A Small Molecule Nanodrug by Self-Assembly of Dual Anticancer

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A small molecule nanodrug by self-assembly of dual anticancer drugs and photosensitizer for synergistic near-infrared cancer theranostics Yan Guo, Kai Jiang, ZhiChun Shen, Guirong Zheng, Lulu Fan, Ruirui Zhao, and Jingwei Shao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14755 • Publication Date (Web): 24 Nov 2017 Downloaded from http://pubs.acs.org on November 24, 2017

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ACS Applied Materials & Interfaces

A small molecule nanodrug by self-assembly of dual anticancer drugs and photosensitizer for synergistic near-infrared cancer theranostics Authors: Yan Guo, Kai Jiang, Zhichun Shen, Guirong Zheng, Lulu Fan, Ruirui Zhao, Jingwei Shao * Affiliations: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350116, China. * Corresponding author: Jingwei Shao Address: 2 Xueyuan Road, Sunshine Technology Building, 6FL, Fuzhou University, Fuzhou, Fujian 350116, China E-mail: [email protected] (J.W. Shao); [email protected] (J.W. Shao) Telephone: + 86-13600802402

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ABSTRACT Phototherapy including photodynamic therapy (PDT) and photothermal therapy (PTT) has been attracted great attention. However, applications of some photosensitizers remain obstacle by their poor photostability. To enhance the treatment efficiency of photosensitizers and tumor theranostic effect, herein, we reported a novel carrier-free, theranostic nanodrug by self-assembly of small molecule dual anticancer drugs and photosensitizer for tumor targeting. The developed carrier-free small molecule nanodrug delivery system was formed by hydrophobic UA, PTX and amphipathic ICG associated with electrostatic, π-π stacking and hydrophobic interactions exhibiting water stability. The self-assembling of ICG on the dual anticancer nanodrug significantly enhanced water solubility of hydrophobic anticancer drugs and ICG photostability contributing to long term near-infrared (NIR) fluorescence imaging and effective chemophototherapy of tumor. The in vivo NIR fluorescence imaging showed that the theranostic nanodrug could be targeted to tumor site via a potential enhanced permeability and retention (EPR) effect proving the efficient accumulation of NPs in the tumor site. Dramatically, chemophototherapy of tumor-bearing mice in vivo almost completely suppressed tumor growth and no tumor recurrence was observed. Encouraged by its carrier-free, prominent imaging and effective therapy, the small molecule nanodrug via self-assembly will provide a promising strategy for synergistic cancer theranostics. Keywords: Carrier-free, self-assembly, NIR fluorescence imaging, chemophototherapy, cancer theranostics


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1. Introduction Cancer has become the dominating factor resulting in death all over the world.1 Chemotherapy is still the main measures of human to fight against cancer. However, most of traditional chemotherapeutic drugs as single agents haven’t met clinical requirements due to their poor solubility, multidrug resistance (MDR) and toxic side effects.2 To address these handicaps, many nanocarriers based on drug delivery system have been developed and designed, such as water-soluble polymers,

3-8

amphiphilic block copolymers,9 vesicles,10-12 lipidosomes,13-15 and inorganic materials.16-17 With the help of the nanocarriers, most anti-tumor drugs can be delivered to sites of tumor exerting effective anti-tumor activity. Nevertheless, most of nanocarriers remain severe challenges, such as potential toxicity to healthy organs, unclear metabolism and biodegradation problems because of their complicated preparation processes and added into toxic organic solvents

18-19

consequently limited

to clinical applications. In recent years, drug delivery system of carrier-free and phototherapy including photodynamic therapy (PDT) and photothermal (PTT) have been reported. Liang et. al

initiated

a

self-assembly

drug

delivery

system

with

hydrophobic

10-hydroxycamptothecin (HCPT) and water-soluble doxorubicin (DOX) exhibited a synergistic effect.20 Zhou et. al constructed a robust and smart “all-in-one” protoporphyrin-based

polymer

nanoplatform

with

a

step-by-step

multiple

stimuli-responsive function for enhancing the combined chemo-PDT.21 Cui and co-workers successfully prepared gold nanoclusters for simultaneous fluorescence imaging and targeted photodynamic therapy with photosensitizer Ce6, which showed that the fabricated nanoprobes had satisfactory PDT therapy in vitro and enhanced PDT efficacy in vivo.22 Yan and co-workers developed a nanodrug by using photosensitizer Ce6 and water-soluble small molecule doxorubicin hydrochloride (DOX) to achieve purpose of chemotherapy and phototherapy combination.23 These drug delivery systems not only improve water solubility of hydrophobic drugs but also enhance phototherapeutic effect. Thus, based on above ideas, it is impending to



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develop a simple, safe, effective and pure nanodrug delivery system that could integrate chemotherapy and phototherapy achieving theranostic capacity and improved water solubility of hydrophobic drugs. Here, we developed a novel pure, carrier-free nanodrug delivery system (ICG@UA/PTX NPs) with ursolic acid (UA), paclitaxel (PTX) and indocyanine green (ICG) based on electrostatic and π-π stacking interactions via self-assembly. UA, a natural compound that is regarded as a prominent anticancer drug in respect of cancer cells

proliferation,24 induction

of

cancer

cell

apoptosis,25

prevention

of

tumorigenesis26 and cancer metastasis prevention,27 is not clinically due to poor water solubility. PTX is one of the most clinically used chemotherapeutic drugs against many cancers

28

with a poor solubility and low bioavailability. ICG is used as an

excellent tissue penetrating agent based on the penetration depth of near-infrared light in vivo,29 which has been approved by the U.S. Food and Drug Administration (FDA) to image and diagnose human disease.30 Meanwhile, it can convert optical energy into thermal energy or generate single oxygen after absorption optical energy is used for photothermal therapy or photodynamic therapy.29, 31-32 The constructed carrier-free ICG@UA/PTX NPs had a uniform size dramatically improving the water solubility of UA and PTX. In addition, the nanodrug delivery system exhibited excellent stability in PBS (pH=7.4) (Fig. 1) without disintegration in the blood circulation resulting in efficient accumulation, long retention and NIR imaging in tumor site due to EPR effect 33 and enhanced chemophototherapy activity.

2. Experimental Section 2.1 Materials Ursolic acid (UA) was purchased from Sartorius Scientific Instruments Co. Ltd. (Beijing, China). Paclitaxel (PTX) was provided by Southern Pharmaceutical Co. Ltd (Fuzhou, China). Indocyanine green (ICG) was bought from Dalian Mellon Biological Technology Co. Ltd (Dalian, China). MTT ([3-(4,5-dimethylthiazol-2-yl)-，5-diphenyl


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tetrazolium bromide]), Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, fetal bovine serum (FBS) were provided by Life Technologies GMbH (Darmstadt, Germany). Reactive Oxygen Species Assay Kit was obtained from Wanlei Biological Technology Co. Ltd (Shenyang, China). HeLa, and HepG2 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 2.2 Preparation and characterization The nanodrug delivery system was prepared in a green and simple self-assembly approach between these three molecules. Briefly, UA and PTX respectively was dissolved in methanol. Then, UA methanol solution was mixed with PTX methanol solution. The mixture was gradually added into 2 mL deionized water (UA/PTX molar ratio =1:1, concentration respectively 500 µM in solution) by stirring with a magnetic stirrer for around 5min at room temperature. Then, 50 µL ICG methanol solution with a concentration 8 mg/mL was gradually added into the mixed solution by gently stirring with a magnetic stirrer about 1000 r/min for 3 h. Then, the methanol was remove from the solution. Finally, the ICG@UA/PTX NPs were prepared. Meanwhile, different levels of pilot were performed to scale up a test. The size (diameter, nm) and zeta potential (mv) of ICG@UA/PTX NPs were measured by dynamic light scattering (DLS) using Zetasizer Nano-S90 (Malvern Instrument, England). UV data was used to determine ICG@UA/PTX NPs by using an UV-2700 spectrophotometer (Shimadzu, Japan) at room temperature. Fluorescence intensity curves of ICG@UA/PTX NPs, UA/PTX NPs and free ICG were obtained through fluorescence spectrometer (F-7000, Hitachi, Japan). Meanwhile, the morphology of ICG@UA/PTX NPs was observed by atomic force microscope (AFM) (Multimode 8, Brooke, Germany). The encapsulation efficiency (EE) and loading efficiency (LE) was determined by centrifugation (10000r, 10 min) and dialysis method (MWCO 1000). The free drugs were determined and EE and LE were calculated according to the following formula:



EE (%) = total drug-free drug/total drug ×100% LE (%) = weight of drug in nanoparticles/total weight of nanoparticles ×100% 2.3 In vitro stability To evaluate the stability of the NPs, the size and zeta potential were tested by DLS in different time points with stored in water. Meanwhile, photos of NPs dispersed in RPMI-1640 cell media, DMEM cell media, fetal bovine serum (FBS) and phosphate buffer solution (PBS) (pH=7.4) were obtained at different store times. In addition, photostability of NPs was also investigated under NIR laser irradiation (808 nm, 1W/cm2) different temperature points. 2.4 Drug release test Dialysis method was used to conduct the drug release assay. NPs, free UA, and free PTX were put into dialysis bags respectively and immersed in PBS solutions with pH 7.4 and 5.5 contained 10% ethanol. The dialysis bags (MWCO 1000) contained different samples were maintained at magnetic stirrer with stirring 100 rpm. Samples were taken out from dialysis bags at decided time points and then equivalent volume of new release medium replacing. Then, the release rate was detected by using UV spectrophotometer. 2.5 Cell culture HeLa cells were cultured in DMEM medium contained 10% (v/v) fetal bovine serum (FBS) and HepG2 cells were cultured in RPMI 1640 contained 10% (v/v) fetal bovine serum (FBS). Two cell lines were incubated in humidified incubator at 37℃ with 5% CO2. 2.6 Cellular ROS (reactive oxygen species) detection The intracellular ROS generation was monitored with DCFH-DA by incubated cells. The HepG2 cells were seeded in a 12-well plates. After 24 h, cells were incubated with free ICG, PBS and NPs (ICG, 1.6 µg/mL) for another 24 h. Then, the


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cells were washed three times with PBS and incubated with 100 nM DCFH-DA for 30 min. Cells were washed with PBS three times and irradiated using 808 nm laser at a power of 1W/cm2 at different time points. Subsequently, the cells were trypsin digested and collected. Then, ROS was detected by microplate reader. 2.7 In vitro cytotoxicity assay The cytotoxicity of small molecule nanodrug delivery system on HepG2 and HeLa cells was tested by the MTT assay. Briefly, cells were seeded in 96-well plates at a density of 0.8 × 104 cells /well. After 24 h incubation, the cells were incubated with 100 µL fresh complete medium contained various formulations free UA, free PTX, UA+PTX mixture, free ICG (0.8, 1.6, 3.2, 6.4 and 12.8 µg/mL) and ICG@UA/PTX NPs (UA and PTX, 2, 4, 8, 16 and 32 µM) with culture medium (100 µL) for 24 or 48 h. And without any formulations group was control group. After incubation for 12 or 24 h at 37℃ in the dark, the cells of free ICG and NPs groups were irradiated using 808 nm laser (1W/cm2) for 5 min. After extra 12 or 24 h of incubation in dark, other groups were always in dark. After 24 or 48 h, the culture medium contained different formulations was removed and then cells were cultured with 100 µL MTT solution in incubation for another 4 h. Followingly, supernate was removed, and the DMSO (100 µL) was added into each well. The solutions in the wells were incubated for 10 min at room temperature through moving until observed purple precipitate. The absorbance at 490 nm was measured in each well using microplate reader. The cell viability (%) was determined at the absorbance 490 nm compared with control wells contained cell culture medium and blank contained PBS. The equation: cell viability = (OD 490 nm of the experimental group-blank contained PBS group)/ (OD 490 nm of the control group contained blank culture medium-blank contained PBS) ×100%. 2.8 In vitro cellular uptake HepG2 cells with 1 ×105 per well were seeded in 12 wells-plates with slides.



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After incubation 24 h, cells were treated by medium containing NPs and free ICG (ICG, 6.4 µg/mL). After incubation for 3 h, cells were washed three times by PBS. Then each well was added 500 µL paraformaldehyde to fix the cells for 10 min. The nuclear dye cell DAPI was used to stain cell nucleus as a positive control for 15 min, then cells were washed three times with PBS. Finally, the images of cells were acquired using confocal laser scanning microscope (TCS SP8, Leica, Germany). 2.9 In vivo tumor imaging and tumor-targeting efficiency study KM mice obtained from Wu experimental animals with subcutaneous H22 cells xenografts were used as the animal modal. When tumors reached around 150 mm3, the imaging was carried out. The mice were intravenously (i.v.) injected NPs or free ICG (10 mg ICG/kg). Then FL images were acquired on by SI Imaging Amix (USA) using 740 nm excitation wavelength and 790 nm emission wavelength at 0.5, 1, 3, 6, 12 and 24 h (exposure time: 2s). After 24 h imaging, the mice were euthanized. Tumor and the major organs were collected and subjected to image. And tumor and organs imaging parameters were the same with the mentioned above. The relative fluorescence intensity of tumors and organs was obtained using SI Imaging Amix Software. 2.10 Chemo-phototherapy efficacy of NPs in tumor-bearing mice Tumor-bearing mice inoculated subcutaneously in the right flank with H22 tumor cells (2 × 106) were used to evaluate the anti-tumor efficiency in vivo of chemotherapy, phototherapy

and

chemophototherapy.

When

the

tumor

volume

reached

approximately 100 mm3, the mice were randomly divided into 8 groups of 5 animals per group following PBS, free UA (2.67 mg/kg), free PTX (5.0 mg/kg), UA+PTX mixture (2.67 mg/kg+5.0 mg/kg), free ICG (2 mg/kg) without a NIR laser irradiation, free ICG (2.0 mg/kg) with a NIR laser irradiation, NPs (UA 2.67 mg/kg, PTX 5.0 mg/kg and ICG 2.0 mg/kg) without a NIR laser irradiation and NPs with a NIR laser irradiation. All groups were injected into the tail vein. Free ICG with a laser and NPs with a laser groups were irradiated using 808 nm laser (1W/cm2, 5 min) after 12 h


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intravenous injection. The tumor images of H22 tumor-bearing mice were obtained at the day before the tumors irradiated 7 days, 16 days and 21 days. The tumor size and body weight were measured by using a vernier caliper and electronic balance every three days after treatment. The tumor volume was calculated with the formula: volume (mm3) = length × width2/2. Afterwards, the tumor volume and weight of animals were recorded. 2.11 Statistical analysis All experiments were performed in triplicate and the acquired data are presented as mean ± SD. Statistical significance was determined using Student’s t-test. The difference was considered statistically significant as **p

A Small Molecule Nanodrug by Self-Assembly of Dual Anticancer

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