Gold Nanorods Coated with Mesoporous Silica Shell as Drug Delivery System for Remote Near Infrared Light-Activated Release and Potential Phototherapy
涂有介孔二氧化硅壳的金纳米棒作为药物输送系统，用于远程近红外光激活释放和潜在光疗

2.1 Fabrication of GNR@MSNPs Nanovehicles, Drug Loading, and Release Behaviors
2.1 GNR@MSNPs纳米载体的制备、药物负载和释放行为

Gold nanorods coated with mesoporous shell were fabricated according to the classical protocol reported by Matsuura,43 which is known to produce disordered mesopores (≈4 nm) in the silica shell over each individual GNR core. CTAB was used as a soft template to generate the mesopores during the polymerization of silanes around the GNRs, and a shell of ≈35-nm in thickness was obtained (Figure 1a,b). An obvious shifting of ≈35 nm of the surface plasmon resonance (SPR) band was observed in the UV–vis spectrum (Figure 1c), due to the presence of the mesoporous silica shell, which is known to change the surface refractive index.4, 18, 19, 36, 43-46
涂有介孔壳的金纳米棒是根据 Matsuura 报道的经典方案制造的，43 已知该方案会在每个单独的 GNR 核上的二氧化硅壳中产生无序的介孔（约 4 nm）。 CTAB 被用作软模板，在 GNR 周围的硅烷聚合过程中生成介孔，并获得厚度约 35 nm 的壳（图 1a、b）。由于介孔二氧化硅壳的存在，在紫外可见光谱中观察到表面等离子共振（SPR）带的明显位移约 35 nm（图 1c），已知介孔二氧化硅壳会改变表面折射率。 4、18、19、36、43-46

1-tetradecanol has been first reported by Xia and co-workers47 to regulate the guest molecule release from gold nanocages, while treatment with high-intensity focused ultrasound could easily trigger the drug release. More recently, Singamaneni and co-workers34 also reported the use of NIR light irradiation to induce the phase change and activate the drug release, based on the same system. Compared with other gating systems, phase-changing materials, e.g., TD, might bear the advantages of demonstrated biocompatibility, cheapness, facile fabrication without complicate chemical process and post-treatment, and also excellent control over the release behavior.34, 42, 47 Furthermore, compared to AMF, the NIR light might also bear some additional merits, e.g., higher temporal and spatial precision, applicability to patients with metallic implants, etc.1, 2
Xia 和同事 47 首先报道了 1-十四烷醇可以调节金纳米笼中客体分子的释放，而高强度聚焦超声处理可以很容易地触发药物释放。最近，Singamaneni 和同事 34 还报道了基于同一系统，使用近红外光照射来诱导相变并激活药物释放。与其他浇注系统相比，相变材料（例如TD）可能具有生物相容性、价格便宜、易于制造、无需复杂的化学过程和后处理以及对释放行为的良好控制等优点。 34, 42, 47 此外，与 AMF 相比，NIR 光还可能具有一些额外的优点，例如更高的时间和空间精度、适用于金属植入物患者等1, 2

DOX and TD molecules were subsequently loaded into the mesoporous cavities of GNR@MSNPs. The presence of DOX was evidenced by the increased UV–vis absorption at ≈500 nm (Figure 1c), overlaying with the transverse SPR band of GNRs. An overall fraction of TD and DOX was estimated to be ≈16 wt% by TGA measurement (Figure 1d), while the loading amount of DOX was confirmed to be ≈11.4 wt% spectrophotometrically. An incorporated amount of ≈4.6 wt% for TD was confirmed.
随后将 DOX 和 TD 分子加载到 GNR@MSNP 的介孔腔中。 DOX 的存在通过约 500 nm 处紫外可见吸收的增加来证明（图 1c），与 GNR 的横向 SPR 谱带重叠。通过 TGA 测量估计 TD 和 DOX 的总分数约为 16 wt%（图 1d），而分光光度法确认 DOX 的装载量为 11.4 wt%。确认TD的掺入量约为4.6wt%。

To confirm the gating role of the TD molecules, the drug release performance of GNR@MSNPs@DOX@TD (100 mg L⁻¹) suspension in RPMI-1640 medium (phenol red free, W/O PR) was first followed under external heating, i.e., in a bain-marie. In this way, it was observed nearly no release at 25 °C and a very slight release at 37 °C in 48 h (Figure 2a). Moreover, we also performed another experiment on the release of DOX under pH of 4.5. As reported, the interaction between DOX and the dissociated silanols is pH-dependent, and a decrease in pH is known to be in favor of the DOX release.4, 22, 48, 49 However, similar nearly zero premature release was detected again in the presence of TD molecules (Figure S1, Supporting Information), further corroborating the gating efficacy. The release behavior at 45 °C (above T_m), critical temperature for hyperthermia-induced cancer/tumor cell ablation,3, 50 evidenced a sustainable drug release (Figure 2a). Due to the fluid-state of TD molecules above T_m, hydrophilic DOX molecules successfully diffused through the hydrophobic TD fused domains toward the release medium, and the longer the treatment period the higher the release amount. Finally, the release behaviors under alternating heating sequences (on/off 45/37 °C cycles) were also executed by 1-h heating on (45 °C) and subsequent 6-h or 12-h heating off (37 °C). In light of its reversible thermo-induced phase change, an “off/on” control over the release was confirmed (Figure 2b), with a prominent release during the 1-h heating at 45 °C and quenched release along the subsequent period at 37 °C. Therefore, a pulsatile release would be envisaged from this DDS.
为了确认 TD 分子的门控作用，GNR@MSNPs@DOX@TD (100 mg L ⁻¹ ) 悬浮液在 RPMI-1640 介质（不含酚红，W/O PR）中的药物释放性能首先在外部加热下，即在蒸锅中进行。这样，在 48 小时内观察到在 25 °C 时几乎没有释放，在 37 °C 时观察到非常轻微的释放（图 2a）。此外，我们还进行了另一项在pH 4.5下释放DOX的实验。据报道，DOX 和解离的硅烷醇之间的相互作用依赖于 pH 值，并且已知 pH 值的降低有利于 DOX 的释放。 4, 22, 48, 49 然而，在 TD 分子存在的情况下再次检测到类似的几乎为零的过早释放（图 S1，支持信息），进一步证实了门控功效。 45 °C（高于 T _m ）（热疗诱导癌症/肿瘤细胞消融的临界温度 3, 50）时的释放行为证明了可持续的药物释放（图 2a）。由于TD分子在T _m 以上呈流体状态，亲水性DOX分子成功地通过疏水性TD融合域向释放介质扩散，且处理时间越长，释放量越高。最后，交替加热序列（开/关 45/37 °C 循环）下的释放行为也通过 1 小时加热（45 °C）和随后 6 小时或 12 小时加热关闭（37 °C）来执行。 。鉴于其可逆的热诱导相变，确认了对释放的“关/开”控制（图 2b），在 45 °C 加热 1 小时期间有显着的释放，并在随后的 25 °C 温度下淬灭释放。 37°C。因此，可以设想从该 DDS 中进行脉冲释放。

2.2 NIR-Induced Heating and Drug Release
2.2 近红外诱导加热和药物释放

In light of the external heating-triggered release, it is plausible to trigger the drug release via NIR light irradiation, given the high-efficiency of GNRs in photothermal conversion.1 Prior to applying NIR light as a stimulus to activate the drug release, the NIR light-induced heating of GNR@MSNPs suspension in RPMI-1640 medium (culture medium for the afterward-mentioned KB cell line) was recorded first. A NIR wavelength of 802 nm was chosen to match the SPR band of GNR@MSNPs, considering the insufficient heating capability under unmatched wavelengths, as revealed in our previous reports.14, 15] Figure 3a,b shows that the temperature of the pristine RPMI-1640 medium remained of comparable magnitude to the initial stage (ΔT < 2 °C) during the 10-min irradiation (802 nm), despite of the power density, similar to the irradiation of phosphate buffered saline (PBS) buffer.14, 15 In contrast, temperature of the GNR@MSNPs suspension gradually increased, and the heating up (ΔT) within 10 min increases with the GNR@MSNPs concentration, i.e., 20 °C for 200 mg L⁻¹, 12 °C for 100 mg L⁻¹, and 8 °C for 75 mg L⁻¹. Moreover, the NIR-induced photothermal effect is also positively correlated to laser power density. As revealed in our previous reports, the local temperature at the surface of the heating generators, e.g., GNR under NIR light irradiation14, 15 or γ-Fe₂O₃ under AMF, 51-53 is higher than the apparent temperature of the aqueous dispersion. Therefore, in the case of GNR@MSNPs, the temperature of some local sites near the GNR surface, e.g., the mesoporous cavities, should be also much higher than those shown in Figure 3. Therefore, NIR light-triggered release is also envisaged to be faster than that via external heating under the apparent temperature, which will be confirmed in the following section.
鉴于外部加热触发释放，考虑到 GNR 的光热转换效率高，通过近红外光照射触发药物释放是合理的。 1 在应用近红外光作为刺激激活药物释放之前，首先记录 RPMI-1640 培养基（后面提到的 KB 细胞系的培养基）中 GNR@MSNPs 悬浮液的近红外光诱导加热。正如我们之前的报告中所揭示的，考虑到在不匹配的波长下加热能力不足，选择 802 nm 的 NIR 波长来匹配 GNR@MSNP 的 SPR 波段。 [14, 15]图 3a,b 显示，在 10 分钟照射 (802 nm) 期间，原始 RPMI-1640 介质的温度仍与初始阶段 (ΔT < 2 °C) 相当，尽管功率密度不同，类似于磷酸盐缓冲盐水（PBS）缓冲液的照射。 14, 15 相反，GNR@MSNPs 悬浮液的温度逐渐升高，10 分钟内的升温 (ΔT) 随着 GNR@MSNPs 浓度的增加而增加，即 20 °C for 200 mg L ⁻¹ ，100 mg L ⁻¹ 为 12 °C，75 mg L ⁻¹ 为 8 °C。此外，近红外光引起的光热效应也与激光功率密度呈正相关。正如我们之前的报告所揭示的，加热发生器表面的局部温度，例如近红外光照射下的 GNR 14、15 或 AMF 下的 γ-Fe ₂ O ₃ ， 51-53高于水分散体的表观温度。因此，在 GNR@MSNPs 的情况下，GNR 表面附近的一些局部位点（例如介孔腔）的温度也应该比图 3 中所示的温度高得多。 因此，近红外光触发的释放也被认为比表观温度下通过外部加热的释放更快，这将在下一节中得到证实。

The NIR-activated drug release behaviors of the GNR@MSNPs@DOX@TD suspension was also performed in RPMI-1640 medium (W/O PR) under NIR irradiation. The release amount was summarized in Figure 4a, and release of 3.5% and 5.6% was detected after 10-min irradiation at 1.0 and 1.5 W cm⁻² (802 nm), respectively. Subsequently, the release was quenched again in the following 6-h treatment at 37 °C, once the NIR irradiation was switched off. However, only ≈0.5% and 0.2% of the overall DOX were released within 10-min treatment at 45 °C and the following 6-h treatment at 37 °C, respectively. The evolution of temperature of the release system was also followed during the NIR irradiation on/off cycles, as shown in Figure S2, Supporting Information. A prompt heating up of the system was observed, similar to Figure 3, while upon the switching off of NIR irradiation, the temperature quickly decreased to 37 °C, similar to our previous reports.14, 15 The comparison with the release at 50 °C, i.e., nearly the same temperature (T_max 49.6 °C) achieved under NIR light irradiation (1.0 W cm⁻²), evidenced a much higher release, due to the relative higher local temperature as discussed above. Moreover, taking the reversible phase-changing essence of TD into consideration, it is also expected that multiple NIR light irradiation on/off treatment could also impart an “on-demand” control over the drug release. To confirm this assumption, the GNR@MSNPs@DOX@TD suspension was treated with NIR laser (802 nm, 1.0 W cm⁻²) for NIR irradiation on/off alternating treatment. For each cycle, the sample was irradiated for 10 min and then the laser was switched off for few hours. Figure 4b confirmed the activation of DOX release upon irradiation and inhibition of DOX release, once the NIR irradiation was switched off, and ≈20% of the overall DOX loaded were released after four cycles. In contrast, in the work reported by Jiang and co-workers,22 without gating moieties, ≈10% of the loaded DOX was released upon 3-min NIR laser irradiation (3 W cm⁻²) from the GNR@MSNPs@DOX suspension, and then ≈5% DOX molecules were released again in the following 1 h with the NIR light switched off. Moreover, with the presence of gatekeepers, like aptamers reported by Qu and co-workers,35 and poly(N-isopropylacrylamide) by Zhao and co-workers,29 a burst release under NIR irradiation, followed with a sustainable release upon the removal of NIR irradiation, was reported. Here, a remarkable advantage based on the TD phase-changing molecules was disclosed here, promising a fine control over the release via the external manipulation of NIR irradiation.
GNR@MSNPs@DOX@TD 悬浮液的近红外激活药物释放行为也在近红外辐射下的 RPMI-1640 介质（W/O PR）中进行。释放量如图 4a 所示，在 1.0 和 1.5 W cm ⁻² (802 nm) 照射 10 分钟后，分别检测到释放量为 3.5% 和 5.6%。随后，一旦关闭 NIR 照射，在 37°C 下的 6 小时处理中，释放再次被猝灭。然而，在 45°C 10 分钟处理和 37°C 6 小时处理内，分别仅释放了总 DOX 的约 0.5% 和 0.2%。在 NIR 照射开/关循环期间，还跟踪了释放系统的温度演变，如图 S2 支持信息所示。观察到系统迅速升温，类似于图 3，而在关闭 NIR 照射后，温度迅速降至 37°C，与我们之前的报告类似。 14, 15 与 50 °C 下释放的比较，即在近红外光照射（1.0 W cm ⁻² ）下实现的几乎相同的温度（T _max 49.6 °C），如上所述，由于局部温度相对较高，表明释放量要高得多。此外，考虑到TD的可逆相变本质，多次近红外光照射开/关治疗也有望实现对药物释放的“按需”控制。为了证实这一假设，用近红外激光（802 nm，1.0 W cm ⁻² ）对GNR@MSNPs@DOX@TD悬浮液进行近红外照射开/关交替处理。对于每个周期，样品被照射 10 分钟，然后关闭激光几个小时。 图 4b 证实了在 NIR 照射关闭后，照射后 DOX 释放被激活，并且 DOX 释放被抑制，四个周期后，总 DOX 负载量的约 20% 被释放。相比之下，在 Jiang 及其同事报道的工作中，22 个没有门控部分的负载 DOX 在 3 分钟 NIR 激光照射（3 W cm ⁻² ）下从 GNR 中释放出来@MSNPs@DOX悬浮液，然后在关闭近红外光的情况下，在接下来的1小时内再次释放约5％的DOX分子。此外，由于存在守门人，如 Qu 及其同事报道的适体 35 和赵及其同事报道的聚（N-异丙基丙烯酰胺）29，在近红外辐射下会出现突发释放，随后在去除适体后会持续释放。据报道，近红外辐射。在此，公开了基于 TD 相变分子的显着优势，有望通过近红外辐射的外部操作来精细控制释放。

2.3 Targeted Delivery of the GNR@MSNPs with Folate Moieties
2.3 具有叶酸部分的 GNR@MSNP 的靶向递送

Cytotoxicity assay is indispensable to evaluate such kind of GNR- and/or MSNP-based nanovehicles, since the CTAB molecules, structure-directing agents for the GNRs synthesis,12 and soft template for mesoporous silica synthesis,27 are known to be highly cytotoxic, even at the μm scale.14, 15, 42 That is why, a supplementary ionic exchange procedure was performed, in order to remove the CTAB molecules, and an improved cytocompatibility was evidenced through the MTS assay (Figure S3, Supporting Information). Although PEG-decorated nanocarriers are reported to passively target the solid tumor tissue via the “enhanced permeation and retention (EPR)” mechanism, they are still criticized for their incapacity to actively accumulate within some specific cancer cells with an optimal efficiency.54 To improve this, the most widely used approach is to label the surface of the nanovehicles with recognizable targeting ligands, such as antibodies, targeting peptides, or low molecular weight molecules like folate and other vitamins.55 Folate receptor on the cell membrane is regarded as a potential molecular target for tumor-selective drug delivery, based on a folate receptor-mediated endocytosis process.56 Moreover, the folate receptor is highly expressed in a number of epithelial carcinomas, but it occurs only in a limited number of normal cells.56, 57 Herein, we labeled the GNR@MSNPs with folic acid (GNR@MSNPs-FA), and folate receptor overexpressed cancer cells, the KB cell line, were used in order to evaluate the improved targeting efficiency via fluorescence microscope observation (grey images in Figure 5, and corresponding images in Figure S4, Supporting information). A remarkable difference in uptake efficiency was detected after 3-h incubation with targeted GNR@MSNPs-FA (Figure 5a), from those incubated with nontargeted GNR@MSNPs (PEG labeled, Figure 5b). Furthermore, the presence of free folic acid (10 μg mL⁻¹, standard folic acid concentration in those commonly used cell culture media, e.g., RPMI-1640 medium) also obviously inhibited the cellular uptake within KB cells (Figure 5c), through competitive binding of free folic acid to the folate receptors. The relative targeting efficiency was furthermore quantitatively analyzed with a cytoflowmeter, and the visualization results from cytoflowmetric analysis (Figure S5, Supporting Information) were consistent with those presented in the fluorescence microscopic images. Moreover, a quantitative analysis was performed with the treated cells via inductively coupled plasma atomic emission spectroscopy (ICP/OES) measurement (Figure S6, Supporting Information), and the cellular uptake amount of targeted GNR@MSNPs-FA within KB cells reached to ≈14.3 ± 1.3 pg cell⁻¹, compared with ≈0.15 ± 0.09 pg cell⁻¹ for nontargeted GNR@MSNPs within KB cells. Moreover, the presence of folic acid (10 mg L⁻¹) also led to a decrease to ≈7.1 ± 1.3 pg cell⁻¹. All these results corroborated a targeted delivery of GNR@MSNPs-FA via a folate receptor-mediated endocytosis mechanism.57
细胞毒性测定对于评估此类基于 GNR 和/或 MSNP 的纳米载体是必不可少的，因为 CTAB 分子、用于 GNR 合成的结构导向剂 12 和用于介孔二氧化硅合成的软模板 27 已知具有高度细胞毒性，即使在微米尺度上。 14, 15, 42 因此，进行了补充离子交换程序，以去除 CTAB 分子，并且通过 MTS 测定证明了细胞相容性的改善（图 S3，支持信息）。尽管据报道，PEG修饰的纳米载体通过“增强渗透和保留（EPR）”机制被动靶向实体瘤组织，但它们仍然因无法以最佳效率在某些特定癌细胞内主动积累而受到批评。 54为了改善这一点，最广泛使用的方法是用可识别的靶向配体标记纳米载体的表面，例如抗体、靶向肽或叶酸和其他维生素等低分子量分子。 55 基于叶酸受体介导的内吞作用过程，细胞膜上的叶酸受体被视为肿瘤选择性药物递送的潜在分子靶标。 56 此外，叶酸受体在许多上皮癌中高度表达，但仅出现在有限数量的正常细胞中。 56, 57 在此，我们用叶酸标记 GNR@MSNPs (GNR@MSNPs-FA)，并使用叶酸受体过表达的癌细胞 KB 细胞系，通过荧光显微镜观察（灰色图 5 中的图像，以及图 S4 中的相应图像，支持信息）。 使用靶向 GNR@MSNPs-FA（图 5a）与非靶向 GNR@MSNP（PEG 标记，图 5b）孵育 3 小时后，检测到摄取效率存在显着差异。此外，游离叶酸（10 μg mL ⁻¹ ，常用细胞培养基中的标准叶酸浓度，例如RPMI-1640培养基）的存在也明显抑制KB细胞内的细胞摄取（图5c)，通过游离叶酸与叶酸受体的竞争性结合。此外，还使用细胞流率计对相对靶向效率进行了定量分析，并且细胞流率分析的可视化结果（图 S5，支持信息）与荧光显微图像中呈现的结果一致。此外，通过电感耦合等离子体原子发射光谱（ICP/OES）测量对处理后的细胞进行定量分析（图S6，支持信息），KB细胞内靶向GNR@MSNPs-FA的细胞摄取量达到≈ 14.3 ± 1.3 pg 细胞 ⁻¹ ，相比之下，KB 细胞内的非靶向 GNR@MSNP 为 ≈0.15 ± 0.09 pg 细胞 ⁻¹ 。此外，叶酸（10 mg·L ⁻¹ ）的存在也导致细胞 ⁻¹ 减少至约7.1 ± 1.3 pg。所有这些结果证实了 GNR@MSNPs-FA 通过叶酸受体介导的内吞作用机制进行靶向递送。 57

2.4 NIR-Induced Phototherapy and NIR-Activated Chemotherapy
2.4 近红外诱导光疗和近红外激活化疗

As known to us, at temperature of 41–45 °C, tumor cells begin to show signs of apoptosis, while temperature above 50 °C is associated with less apoptosis but more frank necrosis, due to the thermo-induced protein denaturation, revealing potential tumor treatment via hyperthermia.58,59 This temperature-dependent ablation of tumor cells makes the gold GNRs promising in the clinical light-assisted tumor treatment. The KB cell line was again used to assess the phototherapeutic performance of the GNR@MSNPs under 10-min NIR irradiation. To differentiate the living and dead cells, cell staining with calcein AM/propidium iodide (PI) mixture was done before fluorescence microscopy observation.3 It showed that targeted GNR@MSNPs-FA exhibited a more prominent capability in phototherapeutic treatment (Figure 6, corresponding color images shown in Figure S7, Supporting information), compared with the absence of GNR@MSNPs or presence of nontargeted GNR@MSNPs (Figure S8, Supporting Information). Higher concentration of GNR@MSNPs-FA, irradiation power density and/or duration were in favor of higher cancer cells apoptosis (presence of red fluorescence), confirming the role of GNR@MSNPs in the NIR-induced cancer/tumor cells ablation.
我们知道，在41-45°C的温度下，肿瘤细胞开始表现出凋亡的迹象，而温度高于50°C时，由于热诱导的蛋白质变性，细胞凋亡减少，但坏死更明显，这揭示了潜在的潜力。通过热疗治疗肿瘤。 58, 59 这种肿瘤细胞的温度依赖性消融使得金 GNR 在临床光辅助肿瘤治疗中具有广阔的前景。 KB 细胞系再次用于评估 GNR@MSNP 在 10 分钟近红外辐射下的光疗性能。为了区分活细胞和死细胞，在荧光显微镜观察之前用钙黄绿素 AM/碘化丙啶 (PI) 混合物进行细胞染色。 3 结果表明，与不存在 GNR@MSNPs 或存在非靶向 GNR@MSNPs 相比，靶向 GNR@MSNPs-FA 在光疗治疗中表现出更突出的能力（图 6，图 S7 中显示的相应彩色图像，支持信息）（图 S8，支持信息）。较高浓度的 GNR@MSNPs-FA、照射功率密度和/或持续时间有利于较高的癌细胞凋亡（出现红色荧光），证实了 GNR@MSNPs 在近红外诱导的癌症/肿瘤细胞消融中的作用。

One concern should be kept in mind, for tumor treatment via photothermia or magnetic hyperthermia, is that patients rarely die from primary tumors, since many primary tumors can be surgically removed. On the other side, metastatic disease is a common cause of death in advanced cancer, but small metastatic cancers are always not accessible via remote triggers.60 Hence, applications of remote photothermia formulations should be carefully chosen, for reasons of preserving life quality of those patients. On the other side, this heating capacity could be also, and probably more easily, exploited to heat the drug reservoirs, hence, trigger the drug release. Moreover, it could be more attractive to synergistically combine the phototherapeutic and chemotherapeutic modalities to achieve a more prominent therapeutic efficiency. Having established that the drug release from the GNR@MSNPs@DOX@TD could be activated by NIR irradiation, we tested the NIR-triggered release and cytotoxic effect of this DDS against KB cell line. The cells were first incubated with targeted (GNR@MSNPs-FA@DOX@TD, TD of 4.4 wt% and DOX of 10.5 wt%) and nontargeted (GNR@MSNPs-PEG@DOX@TD, TD of 4.0 wt% and DOX of 9.8 wt%) DDS suspension in RPMI-1640 medium (W/O FA), and nearly no loss of cell viability was detected without NIR irradiation (Figure 7), compared with that of pristine DOX (Figure S9, Supporting Information). These results confirmed that the toxic potential of DOX is inhibited, thanks to the presence of TD gatekeepers, thus most all the chemotherapeutics were entrapped within the cavities with a stable manner. In contrast, cell viabilities continued to decrease once there was no gatekeepers, as revealed in previously reported DOX-loaded γ-Fe₂O₃@MSNPs@DOX26 and GNR@MSNPs@DOX systems.22
对于通过光热或磁热疗进行的肿瘤治疗，应牢记的一个问题是患者很少死于原发性肿瘤，因为许多原发性肿瘤可以通过手术切除。另一方面，转移性疾病是晚期癌症的常见死亡原因，但小型转移性癌症始终无法通过远程触发来发现。 60 因此，出于保护这些患者的生活质量的原因，应谨慎选择远程光热制剂的应用。另一方面，这种加热能力也可以而且可能更容易地被用来加热药物储存器，从而触发药物释放。此外，将光疗和化疗方式协同结合以获得更显着的治疗效果可能更有吸引力。确定 GNR@MSNPs@DOX@TD 的药物释放可以通过近红外辐射激活后，我们测试了该 DDS 对 KB 细胞系的近红外触发释放和细胞毒性作用。首先将细胞与靶向细胞（GNR@MSNPs-FA@DOX@TD，TD 为 4.4 wt% 和 DOX 为 10.5 wt%）和非靶向细胞（GNR@MSNPs-PEG@DOX@TD，TD 为 4.0 wt% 和 DOX）一起孵育。 9.8 wt%）DDS 悬浮液在 RPMI-1640 培养基（W/O FA）中，与原始 DOX 相比（图 S9，支持信息），在没有 NIR 照射的情况下几乎没有检测到细胞活力损失（图 7）。这些结果证实，由于 TD 看门人的存在，DOX 的毒性潜力受到抑制，因此大多数化疗药物都以稳定的方式被捕获在腔内。 相反，一旦没有看门人，细胞活力就会继续下降，正如之前报道的负载 DOX 的 γ-Fe ₂ O ₃ @MSNPs@DOX 26 和 GNR@MSNPs@ 所揭示的那样DOX 系统。 22

On the other side, the cell viabilities of KB cells treated by NIR irradiation in the presence of GNR@MSNPs-FA@DOX@TD were also evaluated, and here we chose the irradiation power density of 1 W cm⁻² within 10-min irradiation, in light of the optimal cell killing effect revealed in Figure 4. As shown in Figure 7, cell viabilities decreased obviously upon NIR-light irradiation, and a significant difference between the cell-killing efficiency for GNR@MSNPs-FA@DOX@TD combined with NIR irradiation and GNR@MSNPs-FA, even with the same GNR@MSNPs-FA concentration. This further decrease in cell viability should come from the activation of DOX release, as well as slight enhanced toxicity of DOX under higher temperatures.10 The relative lower cell viability for GNR@MSNPs-FA@DOX@TD (10-min NIR irradiation), compared with the pristine DOX at the same concentration, could be attributed to the incomplete drug release (Figure 4b, <10%). However, a comparable cell killing behavior was confirmed here, thank to the synergistic combination of phototherapy and chemotherapy. Moreover, to confirm the targeting moieties on therapeutic performance, we also investigated the cell-killing efficacy of GNR@MSNPs@DOX@TD nanoparticles (nontargeted) with NIR light, and very slight change in the cell viability was detected (Figure S10, Supporting Information), due to the ultralow delivered amount (Figure S6, Supporting Information).
另一方面，我们还评估了在GNR@MSNPs-FA@DOX@TD存在下经NIR照射处理的KB细胞的细胞活力，这里我们选择1 W cm的照射功率密度 ⁻² 照射10分钟内，根据图4显示的最佳细胞杀伤效果。如图7所示，近红外光照射后细胞活力明显下降，与GNR@MSNPs的细胞杀伤效率有显着差异-FA@DOX@TD 与 NIR 照射和 GNR@MSNPs-FA 相结合，即使 GNR@MSNPs-FA 浓度相同。细胞活力的进一步降低应该是由于 DOX 释放的激活，以及 DOX 在较高温度下毒性的轻微增强。 10 与相同浓度的原始 DOX 相比，GNR@MSNPs-FA@DOX@TD（10 分钟近红外照射）的细胞活力相对较低，可能是由于药物释放不完全所致（图 4b，<10%） 。然而，由于光疗和化疗的协同组合，这里证实了类似的细胞杀伤行为。此外，为了确认靶向部分对治疗性能的影响，我们还研究了 GNR@MSNPs@DOX@TD 纳米颗粒（非靶向）在近红外光下的细胞杀伤功效，并检测到细胞活力的微小变化（图 S10，支持）信息），由于交付量超低（图 S6，支持信息）。

As reported, a great variety of antitumor therapeutics efficiently function within the nuclei, for example, DOX, the mechanism of which is to inhibit the replication of DNA in nucleus, where the genetic information and the transcription machinery reside.61 As shown in Figure S11, Supporting Information, we took a fluorescence microscopic image of the DOX-treated KB cells (cell viability of ≈6%), and the red fluorescence (DOX) mostly distributed within the nuclei, while part of them in the cytoplasm, due to a concentration gradient diffusion mechanism of DOX. Herein, we also used fluorescence microscope to visualize the NIR-triggered release of DOX from the GNR@MSNPs-FA@DOX@TD within the KB cell culture. Without any NIR irradiation, we could easily observe the accumulation of FITC-labeled GNR@MSNPs-FA@DOX@TD within the cells (Figure 8a-2). Whereas, as shown in the merged image of Figure 8a-5, the green and red fluorescence colocalized and emitted yellow fluorescence, and essentially located in the cytoplasm (Figure 8a-4,a-6), rather than in the nuclei. This observation is also in coherence with our previous finding on the uptake of γ-Fe₂O₃@MSNPs@DOX@TD within human melanoma MEL-5 cells, corroborating the gating role of the TD molecules.26 Then we also examined the treated cells after 10-min NIR irradiation, followed with a further 24-h incubation in fresh medium. It was observed that, in contrast to that in Figure 8a-3, the red fluorescence became more scattered (Figure 8b-3), and some red fluorescence diffused outside the green luminance (Figure 8b-5) and did not completely colocalize with the latter any more. Some red fluorescence was also detected in the nuclei, in the merged images of Figure 8b-4,b-6, due to the NIR-triggered DOX release and further diffusion into the nuclei, in agreement with function mechanism of DOX61 and the cell death detected in Figure 7. To further distinguish the role of targeting moieties in the NIR-triggered release within cell culture, similar experiments were also performed on the KB cells with GNR@MSNPs@DOX@TD (nontargeted). Scared green and red fluorescence were also evidenced to overlay at the same spots before NIR irradiation (Figure S12a-5, Supporting Information), while no clear diffusion of red luminance could be observed. Moreover, the release of DOX could not obviously been observed (Figure S12b, Supporting Information) after NIR irradiation, due to the limited delivered as well as released amount of DOX, leading to unobvious decrease in cell viability (Figure S10, Supporting Information). For this kind of photothermia-triggered drug release, there would be some concerns about the potential thermo-induced degradation of the drug. However, as far as we know, there is no specific report on the exact value of the temperature in close proximity of the GNPs. The power density of 1 W cm⁻² used here is a very mild irradiation compared with those commonly used, i.e., 20 W cm⁻²4 or even higher. Because not only the self-fluorescence of the DOX (Figure 8) but also its cytotoxicity were not disturbed after near-IR irradiation, we conjectured that the fraction of DOX molecules possibly degraded was negligible.
据报道，多种抗肿瘤疗法在细胞核内有效发挥作用，例如DOX，其机制是抑制细胞核中DNA的复制，细胞核中存在遗传信息和转录机制。 61 如图 S11（支持信息）所示，我们拍摄了 DOX 处理的 KB 细胞（细胞活力约 6%）的荧光显微图像，红色荧光 (DOX) 大部分分布在细胞核内，部分位于细胞核内。由于 DOX 的浓度梯度扩散机制，细胞质中的在此，我们还使用荧光显微镜观察 KB 细胞培养物中 GNR@MSNPs-FA@DOX@TD 近红外触发的 DOX 释放。在没有任何近红外辐射的情况下，我们可以轻松观察到细胞内 FITC 标记的 GNR@MSNPs-FA@DOX@TD 的积累（图 8a-2）。然而，如图8a-5的合并图像所示，绿色和红色荧光共定位并发出黄色荧光，并且基本上位于细胞质中（图8a-4、a-6），而不是在细胞核中。这一观察结果也与我们之前关于人黑色素瘤 MEL-5 细胞内 γ-Fe ₂ O ₃ @MSNPs@DOX@TD 摄取的发现一致，证实了门控作用TD 分子。 26 然后，我们还检查了经过 10 分钟 NIR 照射后的处理细胞，然后在新鲜培养基中进一步孵育 24 小时。据观察，与图 8a-3 相比，红色荧光变得更加分散（图 8b-3），并且一些红色荧光扩散到绿色亮度之外（图 8b-5），并且没有完全与绿色亮度共定位。后者不再是了。 在图8b-4、b-6的合并图像中，由于近红外触发的DOX释放并进一步扩散到细胞核中，细胞核中也检测到了一些红色荧光，这与DOX 61和细胞的功能机制一致图 7 中检测到的死亡。为了进一步区分细胞培养物中 NIR 触发释放中靶向部分的作用，还在使用 GNR@MSNPs@DOX@TD（非靶向）的 KB 细胞上进行了类似的实验。还证明，在近红外辐射之前，绿色和红色荧光会重叠在相同的点上（图 S12a-5，支持信息），而没有观察到红色亮度的明显扩散。此外，近红外辐射后，由于 DOX 的递送和释放量有限，无法明显观察到 DOX 的释放（图 S12b，支持信息），导致细胞活力不明显下降（图 S10，支持信息）。对于这种光热触发的药物释放，人们会担心药物潜在的热诱导降解。但据我们所知，目前还没有关于GNP附近温度准确值的具体报道。这里使用的1 W cm ⁻² 的功率密度与常用的20 W cm ⁻² 4 甚至更高的功率密度相比是非常温和的。由于近红外辐射后，DOX 的自身荧光（图 8）及其细胞毒性均未受到干扰，因此我们推测可能降解的 DOX 分子的比例可以忽略不计。

The finding from the present study indicates that, the synergistic combination of phototherapy, light-activated chemotherapy, and targeting delivery to tumor cells, did in fact result in improved cancer cell killing performance, compared to the phototherapy alone. It is well known that DOX is highly toxic and can cause severe cardiac damage,61 thus a good trapping, in order to avoid premature drug release, might be mandatory. Herein, the introduction of 1-tetradecanol as gating moieties effectively regulated the release behaviors of DOX from the mesoporous cavities, with a prominent nearly zero premature drug release under physiological temperature; therefore, the toxicity of the chemotherapeutics could be highly quenched before remote trigger was applied. However, triggered release could be achieved under external heating or internal heating generated under NIR irradiation. Remarkably enhanced cytotoxicity for the synergistic therapy in the KB cell line can be attributed to three effects: (1) photothermal contribution, by which NIR light energy is converted into thermal energy thanks to GNR@MSNPs; (2) increased intracellular concentration of DOX and GNR@MSNPs with the aid of targeting moieties, with which lower NIR power density is needed to reach an optimal temperature and subsequent triggered drug release, and (3) a synergistic combination between the photothermal effect and DOX's cytotoxicity, which was well documented in previous reports and also proved to be useful in those clinical practice of tumor treatment.4-6 Moreover, the synergistic contribution from GNRs-assisted phototherapy and chemotherapy could overcome those deficiencies, which are encountered in those regional or over body hyperthermia treatments, such as discomfort to the patient, high risk of damage to normal tissues.3 On the other side, the GNRs modality has been well documented in some previous reports as a photoacoustic imaging agent,19, 29 developed for in vivo and/or in vitro biomedical imaging or tracking of drug delivery systems. Herein, in the case of GNR@MSNPs@DOX@TD, aside by the local heating generation capability under NIR irradiation, the GNR cores could also impart the integrity with some supplementary functions, such as the as-referred biomedical photoacoustic imaging or photoacoustic-assisted in vivo DDS tracking, which could be of great interest for our following research.
本研究的结果表明，与单独的光疗相比，光疗、光激活化疗和靶向递送至肿瘤细胞的协同组合实际上确实提高了癌细胞的杀伤性能。众所周知，DOX 具有剧毒，可导致严重的心脏损伤，61 因此，可能必须进行良好的捕获，以避免药物过早释放。在此，引入1-十四烷醇作为门控部分有效地调节了DOX从介孔腔中的释放行为，在生理温度下具有显着的近乎零的过早药物释放；因此，在应用远程触发之前，化疗药物的毒性可以被高度抑制。然而，在外部加热或近红外辐射下产生的内部加热下可以实现触发释放。 KB细胞系中协同治疗的细胞毒性显着增强可归因于三个效应：（1）光热贡献，通过GNR@MSNP，近红外光能转化为热能； （2）借助靶向部分增加 DOX 和 GNR@MSNP 的细胞内浓度，需要较低的近红外功率密度才能达到最佳温度并随后触发药物释放，以及（3）光热效应和DOX的细胞毒性，在之前的报道中有详细记录，并且在肿瘤治疗的临床实践中也被证明是有用的。 4-6 此外，GNR 辅助光疗和化疗的协同作用可以克服局部或过度全身热疗中遇到的缺陷，例如患者不适、正常组织损伤风险高。 3 另一方面，GNR 模式已在之前的一些报告中作为光声成像剂得到充分记录，19, 29 是为体内和/或体外生物医学成像或药物输送系统跟踪而开发的。在此，在 GNR@MSNPs@DOX@TD 的情况下，除了近红外辐射下的局部发热能力之外，GNR 核还可以赋予一些补充功能的完整性，例如所提到的生物医学光声成像或光声-辅助体内 DDS 跟踪，这对我们后续的研究可能会有很大的兴趣。