Bacterial infections have caused many human diseases, so effective treatment of bacterial infections is an urgent problem that needs to be solved. In this work, a multifunctional therapeutic core–satellite nanoplatform (ASAN NPs) with antibacterial and anti-inflammatory properties was designed and synthesized. First, mesoporous silica-coated silver nanoparticles (Ag NPs) with different sizes were synthesized via a simplified method that improved the stability and drug-carrying capacity of the Ag NPs. Then, the NPs were loaded with anti-inflammatory and antibacterial naringin. The characterization results showed that the ASAN NPs were successfully synthesized, with an average particle size of 110 ± 15 nm. In vitro antibacterial test results showed that the synergistic effect of Ag NPs and naringin enhanced the antibacterial activity in a dose-dependent manner. The ASAN NPs achieved high-efficiency antibacterial activity by increasing the permeability of the cell membrane, destroying the integrity of the bacteria, and significantly increasing the level of bacterial reactive oxygen species. The in vivo antibacterial results showed that the ASAN NPs had highly effective anti-infective and anti-inflammatory activities and did not show obvious side effects. Thus, ASAN NPs appear to be promising antimicrobial agents with good biocompatibility, potent antibacterial activity, and anti-inflammatory activity.
Pathogenic bacteria invade the blood and produce toxins that can cause local inflammation or systemic infections.1,2 Antibiotics are the most widely used method to treat such bacterial infections, but the frequent use of antibiotics has reduced their effectiveness;2–4 therefore, the design of new highly effective antibacterial and anti-inflammatory drugs is a clinical problem that must be solved.
Nano-copper, nano-zinc oxides, and nano-selenium are potential drugs for the treatment of bacterial infections.5–8 In many cases, these materials are still in the research stage due to their unclear antibacterial mechanism and in vivo toxicity;9 however, nano-silver has been widely studied and is considered a safe and efficient antibacterial nanoparticle.10–12 For example, Morones et al. found that nano-silver particles with a diameter of 1–10 nm had a significant antibacterial effect on gram-negative bacteria.13 Shahverdi et al. proposed that the combination of nano-silver and antibiotics to treat bacterial infections improved the antibacterial effect of antibiotics.14 The antibacterial mechanism of nano-silver is generally considered to be a multi-factor, multi-path, and multi-target process; therefore, the physical and chemical properties of nano-silver, including the size, shape, and surface modification, will greatly impact its antibacterial activity.15 Martínez-Castaón et al. found that the antibacterial activity of nano-silver decreased upon increasing the particle size.16 Smaller nano-silver particles will release silver ions faster, resulting in a higher toxicity due to the rapid production of a high concentration of silver ions;17 therefore, methods to balance the silver ion concentration and high-efficiency antibacterial activity are particularly important.
Mesoporous silica nanoparticles have shown excellent stability and biocompatibility, large specific surface areas, and easy modification.18,19 They have been widely used in the surface-stable modification of nanomaterials and the controlled release of drugs.20,21 Tang et al. reported significant achievements in drug delivery using mesoporous silica nanoparticles as drug carriers.22 In particular, the targeted strategy using mesoporous silica to deliver drugs breaks biological barriers. Song et al. coated a copper sulfide nanomaterial with silica shell, which improved its hydrophilicity, biocompatibility, and its biological applications.23 Naringin is a flavonoid compound with a variety of biological properties, especially anti-inflammatory and antibacterial effects. Previous studies have found that naringin and its metabolites can reduce inflammation targets (NF–B, iNOS, and COX-2) and chronic inflammation markers,24,25 but naringin’s extremely low water solubility and easy hydrolysis under acidic conditions limit its biological applications;26,27 therefore, the mesoporous silica delivery strategy may improve the delivery of naringin.
In this work, we used a simple method to synthesize a nano-platform with strong antibacterial and anti-inflammatory effects. First, mesoporous silica was coated with two nano-silver particles of different sizes to improve the stability and drug-carrying capacity of nano-silver. Then, naringin was loaded into the mesoporous silica. The nano-platform uses small nano-silver particles to quickly release silver ions, while the large nano-silver particles continuously release silver ions for antibacterial purposes. The controlled release of silver ions is expected to reduce the toxicity of nano-silver. The rapid release of naringin is the main reason for the antibacterial and anti-inflammatory properties of the nano-platform. Interestingly, we found that the synergy between nano-silver and naringin increased the biological activity of this platform. More importantly, the nano-platform has no toxic side effects and therefore has potential value in clinical applications.
II. MATERIALS AND METHODS
Silver nitrate (AgNO3, 99.8%), polyvinyl pyrrolidone (PVP, K30), tetraethyl orthosilicate (TEOS, 98%), ethylene glycol (EG, 99.5%), absolute ethanol (99.7%), cetyltrimethylammonium bromide (CTAB, 99%), NH4NO3 (98%), and ammonia (28%–32%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Naringin (95%), 2′,7′-dichlorodihydroflfluorescein diacetate (DCFH-DA, 97%), acridine orange (AO, 99%), ethidium bromide (EB, 95%), 3,30-dipropylthiadicarbo cyanine iodide (DISC3-5, 98%), and propidium iodide (PI, 98%) were purchased from Sigma-Aldrich. Escherichia coli (E. coli, ATCC 8739) and Staphyloccocus aureus (S. aureus, ATCC 6538) strains were acquired from the Department of Pharmacy of Yiyang Medical College (China).
B. Synthesis of Ag@mSiO2@Ag NPs
Ag@mSiO2@Ag NPs were fabricated using the following process. AgNO3 (45 mg) and PVP (300 mg) were mixed with 18 ml of EG and then heated at 120 °C for 1 h under vigorous stirring to obtain a yellow-green Ag NP colloidal solution.28 CTAB (0.35 g) and 8.5 ml Ag NP colloidal solution were added to a mixture of 25 ml absolute ethanol and 4 ml deionized water. Ammonia (4.0 ml) and 90 µl TEOS were added separately and stirred vigorously for 1 h at room temperature. After the reaction, unreacted substances were removed by centrifugation (10 000 g, 10 min), and the products were washed three times with ethanol and deionized water. The product was dispersed in 30 ml of NH4NO3 (5 mg/ml) ethanol solution and reacted at 50 °C for 2 h under vigorous stirring to remove the CTAB template by ion exchange. The obtained NPs were washed three times with ethanol and deionized water.
C. Loading and release of naringin
Ag@SiO2@Ag NPs (2 mg) were dispersed in 5 ml of ethanol solution of naringin (2.5 mg/ml) and stirred at room temperature for 48 h. Ag@mSiO2@Ag NPs loaded with naringin were collected by centrifugation (10 000 g, 10 min) and then washed three times with absolute ethanol to remove unbound naringin. All supernatants were collected and measured with an ultraviolet–visible (UV–Vis) spectrophotometer at 284 nm to calculate the naringin payload in Ag@mSiO2@Ag NPs. The naringin loading of the NPs = (weight of naringin loaded in NPs)/(total weight of NPs). The in vitro release of naringin from ASAN NPs was studied by a dynamic dialysis method. Briefly, 1 ml of ASAN NPs (1 mg/ml) was added to a dialysis bag (molecular weight cutoff: 10 kDa). The dialysis bag was immersed in a release medium (pH 7.4, 0.001M PBS containing 0.1% Tween 80/LB liquid medium), and the release study was carried out at 37 °C and 200 rpm. At each fixed time interval, the release medium was refreshed. The naringin in the obtained solution was quantified by a UV–vis spectrophotometer.
The size and morphology of the NPs were investigated via transmission electron microscopy (TEM; HT7700; Hitachi, Japan) and scanning electron microscopy (SEM; S-4800; Hitachi, Japan). The crystal structure of the NPs was determined by x-ray diffraction (XRD; D/max-2550; Rigaku; Japan). X-ray photoelectron spectroscopy (XPS, ESCALAB 250; Thermo Fisher Science, America) was used to identify the chemical composition of the sample surface. The zeta potential and a particle size analyzer (ZetaPALS; Brookhaven, USA) were used to measure the size and potential of NPs. UV–vis (Scinco Co., Korea) and Fourier-transform infrared spectroscopy (FTIR; Nicoletteis50; Thermo Fisher Science, America) were used for structural characterization.
E. In vitro antibacterial activity test of ASAN NPs
Log-phase E. coli and S. aureus (OD600nm = 0.5) were, respectively, inoculated into the LB liquid medium containing 64 µg/ml of naringin/Ag@mSiO2@Ag/ASAN solution (sterile water as the control group) and cultured at 37 °C for 12 h. The colony-forming unit (CFU) was used to analyze the inhibitory power of the sample against bacterial reproduction, and all tests were repeated at least three times.
Log-phase E. coli and S. aureus were diluted to 1 × 10−6 CFU/ml and then added to a 96-well plate and incubated with 0–64 µg/ml naringin/Ag@mSiO2@Ag/ASAN solution at 37 °C for 12 h. Bacterial viability was measured at OD600nm by a multifunctional microplate reader (Tecan Infinite, 200Pro, Austria), and each concentration was measured at least three times.
100 µl of the diluted E. coli and S. aureus solutions was evenly spread on the LB solid medium. Paper pieces containing 64 µg/ml of different samples were placed on the culture medium and incubated overnight at 37 °C. This measurement was repeated at least three times. The size of the inhibition zone was used to evaluate the inhibitory effect of the sample.
F. Fluorescence microscope observation
Log-phase E. coli and S. aureus (OD600nm = 0.5, 1 ml) cells were collected by centrifugation (3000 rpm, 5 min), washed three times with phosphate buffer solution (PBS, 0.01M, pH 7.4), and then re-suspended in 1 ml PBS. Different samples (64 µg/ml) were used to treat bacteria for 12 h. Live/dead staining was used according to the method provided by the AO/EB cell staining kit. Bacterial cells were imaged using fluorescence microscopy (Ti-E, Nikon, Japan).
Bacterial cells were collected in the same method. Bacterial cells were treated with naringin/Ag@mSiO2@Ag/ASAN solution for 0–2 h, stained with 10 µM DCFH-DA in the dark at room temperature for 30 min, and washed twice with PBS. The total bacterial reactive oxygen species (ROS) was detected by fluorescence microscopy and a multifunctional microplate reader (Tecan Infinite, 200Pro, Austria).
G. Bacterial integrity assay
The membrane permeability test samples were prepared by a reported method.18,29 Log-phase E. coli and S. aureus (OD600nm = 0.5, 100 µl) were incubated with 64 µg/ml naringin/Ag@mSiO2@Ag/ASAN solution at 37 °C for 0–12 h, respectively, and stained with PI (3 µM). After the bacterial solution was diluted tenfold, it was incubated with 4 µM DISC3-5 for 1 h, and then, 100 mM KCl was immediately used for ion balance. A multifunctional microplate reader (excitation: Ex = 622 nm; emission: Em = 670 nm) was used to measure the fluorescence of the samples.
Bacterial samples were prepared for SEM observations by a reported method.12 Briefly, log-phase E. coli and S. aureus (OD600nm = 0.5, 3 ml) were incubated with 64 µg/ml naringin/Ag@mSiO2@Ag/ASAN solution at 37 °C for 12 h. The bacteria were collected by centrifugation (3000 g, 5 min) and fixed with 5% glutaraldehyde solution for 4 h at 4 °C. The cells were dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol and acetone for 15 min. The bacterial cells were dried by supercritical carbon dioxide and observed by SEM.
H. Evaluation of anti-infective activity in vivo
ASAN NPs are used for the anti-infection and anti-inflammatory treatment of infected wounds in Kunming mice (KM-mice). Briefly, eight-week-old KM-mice (male, 25 g) were grouped (n = 5) and adaptively fed for one week. The mice were anesthetized by intraperitoneal injection of 1% pentobarbital. Then, a round skin wound with a diameter of 2 cm was cut out from the left and right armpits, and a PBS suspension of E. coli and S. aureus (OD600nm = 0.5, 200 µl) was injected into the left and right wounds, respectively (injection depth: 5 mm). Naringin/Ag@mSiO2@Ag/ASAN solution (20 mg/kg) was injected through the tail vein every day for treatment. The treatment continued for 12 days, and the mice were euthanized on the 14th day. Mouse wound tissues were collected for immunohistochemistry (IHC) and hematoxylin-eosin (H&E) staining analysis. All animals were cared for in accordance with the guidelines outlined in the Guidelines for the Care and Use of Laboratory Animals. All animal experiments were conducted according to the protocols approved by the Yiyang Medical College Animal Center (Permit Number: yyyz2020-1102).
I. Toxicity evaluation of ASAN NPs
The in vitro toxicity of the drug to mouse embryonic fibroblasts (NIH/3T3 cells) and rat cardiomyocytes (H9C2 cells) was assayed by MTT assay. Briefly, cells seeded in the 96-well plate were treated with different concentrations of naringin, Ag@mSiO2@Ag NPs, and ASAN NPs at 37 °C for 24 h. Then, 20 µl MTT (5 mg/ml) was added and incubated for another 5 h. Then, the supernatant was removed and 150 µl DMSO was added to solve the formazan crystals. Absorbance was detected at 570 nm by a microplate reader to reflect the cell viability.
KM-mice (n = 5) were treated with 20 mg/kg ASAN NP solution via tail vein injection every day. After 21 days, all mice were euthanized, and the main organs of the mice (heart, liver, spleen, lung, and kidney) were collected and stained by H&E staining for histopathological observation. Blood samples were also collected from mice, and the blood biochemical indicators (urea nitrogen and cerealthirdtransaminase; ALT) were measured with an ACL-200 automatic blood analyzer. All experiments were repeated at least three times.
We studied the effects of different doses of ASAN NPs on the survival of mice and evaluated their potential toxicity. Briefly, KM-mice (n = 5) were injected with 20–200 mg/kg of ASAN NP solution through the tail vein every day for 21 consecutive days. Changes in behavior were recorded daily, and then the survival rate was calculated. All experiments were repeated at least three times.
J. Statistical analysis
All data and images were obtained from three independent experiments. Data were presented as mean ± SD. Statistical analysis was performed by SPSS 13.0 (SPSS, Inc.). An independent t-test was used for two-group comparisons, and one-way ANOVA is used for multiple-group comparisons, with suitable post hoc analysis. Significant difference was defined as #/&/*P < 0.05, and extremely significant difference was defined as ##/&&/**P < 0.01.
III. RESULTS AND DISCUSSION
A. Characterization of nanomaterials
Mesoporous silica shell cannot coat directly on such electronegative nanoparticles because of the hydrolysate of TEOS with negative charge. Hence, the surfactant-CTAB was used to change the chemistry of the surfactant head groups on the surface of the Ag NPs. The bilayer of cationic CTAB coats around the Ag NPs by electrostatic interaction and hydrophobic interaction and changes the nanocrystals from negative charge to positive charge. The silica shell layer was formed by the hydrolysis of the ethyl orthosilicate on the silver nanoparticles, and the remaining small size silver nanoparticles in the reaction medium form a satellite structure (about 5 nm in size) inside and outside the SiO2. The structure and morphology of ASAN NPs were characterized by TEM and SEM. Figure 1(a) shows spherical nano-silver with a diameter in the range of 0–80 nm [Fig. 2(c)]. Figure 1(b) clearly reveals that Ag@mSiO2@Ag NPs had a core–satellite structure with large Ag NPs as the core and a hybrid arrangement of small Ag NPs in mesoporous silica. Figures 1(c) and 1(d) show the SEM and TEM images of ASAN NPs, respectively. The average diameter was 110 ± 15 nm [Fig. 2(c)], and the average thickness of mesoporous silica was 23 ± 5 nm, which gives the NPs excellent drug-carrying capacity. Elemental analysis was used to determine the elemental composition of ASAN NPs. As shown in Fig. 1(e), ASAN NPs are mainly composed of C, O, Si, and Ag elements.
XPS was used to analyze the valency of Ag and Si atoms in Ag@mSiO2@Ag NPs [Fig. 2(a)]. The binding energy region from 365 to 377 eV was fitted to a bimodal function. The peaks at 368.5 and 374.2 eV correspond to the atomic orbitals of Ag 3d5 and Ag 3d3, respectively, which correspond to Ag0. The peak at 103.5 eV corresponds to the Si 2p atomic orbital, which is consistent with the atomic orbital of Si in silicon dioxide. The XRD characteristic peaks of the NPs [Fig. 2(b)] were consistent with the characteristic peaks of cubic Ag (JCPSD 00-004-0783). In addition, 2θ has an obvious dispersion peak at 20°–30°, which is a typical feature of amorphous SiO2, which helps in stabilizing the performance of Ag. In the UV–vis spectrum of the ASAN NPs [Fig. 2(d)], the characteristic peaks of naringin and Ag NPs are visible at 230 and 276 nm, respectively. The characteristic absorption peak of ASAN NPs at 460 nm was attributed to the red-shift in the absorption peak of Ag NPs. As shown in the FTIR spectrum in Fig. 2(e), the characteristic peaks of ASAN NPs at 1052, 1579, and 1649 cm−1 are attributed to the C–OH stretching vibration, C=C stretching vibration, and C=O stretching vibration of naringin, respectively. ASAN NPs exhibited a typical O–Si–O stretching vibration at 1074 cm−1. The zeta potential of Ag NPs is about −19.8 mV, and the zeta potentials of Ag@mSiO2@Ag NPs and ASAN NPs are −30.5 and −10.2 mV, respectively [Fig. 2(f)]. The change in potential indicates that each step of the modification was successful. These results indicate that ASAN NPs were successfully synthesized.
The regression equation shown in Fig. 2(g) was used to estimate the naringin content in the supernatant. The final drug loading of ASAN NP was 29.2%. As shown in Fig. 2(h), naringin was quickly released from the dialysis bag, indicating that the dialysis bag did not affect naringin diffusion. The cumulative release of naringin in PBS and LB media for 12 h was higher than 55%. As shown in Fig. 2(i), the transmittance of the NP changed less than 5% over 72 h, indicating that it has high dynamic stability and almost no particle sedimentation.
B. Analysis of the antibacterial activity of ASAN NPs
The minimum inhibitory concentration (MIC) was determined to initially analyze the in vitro antibacterial activity of the samples.30 As shown in Table I, the MIC value of ASAN NPs (5.3 ± 0.14 µg/ml) for E. coli was similar to that of ampicillin (4.8 ± 0.15 µg/ml), and the MIC value for S. aureus was significantly higher than those of naringin (6.4 ± 0.10 µg/ml) and Ag@mSiO2@Ag NPs (14.2 ± 0.25 µg/ml). This may be attributed to the synergistic antibacterial activity of Ag NPs and naringin. Compared with other antibacterial agents, ASAN NPs significantly inhibited the growth of gram-positive and gram-negative bacteria at low doses.
|Bacterial .||Ag NPs (μg/ml) .||Ag@mSiO2@Ag (μg/ml) .||Naringin (μg/ml) .||ASAN NPs (μg/ml) .||Ampicillin (μg/ml) .|
|E. coli||10.2 ± 0.21||10.5 ± 0.14||28.4 ± 0.32||5.3 ± 0.14##||4.8 ± 0.15|
|S. aureus||5.7 ± 0.13||6.4 ± 0.10||14.2 ± 0.25||3.6 ± 0.12&&||0.7 ± 0.08|
|Bacterial .||Ag NPs (μg/ml) .||Ag@mSiO2@Ag (μg/ml) .||Naringin (μg/ml) .||ASAN NPs (μg/ml) .||Ampicillin (μg/ml) .|
|E. coli||10.2 ± 0.21||10.5 ± 0.14||28.4 ± 0.32||5.3 ± 0.14##||4.8 ± 0.15|
|S. aureus||5.7 ± 0.13||6.4 ± 0.10||14.2 ± 0.25||3.6 ± 0.12&&||0.7 ± 0.08|
The CFU method was used to analyze the antibacterial activity of the ASAN NPs. As shown in Fig. 3(a), the antibacterial activities of naringin and Ag@MSiO2@Ag NPs against E. coli and S. aureus were limited compared with the control group. ASAN NPs had the greatest antibacterial activity (control group: E. coli 3.41 × 108 CFU/ml; S. aureus 4.03 × 108 CFU/ml, ASAN NPs: E. coli 3.9 × 107 CFU/ml; S. aureus 2.75 × 107 CFU/ml). The Ag@mSiO2@Ag NPs enhanced the antibacterial activity of naringin, and the two showed coordinated antibacterial activity. Figure 3(b) shows that the ASAN NPs significantly inhibited bacterial viability in a dose-dependent manner, and the bacterial viability was less than 10% at a concentration of 64 µg/ml. The size of the inhibition zone produced by the drug-sensitive pieces on a bacteria-containing LB solid medium further demonstrated the inhibitory effect of the sample on E. coli and S. aureus. As shown in Figs. 3(c) and 3(d), the inhibitory radius depends on the sample being tested. The ASAN NPs had a stronger inhibitory effect than naringin alone and Ag@mSiO2@Ag NPs. The results showed the same conclusion as that of the CFU and bacterial survival tests.
C. Dead/live bacteria stain
Dead/live staining of E. coli and S. aureus was performed using the fluorescent dye EB/AO to further determine the bactericidal ability of the sample. Living cells were labeled with AO and emit green fluorescence, while EB only penetrates damaged cells and emits red fluorescence. Figures 4(a) and 4(b) shows that almost all bacteria in the control group showed green fluorescence, which indicates that many bacteria survived. Part of the bacteria treated with naringin and Ag@mSiO2@Ag NPs were stained red by EB, indicating that naringin and Ag@mSiO2@Ag NPs have limited ability to kill E. coli and S. aureus. ASAN NPs caused significant bacteria death, as indicated by the significant red fluorescence, and the number of deaths was higher than by naringin and Ag@mSiO2@Ag NPs, which is consistent with the previous results.
D. Preliminary study on the antibacterial mechanism
SEM was used to observe the morphological changes and cell wall integrity of E. coli and S. aureus treated by naringin/Ag@mSiO2@Ag NPs/ASAN NPs. The SEM results [Fig. 5(a)] showed that untreated E. coli and S. aureus had intact cell morphologies and cell walls. Adding naringin caused the two bacteria to show signs of atrophy and depression. After incubating the bacteria with Ag@mSiO2@Ag NPs for 12 h, the bacteria’s cell walls became wrinkled and damaged. After treatment with ASAN NPs, the bacteria were severely deformed, their contents flowed out, and the integrity of the cell membrane and cell wall was destroyed. This shows that ASAN NPs simultaneously destroyed the cell walls of gram-positive bacteria and gram-negative bacteria and killed them by destroying their integrity.
To further explore the degree of destruction of bacterial integrity by ASAN NPs, the cells were stained with fluorescent dyes DISC3-5 and PI. Fluorescence is quenched when DISC3-5 binds to the cytoplasmic membrane of the bacteria. When the bacteria are destroyed, DISC3-5 is released, and the fluorescence is recovered. PI binds to the nucleic acid of the destroyed bacteria and emits red fluorescence;31,32 therefore, the higher the fluorescence intensity of DISC3-5 and PI detected, the more severe the bacterial damage. Figures 5(b) and 5(c) show that the fluorescence intensity of the control group did not increase significantly. In contrast, the fluorescence intensity of the bacteria treated with ASAN NPs was much stronger than that of bacteria treated with naringin and Ag@mSiO2@Ag NPs. This shows that the cytoplasmic membrane permeabilities of the bacteria treated with ASAN NPs were greatly enhanced. This was attributed to the synergistic effect of naringin and Ag@mSiO2@Ag, which killed bacterial cells by destroying the permeability of the bacterial membrane and cell integrity.
To further explore what kind of biological changes lead to bacterial death after naringin/Ag@mSiO2@Ag NPs/ASAN NPs treatment, the total reactive oxygen species (ROS) concentration in bacteria was evaluated by an ROS probe. As shown in Fig. 6(a), the number of bacteria-producing ROS and the fluorescence intensity after treatment with ASAN NPs were significantly higher than those in the control group. Figures 6(b) and 6(c) indicate that the bacteria in the naringin treatment group produced a lower level of ROS similar to the control group, while the ROS levels of the bacteria treated with ASAN NPs were significantly higher. The increase in ROS may be due to the destruction of the integrity of the bacterial cell membrane and cell wall by ASAN NPs, which destroys the enzymes related to the respiratory chain of bacteria, produces a large number of ROS, and further destroys the bacteria.33 These results indicate that the antibacterial activity of NPs was related to an increase in ROS.
E. Anti-infection and anti-inflammatory efficiency of ASAN NPs in vivo
To compare the relative effectiveness of the three samples in vivo, their ability to heal infected wounds and anti-inflammatory properties were analyzed. The construction of the in vivo model is shown in Fig. 7(a). The left and right armpit wounds of the mouse were infected by E. coli and S. aureus, respectively. Different samples were injected through the tail vein for treatment, and the subsequent H&E staining and immunohistochemistry (IHC) were used to evaluate the anti-infection and anti-inflammatory capabilities of the samples. Figure 7(b) shows a comparison of the H&E-stained images of the treated group and the untreated group. Sections of the untreated infected group showed the most severe infection compared with the healthy tissue. Sections of the infected group treated with naringin and Ag@mSiO2@Ag NPs showed different degrees of infection. In contrast, no obvious infection was observed in the sections of the infected group treated with ASAN NPs, similar to the tissues of healthy mice. Bacterial infections cause severe inflammation, which is evidenced by an increase in 1L-1β [Fig. 7(c)]. Naringin and Ag@mSiO2@Ag NPs reduced the inflammation caused by bacterial infections to varying degrees; however, the ASAN NPs significantly reduced the inflammatory response, which appeared to be similar to the control group. These results indicate that naringin and Ag@mSiO2@Ag NPs synergistically reduced the wound infection and inflammation caused by bacteria, which can help accelerate wound healing.
F. Biological toxicity evaluation of ASAN NPs
MTT assay was used to analyze the effect of ASAN NPs (0–128 µg/ml) on the survival rate of rat cardiomyocytes (H9C2) and mouse embryonic cells (NIH/3T3) and to evaluate its toxicity in vitro. As shown in Figs. 8(a) and 8(b), naringin has almost no toxicity, while Ag@mSiO2@Ag NPs was the most toxic. At the experimental concentration of ASAN NPs (64 µg/ml), the cell survival rate was close to 90%, suggesting that ASAN NPs had no significant toxicity in vitro. ASAN NPs (20 mg/kg) were continuously injected to further evaluate their in vivo toxicity, and the physiological and histological changes of mice were observed. As shown in Figs. 8(c) and 8(d), after 21 days of continuous injection of ASAN NPs, the weight of the mice did not change significantly, and no loss of appetite or abnormal behavior was observed. The survival rate of the mice was 100%. Urea nitrogen and ALT are important indicators of kidney and liver functions. Blood analysis showed [Figs. 8(e) and 8(f)] that ASAN NPs had no significant effect on the blood urea nitrogen and ALT levels in mice, indicating that the liver and kidney functions of mice were normal. The histopathological results of H&E staining showed [Fig. 8(g)] that the in vivo administration of ASAN NPs did not cause obvious pathological changes in the heart, liver, spleen, lung, or kidney. Pulmonary fibrosis was not observed in the lung tissue specimens, the structure of the glomerulus was normal, and no obvious histopathological lesions were observed in the liver. Taken together, these results revealed that ASAN NPs showed good in vitro and in vivo safety and low toxicity, demonstrating their suitability for potential clinical applications.
We synthesized a highly effective antibacterial and anti-inflammatory nano-platform based on a satellite–core structure with two size distributions composed of nano-silver, mesoporous silica, and naringin. The advantage of this platform is that it utilizes the synergistic effect between nano-silver with different size distributions and naringin, thereby exhibiting highly effective bacteriostasis, rapid sterilization, and excellent anti-inflammatory and anti-infective activities. ASAN NPs are highly stable, and the preliminary toxicity analysis showed that they have no obvious in vivo toxicity. In the antibacterial experiments, the results of OD600, CFU, MIC, and drug-sensitive tablets proved that ASAN NPs have significant antibacterial activity. Preliminary evaluation of the antibacterial mechanism of ASAN NPs showed that NPs changed the permeability of the bacterial cell membrane, allowing ASAN NPs to enter the bacteria and to significantly increase their ROS, which destroyed the integrity of the bacteria; thus, they exhibited highly effective antibacterial activities. The results of in vivo antibacterial experiments showed that bacterial infection wounds could be treated by ASAN NPs, which significantly reduced inflammation and accelerated wound healing. In conclusion, ASAN NPs can be used as multifunctional biomedical materials with strong antibacterial activity and high anti-inflammatory activity. As such, ASAN NPs have substantial potential for use in clinical applications as a new generation of effective antibacterial agents.
This work was supported by the Scientific Research Project of the Education Department of Hunan Province (Grant No. 20B590), the Provincial Natural Science Foundation of Hunan (Grant No. 2017JJ3234), the Foundation of Zhejiang Educational Committee (Grant No. Y201839522), and the Jinhua Science and Technology Project (Grant No. 2020-4-096).
There are no conflicts to declare. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
M.T. and D.H. contributed to this experiment design. L.W. and D.Z. performed the experiments. M.T. and L.W. wrote this manuscript. All authors contributed to the data analysis and revised the manuscript.
The data that support the findings of this study are available from the corresponding authors upon reasonable request.