed in Figure 1. At this λmax

of ClO2, a standard graph of absorbance vs. concentration (mg/lit) was plotted, as showed in Figure 2. The regression equation is y= 0.0022x-0.0139 (n=6, R2= 0.9949). This plot was uti- Figure 2. The standard curve of ClO . 2 lized to determine the percentage entrapment and

release study of ClO2 at different time from the encapsulated materials. The total percentage entrapment of

ClO2 was found to be in the range of 80-85%.

IR analysis

IR spectroscopy was used to confirm the presence

of ClO2 in the encapsulated form. Separate spectrums were taken for gelatin and gelatin microspheres with

ClO2 for comparison. Figure 3 shows a comparative IR spectrum of void and en-

capsulated ClO2. The void here is a polymer matrix of gelatin. The presence of chlorine in the encapsulat- Figure 3. Comparison of IR spectrum of gelatin void and encapsulated ClO2. ed sample was confirmed by the characteristic peaks 6 liquid (10 cfu/ml, Escherichia coli and Staphy- of ClO2-like stretching peaks at 1384.8, 1120.6 lococcus aureus) was added into each tube cm-1. respectively. They were cultured at 37°C for 48 hours. The minimum inhibitory concentration Dynamic light scattering analysis (MIC) was determined. The particle size and size distribution of the

The simulated air disinfection test was done in prepared microspheres of ClO2-gelatin were sealed disinfection cabinet. They were divided measured using a dynamic light scattering into control group (naked chlorine dioxide) and instrument (Malvern S-90 series) with a He-Ne

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chlorine oxide-gelatin microspheres. The sample was vacuum dried to remove the excess water. The morphology image of the chlorine oxide-gelatin microspheres are showed in Figure 5. The sample was observed to be spherical and distributed with an average particle size of 2000 nm. This size range was lower as compared to DLS size measurement (1400-1900 nm).

Slow release study

The release of ClO2 from the encapsulated polymer matrix in water was ana- Figure 4. Particle size distribution of microspheres of ClO2-gelatin using DLS. lyzed at different time inter- vals using a UV-Vis spectrophotometer at 358 nm. For analysis, the same stan-

dard curve for ClO2 was used. It was observed that within 48 hours, 7.3% of

ClO2 was released. On fur- thering the time, a maximum of 38.68% release of

ClO2 from the encapsulated polymer matrix was observed at 10 days, as shown in Figure 6.

Antibacterial test

As shown in Table 1, we

detected the MIC of ClO2 Figure 5. SEM morphology image of ClO -gelatin microspheres. in Escherichia coli and Sta- 2 phylococcus aureus and found that the MIC was 3.5 laser beam at a wavelength of 633 nm at 25°C mg/L. The results of simulated air disinfection and at a scattering angle of 90°. Aliquots from test were shown in Table 2. each preparation batch were sampled in DLS cuvettes and microcapsules were then exam- Discussions ined for equivalent diameters, size distribution, and polydispersity. The particle size obtained ClO2 is a fast-acting biocide, which is effective was around 1400-1900 nm with uniform size against a broad spectrum of micro-organisms, distribution as shown in Figure 4. such as bacteria, fungi, spores, mold and virus- es [18]. Previous study developed a polymer

Scanning electron microscopy micro-encapsulating liquid ClO2 coating that could provide long-term surface disinfection

Scanning electron microscopy (SEM, Hitachi through the sustained release of gaseous ClO2

4300-S) was used for a morphology study of from encapsulated liquid ClO2 [19]. In this study,

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Figure 6. A plot of time (h) vs entrapment and release rate of ClO2 from encapsulated material. A: Entrapment; B: Release rate.

Within 48 hours, 7.3% of ClO Table 1. MIC of slow released ClO2 2 ClO content (mg/L) was released and, after that, a Bacterium 2 maximum of 38.68% release 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 of ClO2 from the encapsulated Escherichia coli - - - - + + + + + + polymer matrix was observed Staphylococcus aureus - - - - + + + + + + at 10 days. The release rate of

ClO2 from gelatin microspheres was 6.25 ppm/h in the Table 2. The results of simulated air disinfection first two hours, and then, which was declined test up to 1.58 ppm/h in 39 h. After that, the release Average killing rate (%) rate of ClO2 from gelatin microspheres was kept little change. The release is due to the diffusion Time (h) Chlorine dioxide Chlorine dioxide-gelatin (9.1 mg/m3) microspheres (2.51 ppm/h) of ClO2 from the polymer matrix. The initial hour fast release may be due to the release of loose- 2 89.35 90.25 ly surface-bound ClO on the microspheres. 4 99.95 99.35 2 After 39 hours, the release is mainly due to the 8 100.00 100.00 release of inter-bound ClO2 in the microspheres. However, the release study shows the possibil- we firstly reported ClO -gelatin microspheres ity for slow and extended release formulation of 2 ClO to be able to make it stable, effective and decrease ClO gas release rate. The delayed- 2 2 long-lasting role. There were different suscepti- release ClO2-gelatin microspheres extended the role of the effect, reduced the number of bilities of the bacteria to polymer-encapsulated drug use on the prevention and treatment of ClO2 [19-21], we found that the MIC of ClO2 various types of infection. Using gelatin as an entrapped in a hydrophilic biodegradable poly- encapsulating material can make the final for- mer gelatin was 3.5 mg/L. mulation cost-effective because gelatin is cheap, readily available, and can be solubilized Conclusions easily in water. The emulsion in-situ gelling technique was used here. Rate of addition of From the above results, it can be concluded cross-linker and stirring speed has a significant that it is possible to make a slow release formu- effect on particle size distribution. lation of ClO2 by entrapped in a hydrophilic biodegradable polymer gelatin. ClO2-gelatin micro-

We found that the ClO2 entrapment efficiency spheres can stable release low concentration was 80-85%, the release study of ClO2 from ClO2 gas over an extended period, up to 10 gelatin microspheres showed a biphasic nature. days. The delayed-release ClO2-gelatin micro-

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