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2018 Jan 16; 115(3): 501–506.
美国国家科学院院刊，2018 年 1 月 16 日； 115（3）：501-506。
Published online 2018 Jan 2. doi: 10.1073/pnas.1714421115
2018 年 1 月 2 日在线发布。doi：10.1073/pnas.1714421115
PMCID: PMC5776980 PMCID：PMC5776980
PMID: 29295927 电话号码：29295927

Novel concept of the smart NIR-light–controlled drug release of black phosphorus nanostructure for cancer therapy
用于癌症治疗的智能近红外光控制黑磷纳米结构药物释放的新概念

Meng Qiu,a,b,1 Dou Wang,c,1 Weiyuan Liang,a,b,1 Liping Liu,c Yin Zhang,d Xing Chen,a,b David Kipkemoi Sang,a,b Chenyang Xing,a,b Zhongjun Li,a,b Biqin Dong,e Feng Xing,e Dianyuan Fan,a,b Shiyun Bao,c,2 Han Zhang,a,b,2 and Yihai Caod,2
孟秋， a, b, 1 王斗， c, 1 梁伟远， a, b, 1 刘丽萍、 c 张银、 d 陈星、 a, b b 邢晨阳， a, b 李中军， a, b 董碧琴， e 冯兴， e 范殿元， a, b 包诗韵， c, 2 张瀚、 a, b, 2 和曹益海 d, 2
Author information Copyright and License information PMC Disclaimer
作者信息 版权和许可信息 PMC 免责声明

Associated Data 相关数据

Supplementary Materials  补充材料

Significance 意义

Precision delivery of cancer drugs to tumor site is crucial for improving therapeutic efficacy and minimizing adverse effects. Despite tremendous efforts, current drug delivery systems remain an unmet clinical need for cancer therapy. Herein, we propose a unique concept of applying external light to control drug delivery in cancer tissues. In preclinical cancer models, we demonstrate that the near-infrared light-induced decomposition of black phosphorus hydrogel accurately releases drugs in tumor tissues to eradicate subcutaneous breast and melanoma cancers without causing any adverse effects. We believe that our therapeutic system can be used for effective treatment of most cancer types. Our findings may likely bring about a paradigm shift in clinical treatment of cancer and millions of cancer patients will benefit from our findings.
将癌症药物精确输送到肿瘤部位对于提高治疗效果和最大限度地减少不良反应至关重要。尽管付出了巨大的努力，当前的药物输送系统仍然未能满足癌症治疗的临床需求。在此，我们提出了应用外部光来控制癌症组织中的药物输送的独特概念。在临床前癌症模型中，我们证明近红外光诱导的黑磷水凝胶分解可以准确地在肿瘤组织中释放药物，从而根除皮下乳腺癌和黑色素瘤，而不会造成任何副作用。我们相信我们的治疗系统可用于有效治疗大多数癌症类型。我们的研究结果可能会带来癌症临床治疗的范式转变，数百万癌症患者将从我们的研究结果中受益。

Keywords: black phosphorus, drug delivery, NIR-light responsive, hydrogel, cancer therapy
关键词：黑磷，药物递送，近红外光响应，水凝胶，癌症治疗

Abstract 抽象的

A biodegradable drug delivery system (DDS) is one the most promising therapeutic strategies for cancer therapy. Here, we propose a unique concept of light activation of black phosphorus (BP) at hydrogel nanostructures for cancer therapy. A photosensitizer converts light into heat that softens and melts drug-loaded hydrogel-based nanostructures. Drug release rates can be accurately controlled by light intensity, exposure duration, BP concentration, and hydrogel composition. Owing to sufficiently deep penetration of near-infrared (NIR) light through tissues, our BP-based system shows high therapeutic efficacy for treatment of s.c. cancers. Importantly, our drug delivery system is completely harmless and degradable in vivo. Together, our work proposes a unique concept for precision cancer therapy by external light excitation to release cancer drugs. If these findings are successfully translated into the clinic, millions of patients with cancer will benefit from our work.
可生物降解的药物输送系统（DDS）是最有前途的癌症治疗策略之一。在这里，我们提出了一种独特的概念，即光激活水凝胶纳米结构中的黑磷（BP），用于癌症治疗。光敏剂将光转化为热量，软化并熔化基于水凝胶的载药纳米结构。药物释放速率可以通过光强度、暴露时间、BP浓度和水凝胶成分来精确控制。由于近红外 (NIR) 光对组织的穿透足够深，我们的基于 BP 的系统在皮下治疗方面表现出很高的治疗效果。癌症。重要的是，我们的药物输送系统在体内完全无害且可降解。我们的工作共同提出了通过外部光激发释放癌症药物的精准癌症治疗的独特概念。如果这些发现成功转化为临床，数百万癌症患者将从我们的工作中受益。

Cancer is a life-threatening disease worldwide and cancer-related deaths are higher than those caused by AIDS, malaria, and tuberculosis combined. About one-fifth of human deaths are caused by cancer (). Several standard approaches, such as surgery, chemotherapy, and radiotherapy, etc., are clinically approved for cancer therapy; however, these modalities suffer from low therapeutic efficacy, due to the incomplete excision of the solid tumor as well as the remaining circulation tumor cells. Therefore, therapeutic strategies that are convenient for application, with high specificity, high efficacy, and low adverse effects are urgently needed.
癌症是世界范围内威胁生命的疾病，与癌症相关的死亡人数比艾滋病、疟疾和结核病造成的死亡人数总和还要高。大约五分之一的人类死亡是由癌症引起的 (1)。几种标准方法，如手术、化疗和放疗等，已被临床批准用于癌症治疗；然而，由于实体瘤以及剩余的循环肿瘤细胞的不完全切除，这些方式的治疗效果较低。因此，迫切需要一种应用方便、特异性高、疗效高、不良反应低的治疗策略。

Localized therapeutic approaches would be very attractive for cancer treatment, due to the strong targeted features to change the redistribution of drug in vivo, compared with i.v. injection (). However, this treatment requires frequent injections of chemotherapy drugs. This is a particular problem for cancer treatment as the invasive injection of drugs to the cancerous tissue and organ frequently brings pain to the patients and may lead to many postoperative complications. Consequently, it calls for a shift of researchers’ attentions toward a drug delivery platform that enables sustained and controlled drug release during the treatment cycle (). Many polymer-based drug delivery systems (DDSs) have been investigated to allow direct targeting of the tumor and the delivering of drugs that can be released during the natural degradation process of the polymers (, ). However, the release rate can hardly be controlled, bringing up double consequences. That is, there is no therapy effect due to failing to reach the minimum therapeutic concentration and, even worse, increasing the risk of resistance in cancer cells.
与静脉注射相比，局部治疗方法对于癌症治疗非常有吸引力，因为它具有改变药物体内重新分布的强大靶向特性。注射（2）。然而，这种治疗需要频繁注射化疗药物。这是癌症治疗中的一个特殊问题，因为向癌组织和器官侵入性注射药物经常给患者带来疼痛，并可能导致许多术后并发症。因此，它要求研究人员将注意力转向能够在治疗周期期间持续和受控药物释放的药物递送平台（3）。人们已经研究了许多基于聚合物的药物递送系统 (DDS)，以允许直接靶向肿瘤并递送可在聚合物自然降解过程中释放的药物 (4, 5)。然而，释放速度难以控制，带来双重后果。也就是说，由于达不到最低治疗浓度而没有治疗效果，甚至增加了癌细胞产生耐药性的风险。

Therefore, there is an urgent need for one-time injection and controlled release of drug for cancer treatment, as an effective therapeutic strategy to improve the treatment effect and reduce patient pain. A delivery nanoplatform that can simultaneously function as a drug depot, enabling sustained release or controlled burst release of therapeutic agents (–), would greatly benefit patients and increase the availability of clinical treatments.
因此，迫切需要一次性注射和控释药物用于癌症治疗，作为一种有效的治疗策略，以提高治疗效果，减轻患者痛苦。一个可以同时充当药物仓库的递送纳米平台，能够实现治疗药物的持续释放或受控突发释放（6-19），将极大地造福患者并增加临床治疗的可用性。

A light-responsive hydrogel is an ideal controlled drug delivery platform, due to its minimal invasiveness and potential for controlled release (). The light-controlled reversible phase transition of the hydrogel can be used to deliver drug repeatedly. Moreover, the release rate can be tuned remotely by several light parameters, such as wavelength, power density, and exposure time, etc. Generally, gold nanoparticles (–) and the emerging 2D nanomaterials, such as graphene (–) and MXenes (), and transition-metal dichalcogenides (TMDs), such as molybdenum disulfide (MoS2) (, ) and tungsten disulfide (WS2) (–), are the frequently used photothermal transducing agents (PTAs), due to their near-infrared (NIR) light response characteristics, ease of fabrication, and tunability in optical properties. However, most of them are subject to serious limitations such as a weak photothermal conversion efficient, relatively low biosafety and biodegradability, difficulty in metabolizing out of the human body, undegradability, and cytotoxicity that remain a practice challenge for clinical applications (, ). Therefore, there is a strong motivation to explore novel PTAs with a required comprehensive performance which is of significant importance for clinical application.
光响应水凝胶是一种理想的受控药物递送平台，因为它具有最小的侵入性和受控释放的潜力（20）。水凝胶的光控可逆相变可用于重复递送药物。此外，释放速率可以通过几个光参数远程调节，例如波长、功率密度和曝光时间等。通常，金纳米粒子（21-23）和新兴的2D纳米材料，例如石墨烯（24-26）和 MXenes ( 27) 以及过渡金属二硫属化物 (TMD)，例如二硫化钼 (MoS 2 ) ( 28, 29) 和二硫化钨 (WS 2 ) ( 30– 32）是常用的光热转换剂（PTA），因为它们具有近红外（NIR）光响应特性、易于制造以及光学特性的可调性。然而，它们中的大多数都受到严重的限制，例如光热转换效率弱、生物安全性和生物降解性相对较低、难以在人体内代谢、不可降解性和细胞毒性，这些仍然是临床应用的实践挑战(33, 34) 。因此，有强烈的动机去探索具有所需综合性能的新型PTA，这对于临床应用具有重要意义。

Recently, black phosphorus (BP), a newly discovered 2D material, has shown many novel properties, such as a tunable and direct energy band gap (–), a high photothermal conversion efficient (, ), easy fabrication and relatively high chemical reactivity that may allow for high drug-loading ability and increased loading capacity (), and excellent biocompatibility and biodegradation () that distinguished it from other materials for biomedical application. BP nanosheets (BPNSs) and BP quantum dots (BPQDs) have already been used for bioimaging (–), photothermal therapy (, ), photodynamic therapy (, –), and drug delivery platforms (, ). Due to the abovementioned unique advantages that graphene or TMDs cannot compete with, BP is considered as a kind of rising biomaterial with promising biomedical-related applications. However, to the best of our knowledge, there is as yet no example of a NIR-light–controlled hydrogel drug delivery platform reported.
最近，新发现的二维材料黑磷（BP）表现出了许多新颖的特性，例如可调谐和直接的能带隙（35-41）、高光热转换效率（42、43）、易于制造和相对高化学反应性可实现高载药能力和增加的负载能力（11），以及优异的生物相容性和生物降解性（42），使其区别于其他生物医学应用材料。 BP 纳米片 (BPNS) 和 BP 量子点 (BPQD) 已用于生物成像 (44–46)、光热疗法 (42, 43)、光动力疗法 (11, 47–49) 和药物输送平台 (10, 11) ）。由于具有石墨烯或TMDs无法比拟的上述独特优势，BP被认为是一种新兴的生物材料，具有广阔的生物医学相关应用前景。然而，据我们所知，目前还没有近红外光控制的水凝胶药物递送平台的例子被报道。

Herein, we report a more facile system, namely BP@Hydrogel which is the nanocomposite of BPNSs and hydrogel, that can be used to modulate the release of anticancer drugs under NIR light exposure, shown in Fig. 1. [Agarose is generally recognized as a safe material approved by American Food and Drug Administration (FDA) while BP has been justified as a harmless and degradable biomaterial.] A BP PTA can convert light to thermal energy and lead to the increasing temperature of the hydrogel matrix. Subsequently, the agarose hydrogel undergoes reversible hydrolysis and softening, leading to the accelerated diffusion of drug from the matrix to the environment. Controlled burst release may be advantageous to keep the released drugs within its therapeutic window, especially as therapeutic drugs often have nonlinear pharmacokinetics. More importantly, the hydrogel further hydrolyzes and melts under enhanced laser power and finally degrades into oligomers to be excreted through urine after the treatment. Therefore, this BP@Hydrogel drug delivery platform may not only enlarge the application fields of BP, but also exhibit potentials for clinical treatment of the cancer tumor.
在此，我们报告了一种更简便的系统，即BP@Hydrogel，它是BPNSs和水凝胶的纳米复合材料，可用于在近红外光照射下调节抗癌药物的释放，如图1所示。[琼脂糖通常被认为是一种经美国食品和药物管理局 (FDA) 批准的安全材料，而 BP 已被证明是一种无害且可降解的生物材料。] BP PTA 可以将光能转化为热能，并导致水凝胶基质的温度升高。随后，琼脂糖水凝胶经历可逆水解和软化，导致药物从基质加速扩散到环境中。受控突发释放可能有利于将释放的药物保持在其治疗窗内，特别是因为治疗药物通常具有非线性药代动力学。更重要的是，水凝胶在增强的激光功率下进一步水解和熔化，最终降解为低聚物，并在治疗后通过尿液排出体外。因此，这种BP@Hydrogel药物递送平台不仅可以扩大BP的应用领域，而且还具有临床治疗癌症肿瘤的潜力。

An external file that holds a picture, illustration, etc. Object name is pnas.1714421115fig01.jpg

Schematic diagram of the working principle of BP@Hydrogel. BP@Hydrogel released the encapsulated chemotherapeutics under NIR-light irradiation to broken the DNA chains, leading to the apoptosis induction.
BP@Hydrogel工作原理示意图。 BP@Hydrogel 在近红外光照射下释放封装的化疗药物，破坏 DNA 链，从而诱导细胞凋亡。

Results 结果

Morphology and Characterization.
形态和表征。

BPNSs were prepared by a modified liquid exfoliation method from bulk BP (Fig. 2A). Briefly, the cup ultrasound sonication with an ice bath of BP powder in isopropanol (IPA) was used to get ultrathin BPNSs with no sample contamination (Fig. 2B). As reported in a recent study, ultrasound probe sonication in NMP could easily achieve a large quantity of few-layered BP with high quality (, ). But the sample prepared from the above mentioned method was contaminated by the impurity from the tip during the probe sonication process, while our sample didn't direct contact with the tip during the cup sonication, to avoid the contamination of the sample, which is of benefit in biological applications. Finally, ultrathin BPNSs were obtained after centrifugation. BPNSs were functionalized with positively charged polyethylene glycol–amine (PEG-NH2) via electrostatic adsorption to improve their biocompatibility and physiological stability. After surface modification, the zeta potential of the BPQDs changed from −28.2 mV to −16.7 mV. The BPNSs exhibited enhanced stability in PBS solution after PEGylation. And the size distribution of BPNSs and PEGylated BPNSs measured from dynamic light scattering (DLS) analysis, with average diameters of 155.6 nm and 160.3 nm, respectively, indicated a slight increase in size after PEGylation of BPNSs.
BPNS 是通过改进的液体剥离方法从块状 BP 中制备的（图 2A）。简而言之，使用异丙醇 (IPA) 中的 BP 粉末冰浴进行杯式超声处理，以获得无样品污染的超薄 BPNS（图 2B）。正如最近的一项研究报道，NMP 中的超声探头超声处理可以轻松获得大量高质量的多层 BP (42, 43)。但上述方法制备的样品在探头超声处理过程中受到了针尖杂质的污染，而我们的样品在杯式超声处理时没有直接接触针尖，避免了样品的污染，这对于在生物应用中受益。最后，离心后获得超薄BPNS。通过静电吸附将带正电荷的聚乙二醇胺（PEG-NH 2 ）对BPNS进行功能化，以提高其生物相容性和生理稳定性。表面修饰后，BPQD的zeta电位从-28.2 mV变为-16.7 mV。聚乙二醇化后，BPNS 在 PBS 溶液中表现出增强的稳定性。通过动态光散射（DLS）分析测量BPNS和聚乙二醇化BPNS的尺寸分布，平均直径分别为155.6 nm和160.3 nm，表明聚乙二醇化后BPNS的尺寸略有增加。

An external file that holds a picture, illustration, etc. Object name is pnas.1714421115fig02.jpg

Characterization of BPNSs. (A) Biography of bulky BP and cup sonication device. (B) TEM of BP. (C) HR-TEM and electron diffraction of BP. (D) AFM, height profile, and thickness distributions of BPNSs. (E) XPS of BPNSs. (F) Raman spectra of BPNSs. (G) Absorbance spectra of BPNSs dispersed in IPA at different concentrations. G, Inset shows the normalized absorbance at concentrations of 3.25 ppm, 6.5 ppm, 13 ppm, and 26 ppm, respectively.
BPNS 的表征。 (A) 大型 BP 和杯式超声处理装置的简介。 (B) BP 的 TEM。 (C) HR-TEM 和 BP 的电子衍射。 (D) AFM、BPNS 的高度剖面和厚度分布。 (E) BPNS 的 XPS。 (F) BPNS 的拉曼光谱。 (G) 不同浓度的 BPNS 分散在 IPA 中的吸收光谱。 G，插图分别显示浓度为 3.25 ppm、6.5 ppm、13 ppm 和 26 ppm 时的归一化吸光度。

A BP-containing hydrogel depot named BP@Hydrogel was fabricated by using a low–melting-point agarose and PEGylated BPNSs. Here, BPNSs were mixed with an agarose aqueous solution at 60 °C, followed by the loading of different concentrations of doxorubicin (DOX) and rapid cooling down to room temperature to form the hydrogel matrix (Fig. 3A). It is worth noting that this BP@Hydrogel softens at 40∼45 °C and melts at 45∼50 °C. And the zeta potential of the BP@Hydrogel was −12.3 mV.
使用低熔点琼脂糖和聚乙二醇化 BPNS 制备了一种名为 BP@Hydrogel 的含 BP 水凝胶库。在这里，BPNS与60℃的琼脂糖水溶液混合，然后加载不同浓度的阿霉素（DOX）并快速冷却至室温以形成水凝胶基质（图3A）。值得注意的是，这种BP@Hydrogel在40∼45℃时软化，在45∼50℃时熔化。 BP@Hydrogel的zeta电位为-12.3 mV。

An external file that holds a picture, illustration, etc. Object name is pnas.1714421115fig03.jpg

NIR-light–controlled BP@Hydrogel drug delivery platform. (A) Schematic representation of the BP@Hydrogel drug delivery platform and the physical map shown in Inset. (B) Absorbance spectra of released DOX. (C) Photocontrolled temperature increase and release of DOX from BP@Hydrogel depot. (D) Rheological curves (blue line) and corresponding temperature curves (red line) of BP@Hydrogel with different BP concentrations under 1 W⋅cm−2 NIR-light irradiation. (E) Release rate of DOX with and without laser exposure. (F) Temperature change vs. time under 808 nm laser with power of 1 W⋅cm−2 during 0∼60 min, 2 W⋅cm−2 during 60∼120 min, and 3 W⋅cm−2 during 120∼180 min. (G) BP@Hydrogel under different laser exposures. (H) Absorbance spectra of DOX under 808-nm laser exposure.
近红外光控制的 BP@Hydrogel 药物递送平台。 (A) BP@Hydrogel 药物递送平台的示意图和插图中所示的物理图。 (B) 释放的 DOX 的吸收光谱。 (C) 光控温度升高和 DOX 从 BP@Hydrogel depot 的释放。 (D) 1 W·cm −2 近红外光照射下不同 BP 浓度的 BP@Hydrogel 的流变曲线（蓝线）和相应的温度曲线（红线）。 (E) 有和没有激光照射时 DOX 的释放率。 (F) 808 nm 激光功率 1 W⋅cm −2 下 0∼60 分钟、2 W⋅cm −2 下 60∼120 分钟的温度变化随时间变化，以及3 W⋅cm −2 在 120∼180 分钟内。 (G) 不同激光照射下的 BP@Hydrogel。 (H) DOX 在 808 nm 激光照射下的吸收光谱。

The morphology of BPNSs was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The TEM images in Fig. 2B revealed that the lateral sizes of BPNSs were about 100∼200 nm. The diameter distribution of BPNSs measured from DLS analysis is shown in Fig. S1. The crystallinity of the few-layer BP (or phosphorene) was studied by high-resolution TEM (HR-TEM) (Fig. 2C) and selected-area electron diffraction (SAED) (Fig. 2C, Inset). Clear lattice fringes were observed from the BP atomic layer and those of 0.34 nm and 0.42 nm corresponding to the (021) and (014) planes of the BP crystal, respectively, which is consistent with well-known BP lattice parameters. The uniform lattices suggest that the phosphorene produced by IPA exfoliation retains the original crystalline state. According to the statistical AFM analysis of 200 BPNSs, shown in Fig. 2D, the average thickness was 2.6 ± 1.5 nm, corresponding to approximately one to eight layers of BPNSs.
通过透射电子显微镜（TEM）和原子力显微镜（AFM）对BPNS的形态进行了表征。图2B中的TEM图像显示BPNS的横向尺寸约为100∼200 nm。 DLS 分析测得的 BPNS 直径分布如图 S1 所示。通过高分辨率TEM（HR-TEM）（图2C）和选区电子衍射（SAED）（图2C，插图）研究了少层BP（或磷烯）的结晶度。从BP原子层以及分别对应于BP晶体的(021)和(014)面的0.34 nm和0.42 nm处观察到清晰的晶格条纹，这与众所周知的BP晶格参数一致。均匀的晶格表明 IPA 剥离产生的磷烯保留了原始的晶态。根据对200个BPNS的统计AFM分析，如图2D所示，平均厚度为2.6±1.5 nm，相当于大约1到8层BPNS。

X-ray photoelectron spectroscopy (XPS) was employed to determine the chemical composition of the BPNSs. The BPNSs show the 2p3/2 and 2p1/2doublets at 129.3 eV and 130.2 eV, respectively, which are characteristic of crystalline BP. Furthermore, the weak subband corresponding to oxidized phosphorus (i.e., POx) is apparent at 133.9 eV, indicating BPNSs could be well protected from being oxidized in IPA solvent.
X 射线光电子能谱 (XPS) 用于确定 BPNS 的化学成分。 BPNS 分别在 129.3 eV 和 130.2 eV 处显示 2p 3/2 和 2p 1/2 双峰，这是晶体 BP 的特征。此外，对应于氧化磷的弱子带（即 PO x ）在 133.9 eV 处很明显，表明 BPNS 可以很好地防止在 IPA 溶剂中被氧化。

A Raman spectrum was performed to characterize bare BP, PEGylated BP, and BP@Hydrogel, respectively (Fig. 2F). All of the samples showed nearly the same three characteristic Raman peaks attributed to one out-of-plane phonon mode, A1g at ∼361.0 cm−1, and two in-plane modes B2g and A2g at 438.0 cm−1 and 465.3 cm−1, respectively, suggesting that the prepared BP with organic modification did not lead to structural transformations compared with the corresponding bare BPNSs. Compared with bare BP, the A1g, B2g, and A2g modes of PEGylated BP and BP@Hydrogel were red shifted by about 2.2 cm−1, 4.5 cm−1, and 3.3 cm−1 and 4.5 cm−1, 9 cm−1, and 6.8 cm−1, respectively. A slight red shift was found after PEGylation and hydration with agarose, due to the adsorption after the addition of the PEG coating and hydration with agarose that hindered the oscillation of phosphorus atoms to some extent, thus decreasing the corresponding Raman scattering energy and leading to the red shift of the three Raman peaks of BPNSs.
拉曼光谱分别用于表征裸BP、聚乙二醇化BP和BP@Hydrogel（图2F）。所有样品都显示出几乎相同的三个特征拉曼峰，归因于一种平面外声子模式，A 1 g 在~361.0 cm −1 处，以及 438.0 cm −1 和 465.3 cm 处的两个面内模式 B 2 g 和 A 2 g −1 分别表明，与相应的裸BPNS相比，制备的有机修饰BP不会导致结构转变。与裸BP相比，A 1 g 、 B 2 g 和 A 2 g PEG化 BP 和 BP@Hydrogel 的模式红移约 2.2 cm −1 、4.5 cm −1 、3.3 cm −1 和 4.5 cm分别为 −1 、 9 厘米 −1 和 6.8 厘米 −1 。聚乙二醇化和琼脂糖水合后发现有轻微的红移，这是由于添加PEG涂层和琼脂糖水合后的吸附在一定程度上阻碍了磷原子的振荡，从而降低了相应的拉曼散射能，导致BPNS 的三个拉曼峰的红移。

The strong absorption in the NIR region is a prerequisite for the photothermal conversion. As is shown in Fig. 2G, the BPNSs from IPA exhibited broader and stronger absorption ranging from UV to NIR wavelengths. The extinction coefficient at 808 nm was estimated to be 20.6 L⋅g−1⋅cm−1 according to Beer–Lambert law (Fig. 2G, Inset), compared with 14.8 L⋅g−1⋅cm−1 in NMP, indicating its longer penetration depth and potential application for in-depth clinical treatment.
近红外区域的强吸收是光热转换的先决条件。如图 2G 所示，IPA 的 BPNS 在 UV 到 NIR 波长范围内表现出更宽、更强的吸收。根据比尔-朗伯定律（图 2G，插图），808 nm 处的消光系数估计为 20.6 L⋅g −1 ⋅cm −1 （图 2G，插图），相比之下为 14.8 L⋅g −1 ⋅cm −1 NMP，表明其具有更长的渗透深度和深入临床治疗的潜在应用。

NIR-Light–Controlled Drug Releasing.
近红外光控制的药物释放。

As shown above, the absorption spectra of BPNSs are stronger than those of BPQDs; therefore, an enhanced photothermal conversion efficiency is expected. The photothermal property of BPNSs was evaluated under an 808-nm NIR laser with a power density of 1.0 W⋅cm−2. According to the photothermal conversion efficiency calculation put forward by Roper et al. (), the photothermal conversion efficiency (PTCE) of BPNSs is as high as 38.8%, which is stronger than that of the previous BPQDs at 28.4% (), due to the larger extinction coefficient of BPNSs compared with BPQDs (details in SI Materials and Methods). Subsequently, NIR light with a wavelength of 808 nm was employed to investigate the photothermal effect of the embedded BPNSs on the release rate of the drug. The concentration of released drug in the PBS solution (pH 7.4) was monitored in real time, using a UV/Vis spectrometer. The light intensity was set to 1 W⋅cm−2 with an exposure time of 5 min (“ON”), followed by another 5 min under dark (“OFF”). The release rates, rON and rOFF, were calculated from the concentration–time slope with and without irradiation, respectively. A thermocouple was inserted into the BP@Hydrogel matrix (T1), while another one was affixed above the matrix (T2) and the temperature difference (ΔT) was measured over time. The experimental device is shown in Fig. 3A.
如上图所示，BPNSs的吸收光谱强于BPQDs；因此，预计光热转换效率会提高。在功率密度为 1.0 W·cm −2 的 808 nm 近红外激光下评估 BPNS 的光热性能。根据Roper等人提出的光热转换效率计算。 （49），BPNSs的光热转换效率（PTCE）高达38.8％，强于之前的BPQDs的28.4％（43），这是由于BPNSs与BPQDs相比具有更大的消光系数（详细信息参见SI 材料和方法）。随后，利用波长为808 nm的近红外光研究嵌入的BPNS对药物释放速率的光热效应。使用紫外/可见分光光度计实时监测 PBS 溶液（pH 7.4）中释放的药物浓度。光强度设置为 1 W·cm −2 ，曝光时间为 5 分钟（“ON”），然后在黑暗下再曝光 5 分钟（“OFF”）。释放速率 r ON 和 r OFF 分别根据有照射和无照射的浓度-时间斜率计算。将热电偶插入 BP@Hydrogel 基质 (T1)，同时将另一个热电偶固定在基质上方 (T2)，并测量随时间变化的温差 (ΔT)。实验装置如图3A所示。

UV-Vis calibration curves were used to calculate the drug concentration from the UV-Vis absorbance. As can be seen in Fig. 3B, the absorbance spectra of DOX at 480 nm gradually increased, indicating the concentration of released DOX with the evolution of irradiation time. The photocontrolled temperature increase and release of DOX from the BP@Hydrogel depot is depicted in Fig. 3C, showing that the concentration of DOX increases dramatically under irradiation, compared with the unchanged concentration without irradiation. Rheology measurement of BP@Hydrogel with different BP concentrations indicated the reduction of the storage modulus at increasing BP concentration, shown in Fig. 3D. The release rates (rON and rOFF, in ppm⋅ml−1⋅min−1) during the presence or absence of visible light exposure were measured by the slope of the drug release profile. This calculated release rate is essentially the slope from the linear regression of the protein release profile at the start and the end of photomodulation. The drug release rates within the first four consecutive ON–OFF cycles are depicted in Fig. 3E. It can be seen that rON is much higher than rOFF, indicating that BP@Hydrogel could operate as an effective optical switch of drug release.
UV-Vis 校准曲线用于根据 UV-Vis 吸光度计算药物浓度。从图3B中可以看出，DOX在480 nm处的吸收光谱逐渐增加，表明释放的DOX浓度随着照射时间的变化而增加。图 3C 描绘了光控温度升高和 BP@Hydrogel 库中 DOX 的释放，表明与未照射时浓度不变的情况相比，在照射下 DOX 的浓度急剧增加。不同 BP 浓度的 BP@Hydrogel 的流变学测量表明，随着 BP 浓度的增加，储能模量降低，如图 3D 所示。存在或不存在可见物质时的释放速率（r ON 和 r OFF ，单位为 ppm⋅ml −1 ⋅min −1 ）通过药物释放曲线的斜率来测量光暴露。该计算的释放速率本质上是光调制开始和结束时蛋白质释放曲线的线性回归的斜率。前四个连续 ON-OFF 循环内的药物释放速率如图 3E 所示。可以看出，r ON 远高于r OFF ，表明BP@Hydrogel可以作为药物释放的有效光学开关。

NIR-Light–Controlled BP@Hydrogel Degradation.

NIR-light–controlled degradability of BP@Hydrogel could improve its potential for clinical applications. To further assess the biodegradation of BP@Hydrogel, the temperature of the gel upon irradiation with a NIR laser was monitored over time. As can be seen from Fig. 3F, this BP@Hydrogel works well under low laser power with the temperature increased by more than 10 °C compared with an environmental temperature under 1 W⋅cm−2 irradiation. These cycles were repeated six times stably, with the hydrogel undergoing reversible softening. With the power of the laser increased to 1.5 W, the temperature increased dramatically, and the hydrogel became molten, leading to a gradually decreased temperature increase compared with the environmental temperature, as shown in Fig. 3F. In the meantime, both BP and DOX spread out and diffused all over the solution. And the BP@Hydrogel melted completely under 2 W⋅cm−2 irradiation. The NIR-light–controlled drug release and degradation process is illustrated in Fig. 1. When the BP@Hydrogel is exposed under NIR light, the hydrogel warms up and become softer under the photothermal effect of BPNSs due to the hydrolysis of cross-linking, leading to the release of drug. Further increasing the laser power results in the melting of the hydrogel and polymer degradation due to hydrolysis of the ester linkage into segments (reduced molecular weight), oligomers, and monomers and finally carbon dioxide and water. Degradation of the hydrogel disrupts the BPNSs and triggers release of the interior BPNSs which degrade rapidly if they are not protected by the hydrogel. The final degradation products from the BPNSs are nontoxic phosphate and phosphonate (, ), both of which are commonly found in the human body (, ). As shown in Fig. 3G, the dark colors of BP@Hydrogel gradually faded until nearly transparent under persistent irradiation, indicating that BP@Hydrogel almost degraded, which could be confirmed by the UV-Vis absorption spectra shown in Fig. 3H. Therefore, BP@Hydrogel could be degraded after the treatment, highlighting its large potential for clinical applications.


近红外光控制的 BP@Hydrogel 降解。 BP@Hydrogel 的近红外光控制降解性可以提高其临床应用潜力。为了进一步评估 BP@Hydrogel 的生物降解性，随着时间的推移，监测近红外激光照射时凝胶的温度。从图3F可以看出，这种BP@Hydrogel在低激光功率下工作良好，与1 W·cm −2 照射下的环境温度相比，温度升高了10°C以上。这些循环稳定地重复六次，水凝胶经历可逆软化。随着激光功率增加到1.5 W，温度急剧升高，水凝胶开始熔化，导致温度升高与环境温度相比逐渐下降，如图3F所示。与此同时，BP 和 DOX 都扩散并扩散到整个溶液中。 BP@Hydrogel 在 2 W·cm −2 照射下完全熔化。近红外光控制的药物释放和降解过程如图1所示。当BP@Hydrogel暴露在近红外光下时，由于交联的水解，水凝胶在BPNS的光热作用下升温并变得更软。 ，从而导致药物的释放。进一步增加激光功率会导致水凝胶熔化和聚合物降解，因为酯键水解成链段（分子量降低）、低聚物和单体，最后形成二氧化碳和水。水凝胶的降解会破坏 BPNS 并触发内部 BPNS 的释放，如果不受水凝胶的保护，内部 BPNS 会迅速降解。 BPNS 的最终降解产物是无毒的磷酸盐和膦酸盐 (47, 51)，这两种物质在人体内都很常见 (52, 53)。如图3G所示，在持续照射下，BP@Hydrogel的深色逐渐褪色直至接近透明，表明BP@Hydrogel几乎降解，这可以通过图3H所示的紫外-可见吸收光谱来证实。因此，BP@Hydrogel在治疗后可以被降解，凸显了其巨大的临床应用潜力。

In Vitro Cell Experiments.
体外细胞实验。

Biocompatibility is an important requisite for nanomaterials used in biomedicine. To verify the cytotoxicity of BPNSs, four types of human cells, including MDA-MB-231 (human breast cancer cells), A549 (A549 human lung carcinoma cells), HeLa (human cervical cancer cell), and B16 (mouse melanoma cells), were tested systematically. These cells were cultured with Roswell Park Memorial Institute (RPMI)-1640 medium containing different concentrations of BPNSs for 48 h, and the relative viabilities of cells were detected by Cell Counting Kit-8 (CCK-8) cell cytotoxicity assays. As shown in Fig. 4D, no obvious cytotoxicity could be observed for all cells selected even at a high concentration of 200 μg/mL, indicating a potential biosafety of this material.
生物相容性是生物医学中使用的纳米材料的重要要求。为了验证 BPNS 的细胞毒性，使用了四种类型的人类细胞，包括 MDA-MB-231（人乳腺癌细胞）、A549（A549 人肺癌细胞）、HeLa（人宫颈癌细胞）和 B16（小鼠黑色素瘤细胞） ，进行了系统测试。将这些细胞与含有不同浓度BPNS的Roswell Park Memorial Institute（RPMI）-1640培养基培养48小时，并通过细胞计数试剂盒8（CCK-8）细胞毒性测定检测细胞的相对活力。如图4D所示，即使在200μg/mL的高浓度下，对于所有选择的细胞也没有观察到明显的细胞毒性，表明该材料具有潜在的生物安全性。

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In vitro cell viability experiment. (A) Photograph of MDA-MB-231 cell culture dish with BP@Hydrogel loaded with DOX. (B) Fluorescent images of acridine orange (green, live cells) and propidium iodide (red, dead cells) costained MDA-MB-231 cells after addition of BP@Hydrogel and exposure to laser irradiation for 0 min, 5 min, 10 min, and 20 min. (Scale bars: 100 μm.) (C) Relative cell viabilities of MDA-MB-231 cells treated with different laser irradiation times. (D) Relative cell viabilities of MDA-MB-231, A549, HeLa, and B16 cells with different BP concentrations (10 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL). (E) Representative annexin V-FITC/PI scatter plots of 1 × 104 MDA-MB-231 cells after 24 h of treatment.
体外细胞活力实验。 (A) 带有负载 DOX 的 BP@Hydrogel 的 MDA-MB-231 细胞培养皿的照片。 (B) 添加 BP@Hydrogel 并暴露于激光照射 0 分钟、5 分钟、10 分钟后，吖啶橙（绿色，活细胞）和碘化丙啶（红色，死细胞）共染色 MDA-MB-231 细胞的荧光图像，20分钟。 （比例尺：100 μm。）（C）用不同激光照射时间处理的 MDA-MB-231 细胞的相对细胞活力。 (D) 不同 BP 浓度（10 μg/mL、50 μg/mL、100 μg/mL 和 200 μg/mL）的 MDA-MB-231、A549、HeLa 和 B16 细胞的相对细胞活力。 (E) 处理 24 小时后 1 × 10 4 MDA-MB-231 细胞的代表性膜联蛋白 V-FITC/PI 散点图。

After confirming the biocompatibility of BPNSs, we next investigated their therapeutic effect in vitro. In the cell experiments, acridine orange/propidium iodide (live cells, green fluorescence; dead cells, red fluorescence) was used to differentiate the live/dead cells by costaining the cells. Under NIR-light laser for different times, the MDA-MB-231 cells were killed gradually by the released drugs as shown in Fig. 4B, and the relative cell viabilities were tested with the CCK-8 assay (Fig. 4C). Annexin V-FITC/propidium iodide (PI) staining and FACS analysis demonstrated that the majority of the MDA-MB-231 cell death is due to apoptosis (Fig. 4E), and the difference between cell death and apoptosis may result from nonspecific cell death. These results demonstrated the potential applications for the NIR-light–controlled drug release to clinical cancer treatment. As shown in Fig. 4D, no obvious cytotoxicity could be observed for all cells selected, indicating a potential biosafety of this material.
在确认了 BPNS 的生物相容性后，我们接下来研究了它们的体外治疗效果。细胞实验中，使用吖啶橙/碘化丙啶（活细胞，绿色荧光；死细胞，红色荧光）通过共染细胞来区分活细胞/死细胞。在近红外激光不同时间下，MDA-MB-231细胞逐渐被释放的药物杀死，如图4B所示，并用CCK-8法检测相对细胞活力（图4C）。膜联蛋白V-FITC/碘化丙啶（PI）染色和FACS分析表明，大多数MDA-MB-231细胞死亡是由于细胞凋亡（图4E），并且细胞死亡和细胞凋亡之间的差异可能是由于非特异性细胞所致死亡。这些结果证明了近红外光控制药物释放在临床癌症治疗中的潜在应用。如图4D所示，所有选定的细胞均未观察到明显的细胞毒性，表明该材料具有潜在的生物安全性。

In Vivo Tumor Eradication.
体内肿瘤根除。

We carried out animal experiments to test the possibility of the BP@Hydrogel platform for in vivo application. We first studied the in vivo photothermal effects of our BP@Hydrogel platform. After intratumor injection of BP@Hydrogel and free DOX, respectively, animal NIR images and ΔT were monitored by a thermal camera during 5 min irradiation. ΔT of free DOX was only ∼5 °C while that of BP@Hydrogel was more than 13 °C, reaching more significant temperature rises throughout the irradiation period (Fig. 5 A and B). IVIS Spectrum was used to take the in vivo images and monitor the dynamic change of fluorescence at 1 h, 12 h, and 24 h postirradiation after the in vivo photothermal assay. As presented in Results, local intratumoral injection of BP@Hydrogel exhibited a localized drug distribution around the tumor site and more sustained release over 12 h than intratumoral injection of DOX only (Fig. 5C). Based on the in vivo therapeutic effects, we carried out an in vivo antitumor study to validate the enhanced therapy of cancer. The tumor-bearing nude mice were treated as follows: group 1, with saline (control); group 2, with DOX only; group 3, with BP@Hydrogel only; and group 4, with BP@Hydrogel with laser irradiation. The tumor volumes were calculated by the width and length every 2 d. At the end point of this experiment, all nude mice were killed and the tumors were collected. In line with the results of the tumor growth curve shown in Fig. 5D, the tumor sizes of the BP@Hydrogel depot with laser irradiation group were notably smaller than those of the other three groups. Consequently, these results demonstrated that the laser irradiation-controlled BP@Hydrogel-based drug delivery platform with DOX loaded possesses an excellent tumor ablation effect in vivo. Meanwhile, the body weights of nude mice were not significantly affected, demonstrating that there were no acute side effects in our combined therapy (Fig. 5E). To further evaluate the in vivo toxicity of BP@Hydrogel, major organs of the mice were sliced and stained by hematoxylin and eosin (H&E) for histology analysis. As shown in Fig. 5F, the treated mice that were killed 2 wk after BP@Hydrogel injection with NIR irradiation exhibited no significant damage to the normal tissues, including the heart, liver, spleen, lung, and kidney, indicating that BP@Hydrogel treatment had no observable side effect or toxicity to the normal tissues. Moreover, another s.c. tumor model of malignant melanoma in nude mice was also tested and validated the excellent in vivo therapeutic effect and biodegradability (Fig. S7 and SI Materials and Methods).
我们进行了动物实验来测试 BP@Hydrogel 平台在体内应用的可能性。我们首先研究了 BP@Hydrogel 平台的体内光热效应。分别在肿瘤内注射 BP@Hydrogel 和游离 DOX 后，在 5 分钟照射期间通过热像仪监测动物 NIR 图像和 ΔT。游离DOX的ΔT仅为~5°C，而BP@Hydrogel的ΔT超过13°C，在整个辐照期间达到更显着的温度升高（图5A和B）。 IVIS Spectrum 用于拍摄体内图像并监测体内光热测定后辐照后 1 h、12 h 和 24 h 的荧光动态变化。如结果所示，局部瘤内注射 BP@Hydrogel 在肿瘤部位周围表现出局部药物分布，并且比仅瘤内注射 DOX 更能持续释放 12 小时（图 5C）。基于体内治疗效果，我们开展了体内抗肿瘤研究，以验证癌症的强化治疗。荷瘤裸鼠处理如下：第1组，生理盐水（对照组）；第 2 组，仅使用 DOX；第 3 组，仅使用 BP@Hydrogel；第 4 组，使用 BP@Hydrogel 进行激光照射。每2 d通过宽度和长度计算肿瘤体积。实验结束时处死所有裸鼠并收集肿瘤。与图5D所示的肿瘤生长曲线结果一致，激光照射组的BP@Hydrogel depot的肿瘤尺寸明显小于其他三组。因此，这些结果表明，负载DOX的激光辐照控制BP@Hydrogel基药物递送平台在体内具有优异的肿瘤消融效果。 同时，裸鼠的体重没有受到显着影响，表明我们的联合治疗没有急性副作用（图5E）。为了进一步评估 BP@Hydrogel 的体内毒性，将小鼠的主要器官切片并用苏木精和伊红 (H&E) 染色进行组织学分析。如图5F所示，注射BP@Hydrogel并接受近红外照射2周后处死的处理小鼠没有表现出对正常组织的明显损伤，包括心脏、肝脏、脾脏、肺和肾脏，表明BP@Hydrogel治疗对正常组织没有明显的副作用或毒性。此外，另一个 s.c.还对裸鼠恶性黑色素瘤模型进行了测试，验证了其优异的体内治疗效果和生物降解性（图S7和SI材料和方法）。

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In vivo imaging and anticancer tumor effect of BP@Hydrogel drug delivery platform. (A) Thermal images of mice bearing tumors after injection of DOX or BP@Hydrogel, followed by exposure to 808-nm laser irradiation (1.0 W/cm2, 5 min). (Scale bars: 1 cm.) (B) Tumor temperature changes of mice bearing MDA-MB-231 tumors during laser irradiation as indicated in A. (C) Fluorescence images of mice at 1 h, 12 h, and 24 h postirradiation after the in vivo photothermal assay. The 485-nm wavelength light was used as the excitation source and 590 nm was detected as the emitted light. (Scale bars: 1 cm.) (D) Corresponding growth curves of tumors in different groups of mice treated with PBS solution, DOX, BP@Hydrogel depot only, and BP@Hydrogel depot with laser irradiation. The relative tumor volumes were normalized to their initial size. D, Inset shows representative photographs of tumors removed from the killed nude mice. (E) Body weight of nude mice recorded every other day after various treatments. (F) H&E-stained images of major organs from treated mice. (Scale bars: 50 μm.)
BP@Hydrogel给药平台的体内成像及抗癌肿瘤作用。 (A) 注射 DOX 或 BP@Hydrogel，然后暴露于 808 nm 激光照射（1.0 W/cm 2 ，5 分钟）后，荷瘤小鼠的热图像。 （比例尺：1 cm。）（B）携带 MDA-MB-231 肿瘤的小鼠在激光照射期间的肿瘤温度变化，如 A 所示。（C）小鼠在照射后 1 小时、12 小时和 24 小时的荧光图像体内光热测定。使用 485 nm 波长的光作为激发源，检测到 590 nm 的发射光。 （比例尺：1 cm。）（D）用PBS溶液、DOX、仅BP@Hydrogel depot和激光照射的BP@Hydrogel depot治疗的不同组小鼠的肿瘤相应生长曲线。将相对肿瘤体积标准化为其初始大小。 D，插图显示了从处死的裸鼠中取出的肿瘤的代表性照片。 (E)各种处理后每隔一天记录裸鼠的体重。 (F) 治疗小鼠主要器官的 H&E 染色图像。 （比例尺：50 μm。）

Discussion 讨论

A smart NIR-light–controlled drug-release BP@Hydrogel nanostructure is fabricated by using a low–melting-point agarose and PEGylated BPNSs. The solid state of BP@Hydrogel transforms to a gel state after injection into the cancerous tissue because of the lower body temperature, resulting in the phase transition. The BP@Hydrogel underwent controllable softening and molten states under NIR laser power, due to the high photothermal conversion efficiency of BPNSs, leading to a controllable light-triggered drug release and hydrogel degradation. More importantly, the drug release rate can be precisely tuned by internal (e.g., agarose, BP, and drug concentration) and external parameters as well (e.g., light intensity and exposure duration), which is beneficial for clinical application to maintain an effective blood drug concentration for anticancer therapy. Both in vitro and in vivo experiments show that BP@Hydrogel possesses an excellent cancer cell killing ability and tumor ablation effect. Notably, BP@Hydrogel has negligible toxicity for various classes of cells, and both the agarose hydrogel and BPNSs were degradable when the treatment was accomplished, which makes them promising for clinical translation. Consequently, we are able to conclude that the BP@Hydrogel-based drug delivery platform with low cytotoxicity and high biodegradability has a potential for on-demand and in-depth viscera therapeutics, such as breast and melanoma cancers. However, aiming at final clinical and translational applications of light-activated BP@Hydrogel and successful bench-to-bedside transition, future investigations concerning safety and facile inexpensive and controlled synthesis methods of high-quality BP nanomaterials with much higher efficiency and fewer side effects are crucially needed.
使用低熔点琼脂糖和聚乙二醇化 BPNS 制备了智能近红外光控制药物释放 BP@Hydrogel 纳米结构。 BP@Hydrogel的固态在注射到癌组织后由于体温较低而转变为凝胶态，导致相变。由于BPNSs的高光热转换效率，BP@Hydrogel在近红外激光功率下经历了可控的软化和熔融状态，从而导致可控的光触发药物释放和水凝胶降解。更重要的是，药物释放速率可以通过内部参数（如琼脂糖、血压和药物浓度）和外部参数（如光强度和暴露时间）精确调节，这有利于临床应用以维持有效的血液浓度。抗癌治疗的药物浓度。体外和体内实验均表明BP@Hydrogel具有优异的癌细胞杀伤能力和肿瘤消融效果。值得注意的是，BP@Hydrogel 对各类细胞的毒性可以忽略不计，并且琼脂糖水凝胶和 BPNS 在治疗完成后均可降解，这使得它们具有临床转化的前景。因此，我们可以得出结论，基于 BP@Hydrogel 的药物递送平台具有低细胞毒性和高生物降解性，具有按需和深入内脏治疗的潜力，例如乳腺癌和黑色素瘤。然而，针对光激活 BP@Hydrogel 的最终临床和转化应用以及成功的从实验室到临床的转变，未来的研究涉及高质量 BP 纳米材料的安全性、简便、廉价和受控合成方法，具有更高的效率和更少的副作用是迫切需要的。

Materials and Methods 材料和方法

Reagents.

The BP crystals (Fig. 2A, Inset) were purchased from a commercial supplier (Smart-Elements) and stored in a dark Argon glovebox. NMP and IPA (99.5%, anhydrous) were purchased from Aladdin Reagents. PEG-NH2 and low–melting-point agarose were purchased from Yare Shanghai. Doxorubicin hydrochloride was purchased from Sigma-Aldrich. The AO/PI assay kit was obtained from Logos Biosystems. PBS (pH 7.4), FBS, RPMI-1640 medium, trypsin-EDTA, and penicillin/streptomycin were purchased from Gibco Life Technologies. All other chemicals used in this study were analytical reagent grade and used without further purification. Ultrapure water (18.25 MΩ/cm, 25 °C) was used to prepare all of the solutions.


试剂。 BP 晶体（图 2A，插图）购自商业供应商 (Smart-Elements)，并储存在黑暗的氩气手套箱中。 NMP 和 IPA（99.5%，无水）购自 Aladdin Reagents。 PEG-NH 2 和低熔点琼脂糖购自上海亚热。盐酸阿霉素购自 Sigma-Aldrich。 AO/PI 测定试剂盒购自 Logos Biosystems。 PBS (pH 7.4)、FBS、RPMI-1640 培养基、胰蛋白酶-EDTA 和青霉素/链霉素购自 Gibco Life Technologies。本研究中使用的所有其他化学品均为分析试剂级，无需进一步纯化即可使用。使用超纯水（18.25 MΩ/cm，25 °C）来制备所有溶液。

In Vitro BP@Hydrogel Therapy Study.

Typically, MDA-MB-231cells were incubated in six-well plates at 37 °C with 5% CO2 for 24 h; afterward, the culture medium was replaced by new culture medium and the BP@Hydrogels (500 μg/mL BP, 200 μg/mL DOX, 1% LA) were put at the center of the wells. BP@Hydrogels were irradiated with an 808-nm laser at a power density of 1 W/cm2 for different times (0 min, 5 min, 10 min, 15 min). After incubation for another 6 h, both acridine orange and propidium iodide (live cells, green fluorescence; dead cells, red fluorescence) were used to costain the cells to determine the effect of BP@Hydrogel. To quantitatively analyze the therapy effect of BP@Hydrogel, cells were plated and incubated in 96-well plates at 37 °C in an atmosphere of 5% CO2 and 95% air for 24 h. BP@Hydrogels were added and the cells continued incubating for another 6 h. Subsequently, the cells were exposed to an 808-nm NIR laser irradiation for 5 min. Finally, the viability of MDA-MB-231 cells was determined by a CCK-8 cell cytotoxicity assay. The cell viability was normalized by control group without any treatment.


体外 BP@水凝胶疗法研究。通常，MDA-MB-231细胞在六孔板中于37°C、5%CO 2 下孵育24小时；随后，更换新培养基，并将BP@Hydrogels（500 μg/mL BP、200 μg/mL DOX、1% LA）置于孔中央。用808 nm激光以1 W/cm 2 功率密度照射BP@Hydrogels不同时间（0分钟、5分钟、10分钟、15分钟）。再孵育6小时后，使用吖啶橙和碘化丙啶（活细胞，绿色荧光；死细胞，红色荧光）对细胞进行共染色以确定BP@Hydrogel的效果。为了定量分析 BP@Hydrogel 的治疗效果，将细胞铺板在 96 孔板中，在 37 °C、5% CO 2 和 95% 空气的气氛中孵育 24 小时。添加 BP@Hydrogels，细胞继续孵育 6 小时。随后，将细胞暴露于 808 nm NIR 激光照射 5 分钟。最后，通过 CCK-8 细胞毒性测定测定 MDA-MB-231 细胞的活力。对照组细胞活力正常化，无需任何治疗。

In Vivo Photothermal Assay.

Tumor-bearing nude mice were injected with 100 μL of 200 μg/mL DOX (group 1) or 100 μL of BP@Hydrogel (500 μg/mL BP, 200 μg/mL DOX, 1% LA, group 2). After 1 h injection, the nude mice were irradiated with an 808-nm laser at 1 W/cm2 for 5 min. During the course of irradiation, an IR thermal camera (FLIR E50) was utilized to monitor the temperature changes of the tumor sites. All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Shenzhen University (Approval ID: 2017003).


体内光热测定。荷瘤裸鼠注射 100 μL 200 μg/mL DOX（第 1 组）或 100 μL BP@Hydrogel（500 μg/mL BP、200 μg/mL DOX、1% LA，第 2 组）。注射1 h后，用808 nm激光以1 W/cm 2 照射裸鼠5 min。在照射过程中，利用红外热像仪（FLIR E50）监测肿瘤部位的温度变化。所有动物研究均按照深圳大学动物福利与研究伦理委员会批准的实验实践和标准（批准号：2017003）进行。

In Vivo Biodistribution Analysis.

IVIS Spectrum (PerkinElmer) was used to take the in vivo images of mice at 1 h, 12 h, and 24 h postirradiation after the in vivo photothermal assay, respectively. A 485-nm wavelength light was used as the excitation source and 590 nm was detected as the emitted light.


体内生物分布分析。使用IVIS Spectrum (PerkinElmer)分别拍摄体内光热测定后照射后1小时、12小时和24小时的小鼠体内图像。使用 485 nm 波长的光作为激发源，检测到 590 nm 的发射光。

Supplementary Material 补充材料

Supplementary File 补充文件

Click here to view.(1.2M, pdf)
点击此处查看。 (1.2M, pdf)

Acknowledgments 致谢

This research is partially supported by the National Natural Science Fund (Grants 61435010 and 61575089), the Science and Technology Innovation Commission of Shenzhen (Grants KQTD2015032416270385 and JCYJ20150625103619275), and the China Postdoctoral Science Foundation (Grant 2017M610540).
该研究得到国家自然科学基金（61435010和61575089）、深圳市科创委（KQTD2015032416270385和JCYJ20150625103619275）和中国博士后科学基金（2017M610540）的部分支持。

Footnotes 脚注

The authors declare no conflict of interest.
作者声明不存在利益冲突。

This article is a PNAS Direct Submission.
本文是 PNAS 直接投稿。

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1714421115/-/DCSupplemental.
本文包含在线支持信息：www.pnas.org/lookup/suppl/doi:10.1073/pnas.1714421115/-/DCSupplemental。

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Shiyun Bao

cDepartment of Hepatobiliary and Pancreatic Surgery, Shenzhen People’s Hospital, Second Clinical Medical College of Jinan University, Shenzhen 518020, Guangdong Province, People’s Republic of China;

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