Black Phosphorus-Based Drug Nanocarrier for Targeted and

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Biological and Medical Applications of Materials and Interfaces

Black Phosphorus-Based Drug Nanocarrier for Targeted and Synergetic Chemo-Photothermal Therapy of Acute Lymphoblastic Leukemia Shenfei Zong, Lingling Wang, Zhaoyan Yang, Hong Wang, Zhuyuan Wang, and Yiping Cui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22563 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Applied Materials & Interfaces

Black Phosphorus-Based Drug Nanocarrier for Targeted and Synergetic ChemoPhotothermal Therapy of Acute Lymphoblastic Leukemia Shenfei Zong #1, Lingling Wang #1, Zhaoyan Yang 1, Hong Wang 2, Zhuyuan Wang*1, Yiping Cui* 1 1 2

Advanced Photonics Center, Southeast University, Nanjing 210096, China

Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China # These

authors contributed equally to this work

*[email protected]

[email protected]

Abstract As one of novel two-dimensional nanomaterials, black phosphorus nanosheets (BP NS) have been proven to be excellent carrier materials for drugs owing to their fine optical properties and biocompatibility. In this work, a composite drug nanocarrier based on BP NS is proposed, which can perform synergetic and targeted chemo-photothermal therapy of acute lymphoblastic leukemia (ALL). First, BP NS were prepared by an improved liquid exfoliation technique. Then, polyethylene glycol (PEG) was modified on the surfaces of BP NS through electrostatic adsorption. Drug molecules can also be loaded onto the BP NS via electrostatic adsorption. The PEG layer can effectively protect the interior BP NS from water and air to enhance their physiological stability. The obtained PEGylated BP NS (BP NS@PEG) not only demonstrated an excellent photothermal conversion efficiency and photothermal stability, but also exhibited a good pH and photothermal dual-responsive drug release behavior. In addition, the BP NS@PEG were further modified with Sgc8 aptamers through covalent bonding. The aptamers provided an efficient specificity toward ALL cells (CCRF-CEM) and greatly increased the endocytosis of the nanocarriers through a receptor mediated manner, which can further improve the therapeutic effect. Hence, the presented BP NS based multifunctional nanocarrier can achieve targeted and synergetic chemo-photothermal therapy of ALL, which shows a promising potential in improving the curative efficiency. Key words：black phosphorus, drug nanocarrier, synergetic therapy, leukemia

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Introduction Acute lymphoblastic leukemia (ALL) is one of the malignant tumors with the highest incidence among children. 1 Moreover, the incidence rate of ALL keeps growing at a high speed every year, which seriously threatens the health of human. Conventional cancer treatments, including surgical resection, chemotherapy and radiotherapy, remain far from optimal given their low curative efficiency and strong systemic side effects. 2-3

Also, present clinical anticancer drugs still suffer from low bioavailability, impaired target specificity,

poor stability, organ toxicity and so on. Considerable efforts have been devoted to improve tumor therapies. With the rapid development of biomedicine and nanotechnology, drug nanocarriers have become popular due to their properties such as controllable drug release, improved blood circulation half-life and so on. In addition, photothermal therapy (PTT) has attracted increasing interest which offers many advantages such as high efficiency and minimal invasiveness.4-5 PTT employs optical absorbing agents to convert the photon energy to heat under light irradiation, leading to thermal ablation of cancer cells.6-7 Owing to their good near infrared (NIR) absorption, nanomaterials such as gold nanorods and graphene have been employed as PTT agents or drug release system in cancer therapy.8-9 However, PTT nanoagents generally have poor biodegradability and would remain in the body for a long period, leading to deleterious risk. Moreover, the current therapeutic nanoagents for PTT often consist of complex composites, making them relatively difficult to be used in large-scale. Therefore, it is highly desirable to develop new PTT agents which not only have excellent optical performance but also good biodegradability. Recently, black phosphorus (BP) has attracted considerable attention owing to its unique physical structure and excellent optical properties.10-11 As a metal-free layered semiconductor, BP is constructed by puckered layers of phosphorous via weak van der Waals force, which can be exfoliated to form ultrathin twodimensional (2D) nanosheets. Compared with other 2D nanomaterials (such as graphene, MXenes and transition-metal dichalcogenide), BP NS have some prominent properties that make them standout.12-15 For example, graphene has a zero bandgap and Mxenes have limited bandgap. Meanwhile, although transitionmetal dichalcogenide have finite bandgaps, they possess a low carrier mobility. These features sometimes make them unsuitable for biological applications such as imaging and sensing. On the contrary, BP nanosheets (BP NS) exhibit sufficient carrier mobility and layer-dependent band gaps, thereby allowing broad absorption across the ultraviolet and infrared regions.16-17 What’s more, BP NS have a large NIR 2


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extinction coefficient and a high photothermal conversion efficiency, as a result, they can serve as effective PTT agents.18 In addition, BP NS have much larger surface-to-volume ratio compared with other 2D materials due to their special puckered structure, hence, BP NS are quite efficient for loading therapeutic drugs.19-25 Moreover, since phosphorus is one of the vital elements of our body, BP NS should show excellent biocompatibility. Indeed, researchers have proven that BP shows no observable toxicity in various cell lines.26-27 BP NS, especially the ones with small thickness and size, could irreversibly degrade into nontoxic phosphate and phosphonate when exposed to water and oxygen, both of which can be tolerated by human body.28-31 Surface modification of BP NS (e.g. using polyethylene glycol, PEG) could partially isolate the interior BP NS from the outer water and oxygen to enhance the physical stability and effectively suppress the chemical degradation.32-33 Herein, a multifunctional drug delivery system based on BP NS for ALL therapy is proposed. BP NS were synthesized by an improved liquid exfoliation technique and then modified with PEG through electrostatic adsorption.27 The PEG layer can effectively isolated the interior BP NS from the outer environment to enhance the physiological stability of BP NS. The PEGylated BP NS (BP NS@PEG) showed excellent biocompatibility and could load anti-cancer drugs (e.g. doxorubicin, DOX) with a high efficiency. They also demonstrated an excellent photothermal conversion efficiency and photothermal stability, as well as a good pH and photothermal dual-responsive drug release behavior. Furthermore, to endow the BP NS@PEG with specific targeting ability toward ALL cells, Sgc8 aptamers were attached to the surface of the BP NS@PEG. Experimental results showed that the aptamers can enhance cellular uptake of the nanocarriers.

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Figure 1. Schematic illustration of the fabrication process and structure of the BP NS based nanocarrier.

Results and Discussion The fabrication procedures of the BP NS based nanocarriers are illustrated in Figure 1. When the BP NS were obtained from BP crystal powders, amino functionalized PEG was wrapped onto the surfaces of BP NS via electrostatic interactions. The amino groups of PEG can further conjugated with Sgc8 aptamers, which can specifically recognize the PTK7 proteins overexpressed on the membranes of an ALL cell line (CCRF-CEM). After loading anti-cancer drugs (DOX), the nanocarriers were ready to perform NIR or pH triggered synergetic chemo-photothermal therapy. Characterization of the BP NS The morphology of BP NS is revealed by the transmission electron microscopy (TEM) image (Figure 2a), where the BP NS demonstrate a two-dimensional flaky structure with lateral size of about 190 nm. The large surface-to-volume ratio would endow the BP NS with a high drug loading capacity. Besides, there are some small black dots on the BP NS. The reason might be that the BP NS were fabricated using a liquid sonication exfoliation technique. BP crystal powders were sonicated using an ultrasonic cell disruption system. In addition to sheet shaped BP NS, some BP debris might also be formed during the ultra-sonication procedures. The small black dots on the BP NS should be these debris adhered on them. Due to their small size and small quantity, these debris would not influence the drug delivery behavior of the BP NS. The TEM image of BP NS@PEG is similar to that of BP NS since the PEG layer is relatively hard to see in TEM. Figure 2b illustrates 4


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the absorbance spectra of BP NS and BP NS@PEG, both spectra exhibit a broad absorption band across the UV and NIR regions, indicating that BP NS and BP NS@PEG can absorb NIR light. Figure 2c provides the zeta-potential of BP NS and BP NS@PEG. The original BP NS are negatively charged with a zeta-potential of -43.9 mV. After modification of PEG, the zeta-potential of BP NS@PEG changed to -29.9 mV. This is reasonable since the positively charged amino groups in PEG neutralized some of the negative charges of BP NS. Figure 2 (d) demonstrates the size distributions of BP NS and BP NS@PEG measured by dynamic light scattering (DLS). As can be seen, there is a slight increase on the size after modification of PEG, which is also rational because the PEG layer can increase the hydrodynamic diameter of the BP NS. The changes in zeta-potential and size after PEG modification proved that PEG has been successfully adsorbed onto the BP NS. The PEG layer has two functions. First, it can effectively isolate the interior BP NS from the outer water and air to enhance the stability and dispersity of BP NS. Indeed, in the experiment, no significant color change of BP NS@PEG solution was observed within 7 days, indicating that BP NS@PEG remained stable during this period. Second, together with PEG, amino groups are introduced onto the surface of BP NS, which facilitates further surface modifications of the BP NS. For instance, later, ALL specific aptamers will be linked to the BP NS via these amino groups. The BP NS based nanocarriers also exhibited a good stability. When stored in refrigerator, the nanocarrier can remain stable for 4-5 days with no obvious precipitations. Longer storage would cause oxidation of the BP NS. When studying the photothermal effect and drug release behavior of BP NS@PEG under various pH and illumination conditions, no aggregation of the nanocarriers was observed during the whole experiment. While incubated with cancer cells for 24 h, the nanocarriers also did not aggregate. Although we did not perform in vivo treatment of cancers using the BP NS based nanocarrier, some literature reported that the BP based nanocarriers can remain functional in vivo for one to several days.22, 24, 34 The fine stability of the nanocarriers might attribute to PEG attached on their surfaces, which is frequently used to improve the in-vitro and in-vivo stability of nanomaterials.35-36

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Figure 2. (a) TEM image of BP NS. (b) Absorbance spectra of BP NS and BP NS@PEG. The peak at 9501000 nm is caused by the absorbance of water. (c) Zeta potential of BP NS and BP NS@PEG. (c) DLS size distribution of BP NS and BP NS@PEG.

Photothermal effect and drug release behavior of BP NS@PEG After successfully fabrication of BP NS@PEG, the next step would be to test their photothermal effect and drug release behavior. First, to evaluate the photothermal effect of BP NS@PEG, different concentrations of BP NS@PEG as well as BP NS were respectively exposed to 808 nm NIR light of a femtosecond laser for 10 min (power density: 3.11 W/cm2). The results are shown in Figure 3a. First, the temperatures of BP NS and BP NS@PEG solutions increased similarly upon NIR illumination, indicating that the PEG layer did not influence the inherent photothermal effect of BP NS. Second, larger concentrations of BP NS and BP NS@PEG induced higher temperature increments, which is reasonable since with more BP NS present, more photon energy would be converted to heat. Meanwhile, the temperature of pure water only exhibited a slight increase due to the lack of efficient photothermal agents. To further assess the photothermal stability of BP NS and BP NS@PEG, they were exposed to NIR laser for 10 min (laser on), followed by spontaneous cooling 6


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to room temperature after the NIR laser was turned off (laser off). This procedure was repeated for five times and the temperature variations of the solution were recorded and shown in Figure 3b. As can be seen, both the BP NS and BP NS@PEG demonstrated a similar reversible photothermal effect.

This means that, first,

the PEG layer did not influence the photothermal response of the inner BP NS. Second, the BP NS and BP NS@PEG possess an excellent photothermal stability, which is ideal to be utilized as PTT agents. Besides, the photothermal conversion efficiency (η) of BP NS@PEG can be acquired from the data (when the laser is turned off) in Figure 3b, detailed calculation procedures are presented in the Supporting Information. η is determined to be 26.9%, which indicates a high photothermal conversion efficiency.

Figure 3. (a) Temperature of solutions containing different concentrations of BP NS or BP NS@PEG when exposed to 808 nm laser at a power density of 3.11 W/cm2 for increasing time. (b) Temperature variations of BP NSs and BP-PEG NSs under five on/off cycles of NIR light. (c) Loading efficiencies of DOX corresponding to different feeding ratios of BP NS@PEG:DOX (the concentration of BP NS@PEG is 50 μg/mL). (d) Drug release profiles of BP NS@PEG@DOX at pH 5.0 and 7.4 with or without 808 nm NIR irradiation (3.11 W/cm2). The 2D BP NS has been reported to be able to carry an enormous amount of drug molecules.20 Hence, next, 7



we tested the drug loading capacity of BP NS@PEG. We used DOX as the model drug molecule since it is a popular anti-cancer drug and can fluoresce under a proper laser illumination, which would facilitate the intracellular monitoring of the nanocarriers. The BP NS@PEG were negatively charged (Figure 2c), so the positively charged DOX molecules can load onto the BP NS@PEG via electrostatic interactions (the product is denoted as BP NS@PEG@DOX). BP NS@PEG was mixed with DOX at different feeding ratios (BP NS@PEG: DOX=1:1, 1:2, 1:3 or 1:4) and then stirred overnight. Excess DOX were removed by centrifugation. By comparing the absorbance of excess unloaded DOX with the absorbance of the total added DOX, the loading efficiency of DOX can be calculated. Details on how to calculate the loading efficiency of DOX are provided in the Supporting Information. As shown in Figure 3c, the loading efficiency of DOX gradually decreased with increasing amount of DOX. Hence, the optimal feeding ratio of BP NS@PEG : DOX is 1:1. After successful loading of the DOX molecules, the pH and NIR trigger drug release behaviors of BP NS@PEG@DOX were investigated. The results are presented in Figure 3d. As we can see, more drugs were released under lower pH or with NIR illumination. To be specific, with no NIR illumination, the final DOX release percentage at pH=7.4 was about 9.86%, which increased to 22.34% at pH=5.0. Higher drug release efficiency at a lower pH value is attributed to the electrostatic interaction between BP NS and DOX.20 As can be seen from Figure 3d (the green curve and the yellow curve), a higher drug release ratio was achieved with a lower pH value, which means lower pH value helps to trigger the drug release. In another word, by changing the pH value, we can control the release rate of drugs. Since tumor microenvironments are acidic, such a pH-dependent drug release behavior can be used to realize selective drug release at tumor sites and helps to prevent drug leakage. Moreover, after being incorporated into the cancer cells, the nanocarriers are usually trapped inside lysosomes. The low pH value of the lysosomal environment also can promote the drug release inside cancer cells, which is beneficial for improving the therapeutic efficiency. When illuminated by NIR laser, the drug release efficiency increased dramatically. For example, the DOX release percentage at pH=7.4 increased to 36.17% and the release percentage at pH=5.0 increased to 55.68%, indicating that the drug release can be accelerated significantly by NIR light due to the excellent photothermal effect of BP NS. The above experimental results confirmed that the fabricated nanocarrier can realize both pH and NIR 8


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triggered drug release.

Targeted and synergetic chemo-photothermal therapy of ALL cells After confirming the pH and photothermal dual responsive drug release behavior of the nanocarrier. The next step is to evaluate their performances in living cells. In the experiment, CCRF-CEM cells were used as the model ALL cell line. They overexpress tyrosine kinase 7 (PTK7) on their membranes. To ensure that the nanocarriers can specifically recognize CCRF-CEM cells, Sgc8 aptamers (which has a strong affinity to PTK7) were attached to the BP NS@PEG (the product is denoted as BP NS@PEG@Sgc8). With Sgc8 aptamers present, the BP NS@PEG@Sgc8 can be taken up by CCRF-CEM cells through receptor-mediated endocytosis. The absorbance spectra of BP NS@PEG before and after the attachment of Sgc8 were measured and shown in Figure 4a. Compared with the spectrum of BP NS@PEG, a prominent peak appears at 260 nm in the spectrum of BP NS@PEG@Sgc8, which is caused by the absorbance of the DNA aptamer. As a result, the Sgc8 aptamers have been successfully modified onto the BP NS@PEG. After attaching the aptamers, DOX can subsequently be loaded onto the nanocarriers using a similar electrostatic interaction assisted manner (the product is denoted as BP NS@PEG@Sgc8@DOX). Sgc8 aptamers used in this work functionalize as the specific targeting ligand toward the ALL cell line (CCRF-CEM). CCRF-CEM cells overexpress PTK7 protein (which is a specific biomarker for ALL) on their membranes. Hence, due to the specific recognition between Sgc8 on the nanocarriers and PTK7 on the CCRF-CEM cells, the nanocarriers can be guided to the cells and perform selective drug delivery toward the target cells.37-38 The targeting ability of BP NS@PEG@Sgc8@DOX toward CCRF-CEM cells was investigated. BP NS@PEG@DOX (without aptamers) were used as the control. The BP NS@PEG@Sgc8@DOX or BP NS@PEG@DOX were added into different culture dishes containing CCRFCEM cells, respectively. DOX molecules can fluoresce under 488 nm excitation, hence, if the nanocarriers are taken up by the cells, the cells would also fluoresce. The fluorescence images of these cells were collected and shown in Figure 4b-c. It is quite obvious that the cells incubated with BP NS@PEG@Sgc8@DOX show much stronger fluorescence signals of DOX as compared with the cells incubated with BP NS@PEG@DOX. This means more BP NS@PEG@Sgc8@DOX were incorporated by CCRF-CEM cells than BP 9



NS@PEG@DOX and the Sgc8 aptamer helps to increase the cellular uptake of the nanocarriers. Consequently, the BP NS@PEG@Sgc8@DOX can indeed target the CCRF-CEM cells owing to the Sgc8 aptamers.

Figure 4. (a) Absorbance spectra of BP NS@PEG and BP NS@PEG@Sgc8. (b-c) Fluorescence images of CCRF-CEM cells incubated with BP NS@PEG@DOX (b) and BP NS@PEG@Sgc8@DOX (c). Next, before testing the synergetic therapy of ALL cells using the nanocarriers, we first evaluated the cytotoxicity of BP NS@PEG. The viabilities of three different kinds of cancer cells (CCRF-CEM, HeLa and MCF7) after incubation with BP NS@PEG were measured using CCK-8 assay. The results are presented in Figure 5a. As can be seen, even when the concentration of BP NS@PEG reached 100 μg/mL (which is quite high), the viabilities of these cells are still higher than 85%, confirming that the BP NS@PEG are biocompatible and have negligible cytotoxicity. In addition, we also tested the toxicity of BP NS@PEG toward Zebrafish embryos. BP NS@PEG were injected into the embryos and the survival rate of these embryos after 24 h was found to be 87%, which again proved that the BP NS@PEG are non-toxic. The synergetic therapy function of the nanocarriers was investigated using CCRF-CEM cells. The cells were incubated with different types of nanocarriers (e.g. BP NS@PEG@Sgc8, BP NS@PEG@Sgc8@DOX) and treated with or without NIR illumination. The viabilities of these cell samples were provided in Figure 5b. For cells incubated with BP NS@PEG@Sgc8 and treated without NIR illumination, negligible cell death was observed. This is rational since BP NS@PEG@Sgc8 are non-toxic. For cells incubated with BP NS@PEG@Sgc8 and illuminated with NIR laser, the viability decreased by about 20%. Here, the cell death was probably caused by the photothermal effect or photodynamic activity of BP NS, which corresponds well 10


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with previously published literature.20 For cells incubated with BP NS@PEG@Sgc8@DOX and treated without NIR illumination, the viability decreased to only about 30%. The cell death was mainly caused by the chemo-therapy effect of DOX molecules which could be gradually released. While for cells incubated with BP NS@PEG@Sgc8@DOX and illuminated with NIR laser, the viability falled to less than 20%. Such a high cell death rate was induced by the synergetic chemo-photothermal effect of the nanocarriers. Because the NIR light not only triggered the photothermal or photodynamic activity of BP NS, but also improved the release efficiency of DOX, both of which helped to kill the ALL cells. Live-dead staining of CCRF-CEM cells using propidium iodide (PI) and calcein was also conducted to more vividly show the increased cell death upon NIR illumination. PI is a cell membrane impermeable dye which can only bind with the nuclear DNA of dead or apoptotic cells with strong red fluorescence, and calcein is able to be hydrolyzed by endogenous esterases to generate green fluorescence in the cytoplasm of living cells. CCRF-CEM cells were first incubated with BP NS@PEG@Sgc8@DOX, followed by NIR illumination and subsequent live-dead staining. The results are shown in Figure 5c-d, CCRF-CEM cells incubated with BP NS@PEG@Sgc8@DOX and without NIR illumination were used as the control (Figure 5c). As can be seen, much more dead cells (the red ones) were observed in the NIR treated sample (Figure 5d) as compared with the control sample shown in Figure 5c. This experimental result clearly revealed the NIR assisted cell killing ability of the nanocarriers and the nanocarriers indeed can fulfill synergetic chemo-photothermal therapy of ALL cells.

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Figure 5. (a) Viability of CCRF-CEM, HeLa, MCF-7 cells incubated with various concentrations of BP NS@PEG. (b) Viability of CCRF-CEM cells under different treatments. BP NS@PEG: 50 μg/mL, NIR irradiation: 808 nm, 30 min, 3.11 W/cm2. Cells without any treatment were used as the control. (c-d) Fluorescence images of CCRF-CEM cells incubated with BP NS@PEG@Sgc8@DOX without (c) and with NIR irradiation (d). Irradiation time: 30 min, laser power: 3.11 W/cm2, BP NS@PEG@Sgc8@DOX: 100 μg/mL, green fluorescence: live cells, red fluorescence: apoptotic or dead cells.

Conclusions In summary, we have successfully fabricated a multifunctional drug delivery nanocarrier based on BP NS. The drug release of the nanocarriers can be triggered by both pH and NIR laser. This kind of stimuliresponsive drug release behavior can greatly prevent drug leakage and thus reduce side effect. More importantly, by attaching a specific aptamer onto the nanocarriers, they can realize synergetic chemophotothermal therapy of ALL cells. Although the nanocarriers themselves are highly biocompatible, the 12


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synergetic therapeutic effect successfully induced a significant cell death. By simply changing the targeting ligand on the nanocarriers, we can obtain specificity toward different kinds of cancers. We believe that such a multifunctional nanocarrier is a promising candidate to develop new types of efficient anti-cancer nanodrugs.

Materials and Methods Materials BP crystal powder was purchased from Smart Elements (a commercial supplier) and stored in a dark vacuum glovebox. Amino groups functionalized PEG (NH2-PEG2000-NH2) was purchased from JenKem Technology Co., Ltd. Phosphate buffer (PBS, pH=7.4) was purchased from Nanjing Bookman Biotechnology Co., Ltd. Citric acid monohydrate was purchased from Shanghai Chemical Reagent Co., Ltd. Disodium hydrogen phosphate dodehydrate (Na2HPO4·12H2O) was purchased from Nanjing Chemical Reagent Co., Ltd. DOX, N-(3-Dimethylaminopropyl)-N-ethylcarbod Ⅱ mide (EDC) and N-Hydroxysuccinimide (NHS) were purchased from Aladdin Industrial Corporation. Cell Counting Kit-8 (CCK-8), calcein and PI were purchased from Nanjing KeyGEN BioTECH Co., Ltd. Sgc8 aptamer was synthesized by Sangon Biotech (Shanghai) Co.,

Ltd.

and

its

sequence

is

5

'COOH-

ATCTAACTGCTGCGCCGCCGGGAAAATACTGATCGGTTAGA-3'. Deionized water used in all the experiments was obtained from an ultrapure water system with a resistivity of 18 MΩ/cm. Synthesis of BP NS The BP NS were prepared using a simple liquid sonication exfoliation technique. In brief, BP crystal powders (25 mg) were dispersed in water (50 mL), then the mixture solution was sonicated using an ultrasonic cell disruption system (ultrasonic frequency: 19~25 kHz) for 12 h (period of 3 s with an interval of 4 s) in ice bath (~ 4℃) with a power of 1000 W. The resultant brown solution was centrifuged at 1500 rpm for 15 min to remove large particles, and the supernatant was centrifuged at 7800 rpm for 20 min. The precipitate was resuspended in water. Preparation of BP NS@PEG@Sgc8 BP NS@PEG were prepared as follows. PEG (20 mg) was dispersed in BP NS solution (20 mL, 200 μg/mL) 13



and sonicated for 30 min. After being stirred for 4h, the mixture was centrifuged and washed for 2 times at 7800 rpm, 20 min to remove the excess PEG molecules. The product obtained here is BP NS@PEG. To prepare BP NS@PEG@Sgc8, Sgc8 aptamer (100 μL, 10 μM), EDC (3.6 mg) and NHS (2 mg) were added into BP NS@PEG (5 mL, 100 μg/mL) and shook gently for 4 h at room temperature. Finally, the mixture solution was centrifuged for 2 times at 7800 rpm for 20min to remove the unreacted Sgc8 aptamers. Thus the BP NS@PEG@Sgc8 were obtained. Evaluating the photothermal effect of BP NS@PEG BP NS or BP NS@PEG (2 mL) with various concentrations (10 μg/mL, 50 μg/mL and 100 μg/mL) were respectively added into a transparent quartz cell (4 mL) and irradiated with 808 nm laser at a power density of 3.11 W/cm2 for a certain time. To explore the photothermal stability of BP NS and BP NS@PEG, the solutions were exposed to five laser on/off circles. A temperature probe was used to monitor the temperature change of the solutions. Preparation of BP NS@PEG@DOX or BP NS@PEG@Sgc8@DOX DOX (200 μL, 5 mg/mL) was added into BP NS@PEG (10 mL, 100 μg/mL) or BP NS@PEG@Sgc8 (10 mL, 100 μg/mL) and vigorously stirred in the dark overnight. The obtained BP NS@PEG@DOX or BP NS@PEG@Sgc8@DOX were collected by centrifugation at 7800 rpm for 20min and washed once with water. To determine the drug loading capacity, DOX with various concentrations (50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL) was added into BP NS@PEG (50 μg/mL) following the above similar procedures. By comparing the absorbance of excess unloaded DOX with the absorbance of the total added DOX, the loading efficiency of DOX can be calculated. pH and NIR triggered drug release To test the pH-responsive DOX release kinetics within 24 h, BP NS@PEG@DOX (1 mL) was packaged in a dialysis bag (MW 3500) and incubated in PBS (10 mL) at different pH values (pH=7.4 and pH=5). The outside solution (3 mL) was collected at desired time points to measure the concentrations of released drugs via absorbance spectrometer. The solution was added back after the measurement. To test the pH and NIR dual responsive drug release, the BP NS@PEG@DOX were suspended in PBS with pH values of 7.4 or 5.0, respectively. And illuminated with or without NIR laser (3.11 W/cm2). The samples 14


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were taken out at desired time points and centrifuged at 7800 rpm. The absorption of DOX in the supernatant was measured and the released DOX concentration was calculated according to a standard curve corresponding to different concentrations of DOX. Cell culture The human ALL cells (CCRF-CEM cells), human breast cancer cells (MCF-7 cells) and human cervical cancer cells (HeLa cells) were all purchased from Shanghai cell bank of the Chinese Academy of Sciences. CCRF-CEM cells were cultured in RPMI1640 medium, HeLa cells and MCF-7 cells were cultured in DMEM medium. All the cell culture media were supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin. Cells were incubated under a moist atmosphere with 5% CO2 and 95% air at 37℃. Cell targeting experiment Briefly, CCRF-CEM cells were seeded into two petri dishes and incubated for 24 h at 37 ℃. Then the cells were incubated with BP NS@PEG@DOX or BP NS@PEG@Sgc8@DOX for 4 h at 37 ℃, respectively. Afterwards, the cells were centrifuged at 2000 rpm for 5min and washed with PBS. The fluorescence images of the cells were collected by a confocal laser scanning microscope (CLSM). Cytotoxicity assays The cytotoxicity of the nanocarriers were determined by CCK-8 assay. CCRF-CEM cells, MCF-7 cells and HeLa cells were seeded into 96-well plates with a density of 1×104 cells/mL and cultured for 24 h at 37 ℃. Then the cells were incubated with BP NS@PEG at different concentrations (5 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL) for another 24 h at 37 ℃, respectively. Subsequently, 10 μL of CCK-8 solution was dropped into each well, and then the plates were incubated for another 4 h. Afterwards, the 96-well plates were placed in the Microplate Reader (Bio-Rad model 680) to measure the optical density (OD) of each well at 450 nm. Cells incubated without nanomaterials were used as control. The toxicity of the nanocarriers toward Zebrafish embryos was evaluated by Nanjing YSY Biotech Company Ltd. The nanocarriers (1 nL, 50 μg/mL) were injected into each embryo and the embryos were subsequently incubated for 24 h before counting the survival rate. Embryos injected with PBS were used as the control. Synergetic chemo-photothermal therapy of ALL cells CCRF-CEM cells were seeded into 96-well plates with a density of 1×104 cells/mL and cultured for 24 h at 15



37 ℃. Then the cells were incubated with BP NS@PEG@Sgc8 or BP NS@PEG@Sgc8@DOX (50 μg/mL). For NIR irradiated cells, the illumination time is 30 min by a 808 nm laser with a power density of 3.11 W/cm2. After additional incubation for 24 h, the cell viability was measured by CCK-8 assay. The cells without any treatment were used as control. Living and dead cell staining CCRF-CEM cells were seeded into 96-well plates with a density of 1×104 cells/mL and cultured for 24 h at 37 ℃. Then BP NS@PEG@Sgc8@DOX solution (100 μg/mL) was added to the wells. For NIR treated cells, they were illuminated by 808 nm NIR laser for 30 min at power density of 3.11 W/cm2, cells with no irradiation treatment were used as the control. After that, cells were continuously incubated for 24 h, then, calcein (0.5 μL, 10 nM) was added to each well and shaken gently followed by incubating for 5 min in the dark. Then CCRF-CEM cells were collected by centrifugation at 2000 rpm for 5 min and PBS buffer was added to make the cell concentration to be 106/mL. Next, PI dye (5 μL) was added to the cell suspension (95 μL) and shaken gently followed by incubating for 5 min in the dark. Finally, the fluorescence image of the cells were collected using CLSM. The excitation and emission wavelengths of calcein are 490 nm and 515 nm, while the excitation and emission wavelengths of PI are 436 nm and 617 nm. Instruments TEM images were taken with a JEM-2010HR transmission electron microscope (JEOL, Japan). Hydrodynamic diameter and zeta potential of the BP NSs were obtained by a Zetasizer Nano instrument (ZEN3690, Malvern Instruments). Absorption spectra were obtained using a UV-vis-NIR spectrometer (UV3600PC, Shimadzu). CLSM images were obtained using a microscope (FluoViewTM FV1000, Olympus). The photothermal conversion of BP NS was realized by a femtosecond laser (Mira Model 900-F, Coherent).

A schematic illustration of the experimental lay out when performing NIR triggered drug release can be found in the Supporting Information. The optical density (OD) of 96-well plates at 450 nm in CCK-8 assay was measured by Microplate Reader (Bio-Rad model 680).

Acknowledgement This work was supported by the Natural Science Foundation of China (NSFC) (61535003, 61822503, 16


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61675042, 61505027), the National Key Basic Research Program of China (2015CB352002) and the Fundamental Research Funds for the Central Universities (2242017K41010, 2242018K3DN11).

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TOC figure

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Black Phosphorus-Based Drug Nanocarrier for Targeted and

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