Preparation of Silica Radiation Pore and Its Application as Antimicrobial Carrier

HUANG Jiangxi, ZHA Xinlin, FAN Hui, LU Zhentan, CHEN Yuanli

(Key Laboratory of Textile Fiber and Products (Wuhan Textile University), Ministry of Education, Wuhan 430073, China)

Abstract: Hollow silica nanospheres with radical pore on the surfaces were prepared using the assemblies of valine amphiphilic small molecule and benzene as double-templates through sol-gel method in tetramethylammonium hydroxide (25wt%) solution at the stirring rate of 1 000 rpm. There are a lot of vertical pores on the surfaces of the hollow spheres after removing the templates in Muラe furnace at 550 ℃ for 5 h. The sample was characterized using field-emission scanning electron microscopy, transmission electron microscopy, Brunauer-Emmett-Teller (BET), X-ray diffraction, etc. The diameter, the vertical pore size of the nanospheres and the BET surface areas are 30-100 nm, 4.2 nm, and 570.5 m²/g, respectively. Because the high porosity and specific surface area, this kind of hollow sphere is the excellent antimicrobial carrier. The antibacterial activities of the silica nanospheres were evaluated by using a bacterial growth inhibitory assay. The experimental results show that the silica hollow spheres loaded with Ag+ have a good bactericidal effect.

Key words: sol-gel chemistry; nanostructures; radical pores; silica nanospheres; antimicrobial carrier

1 Introduction

Own to the well-developed supramolecular selfassemble and sol-gel chemistry, the mesoporous silicas with varieties of pore architectures and morphologies have been successfully synthesized in the last years. Recently, scientists have focused their research on controlling the pore structure and morphology which directly influence on their applications[1-6].

During the last decades, the ordered mesoporous silica nanostructure with uniform pore diameter and pore morphology has attracted people’s attention due to its wide application in the fields of adsorption, separation and antimicrobial carrier[7-10]. Especially, hollow silica nanostructures have attracted extensive attention because of their advantages such as large cavities, high temperature resistance, high hardness, no physiological toxicity, high pore volume and permeable pore walls[11-13]. Among them, silica hollow nanospheres with pores vertical to their surfaces attracted interests for researchers due to their applications in the fields of adsorption, drug storage and release, and antibacterial loading[14-16]. Therefore, designing a simple method to fabricate silica nanostructures with pore channels perpendicular to the walls and high BET is highly desirable for increasing drug release and antimicrobial carrier.

Up to now, several synthetic strategies for designing and fabricating silica nanostructures with pores vertical to the wall surfaces have been reported[17-21]. Generally, they were fabricated via dual-templating approaches. Oil droplets[10], bubbles[22], or vesicles[23]mixed with amphiphiles were usually used as the double templates. Yang’s group used the dual template method to prepare single-handed twisted silica nanotubes with mesopores on the surface by adjusting the molar ratio of the chiral small molecule derivatives of valine and the F127 triblock copolymer[24].

Herein, silica nanospheres with holes overtically oriented mesochannels in the wall were fabricated by using a double-templating route. This sample is formed by a phase transition process during the sol-gel transcription process[25]. The diameter of the nanosphere is about 30-100 nm, and the vertical pore diameter in the walls is about 4.2 nm. Silver ions is a widely used antibacterial agent, however silver ions are very soluble in water, the rapid dissolution and diffusion of silver ions in water will affect the antibacterial effect of the material. Immobilizing silver ions on a suitable carrier is an ideal way to solve this problem. When silver ions were loaded on the vertical pores with high specific surface area, it’s found that the loaded nanospheres have a good antibacterial effect.

2 Experimental

2.1 Materials

Staphylococcus aureus ATCC 29213 (S. aureus), and Escherichia coli ATCC 25922 (E. coli) were purchased from Nanjing Lezhen Biotechnology Co. Ltd. Polypepton, yeast extract were purchased from Beijing AOBOXING Bio-Tech Co. Ltd. Agar, glutaraldehyde, ethanol, isoamyl acetate, KH2PO4, Na2HPO4, NaCl, KCl, HCl (35wt%-38wt%), tetraethoxysilaneand and benzene were purchased from Sinopharm Group Chemical Reagent Co., Ltd. L-valine (99%) was purchased from Shanghai Hanhong Chemical Co. Ltd. 6-bromine hexanoic acid (99%), tetramethylammonium hydroxide (25wt%) were purchased from the Aldrich Chemical. All chemicals were used directly without further purification.

2.2 Specimen processing

The typical experimental process is as follows: 100 mg of L-16ValPy6Br (0.0163 mmol) was added into in 100 mL of deionized water, and then 1 mL of benzene was added and stirred to form a homogeneous solution at 80 ℃ for 15 min. Then 350 mL tetramethylammonium hydroxide and 1 mL TEOS were added to the above solution and reacted for 2 h with a rate of 1 000 rpm. Finally, the obtained product was boiled 3 times in a mixed solution of 5 mL of 36.0wt% HCl aq. concentrated hydrochloric acid and 50 mL ethanol, and calcined in a muffle furnace to 550 ℃ for 5 h.

The clean glass slides were washed by the mixture solutions of saturated NaOH-ethanol and water. All the bacterial samples were fixed on the prepared clean glass slides with 2.5% glutaraldehyde for 12 h. Then, these bacterial samples were washed with deionized water, dehydrated with mixed solutions of different volumes of ethanol and water (30%, 50%, 70%, 90%, 95%, and 100%) and dried in a freeze dryer and finally lyophilized on a lyophilizer.

2.3 Test methods

The morphology of the as-prepared material was recorded with the field-emission scanning electron microscopy (FESEM, JEOL-7800F) and transmission electron microscopy (TEM, JEM-2010) operated at 200 kV. Specific surface area and pore-size distribution were determined by the Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods were measured by the Micromeritics ASAP 2460 instrument using N2Nitrogen adsorption and desorption isotherm. Small-angle X-ray diffraction (SAXRD) patterns of as-silica nanospheres were measured with a X-ray diffractometer (Smartlab9k, Cu Kα, λ=1.5418 Å). The absorbance at 630 nm was measured on the enzyme analyzer HEALES MB-530. The particle size distribution and Zeta potential were measured on the Zetasizer Nano ZS ZEN3600.

The antibacterial activity of the membranes was tested usingS. aureusATCC 6538 andE. coliATCC 8099 as model bacteria.S. aureusandE. colistrain were stored under -20 ℃, the frozen bacteria were inoculated into Luria-Bertani (LB) broth where beef extracts, peptone and NaCl were 3 g/L, 5 g/L, and 5 g/L, respectively. Then, the LB broth, mixed with bacteria, were cultured at 37 ℃ for 12 h. At last, the concentration of the bacterial suspension was adjusted to approximately 1.0×105CFU/mL. The bacterial suspension (150 µL) was mixed with an equal volume of 2-fold diluted mesoporous silica ball solution (150 µL, 3.9 µg/mL-500 µg/mL) which was loaded with Ag+, then the cells were seeded into 24-well plates and incubated at 37 ℃. The solutions (1 mL) were added to the bacterial suspension (1 mL). The growth inhibition of the bacterial cells was assessed by measuring OD600 at different time interval. Samples, incubated with pure broth, were used as the control and three independent experiments were carried out.

3 Results and discussion

3.1 Characterization of compound L-16Val6-PyBr

The valine amphipathic small molecule L-16Val6PyBr is shown in Fig.1, which was synthesized according to the Ref.[26]. 4.3 g (0.0072 mol) of valine bromide was added to 80 mL of pyridine solution under the protection of nitrogen, and refluxed at 100 ℃ for 12 hours. The obtained sample was recrystallized three times with a mixture of ethanol and ether, and finally filtered and dried to obtain the product. (yield 4.3 g, 86.0%). The amphiphile L-16Val6PyBr can physically gel benzene, nitrobenzene, toluene and deionized water.

Fig.1 Molecular structure of the amphiphile L-16Val6PyBr

3.2 Characterization of the silica nanospheres

In order to observe the surface morphologies of silica, field emission scanning electron microscopy (FESEM) was used. The specimen was sputtered with 10 nm Pt before taking SEM pictures. As shown in Fig.3(a), SEM images of the prepared sample (Figs.2(a)-2(b)) show silica nanospheres morphology. The corresponding TEM image is shown in Figs.2(c,b). TEM images of these nanospheres confirm that they are all hollow morphology (Figs.2(c)-2(d)). The diameter of the nanospheres is about 30-100 nm. It is interesting to note that the silica nanospheres with vertical pore were found in the walls. The pore size is about 4 nm.

Fig. 2 SEM ( a, b) and TEM (c, d) images of silica nanospheres

Fig.3(a) shows N2nitrogen adsorptiondesorption isothermal plots for the mesoporous silica nanospheres which show a typical type IV adsorption isotherm with a H2-like hysteresis loop, the relative pressure fromP/P0= 0.1 to 1.0, which indicated that, besides mesopores, there were some micropores and macropores. The macropores should be the hollow cavity of the nanospheres, which were formed by the adding of benzene and meanwhile the H2hysteresis loop is shown in Fig.3(a). Mesopores are formed by removing the self-assembly of small chiral molecules. In our system, the self-assemblies of amphiphile L-16Val6PyBr were used as the templates, which were removed after calcination, the BET surface areas of the obtained silica nanospheres reached 570.5 m²/g. Such high surface areas are suitable to be attached to silver ion for sterilization. The vertical pores on the surface of the silica nanospheres have a high degree of thermal stability. Even if calcined at a high temperature of 550 ℃ for 5 h, the morphology of the vertical pores can still be maintained. The pore-size distribution plot from desorption branch shows peaks at 4.2 nm presented in Fig.3(b), which are consistent with the pore size of the silica surfaces.

The small-angle X-ray diffraction (SAXRD) pattern of silica nanospheres with pores vertical to the surfaces are shown in Fig.4(a). There is no sharp peak except broad diffraction peaks at 2θof 1.68°, which shows that the vertical pores on the surface of the silica nanospheres have a certain degree of order. The values ofd-spacing calculated from the diffraction peaks is 5.3 nm. As shown in Fig.4(b), the average particle size of the sample is 50.2 nm.

As shown in Fig.4(c), theZ-potential of silica nanospheres is -5.1 mV, the surface of the negatively charged silica surface can effectively adsorb positively charged silver ions.

Fig. 3 Nitrogen adsorption-desorption isotherm of the nanospheres (a) and pore-size distribution plot (b)

Fig.4 SAXRD patterns of the nanospheres(a), particle size distribution chart(b) and Zeta potential values of silica nanospheres(c)

3.4 Antibacterial activity test

The antibacterial activities of the silica nanospheres were evaluated by using a bacterial growth inhibitory assay. Based on these results (Fig.5), the vertical mesoporous silica shows obvious antibacterial activity againstE. coli(Fig.5(a)) andS. aureus(Fig.5(b)), The MIC value is 15.6 µg/mL, and the mesoporous silica also shows antibacterial activity even the concentration as low as 3.9 µg/mL. The antibacterial mechanism of mesoporous silica,E. coliandS. aureusas the model bacteria was observed through SEM images.

Fig.5 Antibacterial activity of mesoporous silica against E. coli (a) and S. aureus (b)

As shown in Fig.6, the bacteria surface morphologies have changed greatly compared with the negative control. The bacterial walls ofE. coli(Figs.6(a)-6(b)) andS. aureus(Figs.6(c)-6(d)) are distorted after co-culture with vertical pore channels silica for 30 min, compared with the smooth surfaces of the negative control. As shown in SEM images, the walls of the bacteria are destroyed by silver ions, and ultimately caused the death of bacteria. These results show the mesoporous silica has the potential use as drug carrier, such as loading antibacterial agent.

Fig.6 SEM images of E. coli (a, b) and S. aureus (c, d) before (a, c) and after (b, d) culturing with mesoporous silica

4 Conclusions

In this study, we have synthesized silica nanospheres with pore channels vertical to the wall surfaces formed via phase transition using double-template approach. The formation of hollow silica nanospheres with vertical pores on the shells with a cooperation self-assembly mechanism. This porous silica nanosphere has high porosity and specific surface area, which indicated that the nanosphere was the potential drug carrier. And, the silica nanospheres with radical pore loaded with Ag+showed good bactericidal effect. Further research will be carried out in these fields.