Preparation of Vanillic Acid-Loaded Core-Shell Gold Nanospheres/Mesoporous Silica Nanoparticles for the Treatment of Orthopedic Infection.

Orthopedic infection is a serious complication in surgeries and remains a great challenge in clinics. Here, the natural antimicrobial compound vanillic acid-loaded gold nanospheres/mesoporous silica nanoparticles (VA@Au-MSNs) were fabricated for chemo-photothermal synergistic therapy to orthopedic infections. The shape and morphology of Au-MSN and VA@Au-MSN were observed by scanning electron microscopy and transmission electron microscopy. The properties of VA@Au-MSN or related components were characterized by dynamic light scattering, thermogravimetric analysis, Brunauer-Emmett-Teller (BET) analysis, and photothermal analysis. Vanillic acid released from VA@Au-MSN was detected in phosphate-buffered saline. A cytotoxicity test and an antibacterial assessment were performed to explore the biosafety and antibacterial activity of VA@Au-MSN, respectively. The results showed that Au-MSN possessed a high BET surface area (458 m2/g). After loading vanillic acid, the BET surface area reduced to 72 m2/g. The loading efficiency of Au-MSN was 18.56%. Under 808 nm laser irradiation, the temperature at the wound site injected with the Au-MSN solution in the mouse increased from 24 to 60 °C within about 12 s. Also, the high temperature could promote the release of vanillic acid from VA@Au-MSN. Additionally, VA@Au-MSN has no obvious cytotoxicity to MC3T3-E1 cells, but the generated local hyperthermia and the VA released from VA@Au-MSN had excellent antibacterial activity against Staphylococcus aureus in a synergistic way. In conclusion, the VA@Au-MSN with biosafety and excellent antibacterial performance might be applied for the treatment of orthopedic infection.

Introduction

Medical device-associated infection is an extremely serious complication after orthopedic surgery and remains a challenging and troubling problem in clinics.[1,2] Antibiotic therapy is a conventional and widely used approach for orthopedic infection. However, due to the emergence of antibiotic-resistant strains and the formation of bacterial biofilms, the control of infection by antibiotics is always a failure.[3,4] Otherwise, antibiotic application contributed to fever in orthopedic patients.[5] Thus, it is essential and meaningful to develop novel treatment approaches for the management of orthopedic infection.

Vanillic acid, a natural phenolic compound, can be extracted from numerous edible fruits and plants, including Amburana cearensis AC Smith,[6]Actinidia deliciosa,[7] and Panax ginseng CA Meyer.[8] Stanely et al. disclosed that vanillic acid could protect the rats against cardiotoxicity induced by isoproterenol due to its anti-inflammatory and antioxidant properties.[9] In the typical inflammatory disease—ulcerative colitis, vanillic acid also had beneficial effects.[10] In addition, vanillic acid posed excellent antimicrobial effects against several strains of Listeria spp., including Listeria monocytogenes, which could cause infections in humans.[11−13] Vanillic acid isolated from kiwifruit is nontoxic and could impede virulence and infection caused by Serratia marcescens.(7)

Purified gold nanoparticles had no detectable cytotoxicity and immunogenicity and are biocompatible in macrophage cells.[14,15] Gold nanoparticles had been reported to be used for the diagnosis and therapy of diverse diseases.[16−18] Mesoporous silica nanoparticles (MSNs) have attracted the attention of most researchers working in the biomedical domain due to their excellent properties for drug delivery.[19,20] Both gold nanoparticles and MSNs are candidate nanoscale materials for drug delivery systems.[21,22] For example, MSNs have been used for integrating chemo-, gene- and photothermal substances to prepare a multifunctional antitumor nanoplatform.[23,24] Additionally, gold-modified porous silicon nanopillars had excellent antibacterial performance against S. aureus.(25) In this study, core–shell nanocomposite Au-MSNs were prepared by coating MSN onto gold nanoparticles. The natural antimicrobial compound vanillic acid was loaded into Au-MSN, and the obtained composites were termed VA@Au-MSN. The shape, morphology, particle size, Brunauer–Emmett–Teller surface area thermogravimetric property, and drug-loading efficacy of Au-MSN or VA@Au-MSN were detected according to responding methods. The cytotoxicity of VA@Au-MSN was determined in MC3T3-E1 mouse calvaria-derived cells. A clone formation assay and a live/dead bacterial observation test were performed to assess the antibacterial capacity of VA@Au-MSN.

Results and Discussion

Characterization of Au-MSNs and VA@Au-MSNs

The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that Au-MSN displayed a uniform spherical shape with a relatively smooth surface; while after loading vanillic acid, the surface of VA@Au-MSN was uneven and much rougher (Figure a,b). The diameter of the fabricated Au-MSN was 101.1 ± 8.5 nm (Figure c). N2 adsorption–desorption isotherms of Au-MSN presented a typical type-IV isotherm, which exhibited a mesoporous property[26] (Figure d). The BET surface area and pore size of Au-MSN were 458 m2/g and 2.65 nm, respectively, which were consistent with those of MSN.[27] MSN has been reported to have high drug-loading efficiency.[28,29] Herein, after loading vanillic acid, the BET surface area of VA@Au-MSN reduced to 72 m2/g and the diameter of VA@Au-MSN increased to 116.7 ± 9.2 nm. The TGA analysis showed that the loading efficiency of Au-MSN was 18.56% (Figure e).

Figure 1

Characterization of Au-MSN and VA@Au-MSN. Representative SEM and TEM images of Au-MSN (a) and VA@Au-MSN (b). Particle sizes (c) and N2 adsorption–desorption isotherms (d) for Au-MSN and VA@Au-MSN. (e) TGA curves recorded for VA, Au-MSN, and VA@Au-MSN. Au-MSN, gold-plated mesoporous silica nanoparticles; VA@Au-MSN, vanillic acid-loaded Au-MSN.

The heat conversion capacity of Au-MSN was assessed by a photothermal effect assay. As shown in Figure a,b, the temperature of the Au@MSN suspension could increase rapidly up to about 60 °C within 40 s under NIR 808 nm laser irradiation at a power density of 1.0 W/cm2 in vitro. The local temperature at the site injected with the Au-MSN solution in the mouse increased from 24 to 60 °C after 12 s of irradiation by the NIR 808 nm laser light (Figure c). The results indicated the great heat conversion capacity of Au-MSN and were consistent with Zhao et al.’s report that gold nanorod-loaded-multicompartment MSNs produced hyperthermia under NIR irradiation.[30]

Figure 2

Photothermal effect was assessed in vitro and in vivo. (a) Images of the temperature change under 808 nm NIR irradiation in vitro and in vivo. Temperature curves of Au-MSN under 808 nm NIR irradiation in vitro (b) and in vivo (c).

Measurement of Vanillic Acid Release

To determine the effects of temperature on the release performance of vanillic acid from VA@Au-MSN, vanillic acid release assays were performed at different temperatures. After 72 h of incubation, the cumulative release rate of vanillic acid from VA@Au-MSN was 78.95 ± 1.41% at 42 °C, while the release rate was 42.61 ± 1.71% at 37 °C (Figure ). The amount of vanillic acid released from VA@Au-MSN at 42 °C was significantly higher than that at 37 °C. These findings revealed the highly effective vanillic acid release triggered by the temperature.

Figure 3

Vanillic acid release curves from VA@Au-MSN at 37 and 42 °C.

Cytotoxicity of VA@Au-MSNs in MC3T3-E1 Cells

To investigate the cytotoxicity of Au-MSN and VA@Au-MSN in MC3T3-E1 cells, MC3T3-E1 cells were incubated with Au-MSN or VA@Au-MSN and the growth of the cells was observed by CLSM. After 24 h of incubation, there was no significant difference in the nuclear morphology and cell number of MC3T3-E1 cells among the control group, the group incubated with Au-MSN, and the group incubated with VA@Au-MSN (Figure ). After treating with Au-MSN or VA@Au-MSN, the cell viability was more than 85% even at a concentration of 0.5 mg/mL. All of these results indicated that Au-MSN and VA@Au-MSN had no obvious cytotoxicity to MC3T3-E1 cells and suggested its biosafety in the biological application of VA@Au-MSN for orthopedic infections.

Figure 4

Cytotoxicity of Au-MSN and VA@Au-MSN to MC3T3-E1 cells. (a) Confocal laser scanning microscopy observations of MC3T3-E1 cells incubated with Au-MSN or VA@Au-MSN for 24 h. For each panel, the images from left to right represented cell nuclei (blue fluorescence) stained with DAPI, F-actin (green fluorescence) stained with FITC, and the merged images of the left two. (b) Cell viability of MC3T3-E1 cells incubated with different concentrations of Au-MSN or VA@Au-MSN for 24 h.

Antibacterial Potential of VA@Au-MSNs

As the most common causative pathogen of orthopedic and device-related infections, Staphylococcus aureus (S. aureus) has been widely used in research studies for orthopedic infections.[31,32] Thus, the antibacterial potential of VA@Au-MSN was assessed by coculturing with S. aureus here. Compared with the control group and the group cocultured with Au-MSN, VA@Au-MSN and VA@Au-MSN + NIR groups had a significantly lower number of colony-forming units (Figures a and S1). Moreover, fewer colony-forming units obviously appeared in the VA@Au-MSN + NIR group than in the VA@Au-MSN group, suggesting NIR had a synergistic effect against the bacterial growth.

Figure 5

Antibacterial performance assessment of VA@Au-MSN against Staphylococcus aureus. (a) Antibacterial potential of Au-MSN and VA@Au-MSN with and without 808 nm NIR irradiation was assessed by the colony-forming assay. (b) Live (green fluorescence) and dead (red fluorescence) bacteria-incubated Au-MSN or VA@Au-MSN with and without 808 nm NIR irradiation. The scale bar is 50 μm.

As shown in Figure b, the control group and the group cocultured with Au-MSN had a large number of live bacteria (green fluorescence) and a few dead bacteria (red fluorescence), suggesting no antibacterial performance. VA@Au-MSN and VA@Au-MSN + NIR groups had a markedly reduced number of live bacteria and an increased number of dead bacteria. Additionally, the VA@Au-MSN + NIR group had fewer live bacteria and more dead bacteria than the VA@Au-MSN group. The results were consistent with those of the colony formation assay. NIR light irradiation could not only promote the release of antibacterial drugs from the thermosensitive hydrogel-based drug reservoir, but the resulting local hyperthermia induced the destruction of bacterial integrity.[33] Here, the generated local hyperthermia by the nanoparticles under NIR irradiation accelerated the release of VA, which blocked quorum sensing and thus inhibited biofilm production and bacterial virulence.[7] Finally, VA and the local hyperthermia exerted antibacterial effects in a synergistic way.

Conclusions

In conclusion, the core–shell gold nanospheres/mesoporous silica nanoparticles (Au-MSNs), which had great heat conversion capacity and high drug-loading property, were prepared by coating MSN onto gold nanospheres. Higher temperatures promoted vanillic acid release from vanillic acid-loaded Au-MSN (VA@Au-MSN), which had no detectable cytotoxicity to MC3T3-E1 mouse calvaria-derived cells. Notably, VA released from VA@Au-MSN and the local hyperthermia under NIR irradiation had excellent antibacterial activity against Staphylococcus aureus in a synergistic way. Therefore, VA@Au-MSN might be used for chemo-photothermal therapy of orthopedic infections.

Materials and Methods

Preparation of Au-MSNs and Vanillic Acid-Loaded-Au-MSNs

Au-MSNs were fabricated according to the reported method.[34,35] First, 1.5 mL of cetyltrimethylammonium bromide (CTAB, 0.2 M) and 5 mL of chloroauric acid (HAuCl4·4H2O, 0.05 mM) were mixed. After adding 0.6 mL of NaBH4 (0.01 M), the above solution was stirred for 3 min and maintained at 25 °C for 2 h. The obtained standby solution was stored for usage in the following steps. Second, 200 mL of CTAB (0.1 M), 10 mL of HAuCl4·4H2O (0.01 M), and 2 mL of AgNO3 (0.01 M) were mixed. After 3 min of reaction, the solution was supplied with 1.6 mL of ascorbic acid (17.6 mg/mL) and stirred for 5 s. Then, 4 mL of H2SO4 solution was added to the above mixture followed by 30 min of reaction. In the end, the reaction mixture and 0.5 mL of the above standby solution were mixed and kept for 12 h at 30 °C to prepare Au nanoparticles.

Subsequently, the Au nanoparticles were dispersed in 15 mL of ultrapure water. NaOH (150 μL, 0.1 M) was added to the Au nanoparticle solution. Then, the Au-MSN was prepared by adding 20% tetraethylorthosilicate three times. Vanillic acid-loaded-Au-MSN (VA@Au-MSN) was fabricated by adding the prepared Au-MSN to the vanillic acid solution.

Characterization of Au-MSNs and VA@Au-MSNs

Transmission electron microscopy (TEM, JEM-2100, JEOL, Japan) and scanning electron microscopy (SEM, JSM-7001F, JEOL, Japan) were performed to observe the shape and morphology of Au-MSN and VA@Au-MSN. The particle sizes of Au-MSN and VA@Au-MSN were detected by dynamic light scattering on a Malvern Zetasizer nanoZS 90 (Malvern, UK). N2 adsorption–desorption analysis was performed and the Brunauer–Emmett–Teller (BET) surface area was calculated. The weight loss of Au-MSN, vanillic acid, and VA@Au-MSN was measured by thermogravimetric analysis from 25 to 800 °C at a temperature rate of 20 °C/min in a nitrogen atmosphere at a rate of 20 mL/min.

Photothermal Effect Measurement

In a 1.5 mL Eppendorf tube or wound on the back of 4-week-old BALB/c mice, 1 mL of nanoparticle dispersion in phosphate-buffered saline (PBS, pH = 7.0) was added to the tube or 100 μL of nanoparticles in the wound and then irradiated by a near-infrared ray (NIR) 808 nm laser for 5 min. During each 30 s interval, thermal images and the temperature of the tube were recorded using a NIR thermal imaging camera (Fluke, Ti400) with a laser power density of 1.0 W/cm2.

Vanillic Acid Release Assay

Vanillic acid released from VA@Au-MSN was detected according to the reported high-performance liquid chromatography (HPLC) method.[36] VA@Au-MSN was dispersed in PBS and maintained at 37 or 42 °C. The vanillic acid concentration in PBS at different time points was measured by HPLC. The ratio of the vanillic acid amount in PBS to the total amount of vanillic acid was as the cumulative vanillic acid released from VA@Au-MSN.

Cell Culture

The MC3T3-E1 mouse calvaria-derived cell line was purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). MC3T3-E1 cells were cultured in Gibco minimum Eagle’s medium α (11900024, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humid atmosphere of 5% CO2 and 95% air. The medium was replaced every other day.

Cytotoxicity Test

MC3T3-E1 cells (5 × 103 cells/cm2) were incubated with Au-MSN and VA@Au-MSN for 24 h. The cells without any treatment were used as a control. After half an hour of fixation using 4% paraformaldehyde, MC3T3-E1 cells were stained with DAPI and FITC-phalloidin, respectively, for 15 min. Then, the fluorescence in MC3T3-E1 cells was observed using confocal laser scanning microscopy (CLSM, Leica Fluoview FV 1000, Olympus, Japan) with 405 and 562 nm laser excitation, respectively.

The cytotoxicity of Au-MSN and VA@Au-MSN to MC3T3-E1 cells was also determined by a cell counting kit-8 (CCK-8) assay. After treating with different concentrations of Au-MSN or VA@Au-MSN (0, 0.025, 0.0625, 0.125, 0.25, and 0.5 mg/mL) for 24 h at 37 °C, 100 μL of cell suspension (3 × 104 cells/mL) was mixed with 10 μL of CCK-8 reagent for 1 h at 37 °C. The absorbance at 450 nm was detected. The cells without adding Au-MSN or VA@Au-MSN were considered as a control.

Antibacterial Assessment

S. aureus (ATCC 25923) was purchased from the American Type Culture Collection. S. aureus (1 × 106 colony-forming units/mL) was incubated with Au-MSN or VA@Au-MSN for 24 h at 37 °C in Mueller-Hinton Broth medium. The bacteria treated with VA@Au-MSN with 808 nm NIR irradiation were termed as VA@Au-MSN + NIR group. S. aureus without any treatment was used as a control. Then, S. aureus suspension was plated onto tryptic soy agar and cultured at 37 °C for 24 h. The formative colonies on the tryptic soy agar reflected the antibacterial potential of Au-MSN or VA@Au-MSN against S. aureus.

After 24 h of coculture, S. aureus in the control, Au-MSN, VA@Au-MSN, and VA@Au-MSN + NIR groups were stained using the Live/Dead BacLight Bacterial Viability Kit (L-7012, Thermo Fisher Scientific, Waltham, MA). The fluorescence images were obtained by CLSM.

Statistical Analysis

The data was represented as mean ± standard deviation. The statistical analysis was performed using the Student t-test in SPSS software (version 20.0, SPSS, Chicago, IL). The P value < 0.05 was considered a statistically significant difference.