Cetyltrimethylammonium bromide (CTAB), absolute ethanol, silver nitrate solution (AgNO3, ≥ 99.0%), formaldehyde (HCHO), branched PEG-g-PEI, ribonuclease A (RNase A), and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, USA). Sodium hydroxide solution (NaOH), formaldehyde solution (37%), ethyl acetate (EA), and tetraethyl orthosilicate (TEOS) were obtained from Macklin Co., Ltd (Shanghai, China). Ammonium nitrate (NH4NO3) and CFL were from Aladdin Reagent Co., Ltd (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), luria-bertani (LB) broth, penicillin-streptomycin solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and lipofectamineTM 3000 (Lipo3000) were purchased from Gibco (Thermo Fisher, USA). Cell Counting Kit-8 (CCK-8) and lipopolysaccharide (LPS) were from Biosharp Co., Ltd (Beijing, China). siTNF-α, Cy3-siRNA-negative control (Cy3-siNC) was synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Trypsinization (0.25%, without EDTA) was obtained from Solarbio Biotech Co., Ltd (Beijing, China). TRIcom reagent was from TIANMO BIOTECH Co., Ltd (Beijing, China). Evo M-MLV RT kit was purchased from Accurate Biology Co., Ltd (Hunan, China). Stormstar Sybr Green qPCR Master Mix was from DBI Bioscience Co., Ltd (Shanghai, China). Mouse TNF-α ELISA kit was purchased from Abcam (ab208348, UK). Escherichia coli (E. coli, CMCC44103) and Staphylococcus aureus (S. aureus, ATCC6538P) were obtained from the China General Microbiological Culture Collection Center.
Preparation of PEG-g-PEI-modified mesoporous silica-coated silver (AMP)
AM was prepared as described Song et al.  and Wang et al.  with minor modifications. Firstly, 0.3 mL of NaOH aqueous solution (2 M) and 0.1 g of CTAB were added to 50 mL of deionized water for incubation at 37°C for 30 min. And then, 0.3 mL of HCHO solution (1 M) and 1 mL of AgNO3 solution (0.1 M) were added under stirring. Subsequently, 0.5 mL of TEOS was dropped into the reactive mixture. All the ingredients were then continuously stirred at 80°C and refluxed for 2 h. The resultant precipitate was collected via centrifugation at 8000 rpm for 10 min and washed with ethanol for three times. In order to remove the surfactant template CTAB, 0.06 g of NH4NO3 was added to the NPs dispersed in 60 mL of ethanol solution under a sonic bath for 2 h. After drying for 120 min at 60°C, the AM was obtained without the template. And then, 10 mg of AM was dissolved in 10 mL of deionized water, and 0.5 mL of PEG-g-PEI solution (100 mg/mL) was dropped into the solution under stirring (300 rpm/min, 25°C) overnight. Finally, AMP (1 mg/mL) was obtained through centrifuging at 12000 rpm/min for 15 min, discarding the supernatant, and washing the precipitation with deionized water for three times to remove the excess PEG-g-PEI.
Ciprofloxacin loading by AMP
In order to load CFL into drug carriers, AMP (5 mg) was mixed with CFL aqueous solution (500-4000 μg/mL, 5 mL) under stirring overnight at 25°C. Then, the mixture was separated by centrifugation (8000 rpm/min, 5 min) and washed several times until there was no free CFL in the supernatant. The amount of free CFL in the supernatant was calculated from a calibration curve based on the absorbance intensity at 275 nm by UV-vis (TP-720 spectrometer, Tianjin Tuopu Instrument Co., Ltd). The percentage of CFL loading into AMP was calculated as follows:
Where the moriCFL, msupCFL, and mAMP represent the mass of original CFL, CFL in the supernatant, and AMP, respectively. The LE represents the loading efficiency.
Drug release from AMP loaded with CFL (AMPC)
To detect the release of CFL from AMPC, the AMPC (2 mg) were dispersed in PBS (pH 7.4, 2 mL) and transferred into a dialysis bag with a molecular weight cut-off of 1000 Da and kept in PBS (50 mL) on a shaking table at 37°C for 48 h. After 2 mL of the solution was removed at different time points, the drug release efficiency was measured by UV-vis at 275 nm. In order to keep the solution volume constant, 2 mL of fresh PBS needed to be added after each sampling.
To study the release of Ag from AMPC, the AMPC was suspended in an LB culture medium. After the mixture was incubated at 37°C, the UV-vis adsorption of the AMPC solution was monitored over a time period. The amount of consumed Ag was detected at 417 nm using a microphone reader (Bio-teak, Epoch-2).
Preparation and characterization of AMP loaded with siRNA ([email protected])
First, AMP and siNC (sense: 5′-CGAAGUGUGUGUGUGUGGC-3′, antisense: 5′-GCCACACACACACACUUCG-3′) with different weight ratios (0:1, 7.5:1, 15:1, 30:1, 60:1, and 120:1) were mixed at 25°C for 30 min. and then the binding capacity was evaluated by the agarose gel electrophoresis (110 V, 8 min), the gel was imaged under a UV transillumination (FlourChem E, ProteinSimple, San Jose, CA, USA) and the gray value was calculated by Image J (Bethesda, Maryland, USA). The zeta potential and hydrodynamic diameter of [email protected] were then measured by Zetasizer Nano-ZS90 (Malvern Panalytical, Ltd). Their morphological properties were detected by transmission electron microscopy (TEM, HT7700, Hitach, Ltd).
Serum enzymatic protection test
To determine the ability of AMP to protect siRNA from RNase A, the AMP and siNC (weight ratio of 15:1) were incubated at a 2 μL of RNase A (0.5 μg/mL) for 0, 5, 10, 15, 20, 25, and 30 min respectively. Subsequently, the solution was mixed with 1% SDS at 4°C for 3 min. Then the remaining siRNAs were detected by agarose gel electrophoresis (110 V, 8 min) and quantified based on the fluorescence intensity.
The cytotoxicity and hemolysis assay of AMP
To evaluate the cytotoxicity of AMP in vitro, 100 μL of RAW 264.7 cells with a density of 5000 cells/well were seeded into 96-well plates. After culturing for 24 h, AMP with different concentrations (5, 10, 20, 40, 60, 80, 100, 120, and 140 ppm) were placed in the wells and co-cultured for another 24 h. Then, the culture medium was removed, and the wells were washed twice with PBS. For each well, 100 μL of 10% CCK-8 solution diluted in culture medium was added, and the plate was incubated in an incubator (37°C, 5% CO2) for 1 h. Subsequently, the cell viability was measured at the absorbance of 450 nm by a microplate reader (Bio-teak, Epoch-2) and calculated according to the following formula:
Where Abg and Ang represent the absorbance of cell- and AMP-free medium with CCK-8 solution, respectively. Aeg represents the absorbance of medium with cells, CCK-8, and AMP solution.
To investigate the hemolytic effects of AMP to red blood cells (RBCs), 500 μL of blood was diluted 10-fold with PBS. The blood was mixed gently and centrifuged at 10000 g for 5 min. The supernatant was discarded, and RBCs were washed a few times by suspending them in a PBS solution (pH 7.4) until the supernatant was clear. Finally, RBCs were resuspended with 10 mL of PBS. To evaluate the hemolytic effects, 200 µL of RBCs were incubated with 800 µL of H2O (as positive control), 800 µL of PBS (as negative control), and AMP with different concentrations for 4 h in a 37 °C incubator. After incubation, the samples were further centrifugated at 10000 g for 5 min, and 100 µL of supernatants were extracted to quantify hemoglobin by recording the absorbance at 577 nm. The percentage of hemolysis rate was calculated as follows.
Where the Asam, Aneg, and Apos represent the absorbance value of treatment, negative and positive groups, respectively.
RAW264.7 cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin (100 μg/mL), and streptomycin (100 μg/mL) in an atmosphere with 5% CO2 at 37°C. Subsequently, RAW264.7 cells were seeded onto 24-well plates with a density of 3×104 cells/well, and cultured for 24 h. And then, cells were activated with 1 μg/mL of LPS. After 4 h, the maintenance medium was replaced with serum-free DMEM. Meanwhile, the AMP (1 mg/mL) and Cy3-siNC (100 pM) were mixed at a weight ratio of 15:1 and 30:1 at 25°C for 40 min. Then, the above [email protected] were added to the 24-well plates and incubated for 4 h.
To examine the uptake efficiency, these cells were imaged using fluorescent microscopy and assessed by flow cytometry, respectively. Additionally, to study the gene TNF-α expression, some cells were cultured for 72 h post-transfection in DMEM medium with 10% FBS after removing the old medium-containing material. The sense and antisense sequences of siTNF-α were listed as follows: sense: 5′-GUCUCAGCCUCUUCUCAUUdTdT-3′, antisense: 5′- AAUGAGAAGAGGCUGAGACdTdT-3′.
Fluorescence imaging and siRNA transfection efficiency
After being treated with [email protected] for 4 h, cells were washed three times with PBS (pH 7.4) and fixed with 4% formaldehyde for 15 min. Cells were then stained with DAPI for 20 min. The filters of the inverted microscope were set for DAPI (excitation at 405 nm and the emission was collected with a 450/50 nm band pass filter) and Cy3 (excited with 543 nm and emission was collected with a band pass filter 605/50 nm). To quantify cell internalization, the post-transfection cells were washed three times with PBS and collected by trypsinization (0.25%, without EDTA). Cy3 was used as a fluorescent marker (filter set for ECD was applied) to quantify the fluorescence intensity. The samples were evaluated by a flow cytometer (CytoFLEX, Beckman).
Anti-inflammatory activity of [email protected]α
To demonstrate the anti-inflammatory, LPS-activated macrophages were used to elicit the release of the inflammatory mediator TNF-α [28, 29]. The transcription level of TNF-α gene was investigated by qRT-PCR according to previous experiences . In brief, the total RNA from RAW264.7 cells was extracted using a TRIzol reagent (Invitrogen) and quantified using a micro-spectrophotometer (Epoch2, Biotek Instruments). Total RNA (800 ng) was reverse-transcribed to cDNA using PrimeScriptTM RT reagent Kit (AG11705, Aikerui Biological Engineering Co., Ltd, Hunan, China). The mRNA level of TNF-α gene was measured by qRT-PCR using the SYBR green dye (DBI-Bioscience 2143) in a QuanStudio 1 applied biosystem. The qRT-PCR was performed in a 20 µL reaction volume containing SYBR Premix Ex Taq II (10 µL), forward prime (10 µM, 0.8 µL), reverse primer (10 µM, 0.8 µL), cDNA template (5 ng/µL, 2 µL), and ddH2O (6.4 µL). The PCR conditions were denaturation at 95°C for 30 s, followed by 40 cycles of amplification (95°C for 5 s, 60°C for 30 s). The melting curves were measured at 95°C for 5 s and 60°C for 1 min. The β-actin gene was used as the internal control reference gene. Finally, gene expression was calculated using the 2−∆∆Ct method . The primer sequences were as follows: β-actin F: 5′-GGTCATCACCATTGGCAATG-3′, R: 5′-TAGTTTCGTGGATGCCACAG-3′; TNF-α F: 5′-GTCTGGGCAGGTCTACTTTGG-3′, 5′-GGTTGAGGGTGTCTGAAGGAG-3′. Furtherly, TNF-α content in the cell-free supernatants was determined using the TNF-α ELISA kit according to the manufacturer’s instructions.
In vitro antibacterial activity of AMPC
The minimum inhibitory concentrations (MICs) of the different NPs for E. coli and S. aureus were determined by a micro broth dilution method. The strains were cultured in LB medium at 37°C to the logarithmic phase. And then, the bacterial fluid was diluted to a concentration of 5 × 105 colony-forming units per mL (CFU/mL). Subsequently, AM, AMP, and AMPC were separately added into tubes with 4 mL of bacterial cultures and shaken for 24 h at 37°C. After naked eye observation, the lowest concentration of the NP in the tube without bacteria growth was determined as MIC.
To further evaluate the antibacterial activity of these NPs, E. coli and S. aureus in the exponential phase were serially diluted with LB medium to a concentration of 5 × 105 CFU/mL. Then, the bacterial suspension was added to 96-well plates and treated with AM, AMP, and AMPC (50 μg/mL). At different time intervals, the OD600 of bacterial suspensions was determined using a microphone reader (Bio-teak, Epoch-2) to obtain killing curves. Additionally, after incubation at 37°C for 12 h, 10 µL of the diluted bacterial solution was spread on LB agar plates. After incubation at 37°C for another 12 h, digital images of each plate were captured, and the CFU/mL and antibacterial ratio were obtained. CFU/mL was calculated according to the following equation respectively:
In vivo wound healing and safety evaluation
The in vivo antibacterial efficacy of [email protected]α was examined on the E. coli infection model in terms of wound recovery and histological analysis. All experiments involving animals were approved by the Institutional Animal Ethical Committee at the Laboratory Animal Research Center at Shenzhen University (Shenzhen, China). Briefly, 6-8-week-old BALB/c mice (18-22 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China). Mice were anesthetized by intraperitoneal injection of 4% pentobarbital sodium (1.0 mL/kg). Round skin wounds were created on the back with a biopsy puncture of 8 mm diameter, and then 10 μL of E. coli suspension (107 CFU/mL) was added to the wound surface. One day later, 200 μL of AM, AMP, AMPC, [email protected]α, and [email protected]α suspensions in PBS (50 μg/mL) were placed on the wounds. The wounds were treated with PBS and levofloxacin (LEVO) as the negative and positive controls, respectively. The area and images of the wound were recorded from 0 to 12 days. After 12 days of treatment, wound tissues were collected and dipped in fixative (4% paraformaldehyde). Wound tissues were sectioned and stained at Wuhan Service Biotechnology Co., Ltd., and the images were then recorded and analyzed using a Pathology Sectioning Scanner (LEICA-Aperio, carbon disulfide).
All experiments were conducted at least three times, and the data were shown as mean ± standard deviation (SD). T-test were used to evaluate the significance of different data. It was considered as statistically significant when p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).