Targeted Imaging and Chemo-Phototherapy of Brain Cancer by a Multifunctional Drug Delivery System.

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Targeted Imaging and Chemo-Phototherapy of Brain Cancer by a Multifunctional Drug Delivery System Yongwei Hao, Lei Wang, Yalin Zhao, Dehui Meng, Dong Li, Haixia Li, Bingxiang Zhang, Jinjin Shi, Hongling Zhang, Zhenzhong Zhang,* Yun Zhang*

The aim of this study was to develop multifunctional poly lactide-co-glycolide (PLGA) nanoparticles with the ability to simultaneously deliver indocyanine green (ICG) and docetaxel (DTX) to the brain by surface decoration with the brain-targeting peptide angiopep-2 to achieve combined chemo-phototherapy for glioma under near-infrared (NIR) imaging. ICG was selected as a near-infrared imaging and phototherapy agent and DTX was employed as a chemotherapeutic agent. ICG and DTX were simultaneously incorporated into PLGA nanoparticles with higher stability. These nanoparticles were further decorated with angiopep-2 via the outer maleimide group of 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000]-maleinimide incorporated in the nanoparticles. The NIR image-guided chemo-phototherapy of the angiopep-2 modified PLGA/DTX/ICG nanoparticles (ANG/PLGA/DTX/ICG NPs) not only highly induced U87MG cell death in vitro, but also efficiently prolonged the life span of the brain orthotopic U87MG glioma xenograft-bearing mice in vivo. Thus, this study suggests that ANG/PLGA/DTX/ICG NPs have the potential for combinatorial chemotherapy and phototherapy for glioma. 1. Introduction Glioblastoma is the most aggressive and frequent primary malignant brain tumor in humans, and the life times of patients are quite short.[1] Treatments for gliomas usually involve combined therapies of surgery, radiotherapy, and Y. Hao, L. Wang, Y. Zhao, D. Meng, D. Li, H. Li, B. Zhang, J. Shi, H. Zhang, Prof. Z. Zhang, Prof. Y. Zhang School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, P.R. China E-mail: [email protected]; [email protected] Yongwei Hao and Lei Wang contributed equally to this work. Macromol. Biosci. 2015, 15, 1571–1585 © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

chemotherapy, depending on the location and degree of malignancy.[2] The combination of several types of therapeutic approaches with distinct mechanisms is considered to be a potential strategy for the effective treatment of cancers.[3] Chemotherapy is one of the most common therapies employed in oncology. Docetaxel (DTX), a microtubule-stabilizing taxane, is commonly used to treat systemic cancers. Despite its great efficacy in treatment of breast cancer, ovarian cancer, and non-small cell lung cancer, DTX failed to treat glioma because of the BBB restricting its access to the brain.[4] One way to overcome the limitation of DTX application in glioma therapy is to develop novel delivery systems that significantly optimize

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DOI: 10.1002/mabi.201500091

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the treatment strategy for glioma, including brain targeting, increased delivery to sites of glioma, and combination with other therapy strategy. Phototherapy, including photothermal and photodynamic therapy, has increasingly attracted much attention for the treatment of various cancers, including glioma, due to its many advantages such as low cost, highly localized and specific tumor treatment, fewer side effects compared with traditional radiation therapy and chemotherapy.[5,6] While synergistic effects were achieved when phototherapy was combined with other therapy, such as chemotherapy[7] and gene therapy,[8] there is more room for improvement in combination therapy of chemotherapy and phototherapy, especially for glioma treatment. Indocyanine green (ICG) is a tricarbocyanine dye that has been approved by the US Food and Drug Administration (FDA) for medical diagnostic application for over 50 years.[9] It has also proved to be an effective photosensitive agent by laser-excitation heat for photothermal therapy, generation reactive oxygen species for photodynamic therapy.[10–12] In addition, ICG could also serve as an imaging tracer to directly understand the distribution characteristic of drug delivery system in vivo owing to its absorptance of near-infrared light. Keller et al. demonstrated that ICG at a dose of 5 mg kg1 following 1 h NIR-light exposure is safe to the mouse brain.[13] However, poor aqueous stability and rapid clearance from the blood limit its use for long-term tracing applications.[14] This restriction could be well solved by incorporating ICG in nano-carrier delivery systems for increased stability in vitro and in vivo. In order to deliver more therapeutic agent into the glioma, brain targeting, or/and tumor targeting are usually necessary for achieving better anti-glioma effect. Of several BBB-penetrating peptides, angiopep-2, the substrate of lowdensity lipoprotein receptor-related protein-1 (LRP-1) expressed on brain capillary endothelial cells and malignant tumor cells, has been used to transport many kinds of NPs across the BBB to treat glioma.[15,16] Considering the FDA approved poly (lactic-co-glycolic acid) (PLGA) with outstanding biocompatibility and biodegradability,[17] we prepared angiopep-2 modified PLGAbased nanoplatform decorated with angiopep-2 and simultaneously loaded with DTX and ICG with multiple functions, such as chemo-phototherapy and optical imaging, thereby enhancing the inhibitory effect on glioma under NIR imaging.

2. Experimental Section 2.1. Materials PLGA (Lactide/glycolide ¼ 50:50, MW: 17 000 Da) was purchased from Jinan Daigang Biomaterial Co., Ltd. Docetaxel (DTX, purified >98%)

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was purchased from Dalian Meilun Biotech. Co., Ltd. 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DSPE-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleinimide(DSPEPEG2000-Mal) were purchased from Sigma–Aldrich, Inc. (St Louis, MO, USA). U87MG cells were obtained from Chinese Academy of Sciences Cell Bank (Catalog NO. Tchu138). The water used was pretreated with the Milli-Q Plus System (Millipore Corporation, Bedford, USA). All other chemicals were of analytical grade and used without further purification. Experimental animals were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and all the in vivo experiments were performed in accordance with the protocols evaluated and approved by the Ethical Committee of Zhengzhou University.

2.2. Preparation of ANG/PLGA/DTX/ICG NPs The NANG/PLGA/DTX/ICG NPs were the PLGA/DTX/ICG NPs without conjugation of the targeting peptide angiopep-2, which were fabricated by a modified oil-in-water single-emulsion solvent evaporation method.[18] In brief, BSA was dissolved in deionized water as the water phase. Organic phase was prepared by dissolving 40 mg PLGA, 6 mg DTX, 1 mg ICG, 2 mg DSPE-PEG2000, and 2 mg DSPE-PEG2000-Mal in 4 mL acetone/ methanol solution (2:1, V/V). Then, the 4 mL organic phase was added to the 16 mL water phase gradually under a stirring condition at room temperature. The mixture of the water phase and organic phase was stirred for 4 h at room temperature in order to ensure complete evaporation of the organic solvent. After preparation of NANG/PLGA/DTX/ICG NPs, the maleimide concentration was determined by using AmpliteTM colorimetric maleimide quantitation kit (Qianchen Biotechnology. Co., Ltd. Shanghai, China). For preparation of angiopep-2-conjugated nanoparticles (ANG/ PLGA/DTX/ICG NPs), the NANG/PLGA/DTX/ICG nanoparticles were reacted with angiopep-2 in PBS buffer (pH 7.0) for 12 h under nitrogen flow at room temperature. The outer maleimide groups of NANG/PLGA/DTX/ICG nanoparticles were specifically reacted with the thiol groups of angiopep-2 and the molar ratio of angiopep-2 to maleimide was 1:3.[19] The reaction mixture was then centrifuged at 15 000 rpm for 30 min at 4 8C and washed twice by PBS buffer (pH 7.4). The pellets were re-suspended in PBS (pH 7.4) and kept at 4 8C for further use.

2.3. Characterization of ANG/PLGA/DTX/ICG NPs 2.3.1. Size Distribution, Polydispersity Index and Zeta Potential and Morphology The nanoparticle size, polydispersity index, and zeta potential were measured by a Nano ZS-90 (Malvern instruments, Worcestershire, UK) at 25 8C. The dispersion of NPs was diluted to a suitable concentration with ultrapure water before measurement. The morphology of NPs was observed by transmission electron microscopy (TEM). For TEM, nanoparticle suspension (2 mg mL1) was stained with 2% (w/v) phosphotungstic acid, and the mixture was then dropped on a 200 mesh copper grid coated with carbon,

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dried at room temperature, and observed by a TEM instrument at 100 kV (JEOL-1230, Japan).

2.3.2. Encapsulation Efficiency The drug encapsulation efficiency was expressed as the percentage of entrapped drug with respect to the total amount of drug added. The DTX encapsulation efficiency in the DTXloaded PLGA NPs was investigated as reported.[18] An HPLC (Angilent1200, USA) equipped with a reverse-phase Intertex C18 column (Sciencehome, 5 mm, 4.6 250 mm) and an UV–Vis detector was used. The mobile phase consisted of methanol: water (73:27). The eluent was monitored at 230 nm with a flow rate of 1 mL min1. The ICG loading efficiency was evaluated to determine the amount of ICG dye encapsulated in NPs indirectly. ICG standards (0.5–10 mg mL1 of ICG solution) were freshly prepared by dispersing ICG in ultrapure water. An UV–Vis spectrophotometer (Shimadzu, Tokyo, Japan) was used to measure the absorption intensity of the ICG standard solutions and centrifugal supernatant after NPs formation, respectively. The absorption intensity of the solutions was measured at 784 nm. A calibration curve was established between different ICG concentrations and their absorption intensity at 784 nm. The calibration curve was, thus, used to measure the ICG amount present in the supernatant. Loading efficiency of ICG was quantified by an indirect method by measuring the amount of ICG present in the supernatant, as shown in the following formula. Encapsulation ratio % ¼

MICGprep MICGsupernatant 100% MICGprep

where MICG-prep is the initial amount of ICG for preparation and MICG-supernatant is the ICG recovered from the supernatant, which represents the free drug.

2.3.3. Stability of Nanoparticles and Differential Scanning Calorimetry (DSC) The absorption spectra of freely dissolved ICG and ANG/PLGA/DTX/ ICG NPs at different times were obtained using an UV–Vis spectrometer (Shimadzu, Tokyo, Japan). The physical state of drug entrapped in the NPs was characterized by DSC. A sample of 10 mg of DTX, PLGA, freezedried ANG/PLGA/DTX NPs, freeze-dried blank ANG/PLGA NPs or a mixture of freeze-dried blank ANG/PLGA NPs, DTX, and ICG was sealed in a standard aluminum pan with a lid. The temperature ramp speed was set at 10 8C min1 from 30 to 350 8C in a differential scanning calorimeter (Shimadzu DSC-60A, Kyoto, Japan). An empty aluminum pan was used as a reference.

mass 12 000 D). Then the dialysis bag was maintained in 40 mL PBS containing 1% Tween-80 and agitated at 100 rpm at 37 8C. An aliquot of 2 mL was taken from the release medium at the predetermined time points, followed by addition of the same volume of fresh medium into the system. The concentration of DTX in each sample was determined by HPLC after dilution with methanol. The HPLC assay was conducted as described above.

2.4. Cell Culture and Uptake U87MG cells were cultured at 37 8C in a humidified 5% CO2 atmosphere. MEM culture medium was supplemented with 10% Hyclone fetal bovine serum (FBS, Hyclone, Thermo Fisher Co., UT, USA), 0.1 mg mL1 streptomycin and 100 U mL1 penicillin (Beijing Dingguo Changsheng Biotech Co. Ltd., China). U87MG cells were seeded at a concentration of 2.5 105 per well in 6-well plates (Coring Inc., Corning, NY, USA). After 24 h incubation, the adherent cells were treated with different formulations at a concentration of 5 mg mL1 ICG in complete medium. The cells were trypsinized at different times and collected, washed with PBS, and then resuspended in 0.5 mL of PBS. Samples were analyzed by flow cytometry (BD FACSCalibu, San Jose, CA) with a 633 nm laser for ICG excitation. The fluorescence from ICG was measured with a FL3 filter. Normally, cultured cells without treatment of drug-loaded NPs were used as a control for background calibration. CellQUEST Pro software (BD Biosciences, San Jose, CA) was used to analyze the data. U87MG cells were seeded at a concentration of 2.5 105 per well in 6-well plates (Coring Inc., Corning, NY, USA) with a glass coverslip in each well. After 24 h incubation, the adherent cells were treated with different formulations at a concentration of 5 mg mL1 ICG in complete medium. After incubation at 37 8C for 2 h, the cells were washed with PBS, and then counterstained with 40 , 6-diamidino-2phenylindole (DAPI, 1 mg mL1) for 15 min to stain the cell nuclei, and then imaged with a laser confocal microscope (Olympus Flowview V1000, USA).

2.5. Cytotoxicity and Cell Viability Assay U87MG cells were plated in 96-well plates at a density of 5 103 per well in 0.2 mL of MEM culture medium with 10% fetal bovine serum, 0.1 mg mL1 streptomycin and 100 U mL1 penicillin, and then cultured at 37 8C for 24 h. After the culture, the growth medium was removed and the MEM medium containing blank PLGA NPs, ANG/PLGA/DTX NPs, or NANG/PLGA/DTX/ICG NPs with different drug concentrations was added, respectively. The cells were then incubated for 48 h. Cell viability was examined by the sulforhodamine B assay (SRB). All experiments were conducted in triplicate.

2.3.4. In vitro Release of DTX from Nanoparticles Since DTX is a hydrophobic drug, in vitro release of DTX from nanoparticles was performed in phosphate buffer saline (PBS) containing 1% Tween-80 at 37 8C. The surfactant Tween-80 was used to increase DTX solubility and stability in the release medium. In brief, 1 mL of nanoparticle suspension (final concentration of DTX was 1.5 mg mL1) was enclosed into a dialysis bag (Cut off


2.6. Cell Cycle and Apoptosis Assay U87MG cells were seeded in 6-well plates at a density of 2.5 105 per well. After the cells were cultured for 24 h, the different preparations in serum-free culture medium were added. The concentrations of DTX and ICG were kept at 0.5 mg mL1 and


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5 mg mL1, respectively. After the cells were treated for 4 h, the cell culture medium in each well was replaced with the same volume of serum-containing MEM medium, and the cells were further incubated for 24 h. The treated cells were fixed in chilled 70% ethanol and were labeled with PI (50 mg mL1) and RNase (100 mg mL1) for 30 min before flow cytometry. For analysis of cell apoptosis, the staining of the cells was performed with Annexin V-FITC/PI per manufacturer’s instruction. The concentrations of DTX and ICG were kept at 0.5 mg mL1 and 5 mg mL1, respectively. Samples were analyzed by flow cytometry. Cell QUEST Pro software (BD Biosciences, San Jose, CA) was used to analyze the data.

with Kodak In Vivo Imaging System FX PRO (Carestream Health, Inc., USA) with an excitation bandpass filter at 720 nm and an emission at 830 nm. After tumor implantation for a week, the glioma-bearing mice were intravenously injected with NANG/ PLGA/DTX/ICG and ANG/PLGA/DTX/ICG NPs. The doses of DTX and ICG were kept at 15 mg kg1 and 1.6 mg kg1, respectively. At the scheduled time, the mice was anesthetized and placed in the In Vivo imaging system for capturing the NIR images. For each NIR image, a corresponding X-ray image was captured to identify the location of the fluorescence. After in vivo imaging, the mice were sacrificed and the major organs were collected. Images were analyzed using the Bruker software.

2.7. Measurement of Temperature and Intracellular ROS under NIR Irradiation

2.10. In vivo Infrared Thermal Imaging

ANG/PLGA/DTX/ICG NPs and free ICG at a concentration of 10 mg mL1 ICG was irradiated by an 808 nm laser (Changchun Laser Research Center, China) at 2.5 W cm2 for a total of 5 min. After irradiation, the temperature was recorded immediately by a thermal camera (Ti 200, Fluke). U87MG cells growing in 6-well plate were exposed to ICG in the form of targeting ANG/PLGA/DTX/ICG NPs or free ICG in each well at a concentration of 10 mg mL1 for 4 h, rinsed with PBS and replaced with fresh culture medium. All cancer cells were then exposed to an 808 nm laser irradiation at 2.5 W cm2 for a total of 5 min. The temperature of plate dishes was recorded by a thermal camera (Ti200 fluke). For analysis of intracellular ROS, the redox-sensitive fluorescent probe 5-(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) was used with a reactive oxygen species assay kit (Beyotime Ins, China). U87MG cells were incubated with ICG, NANG/PLGA/DTX/ICG, or ANG/PLGA/DTX/ ICG at an equivalent concentration of 10 mg mL1 ICG for 4 h, followed by addition of 1 mg mL1 CM-H2CDFDA in complete medium and further incubation at 37 8C for 30 min. The cells were washed with phosphate buffered saline (PBS), and then exposed to an 808 nm laser for 5 min. A fluorescence microscope (Nikon, Kyoto, Japan) was used to image the cells.

2.8. Tumor Implantation BALB/c nude mice were anesthetized via subcutaneous injection of diazepam (1 mg/kg) and intraperitoneal injection of pentobarbital sodium (40 mg kg1). A burr hole was drilled 2.5 mm to the right of midline and 1 mm anterior to bregma. U87MG cells were suspended in serum-free MEM containing 1.2% carboxymethylcellulose. 10 mL of cell suspension (5 105 cells) were injected into the right striatum at a depth of 3.5 mm over 5 min.[20]

2.9. In vivo and ex vivo NIR Fluorescence Imaging In order to determine the whole body distribution of ANG/PLGA/ DTX/ICG NPs and NANG/PLGA/DTX/ICG NPs, in vivo imaging was performed by in vivo imaging system (Bruker, Germany) equipped

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The glioma-bearing nude mice were anaesthetized as described before and then intravenously injected with 200 mL of saline or ANG/PLGA/DTX/ICG in saline. The doses of DTX and ICG were kept at 15 mg kg1 and 1.6 mg kg1, respectively. After 2 h injection, the glioma areas of mice were subject to an 808 nm laser irradiation for 5 min. The temperature changes of the mice were recorded by a thermal camera (Ti 200, Fluke).

2.11. In vivo Efficacy of Drug-Loaded NPs Intracranial glioma-bearing mice were randomly assigned to five groups. On the seventh, ninth, and eleventh day after the implantation, the model mice were repeatedly treated by intravenous administration of saline, ANG/DTX NPs, NANG/ PLGA/DTX/ICG NPs, ANG/PLGA/DTX/ICG NPs, ANG/PLGA/ DTX/ICG NPs þ 808 nm laser irradiation with 1.5 W cm2 at 15 mg kg1 DTX and 1.6 mg kg1 ICG. The irradiation was performed after 2 h drug administration. The survival time of each mouse was recorded. At 25 d after the implantation, one mouse were randomly picked from each group and sacrificed to collect brain. The collected brain samples were dipped into the formalin solution for 24 h, and sliced into coronal sections using a sliding microtome. The brain slices were stained with Nissel to roughly estimate and compare the tumor size because Nissel stains glioma tissue blue. For quantitative analysis of the tumor volume, the area of tumor was defined and the tumor volume was calculated according to the formula reported in the literature:[14] Tumor volume ðmm3 Þ ¼

n X ðareaÞ 0:35 mm 1

3. Data Analysis Data were analyzed by an F-test with subsequent t-test (equal variance) for comparison between two different groups. For three or more groups, ANOVA was performed followed by Dunnett post test. Survival curves were plotted with the Kaplan–Meier method and statistical analysis of the survival period was performed using a nonparametric




log-rank test. Results were considered statistically significant at p < 0.05. All data reported are mean value S.D., unless otherwise noted.

4. Results and Discussion 4.1. Preparation and Characterization of Size and Zeta Potential Although, there are previous reports on combining phototherapy with cytotoxic agents using single nanocarriers,[21] efficient loading of two different drugs into a single carrier is a challenge because of varied hydrophobic/hydrophilic properties and possible drug–drug interactions. Furthermore, in a single system both drugs need to be compatible with the processing steps appropriate for the carrier material. Considering that ICG easily binds to proteins in blood,[22] we utilized BSA as the emulsion agent as well as DSPEPEG for preparing the PEG modified PLGA-based nanoparticles with high drug encapsulation efficiency. The basic principle of (oil/water) single-emulsion solvent evaporation is shown in Figure 1A.[23] When the organic phase containing dissolved PLGA, DTX, ICG, DSPE-PEG2000, and DSPE-PEG2000-Mal Figure 1. Schematic illustration of the ANG/PLGA/DTX/ICG nanoparticles and their was added dropwise into the aqueous characterization. (A) A schematic diagram of (O/W) single-emulsion solvent phase containing 1% BSA, a large interevaporation; (B) schematic illustration of the ANG/PLGA/DTX/ICG NPs structure; (C) zeta potential of ANG/PLGA/DTX/ICG NPs; (D) TEM image of the ANG/PLGA/DTX/ICG; face between water and ‘‘oil’’ was and (E) TEM image of the ANG/PLGA/DTX/ICG at a higher magnification. spontaneously and rapidly created, and nanoscale organic emulsion drops were formed (Phase 1). With the organic NPs apart from the physical packaging of the materials solvent spreading from the emulsion drops to the in the matrix. The content of maleimide in the nonexternal phase quickly (Phase 2), there was a reduction targeting nanoparticles without angiopep-2 was deterin the surface tension and a sharp decrease in the droplet mined to be 0.1 mmol mg1, which further confirmed diameter. At this point, the increased organic concentration at the interface would also increase the vicinity of the interaction between DSPE-PEG and PLGA. Therefore, emulsion drops, leading to flocculation of BSA (Phase 3). as expected, the outer hydrophobic groups coming from As diffusion of the organic solvent occurred in the BSA and DSPE-PEG2000 (with or without maleimide) not emulsion droplets, the concentration of drugs and other only stabilized the nanoparticles but also supplied the materials, including PLGA, DSPE-PEG2000 or DSPEtips for anchoring the targeting group, angiopep-2. PEG2000-Mal, increased and the nanoparticles gradually Schematic illustration of the ANG/PLGA/DTX/ICG NPs solidified. Due to the amphipathic property of DSPEstructure was shown in Figure 1B. Although ICG is a hydrophilic compound, PLGA and ICG were co-dissolved PEG2000 (with or without maleimide) and BSA, the surface modification of our system may be achieved by in acetone/methanol solution to form the organic phase. hydrophobic interactions between them and the PLGA After the organic phase was added to the water phase



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gradually under a stirring condition at room temperature, it is inevitable to incorporate some ICG inside the particle (PLGA materix). Furthermore, the interaction of ICG with BSA as well as DSPE-PEG would also result in the entrapment of ICG in the stabilizing shell (BSA/ DSPE-PEG200 molecules). Therefore, ICG was probably incorporated inside both the particle and the stabilizing shell. As shown in Table 1, the size of the drug-loaded nanoparticles with the targeting peptide was 211.1 4.8 nm, 199.0 2.9 nm, and 221.7 1.6 nm for ANG/PLGA/DTX NPs, ANG/PLGA/ICG NPs, and ANG/PLGA/DTX/ICG NPs, respectively. It can be seen that there was a size increase tendency for nanoparticles simultaneously encapsulating two drugs, and that other three kinds of non-targeting NPs without angiopep-2 displayed the similar size changing trend. Surface charge is an important indication for the stability of a colloidal nanoparticle system in medium. The repulsion among the nanoparticles with the same type of surface charge provides extra stability. The zeta potentials of all the kinds of NPs were negative and below 30 mv, which is beneficial to keep good stability. It could be attributed to the overall environment since the absolute value of the negative charge was from the BSA and DSPE molecules. The surprisingly high DTX encapsulation efficiency could be attributed to its hydrophilic property, resulting in easily entrapped in the PLGA nanocore when the diffusion of the organic solvent occurred in the emulsion process. While ICG was a hydrophilic drug, its encapsulation efficiency amazingly reached more than 40% because of the interaction of ICG with BSA as well as DSPE-PEG, which formed a lipid shell around the PLGA nanocore. TEM was used to image the morphology of the nanoparticles. As can be seen in the Figure 1D, the ANG/PLGA/DTX/ICG NPs were generally spherical in

shape with good monodispersity. As shown in Figure 1E, there is a low contrast ring around the PLGA core. This phenomenon may result from the interactions, such as hydrogen bonding or hydrophilic–hydrophobic interactions, between the DSPE and the backbone of the BSA molecules. What is more, the lipid shell is critical for connecting angiopep-2 to the NPs. The maleimide group in the DSPE-PEG20000-MAL was located at the terminal end of the hydrophilic PEG block; therefore, upon NPs formulation, the PEG should facilitate the presentation of the maleimide groups on the surface making them available for surface modification. As a result, angiopep-2 could be easily grafted to the NPs through the DSPE-PEG-Mal. 4.2. Appearance and Stability The appearance of ANG/PLGA/DTX NPs and blank NPs without any drug was milk-white while ANG/PLGA/ DTX/ICG NPs looked green due to the original color of ICG (Figure 2A). As seen in UV–Vis spectra of DTX, ICG, Blank NPs and ANG/PLGA/DTX (Figure 2B), an absorption peak at 230 nm was observed in both DTX and ANG/PLGA/DTX/ICG NPs. In addition, the typical absorption of ICG at 784 nm was red-shifted to 808 nm in ANG/ PLGA/DTX/ICG NPs. All the results indicated that DTX and ICG were simultaneously entrapped into the NPs and there were some molecule interactions between each other. In order to determine whether the ICG formulated in NPs had a higher stability than freely dissolved ICG, absorption spectra of ICG and ANG/PLGA/DTX/ICG NPs were measured at different time. As shown in Figure 2C and D, the absorbance value of free ICG at 784 nm after 4 d decreased by 84.0%, while that of ICG in ANG/PLGA/DTX/ICG only decreased by 10.92% even after 30 d. The changes observed in absorption spectrum of

Table 1. Physicochemical characteristics of nanoparticles (mean SD, n ¼ 3).

Groups

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Size [nm]

Zeta [mv]

PDI

Encapsulation efficiency [%] ICG

DTX

Blank NPs

161.2 2.1

22.8 1.2

0.097 0.02

/

/

NANG/PLGA/DTX

214.3 6.5

24.6 2.3

0.233 0.05

/

92.3 2.1

ANG/PLGA/DTX

211.1 4.8

20.4 1.6

0.240 0.02

/

93.2 3.4

NANG/PLGA/ICG

198.8 6.8

23.1 2.4

0.127 0.04

46.3 4.2

/

ANG/PLGA/ICG

199.0 2.9

20.5 2.6

0.115 0.03

45.6 3.8

/

NANG/PLGA/DTX/ICG

218.1 2.9

24.4 2.8

0.188 0.04

41.2 3.2

92.3 3.2

ANG/PLGA/DTX/ICG

221.7 1.7

22.2 2.1

0.233 0.02

42.3 2.4

93.1 2.2




Figure 2. Appearance of drug-loaded NPs, stability characterization and DSC curves. (A) Photos of drug-loaded NPs; (B) UV–Vis absorption spectra of different formulations; (C) UV–Vis absorption spectra of ICG at different times; (D) UV–Vis absorption spectra of ANG/PLGA/DTX/ ICG at different times; and (E) DSC curves of different formulations.

ICG with respect to time are attributed to self-aggregation between ICG monomers and dimers.[24] However, these changes were not found in ANG/PLGA/DTX/ICG NPs since ICG monomers were encapsulated into PLGA NPs, and no self-aggregation occurred. Furthermore, DSC was implemented to investigate the state of DTX and ICG in NPs. As shown in Figure 2E, ICG had a melting point around 52.70 8C presented as an exothermic peak, which could be attributed to crystallization. Besides, a melting point around 240 8C presented as endothermic peak was attributed to decomposition. DTX had a melting point around 97.54 8C presented as an exothermic peak. However, the obvious decrease of the melting point peaks of DTX and ICG in ANG/PLGA/DTX/ICG NPs may result from the encapsulation process since the peaks could be seen in the mixture of blank ANG/PLGA nanoparticle, DTX and ICG. Therefore, it can be deduced


that DTX and ICG in the NPs might be converted from the crystalline phase to the amorphous phase. In other words, the encapsulation process might interfere with the formation of DTX and ICG crystals, which was beneficial to keep the drugs’ stability in NPs. These results strongly supported that the ICG formulated in NPs had a high stability compared to freely dissolved ICG. 4.3. In vitro Release To investigate the release feature of entrapped drug from nanoparticles, we selected DTX instead of ICG for in vitro release experiment duo to the released ICG was unstable under the release medium. As seen in Figure 3, DTX release from ANG/PLGA/DTX was sustained over 200 h. In contrast, the release of free DTX was very fast in the Tween-80 contained medium, indicating that


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Figure 3. Release profiles of DTX from ANG/PLGA/DTX NPs and DTX solution.

drug-loaded NPs could diminish the burst release of the drug. 4.4. Cellular Uptake Based on the auto-fluorescence feature of ICG, the effects of the ANG-targeting ligand on the cellular uptake and intracellular distribution of PLGA-based nanocarriers in U87MG cells were analyzed using both flowcytometry and CLSM. As shown in Figure 4, quantitative flow cytometric analysis showed that the cellular uptake of ANG/PLGA/DTX/ICG (targeted) at 0.5 and 2 h was 1.98 and 4.98 times of that of NANG/PLGA/DTX/ICG (non-targeted), respectively, while there was no significant difference at 5 h, which may be due to the saturation of ICG in cells after a longer time incubation with the drug system. DTX has to reach the nucleus because it destroys the cancer cell by binding to microtubules and inhibiting microtubule depolymerization to free tubulin.[25] Therefore, the specific distribution of drug system was conducted by using CLSM. After incubation with NANG/PLGA/DTX/ICG or ANG/PLGA/DTX/ICG for 2 h (Figure 5A and B), we observed that ICG fluorescence signals were distributed inside cells, indicating the efficient uptake of NANG/PLGA/DTX/ICG. What is more, the red fluorescence signals inside cells exhibited much higher, clearly suggesting that more ICG molecules were delivered into cells. Furthermore, the colocalization of ICG and DAPI signals in the overlaid images indicated that more ICG molecules penetrated into nuclei after ANG/PLGA/DTX/ICG treatment. Because ICG and DTX were loaded simultaneously into the delivery system, it was reasonable to infer that DTX was also efficiently delivered into the nuclei. In contrast, less ICG molecules accumulated inside nuclei in the free ICG group (Figure 5B). Therefore, the prepared angiopep-2 modified

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Figure 4. Flow cytometry histogram and bar graph representations of ICG-positive cells percentages. (A) Flow cytometry analysis of the endocytosis of U87MG cells incubated with NANG/PLGA/ DTX/ICG NPs for different times; (B) flow cytometry analysis of the endocytosis of U87MG cells incubated with ANG/PLGA/DTX/ICG NPs for different times; and (C) bar graph representations of ICGpositive cells percentages of NANG/PLGA/DTX/ICG NPs and ANG/ PLGA/DTX/ICG NPs.




system with an 808 nm laser irradiation showed higher inhibition efficiency than the system without irradiation. These results indicated that the delivery system could deliver more drugs into tumor cells with the help of angiopep-2 and enhance the inhibitory effect on U87MG cells, especially when the system was exposed to an 808 nm laser irradiation. 4.6. Cell Cycle and Apoptosis Analysis In order to study whether the enhanced inhibition rate caused by the co-delivery system under 808 nm laser irradiation correlated with cell cycle progression, a cell cycle determination was conducted by flow cytometry. Compared with the control group (Figure 7A), U87MG cells treated with blank NPs showed the least alteration Figure 5. Intracellular distribution of ICG. Nucleus was stained blue by DAPI as a control (1), and it was overlapped with the fluorescence of ICG (2), and the overlaid in the cell cycle phase distribution images were displayed in the panel (3) to show the intracellular distribution of ICG (Figure 7B), suggesting good biocompatieither formulated in ANG/PLGA/DTX/ICG NPs (A) or NANG/PLGA/DTX/ICG NPs (B), bility. In contrast, DTX or DTX-loaded NPs and free ICG (C). caused dramatic changes in cell-cycle profiles. The G2/M phase of U87MG cells was significantly increased after the treatment with DTX (Figure 7C) or ANG/PLGA/DTX (Figure 7D) drug-loaded PLGA nanoparticles exhibited strong ability to bind to and enter U87MG cells, which may duo to the for 24 h compared with control group with a ratio of 17.63 3.1%, 82.7 1.9%, and 84.6 4.1% for control, DTX receptor-mediated endocytosis between the angiopep-2 and ANG/PLGA/DTX, respectively, indicating that DTX and LPR1 expressed on the U87MG cells. caused the cell-cycle arrest at G2/M phase. As shown in 4.5. Cell Viabilities Biocompatibility is an important concern when it comes to the development of nanomaterials for biomedical application. Prior to application of ANG/PLGA NPs, the in vitro cytotoxicity of the system was assessed by a traditional SRB assay. Figure 6A showed the in vitro cell viability of U87MG cells exposed to blank ANG/PLGA NPs at a concentration ranging from 1.56 to 25 mg mL1 for 24 h. The cell viabilities were higher than 90% for U87MG cells, even at the high concentration of 25 mg mL1. These data suggested that ANG/PLGA NPs did not show significant cytotoxicity at all dosages tested. Good biocompatibility and low cytotoxicity implied that ANG/PLGA NPs could serve as a multifunctional drug delivery system for simultaneous NIR imaging and combinatorial chemo-phototherapy of cancer. The cell growth inhibitory effects of drug-loaded NPs on U87MG cells were evaluated at 48 h. In general, the cell viability decreased when the drug concentration increased (Figure 6B). ANG/PLGA/DTX/ICG NPs exhibited higher inhibitory effect than NANG/PLGA/DTX/ICG NPs (P < 0.05) at 48 h. It was also found that DTX and ICG co-delivery


Figure 6. Cell viabilities of blank ANG/PLGA NPs (A) at 24 h and different formulations (B) at 48 h.


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Figure 7. Cell cycle analysis. (A) Control; (B) blank drug system; (C) DTX; (D) ANG/PLGA/DTX NPs; (E) ANG/PLGA/ICG NPs; (F) ANG/PLGA/ICG NPs with 808 nm irradiation; (G) ANG/PLGA/DTX/ICG NPs; and (H) ANG/PLGA/DTX/ICG NPs with 808 nm irradiation.

Figure 7 E and F, treatment with ICG induced more cells to enter the sub-G0/G1 phase of the cell cycle. The sub-G0/G1 phase of the cell cycle of ANG/PLGA/ICG NPs and ANG/PLGA/ICG NPs with irradiation was around 13.8 1.2% and 22.08 2.1, respectively, indicating that irradiation could enhance the sub-G0/G1 arrest effect of ICG. Thus, as expected, the combinational effect of ICG and DTX on U87MG cells induced both sub-G0/G1 and G2/M arrest. More importantly, after being treated with both ICG and DTX loaded NPs, much more cell cycle arrest effect was achieved for ANG/PLGA/DTX/ICG with irradiation than without irradiation. We also measured the induction of apoptosis by different formulations. As can be seen from Figure 8, compared to the control group, drug-loaded NPs induced cell apoptosis obviously, especially for the co-delivery system under an 808 nm laser irradiation. Compared to the non-targeting codelivery system (NANG/PLGA/DTX/ICG NPs) of DTX and

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ICG under the same laser irradiation, the targeting codelivery system (ANG/PLGA/DTX/ICG NPs) of DTX and ICG without or with an 808 nm irradiation induced more severe cell apoptosis (P < 0.05) with a apoptosis rate of 14.5 2.1% and 28.6 1.8%, respectively. All the results confirmed not only the combinational antitumor effect with the help of angiopep-2, but also the enhanced effect under the 808 nm laser irradiation. 4.7. Measurement of Temperature and Intracellular ROS Under NIR Irradiation An infrared thermal imaging camera was employed to record the temperature changes of the ANG/PLGA/DTX/ICG under laser irradiation. As shown in Figure 9A, after an 808 nm laser irradiation for 5 min at 1.5 W cm2, the maximum temperature of the ANG/PLGA/DTX/ICG NPs and free ICG solution reached 52.9 and 55.6 8C, respectively,




Figure 8. Cell apoptosis analysis. (A) Induction of apoptosis on U87MG cells by different treatments; (B) Quantitative apoptotic rate of each group. (a) Control; (b) ANG/PLGA/DTX NPs; (c) NANG/PLGA/ICG NPs; (d) NANG/PLGA/ICG NPs with an 808 nm laser irradiation; (e) NANG/ PLGA/DTX/ICG NPs; (f) NANG/PLGA/DTX/ICG NPs with an 808 nm laser irradiation; (g) ANG/PLGA/DTX/ICG NPs; (h) ANG/PLGA/DTX/ICG NPs with an 808 nm laser irradiation. ( P < 0.05).

indicating that there is no obvious influence on heat production after drug entrapment, but the PBS under the same treatment only increased to 24.0 8C. In addition, the temperature of the cell culture dishes growing the U87MG cells exposed to the treatment of ICG, NANG/PLGA/DTX/ ICG, and ANG/PLGA/DTX/ICG followed by an 808 nm irradiation was recorded. As shown in Figure 9B, although 808 nm irradiation alone without addition of any drug could also increase the temperature of the control dish to 40 8C, the cell culture dishes treated by ICG, NANG/PLGA/ DTX/ICG, and ANG/PLGA/DTX/ICG showed a striking temperature increase by 7.1, 12.5, and 21.2 8C, respectively, revealing that the ANG/PLGA/DTX/ICG was an effective phototherapy agent in damaging tumor cells. Figure 9C shows the fluorescence micrographs of U87MG cells incubated with the H2DCFDA probe after treatment without or with ICG and ICG-loaded NPs (ICG concentration was 5 mg mL1) for 2 h. We observed little green fluorescence in cells without addition of drug but strong


fluorescence evenly distributed throughout cells treated with ICG-loaded NPs, especially for ANG/PLGA/DTX/ICG NPs, suggesting that generation of intracellular ROS was induced by ICG under the 808 nm irradiation. This result is consistent with the literature reports.[26] The entrapment of ICG in nanoparticles did not change the property of ICG. Therefore, ICG-loaded NPs was cytotoxic, especially under the 808 nm irradiation, and could be used as a cancer therapeutic photosensitizer. 4.8. In vivo and ex vivo Imaging In order to determine the potential use of this kind of nanoplatforms as fluorescence imaging contrast agents, in vivo biodistribution tests were performed. As shown in Figure 10, the targeting nanoparticles were observed to be extensively retained at the brain area, especially at 2 h (Figure 10B), as denoted by the intense fluorescence intensity compared with that of NANG/PLGA/DTX/ICG


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Figure 9. Infrared thermal maps and intracellular ROS determination. (A) Infrared thermal maps of centrifuge tubes with PBS, free ICG or ANG/PLGA/DTX/ICG NPs. The maps were captured at 5 min after continuous irradiation by an 808 nm laser. (B) Infrared thermal maps of cell culture dishes growing with U87MG cells exposed to PBS, free ICG or ANG/PLGA/DTX/ICG NPs. The maps were captured at 5 min after continuous irradiation by an 808 nm laser. (C) Fluorescence microscopic images of U87MG cells treated with

NPs. In addition, ex vivo fluorescence evaluation of dissected major organs at 4 h post-injection (Figure 10D) also confirmed the brain accumulation of ANG/PLGA/DTX/ ICG NPs duo to the combination of an enhanced permeation and retention effect (EPR effect) and a receptor-mediated endocytosis process compared to the NANG/PLGA/DTX/ICG NPs.

laser irradiation (Figure 11D), we finally chose 30 s as the irradiation time because too high temperature may result in undesirable side effect to animals. In comparison, the glioma area of the mice administered with saline under the

4.9. In vivo Infrared Thermal Imaging and Anti-Glioma Therapy It was reported that photothermal ablation of brain tumor was effective and safe.[27] Therefore, an 808 nm laser irradiation was used to laser the tumor area for ICG phototherapy and chemotherapy with delivered DTX was also applied (Figure 11A). When the mice were i.v. injected with ANG/PLGA/DTX/ICG NPs, the temperature of their glioma rapidly increased from 29.9 (Figure 11B) to 35.2 8C (Figure 11C) within 30 s of laser irradiation. Previous researches reported that efficient tumor cell destruction would be induced when the temperature of tumor focus increased by 6 8C.[28,29] Although, the temperature would continue to increase to 43.5 8C after 60 s of

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Figure 10. In vivo and ex vivo NIR fluorescence imaging. (A) Representative in vivo fluorescent images of glioblastoma-bearing nude mice following i.v. administration of NANG/PLGA/DTX/ICG NPs (left) and ANG/PLGA/DTX/ICG NPs (right) at 1 h (A), 2 h (B), 4 h (C); (B) Ex vivo imaging of major organs dissected from mice 4 h post-injection.




Figure 11. The process of irradiation treatment (A), the surface temperature of U87 MG glioma-bearing mice at different times (B: 0 s; C: 30 s; D: 60 s.) under an 808 nm NIR laser irradiation, and survival curves of U87MG glioma-bearing mice (E).

under an 808 nm laser irradiation resulted in significantly (p < 0.05) longer median survival (Table 2). The anti-glioblastoma effect of co-delivery was also confirmed by glioma volume reduction under Nissl staining. Figure 12A–E showed the glioma size after three times of treatment with different formulations. Without drug treatment (the control), the glioma grew rapidly. It was noted that the NANG/PLGA/DTX/ICG group also displayed the similar tumor size compared to the control group duo to the enhanced permeability and retention effect. The two groups that were treated with ANG/ PLGA/DTX and ANG/PLGA/DTX/ICG had smaller tumor volumes than the control group. In contrast, the group treated with ANG/PLGA/DTX/ICG with an 808 nm irradiation displayed significant tumor reduction due to the synergistic effects from DTX and phototherapy generated by the ICG with an 808 nm irradiation. To account for the different glioma size, we utilized the tumor inhibition rate instead of dissection of the glioma

same irradiation condition displayed little temperature change. Therefore, the results strongly suggested that when mice were treated with ANG/PLGA/DTX/ICG NPs followed by an 808 nm laser irradiation, tumors would possibly obtain enough heat energy to be destructed, while treated with saline it would be insufficient to achieve tumor destruction. To test the combinational anti-glioma effect of DTX and phototherapy generated by ICG under an 808 nm laser irradiation in vivo, we administrated the glioma-bearing mice with the co-delivery system as well as other kinds of formulations at the early stage of intracranial glioblastoma (7 d post implantation) to facilitate brain drug transport. The survival time of each group was recorded (Figure 11E). The combinational effect of co-delivery of DTX and phototherapy generated by ICG in vivo was illustrated by the prolonged survival (37.3 d). Compared to saline (23.2 d) or ANG/PLGA/DTX/ICG (34.0 d), co-delivery of DTX and ICG

Table 2. Median survival time of mice bearing intracranial U87MG glioma treated with different drug systems.

Treatment

P value

Number of mice

Median survival time

Standard error

Saline

6

23.2

3.1

NANG/PLGA/DTX/ICG

6

24.3

1.7

>0.05a)

ANG/PLGA/DTX

6

33.3

0.6

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