PEGylated Polydopamine Nanoparticles Incorporated with

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PEGylated Polydopamine Nanoparticles Incorporated with Indocyanine Green and Doxorubicin for Magnetically-guided Multimodal Cancer Therapy Triggered by Near-infrared Light Lihong Sun, Qian Li, Lei Zhang, Zhigang Xu, Yuejun Kang, and Peng Xue ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00176 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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ACS Applied Nano Materials

PEGylated Polydopamine Nanoparticles Incorporated with Indocyanine Green and Doxorubicin for Magnetically-guided Multimodal Cancer Therapy Triggered by Near-infrared Light Lihong Sun†,‡, Qian Li†,‡, Lei Zhang§, Zhigang Xu†,‡, Yuejun Kang*,†,‡, Peng Xue*,†,‡ †

Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy,

Southwest University, Chongqing 400715, China. ‡

Chongqing Engineering Research Center for Micro-Nano Biomedical Materials and

Devices, Chongqing 400715, China. §

State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing

400716, China.

ABSTRACT Highly efficient cancer therapeutics have been extensively sought after using versatile multifunctional nanoagents, which are desirable to be biocompatible and produce synergistic effects for multimodal treatments. Herein, we propose a facile strategy to synthesize a magnetic nanosystem comprising a core of Fe3O4, a PEGylated shell of polydopamine, and loaded with Indocyanine green and doxorubicin. This system may enable combination of photothermal therapy (PTT), photodynamic therapy (PDT) and chemotherapy under magnetic guidance. The obtained nanocomposites exhibited strong superparamagnetism, facilitating cell internalization of drugs under a localized magnetic field. As a tumor-specific environmental cue, the acidic condition was able to trigger a burst mode of drug release from the nanocarrier. Experimental studies in vitro based on HeLa cells demonstrated notable effects on cell viability, apoptosis and uptake efficiency of drugs under the combination of PDT/PTT and chemotherapy. This nanosystem may work as a promising therapeutic tool for combination cancer treatments.

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Keywords Combination therapies; magnetic guidance; photothermal therapy; photodynamic therapy; chemotherapy

INTRODUCTION Conventional cancer therapies relying on a single therapeutic modality are difficult to achieve optimal efficacy during clinical applications1. Recently, therapeutic nanoagents have drawn much attention due to their potential to be multifunctionalised and realize combined treatments with minimal side effects and high efficiency2-4. These nanoplatforms are usually responsive to diverse external or internal environmental factors, such as light5, heat6, 7, ultrasound8, magnetic field9, pH conditions10 and oxidative chemicals11.

In particular, minimally invasive treatments based on light-induced

therapeutic effects have become promising therapies against cancer12, 13, mainly including photothermal therapy (PTT) and photodynamic therapy (PDT). Typically, PTT achieves the tumour destruction through rapid heat generation of photothermal agents under near infrared (NIR) light irradiation. PTT has been widely investigated and also combined with other treatments for synergistic therapeutic effects14, 15. For example, the efficacy of chemotherapy can be considerably improved by hyperthermia that enhances the cellular uptake of drugs and thereby induces a higher level of tumour cell necrosis16,

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.

Meanwhile, PDT utilizes photosensitizers to produce free radicals or reactive oxygen species (ROS), such as singlet oxygen (1O2), under laser irradiation for tumour ablation with spatiotemporal selectivity18, 19. Moreover, therapeutic nanoagents have prolonged blood circulation time and may passively accumulate at tumour site via enhanced permeability and retention (EPR) effect20, 21. Therefore, integration of chemotherapeutic drugs with PDT and PTT nanoagents may provide a multimodal solution for thorough ablation of tumour cells with minimal side effects. Among various therapeutic nanoagents, polydopamine (PDA)-based nanoparticles (NPs) have been used as a NIR-induced PTT agent in cancer therapies22, 23. PDA NPs can be synthesized through spontaneous polymerization under mild alkaline conditions, and have proved biocompatibility and biodegradability in human body23. However, PTT relying on PDA-based NPs alone is insufficient to eradicate solid tumours due to

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inevitable laser light scattering and limited adsorption24. Meanwhile, indocyanine green (ICG) is an FDA-approved dye used in bioimaging for medical diagnosis, and can also generate a photothermal effect and cytotoxic ROS simultaneously25, 26. However, several critical problems of ICG molecules, including poor photostability, rapid blood clearance, lack of specificity and liable to aggregation, have restrained practical applications of ICG in PDT for cancer therapies27, 28. Therefore, many nanocarriers have been developed to assist the delivery of ICG and thus avoid the problems of using free ICG molecules29. Recently, multimodal strategies combining PDT and PTT have been proposed to achieve an improved therapeutic index30. For this purpose, the combination of ICG and PDA represents a promising option due to their unique NIR-induced responses. For example, Hu and co-workers loaded ICG on a novel reduced graphene oxide (rGO)-PDA nanocomposite for photoacoustic imaging and PTT31, which showed a prominent suppression effect in orthotopic tumour models in vivo. On the other hand, ICG molecules are usually conjugated with PDA through physical adsorption, which may be subject to disruption due to the weak bonding and thus lead to early degradation of the composites and premature blood clearance. Moreover, the biocompatibility of these nanocomposites is still suboptimal for clinical translations. Therefore, it is essential to functionalize the PDA/ICG nanocomplex for enhanced multimodal therapeutics with controllable therapeutic effects. As a critical component for multimodal cancer treatments, chemotherapeutic drugs work in a biomolecular level to inhibit tumour cell proliferation and induce apoptosis32. However, most chemotherapeutic drugs can also damage normal cells and thus cause serious side effects. A more advantageous strategy of drug delivery is to make use of tumour-specific cues, such as the acidic33 or reductive34 tumoral microenvironment. Furthermore, magnetic guidance is desirable to rapidly transport and concentrate the drugs into tumour sites and thus minimize the adverse effects during the blood retention. For example, superparamagnetic Fe3O4 NPs have been utilized as the core structure for delivery of drugs to tumour cells under local magnetic guidance35. Moreover, surface functionalization with polyethylene glycol (PEG) has proved to be an effective approach to enhance the colloidal stability and biocompatibility of nanocarriers36. Based on these typical strategies reported previously, it is a rational design to functionalize the PDA/ICG

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complex with a magnetic Fe3O4 nanocore, a PEG shell and a chemotherapeutic drug load, which may lead to an integrated nanosystem for combination of PDT, PTT and chemotherapy under magnetic guidance.

Figure 1. (a) Design of FPPI-D NPs and (b) the scheme of combining PTT, PDT and chemotherapy under magnetic guidance for ablation of tumour cells.

In this work, we constructed a multifunctional nanoplatform comprising a PEGylated PDA nanoshell with a Fe3O4 nanocore (Fe3O4@PDA/PEG). The PDA layer was developed through oxidation-induced self-polymerization of dopamine on the surface of Fe3O4 nanocore under alkaline conditions37. The Fe3O4@PDA NPs was conjugated with thiol-polyethylene glycol (SH-PEG) covalently after spontaneous reaction between catechol groups on PDA and thiols via Michael addition38. Finally, ICG and an anti-tumour drug doxorubicin (DOX) were loaded on the Fe3O4@PDA/PEG NPs (Figure 1a) via hydrophobic interaction and π-π stacking39, 40. We hypothesize that ICG may exist in a more stable aggregates after adsorption on PDA, thus preventing undesired degradation and rapid blood clearance. The physicochemical properties of the nanoagents, 4


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including surface morphology, optical absorbance, superparamagnetism and pH-triggered drug release kinetics were characterized systematically. The cellular uptake and tumour ablation effect under combination treatments were further explored in vitro. This therapeutic nanoagent of Fe3O4@PDA/PEG/ICG-DOX, abbreviated as FPPI-D NPs, may provide an integrated system taking advantage of combination therapies benefited from laser-induced PDT/PTT and chemotherapy under magnetic guidance (Figure 1b).

EXPERIMENTAL SECTION Materials Sodium hydroxide (NaOH) was purchased from Guoyao Group Chemical Reagent Co., Ltd (China). Hydrochloric acid (HCl, 37%) was obtained from Chongqing Chuandong Chemical (Group) Co., Ltd (China). Iron (III) chloride hexahydrate (FeCl3·6H2O), NH3·H2O

(28~30%),

iron

(II)

sulfate

heptahydrate

(FeSO4·7H2O),

tris(hydroxymethyl)aminomethane (Tris, 99.0%), dopamine hydrochloride (99.0%), 1,3diphenylisobenzofuran

(DPBF),

indocyanine

green

(ICG)

and

2’,7’-

dichlorodihydrofluorescein diacetate (DCFH-DA) were provided by Sigma-Aldrich (USA). Thiol−polyethylene glycol (SH-PEG, Mw=2000Da) was acquired from Xi’an Ruixi Biological Technology Co., Ltd (China). Doxorubicin hydrochloride (DOX·HCl) was supplied with Hua Feng United Technology Reagent Co., Ltd (Beijing, China). Dulbecco’s modified eagle’s medium (DMEM), penicillin/streptomycin mixture, fetal bovine serum (FBS), phosphate buffered saline (1×PBS), TrypLETM Express enzyme, phosphate buffered saline (PBS), propidium iodide (PI), DAPI, Alexa Fluor 633 phalloidin, Annexin V-FITC/PI Apoptosis Detection Kit, PrestoBlue cell viability assay and Calcein AM purchased from Life Technologies (USA). A water purification system (Milli-Q, Molsheim, France) supplied deionized (DI) water for all experiments.

Synthesis of Fe3O4 NPs Fe3O4 NPs were produced based on a traditional alkaline precipitation method33. Briefly, FeCl3·6H2O and FeSO4·7H2O (2:1 M/M) were added into 5 mL of DI water and subsequently added with 170 µL of concentrated hydrochloric acid. The above mixture was introduced dropwise into 50 mL of NaOH solution with a concentration of 1.5 M and

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gently stirred at 80°C. After magnetic separation, the final product was obtained and rinsed three times with DI water.

Synthesis of Fe3O4@PDA magnetic NPs 100 mg Fe3O4 NPs were uniformly introduced in 50 mL of Tris buffer with a concentration of 10 mM and pH = 8.5, and then added with 200 mg dopamine monomer. This mixture was stirred continuously for 12 h. Finally, as-synthesized Fe3O4@PDA composites were separated magnetically and washed three times with DI water.

Preparation of PEGylated Fe3O4@PDA NPs As-prepared Fe3O4@PDA NPs were dispersed into 20 mL DI water (1 mg mL-1), followed by introducing 50 mg of thiolpolyethylene glycol (SH-PEG) under vigorous stirring for 5 min. Afterwards, 100 µL of NH3 ·H2O (28~30%) was added into the previous mixture and stirred for 1 h. Finally, PEGylated Fe3O4@PDA NPs were obtained through magnetic separation and washed with DI water three times.

Synthesis of FPPI-D NPs The Fe3O4@PDA/PEG/DOX-ICG (FPPI-D) NPs were synthesized following a one-step reaction. Firstly, 5 mg of ICG and 10 mg of DOX were added into 10 mL of Fe3O4@PDA/PEG solution with a concentration of 1 mg mL-1 at pH = 8, and stirred continuously for 12 h. The FPPI-D NPs were obtained by magnetic separation, and washed with DI water thoroughly to remove excess DOX and ICG. The produced FPPI-D NPs were dispersed in DI water for following experiments. The DOX loading efficiency was evaluated based on the optical absorbance at 490 nm. The ICG loading efficiency was determined by measuring the fluorescence intensity (excitation = 780 nm, emission = 810nm). Specifically, the drug loading efficiency were calculated using the following equation: Drug loading efficiency (%) = (Weight of loaded drug) / (Total weight of drug and carriers) × 100%.

Characterization of FPPI-D NPs

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The absorbance spectra were measured by a Shimadzu UV-1800 UV-vis-NIR spectrometer (Japan). The phase quantification and crystal structures were analysed using X-ray diffraction (XRD, Shimadzu XRD-7000). The surface morphology was measured using a Carl Zeiss Libra 120 transmission electron microscope (USA). The magnetization of the dried NPs was measured at 25oC using a Lakeshore 7400 magnetometer (USA). The Fourier transform infrared (FTIR) spectrum was acquired from a Perkin Elmer spectrophotometer (USA).

Drug release in vitro 2 mL of FPPI-D solution at a concentration of 1 mg mL-1 was loaded into each of three dialysis bags (MWCO = 3500), and immersed in 80 mL of buffer solutions under different pH conditions (pH = 7.4, 5.0). The releasing medium was placed in the dark at 37 °C under gently shaking. After that, 3 mL of medium was collected for analysis at designated time, and replenished with fresh medium. The free DOX released was detected using fluorescence spectroscopy (excitation at 488 nm; emission at 555 nm).

Photothermal property of FPPI-D NPs The aqueous solution of Fe3O4 NPs (100 µg mL-1), Fe3O4@PDA NPs (100 µg mL-1) and FPPI-D NPs with various concentrations were subject to 10 min of NIR irradiation (λ = 808 nm, power = 2 W). The temperature of the solutions was monitored at a time interval of 10 seconds using a digital thermometer during laser irradiation, and the thermographic images of the sample-laden cuvettes were captured with pseudo colouring using a thermal imager (Fluke, TiS55). Four irradiation cycles were performed to characterize the photothermal stability of FPPI-D NPs. Briefly, the FPPI-D NP suspensions were exposed for 10 min and then cooled down to the ambient temperature in each cycle.

Photodynamic property of FPPI-D NPs To evaluate the photodynamic properties of FPPI-D NPs, 1,3-diphenylisobenzofuran (DPBF) was used as a 1O2 probe to evaluate the quantum yield of singlet oxygen41. Briefly, 3 mL of FPPI-D NP suspension containing 20 µL of DPBF dissolved in DMSO (1.5 mg mL-1) was exposed to a NIR irradiation (λ = 808 nm, power = 2 W) for different

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period of time. Then, the absorption of DPBF at 417 nm was analysed using a spectrophotometer. The intracellular 1O2 generation was characterized using DCFH-DA fluorescent probe. Briefly, HeLa cells were seeded in 12-well plates at 1 ×105 cells per well. After 12 h of incubation, FPPI or free ICG were added to the HeLa cells at an equivalent ICG concentration of 10 µg mL-1. After further incubation for 5 h, the culture medium was replaced with 1 mL of fluorescent probe DCFH-DA (10 µM). 30 min later, the cells were exposed to NIR laser irradiation (λ = 808 nm, power = 2 W) for 5 min. Then, the cells were stained with DAPI (1 µg mL-1) for 5 min, followed by imaging with a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany).

Biocompatibility We used human umbilical vein endothelial cells (HUVECs) to evaluate the biocompatibility of DOX-free carrier Fe3O4@PDA/PEG/ICG NPs using PrestoBlue cell viability assay. Firstly, the HUVECs were cultured at 37oC for 12 h with approximately 1 ×104 cells in each well. 200 µL of fresh medium containing a range of Fe3O4@PDA/PEG/ICG NP concentrations (0 to 80 µg mL-1) was introduced to replace the old medium. After 24 h of incubation, cells were washed with PBS, followed by adding 100 µL of PrestoBlue reagent (10%, dissolve in DMEM). Meanwhile, PrestoBlue reagents of the same volume were added into the wells without seeded cells as a control group. After 1 h of reaction, and optical absorbance of the reduced PrestoBlue reagent was measured at 570 nm and 600 nm using a microplate reader (SPARK 10M, TECAN). Since the reduction rate of PrestoBlue reagent was positively correlated with the amount of viable cells, the recorded absorbance were normalized to obtain the relative cell viability following a commercial protocol.

Magnetically enhanced PTT/PDT The photothermal ablation effect of FPPI-D NPs under the guidance of magnetic field was investigated in vitro using HeLa cells. Specifically, HeLa cells were cultured with 1 mL of FPPI-D NP suspension (50 µg mL-1) at a density of 1×105 cells in each well. The cells were cultured for 2 h in the presence of a magnetic field. The cells without magnetic guidance worked as a negative control. Then, all cell samples were subject to 10 min

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exposure to a laser irradiation (λ = 808 nm, power = 2 W). After further incubation for 1 h, Calcein AM and PI were used for live and dead cell staining, respectively.

Cellular uptake and internalization A confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany) and a flow cytometer (Novocyte, ACEA, USA) were used to characterize the cellular uptake efficiency of FPPI-D NPs. For confocal microscopy, cells were incubated for 2 h with FPPI-D NPs at an equivalent DOX concentration of 10 µg mL-1. Then, the cells were washed with PBS, fixed with formalin, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA. After that, cells were treated with Alexa Fluor 633 phalloidin for 1 h and DAPI for 1 min. For flow analysis, the cells cultured under various conditions were rinsed with PBS thoroughly, trypsinized, centrifuged and redispersed in PBS for testing. The data acquired were processed using FlowJo software. The internalization of FPPI-D NPs was visualized using Prussian blue staining method. Briefly, cells were treated with FPPI-D NPs for 4 h and fixed with 4% paraformaldehyde. Subsequently, cells were stained for 25 min with a mixture containing equal fractions of 2% HCl and 2% K4[Fe(CN)6], and thoroughly washed prior to fluorescence imaging.

Cytotoxicity study For cytotoxicity assay, HeLa cells were cultured under a range of FPPI-D NP concentrations. Afterwards, all the cells were divided into four groups, including a control group without any treatment, a magnetically guided group, a laser irradiated group, and a group treated with both magnetic guidance and laser irradiation. The magnetically guided groups were treated for 4 h. For laser irradiated group, cells were treated for 5 min under a NIR laser with a wavelength of 808 nm and power of 2 W. Cell viability was evaluated using PrestoBlue assays after 24 h of incubation.

Cell apoptosis assay The cell apoptosis induced by combined PDT and PTT was analysed by flow cytometry. Briefly, HeLa cells were incubated with Fe3O4@PDA/PEG-ICG (FPPI) NPs with a range

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of concentrations. Afterwards, the cells were subject to a range of laser irradiation time or nanoagent concentrations. Specifically, for a fixed equivalent ICG concentration of 10 µg mL-1, the laser irradiation time varied from 2 min to 10 min; while for a fixed laser irradiation time of 10 min, the equivalent ICG concentrations varied from 2 µg mL-1 to 10 µg mL-1. After further incubation for 12 h, cells were trypsinized and resuspended in 1×PBS. Finally, the cells were stained with Alexa Fluor 488 annexin V-FITC and PI for 15 min prior to flow cytometry analysis.

Statistical analysis The statistical analysis was based on one-way analysis of variance (ANOVA). Statistical significance was confirmed if the probability value (p-value) was found less than 0.05 (*p

PEGylated Polydopamine Nanoparticles Incorporated with

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