A Near-Infrared Light-Triggered Nanocarrier with Reversible DNA Valves for Intracellular Controlled Release
具有可逆 DNA 阀的近红外光触发纳米载体，用于细胞内控制释放

1. Introduction 一、简介

Multifunctional nanocarriers combining several useful properties in a single nanostructure have become one of the dominant strategies in drug delivery systems.1 The nanocarrier can encapsulate the drugs with high loading amount and efficiency, while simultaneously reducing side effects. Recently, many efforts have been made to develop stimuli-triggered drug carriers that can regulate the release of the loaded drug effectively in response to a given stimulus, such as pH change,2 temperature change,3 redox activation,4 enzymatic activity,5 competitive binding,6 and photoirradiation.7 Among these approaches, light-triggered drug delivery has attracted much attention because it does not rely on changes in specific chemical properties of the environment, which would be expected for intracellular or in vivo applications. A variety of light-responsive nanocarriers have been designed based on ultraviolet light or visible light excitation to liberate the entrapped cargo molecules,8 which may cause damage to the biological samples and is suitable only for in vitro studies because of its quick attenuation in tissue.9 As a promising candidate, a near-infrared (NIR) light-triggered nanocarrier brings new opportunities to improve the efficiency for drug delivery due to minimal absorbance and maximum penetration for tissues and organs.
将多种有用特性结合在单个纳米结构中的多功能纳米载体已成为药物输送系统的主要策略之一。 1 纳米载体可以高负载量和高效率地封装药物，同时减少副作用。最近，人们做出了许多努力来开发刺激触发的药物载体，该载体可以响应给定的刺激有效地调节负载药物的释放，例如pH变化，2温度变化，3氧化还原激活，4酶活性，5竞争性结合，6 和光照射。 7 在这些方法中，光触发药物递送引起了广泛关注，因为它不依赖于环境特定化学性质的变化，而这在细胞内或体内应用中是预期的。基于紫外光或可见光激发，设计了多种光响应纳米载体，以释放捕获的货物分子，8这可能会对生物样品造成损害，并且由于其在组织中快速衰减，仅适合体外研究。 9 作为一种有前途的候选者，近红外 (NIR) 光触发纳米载体由于对组织和器官的最小吸光度和最大渗透性，为提高药物输送效率带来了新的机会。

The gold nanorods (AuNRs) have strong absorption in the NIR region and can be employed as the local heat sources when irradiated with an NIR laser through the photothermal effect.10 Mesoporous silica nanoparticles (MSNs) are considered to be ideal candidates for drug delivery because of their high surface area, tunable size, good biocompatibility, and easy functionalization.11 Although inorganic materials and organic molecules have been widely used as gatekeepers for MSNs, the utility of the biomolecules as valve provides many advantages, such as good biocompatibility and better cellular uptake. Recently, DNA-capped MSNs as the controlled-release platforms have been intensively studied.9, 12 Most of these platforms performed well in opening the valve to release the cargo molecules triggered by stimulus. However, the valves were not reversible, which may lead to uncontrollable release after the gates are opened. Therefore, it is highly desirable to develop an on-command delivery system that the valve can be opened and closed at will, which could prevent unexpected release and deliver the cargo molecules accurately.
金纳米棒（AuNR）在近红外区域有很强的吸收，当受到近红外激光照射时，通过光热效应可以用作局部热源。 10 介孔二氧化硅纳米颗粒 (MSN) 因其高表面积、可调尺寸、良好的生物相容性和易于功能化而被认为是药物递送的理想候选者。 11 尽管无机材料和有机分子已广泛用作 MSN 的看门人，但生物分子作为阀门的用途提供了许多优点，例如良好的生物相容性和更好的细胞摄取。近年来，DNA加帽的MSNs作为控释平台得到了深入研究。 9, 12 大多数这些平台在打开阀门以释放刺激触发的货物分子方面表现良好。然而，阀门是不可逆的，这可能会导致闸门打开后无法控制的释放。因此，非常需要开发一种可以随意打开和关闭阀门的按需输送系统，这可以防止意外释放并准确地输送货物分子。

Herein, we develop a novel NIR light-triggered nanocarrier in which the cargo molecules can be loaded into mesoporous silica coated gold nanorods and then capped with reversible single-stranded DNA valves. The gold nanorods (AuNRs) were prepared and employed as a template to build mesoporous silica shell. The cargo molecules were then loaded into the mesoporous silica shell. The single-stranded DNA was anchored to mesoporous silica shell by amide bonds and the bases of DNA were absorbed on the surface of the silica shell via electrostatic interaction,12 resulting in the “off” state of the valves. Upon irradiation with the NIR laser with a wavelength that matches the absorption peak of the nanocarrier, the light will be absorbed and converted into heat through the photothermal effect. The heat will dissipate into the surroundings and destroy the electrostatic interaction between DNA and silicon shell, leading to the “on” state of the valves and the release of the cargo molecules from the nanocarrier. When the laser is turned off, the heating will immediately stop and the DNA valves will be back to its original state. Then, the nanocarrier stops releasing the cargo molecules. The details of this approach are described in Figure 1.
在此，我们开发了一种新型近红外光触发纳米载体，其中货物分子可以装载到介孔二氧化硅涂覆的金纳米棒中，然后用可逆单链 DNA 阀封盖。制备金纳米棒（AuNR）并用作构建介孔二氧化硅壳的模板。然后将货物分子装载到介孔二氧化硅壳中。单链 DNA 通过酰胺键锚定在介孔二氧化硅壳上，DNA 碱基通过静电相互作用吸附在二氧化硅壳表面，12 导致阀门处于“关闭”状态。当用与纳米载体的吸收峰相匹配的波长的近红外激光照射时，光将被吸收并通过光热效应转化为热量。热量将消散到周围环境并破坏 DNA 和硅壳之间的静电相互作用，导致阀门处于“开启”状态并从纳米载体中释放货物分子。当激光关闭时，加热将立即停止，DNA 阀门将恢复到原来的状态。然后，纳米载体停止释放货物分子。图 1 描述了该方法的详细信息。

2. Results and Discussion
2 结果与讨论

The AuNRs were typically synthesized using a seed-mediated growth procedure according to a reported protocol with some modifications.13 As shown in Figure 2A, the average length and width of the Au NRs are about 52 and 14 nm, respectively. The modified co-condensation method14 was employed for the preparation of the amino-functionalized mesoporous silica coated Au NRs (AuNRs@MS-NH₂). As can be seen from Figure 2B, the silica shell of AuNRs@MS-NH₂ is estimated to have a homogeneous thickness of about 22 nm and is composed of disordered mesopores, offering an opportunity for AuNRs@MS-NH₂ to be used as a general drug carrier. N₂ adsorption-desorption isotherms of AuNRs@MS-NH₂ showed a typical Type IV curve with a specific surface area of 137 m² g⁻¹ and average pore diameter of 2.2 nm with a narrow pore-size distribution (Supporting Information Figure S1). The rhodamine B (RhB) cargo was then loaded in the mesopores of AuNRs@MS-NH₂. Single-stranded DNA (5′-COOH-(CH₂)₆-CTCCTGTAATGAAGCGCTAAGTGTAATGG-3′) was used as gatekeeper to cap the pores to prevent the cargo from leaking. The AuNRs@MS-DNA was fabricated through the amidation reaction of AuNRs@MS-NH₂ with carboxyl-functionalized DNA. The morphology of AuNRs@MS-DNA did not show obvious change compared with AuNRs@MS-NH₂ (Figure 2C). The UV-vis absorption spectra of AuNRs, AuNRs@MS-NH₂ and AuNRs@MS-DNA were shown in Figure 2D. The maximum absorption of the Au NRs is at 780 nm and it is red shifted to 805 nm after coating with the silica shell. This is due to the local increase of refractive index and the scattering from the silica shells.15 After the DNA was modified on the surface of AuNRs@MS-NH₂, the maximum absorption further shifted to 813 nm. Moreover, the appearance of the peak at 260 nm was attributed to the DNA absorbance, indicating the successful modification of DNA on the AuNRs@MS-NH₂ surface. Zeta potential experiments further verified the successful treatment of the nanocarrier in different stages, i.e., –19.3 ± 0.4 mV (AuNRs@MS), 13.1 ± 0.9 mV (AuNRs@MS-NH₂), and –23.1 ± 0.4 mV (AuNRs@MS-DNA).
AuNR 通常是根据已报道的方案并进行一些修改，使用种子介导的生长程序来合成的。 13 如图 2A 所示，Au NR 的平均长度和宽度分别约为 52 和 14 nm。采用改进的共缩合方法14制备氨基功能化介孔二氧化硅包覆的Au NRs（AuNRs@MS-NH ₂ ）。从图2B中可以看出，AuNRs@MS-NH ₂ 的二氧化硅壳估计具有约22 nm的均匀厚度，并且由无序介孔组成，这为AuNRs@MS-NH提供了机会。 NH ₂ 用作通用药物载体。 AuNRs@MS-NH ₂ 的N ₂ 吸附-脱附等温线表现出典型的IV型曲线，比表面积为137 m ² g ⁻¹ 平均孔径为 2.2 nm，孔径分布较窄（支持信息图 S1）。然后将罗丹明 B (RhB) 货物装载到 AuNRs@MS-NH ₂ 的中孔中。单链DNA (5'-COOH-(CH ₂ ) ₆ -CTCCTGTAATGAAGCGCTAAGTGTAATGG-3') 用作守门人，封闭孔以防止货物泄漏。 AuNRs@MS-DNA 通过 AuNRs@MS-NH ₂ 与羧基功能化 DNA 的酰胺化反应制备。与AuNRs@MS-NH ₂ 相比，AuNRs@MS-DNA的形貌没有表现出明显的变化（图2C）。 AuNRs、AuNRs@MS-NH ₂ 和 AuNRs@MS-DNA 的紫外-可见吸收光谱如图 2D 所示。 Au NRs 的最大吸收位于 780 nm，涂覆二氧化硅壳后红移至 805 nm。这是由于折射率的局部增加和二氧化硅壳的散射。 15 DNA在AuNRs@MS-NH ₂ 表面修饰后，最大吸收进一步移动至813 nm。此外，260 nm处峰的出现归因于DNA吸光度，表明AuNRs@MS-NH ₂ 表面上DNA的成功修饰。 Zeta电位实验进一步验证了纳米载体在不同阶段的成功处理，即–19.3 ± 0.4 mV (AuNRs@MS)、13.1 ± 0.9 mV (AuNRs@MS-NH ₂ )和–23.1 ± 0.4 mV（AuNR@MS-DNA）。

The stability of the nanocarrier was evaluated by time-dependent fluorescence changes at room temperature. As shown in Figure 3A, about 38% RhB was leaked after 168 h for AuNRs@MS-NH₂, while less than 7% RhB was leaked after 168 h for AuNRs@MS-DNA (Figure 3B), indicating that the DNA valves could prevent the cargo molecules from leaking effectively. After the nanocarrier was irradiated with a continuous-wave NIR diode laser (808 nm) for 90 min, more than 70% cargo molecules were released, suggesting that the DNA valves could open as expected because of the photothermal effect. To confirm that the release of cargo molecules was indeed induced by the opening of the DNA valves instead of the thermal diffusion upon the NIR irradiation, FAM labeled DNA (5′-CTCCTGTAATGAAGCGCTAAGTGTAATGG-(CH₂)₆-FAM-3′) was used as the gatekeeper instead of the carboxylated DNA to cap the pores of AuNRs@MS-NH₂. After being irradiated with the NIR laser, the AuNRs@MS-DNA-FAM solution was centrifuged and the fluorescence intensity of the supernatant was measured. The fluorescence intensity of AuNRs@MS-DNA-FAM without irradiation was used as the control. Supporting Information Figure S2 shows that the fluorescence intensity of irradiated AuNRs@MS-DNA-FAM solution was much higher than that of AuNRs@MS-DNA-FAM without irradiation. This suggests that the electrostatic interaction between DNA and the silicon shell could be destroyed upon irradiation by NIR laser and that the DNA could detach from the surface of the silicon shell. Zeta potential experiments further verified the above results, i.e., –19.0 ± 1.4 mV (AuNRs@MS-DNA-FAM without irradiation after centrifugation) and –7.4 ± 0.9 mV (AuNRs@MS-DNA-FAM with irradiation after centrifugation).
通过室温下随时间变化的荧光变化来评估纳米载体的稳定性。如图3A所示，AuNRs@MS-NH ₂ 在168小时后泄漏了约38％的RhB，而AuNRs@MS-DNA在168小时后泄漏了不到7％的RhB（图3B） ，表明DNA阀门可以有效防止货物分子泄漏。用连续波近红外二极管激光（808 nm）照射纳米载体90分钟后，超过70%的货物分子被释放，这表明由于光热效应，DNA阀门可以按预期打开。为了确认货物分子的释放确实是由 DNA 阀门的打开而不是近红外辐射时的热扩散诱导的，FAM 标记了 DNA (5'-CTCCTGTAATGAAGCGCTAAGTGTAATGG-(CH ₂ ) ₆ -FAM-3') 代替羧化 DNA 作为看门人来封闭 AuNRs@MS-NH ₂ 的孔道。用近红外激光照射后，将 AuNRs@MS-DNA-FAM 溶液离心并测量上清液的荧光强度。以未经照射的 AuNRs@MS-DNA-FAM 的荧光强度作为对照。支持信息图S2显示，经过辐照的AuNRs@MS-DNA-FAM溶液的荧光强度远高于未经辐照的AuNRs@MS-DNA-FAM溶液。这表明DNA和硅壳之间的静电相互作用在近红外激光照射下可能被破坏，并且DNA可能从硅壳表面分离。 Zeta电位实验进一步验证了上述结果，即–19.0 ± 1.4 mV（AuNRs@MS-DNA-FAM离心后无辐照）和–7.4 ± 0.9 mV（AuNRs@MS-DNA-FAM离心后辐照）。

The effect of temperatures on the opening of the DNA valves of the nanocarrier was also investigated. As shown in Figure 4, the release amount of the cargo molecules did not show obvious change when the temperature was lower than 44 °C. This demonstrates that the electrostatic interaction between DNA and the silicon shell still existed and that the DNA valves were closed. When the temperature was ≥44 °C, the release amount of the cargo molecules sharply increased within 90 min, suggesting the opening of the DNA valves.
还研究了温度对纳米载体 DNA 阀门打开的影响。如图4所示，当温度低于44℃时，货物分子的释放量没有表现出明显的变化。这表明DNA和硅壳之间的静电相互作用仍然存在并且DNA阀门是关闭的。当温度≥44℃时，货物分子的释放量在90分钟内急剧增加，表明DNA阀门打开。

Prior to applying NIR light as a stimulus to trigger the cargo release, the effect of laser activated temperature increase of the DNA-MS@AuNRs (RhB) solution was investigated under different laser wavelengths and different power densities with increasing irradiation time. Figure 5 shows that the solution temperature remained unchanged under irradiation at 655 nm (3 W·cm⁻²) and was of comparable magnitude to the initial temperature. When irradiated with at 808 nm at different power densities, the solution temperature gradually increased up to 500 s, after which the solution temperature remained constant. The final temperature increased with increasing laser power density, i.e., 36 °C for 2 W·cm⁻², 45 °C for 2.7 W·cm⁻², 48 °C for 3 W·cm⁻², and 58 °C for 4 W·cm⁻². These results indicate that the nanocarrier was efficiently responsive to the NIR light matched to its absorption peak and the generated photothermal effect was positively correlated with laser power density. In order to open the DNA valves (T ≥ 44 °C) and minimize the chance of apoptosis by hyperthermia (T ≤ 45 °C),12 a laser power of 2.7 W·cm⁻² was chosen in the following experiments.
在应用近红外光作为刺激来触发货物释放之前，研究了在不同激光波长和不同功率密度下随着照射时间的增加，激光激活 DNA-MS@AuNRs (RhB) 溶液温度升高的影响。图5显示，在655 nm（3 W·cm ⁻² ）照射下，溶液温度保持不变，与初始温度相当。当用不同功率密度的808 nm照射时，溶液温度逐渐升高至500 s，之后溶液温度保持恒定。最终温度随着激光功率密度的增加而升高，即2 W·cm ⁻² 为36 °C，2.7 W·cm ⁻² 为45 °C，3 W为48 °C ·cm ⁻² ，4 W·cm ⁻² 为 58 °C。这些结果表明纳米载体能够有效地响应与其吸收峰匹配的近红外光，并且产生的光热效应与激光功率密度正相关。为了打开 DNA 阀门（T ≥ 44 °C）并最大程度地减少热疗导致细胞凋亡的机会（T ≤ 45 °C），12 选择了 2.7 W·cm ⁻² 的激光功率。以下实验。

The controlled release of the AuNRs@MS-DNA (RhB) triggered by NIR laser was determined with fluorescence spectroscopic analysis. To evaluate the repeatability and triggered nature of the release, the nanocarrier solutions were manipulated with the NIR laser (808 nm, 2.7 W·cm⁻²) for several laser on/off cycles. For each cycle, the sample was irradiated for 15 min and then the laser was turned off for 12 h. Figure 6 shows that the cargo molecules could be released upon irradiation (laser on), indicating the DNA valves were open after irradiation. When the laser was turned off (laser off), the release of the cargo molecules was inhibited, suggesting the DNA valves were close again. The results demonstrated the nanocarrier was reversible after every laser on/off cycle as expected. Approximately 70% RhB was released after seven cycles. When the nanocarrier solution was irradiated by NIR laser continuously, the release amount of cargo was increased with the irradiation time and nearly 70% RhB was released within 100 min (Supporting Information Figure S3). In contrast, about 21% cargo molecules were released upon the first irradiation for 15 min of the AuNRs@MS-NH₂ (RhB) solution and about 20% cargo molecules were still released when the laser was turned off (Supporting Information Figure S4). This indicates that the controlled release could not be achieved for the nanocarrier without the DNA valves. The results show that the current nanocarrier is capable of controlling the cargo release accurately by adjusting the irradiation time and laser on/off states.
通过荧光光谱分析确定近红外激光触发的 AuNRs@MS-DNA (RhB) 的受控释放。为了评估释放的可重复性和触发性质，使用近红外激光（808 nm，2.7 W·cm ⁻² ）操纵纳米载体溶液几个激光开/关循环。对于每个循环，样品被照射 15 分钟，然后关闭激光 12 小时。图 6 显示货物分子可以在照射（激光开启）时释放，表明 DNA 阀门在照射后打开。当激光关闭（激光关闭）时，货物分子的释放受到抑制，表明 DNA 阀门再次关闭。结果表明，纳米载体在每次激光开/关周期后都是可逆的，正如预期的那样。七个循环后大约释放了 70% RhB。当纳米载体溶液受到近红外激光连续照射时，货物的释放量随着照射时间的增加而增加，在100分钟内释放了近70%的RhB（支持信息图S3）。相比之下，AuNRs@MS-NH ₂ (RhB) 溶液第一次照射 15 分钟时，约 21% 的货物分子被释放，当激光关闭时，约 20% 的货物分子仍被释放。 （支持信息图S4）。这表明如果没有DNA阀，纳米载体就无法实现受控释放。结果表明，当前的纳米载体能够通过调节照射时间和激光开关状态来精确控制货物释放。

The nuclease resistance ability is critical for nanocarriers when used in living cells and this was therefore evaluated under physiological conditions. Enzyme deoxyribonuclease I (DNase I),16 a common endonuclease, was employed to assess the nuclease stability of the nanocarrier. Figure 7 showed that the nanocarrier treated with DNase I exhibited slight degradation compared with the case without DNase I. When the nanocarrier only and nanocarrier/DNase I solutions were irradiated with the NIR laser (808 nm) for 90 min, the fluorescence intensities of the two solutions increased greatly (Figure 7, inset). The results indicate the nanocarrier possessed high resistance to nuclease and the release of cargo molecules was indeed due to the irradiation. The nanocarrier could avoid unexpected release of cargo molecules caused by nuclease degradation and it was shown to be viable for application in living cells.
当纳米载体用于活细胞时，核酸酶抗性能力至关重要，因此在生理条件下进行评估。酶脱氧核糖核酸酶 I (DNase I) 16 是一种常见的核酸内切酶，用于评估纳米载体的核酸酶稳定性。图7显示，与没有DNase I的情况相比，用DNase I处理的纳米载体表现出轻微的降解。当用NIR激光（808 nm）照射仅纳米载体和纳米载体/DNase I溶液90分钟时，纳米载体的荧光强度两种解决方案都大大增加了（图 7，插图）。结果表明，纳米载体对核酸酶具有较高的抵抗力，并且货物分子的释放确实是由于辐射所致。该纳米载体可以避免由核酸酶降解引起的货物分子的意外释放，并且它被证明可以在活细胞中应用。

For the application of NIR laser triggered nanocarrier for controlled delivery, intracellular release experiments were carried out in a human breast cancer cell line (MCF-7). As shown in Figure 8, nearly no fluorescence signal was observed under confocal laser scanning microscopy when the MCF-7 cells were without irradiation. In order to confirm the controlled release in living cells, three laser on/off cycles were performed in the same cells. For every cycle, the laser on time was chosen to be 10 min to ensure that the final temperature is 45 °C (the irradiation time needs to be more than 8 min, as shown in Figure 5), which makes the DNA valves open and minimizes the potential damage to cells by hyperthermia for a long time. After the first irradiation, a faint fluorescence signal appeared, indicating the successful release of RhB. The fluorescence intensity was gradually enhanced when the laser on/off cycles increased. This demonstrates that the nanocarriers could be triggered by the laser and that the controlled delivery could be achieved in living cells. The bright-field images revealed that the cells were viable throughout the whole imaging experiments of three cycles.
为了应用近红外激光触发纳米载体进行控制递送，在人乳腺癌细胞系（MCF-7）中进行了细胞内释放实验。如图8所示，当MCF-7细胞未受到照射时，在共焦激光扫描显微镜下几乎观察不到荧光信号。为了确认活细胞中的受控释放，在同一细胞中进行了三个激光开/关循环。对于每个循环，激光开启时间选择为10分钟，以确保最终温度为45℃（照射时间需要超过8分钟，如图5所示），这使得DNA阀门打开并最大限度地减少长时间高温对细胞的潜在损害。第一次照射后，出现微弱的荧光信号，表明RhB释放成功。当激光开/关周期增加时，荧光强度逐渐增强。这表明纳米载体可以被激光触发，并且可以在活细胞中实现受控递送。明场图像显示，细胞在三个周期的整个成像实验中都是存活的。

To evaluate the cytotoxicity of the nanocarrier and the photothermal effect by irradiation, an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide) assay in MCF-7 cells was performed. The absorbance of MTT at 490 nm is dependent on the degree of activation of the cells. The cell viability was expressed by the ratio of absorbance of the treated cells (incubated with the nanocarrier or irradiated with NIR laser) to that of the untreated cells. The results indicated that the nanocarrier showed almost no cytotoxicity or side effects in living cells (Figure 9A). The apoptosis induced by irradiation and the photothermal effects was also investigated. Figure 9B indicated that no obvious decrease in the cell viability was observed after each irradiation, which further confirmed, as expected, that the NIR laser with a power density of 2.7 W·cm⁻² exerted almost no damage on the living cells.
为了评估纳米载体的细胞毒性和辐射的光热效应，在 MCF-7 细胞中进行了 MTT（3-（4, 5-二甲基噻唑-2-基）-2, 5-二苯基四唑溴化物）测定。 MTT 在 490 nm 处的吸光度取决于细胞的活化程度。细胞活力通过处理的细胞（与纳米载体一起孵育或用近红外激光照射）与未处理的细胞的吸光度之比来表示。结果表明，纳米载体在活细胞中几乎没有表现出细胞毒性或副作用（图9A）。还研究了辐射诱导的细胞凋亡和光热效应。图9B表明，每次照射后细胞活力均未观察到明显下降，这进一步证实，正如预期的那样，功率密度为2.7 W·cm ⁻² 的近红外激光对细胞几乎没有造成损伤。活细胞。

For further application of the nanocarrier, a chemotherapeutic drug doxorubicin (Dox) was loaded to investigate if the therapeutic effect could be controlled using the current nanocarrier. Figure 9C shows that the cell viability is about 95% without the irradiation when the nanocarrier was incubated with the MCF-7 cells, suggesting that the nanocarrier indeed performed well in preventing the Dox molecules from leaking. The cells were then treated with different laser on/off cycles; the cell viability decreased with increasing number of irradiation cycles. After three cycles of irradiation, only about 50% cells survived. The result is consistent with the in vitro release experiment and intracellular imaging, which further confirmed that the release amount of cargo molecules could be controlled using the nanocarrier.
为了进一步应用纳米载体，负载化疗药物阿霉素（Dox）以研究是否可以使用当前的纳米载体来控制治疗效果。图9C显示，当纳米载体与MCF-7细胞一起孵育时，在没有照射的情况下，细胞活力约为95%，这表明纳米载体确实在防止Dox分子泄漏方面表现良好。然后用不同的激光开/关周期处理细胞；细胞活力随着照射周期次数的增加而降低。经过三个周期的照射后，只有约 50% 的细胞存活。该结果与体外释放实验和细胞内成像一致，进一步证实了纳米载体可以控制货物分子的释放量。

3. Conclusions 3. 结论

We have presented a novel NIR laser-triggered nanocarrier based on mesoporous, silica-coated gold nanorods with reversible DNA valves. The reversible DNA valves of the nanocarrier were manipulated by switching the laser on/off states, which was capable of controlling the release amount of cargo molecules. Moreover, the nanocarrier possesses good stability, high nuclease resistance and good biocompatibility. Intracellular imaging experiments indicated the nanocarrier could be triggered with a NIR laser and the controlled release could be achieved in living cells. When doxorubicin was loaded into the nanocarrier, the therapeutic effect also could be controlled. Compared to the reported nanocarriers, the current approach could deliver the cargo molecules accurately in a controlled manner, which is indispensable for the treatment of some diseases with precise dosage at a desired time in a specified area. We anticipate that the nanocarrier could provide new insights for designing the on-command drug delivery systems.
我们提出了一种新型近红外激光触发纳米载体，其基于具有可逆 DNA 阀的介孔二氧化硅涂层金纳米棒。通过切换激光开/关状态来操纵纳米载体的可逆DNA阀，从而能够控制货物分子的释放量。此外，该纳米载体还具有良好的稳定性、高核酸酶抗性和良好的生物相容性。细胞内成像实验表明，纳米载体可以用近红外激光触发，并且可以在活细胞中实现受控释放。当阿霉素负载到纳米载体上时，治疗效果也可以得到控制。与报道的纳米载体相比，目前的方法可以以受控的方式准确地递送货物分子，这对于在特定区域在所需时间以精确剂量治疗某些疾病是必不可少的。我们预计纳米载体可以为设计按需给药系统提供新的见解。

4. Experimental Section 4. 实验部分

Materials: DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co., Ltd (Shanghai, China). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin (Dox) and rhodamine B (RhB) were purchased from Sigma Chemical Company; deoxyribonuclease I (DNase I) was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China); sodium borohydride (NaBH₄), cetyltrimethyl ammonium bromide (CTAB), silver nitrate (AgNO₃), ascorbic acid, tetraethyl orthosilicate (TEOS), and hydrogen tetrachloroaurate (III) (HAuCl₄·4H₂O) were purchased from China National Pharmaceutical Group Corporation (Shanghai, China); (3-aminopropyl) triethoxysilane (APTES) and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) were purchased from Alfa Aesar Chemical Ltd (Tianjin, China). All the chemicals were of analytical grade and used without further purification. Sartorius ultrapure water (18.2 MΩ cm) was used throughout the experiments. The human breast cancer cell line (MCF-7) was purchased from KeyGEN biotechnology Company (Nanjing, China).
材料：DNA寡核苷酸由生工生物技术有限公司（中国上海）合成和纯化。 3-(4,5-二甲基-噻唑-2-基)-2,5-二苯基溴化四唑(MTT)、阿霉素(Dox)和罗丹明B(RhB)购自Sigma化学公司；脱氧核糖核酸酶I（DNase I）购自索拉宝科技有限公司（中国北京）；硼氢化钠 (NaBH ₄ )、十六烷基三甲基溴化铵 (CTAB)、硝酸银 (AgNO ₃ )、抗坏血酸、原硅酸四乙酯 (TEOS) 和四氯金酸 (III) (HAuCl) ₄ ·4H ₂ O)购自中国医药集团公司（中国上海）； (3-氨基丙基)三乙氧基硅烷(APTES)和1-乙基-3-(3-二甲基氨基-丙基)碳二亚胺盐酸盐(EDC)购自阿法埃莎化学有限公司(中国天津)。所有化学品均为分析纯，无需进一步纯化即可使用。整个实验使用 Sartorius 超纯水 (18.2 MΩ cm)。人乳腺癌细胞系（MCF-7）购自KeyGEN生物技术公司（中国南京）。

Instruments: Near infrared (NIR) lasers with irradiation wavelength of 655 nm (MLL-III-655) and 808 nm (MDL-III-808) were purchased from Changchun New Industries Optoelectronics Tech. Co., Ltd (Changchun, China). Transmission electron microscopy (TEM) was carried out on a JEM-100CX II electron microscope and high resolution transmission electron microscopy (HRTEM) was carried out on a JEM-2100 electron microscope, respectively. N₂ adsorption-desorption isotherms were recorded on a Micromeritics ASAP2020 surface area and porosity analyzer. The samples were degassed at 150 °C for 5 h. The specific surface areas were calculated from the adsorption data in the low pressure range using the BET model and pore size was determined using the Barrett–Joyner–Halenda (BJH) method. Absorption spectra were measured on a Pharmaspec UV-1700 UV-vis spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained with FLS-920 Edinburgh fluorescence spectrometer with a xenon lamp and 1.0 cm quartz cells at the slits of 3.0/3.0 nm. All pH measurements were performed with a pH-3c digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) with a combined glass-calomel electrode. Absorbance was measured in a microplate reader (RT 6000, Rayto, USA) in the MTT assay. Confocal fluorescence imaging studies were performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (40×).
仪器：近红外（NIR）激光器，照射波长为655 nm（MLL-III-655）和808 nm（MDL-III-808），购自长春新产业光电科技有限公司。有限公司（中国长春）。透射电子显微镜（TEM）分别在JEM-100CX II电子显微镜上进行，高分辨率透射电子显微镜（HRTEM）在JEM-2100电子显微镜上进行。 N ₂ 吸附-解吸等温线在 Micromeritics ASAP2020 表面积和孔隙率分析仪上记录。样品在150℃下脱气5小时。使用 BET 模型根据低压范围内的吸附数据计算比表面积，并使用 Barrett-Joyner-Halenda (BJH) 方法确定孔径。吸收光谱在 Pharmaspec UV-1700 紫外可见分光光度计（Shimadzu，日本）上测量。使用FLS-920 Edinburgh荧光光谱仪、氙灯和1.0 cm石英池在3.0/3.0 nm狭缝处获得荧光光谱。所有 pH 测量均使用带有组合玻璃甘汞电极的 pH-3c 数字 pH 计（中国上海雷磁设备厂）进行。 MTT 测定中使用酶标仪（RT 6000，Rayto，USA）测量吸光度。使用带有物镜（40×）的 TCS SP5 共焦激光扫描显微镜（德国徕卡有限公司）进行共焦荧光成像研究。

Preparation of AuNRs: Gold nanorods (AuNRs) were typically synthesized using a seed-mediated growth procedure according to a reported protocol with some modifications.13 Seed solution was first synthesized by mixing HAuCl₄ (10 mM, 0.25 mL) and CTAB (0.1 M, 10 mL). Next, 0.6 mL ice-cold NaBH₄ aqueous solution (0.01 M) was added to the mixture which resulted in formation of a bright brownish-yellow solution. This solution was kept at 25 °C at least 2 h. The growth solution was prepared as follows. CTAB (0.1 M, 40 mL) was added to HAuCl₄ (10 mM, 2 mL) with gentle stirring. Then, AgNO₃ (0.01 M, 320 μL), HCl (1.0 M, 0.8 mL) and ascorbic acid (0.1 M, 0.32 mL) were successively added to the above mixture. Finally, 48 μL seed solution was added to the growth solution and the growth medium was kept at 27 °C for more than 6 h before further use.
AuNR 的制备：金纳米棒 (AuNR) 通常是根据已报道的方案并进行一些修改，使用种子介导的生长程序合成的。 13 首先通过混合 HAuCl ₄ (10 mM, 0.25 mL) 和 CTAB (0.1 M, 10 mL) 合成种子溶液。接下来，将0.6mL冰冷的NaBH ₄ 水溶液(0.01M)添加到混合物中，形成明亮的棕黄色溶液。将该溶液在 25°C 下保存至少 2 小时。如下制备生长溶液。将 CTAB (0.1 M，40 mL) 添加到 HAuCl ₄ (10 mM，2 mL) 中，同时轻轻搅拌。然后，将AgNO ₃ (0.01M，320μL)、HCl(1.0M，0.8mL)和抗坏血酸(0.1M，0.32mL)依次添加至上述混合物中。最后，将48μL种子液加入到生长液中，并将生长培养基在27℃下保存6小时以上，然后再使用。

Preparation of AuNRs@MS-NH₂: Amino modified mesoporous silica coated AuNRs were synthesized using the typical co-condensation method reported previously with some modifications.14 The as-synthesized AuNRs were washed by centrifugation at 10 000 rpm for 10 min twice to remove excess CTAB and redispersed in 100 mL water. NaOH (0.1 M, 0.3 mL) was added to the 30 mL prepared AuNRs upon gentle stirring for 10 min, followed by the addition of 45 μL of 20% TEOS in methanol along with 15 μL of 2% APTES in methanol three times under gentle stirring at 30 min intervals. The mixture was reacted for 24 h to form the mesoporous silica shell. The as-synthesized AuNRs@MS-NH₂ was washed with methanol and water for several times to remove the CTAB remained inside the mesopores. The precipitates were finally dispersed in 6 mL water to get a solution with concentration of 0.2 mg/mL. The AuNRs@MS-NH₂ was dried and weighted for further use.
AuNRs@MS-NH ₂ 的制备：使用先前报道的典型共缩合方法并进行一些修改，合成了氨基改性介孔二氧化硅包覆的 AuNRs。 14 将合成的 AuNR 以 10 000 rpm 离心 10 分钟洗涤两次，以去除过量的 CTAB，然后重新分散在 100 mL 水中。将 NaOH (0.1 M，0.3 mL) 添加到 30 mL 制备的 AuNR 中，轻轻搅拌 10 分钟，然后在温和搅拌下添加 45 μL 20% TEOS 的甲醇溶液以及 15 μL 2% APTES 的甲醇溶液 3 次。每隔 30 分钟搅拌一次。将混合物反应24小时以形成介孔二氧化硅壳。将合成的AuNRs@MS-NH ₂ 用甲醇和水洗涤数次以去除残留在介孔内的CTAB。最后将沉淀分散在6mL水中，得到浓度为0.2mg/mL的溶液。将 AuNRs@MS-NH ₂ 干燥并称重以供进一步使用。

Preparation of AuNRs@MS-DNA (RhB) and AuNRs@MS-DNA (Dox): 2 mL as-prepared AuNRs@MS-NH₂ solution was added to 10 mL RhB solution (0.5 mg/mL). The mixture was stirred for 3 days in darkness to reach the maximum loading. The RhB loaded AuNRs@MS solution was centrifuged (10 000 rpm, 10 min) and washed twice with water to remove the RhB molecules absorbed physically on the outer surface of the silica shell. The precipitates were redispersed in 4 mL MES buffer (10 mM, pH 6.0). AuNRs@MS-DNA was obtained by coupling the carboxl group of the oligonucleotides and the amino group on the surface of AuNRs@MS-NH₂ to form the amido bonds. 17.8 μL EDC (2.8 mM) solution was added to 100 μL of single-stranded DNA (100 μM) solution with the sequence of 5′-COOH-(CH₂)₆-ACTCCTGTAATGAAGCGCTAAGTGTAATGG-3′. The solution was mixed and reacted for 30 min at room temperature to activate carboxylate groups. The mixture was then added to 2 mL RhB loaded AuNRs@MS solution with gentle stirring in darkness. The solution was reacted for 24 h which resulted in the formation of the amido bonds. Following this, the precipitates were centrifuged (10 000 rpm, 10 min) and washed with PBS buffer (10 mM, pH 7.4. 100 mM NaCl, 1 mM MgCl₂) for three times and finally redispersed in 2 mL PBS buffer. The preparation of AuNRs@MS-DNA (Dox) was same as the method mentioned above.
AuNRs@MS-DNA (RhB) 和 AuNRs@MS-DNA (Dox) 的制备：将 2 mL 制备好的 AuNRs@MS-NH ₂ 溶液添加到 10 mL RhB 溶液（0.5 mg/mL）中）。将混合物在黑暗中搅拌3天以达到最大负载。将负载 RhB 的 AuNRs@MS 溶液离心（10 000 rpm，10 分钟）并用水洗涤两次，以去除物理吸附在二氧化硅壳外表面上的 RhB 分子。将沉淀物重新分散在 4 mL MES 缓冲液（10 mM，pH 6.0）中。 AuNRs@MS-DNA是通过寡核苷酸的羧基与AuNRs@MS-NH ₂ 表面的氨基偶联形成酰胺键而得到的。将 17.8 μL EDC (2.8 mM) 溶液添加到 100 μL 单链 DNA (100 μM) 溶液中，序列为 5'-COOH-(CH ₂ ) ₆ - ACTCCTGTAATGAAGCGCTAAGTGTAATGG-3'。将溶液混合并在室温下反应30分钟以活化羧酸酯基团。然后将混合物添加至 2 mL RhB 负载的 AuNRs@MS 溶液中，并在黑暗中轻轻搅拌。溶液反应24小时，形成酰胺键。随后，将沉淀物离心（10 000 rpm，10 分钟）并用 PBS 缓冲液（10 mM，pH 7.4。100 mM NaCl，1 mM MgCl ₂ ）洗涤 3 次，最后重新分散在 2 中。 mL PBS 缓冲液。 AuNRs@MS-DNA(Dox)的制备与上述方法相同。

Quantitation of RhB Loaded into the Nanocarrier: To quantify the RhB loaded into the nanocarrier, 2 mL AuNRs@MS-DNA (RhB) solution was heated in the water bath at 80 °C for 2 h. The sample was centrifuged (10 000 rpm, 10 min) and the supernate was separated. Then, the precipitates were redispersed in 2 mL PBS buffer. The above procedure was repeated at least twice to ensure the RhB release from the pores completely. The fluorescence intensity (λ_ex = 532 nm, λ_em = 575 nm) of the supernate was measured and the concentrations of RhB were determined according to a standard linear calibration curve of RhB (Supporting Information Figure S5). The loading content of RhB was calculated to be 0.0192 mg RhB per 1 mg AuNRs@MS-NH₂.
加载到纳米载体中的 RhB 定量：为了定量加载到纳米载体中的 RhB，将 2 mL AuNRs@MS-DNA (RhB) 溶液在 80 °C 水浴中加热 2 小时。将样品离心（10 000 rpm，10 分钟）并分离上清液。然后，将沉淀物重新分散在 2 mL PBS 缓冲液中。上述过程至少重复两次，以确保RhB完全从孔中释放。测定上清液的荧光强度（λ _ex = 532 nm，λ _em = 575 nm），根据RhB标准线性校准曲线测定RhB的浓度（支持信息图S5）。经计算，RhB 的负载量为每 1 mg AuNRs@MS-NH ₂ 0.0192 mg RhB。

Stability of the Nanocarriers: To evaluate the stability of the nanocarrier, the as-prepared 2 mL AuNRs@MS-NH₂ (RhB) or AuNRs@MS-DNA (RhB) (0.1 mg/mL) was stored at room temperature and the fluorescence intensity of the sample was measured (λ_ex = 532 nm, λ_em = 575 nm) at 0, 24, 48, 72, 96, 120, 144, and 168 h, respectively. After that, this sample was irradiated with NIR laser at 808 nm for 90 min and the fluorescence intensity was also measured. The experiment was repeated three times and the data are shown as the mean ± SD.
纳米载体的稳定性：为了评估纳米载体的稳定性，将制备的 2 mL AuNRs@MS-NH ₂ (RhB) 或 AuNRs@MS-DNA (RhB) (0.1 mg/mL)室温保存，在 0、24、48、72、96、120 处测量样品的荧光强度（λ _ex = 532 nm、λ _em = 575 nm） 、 144 和 168 小时，分别。之后，用808 nm的NIR激光照射该样品90分钟，并测量荧光强度。实验重复3次，数据以平均值±SD表示。

Confirmation of the Opening of DNA Valves Upon NIR Laser Irradiation: To confirm that the DNA valves could be open upon the NIR irradiation, FAM labeled DNA (5′-CTCCTGTAATGAAGCGCTAAGTGTAATGG-(CH₂)₆-FAM-3′) was used as gatekeeper instead of the carboxylated DNA (5′-COOH-(CH₂)₆-CTCCTGTAATGAAGCGCTAAGTGTAATGG-3′) to cap the pores of AuNRs@MS-NH₂. In this case, there was only electrostatic interaction between DNA and the silicon shell and no covalent bond between them, when the electrostatic interaction was destroyed, the DNA would detach from the surface of silicon shell. 100 μL of FAM labeled single-stranded DNA (100 μM) solution was added to 2 mL as-prepared AuNRs@MS-NH₂ solution. The mixture was stirred for 12 h in the darkness to make the DNA absorb on the surface of AuNRs@MS-NH₂ and cap the porous. The mixture was then centrifuged (10 000 rpm, 10 min) and washed with water to remove the excess DNA molecules. The precipitates (AuNRs@MS-DNA-FAM) were finally dispersed in 2 mL PBS buffer. The solution was then divided into two parts. One part (1 mL) was irradiated with the NIR laser (808 nm, 2.7 W·cm⁻²) for 15 min and the other part was kept without treatment as the control. Both the two samples were kept in the darkness to prevent FAM from photobleaching. The two samples were immediately centrifuged (10 000 rpm, 10 min). The fluorescence intensity of the supernate was measured. The precipitates of each sample were redispersed into 1 mL water. The above procedure was repeated twice to make the DNA molecules desorption from the silicon shell and the control group was also centrifuged twice to keep the consistency principle. Finally, the precipitates of the two samples were redispersed into 1 mL water and the zeta potential was measured. Each experiment was repeated at least three times and the data are shown as the mean ± SD.
确认 NIR 激光照射下 DNA 阀门打开：为了确认 DNA 阀门可以在 NIR 照射下打开，FAM 标记 DNA (5′-CTCCTGTAATGAAGCGCTAAGTGTAATGG-(CH ₂ ) ₆ ) ₆ -CTCCTGTAATGAAGCGCTAAGTGTAATGG-3') 作为看门人来封盖孔AuNRs@MS-NH ₂ 。此时，DNA与硅壳之间仅存在静电相互作用，而没有共价键，当静电相互作用被破坏时，DNA就会从硅壳表面脱离。将 100 μL FAM 标记的单链 DNA (100 μM) 溶液添加到 2 mL 准备好的 AuNRs@MS-NH ₂ 溶液中。将混合物在黑暗中搅拌12小时，使DNA吸附在AuNRs@MS-NH ₂ 表面并封盖多孔。然后将混合物离心（10 000 rpm，10 分钟）并用水洗涤以除去多余的 DNA 分子。沉淀物 (AuNRs@MS-DNA-FAM) 最终分散在 2 mL PBS 缓冲液中。然后将溶液分成两部分。一部分（1 mL）用近红外激光（808 nm，2.7 W·cm ⁻² ）照射15 min，另一部分不做任何处理作为对照。两个样品均保存在黑暗中以防止 FAM 发生光漂白。立即将两个样品离心（10 000 rpm，10 分钟）。测量上清液的荧光强度。将每个样品的沉淀物重新分散到1mL水中。重复上述过程两次，使DNA分子从硅壳上解吸，对照组也离心两次，以保持一致性原则。 最后，将两个样品的沉淀物重新分散到1mL水中并测量zeta电位。每个实验至少重复3次，数据以平均值±SD表示。

Determination of the Critical Temperature of the DNA Valves: To determine the critical temperature of the DNA valves to open, six samples of 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) in PBS buffer were heated in water bath with different temperatures (30, 37, 42, 44, 50, and 60 °C). Each sample was heated for 90 min and the fluorescence intensity was measured every 15 min. The final fluorescence intensity of the dye released completely was measured as mentioned above. The percentage of dye release from the nanocarrier was calculated as follows: (fluorescence intensity of each sample at different time)/(the final fluorescence intensity of the sample). Each experiment was repeated at least three times and the data are shown as the mean ± SD.
DNA 阀门临界温度的确定：为了确定 DNA 阀门打开的临界温度，将 PBS 缓冲液中的 6 个 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) 样品在水浴中加热，不同的温度（30、37、42、44、50 和 60 °C）。每个样品加热 90 分钟，每 15 分钟测量一次荧光强度。如上所述测量完全释放的染料的最终荧光强度。染料从纳米载体释放的百分比计算如下：（每个样品在不同时间的荧光强度）/（样品的最终荧光强度）。每个实验至少重复3次，数据以平均值±SD表示。

Effect of Laser Induced Temperature Change: Five samples of 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) were irradiated under different laser and different power densities. The laser power densityies used were 3.0 W·cm⁻² at 655 nm, and 2.0, 2.7, 3.0, and 4.0 W·cm⁻² 808 nm. Each sample was irradiated for 750 s and the temperature was recorded every 30 s. The sample was exposed to the laser light with a beam area of 0.4 cm². Each experiment was repeated at least three times and the data are shown as the mean ± SD.
激光诱导温度变化的影响：在不同激光和不同功率密度下照射 5 个 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) 样品。使用的激光功率密度在 655 nm 处为 3.0 W·cm ⁻² ，在 808 nm 处为 2.0、2.7、3.0 和 4.0 W·cm ⁻² 。每个样品照射 750 秒，每 30 秒记录一次温度。将样品暴露于光束面积为0.4cm ² 的激光下。每个实验至少重复3次，数据以平均值±SD表示。

Controlled Release of the Nanocarrier: The release amount of RhB for seven laser on/off cycles was evaluated to study the reversibility of valve on/off cycle and triggered nature of the release. 2 mL AuNRs@MS-NH₂ (RhB) or AuNRs@MS-DNA (RhB) (0.1 mg/mL) in PBS buffer was irradiated with NIR laser (808 nm, 2.7 W·cm⁻²) for several laser on/off cycles. For each cycle, the sample was first irradiated for 15 min then cooled to room temperature for 15 min and the fluorescence intensity of the sample was measured. The sample was then kept at room temperature for 12 h and the fluorescence intensity of the sample was measured. In addition, to evaluate the relationship of the release amount of cargo and irradiation time, 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) was irradiated with the NIR laser (808 nm, 2.7 W·cm⁻²) for 105 min continuously. Each experiment was repeated at least three times and the data are shown as the mean ± SD.
纳米载体的受控释放：评估七个激光开/关循环的 RhB 释放量，以研究阀门开/关循环的可逆性和释放的触发性质。用近红外激光（808 nm，2.7 W·cm (RhB) 或 AuNRs@MS-DNA (RhB) (0.1 mg/mL) > ）用于多个激光开/关循环。对于每个循环，首先将样品照射15分钟，然后冷却至室温15分钟，并测量样品的荧光强度。然后将样品在室温下保存12小时并测量样品的荧光强度。此外，为了评估货物释放量与照射时间的关系，用近红外激光（808 nm，2.7 W·cm ⁻² ）连续105分钟。每个实验至少重复3次，数据以平均值±SD表示。

Nuclease Assay: Two groups of 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) in PBS buffer were incubated at 37 °C. After allowing the samples to equilibrate (10 min), 1.3 μL of DNase I in assay buffer (2 U/L) was added to one group. The fluorescence signal of the two samples was monitored for 6 h and was collected at intervals during this period. These two groups were then irradiated with the NIR laser (808 nm, 2.7 W·cm⁻²) for 90 min, and the fluorescence was measured after the solution was cooled to room temperature. The experiment was repeated at least three times and the data are shown as mean ± SD.
核酸酶测定：PBS 缓冲液中的两组 2 mL AuNRs@MS-DNA (RhB) (0.1 mg/mL) 在 37 °C 下孵育。使样品平衡（10 分钟）后，将 1.3 μL DNase I 的测定缓冲液 (2 U/L) 添加到一组。监测两个样品的荧光信号6小时，并在此期间每隔一段时间收集一次。然后用近红外激光（808 nm，2.7 W·cm ⁻² ）照射两组90 min，待溶液冷却至室温后测量荧光。实验至少重复3次，数据以平均值±标准差表示。

Confocal Fluorescence Imaging: MCF-7 cells were cultured on chamber slides for 24 h. The AuNRs@MS-DNA (RhB) (0.1 mg/mL) was delivered into the cells in RPMI-1640 culture medium at 37 °C for 12 h. Cells were then washed twice with PBS buffer and 2 mL RPMI-1640 culture medium was added. The cells were examined with confocal laser scanning microscopy (CLSM) with 543 nm excitation. Next, three NIR laser on/off cycles were performed in the same cells. For each cycle, the cells were irradiated for 10 min then examined by CLSM with 543 nm excitation followed by 2 h incubation.
共焦荧光成像：MCF-7细胞在室载玻片上培养24小时。将 AuNRs@MS-DNA (RhB) (0.1 mg/mL) 递送至 RPMI-1640 培养基中的细胞中，37°C 培养 12 小时。然后用 PBS 缓冲液洗涤细胞两次，并添加 2 mL RPMI-1640 培养基。使用 543 nm 激发的共焦激光扫描显微镜 (CLSM) 检查细胞。接下来，在相同的单元中执行三个近红外激光开/关循环。对于每个循环，将细胞照射 10 分钟，然后通过 CLSM 用 543 nm 激发进行检查，然后孵育 2 小时。

MTT Assay: MCF-7 cells were cultured in 96-well microtiter plates and incubated at 37 °C in 5% CO₂ for 24 h. MCF-7 cells were incubated with culture medium, AuNRs@MS-DNA (0.1 mg/mL, 0.2 mg/mL) for different times (6, 12, and 24 h), respectively. Next, 150 μL MTT solution (0.5 mg/mL) was added to each well. After 4 h, the remaining MTT solution was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a RT 6000 microplate reader. (Figure 9A) Then the MCF-7 cells were incubated with culture medium and AuNRs@MS-DNA (0.1 mg/mL) (Figure 9B) or AuNRs@MS-DNA (Dox) (0.1 mg/mL) (Figure 9C) for 12 h. The cells were then irradiated with the NIR laser (808 nm, 2.7 W·cm⁻²) for different times. Each irradiation time lasted for 10 min at 2 h intervals. After irradiation, the cells were incubated at 37 °C for 24 h. The cells were then treated as mentioned above. Each experiment was repeated at least three times and the data are shown as the mean ± SD.
MTT法：将MCF-7细胞培养在96孔微量滴定板中，并在37℃、5%CO ₂ 中孵育24小时。 MCF-7 细胞分别与培养基 AuNRs@MS-DNA (0.1 mg/mL、0.2 mg/mL) 孵育不同时间（6、12 和 24 小时）。接下来，向每孔中添加 150 μL MTT 溶液 (0.5 mg/mL)。 4小时后，除去剩余的MTT溶液，每孔加入150μL DMSO以溶解甲臜晶体。使用 RT 6000 酶标仪在 490 nm 处测量吸光度。 (图 9A) 然后将 MCF-7 细胞与培养基和 AuNRs@MS-DNA (0.1 mg/mL) (图 9B) 或 AuNRs@MS-DNA (Dox) (0.1 mg/mL) (图 9C) 一起孵育12小时。然后用近红外激光（808 nm，2.7 W·cm ⁻² ）照射细胞不同时间。每次照射时间持续10分钟，间隔2小时。照射后，将细胞在37℃下孵育24小时。然后如上所述处理细胞。每个实验至少重复3次，数据以平均值±SD表示。

Supporting Information 支持信息

Supporting Information is available from the Wiley Online Library or from the author.
支持信息可从 Wiley 在线图书馆或作者处获取。