1 Introduction 1 引言

Mesostructured materials such as mesoporous silica have attracted much attention during the past decades since their early reports in the 1990s [1,2,3,4]. Mesoporous silica nanoparticles (MSNs) have been applied in biocatalysis [5, 6], antitumor [7, 8] and specially antibacterial applications [9,10,11,12] due to unique properties including adjustable particle/pore sizes, high pore volume and excellent biocompatibility [13, 14]. MSNs generally act as carriers for the delivery of antibacterial compounds [15,16,17,18,19,20]. To create the mesopores, surfactants are removed for subsequent loading of drug molecules [21, 22]. Recently, a bactericidal reagent, benzalkonium chloride (BAC) was reported to act as the cationic surfactant to template the synthesis of mesostructured silica composite material. The antibacterial activity of BAC is due to the electronic interaction between cationic ammonium head group and negatively-charged bacterial membrane as well as the lipophilic tail enhanced membrane permeability, leading to bacterial membrane rupture and a leakage of cytoplasmic materials [23, 24]. However, the obtained composite particles have an large size of 650–850 nm, a small mesopore size of 18 Å, a low BAC release percentage of < 8% (in acidic condition) thus limited bactericidal efficiency (bacteria alive after 6 h treatment) [9, 25]. Therefore, it is highly desired to design novel silica based antibacterial nanocomposites with controlled structural properties and improved efficacy.
介孔二氧化硅等介观结构材料自 1990 年代早期报道以来，在过去几十年中引起了广泛关注 [ 1， 2， 3， 4]。介孔二氧化硅纳米颗粒（MSNs）因其独特的特性，包括可调节的颗粒/孔径、高孔体积和优异的生物相容性[13,14]，已被应用于生物催化[5,6]、抗肿瘤[7,8]和特殊的抗菌应用[9,10,11,12]。MSN通常作为递送抗菌化合物的载体[15,16,17,18,19,20]。为了产生介孔，去除表面活性剂以随后加载药物分子[21,22]。最近，据报道，一种杀菌试剂苯扎氯铵（BAC）作为阳离子表面活性剂，用于模板化介结构二氧化硅复合材料的合成。BAC的抗菌活性是由于阳离子铵头基团与带负电荷的细菌膜之间的电子相互作用以及亲脂性尾部增强的膜通透性，导致细菌膜破裂和细胞质物质泄漏[23,24]。然而，所获得的复合颗粒具有650–850 nm的大尺寸，18 Å的小介孔尺寸，<8%的低BAC释放百分比（在酸性条件下），从而限制了杀菌效率（处理6 h后细菌存活）[9,25]。因此，非常需要设计一种具有可控结构性能和提高功效的新型二氧化硅基抗菌纳米复合材料。

Among various strategies to combat bacterial infection [26, 27], creating spiky nanotopography has been reported with enhanced bacterial membrane adhesion and physical damage performance [28,29,30]. This strategy has also been applied to engineer antimicrobial nanoparticles with a rough surface topology and enhanced performance [31, 32]. For the synthesis of spiky silica nanoparticles, there are mainly two methods. One approach is using co-assembly of silica and polymer [9], while the other via surfactant cylindrical micelle templating in an oil/water biphasic system [33]. In these reports, extra treatment steps such as calcination or extraction are needed to remove the template to get the spiky surface, similar to most MSNs prepared as nanocarriers for further loading of antibacterial agents [34, 35]. To date, there are rare reports on the preparation of bactericidal surfactant containing silica nanocomposites with a spiky surface.
在对抗细菌感染的各种策略中[26,27]，已经报道了具有增强细菌膜粘附和物理损伤性能的尖刺纳米形貌[28,29,30]。该策略还被应用于设计具有粗糙表面拓扑结构和增强性能的抗菌纳米颗粒[31,32]。对于尖刺二氧化硅纳米颗粒的合成，主要有两种方法。一种方法是使用二氧化硅和聚合物的共组装[9]，而另一种方法是通过表面活性剂圆柱胶束模板在油/水双相体系中[33]。在这些报道中，需要额外的处理步骤，如煅烧或提取，以去除模板以获得尖刺表面，类似于大多数作为纳米载体制备的MSN，用于进一步加载抗菌剂[34,35]。迄今为止，关于制备含有具有尖刺表面的二氧化硅纳米复合材料的杀菌表面活性剂的报道很少见。

Herein, a “dual active templating” strategy is reported to synthesize bactericidal silica nanocomposites with a spiky surface, using cationic and anionic dual templates that are both active antibacterial agents. As shown in Scheme 1, BAC is used as a cationic structure-directing agent and sodium salicylate (NaSal) as an anionic agent. It is noted that sodium salicylate (NaSal) has antibacterial activity [36, 37], and has been utilized as a co-template to finely adjust the structures of MSNs (e.g., with large pores) [38, 39]. However, neither its content nor bactericidal property in silica nanocomposites has been reported. The use of BAC and NaSal as co-templates leads to a spiky silica nanocomposite I. Compared to nanocomposite II with a smooth surface templated by pure BAC, nanocomposite I showed high BAC loading (~ 22.0 wt%), efficient release (BAC and NaSal) (> 75% in 24 h), improved bactericidal activity and enhanced biofilm inhibition (70%) toward Gram-positive bacteria Staphylacoccus Epidermidis (S. Epidermis). The dual active templating strategy developed from this study may pave the way for the designed synthesis of novel functional nanocomposites for antibacterial applications.
本文报道了一种“双活性模板”策略，使用阳离子和阴离子双模板合成具有尖刺表面的杀菌二氧化硅纳米复合材料，这两种模板都是活性抗菌剂。如方案1所示，BAC用作阳离子结构导向剂，水杨酸钠（NaSal）用作阴离子剂。值得注意的是，水杨酸钠（NaSal）具有抗菌活性[36,37]，并已被用作微调MSN结构（例如，具有大孔）的共模板[38,39]。然而，其在二氧化硅纳米复合材料中的含量和杀菌性能均未见报道。与纯BAC模板化表面光滑的纳米复合材料II相比，纳米复合材料I显示出高BAC负载量（~22.0 wt%），高效释放（BAC和NaSal）（24小时内>75%），改善了杀菌活性，增强了对革兰氏阳性菌表皮葡萄球菌（S. Epidermis）的生物膜抑制（70%）。本研究开发的双活性模板策略可能为设计用于抗菌应用的新型功能纳米复合材料的合成铺平道路。

Scheme 1 方案1
scheme 1

Schematic illustration for the synthesis, enhanced antibacterial and anti-biofilm activity of spiky nanocomposite I co-templated by BAC and NaSal
BAC和NaSal共同模板化的尖刺纳米复合材料I的合成、增强抗菌和抗生物膜活性示意图

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2 Experimental Section 2 实验部分

2.1 Materials and Reagents
2.1 材料和试剂

Benzalkonium chloride (≥ 95%), triethanolamine (TEA, 99%), tetraethyl orthosilicate (TEOS, 98%), sodium salicylate (≥ 95%), hydrofluoric acid (HF reagent, 48%), hydrochloric acid (HCI, 37%), crystal violet solution (CV, 1% aqueous solution), phosphate buffer solution (PBS, 10 mM, pH = 7.4), and dead cell staining buffer propidium iodide (PI, Minimum Purities ≥ 95%) were purchased from Sigma-Aldrich. Green-fluorescent nucleic acid stains (SYTOTM 9, 5 mM) were purchased from Thermo Fisher Scientific. S. Epidermis (American Type Culture Collection (ATCC)-12,228) was purchased from ATCC.
苯扎氯铵（≥95%）、三乙醇胺（TEA，99%）、原硅酸四乙酯（TEOS，98%）、水杨酸钠（≥95%）、氢氟酸（HF试剂，48%）、盐酸（HCI，37%）、结晶紫溶液（CV，1%水溶液）、磷酸盐缓冲溶液（PBS，10mM，pH = 7.4）和死细胞染色缓冲液碘化丙啶（PI，最低纯度≥95%）购自Sigma-Aldrich。绿色荧光核酸染料（SYTOTM 9,5 mM）购自赛默飞世尔科技。表皮链球菌（American Type Culture Collection （ATCC）-12,228）购自ATCC。

2.2 Synthesis of Nanocomposite I and II
2.2 纳米复合材料I和II的合成

Nanocomposites I with a spiky morphology containing BAC/NaSal/silica were synthesized using BAC and NaSal as co-templates and TEOS as the silica source. In a typical synthesis, 68 mg of TEA was added into 25 mL of deionized water and stirred at 80 °C for 30 min. Then, around 0.7 mL of 50% aqueous BAC and 80 mg of NaSal were added into the above solution and stirred for 1 h at 80 °C (molar ratio: NaSal/BAC = 0.5). After addition of 3 mL of TEOS, the solution was further stirred at 80 °C for another 2 h. Final nanocomposite I was collected by centrifugation at 25,200 RCF for 5 min, washing with ethanol for three times, and vacuum dried at 50 °C for 12 h. Part of nanocomposite I was calcined at 550 °C under air for 5 h and denoted as I-calcined. Nanocomposite II with a smooth surface was prepared vis a similar method, using only BAC as the template and TEOS as the silica precursor. Specifically, 68 mg of TEA was added into 25 mL of deionized water and stirred at 80 °C for 30 min. Then, 0.7 mL of 50% aqueous BAC was added into the above solution and stirred for 1 h at 80 °C. After the addition of 3 mL of TEOS, the solution was further stirred at 80 °C for another 2 h. The final nanocomposite II was collected by centrifugation at 25,200 RCF for 5 min, washing with ethanol for three times, and vacuum dried at 50 °C overnight.
以BAC和NaSal为共模板，以TEOS为二氧化硅源，合成了含有BAC/NaSal/二氧化硅的尖刺形貌纳米复合材料I.在典型的合成中，将 68 mg TEA 加入 25 mL 去离子水中，并在 80 °C 下搅拌 30 分钟。然后，将约0.7 mL的50%BAC水溶液和80 mg NaSal加入上述溶液中，并在80°C下搅拌1小时（摩尔比：NaSal/BAC = 0.5）。加入 3 mL TEOS 后，将溶液在 80 °C 下进一步搅拌 2 小时。通过在25,200 RCF下离心5分钟，用乙醇洗涤3次，并在50°C下真空干燥12小时来收集最终的纳米复合材料I。将部分纳米复合材料I在550°C空气下煅烧5小时，表示为I-煅烧。采用类似方法制备了表面光滑的纳米复合材料II，仅使用BAC作为模板，TEOS作为二氧化硅前驱体。具体而言，将68 mg TEA加入到25 mL去离子水中，并在80°C下搅拌30分钟。 然后，将0.7 mL的50%BAC水溶液加入上述溶液中，并在80°C下搅拌1小时。 加入3 mL TEOS后，将溶液在80°C下进一步搅拌2小时。通过在25,200 RCF下离心5分钟，用乙醇洗涤3次，并在50°C下真空干燥过夜来收集最终的纳米复合材料II。

2.3 Quantification of BAC and NaSal Contents in Nanocomposites
2.3 纳米复合材料中BAC和NaSal含量的定量

Ultraviolet–visible spectroscopy (UV–Vis) analysis was applied to determine the contents of BAC and NaSal in silica nanocomposite. Nanocomposites were dissolved in HF at room temperature for 4 h and then diluted for UV–Vis analysis. The NaSal and BAC contents were determined and calculated at 299 and 209 nm, respectively (see details in Results and Discussion section).
采用紫外-可见光谱（UV-Vis）分析测定二氧化硅纳米复合材料中BAC和NaSal的含量。将纳米复合材料在室温下溶解在HF中4小时，然后稀释用于紫外-可见分光度计分析。NaSal 和 BAC 含量分别在 299 nm 和 209 nm 处测定和计算（详见结果和讨论部分）。

2.4 Characterization 2.4 表征

Transmission electron microscopy (TEM) study was performed using J HT7700-EXALENS with an accelerated voltage of 80–100 kV. Scanning electron microscope (SEM) measurements were conducted using a JEOL JSM 7800 field-emission scanning electron microscope (FE-SEM). Energy-dispersive X-ray (EDX) mapping analysis was carried out using Hitachi HF5000 Cs-STEM/TEM. Dynamic light scattering (DLS) measurement was conducted at 25 °C using the Zetasizer Nano-ZS from Malvern Instruments. Before measurements, the samples were dispersed in deionized water by ultra-sonication, and all samples were measured for three times. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis was conducted on a ThermoNicolet Nexus 6700 FTIR spectrometer equipped with Diamond ATR Crystal. Nitrogen adsorption–desorption analysis was measured by a Micromeritcs Tristar II system at 77 K. Before the measurement, samples were degassed at 353 K overnight on a vacuum line. The total pore volume was calculated from the adsorbed amount at the maximum relative pressure (P/P0) of 0.99. The pore size of samples was calculated through Barrrett-Joyner-Halenda (BJH) method from the adsorption branches of the isotherms. The Brunauer–Emmett–Teller (BET) method was used to calculate the specific surface areas. Cross-Polarization Magic Angle Spinning 13Carbon Nuclear Magnetic Resonance (13C CP/MAS NMR) spectrum was measured by a solid-state Bruker Avance III spectrometer with 7 T (300 MHz for 1H) magnet, Zirconia rotor, 4 mm, rotated at 7 kHz. ICP-OES was performed to provide the quantitative measurement of silica adhered or uptaken by bacterial after 4 h incubation. A Thermo Scientific iCAP 6500 ICP-OES instrument was used, and the analysis was duplicated. The bacterial suspension and nanocomposite solution were mixed at the same ratio and condition as the antibacterial test. After 4 h culturing, the solution was filtered through 450 nm-pore filter membrane and washed with PBS twice. Then, the filter paper was dissolved in 10% HF solution for 24 h before the ICP-OES quantification of silicon content. The silicon amount was calculated based on each bacteria, and the bacterial number was determined by optical density (OD) reading at 600 nm.
透射电子显微镜 （TEM） 研究使用 J HT7700-EXALENS 进行，加速电压为 80–100 kV。使用JEOL JSM 7800场发射扫描电子显微镜（FE-SEM）进行扫描电子显微镜（SEM）测量。使用Hitachi HF5000 Cs-STEM/TEM进行能量色散X射线（EDX）映射分析。使用马尔文仪器的 Zetasizer Nano-ZS 在 25 °C 下进行动态光散射 （DLS） 测量。测量前，通过超声波将样品分散在去离子水中，所有样品进行三次测量。在配备金刚石 ATR 晶体的 ThermoNicolet Nexus 6700 FTIR 光谱仪上进行了衰减总反射率傅里叶变换红外光谱 （ATR-FTIR） 分析。氮气吸附-脱附分析通过Micromeritcs Tristar II系统在77 K下测量。在测量之前，样品在真空管线上以 353 K 脱气过夜。总孔隙体积由最大相对压力（P/P 0 ）为0.99时的吸附量计算得出。通过Barrrett-Joyner-Halenda（BJH）方法从等温线的吸附分支中计算样品的孔径。采用Brunauer-Emmett-Teller（BET）方法计算比表面积。交叉偏振魔角旋转 13 碳核磁共振 （ 13 C CP/MAS NMR） 光谱由固态布鲁克 Avance III 光谱仪测量，该光谱仪具有 7 T（300 MHz for 1H）磁铁，氧化锆转子，4 mm，以 7 kHz 旋转。进行 ICP-OES 以提供孵育 4 小时后细菌粘附或吸收的二氧化硅的定量测量。使用 Thermo Scientific iCAP 6500 ICP-OES 仪器，并重复分析。 将细菌悬浮液和纳米复合溶液以与抗菌试验相同的比例和条件混合。培养4小时后，将溶液通过450nm孔隙滤膜过滤，并用PBS洗涤两次。然后，将滤纸溶解在10%HF溶液中24小时，然后进行ICP-OES定量硅含量。根据每种细菌计算硅量，并通过600nm处的光密度（OD）读数确定细菌数量。

2.5 Drug Release Study 2.5 药物释放研究

The release study was investigated by dispersing nanocomposite in pH 7.4 or pH 5 PBS solutions shaking at 37 °C at 220 RPM. Released BAC and NaSal were determined by analyzing the collected supernatant at different time points using UV–Vis.
通过将纳米复合材料分散在pH 7.4或pH 5 PBS溶液中，在37 °C下以220 RPM振荡来研究释放研究。通过使用紫外-可见分光分析不同时间点收集的上清液来测定释放的 BAC 和 NaSal。

2.6 Antibacterial Activity
2.6 抗菌活性

The antimicrobial capability of nanocomposite I was tested in S.epidermidis using Luria–Bertani (LB) -agar plates assay. Nanocomposite II, BAC, NaSal, calcined nanocomposite I and mixture of BAC/NaSal were selected as control. The nanocomposites were sterilized by dissolved in 70% (v/v) ethanol, followed by washing with sterilized PBS for three times before bacterial culture based on a reported protocol [40]. All the tests were conducted under acidic LB medium (pH = 5) with the tested BAC concentrations of 1, 2, 4 μg mL−1. The mixture of bacteria suspension (1.0 × 107 CFU mL−1), acidic LB medium and nanocomposites/drug was incubated in 37 °C shaker at 220 RPM for 24 h and examined by LB-agar plate assay. 200 μL of treated bacterial suspensions were spread on sterilized LB-agar plates. After incubation at 37 °C for overnight, photographs were taken, and the bacteria colonies grown in each plate were counted.
使用 Luria-Bertani （LB） -琼脂平板测定在表皮葡萄球菌中测试了纳米复合材料 I 的抗菌能力。选取纳米复合材料II、BAC、NaSal、煅烧纳米复合材料I和BAC/NaSal的混合物作为对照。将纳米复合材料溶解在70%（v/v）乙醇中灭菌，然后用灭菌的PBS洗涤3次，然后根据报道的方案进行细菌培养[40]。所有测试均在酸性LB培养基（pH = 5）下进行，测试的BAC浓度为1,2,4μgmL −1 。将细菌悬浮液（1.0× 7 10CFU mL −1 ）、酸性LB培养基和纳米复合材料/药物的混合物在37°C振荡器中以220RPM孵育24小时，并通过LB-琼脂平板测定进行检查。将 200 μL 处理过的细菌悬浮液铺在灭菌的 LB-琼脂平板上。在37°C下孵育过夜后，拍摄照片，并计数每个板中生长的细菌菌落。

2.7 In vitro Biofilm Inhibition
2.7 体外生物膜抑制

For biofilm inhibition study, 200 μL of S. Epidermidis bacterial suspension (1.0 × 108 CFU mL−1) added with BAC or nanocomposite I at 4 μg mL−1 BAC was added to 24-well plates and cultured at 37 ℃ without shaking. After 12 h incubation, the supernatant was removed. The samples were washed with 85% NaCl aqueous solution three times and then stained by SYTO9 and PI for confocal microscopy. Another plate was prepared for CV staining. The untreated biofilm was denoted as the control, and all experiments were performed in duplicates.
对于生物膜抑制研究，将200μL表皮葡萄球菌细菌悬浮液（1.0× 8 10CFU mL −1 ）与BAC或4μgmL −1 BAC的纳米复合I一起加入到24孔板中，并在37°C下培养而不振荡。孵育12小时后，除去上清液。样品用85%NaCl水溶液洗涤3次，然后用SYTO9和PI染色进行共聚焦显微镜检查。准备另一块板进行CV染色。未经处理的生物膜表示为对照，所有实验一式两份进行。

2.8 Biocompatibility Assay
2.8 生物相容性测定

Cell viability was evaluated in Human Embryonic Kidney (HEK239T) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. HEK cells were seeded in a 96-well flat-bottom plate with a density of 7000–8000 cells per well for 24 h. Then, cell culture medium was replaced with fresh medium containing PBS, nanocomposite I/II and calcined nanocomposite I at a BAC concentration of 1 μg mL−1. After incubation in the incubator at 37 ℃ for 24 h, 20 μL of MTT solution (5 mg mL−1) was added to each well and cells were incubated for another 4 h. Then medium was replaced with 100 μL of dimethyl sulfoxide (DMSO). Then absorbance readings were measured at the wavelength of 570 nm using a microplate reader. The cells incubated with PBS were used as the control. All experiments were performed four times.
使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑 （MTT） 测定法评估人胚胎肾 （HEK239T） 细胞的细胞活力。将HEK细胞接种在96孔平底板中，每孔密度为7000-8000个细胞，持续24小时。然后，用含有PBS、纳米复合材料I/II和煅烧纳米复合材料I的新鲜培养基代替细胞培养基，BAC浓度为1 μg mL −1 。在37°C培养箱中孵育24小时后，向每个孔中加入20μLMTT溶液（5mgmL −1 ），并将细胞再孵育4小时。然后用 100 μL 二甲基亚砜 （DMSO） 替换培养基。然后使用酶标仪在570nm的波长处测量吸光度读数。使用与PBS孵育的细胞作为对照。所有实验均进行四次。

3 Results and Discussion 3 结果与讨论

The nanocomposite I was prepared in an aqueous system using TEOS as the silica precursor, BAC as the cationic surfactant and NaSal as an anionic co-templating agent [38]. Low and high magnification TEM images (Fig. 1a, b) showed that nanocomposite I possesseds a spiky structure and a uniform particle size of 100 ± 10 nm by measuring 50 particles. The spiky surface morphology was further visualized in the SEM image (Fig. 1c). Uniform distribution of nitrogen, silicon and oxygen elements in nanocomposite I was revealed by the energy-dispersive X-ray spectroscopy (EDX)-mapping results (Fig. 1d–g). The observation of nitrogen indicates the existence of BAC in the nanocomposite I. However, the presence of NaSal cannot be confirmed from these results.
以TEOS为二氧化硅前驱体，BAC为阳离子表面活性剂，NaSal为阴离子共模板剂，在水性体系中制备了纳米复合材料I[38]。低倍率和高倍率TEM图像（图1a，b）显示，通过测量50个颗粒，纳米复合材料I具有尖刺结构和100±10 nm的均匀粒径。在SEM图像中进一步可视化了尖刺表面形态（图1c）。能量色散X射线光谱（EDX）映射结果揭示了纳米复合材料I中氮、硅和氧元素的均匀分布（图1d-g）。氮的观察表明纳米复合材料I中存在BAC。然而，从这些结果中无法确认NaSal的存在。

Fig. 1 图1
figure 1

a, b TEM, c SEM and d–g EDX mapping images of nanocomposite I. h FTIR spectra of nanocomposites I and II, calcined nanocomposite I, BAC and NaSal. I. i UV–Vis spectra of nanocomposites I and II. j Time-dependent release profile of BAC and NaSal from nanocomposite I at pH 7.4 and 5.0
纳米复合材料I.h的傅里叶变换红外光谱图（a， b TEM， c SEM）和d-g EDX映射图像，煅烧纳米复合材料I、BAC和NaSal。I. i 纳米复合材料 I 和 II 的紫外-可见光谱。j pH 7.4 和 5.0 下纳米复合材料 I 中 BAC 和 NaSal 的瞬态释放曲线

Full size image 全尺寸图像

To understand the role of NaSal in the synthesis and antibacterial application, the nanocomposite II was fabricated via a similar synthetic protocol without the addition of NaSal. Nanocomposite II exhibited a smooth surface and similar diameter to Nanocomposite I as shown in TEM (Fig. S2a, b) and SEM images (Fig. S2c). EDX mapping results of nanocomposite II also showed uniform distribution of nitrogen, silicon and oxygen elements (Fig. S2d–g). The relatively weak nitrogen signal indicated the content of BAC in nanocomposite II was lower than that in nanocomposite I. FTIR was further conducted to characterize the existence of BAC and NaSal. Compared to the spectrum of BAC (Fig. 1h), nanocomposites I and II exhibited typical peaks at 2950, 2850, and 1480 cm−1 corresponding to C–H stretching bands and benzene ring originated from BAC [23], suggesting the existence of BAC, which is consistent with EDX mapping results. The peaks originated from NaSal at 1300, 810, and 620–690 cm−1 are only observed from nanocomposite I [38]. The typical peaks of BAC and NaSal were not observed in I-calcined, suggesting complete removal of surfactants after calcination. 13C MAS NMR was conducted to characterize the compositions in nanocomposite I. As shown in Fig. S3, The typical peaks at 134 and 137 ppm were originated from benzyl chain of NaSal [41]. The peaks at 51 and 57 ppm are assigned to N(CH3)2 and NCH2 species, respectively; and the peak around 29 ppm is attributed to hydrophobic carbon chain in BAC [42]. These observations indicate the presence of BAC and NaSal in nanocomposite I.
为了了解NaSal在合成和抗菌应用中的作用，在不添加NaSal的情况下，通过类似的合成方案制备了纳米复合材料II。纳米复合材料II具有光滑的表面和与纳米复合材料I相似的直径，如TEM（图S2a，b）和SEM图像（图S2c）所示。纳米复合材料II的EDX映射结果也显示氮、硅和氧元素的均匀分布（图S2d–g）。相对较弱的氮信号表明，纳米复合材料II中BAC的含量低于纳米复合材料I.，进一步进行FTIR表征BAC和NaSal的存在。与BAC的光谱（图1h）相比，纳米复合材料I和II在2950、2850和1480 cm −1 处表现出典型的峰，对应于C-H拉伸带和源自BAC的苯环[23]，表明BAC的存在，这与EDX映射结果一致。NaSal在1300、810和620–690 cm −1 处的峰仅从纳米复合材料I中观察到[38]。在I煅烧中未观察到BAC和NaSal的典型峰，表明煅烧后表面活性剂完全去除。 13 通过C MAS NMR表征纳米复合材料I的组成。如图S3所示，134和137 ppm的典型峰来自NaSal的苄基链[41]。51 ppm 和 57 ppm 处的峰分别分配给 N（CH 3 ） 2 和 NCH 2 物种;29 ppm左右的峰值归因于BAC中的疏水性碳链[42]。这些观察结果表明纳米复合材料I中存在BAC和NaSal。

The nitrogen sorption analysis was conducted to characterize the porous structure. The adsorption–desorption isotherms of nanocomposite I and nanocomposite II are shown in Fig. S2h. The major capillary condensation steps of nanocomposite I occurred at two high relative pressure (P/P0) steps, one around 0.90 and the other at > 0.97. The first capillary condensation step corresponds to a broad pore size distribution (Fig. S2h-inserted) centered at 26.3 nm for nanocomposite I, presumably reflecting the mean pore size of the spiky layer. The second capillary condensation step at higher P/P0 of 0.97 is attributed to packing voids between particles [33], which is also observed in the adsorption–desorption isotherm of nanocomposite II. The physical properties of nanocomposites I and II are summarized in Table S1. Compared to BAC templated nanocomposite II with a specific surface area of 86 m2 g–1 and a pore volume of 0.34 cm3 g−1, nanocomposite I templated by BAC/NaSal showed a higher specific surface area of 290 m2 g–1 and a pore volume of 0.69 cm3 g−1. Considering their difference in morphology (Figs. 1a–c and S2–c), the higher specific surface area of nanocomposite I than nanocomposite II is mainly attributed to the nanostructured spiky rough surface, which contains a portion of mesopores as evidenced in Fig. S2h. The zeta potential of three particles was measured (Table S1). Compared to nanocomposite II, nanocomposite I exhibited a higher surface charge, in accordance with a higher BAC content in nanocomposite I which is beneficial for antibacterial performance. After calcination, calcined-I exhibited the negative surface charge, indicating the successful removal of BAC in the nanocomposite.
通过氮气吸附分析对多孔结构进行表征。纳米复合材料I和纳米复合材料II的吸附-脱附等温线如图S2h所示。纳米复合材料I的主要毛细管缩合步骤发生在两个高相对压（P/P 0 ）步骤下，一个在0.90左右，另一个在0.97>。第一个毛细管缩合步骤对应于纳米复合材料I以26.3 nm为中心的宽孔径分布（图S2h插入），大概反映了尖刺层的平均孔径。在较高的P/P 0 （0.97）时，第二个毛细管冷凝步骤归因于颗粒之间的填充空隙[33]，这在纳米复合材料II的吸附-解吸等温线中也观察到。纳米复合材料I和II的物理性质总结在表S1中。与比表面积为86 m 2 g –1 、孔容为0.34 cm 3 g −1 的BAC模板化纳米复合材料II相比，BAC/NaSal模板化的纳米复合材料I具有更高的比表面积，为290 m 2 g –1 ，孔容为0.69 cm 3 g −1 。考虑到它们在形态上的差异（图1a-c和S2-c），纳米复合材料I的比表面积高于纳米复合材料II，主要归因于纳米结构的尖刺粗糙表面，其中包含一部分介孔，如图S2h所示。测量了三个粒子的zeta电位（表S1）。与纳米复合材料II相比，纳米复合材料I表现出更高的表面电荷，符合纳米复合材料I中较高的BAC含量，有利于抗菌性能。 煅烧后，煅烧-I表面带负电荷，表明纳米复合材料中BAC的去除成功。

Time-dependent TEM images of intermediated structures at different timepoints were collected to investigate the formation mechanism of nanocomposite I (Fig. S4). At the reaction time of 15 min, nanospheres with an average diameter of ~ 100 ± 5 nm were observed (Fig. S4a). A few rod-like structures were found deposited on the nanosphere outer surface. With the reaction time prolonged to 20 and 40 min, the nanoparticles with clear spike structure were observed as shown in Fig. S4b-c. Considering that in the absence of NaSal nanocomposite II a smooth surface formed, it is proposed that the interaction between negatively charged Sal– and positively charged BAC+ decreases the charge density and thus the hydrophilic head group area in the BAC+/Sal–/silicate assembly, leading to an increase in packing parameter (g) and structural transition from spherical composite micelles toward cylindrical structures [38]. As reported by Zhao and co-workers, the epitaxial growth of cylindrical structures contributes to the growth of spiky silica nanocomposite I [33].
收集不同时间点中间结构的瞬态透射电镜图像，研究纳米复合材料I的形成机理（图S4）。在15 min的反应时间，观察到平均直径为~100±5nm的纳米球（图S4a）。在纳米球外表面上发现了一些棒状结构。随着反应时间延长至20和40 min，观察到具有清晰尖峰结构的纳米颗粒，如图S4b-c所示。 考虑到在没有NaSal纳米复合材料II的情况下形成光滑表面，提出带负电荷的Sal – 和带正电荷的BAC + 之间的相互作用降低了电荷密度，从而降低了BAC + /Sal – 中的亲水头基面积/硅酸盐组装，导致堆积参数（g）的增加和结构从球形复合胶束向圆柱形结构的转变[38]。正如Zhao及其同事所报道的那样，圆柱结构的外延生长有助于尖刺二氧化硅纳米复合材料I的生长[33]。

To quantitatively measure BAC and NaSal contents in the nanocomposites, a protocol based on UV–Vis spectroscopy was developed. As shown in Fig. 1i, the UV–Vis absorption spectrum of pure NaSal showed a peak at 299 nm, while at this wavelength pure BAC or nanocomposite II prepared in the absence of NaSal showed negligible absorbance. Therefore, the content of NaSal in nanocomposite I was determined from the standard curve obtained at 299 nm, which was calculated to be 4.28 wt% (Fig. S2j, top). To quantify the BAC content, the standard curves of NaSal and BAC at 209 nm were measured (Fig. S2j middle and bottom). By deducting the absorbance contributed from NaSal, the BAC content in nanocomposite I calculated was determined to be 21.96 wt%. Similarly, the percentage of BAC in nanocomposite II was determined to be 14.92 wt%, lower than that in nanocomposite I. Thermogravimetric analysis (TGA) was further conducted to determine the surfactants loading contents of nanocomposites. The weight loss before 110 °C is attributed to the removal of moisture. The weight loss in the temperature range between 110 and 600 °C should be attributed to the decomposition of templates and silica condensation, which was calculated to be 37.2% for nanocomposite I (mainly BAC and NaSal) and 20.3% (mainly BAC) for nanocomposite II. The difference between nanocomposites I and II is similar to that obtained from UV–Vis analysis. The higher loading contents analyzed from TGA compared to UV–Vis can be attributed to the further condensation of silanol groups.
为了定量测量纳米复合材料中BAC和NaSal的含量，开发了一种基于紫外-可见光谱的方案。如图1i所示，纯NaSal的紫外-可见吸收光谱在299 nm处显示峰值，而在该波长下，在没有NaSal的情况下制备的纯BAC或纳米复合材料II的吸光度可以忽略不计。因此，根据在299 nm处获得的标准曲线测定了纳米复合材料I中NaSal的含量，计算结果为4.28 wt%（图S2j，顶部）。为了量化BAC含量，测量了NaSal和BAC在209 nm处的标准曲线（图S2j中间和底部）。通过扣除NaSal贡献的吸光度，我计算的纳米复合材料中的BAC含量确定为21.96 wt%。同样，纳米复合材料II中BAC的百分比为14.92 wt%，低于纳米复合材料I.，进一步进行热重分析（TGA）以确定纳米复合材料的表面活性剂含量。110°C之前的重量损失归因于水分的去除。在110-600°C的温度范围内，重量损失应归因于模板的分解和二氧化硅的缩合，计算出纳米复合材料I（主要是BAC和NaSal）为37.2%，纳米复合材料II为20.3%（主要是BAC）。纳米复合材料I和II之间的差异与紫外-可见分光度计分析的差异相似。与紫外-可见分光光度计相比，TGA分析的负载含量较高，可归因于硅醇基团的进一步缩合。

Furthermore, the release of BAC and NaSal from nanocomposite I as a function of time was studied by UV–Vis spectroscopy (Fig. 1j). The release test was conducted in PBS at two pH values (7.4 or 5), considering the acidic pH at the bacterial infection site [43]. Compared to the relatively slower release of BAC and NaSal (< 40% within 12 h) at neutral pH, nanocomposite I exhibited a higher release percentage of BAC/NaSal (e.g., >  ~ 60% at 12 h). For BAC release at pH 5, a burst release stage before 12 h and a subsequent sustained release stage (> 12 h) were observed. Moreover, the BAC release percentage at 24 h from nanocomposite I (~ 45% at pH 7.4; ~ 70% at pH 5) was significantly higher than that from nanocomposite II (< 20% at pH 7.4; ~ 36% at pH 5). The dissolved silicon content was also measured by ICP-OES. After 24 h, the concentration of dissolved silicon under pH 5 (23.3 mg L−1) was higher than that at pH 7.4 (10.8 mg L−1). Therefore, the increased release percentage of BAC at acidic pH is presumably due to the faster silica degradation as well as the replacement of benzalkonium ions by H+ under more acidic conditions [20]. The higher BAC release percentage of nanocomposite I compared to nanocomposite II is probably due to the faster degradation rate of large pore sized structure, which is consistent with a literature report where large-pore sized MSNs exhibited faster degradation than MSNs with smaller pore sizes [44]. The increased release of active molecules in nanocomposite I with spiky surface is beneficial for antibacterial applications.
此外，通过紫外-可见光谱研究了纳米复合材料I中BAC和NaSal的释放随时间的变化（图1j）。考虑到细菌感染部位的酸性pH值，在PBS中以两种pH值（7.4或5）进行释放试验[43]。与中性pH下BAC和NaSal的释放相对较慢（12 h内<40%）相比，纳米复合材料I表现出较高的BAC/NaSal释放百分比（例如，12 h时>~60%）。对于pH 5下的BAC释放，观察到12小时前的爆裂释放阶段和随后的缓释阶段（>12小时）。此外，纳米复合材料I在24 h时的BAC释放百分比（pH 7.4时~45%;pH 5时~70%）显著高于纳米复合材料II（pH 7.4时<20%;pH 5时~36%）。 溶解硅含量也通过ICP-OES测定。 24 h后，pH 5 （23.3 mg L −1 ）下溶解硅的浓度高于pH 7.4 （10.8 mg L −1 ).因此，BAC在酸性pH下释放百分比的增加可能是由于二氧化硅降解速度更快，以及 + 在酸性更强的条件下苯扎铎离子被H取代[20]。与纳米复合材料II相比，纳米复合材料I的BAC释放百分比较高，这可能是由于大孔径结构的降解速度更快，这与文献报道一致，即大孔径MSNs比孔径较小的MSNs表现出更快的降解速度[44]。具有尖刺表面的纳米复合材料I中活性分子的释放增加有利于抗菌应用。

To demonstrate the advantage of nanocomposite I prepared by the “dual active templating” approach, its antibacterial activity toward S. epidermis was evaluated using plate counting method. The bacteria were cultured in acidic LB medium for 24 h, using nanocomposite II, I-calcined (see detailed characterization in Fig. S6), BAC/NaSal, BAC and NaSal as control groups due to more efficient release of BAC/Sal at acidic pH tested above. Nanocomposite I, BAC/NaSal, nanocomposite II (Fig. 2a, b), BAC or NaSal (Fig. S6) all showed dose-dependent antimicrobial activity compared to untreated group (Fig. 2c) while no obvious antibacterial activity was observed for calcined I (Fig. S6), suggesting the antibacterial function from silica is minimal. Less bacterial colony grown in BAC/NaSal treated group compared to either BAC or NaSal treated group, suggesting the enhanced antibacterial efficiency of the combination of BAC and NaSal. Least colony was observed for nanocomposite I group at all dosages compared to nanocomposite II or drug controls, suggesting the advantage of the spiky topography and BAC/NaSal compositions (Fig. 2d).
为了证明采用“双活性模板”方法制备的纳米复合材料I的优势，采用平板计数法评估了其对表皮链球菌的抗菌活性。将细菌在酸性LB培养基中培养24小时，使用纳米复合材料II，I-煅烧（见图S6中的详细表征），BAC / NaSal，BAC和NaSal作为对照组，因为在上面测试的酸性pH下更有效地释放BAC / Sal。与未处理组（图2c）相比，纳米复合材料I、BAC/NaSal、纳米复合材料II（图2a、b）、BAC或NaSal（图S6）均显示出剂量依赖性抗菌活性，而煅烧I（图S6）没有观察到明显的抗菌活性，这表明二氧化硅的抗菌功能微乎其微。与 BAC 或 NaSal 处理组相比，BAC/NaSal 处理组中生长的细菌菌落较少，表明 BAC 和 NaSal 组合的抗菌效率更高。与纳米复合材料II或药物对照组相比，在所有剂量下，纳米复合I组的菌落最少，这表明尖峰形貌和BAC/NaSal组合物的优势（图2d）。

Fig. 2 图2
figure 2

a, b Dose-dependent killing of S. epidermidis by BAC/NaSal, nanocomposites I and II. Photographs of plates containing treated culture, where 1/2 represents the BAC concentration of each group. c Photograph of plate containing tenfold serial dilutions of untreated culture. d the average survival percentage of S. epidermidis upon exposure to each group quantified based on 1, 2 and 4.μg BAC mL−1 Statistical significance is calculated using a two-tailed t test with significant p-values shown. “ns” denotes “not significant.” e–h SEM images of BAC/NaSal, nanocomposite I, nanocomposite II treated bacterial and untreated S.Epidermidis. (red arrows refer to a semi-spherical dent on bacterial surface upon adhesion of nanocomposite I; scale bar: 1 μm)
a， b BAC/NaSal、纳米复合材料 I 和 II 对表皮链球菌的剂量依赖性杀伤。含有处理培养物的平板的照片，其中 1/2 代表每组的 BAC 浓度。c 含有十倍连续稀释的未经处理的培养物的平板照片。d 根据 1、2 和 4.μg BAC mL −1 量化每组时表皮链球菌的平均存活率 使用双尾 t 检验计算统计学显着性，显示显着的 p 值。“ns”表示“不显著”。 BAC/NaSal、纳米复合 I、纳米复合 II 处理的细菌和未经处理的表皮链球菌的 e-h SEM 图像。（红色箭头是指纳米复合材料I粘附后细菌表面的半球形凹痕;比例尺：1μm）

Full size image 全尺寸图像

Compared to the bacterial viability of BAC and NaSal treated groups (7.39% and 32.27%, respectively), the survival rate of BAC/NaSal treated group was less (1.25%), implying the synergistic effect of the dual antibacterial agents in bacterial killing. Similar synergistic effect can also be evidenced by the higher survival rate of nanocomposite II treated group (7.59%) than nanocomposite I (1.07%) treated bacteria. The cell viability was also evaluated in Human Embryonic Kidney (HEK239T) cells. Compared to the 41% cell viability of pure drug treated group (BAC/Sal), nanocomposite I, II and calcined nanocomposite I all exhibited ~ 90% cell viability, suggesting excellent biocompatibility of the silica-based nano-formulations.
与BAC和NaSal处理组的细菌活力（分别为7.39%和32.27%）相比，BAC/NaSal处理组的存活率较低（1.25%），表明双抗菌剂在杀灭细菌方面具有协同作用。纳米复合II处理组（7.59%）的存活率高于纳米复合I（1.07%）处理的细菌，也证明了类似的协同作用。还评估了人胚胎肾（HEK239T）细胞的细胞活力。与纯药物处理组（BAC/Sal）的41%细胞活力相比，纳米复合材料I、II和煅烧纳米复合I均表现出~90%的细胞活力，表明二氧化硅基纳米制剂具有优异的生物相容性。

Next, SEM was conducted to visualize the morphology change in bacteria with or without nanocomposite treatment. Compared to smooth and intact membrane in untreated group (Fig. 2h), clear bacterial cell membrane damage with nanocomposite sinking into bacteria was observed in nanocomposite I treated group, creating some dents on bacterial surface (Fig. 2f red arrow), while limited membrane damage was observed in BAC/NaSal (Fig. 2e) or nanocomposite II (Fig. 2g) treated bacteria. These results collectively demonstrated that rough nanocomposite I templated by "dual actives" displayed the most effective bactericidal capability, presumably due to bacterial membrane disruption through spiky surface enhanced adhesion and boosted release of dual antibacterial agents (BAC and NaSal). The silicon content was analyzed by ICP-OES as shown in Fig. S8. Nearly 2 pg of nanocomposite I adhered on/uptaken by each bacteria, which was almost twofold of the silicon content of nanocomposite II or I-calcined treated bacteria. The results are consistent with the observation from SEM images. From Figs. 2d and S6, BAC/NaSal and nanocomposites I treated groups showed 100% bactericidal performance at the concentration of 4 μg BAC mL−1, thus 4 μg mL−1 was chosen for the following biofilm inhibition study.
接下来，进行扫描电镜（SEM）以可视化细菌的形态变化，无论是否进行纳米复合处理。与未处理组（图2h）中光滑和完整的膜相比，在纳米复合I处理组中观察到明显的细菌细胞膜损伤，纳米复合材料沉入细菌中，在细菌表面产生一些凹痕（图2f红色箭头），而在BAC/NaSal（图2e）或纳米复合II（图2g）处理的细菌中观察到有限的膜损伤。这些结果共同表明，由“双活性物质”模板化的粗糙纳米复合材料I显示出最有效的杀菌能力，这可能是由于细菌膜通过尖刺表面增强粘附和促进双重抗菌剂（BAC和NaSal）的释放而破坏。通过ICP-OES分析硅含量，如图S8所示。每个细菌粘附/吸收了近 2 pg 的纳米复合 I，这几乎是纳米复合 II 或 I 煅烧处理细菌硅含量的两倍。结果与SEM图像的观察结果一致。从图2d和S6中可以看出，BAC/NaSal和纳米复合材料I处理组在4 μg BAC mL浓度下表现出100%的杀菌性能 −1 ，因此选择4 μg mL −1 进行以下生物膜抑制研究。

The advantage of nanocomposite I over nanocomposite II and the BAC/NaSal group was further evaluated in their biofilm inhibition performance through examining their efficiency in inhibiting bacterial surface adhesion and biofilm formation. Nanocomposites or BAC/NaSal were added to planktonic bacteria at the concentration of 4 μg BAC mL−1. Crystal violet (CV) staining was applied to visualize remaining biofilm treated with or without nanocomposite [45], where the positively charged dye interacted with the negatively-charged cell wall and resulted in the purple color of CV [46]. As shown from the standard CV colorimetric assay (Fig. 3a), untreated control or nanocomposite II remained dark purple, suggesting more biofilm biomass, the BAC/NaSal or nanocomposite I treated groups exhibited significantly lighter color, suggesting its ability in biofilm inhibition. Quantification of biofilm biomass normalized to untreated group was shown in Fig. 3b, the inhibiting effect of nanocomposite I was significantly better (biofilm formation reduced to 33%) than nanocomposite II (85%) and BAC/NaSal (63%).
通过研究纳米复合材料I组在抑制细菌表面粘附和生物膜形成方面的效率，进一步评估了纳米复合材料I组相对于纳米复合材料II组和BAC/NaSal组的生物膜抑制性能的优势。将纳米复合材料或BAC/NaSal加入到浮游细菌中，浓度为4 μg BAC mL −1 。应用结晶紫（CV）染色来观察用或不用纳米复合材料处理的剩余生物膜[45]，其中带正电荷的染料与带负电荷的细胞壁相互作用，导致CV呈紫色[46]。如标准CV比色法（图3a）所示，未经处理的对照组或纳米复合材料II保持深紫色，表明更多的生物膜生物质，BAC/NaSal或纳米复合I处理组表现出明显较浅的颜色，表明其生物膜抑制能力。将归一化为未处理组的生物膜生物量定量如图3b所示，纳米复合材料I的抑制作用明显优于纳米复合材料II（85%）和BAC/NaSal（63%）（生物膜形成减少至33%）。

Fig. 3 图3
figure 3

a Digital images of biofilms stained by CV. b Quantitative analysis of CV stained biofilm. c 3-D Confocal images of 24 h old biofilm treated by PBS, nanocomposite I, BAC/NaSal or nanocomposite II
a CV染色的生物膜的数字图像。 b CV染色生物膜的定量分析。c 经 PBS、纳米复合材料 I、BAC/NaSal 或纳米复合材料 II 处理的 24 小时旧生物膜的 3-D 共聚焦图像

Full size image 全尺寸图像

To better analyze the biofilm thickness, 3D confocal microscopy was conducted and LIVE/DEAD bacteria was stained and analyzed (Fig. 3c). The thickness of untreated biofilm was estimated to be 25 μm. Much thinner biofilms (less than 10 μm) with significantly increased dead cell populations (> 90%) were observed after 24 h incubation withnanocomposite I (Fig. 3c, I). For comparison, thicker biofilm was formed on BAC/NaSal (~ 15 μm) or nanocomposite II (22 μm biofilm) treated groups. The above results demonstrate that "dual actives templated" nanocomposite I shows excellent synergy in inhibiting biofilm formation.
为了更好地分析生物膜厚度，进行了3D共聚焦显微镜检查，并对活/死细菌进行了染色和分析（图3c）。未经处理的生物膜厚度估计为25μm。在与纳米复合材料I孵育24小时后，观察到更薄的生物膜（小于10μm），死细胞群显著增加（>90%）（图3c，I）。为了进行比较，在BAC / NaSal（~15μm）或纳米复合II（22μm生物膜）处理组上形成较厚的生物膜。以上结果表明，“双活性模板化”纳米复合材料I在抑制生物膜形成方面表现出优异的协同作用。

4 Conclusions 4 结论

In summary, we have reported a “dual active templating” strategy and successfully synthesized spiky silica nanocomposite containing two active agents for antibacterial applications. The dual active templating strategy contributes to not only the formation of a spiky surface that enhances bacterial membrane adhesion and physical damage, but also an effective release of two bactericidal components for synergistically improved bacterial killing and biofilm inhibition. These findings provide a new strategy for the designed synthesis of novel functional silica composite materials with boosted performance in antibacterial applications.
综上所述，我们报道了一种“双活性模板”策略，并成功合成了含有两种活性剂的尖刺二氧化硅纳米复合材料，用于抗菌应用。双重活性模板策略不仅有助于形成增强细菌膜粘附和物理损伤的尖刺表面，还有助于有效释放两种杀菌成分，从而协同改善细菌杀灭和生物膜抑制。这些发现为设计合成新型功能性二氧化硅复合材料提供了新的策略，提高了抗菌应用的性能。