Multifunctional Cancer Phototherapy Using Fluorophore

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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Multifunctional Cancer Phototherapy Using FluorophoreFunctionalized Nanodiamond Supraparticles Yue Yu,†,⊥ Xi Yang,†,⊥ Ming Liu,‡ Masahiro Nishikawa,‡ Takahiro Tei,§ and Eijiro Miyako*,†,⊥ †

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Department of Materials and Chemistry, Nanomaterials Research Institute (NMRI), National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8565, Japan ‡ Corporate Research Center, R&D Headquarters, Daicel Corporation, 1239, Shinzaike, Aboshi-ku, Himeji, Hyogo 671-1283, Japan § Advanced Materials Planning, R&D Headquarters, Daicel Corporation, 2-19-1 Konan, Minato-ku, Tokyo 108-8230, Japan S Supporting Information *

ABSTRACT: In this study, the preparation and characterization of selfassembled supraparticles (SPs) comprising fluorophore-conjugated nanodiamond (ND) nanoclusters are described. The SPs are further used in photohyperthermic, photodynamic, and chemotherapeutic multifunctional tumor therapy systems that use bio-optical-window near-infrared lasers. NDs with surface amino groups are conjugated with various fluorophores via carbodiimide chemistry and are spontaneously transformed into selfassembled ND-based SP (ND−SP) nanoclusters. The fluorophore-functionalized ND−SPs exhibit a uniform particle size, high dispersity in water, and low cytotoxicity. In addition, the energy or electron transfer between fluorophores and NDs can enhance the photothermal conversion efficiency. Further, it is demonstrated that the synthesized ND−SPs strongly fluoresce and penetrate the cellular transmembrane, making them attractive for the targeted delivery of conventional nanomedicines such as albumin-bound paclitaxel (Abraxane) and simple drug-loaded ND conjugates. The near-infrared-light-driven efficiency of the fluorophore-functionalized ND−SPs encapsulating anticancer drugs is confirmed by the targeted eradication of cancer cells both in vitro and in vivo. Thus, the fluorophore-functionalized ND−SP nanoclusters may be proven to be a valuable new therapeutic agent for use in cancer treatment. KEYWORDS: nanodiamond, fluorophore, supraparticle, anticancer, phototherapy

1. INTRODUCTION Cancer is one of the most challenging global healthcare problems. Even though several drugs can be used in cancer treatment, the problem of selectively killing all the cancer cells while reducing the amount of collateral toxicity transferred to the healthy cells remains unresolved.1 Nanoencapsulation of therapeutic anticancer drugs in nanocarriers is an effective strategy for delivering problematic drugs, including those that are insoluble in aqueous solutions and those exhibiting low selectivity against the targeted cancer cells.2 One advantage of the usage of such nanocarriers is that they improve targeting, thereby reducing the dose requirements and systemic toxicity when compared with those caused by nontargeted systemic drug delivery.3 Nanodiamonds (NDs) are less than 10 nm in size and have attracted considerable research attention in biomedical applications because of their low toxicity, excellent mechanical and optical properties, large surface area, and tunable surface structures.4,5 In fact, because of their outstanding characteristics, NDs have significantly contributed to the development of highly efficient and successful drug-delivery systems for cancer treatment.6−8 In the continuation of this study, we recently observed that the perfluorooctanoic-acid-modified © XXXX American Chemical Society

NDs are naturally transformed into biocompatible and water dispersible supraparticle (SP) nanoclusters, which offer improved cellular uptake and excellent tumor chemotherapy.9 However, it remains difficult to synthesize drug-loaded ND− SP nanoclusters with high drug-delivery efficiency, versatile surface functionalization, and tunable optical properties and sizes. Cancer phototherapy by exploiting the near-infrared (NIR) (600−1100 nm) phototherapeutic window is one of the promising technologies that can be used as a drastic tumor remedy because all the targeted cancer cells can be killed just by the usage of NIR irradiation, which can penetrate biological deep tissues.10−16 ND is one of the candidate nanomaterials as a NIR photoactive agent for photoexothermic cancer hyperthermia.17,18 However, the low photothermal conversion efficiency and poor drug-delivering capacity of NDs indicate that effective cancer phototherapy requires further investigation, requiring not only appropriate synthesis strategies for Received: July 6, 2019 Accepted: July 30, 2019

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DOI: 10.1021/acsabm.9b00603 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

2.2. Structural Characterization of ND−SPs. The morphology and structure of the prepared ND−SPs were analyzed by highresolution TEM (EM-002B; Topcon, Tokyo, Japan) (acceleration voltage = 120 kV). The hydrodynamic diameter of the ND−SPs was measured by dynamic light scattering (DLS) (Photal FPAR-1000; Otsuka Electronics, Osaka, Japan). Additionally, the optical absorption spectra and concentrations of the ND−SPs and the fluorescent dyes or nanohybrids with PTX were analyzed by a UV− vis/NIR spectrophotometer. Curve fitting was performed with Origin software (LightStone, Tokyo, Japan). The fluorescence of the ND− SPs, fluorescent dyes, and ND-ori was measured using a fluorescence spectrometer (FP-6500 or FP-8500; Jasco, Tokyo) and a microplate reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland). 2.3. Cell Culturing and in Vitro Assays. The bone osteosarcoma (U2OS) and human ovarian cancer (SKOV3) cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan). These cell lines were basically cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA). DMEM contained 10% fetal bovine serum, 2 mM Lglutamine, 1 mM sodium pyruvate, gentamycin, penicillin− streptomycin (100 IU mL−1), and Hank’s balanced salt solution (Life Technologies, Carlsbad, CA, USA). The cells were cultured and maintained in a 5% CO2 chamber at 37 °C. The cellular viability was tested by a cell counting kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan). The cells were seeded in a 96-well plate (5 × 103 cells well−1). After incubation overnight, the cells were further incubated with anticancer drugs or nanohybrids. Treated cells were washed by a fresh medium, and they were incubated with the CCK-8 solution to monitor the absorbance at 450 nm by a microplate reader. 2.4. In Vitro Fluorescence Bioimaging. The U2OS cells were seeded in imaging dishes and incubated in 5% CO2 chamber at 37 °C overnight. The adhered cells were exposed to fluorophore-functionalized ND−SPs for 6 or 24 h in 5% CO2 humidified chamber at 37 °C. Shortly thereafter, they were stained with 1 μg mL−1 of Hoechst 33342 solution (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. After being washed with fresh PBS three times, the cells were further cultured in RPMI 1640 Phenol Red-free medium (Thermo Fisher Scientific) for live cell imaging. Actin (cytoskeleton) was stained by Phalloidin (Thermo Fisher Scientific) to image PBA− ND−SP. For fixed-cell imaging, cells were either fixed with 4% paraformaldehyde solution or prechilled in a methanol−acetone mixture (v/v = 1:1) for 10 min at 20 °C and subsequently washed with fresh PBS buffer just before observation. The NIR images of ICG−ND−SP were obtained by fluorescence microscopy (IX73; Olympus, Tokyo, Japan). A mirror unit (IRDYE800−33LP-A-U01; Semrock) and a camera with an electron-multiplying charge-coupled device (DP80; Olympus) were incorporated into the microscopy. The intracellular images of the PBA-, BODIPY-, and Alexa568-functionalized ND−SP complexes were obtained by a confocal microscope (LSM 5 PASCAL, Carl Zeiss Inc., Tokyo, Japan). 2.5. Temperature Measurements. The temperature of the laserirradiated ICG−ND−SP suspensions was investigated as follows. ICG−ND−SPs in Milli-Q water (200 μL; ICG and ND concentrations were 0.75 and 3 mg mL−1, respectively) were added to a quartz cuvette (GL Science, Tokyo, Japan). The samples were irradiated for 5 min by a 785 nm laser (spot diameter, ∼4 mm; maximum power, 1 W, ∼80 mW mm−2; BRM-785−1.0−100−0.22SMA; B&W Tek, Newark, DE, USA). The control (PBS buffer without ICG−ND−SPs) was also irradiated by the laser light. The temperature of the solutions was measured over time by a thermocoupler (AD-5601A; A&D, Tokyo, Japan). The ICG and ND concentrations of the ICG−NHS and ND-ori solutions were adjusted to match those of the ICG−ND−SP complex based on their absorbances. The effects of the ICG and ND concentrations on the laser-induced photoexothermic activity of the ICG−ND−SP complexes were also assessed by diluting the original ICG−ND−SP stock solution using the DMEM cell-culture medium.

the production of chemically functionalized NDs but also a better understanding of the unique optical properties of NDs. The objective of this study is to design and prepare selfassembled ND-based SP (ND−SP) nanoclusters using various fluorophores to construct molecule carriers having high drugdelivery efficiency. In addition, we investigate the optical characteristics of the fluorophore-modified ND−SPs to develop an effective cancer phototherapy. Here, we describe the physicochemical properties of these ND constructs, their intercellular permeation and targeted drug-delivery performance, and their NIR-induced photothermal, photodynamic, and chemotherapeutic functions against cancer cells in vitro and in vivo. In addition, we use them for systemic fluorescent bioimaging in an animal model and analyze their biocompatibility.

2. MATERIALS AND METHODS 2.1. Synthesis of ND−SPs. Original ND (ND-ori) (diameter = 4−5 nm) solution was prepared by Daicel Co., Ltd. according to the procedures to previous works.19−22 A 0.1 mL volume of ND-ori (ND concentration = 30 mg mL−1) and 1 mg of ICG−NHS (Goryo Chemical, Hokkaido, Japan) were dissolved in 5 mL of 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (pH 6.0, 100 mM) by bath sonication (80 W, 40 kHz) (USD-2R; AS ONE, Osaka, Japan) for 5 min. After vigorous stirring for another 1.5 h at room temperature, the mixture was centrifuged and washed twice by MilliQ water to eliminate the unreacted chemicals. The obtained pellet was dispersed in 1 mL of Milli-Q water for 10 min using a pulse-type sonicator (VCX-600; Sonics, Danbury, CT, USA). The resulting ICG−ND−SP was subsequently used for performing the studies. The concentrations of ICG (∼0.75 mg mL−1) and ND (3 mg mL−1) in the synthesized product were assessed by UV−sis/NIR spectrophotometry (V-730 BIO; Jasco, Tokyo, Japan) and thermogravimetric analysis (TGA) (Q 500; TA Instruments, New Castle, DE, USA). BODIPY−ND−SP and Alexa568−ND−SP were synthesized in a similar way to the synthesis of ICG−ND−SP. BODIPY FL NHS ester (succinimidyl ester) (1 mg; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 568 NHS ester (succinimidyl ester) (1 mg; Invitrogen) was added to the reaction media instead of ICG−NHS. After the preparation, the BODIPY (ca. 0.04 mg mL−1) and Alexa568 molecules (ca. 0.04 mg mL−1) were covalently bonded onto the ND surface (ca. 3 mg mL−1) according to a calculation performed based on UV−vis/NIR spectrophotometry. Further, PBA−ND−SP was synthesized basically in a manner similar to the synthesis of other fluorescent-dye-functionalized ND− SPs apart from a few factors including the weight of the dye molecule, addition of WSC, volume of the reaction medium, and reaction time. Briefly, ND-ori aqueous solution (1 mL) (concentration of ND is 30 mg mL−1), PBA (10 mg) (Tokyo Chemical Industry, Tokyo, Japan), and WSC (10 mg) were dispersed in MES buffer (9 mL) (100 mM, pH 6.0) using bath-type sonicator for 20 min until the PBA was completely dissolved. The mixture was stirred for 1.5 h at 20 °C and centrifuged and washed twice by Milli-Q water to eliminate the unreacted materials. The formed pellet was dispersed in Milli-Q water (10 mL) for 10 min using pulse-type sonicator. The prepared PBA− ND−SP solution (ND concentration, ∼3 mg mL−1; PBA concentration, ∼0.15 mg mL−1) was subsequently utilized to conduct the investigations. The PTX@ICG−ND−SP complex was prepared as follows. The washed ICG−ND−SP pellet was dispersed with PTX (10 mg) (Wako) in Milli-Q water (10 mL) for 10 min by pulse-type sonicator. The resultant solutions were further washed using Milli-Q water to eliminate the excess chemicals and were stored in fridge at 4 °C just before use. PTX@ND-ori was prepared by mixing 10 mg of PTX, 1 mL of ND-ori, and 9 mL of water by a pulse-type sonication for 10 min. Abraxane was purchased from the Taiho Pharmaceutical Co., Ltd. and was directly utilized without any modification and chemical reactions. B


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derived tumors were generated by injecting 100 μL of culture medium/Matrigel (Dow Corning, Corning, NY, USA) mixture (v/v = 1:1) containing 1 × 106 cells into the right flank of the mice. After 2 weeks, when the tumor volumes became ∼50 mm3, the mice were intraperitoneally injected every other day with 200 μL of sterilized water containing ICG−ND−SP (ICG, 7.5 mg kg−1; ND-ori, 30 mg kg−1), PTX (5 mg kg−1) in 10% Cremophor EL (Sigma-Aldrich), PTX@ICG−ND−SP (PTX, 5 mg kg−1; ICG, 7.5 mg kg−1; ND-ori, 30 mg kg−1), or 200 μL of PBS (vehicle control). The tumor formation and overall health (body weight) were monitored every other day. Further, the tumor volume was calculated using V = L × W2/2, where L and W denote the length and width of the tumor, respectively. 2.9. In Vivo Multiple Cancer Therapy. Eight-week-old nude female mice (average weight = 18 g, n = 3; BALB/cSlc-nu/nu, Japan SLC) were bilaterally implanted with Matrigel (Corning) cell culture media (1:1 volume ratio) containing the SKOV3 cells (1 × 106) via subcutaneous injections into each side of the mouse’s back. Three weeks after implantation, the mice were separated into four groups (three mice per group), and the tumors were injected with PBS (50 μL), sterilized water containing ICG−ND−SP (50 μL) (ICG, 1.88 mg kg−1; ND-ori, 7.5 mg kg−1), sterilized water containing ND-ori (50 μL) (7.5 mg kg−1), or sterilized water containing PTX@ICG− ND−SP (50 μL) (PTX, 1.25 mg kg−1; ICG, 1.88 mg kg−1; ND-ori, 7.5 mg kg−1). The tumors on the right side of the backs were only irradiated for 3 min every 3 days using the 785 nm laser at maximum power (1 W, ∼80 mW/mm2). When the tumors were considerably larger than the laser spot size (diameter ∼4 mm), we thoroughly irradiated each tumor in two locations (3 min per location). The thermographic measurements were conducted during irradiation using IR thermography (i7; FLIR, Nashua, NH, USA). During the treatment period, we recorded the tumor size for each mouse in a manner similar to that used in the in vivo antitumor assay. 2.10. In Vivo Biodistribution of ND−SPs in Tumor Model. The SKOV3 tumor-bearing mice were injected in the tail vein with sterilized water containing PTX@ICG−ND−SP (200 μL) (PTX, 5 mg kg−1; ICG, 7.5 mg kg−1; ND-ori, 30 mg kg−1). At the indicated time points, the NIR fluorescence images of the mice and major organs were collected using an IVIS Imaging Spectrum System (PerkinElmer, MA, USA) with the parameters λex = 740 nm and λem = 800 nm, and the images were analyzed using the IVIS Living Imaging 3.0 software (PerkinElmer). 2.11. Blood Tests. The CBC and biochemical parameters were investigated by Japan SLC and Oriental Yeast Co. (Tokyo, Japan). BALB/cSlc mice (female; 10 weeks; n = 5; average weight = 21 g; Japan SLC) were injected in the tail vein with sterilized water containing ICG−ND−SP (200 μL) (ICG, 7.5 mg kg−1; ND-ori, 30 mg kg−1) or PBS buffer (200 μL). The blood samples were collected from the inferior vena cava of the mouse after 7 and 28 days. 2.12. Statistical Analysis. The results are presented as mean ± standard deviation of at least three independent experiments, with “n” indicating the number of samples per group. The differences between the groups were evaluated using the Student’s t test for two groups and a two-way analysis of variance (ANOVA) for multiple groups. The asterisks ∗, ∗∗, and ∗∗∗ denote p-values of less than 0.05, 0.01, and 0.001, respectively.

The photothermal conversion efficiency of the ICG−ND−SP nanoclusters was determined using the published methods.23−25 Specifically, the efficiency was η=

hS(Tmax − TSurr) − Q Dis 1(1 − 10−A785)

(1)

where h denotes the heat transfer coefficient, S denotes the surface area of the container, and hS is obtained from eq 4 and Figure 4B. The maximum steady-state temperature Tmax of the solution of ICG− ND−SP nanoclusters was 53.8 °C, and the environmental temperature TSurr was 21.5 °C. Thus, the temperature change TMax − TSurr of the solution of ICG−ND−SP nanoclusters was 32.3 °C. The laser power I was 1 W. The absorbance A785 of the ICG−ND−SP nanoclusters at 785 nm was 1.26. The quantity QDis denotes the dissipated thermal energy accumulated from the light absorbed by the solvent and container. To obtain hS, we introduce the following dimensionless parameter θ:

θ=

T − TSurr TMax − TSurr

(2)

A sample system time constant τs can be calculated as follows: t = ‐τs ln(θ )

(3)

where τs = 132.2 s. Finally, hS =

mDc D τs

(4) −1

where mD = 0.2 g and cD = 4.2 J (g °C) . Thus, according to eq 4), hS = 6.35 mW °C−1. The quantity QDis is the dissipated thermal energy accumulated from the light absorbed by the plastic cell itself; QDis was measured independently to be 148.6 mW using a dispersible plastic cuvette cell containing distilled water. Thus, by substituting the values of each parameter into eq 1, the heat-conversion efficiency η at 785 nm for the ICG−ND−SP nanoclusters was calculated to be approximately 58%. We also calculated the 785 nm heat-conversion efficiency η for ICG and NDori to be approximately 38% and 16%, respectively. 2.6. Reactive Oxygen Species (ROS) Detection. ROS analysis was performed in a 96-well plate with black walls and a clear bottom (Thermo Fisher Scientific) using SOSG (Invitrogen). The ICG− ND−SPs in Milli-Q water (100 μL) were diluted by adding the PBS buffer solution that included SOSG. The final concentrations of ICG, ND, and SOSG in the systems were 75 μg mL−1, 300 μg mL−1, and 1 μM, respectively. The samples were further irradiated for 5 min with 1 W at 785 nm (ca. 80 mW mm−2). The control (PBS without nanocomplexes) was also measured. A fluorescence microplate reader (InfiniteF200 PRO; Tecan, Männedorf, Switzerland) was used to detect green fluorescence that occurred by the ROS generation at 485 nm excitation and 535 nm emission. 2.7. Laser-Induced Cytotoxicity of ICG−ND−SPs. To assess the cell viability, SKOV3 cells were preseeded overnight in 96-well plates at 5 × 103 cells per well. The cells were treated with 100 μL DMEM culture medium containing samples (ND-ori, ICG, ICG− ND−SP, and PTX@ICG−ND−SP) or PBS buffer and were then irradiated at maximum power (1 W, 80 mW mm−2) using a 785 nm fiber-coupled continuous-wave laser. After irradiation, the cells were washed and incubated with fresh culture medium. Further, the viability was measured using a CCK-8 kit 0 or 24 h after irradiation. The concentrations of ICG and ND were carefully adjusted to 25 and 100 μg mL−1, respectively. 2.8. In Vivo Antitumor Assay. The animal experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of AIST. Female BALB/cAJc-nu/nu mice (n = 4; 8 weeks old, average weight = 18 g) were obtained from Japan SLC (Hamamatsu, Japan) and housed in a specific pathogen-free environment. Mice bearing the SKOV3 cell-

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of FluorophoreModified ND−SPs. Our strategy for the preparation of fluorophore-modified ND−SPs is based on the covalent attachment of the fluorophore units onto the surface of NDs. In this approach, four types of fluorophores, such as 4phenylbutyric acid (PBA), boron-dipyrromethene N-succinimidyl ester (BODIPY−NHS), Alexa Fluor 568 N-succinimidyl ester (Alexa568−NHS), and indocyanine green succinimidyl ester (ICG−NHS), are bonded to the amino-functionalized ND originals (ND-ori) to generate ND−SP nanoclusters. These modified NDs contain fluorophore units located at a C


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Figure 1. Synthesis and characterization of the ICG−ND−SP. (A) Scheme of the ICG−ND−SP synthesis. Crude ND-ori was functionalized via cross-linking by a condensation reaction between the surface amino groups and the succinimidyl group of ICG−NHS, followed by sonication to promote the self-assembly of the ICG−ND−SP nanoclusters. (B) DLS size distribution of the ICG−ND−SPs. The inset denotes an image of the ICG−ND−SP aqueous solution. The concentrations of ICG and ND are approximately 0.75 and 3 mg mL−1, respectively. (C) Representative TEM images of ICG−ND−SPs. A high-magnification image is denoted in the upper right corner. (D) UV−vis/NIR absorbance spectrum of ICG− ND−SP. The concentrations of ICG and ND are 37.5 and 150 μg mL−1, respectively. (E) Fluorescence (FL) intensity of ICG−ND−SP (ICG concentration = 18.75 μg mL−1, ND concentration = 75 μg mL−1) and of ICG−NHS (ICG concentration = 2 μg mL−1). (F) Fluorescence intensity of ICG−ND−SP and of ICG−NHS at the adjusted ICG concentration (0.75 μg mL−1). The concentration of ND is 3 μg mL−1. Data are presented as mean ± (n = 4), ∗∗∗p < 0.001 (Student’s t test).

short distance above the ND skeleton, thereby allowing efficient and sufficient interactions between the ND and the fluorophore.

We initially chose ICG−NHS as the model molecule to construct the fluorophore-modified ND−SPs because of its attractive optical properties in the biologically transparent NIR D


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Figure 2. Cellular internalization and distribution of the ND−SPs. (A) Evaluation of the cytotoxicity of various ND−SPs. The U2OS cell viability was tested after 24 h of treatment. (B) Fluorescence micrographs of the U2OS cells after incubation with ICG (ICG concentration = 1.5 μg mL−1) and ICG−ND−SP (concentration of ICG and ND is 1.5 and 6 μg mL−1, respectively) for 4 h at 37 °C in 5% CO2 atmosphere. The cells were fixed with 4% PFA before observation.

region.26,27 ICG is also approved by the U.S. Food and Drug Administration (USFDA) as a clinical NIR imaging agent.28−30 The ICG−ND−SP fluorescent nanoclusters were synthesized by adding ICG−NHS to the MES buffer (pH 6.0) containing an amino-functionalized ND original (ND-ori) solution (Figure 1A). We are confident that the aforementioned protocol, which uses a considerable number of ingredients, is suitable for future clinical applications. ICG−NHS triggers the formation of SPs, which can be tentatively attributed to the repulsive Coulomb force destabilizing the electrostatic charge balance of the ND particles when fluorophores are attached onto NDs.31−33 NDs coupled with ICG−NHS precipitate from the solution because of the condensation reaction that occurs between the amine and succinimidyl ester. After being washed several times to eliminate the excess chemicals, the ND nanoclusters are observed to self-assemble upon sonication and are further redispersed in Milli-Q water. The prepared ICG− ND−SPs cause a pale green solution (see image in the inset of Figure 1B) and a well-defined size distribution with an average diameter of 50 nm in accordance with the dynamic light scattering (DLS) measurements (Figure 1B). The transmission electron microscopy (TEM) imaging reveals that the ICG− ND−SPs form a cluster structure with an average diameter of 50 nm (Figure 1C). Interestingly, characterizations using TEM and DLS denote that functionalizing NDs with different fluorophores (PBA, BODIPY−NHS, and Alexa568−NHS) produce ND−SPs having various sizes, indicating that the size of ND may be controlled simply by changing the fluorophores on the ND (Figure S1A and Figure S1B). Thermogravimetric analysis (TGA) results reveal that approximately 25 wt % of the ICG is conjugated to the ND surface (Figure S1C). ND-ori has a smooth UV−vis absorption curve without any characteristic peaks and has no fluorescence properties.9 However, the characteristic optical properties of ICG−ND− SP can be observed the absorption and fluorescence spectra because of the substituted ICG moiety (Figure 1D−F). After the absorption spectra are analyzed by curve fitting, the absorption spectrum of ICG−ND−SP, as compared to ICG, broadens and undergoes red-shifting in 500−770 (Fit Peak 1 in Figure S2A) and 780−920 nm, respectively (Fit Peak 2 Figure S2B), denoting the first hints of intramolecular electronic interactions between ICG and NDs (Figure 1D).

Further insights into the electronic communication between the ICG units and NDs in the SP nanoclusters are observed to originate from fluorescence spectrometry (Figure 1E,F).34,35 The quenching of the 800 nm ICG emission from the ICG− ND−SP indicates that the excited state of ICG is effectively deactivated by NDs via energy or electron transfer (Figure 1E).36−38 Besides, we estimate that 39 mol of ICGs are approximately conjugated with 1 mol of nanodiamond from the results of UV−vis/NIR spectra. Further, the fluorescence (FL) intensity of the ICG−ND− SP is 15-times less than that of ICG at the same ICG concentration (excitation at 750 nm, emission at 820 nm) (Figure 1F). This difference indicates that an efficient photoinduced process between ICG and ND can partially quench the dye emission.38 Similar phenomena were observed in the absorption and FL spectra of the PBA−ND−SP, BODIPY−ND−SP, and Alexa568−ND−SP (Figure S3). These results indicate that various fluorophores tightly bind onto the ND surface by covalent bonding.39 3.2. Cytotoxicity and Cellular Permeation of ND−SPs. Biocompatibility of the ND−SP nanoclusters is an important characteristic not only for controlling the cellular functions but also for future clinical and biomedical applications. Therefore, we verified the viability of the human bone osteosarcoma epithelial cells (U2OS cell line) that were preincubated with various nanoclusters at different concentrations (ND = 0, 2, 4, 8, 15, 30, and 60 μg mL−1) using a water-soluble tetrazolium-1 (WST-1) assay (Figure 2A). More than 78% of the cells remained viable after the treatment was performed for all the tested concentrations of the fluorophore-functionalized ND− SPs. In addition, we used FL microscopy to investigate the internalization and distribution of the fluorophore-functionalized ICG−ND−SPs in the U2OS cells (Figure 2B and Figure S4A). The U2OS cells were seeded in 35 mm glass-bottom dishes and were incubated with ICG−ND−SPs for 4 h. Further, the intracellular uptake of the ICG−ND−SPs was clearly observed. We also confirmed that the nanoclusters were uniformly localized in the cells. In particular, the ICG−ND− SPs exhibited a higher degree of cell permeability when compared to that exhibited by the ICGs, as demonstrated by the higher FL intensity observed from the cells incubated with E


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Figure 3. In vitro anticancer effect of PTX@ICG−ND−SP as a drug-delivery vehicle. (A) Synthesis of PTX@ICG−ND−SP via the noncovalent interaction of the PTX anticancer drug and ICG−ND−SP clusters. (B) UV−vis/NIR absorbance spectra of ICG−ND−SP before (black curve) and after (pink curve) PTX incorporation. (C) DLS analysis of the ICG−ND−SP and PTX@ICG−ND−SP size distributions. (D) Cytotoxicity evaluation of Abraxane (commercially available and FDA approved PTX-based nanomedicine), PTX@ND-ori, and PTX@ICG−ND−SP in the SKOV3 cells after 24 h of treatment. Data are presented as mean ± (n = 5), ∗∗p < 0.01 (two-way ANOVA test).

(72 nm) than that of the ICG−ND−SPs without drug molecules (ca. 50 nm), presumably because of the aggregations that arise owing to the interactions with the water-insoluble PTX molecules (Figure 3C). Regardless, PTX@ICG−ND− SPs produce highly stable dispersions in an aqueous media at least for a week because of the high drug-loading capacity of the ND−SPs (Figure S5). The cytotoxicity of PTX@ICG−ND−SP to the SKOV3 human ovarian cancer cells was evaluated by applying a WST1-based assay (Figure 3D). SKOV3 was also approved by the USFDA as a PTX-targeting cancer type.44 After 24 h, PTX@ ICG−ND−SPs are more toxic than the comparable doses of ND-ori. Notably, PTX@ICG−ND−SPs are more potent against cancer cells (approximately 10% enhancement) when compared to the nanoparticle albumin-bound PTX (Abraxane),45 which is the only commercially available nanomedicine contained in the pharmaceutical PTX. This is likely because the ND−SP nanoclusters efficiently permeate the cells because of strong interactions, which are more effective than those of Abraxane. In any case, these results demonstrate that the ND− SP nanoclusters are critical for enhancing drug efficiency. 3.4. In Vitro Multiple Cancer Elimination by Photoinduced ND−SP Encapsulating Drug. To further explore the manner in which the fluorophore-functionalized ND−SP nanoclusters can be used in advanced cancer therapy, we investigated the photothermal and photodynamic properties of the ND−SPs via NIR irradiation (Figure 4). Interestingly, the results show that NIR irradiation (785 nm) of an aqueous suspension of ICG−ND−SP leads to a significant increase in temperature over time and an increase in the concentration of ICG and ND, whereas the temperature of the control phosphate-buffered saline (PBS) buffer solution (without NDs) increases less, with the water absorbing all the NIR power (Figure 4A,B). In addition, the temperature difference ΔT produced by the NIR irradiation of ND-ori in Milli-Q water for 5 min at 1 W (ca. 80 mW mm−2) is only 16.7 °C

nanohybrids. Indeed, carbon-based nanomaterials can permeate the cells because of their morphology and strong interactions with cells.40−42 In addition, ND-ori itself does not emit strong FL, as denoted by the lack of FL intensity from the cells (Figure S4A). Meanwhile, other fluorophore-modified ND−SPs are also available for in vitro bioimaging (Figure S4B,C). Although the PBA−ND−SPs and Alexa568−ND−SPs were localized in cells, BODIPY−ND−SP permeated more into cells, which was similar to that observed in case of ICG−ND−SP. These results strongly indicate that the fluorophore-functionalized ND−SP nanoclusters denote effective and safe cellular interactions. In particular, the effective permeation of the ICG−ND−SPs and BODIPY−ND−SPs into cells would be useful as a superior drug carrier to replace conventional nanomedicines. 3.3. In Vitro Drug Efficacy. Paclitaxel (PTX) is a major mitotic inhibitor used in cancer chemotherapy and is approved by the USFDA.43 Generally, the commercially available pharmaceutical PTX must be dissolved in an organic solvent (e.g., a mixture of polyoxyethylene castor oil and ethanol) because PTX is completely insoluble in water.38 However, the residual organic solvent in pharmaceutical PTX can potentially cause severe side effects including anaphylactic shock. One of the biggest advantages of ND−SPs is their capacity to encapsulate hydrophobic molecules in the nanocavities between NDs using various interactions including hydrophobic, electrostatic, and π−π interactions or hydrogen bonding.9 Thus, we investigated the use of fluorophorefunctionalized ND−SPs as PTX drug vehicles. PTX molecules are easily incorporated into the ICG−ND− SP nanoclusters (PTX@ICG−ND−SP) by sonication without any organic solvents (Figure 3A). The UV−vis/NIR absorption (Figure 3B) spectrum of PTX@ICG−ND−SPs reveals the characteristic PTX peak, indicating the successful incorporation of the drug into the ND−SP clusters. PTX@ ICG−ND−SPs have a slightly larger hydrodynamic diameter F


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Figure 4. In vitro multiple cancer elimination activities of photoinduced ICG−ND−SPs. (A) Laser-induced increase in temperature in PBS (control), ND-ori, ICG, and ICG−ND−SP for each laser irradiation time. The concentration of ICG and ND is 0.75 and 3 mg mL−1, respectively. (B) Laser-induced temperature increase as a function of the concentration of ICG−ND−SPs for several laser irradiation times. (C) ROS generation by photoinduced PBS (control), ND-ori, ICG, and ICG−ND−SP. The concentration of ICG and ND in the samples is carefully adjusted to 75 and 300 μg mL−1, respectively. Data are presented as mean ± s.d. (n = 3), ∗∗∗p < 0.001 (Student’s t test). (D) Cell viability of SKOV3 treated by laser-irradiated PBS (control), ND-ori, ICG, and ICG−ND−SP as a function of the laser irradiation time. The cell viability was tested at (D) 0 and (E) 24 h after irradiation. The concentration of ICG and ND in the samples is carefully adjusted to 25 and 100 μg mL−1, respectively. Data are presented as mean ± s.d. (n = 3), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test of PBS). (F) Cytotoxicity evaluation of ICG−ND−SP and PTX@ICG−ND−SP in the SKOV3 cells after 24 h of treatment with and without 3 min laser irradiation. The concentrations of PTX, ICG, and ND are 10, 75, and 300 μg mL−1, respectively. Data are presented as mean ± s.d. (n = 3), ∗∗∗p < 0.001 (Student’s t test).

(Figure 4A). Similarly, for ICG, ΔT for the ICG−ND−SP nanocluster solution is greater than that for the ICG solution (Figure 4B). The photostabilities of ICG−ND−SP and ICG were also examined by comparing the UV−vis absorption profiles before and after laser irradiation. The results showed that ICG was completely decomposed after 5 min irradiation, while ICG-ND-SP only showed negligible change, indicating that the stability of ICG to light was significantly enhanced after conjugated to ND-SP (Figure S6). According to our calculations (see the Materials and Methods for more details), the photothermal conversion efficiency η of the ICG−ND−SP,

ICG, and ND-ori is approximately 58%, 38%, and 16%, respectively. These results clearly demonstrate that the photothermal effect of the ICG−ND−SP improved because of energy or electron transfer between the ICGs and NDs. In addition, η of the ICG−ND−SP is better than conventional semiconducting polymer nanoparticles,46,47 which are also candidates as promising photothermic materials because of their attractive features including low toxicity and unique optical properties. ICG is a useful optical agent for performing photodynamic therapy because it can generate reactive oxygen species (ROS) G


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Figure 5. In vivo anticancer effect of ICG−ND−SPs as a drug-delivery vehicle. (A) Schematic of in vivo antitumor experiment using ICG−ND−SP for drug delivery. (B) Average body weight and (C) relative tumor volume of different groups of mice during treatment. PBS, sterilized water dispersions of ND-ori, ICG−ND−SP, or PTX@ICG−ND−SP were intraperitoneally injected every other day. Equivalent doses of PTX (5 mg kg−1), ICG (7.5 mg kg−1), and ND (30 mg kg−1) were applied to each sample. Data are presented as mean ± s.d. (n = 4), ∗p < 0.05, ∗∗p < 0.01 (two-way ANOVA test).

when exposed to NIR light.48,49 Herein, we report the capacity of the ICG−ND−SP nanoclusters to generate ROS in addition to thermal energy when irradiated by a NIR laser (these are useful properties for performing cancer opto-therapy). ROS generation was confirmed using an FL microplate reader with a singlet oxygen sensor green (SOSG) (Figure 4C). Further, the control samples (PBS and ND-ori) generated no significant ROS. However, the FL intensities exhibited a significant increase with NIR power while using both ICG−ND−SP and ICG. We carefully adjusted the concentration of free ICG to become the same as that of the ICG attached to the NDs. However, the average laser-induced FL intensity of SOSG from ICG−ND−SP was less than that obtained from ICG, which is probably because of the competitive relaxation pathway that involves a photoinduced process through either electron or energy transfer from the ICGs to the NDs.31−33 These results indicate that the thermal energy generated by the NDs is enhanced by the good absorbing properties of the ICG molecules; upon the excitation of ICG, a part of the excitation energy is likely to transfer to the lower-energy levels of NDs and subsequently decrease by thermal conversion. Thus, the ICG-labeled ND−SPs generate both thermal energy and ROS. Accordingly, the capacity to generate thermal energy and ROS could be used to destroy the cancer cells. To verify this possibility, we irradiated the SKOV3 cells containing ICG− ND−SPs in a 96-well plate using a 1 W 785 nm fiber-coupled continuous-wave NIR laser (ca. 80 mW mm−2) with different irradiation times (Figure 4D,E). The viability of SKOV3 was measured using the CCK-8 kit at 0 h (Figure 4D) or 24 h post (Figure 4E) irradiation. Although the photoinduced ICG itself can kill the cancer cells, ICG−ND−SP exhibits the highest cancer elimination rate immediately after laser irradiation

because of its significant photothermal effect (Figure 4D). More interestingly, 3 and 5 min laser irradiations of ICG− ND−SP lead to drastic cancer elimination 24 h after irradiation when compared with that of ICG (Figure 4E). We believe that the synergetic effect of ROS and thermal energy effectively eradicates the cancer cells after 24 h of incubation. Note that no cancer cell elimination can be observed in the control samples (PBS and ND-ori) both 0 and 24 h after irradiation (Figure 4D,E). We also assessed the drug efficacy of PTX@ICG−ND−SP when exposed to the NIR laser irradiation to determine whether the laser irradiation of PTX@ICG−ND−SP improves its capacity to destroy the cancer cells. As shown in Figure 4F, more than 99% of the nonirradiated SKOV3 cells incubated with the ICG−ND−SP nanoclusters remain viable, whereas laser irradiation decreases the cell viability to 70% because of the powerful photothermal property of the ICG−ND−SP and the laser-induced generation of ROS in the ICG−ND−SPs. Laser irradiation itself does not affect the cell viability at all or the ICG−ND−SP nanoclusters themselves. The viability of the nanoclusters containing PTX was 67%. The most drastic decrease in cell viability (down to 24%) is caused by the laser irradiation of the PTX@ICG−ND−SP nanoclusters. These results clearly indicate that the laser-induced multifunctionality of the ND−SP nanoclusters, such as the photothermal effect, ROS generation, and drug release, can effectively eliminate the cancer cells. 3.5. In Vivo Multifunctional Phototherapy. Next, we used a tumor model to investigate the capacity of ICG−ND− SP for delivering the anticancer drug PTX. Because U2OS do not yield solid tumor and PTX is an FDA-approved targeted drug for ovarian cancer, SKOV3 is used for in vivo assessments. H


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Figure 6. (A) Thermographic measurement and (B) laser-induced temperature increase as a function of time for tumor on mouse’s body surface for treatment with laser-irradiated (785 nm) PBS, ND-ori, ICG−ND−SP, and PTX@ICG−ND−SP [laser power = 1 W (∼80 mW/mm2), irradiation time = 180 s]. Equivalent doses of PTX (1.25 mg kg−1), ICG (1.88 mg kg−1), and ND (7.5 mg kg−1) were applied to each sample. Measurement began with the mice body surface temperature around 33 °C. Data are presented as mean ± s.d. (n = 3), ∗∗∗p < 0.001 (two-way ANOVA test).

Mice bearing 50 mm3 SKOV3 cell-derived tumors were randomly assigned to one of four groups, and the different groups were administered by the intraperitoneal injection of the PBS, ICG−ND−SP (ICG, 7.5 mg kg−1; ND-ori, 30 mg kg−1), PTX (5 mg kg−1) in 10% Cremophor EL, or an equivalent dose of PTX@ICG−ND−SP (Figure 5A). Intraperitoneal injection was chosen for convenient and repetitive drug administrations into mice. The mice did not lose weight significantly during the treatment (Figure 5B), which demonstrated that the combination of PTX and ICG−ND− SP exhibited low systemic toxicity. After 27 days, the tumor size clearly differed in the four groups (Figure 5C). The mice treated with PTX@ICG−ND−SPs experienced superior tumor suppression over time (Figure 5C), with significant shrinking by the end of the experiment (Figure S7). In contrast, the tumors in the PBS- and ICG−ND−SP-treated groups grew rapidly, with a ∼7.5-fold increase being observed in size on day 27 (Figure 5C). The treatment with PTX alone slowed the progression of tumor, but the tumor volume still increased by 5.6-times (Figure 5C). These results indicate that the ICG−ND−SPs enhance the efficiency of PTX. The in vivo photothermal effect of ND−SP was further investigated to develop a multifunctional cancer therapeutic agent (Figure 6). Further, the body surface temperature of the mice was monitored using a thermographic infrared camera during laser irradiation after the intratumoral administration of each sample (PBS, ND-ori, ICG−ND−SP, and PTX@ICG− ND−SP). We selected intratumoral administration, which can satisfy the sample volume condition, for observing direct temperature elevation and anticancer performance by the laserinduced materials because the injected samples could surely stay in a tumor at least during laser irradiation without diffusion in body of mice by intratumoral administration. The laser-irradiated ICG−ND−SPs and PTX@ICG−ND−SP drastically increase the surface temperature (ΔT = 37 °C) of mice with irradiation time when compared with those observed in case of PBS and ND-ori because of the effective energy or

electron transfer between ICG and NDs. In fact, although laser irradiation resulted in a greater temperature increase for NDori than that for PBS, the value of ΔT for ND-ori was only 14 °C. SKOV3 cells were subcutaneously transplanted into both the left and right sides of the backs of the nude mice to investigate the in vivo photothermal, photodynamic, and anticancer chemotherapeutic effects of the laser-irradiated PTX@ICG− ND−SP. Twenty-one days after transplantation, each tumor was injected once with 50 μL of PBS, 50 μL of sterilized water containing ICG−ND (ICG, 1.88 mg kg−1; ND-ori, 7.5 mg kg−1), 50 μL of sterilized water containing ND-ori (7.5 mg kg−1), or 50 μL of sterilized water containing PTX@ICG−ND (PTX, 1.25 mg kg−1; ICG, 1.88 mg kg−1; ND-ori, 7.5 mg kg−1). After the injection, the tumors on the right sides were irradiated by a laser at 785 nm for 3 min every 3 days (Figure 7A). Further, the tumors that were transplanted on the left side of the back were not irradiated. Surprisingly, ICG−ND−SP and PTX@ICG−ND−SP exhibit clear therapeutic effects; the irradiated solid tumors are observed to completely disappear after laser irradiation (Figure 7B,C). In contrast, without laser irradiation, the tumor volumes increased over time regardless of the substance that was injected into the tumors except for PTX@ICG−ND−SP, which exhibited anticancer drug activity (Figure 7D). For the controls of PBS and ND-ori, the tumor volumes increased over time independent of the laser irradiation (Figure 7B−D). Despite the anticancer effect of ICG−ND−SP and PTX@ICG−ND−SP, the body weights of the mice continuously increased during the test period, which indicated that there were no side effects (Figure 7E). The advantage associated with the usage of nanocarriers is that they enable better targeting of the tumor because of the enhanced permeability and retention (EPR) effect and because they subsequently reduce the dose requirements and systemic toxicity when compared to those observed in the nontargeted systemic delivery of anticancer drugs.50−52 Herein, we further investigate the biological distribution of ND−SP (Figure 8). I


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Figure 7. Photothermal, photodynamic, and chemotherapeutic destruction of tumors in vivo. (A) Experiment design of the ICG−ND−SPmediated combination therapies for xenograft tumor models. (B) Photographs of mice after 7 and 14 days of intratumoral injection of PBS, ND-ori, ICG−ND−SP, and PTX@ICG−ND−SP and laser irradiation of the tumor on the right side of the mouse’s back. Black arrows denote the laser irradiation spots. (C) Relative volumes of tumors on the laser-irradiated right side of mouse’s back. The PBS, sterilized water dispersions of ND-ori, ICG−ND−SP, or PTX@ICG−ND−SP were intratumorally injected and treated with 785 nm laser irradiation [laser power = 1 W (∼80 mW/ mm2); irradiation time = 180 s]. (D) Relative volume of tumors on the left side of the mouse’s back after intratumoral injection of PBS, sterilized water dispersions of ND-ori, ICG−ND−SP, or PTX@ICG−ND−SP but no laser irradiation. Equivalent doses of PTX (1.25 mg kg−1), ICG (1.88 mg kg−1), and ND (7.5 mg kg−1) are applied to each sample. Data are presented as mean ± s.d. (n = 3), ∗p < 0.05, ∗∗p < 0.01 (two-way ANOVA test). (E) Average body weight of the mice after the multidimensional cancer therapy testing period.

The samples were intravenously injected into mice for analyzing systemic pharmacokinetics. Because of the significant NIR FL of PTX@ICG−ND−SPs, in vivo whole-body bioimaging may be performed (Figure 8A). The results denote that PTX@ICG−ND−SPs accumulate in the tumor because of the EPR effect. Figure 8A and B denote the maximum fluorescent intensity 24 h after injection. Further, the collected solid tumor is also obviously stained with bright FL from the ICG−ND−SPs (Figure 8C,D). The capacity of the ND−SPs to target tumors can clearly explain the reason because of which PTX@ICG−ND−SPs have such a potent in vivo therapeutic effect against cancer cells. In addition, we believe

that the ICG−ND−SPs would result in outstanding anticancer therapeutic and imaging agents for cancer theranostics. To further assess the safety of the ND−SPs, mice without tumors were intravenously administered sterilized water containing ICG−ND−SPs (200 μL; ICG, 7.5 mg kg−1; NDori, 30 mg kg−1) or 200 μL of PBS buffer for 1 or 4 weeks, following which we examined the blood tests (Tables S1 and S2). We selected intravenously administration because it is beneficial for one of the systemic toxicological assessments to know the direct toxicity of nanoparticles that are circulating in blood vessels after injection.53,54 No difference can be observed between the complete blood count (CBC) and the J


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Figure 8. Biological distribution of PTX@ICG−ND−SPs. (A) Fluorescence imaging of the SKOV3 tumor-bearing mice after intravenous injection of PTX@ICG−ND−SPs (PTX, 5 mg kg−1; ICG, 7.5 mg kg−1; and ND, 30 mg kg−1). Blue dashed circle denotes the location of the solid tumor. (B) Corresponding radiant efficiency of the tumor at different times after the injection. (C) Ex vivo image of the tumor and major organs at different postinjection times. (D) Quantitative radiant efficiency of the tumor and major organs 24 h after injection.

developing image-guided surgery to efficiently remove the targeted cancer cells by exploiting the optical and chemotherapeutical effects of these nanoclusters.

biochemical parameters of the mice intravenously injected with the ICG−ND−SPs and those injected with the PBS, confirming the absence of the inflammatory response and systemic side effects and underscoring the biocompatibility of the fluorophore-functionalized ND−SPs.

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ASSOCIATED CONTENT

* Supporting Information S

4. CONCLUSION In summary, we developed multifunctional SP nanoclusters by conjugating various fluorophores with NDs for multidimensional cancer theranostics. The synthesized fluorophorefunctionalized ND−SP nanoclusters exhibit an excellent drug-loading capacity, a high water dispersibility, and a low toxicity. In addition, they offer a remarkable intracellular permeation capacity, are highly biocompatible, and emit NIR FL for bioimaging. In addition, the fluorophore-functionalized ND−SPs enhance the anticancer potency of the loaded drugs and inhibit the tumor growth both in vitro and in vivo. Furthermore, the laser-irradiated ICG−ND−SPs effectively accumulate the thermal energy and generate ROS by energy or electron transfer between ICGs and NDs. They also exhibit tumor targeting, which can drastically eliminate the cancer cells in vitro and in vivo and emit NIR FL for marking solid tumors. This multifunctionality of the fluorophore-functionalized ND− SPs makes them of significant interest for developing new therapeutic and diagnostic tools for cancer treatment. Finally, the ND−SPs could prove to be considerably useful for

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00603.

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Additional figures: TEM images; DLS profile; TGA measurements; absorbance and fluorescence spectra of various ND−SPs; in vitro fluorescence bioimaging; dispersing stability of PTX@ICG−ND−SPs; photographs of tumors after examinations; blood tests (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Eijiro Miyako: 0000-0002-1157-6174 Present Address ⊥

Y.Y., X.Y., and E.M., School of Materials Science, Japan Advanced Institute of Science and Technology, 1−1 Asahidai, Nomi, Ishikawa 923-1292, Japan. K


Article

ACS Applied Bio Materials Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Daicel Corporation; a Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (A) [Grant No. 19H00857]; JSPS KAKENHI Grant-in-Aid for Scientific Research (B) [Grant No. 16H03834]; and JSPS KAKENHI Fund for the Promotion of Joint International Research (Fostering Joint International Research) [Grant No. 16KK0117].

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