Preparation and Characteristic of Antibacterial Facemasks with Chinese Herbal Microcapsules

Along with the current increases in human activities, air pollution, especially that generated from bioaerosols, has begun to receive more extensive attention. Among the bioaerosols, bacteria and fungi can result in organisms that are pathogenic to animals and humans. Therefore, a biological protective respirator with an antibacterial function would be a significant method by which to prevent epidemic spread of respiratory infectious diseases and protect human health. In this study, a type of facemask with an antibacterial function was prepared. The structure of the respirator included a respirator body and an inner filter layer that was assembled with herbal microcapsules. A kind of Chinese herb microcapsule developed through a cross-linking reaction method was successfully prepared to serve as an important component of the respirator. Scutellaria baicalensis (SB), a typical Chinese medicine, was employed to prepare the Chinese herb microcapsule. Three kinds of filter cloth (cotton, polyester fiber, and non-woven fabric, respectively) were employed to assemble with Chinese herb microcapsule. Meanwhile, the antibacterial performance and adhesion of the Chinese herb microcapsule for E. coli and S. aureus were investigated. The measurement results revealed that (i) the antibacterial efficiency of the Chinese herb microcapsule and the respirator filter was above approximately 99.999 and 96.000%, respectively; (ii) the adhesion of the herb microcapsules was still excellent after the filter was washed 100 times; (iii) the as-prepared antibacterial filter modified by an O2 plasma-surface (PSM-O2) treatment exhibited excellent antibacterial performance. Finally, a chamber simulation utilizing an Anderson first order sampler was designed to simulate the effect of wearing the antibacterial filter in a real environment. Overall, the respirator exhibited excellent biological protection properties compared with general masks, which will satisfy a vast number of application prospects.


INTRODUCTION
Bioaerosols generated from airborne particles of biological origin include fungi, bacteria, viruses, and fragments of the foregoing or their metabolic products (e.g., endotoxins, mycotoxins) (Chow et al., 2015;Walser et al., 2015;Chen et al., 2016;Zhang et al., 2016;Lal et al., 2017).Environmental health studies have indicated that constant exposure to bioaerosols containing high concentrations of bacteria can lead to respiratory diseases including allergies, infections, and other adverse health effect (Bünger et al., 2007;Lu et al., 2016;Mirhoseini et al., 2016).With the current increases in atmospheric pollution, human respiratory systems have been suffering from more health risks both indoors and outdoors when exposed to biological aerosols.Therefore, it is very necessary for people to wear a biological protective respirator with an antibacterial function.A facemask, as a kind of simple and convenient respirator, is widely used in daily life.It has been demonstrated that wearing facemasks for a long time can reduce the probability of infection from influenza-like illnesses (MacIntyre et al., 2009;Natarajan et al., 2016;Lin et al., 2017).Especially, functional facemasks are recommended for frontline healthcare workers to prevent splashes and sprays of blood, body fluids and the spread of infection from the wearer (Siegel et al., 2007;Agarwal et al., 2016).At present, polypropylene (PP) melt-blown nonwoven fabric has been extensively employed as a filter for biological protective respirators because it resists microparticles and pathogenic microorganisms based on physical effects.However, bacteria, viruses, and other microorganisms remaining on filter materials can survive and multiply under suitable conditions and result in secondary infections in humans (Chow et al., 2005).Hence, a facemask filter with an antibacterial function is urgently needed (Lee et al., 2017;Rissler et al., 2017).
Traditional Chinese herbal medicines, having a long history of treating diseases, serve as an established segment of the public health system in China at present and have attracted much more concern in other parts of Asia (Chen and Zhu, 2013).According to the third national survey on Chinese material resources, there are more than 12,000 medicinal plants (Kong et al., 2004).Literally, traditional Chinese medicine utilizes multiple plant prescriptions made from their extraction agents that have served the health needs of the Chinese general public for over 5000 years (Mahady et al., 2008).For example, Scutellariabaicalensis (SB), a traditional Chinese medicine, comprises several biological activities including anti-oxidative, antiinflammatory, antibacterial, and antiviral activities ( (Duan et al., 2007).Compared to antibiotics, Chinese herbal medicine has mild pharmacology, a high degree of safety, curative effectiveness, is low cost, and is not compromised by drug-resistant bacteria.Therefore, an increasing number of antibacterial respirators employing Chinese herbal medicine has emerged (Chen and Zhu, 2013;Han et al., 2015;Mazzio et al., 2016).
Microcapsule technology offers various significant applications in many fields, including chemical science, bioscience, biotechnology, and drug delivery systems (Singh et al., 2010).It is reported that microcapsules can increase the adhesive strength with fiber and slow down the process of drug release (De Geest et al., 2009;Hebeish et al., 2011).Otherwise, Plasma-surface modification (PSM) is an effective and economical surface treatment technique for many materials and is of growing interest in the field of biomedical engineering (Encinas et al., 2012).In particular, plasma treatment can improve adhesion strength, surface, and coating properties as well as mechanical-and biocompatibility (Chu et al., 2002;Encinas et al., 2012;Armağan et al., 2014).With respect to atmospheric plasma on the poly (lactic acid) (PLA) surface, plasma treatment promotes an increase in the surface energy of 59% from values of around 37.10 mJ m -2 to values close to 58.92 mJ m -2 .This treatment of the PLA surface induces the appearance of new activated species, such as carboxyl (ACOOH), carbonyl (ACO), hydroxyl (AOH), peroxide (AROORA), and other functional groups, which change overtime to achieve a stable state (Jordá-Vilaplana et al., 2016).
In this work, a novel kind of mask with an antibacterial function was prepared.We used the extraction of scutellaria baicalensis (SB) to synthesis the Chinese herb microcapsule.A novel Chinese herb microcapsule method was successfully prepared using a cross-linking reaction during the process of respirator fabrication.Three kinds of filter cloth (cotton, polyester fiber, and non-woven fabric, respectively) were compared to assemble with Chinese herb microcapsule.Meanwhile, the antibacterial performance and adhesion of the Chinese herb microcapsule for E. coli and S. aureus were investigated.Moreover, a chamber simulation utilizing an Anderson first order sampler was designed to simulate the effect of wearing the antibacterial filter in a real environment.Overall, the respirator exhibited excellent biological protection properties compared with general masks, which indicates that it may be significant in the control of bioaerosol and bacterial infections.

Preparation of Chinese Herbal Extraction
Firstly, the extraction of the Chinese herbal medicine used in this study was conducted via the Soxhlet extraction method (Snyder et al., 1992).Secondly, the extraction was centrifuged and filtered to remove any impurities.Thirdly, the pure extraction was concentrated by vacuum decompression rotary concentrator (EYELA).The concentrated solution of the Chinese medicine was frozen in a refrigerator at -20C overnight.Then, the concentrated solution was freeze-dried until no water remained.Finally, the powder samples were stored in a refrigerator at 4C for subsequent microcapsule synthesis.

Preparation of the Chinese Herbal Microcapsules Preparation of Chinese Herb Microcapsule by Cross-Linking Reaction
Chitosan was also employed as a cladding material (Kanmani et al., 2011).Gelatin was used as a substrate in the formation of the microcapsules.The Chinese medicine extraction served as a core material.After treatment involving concentrating and freeze-drying, the Chinese herbal microcapsules were synthesized.The detailed procedures are appended as follows: (i) Chitosan was dissolved in an acetic acid solution (denoted as solution A).Subsequently, gelatin powder and the Chinese herbal medicine powder were dissolved in deionized water and stirred at 2500 rpm (denoted as solution B). (ii) Solution B was transferred to a water bath at 50°C and stirred at 400 rpm (denoted as solution C). (iii) Solutions A and C were then mixed, and the pH value was adjusted to 5.3 with 10% NaOH accompanied by stirring at 400 rpm for 30 min (denoted as solution D). (iv) Solution D was cooled to 5-10°C.The cross-linking reaction was conducted for 30 min after adding 2 mL 25% glutaraldehyde to the cooled solution (denoted as solution E) (Singh et al., 2010).(v) Finally, the Chinese herb microcapsule suspension was obtained after solution E was stirred for 30 min in a 40°C water bath.
For comparison, the Chinese herb microcapsule without the cross-linking reaction method was also prepared following the above procedure without process (iv).

Assembly Filter with the Chinese Herb Microcapsule Pre-Processing with Filter Cloth
Three kinds of filter cloth (cotton, polyester fiber, and non-woven fabric, respectively) were soaked in a 5% detergent solution for 30 min and then ultrasonically oscillated at 45°C for 1 h in order to eliminate the processing pulp material.Afterwards, the cotton fabric was washed and rinsed with deionized water and then soaked in acetone for 10 min to remove any remains on the fabric surface.It was then dried at 75°C in an oven.The treated fabric was tailored into an appropriate size and placed in an ultrasonic concussion machine for 10 min to get rid of the fabric clippings.Finally, the obtained fabric was reserved as a filler material after drying in an oven.

Preparation of the Filter with the Chinese Herbal Microcapsule
The treated fabric was weighed and the mass was recorded as m 1 .Then, it was immediately soaked into a suspension containing the Chinese herb microcapsule for 5 min.and then dried and solidified gradually on the soaked fabric.Finally, the filter with Chinese herbal microcapsule was prepared successfully, and the filter mass was labeled m 2 .Before testing the antibacterial efficiency of the microcapsules, the coating efficiency should verified as stable.The coating efficiency of the Chinese herb microcapsule was calculated by the following equation:

Plasma-Surface Modification (PSM) of the Fabric
The pre-processed fabric was treated using a lowtemperature plasma system.The schematic of the set-up system is shown as Fig. 1.Firstly, the sample table was wiped with acetone after breaking the chamber vacuum.The tailored filter material was then placed on it.Secondly, residual gases and substances in the chamber were removed using a vacuum pump.The pressure was controlled at 600 torr (1 torr = 133 Pa) with argon gas, and the argon and oxygen flow were controlled at 20 sccm (standard cubic centimeter per minute).Thirdly, the tailored filter fabric was treated with low-temperature plasma for 30 seconds to modify the surface.
As a contrast, the filter containing the Chinese herbal microcapsule without plasma treatment was also prepared.

Antibacterial Performance Evaluation Minimum Antibacterial Concentration Test
Due to the fact that E. coli and S. aureus are respectively representative of Gram-positive and Gram-negative bacteria (Fischer et al., 2015), a series of different concentrations of an SB (from 0.0078125 to 2 g mL -1 ) extraction was respectively employed to inhibit the E. coli and S. aureus.In brief, the original bacterial solution (E. coli and S. aureus) was diluted to 2  10 5 CFU mL -1 .Different concentrations of the Chinese herbal extraction were added into the abovementioned solution.The culture medium TSA sterilized at a high temperature was added and mixed uniformly.Then, the culture dish was cultivated in an incubator at 37°C for 24 h.Finally, the number of colonies was calculated.

Antibacterial Performance Test of Chinese Herb Microcapsules
The culture medium TSB mixed with the E. coli or S. aureus colony, respectively, and cultivated at 37°C and 85 rpm for 24 h.Each of the two Chinese herbal microcapsule suspensions was respectively added into the above diluted bacteria solution (2  10 5 CFU mL -1 ).The antibacterial results of the Chinese herb microcapsules was measured after 0, 2 4, 6, 12, 24, 48, 72 hr, orderly).Namely, 1 mL of the mixed solution was respectively taken and solidified.The number of colonies was then counted.For comparison, the blank group (the same bacterial solution without the Chinese herbal extraction or microcapsules) was counted.The antibacterial efficiency was then calculated using the following equation: where N 0 is the number of bacterial colonies for the blank experiment, and N is the number of bacterial colonies in the presence of the Chinese herbal element.

Antibacterial Performance Test of the Filter with the Chinese Herbal Microcapsule
The antibacterial test method for the filter containing the Chinese herbal microcapsule followed AATCC-100 2004 (Gao and Cranston, 2008).The number of colonies was counted and calculated to determine the antibacterial efficiency.

Durability Test of Antibacterial Filter
The filter with the Chinese herbal microcapsule was washed using a 5% detergent and was then vibrated ultrasonically for 3 min and dried circularly 0, 10, 30, 50, 70, 90, and 100 times.The washed antibacterial fabric filter at the different times was investigated with an antibacterial test using the AATCC-100 2004 method.

Simulated Test Filter with the Chinese Herbal Microcapsule Chamber Simulation
For the purpose of testing the effect of wearing the antibacterial filter, a chamber simulation was designed to simulate the human lung (Fig. 2).In this study, an Anderson first order sampler was used for sampling (Heinrich et al., 2003;Lin et al., 2012).The chamber volume was 100 L, and the height of the sampling port was set at 150 cm from the ground.Before sampling, the pumping frequency was adjusted to once every 3 seconds at a pumping volume of 500 mL.

Antibacterial Performance of Filter for Bioaerosol
Firstly, the antibacterial filter with the Chinese medicine microcapsules was placed in the sampling port.A controlled pumping system was used to simulate breathing frequency, which was maintained for 200 min.An Anderson first order sampler was used for a 3-min sampling.The sample was cultivated in a bacterial culture substrate at 37°C for 48 h.After cultivating at 25°C for 5 days, the number of colonies was calculated.For comparison, the filter without any process was also analyzed in the above experiment.

Preparation of the Antibacterial Mask
The structure of the respirator included a respirator body and an inner filter layer, which was assembled with herbal The three layers from inner to outer are medical cotton yarn, the antibacterial filter, and non-woven fabric, respectively.

Antibacterial Performance Evaluation Minimum Antibacterial Concentration Test
Before preparing the Chinese herb microcapsule, the minimum antibacterial concentration of the Chinese herb extraction was tested.Fig. 4(A) shows that SB exhibited  excellent antibacterial performance for both E. coli and S.aureus after treating the bacterium culture solution with different concentrations of SB (0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.003125 g mL -1 ).The results showed that the minimum antibacterial concentrations of SB for E. coli and S. aureus were 0.2 and 0.0125 g mL -1 , respectively.Fig. 4(B) illustrates that the inhibition effect of S. aureus revealed a higher degree of selectivity as compared with E. coli.According to a previously reported work, we concluded that Gram-negative bacteria (E.coli) has both a doublelayer membrane and a lipopolysaccharide layer.These layers act as a barrier to the entry and exit of other objects (Jiang and Zhang, 2016).Therefore, a higher concentration of SB is needed to achieve a better antibacterial effect.

Antibacterial Performance Test for the Chinese Herbal Microcapsules
To make better use of SB to inhibit the multiplication of bacteria, a novel Chinese herbal microcapsule developed using the cross-linking reaction method was successfully prepared.For comparison, the Chinese herb microcapsule without the cross-linking reaction method was prepared.It was only coated with the Chinese herbal medicine chitosan (denoted as CHM 1 ).With respect to the antibacterial efficiency of CHM 1 , the overall release rate of the microcapsules for S. aureus was superior to that of E. coli.After 24 h, the antibacterial efficiency was still maintained at more than 96%, as compared with E. coli (86.29%).Hence, CHM 1 exhibited relatively poor antibacterial performance for S. aureus, as shown in Fig. 5(A).
In the case of the Chinese herbal microcapsule developed using the cross-linking reaction method (denoted as CHM 2 ), Fig. 5(B) shows excellent antibacterial efficiencies for both of E. coli and S. aureus after 24 h of as much as 99.999%.We can therefore conclude that the antibacterial performance of the CHM 2 microcapsule was superior to that of CHM 1 .In order to reduce the cost, different dilution concentrations (10, 100, 1000 and 10000 times) of CHM 2 were tested.As shown in Fig. 5(C), the antibacterial ability for E. coli and S. aureus was still maintained at 99.999% when the Chinese herbal microcapsules were diluted by 100 times.However, the antibacterial capacity started to decline sharply after being diluted more than 100 times, especially in the case of E. coli.Therefore, microcapsules diluted 100 times were employed to test bacterial growth for 48 h.
Fig. 5(D) presents the bacterial multiplication for 48 h in culture solution contained CHM 2 (diluted 100 times).As observed, the antibacterial results for S. aureus showed escalating trend during the initial period.It was because that microcapsule was released slowly.Overall, the antibacterial performance of CHM 2 (diluted 100 times) for E. coli and S. aureus was excellent until 24 h.While the antibacterial capability started to decline down to 60 and 74% (for S. aureus and E. coli, respectively) at 36 h.It was speculated that the amount of released Chinese medicines from the microcapsules was not enough to inhibit multiplication of bacteria.At 48 h, CHM 2 almost showed no antibacterial performance.

Characterization of the Filter Cloth with the Chinese Herbal Microcapsule
Three kinds of readily available filter cloth (cotton, polyester fiber, and non-woven fabric, respectively) were employed to study the antibacterial performance and the adhesion of the herbal medicine after assembly with the Chinese herbal microcapsule.
Fig. 6 shows the morphology and size of the as-prepared filter cloth as characterized by SEM.The results reveal that the Chinese herbal microcapsules (marked in the yellow circle) were successfully assembled with the cotton (Fig. 6(A)), polyester fiber (Fig. 6(C)), and nonwoven fabric (Fig. 6(E)), and these microcapsules aggregated excessively.Also, it can be observed that the structure of the nonwoven fabric was different from that of the other fabrics.The nonwoven fabric presented disordered, unsystematic features.From their corresponding high-magnification images (Figs.6(B), 6(D) and 6(F)), it can be observed that the pattern of the nonwoven fabric was much more loosened and rough than its counterparts.

Antibacterial Performance Test of the Filter Cloth with the Chinese Herbal Microcapsule
As shown in Fig. 7, the antibacterial efficiency and adhesion of the herbal medicine for the different types of filter cloth with CHM 2 were measured after they were washed 100 times.The results showed that the antibacterial efficiency of the filter cloth with CHM 2 (assembled with cotton, polyester fiber, and non-woven fabric, respectively) for E. coli and S. aureus still remained above 96% (96.94, 96.69, 96.99%) and above 95% (95.65, 96.33 and 96.67%), respectively, although there was a slight downward trend after 100 washings.Further, it was proved that all types of filter cloth assembled with CHM 2 can maintain stable adhesion and can be re-used many times.
In order to strengthen overall capacity, the above referenced as-prepared filter cloth with CHM 2 was treated with plasma-surface modification (PSM).Compared with PSM-Ar, the filter cloth with CHM 2 modified by the O 2 plasma-surface (PSM-O 2 ) treatment exhibited a much higher microcapsule residual rate after 100 washings, as calculated using the following equation: where m 0 is the initial weight of filter cloth; m 1 is the weight of filter cloth after coating with antibacterial microcapsules, and m 2 is the weight of the above-mentioned filter after 100 washings.We speculated the reason this outcome was that modification by PSM-O 2 not only improves the physical properties of the surface, but also increases the oxygen functional groups and hydrophilic performance (Jordá-Vilaplana et al., 2016).It has been reported that the hydrophilicity of a biomaterial is related to the adhesion of microbial proteins, which provide an additional hydration layer that prevents protein adhesion (Wei et al., 2014).This would suppress the breeding of microorganisms on the surface of such biomaterials.After being treated with PSM-O 2 , the antibacterial efficiency and adhesion of the herbal medicine for the different types of filter cloth with CHM 2 were measured after washing 100 times.As shown in Figs.8(A) and 8(B), the results show that the antibacterial efficiency of the non-woven fabric with CHM 2 for E. coli and S. aureus could be maintained above 99% and 96%, respectively, after 100 washings.In the case of the cotton fabric, the increase in antibacterial efficiency was not remarkable (97 and 95%) while the antibacterial efficiency of the polyester fiber fabric with CHM 2 for E. coli and S. aureus declined sharply (78.6 and 73.4%).This is because the fabric fibers were so close together that there were not enough holes for microcapsule preservation.Therefore, the microcapsules fell off the surface of the fabric when it was treated with PSM-O 2 .

Simulation Test of the Filter with the Chinese Herbal Microcapsule
Both indoors and outdoors, bacteria and fungi always give rise to bioaerosols, which influences air quality and in turn the human respiratory system.In order to test the effect of wearing the antibacterial filter in a real environment, a chamber simulation was designed to simulate the human lung (Fig. 2).In this study, an Anderson first order sampler was used for sampling.We recorded the number of bacterial colonies for the three types of filter cloth with CHM 2 for 24 h.For comparison purposes, a blank sample was also employed to calculate the multiplication of bacterial colonies.The three sampling results indicate that the number of bacterial colonies in the cotton coated with the Chinese herbal microcapsules was 17, 20 and 19 CFU m -3 , respectively.For the polyester fiber, the number of bacterial colonies was 33, 35 and 30 CFU m -3 .For the nonwoven fiber, the number of bacterial colonies was 10, 11 and 12 CFU m -3 .However, the number of bacterial colonies was 408, 417 and 405 CFU m -3 in the blank group.Fig. 9(A) shows the antibacterial performance results.As can be seen from the bar chart, the nonwoven fiber with the Chinese herbal microcapsules exhibited the best inhibition of multiplication of bacterial colonies.The average value of the antibacterial efficiency for the nonwoven fiber (97.31%) was higher than that of both the cotton (95.44%) and polyester fibers (91.7%).At the same time, we also recorded the number of fungi.The three sampling results show that the number of fungi in the cotton coated with the Chinese herbal microcapsules was 62, 60, and 62 CFU m -3 , respectively.For the polyester fiber, it was 62, 60 and 61 CFU m -3 .For the nonwoven fiber, it was 51, 50 and 55 CFU m -3 .For the blank fiber, it was 716, 724 and 715 CFU m -3 .Fig. 9(B) shows the antibacterial performance results.As was previously observed, all of the filters with the Chinese herbal microcapsules exhibited the same performance related to inhibiting the multiplication of fungi.The average antibacterial efficiency for the nonwoven fiber (92.66%) was slightly higher than that for the cotton (91.45%) and polyester fibers (91.04%).Overall, these three types of filters exhibited excellent antibacterial capability and stability.

CONCLUSION
In summary, a novel type of mask with an antibacterial function was prepared.We used an extractant of scutellaria baicalensis (SB) to synthesize the Chinese herbal microcapsule.A novel Chinese herbal microcapsule developed using a cross-linking reaction method was successfully prepared during the process of respirator fabrication.Three types of filter cloth (cotton, polyester fiber, and non-woven fabric, respectively) were compared when assembles with the Chinese herbal microcapsule.Meanwhile, the antibacterial performance and adhesion of the Chinese herbal microcapsule for E. coli and S. aureus was investigated.The experimental results are summarized as follows: (a) the antibacterial efficiency of Chinese herb microcapsule and the respirator filter was as high as approximately 99.999 and 96.00%, respectively; (b) the adhesive strength of the herbal microcapsules maintained excellent performance after the filter was washed 100 times; (c) the as-prepared antibacterial filter modified using an O 2 plasma-surface (PSM-O 2 ) treatment exhibited much higher antibacterial performance.A chamber simulation utilizing an Anderson first order sampler was designed to simulate the effect of wearing the antibacterial filter in the real environment.Overall, these three types of filters showed excellent antibacterial capability and stability, and are predicted to achieve further applications in real-life situations.

Fig. 1 .
Fig. 1.Schematic illustration of plasma set-up system for treating pre-processed fabric.

Fig. 5 .
Fig. 5. Antibacterial efficiency of Chinese herb microcapsule without cross-linking reaction method (A) and by crosslinking reaction method (B) for 24 h; (C)Antibacterial efficiency of Chinese herb microcapsule by cross-linking reaction method after diluted by different times (10, 100, 1000 and 10000 times).(D) Antibacterial performance evaluation of Chinese herb microcapsule by cross-linking reaction method (after diluted 100 times) for 48 h.

Fig. 6 .
Fig. 6.SEM images of the filter cloth with Chinese herb microcapsule by cross-linking reaction method: cotton (A, B), polyester fiber (C, D) and non-woven fabric (E, F).B, D and F are the high-magnification SEM images for A, C and E, respectively.

Fig. 7 .
Fig. 7. Antibacterial performance of three kinds of filter cloth (cotton, polyester fiber and non-woven fabric, respectively) coated with Chinese herb microcapsule after washed by 100 times.The top of the graph is antibacterial efficiency for E.coli, and the bottom for S. aureus.

Fig. 8 .
Fig. 8. Antibacterial efficiency of different kinds of filter cloth with CHM 2 treated with PSM-O 2 after washed by 100 times.The left (A) antibacterial efficiency for E.coli, and the right (B) for S. aureus.

Fig. 9 .
Fig. 9. Antibacterial capability of three kinds of filter cloth with CHM 2 in the real environment for bacteria (A) and fungi (B) to simulate the effect of wearing the antibacterial filter.