Polydopamine-Decorated Orlistat-Loaded Hollow Capsules with an

Apr 23, 2019 - Orlistat, an FDA-approved antiobesity drug, has recently been shown to have anticancer effects. However, orlistat is extremely hydropho...
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Polydopamine decorated orlistat-loaded hollow capsules with enhanced cytotoxicity against cancer cell lines Xiaqing Zhou, Tzu-lan Chang, Shuang Chen, Tianchi Liu, Haoyu Wang, and Jun F. Liang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00116 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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Molecular Pharmaceutics

Polydopamine decorated orlistat-loaded hollow capsules with enhanced cytotoxicity against cancer cell lines Xiaqing Zhou, Tzu-Lan Chang, Shuang Chen, Tianchi Liu, Haoyu Wang, Jun F. Liang* Department of Chemistry and Chemical Biology, Charles V. Schaefer School of Engineering and Sciences, Stevens Institute of Technology, Hoboken, New Jersey 07030, USA *Corresponding author E-mail address: [email protected]; Phone number: (+1)2012165640

Abstract: Orlistat, an FDA approved anti-obesity drug, has recently been shown to have anti-cancer effects. However, orlistat is extremely hydrophobic with low absorption. Therefore, new approaches are needed to effectively deliver orlistat for cancer therapy. Herein, we developed a fast and simple method to use polydopamine coated hollow capsule (PHC) as a drug nanocarrier for enhancing the therapeutic effects of orlistat. Orlistat-loaded PHC had an average size of 200 nm, which was characterized by using dynamic light scattering and scanning electron microscope. Furthermore, the polydopamine layer provided excellent control of orlistat release because it was extremely sensitive to pH values. The cellular uptake and cytotoxicity experiments were performed to show that orlistat packaged in PHC could be endocytosed into cells and then significantly improved the cytotoxic activity against cancer cell lines in a short time compared with free orlistat. Moreover, dynamic study of cell membrane lysis was performed by stained with the LIVE/DEAD kit to demonstrate the cancer killing mechanism. The size of the cell surface area has also been proven to be a key parameter which affected drug efficacy. Taken all together, these results present that orlistatloaded PHC is a very promising formula for cancer treatments. 1

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Keywords: Orlistat, polydopamine, nanoparticle, hollow capsule, cancer therapy, controlled release

1. Introduction Orlistat (Xenical), an anti-obesity drug approved by the US Food and Drug Administration, inhibits gastric and pancreatic lipases when taken orally.1 Treatment with orlistat combined with healthy lifestyle intervention exhibits more weight loss and better glycemic control compared with lifestyle changes alone, especially in overweight and obese patients.2 However, until 1994, Kridel et al. found that in addition to controlling weight, orlistat also has the anticancer ability because it is an irreversible inhibitor of Fatty acid synthase (FASN) by binding to the thioesterase domain of the enzyme.3 FASN is a 272 kDa cytosolic multifunctional enzyme, which is overexpressed in many types of malignancies, but has low expression in all normal tissue cells, suggesting that the inhibition of FASN might be considered as a therapeutic target in patients with cancer.4-6 Several experimental studies demonstrated that orlistat displayed excellent anticancer activity on different types of cancers. For example, orlistat could reduce proliferation and enhance apoptosis in human pancreatic cancer cells;7 orlistat could inhibit the growth of HT-29/tk-luc human colorectal carcinoma by arresting cells at G1 stage;8 orlistat could also inhibit the growth of PC-3 tumors in nude mice.9 Although orlistat exerts an anticancer effect in both humans’ and animals’ cancer cells, it is established on the foundation of very high concentration, because the drug is extremely hydrophobic.10 Due to orlistat’s high hydrophobicity and limited absorption, it is important to achieve an improvement in drug delivery efficiency and prevention of 2

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Molecular Pharmaceutics

negative effects through effectively delivering orlistat to tumor sites. In this respect, the use of nanoparticle (NP)-based drug delivery carrier has generated considerable interest because of its advantages of small size, high stability, high carrier capacity, and controllable drug release.11 Recently, Hill et al. reported the development of orlistat NP, in which orlistat was loaded into hydrophobic regions derived from hyaluronic acid.12 Loading orlistat into the drug delivery carrier allowed it to decrease the survival rate of human prostate and breast cancer cell lines to 55% and 57%, respectively. In comparison, free orlistat reduced the survival rate of these two cell lines to 71% and 79%. In addition, Shah et al. explored the use of PLGA-PEG-NP as a delivery system to improve the antitumor effect of orlistat for triple-negative breast cancer therapy.13 In SK-BR-3 cell line, the IC50 for free orlistat treatment of 4.70 μM decreased to 1.10 μM for orlistat NP treatment at 48 h. These orlistat formulations significantly improved the cytotoxic activity of orlistat compared with free orlistat. However, there were still some issues that needed to be addressed. First, the orlistatloaded NP synthesis was time-consuming, and the preparation procedures were very complicated. Second, particle sizes were too big to be absorbed, and the blood had cleared many of them before they reached the target position. As a result, most of the drugs were lost during body circulation. Third, drug-release from NP was uncontrollable. In order to overcome the above drawbacks, we developed a polydopamine (PDA) coated hollow capsule (PHC) as a drug nanocarrier for facilitating delivery of active orlistat to tumor cells. The advantage of this carrier was simple and time-saving; only 3

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water, oil, and OH- were involved.14 After dissolving orlistat in the oil solution, the oilin-water emulsion was obtained by the intense sonication method, thereby wrapping orlistat into the oil droplets. This emulsion was then used as a template for dopamine polymerization, which allowed the hydrophobic orlistat to be loaded into the resulting PHC (scheme 1). PDA is the product of dopamine self-polymerization that gives place to synthetic melanin similar to natural eumelanin.15,

16

Recently, PDA has attracted

great attention due to its optical property, multifunctional biological capabilities, and strong adhesion strength. Additionally, PDA has also been widely used in the fields of sensing discipline, environment, energy, and biomedical fields.17-20 PDA coating can be applied as a quality gatekeeper for oil droplet surfaces because they are highly sensitive to pH values.21-23 With PDA layer, orlistat was encapsulated and protected in the NPs under the physiological pH conditions and released under acidic pH conditions. This provided an opportunity for controlled drug release, ensuring that the drug reached the target cancer cells efficiently. Moreover, the PDA coating allowed nanoparticles to bind with specific proteins on the surface of cell membranes and increased the chance that nanoparticles would be endocytosed into cells. Ding et al. demonstrated that PDA coated NPs could be efficiently internalized into Hela cells through three pathways, which were, Caveolae, Arf6, and Rab34.24 More importantly, H2O2 would be generated during the dopamine polymerization process. The formation of H2O2 constituted a potential source of toxic reactive oxygen species (ROS), leading to apoptotic cellular responses.25 Therefore, we predicted that PDA coated nanoparticles could have a good synergistic effect with fatty acid synthase inhibitors, orlistat, to exhibit strong 4

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Molecular Pharmaceutics

anticancer activity, especially to drug-resistant cancers. The physicochemical properties of orlistat-loaded PHC and orlistat loading efficiency were analyzed. In addition, we detected the anti-proliferative and apoptotic effects of orlistat-loaded PHC on seven different cell lines (six cancer cell lines and one normal cell line). The results indicated that orlistat packaged in PHC distinctly increased its biological efficiency and cytotoxicity.

2. Materials and Methods 2.1. Materials and cell cultures. Orlistat powders (Purity>98%), dopamine hydrochloride powders (Purity>99%), sodium hydroxide pellets (ACS reagent, > 97.0%), nile red, and octane (anhydrous >99%) were supplied by Sigma Aldrich Company (St. Louis, MO, USA). Whatman nucleopore track-etched polycarbonate membranes were obtained from Whatman Company (Pittsburgh, PA, USA). LIVE/DEAD cell staining kit was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Cells and cell culture related products were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from SigmaAldrich Company (St. Louis, MO, U.S.A) and used as received. Deionized water (D.I. water) and 0.10 M phosphate buffer were used for solution preparation. The MDA-MB-231, MCF7, PC-3, VCaP, Hela, A549 cells were maintained in DMEM high glucose, DMEM low glucose, RPMI-1640, DMEM high glucose, MEM, F-12K medium, respectively. All the above cell culture mediums were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin.26 Normal human breast cell 5

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MCF 10A was cultured in DMEM/F-12 medium containing 1% penicillin/ streptomycin, epidermal growth factor (EGF) (20 ng/ml), 5% horse serum, hydrocortisone (0.5 mg/ml), cholera toxin (100 ng/ml) and insulin (10 μg/ml). All cell lines were maintained at 37 °C with 5% CO2 in a humid atmosphere.

2.2. Synthesis of orlistat-loaded PHC by oil in water emulsion method. The orlistat powder was dissolved in octane to prepare a 25 mg/mL orlistat stock solution. In a typical experiment, 0.4 mL of octane, which contains orlistat, and 0.6 mL of the aqueous solution of NaOH (0.02 M) were gradually added to 9.8 mL of D.I. water. Homogenization of the mixed solution was carried out by intense sonication under 300 rpm stirring at room temperature for 30 mins. Afterward, a hand-driven mini-extruder containing two 1 mL syringes was used for uniforming particle size.27 Extrusions were achieved using two 200 nm track-etch membranes together without any internal membrane support. The oil in water emulsions were drawn into the syringes and pushed back and forth through the track-etch membranes several times. Subsequently, dopamine (0.5 mg / mL) was added to form an emulsion. As the pH of the emulsion was adjusted to 8.0, it was then shaken for 1 h. The polymerization step was terminated by adjusting the pH back to 7.4. A schematic of the nanoparticle preparation process is shown in Scheme 1. Nile-red loaded PHC were prepared following the same procedure of orlistat-loaded PHC, where hydrophobic dye nile-red (2 mM) was dissolved in octane solution before mixing with the aqueous phase. 6

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Molecular Pharmaceutics

Scheme 1. Schematic illustration of the step-by-step preparation of orlistat-loaded PHC.

2.3. Characterization of orlistat-loaded PHC. Scanning electron microscope (SEM) was conducted to observe the size and morphology of orlistat-loaded PHC. To be specific, a small volume (about 30 μL) of orlistat nanoparticles were prepared on the silicon wafer and air-dried with argon. The specimen was sputter-coated with a 1.5 nm thick Au layer and micrographs were acquired with an Auriga Modular Cross Beam workstation (Carl Zeiss, Inc.) at 3.00 kV. The particle size and zeta potential of orlistat nanoparticles were measured by dynamic light scattering (DLS) using Zetasizer (Nano ZS 90, Malvern Ltd., UK). All the DLS results were averaged from three measurements, and the same calculation was repeated six times. In order to prove that PDA was successfully coated on the surface of the oil droplet, ultraviolet-induced fluorescence of PDA was conducted as described before.28 In brief, prepared orlistat-loaded PHC was induced upon ultraviolet A (UVA) at 365 nm for 5 mins, and then a drop of sample solution was placed in the middle of a clean microscope slide and a coverslip was gently lowered over the sample at an angle allowing the liquid 7

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to spread out between the two pieces of glass. The fluorescence microscopy images were taken under a Nikon Eclipse 80i epifluorescence microscope.

2.4. Drug loading capacity. A fluorescence microplate reader (Synergy HT, BioTek Instruments, Inc., Vermont, U.S.A) was used to measure the drug encapsulation efficiency (DEE). DEE (wt%) = (Amount of loaded nile red) / (Initial amount of nile red) × 100% Briefly, after obtaining the nile red-loaded PHC, the oil and water phases were separated by gentle centrifugation at 1000 rpm for 5 mins to measure the concentration of free nile red, which was not packaged in the nanoparticles but dispersed in water. 1 mL of the solution from the aqueous phase was mixed with 10 mL of octane solution to extract free nile red from water to octane. The fluorescence microplate reader (Excitation wavelength: 485 nm, Emission wavelength: 570 nm) was used to measure the concentration of free nile red in the aqueous phase so that the amount of nile red encapsulated within the nanoparticles could be calculated. The standard curve of nile red was prepared by dissolving nile red in octane with various concentrations. (Figure S2A). The properties of nile red-loaded PHC were evaluated by a Nikon Eclipse 80i epifluorescence microscope.

2.5. In vitro orlistat release. Nile red was used as a model of lipophilic drug: orlistat, and in vitro release studies of nile red from PHC were performed using the dialysis method.29 Specifically, nile red-loaded PHC solution (1 mL) was placed into a dialysis bag (MWCO=3500 Da) immersed in 20 mL of release medium (pH 5.0, 6.0 or 7.4) and 8

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stirred at 37 °C. Then, 1 mL of the sample was taken from the release medium at predetermined time intervals (from 0.5 to 55 hours) and replaced with the same volume of fresh medium. This 1 mL sample solution was then mixed with 10 mL of octane solution to extract free nile red from water to octane. The cumulative amount of nile red released from nile red-loaded PHC in different buffer solutions was monitored by a fluorescence microplate reader (Emission wavelength: 570 nm).

2.6. In vitro cytotoxicity test. To evaluate the cytotoxicity of different groups: orlistatloaded PHC, empty PHC, and free orlistat, both cancer cell and normal cell viabilities were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded at the same density of 1.0×104 cells/well in 100 μL growth medium in 96-well plates and grown for 6 (not fully spread) or 18 h (fully spread). Then, the cells were added with different concentrations of drugs for 4 h and 24 h by using untreated cells as blank. Cell viability was determined after 4 h of incubation by dissolving crystallized MTT with 10% sodium dodecyl sulfate (SDS) solution containing 5% isopropanol and 0.1% HCl. Then, the absorbance of the solution was measured at 570 nm by using μQuant microplate spectrometer (BioTek Instruments, Inc., Vermont, U.S.A). The IC50 values, which were determined at the half-maximal inhibitory concentration, were calculated by CalcuSyn software (Biosoft, Cambridge, UK).

2.7. Kinetic studies of orlistat-loaded PHC/free orlistat induced cell membrane 9

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damage using LIVE/DEAD kit. Freshly trypsinized A549 and MDA-MB-231 cells were separately seeded in a 6-well plate (2.0×105 cells/well) and cultured in F-12K and DMEM high glucose mediums, respectively. Prior to the assay, cell were triple rinsed with PBS and treated with 50 µg/mL orlistat-loaded PHC and free orlistat dissolved in DMSO as control, respectively. The cell viability was examined with LIVE/DEAD staining kit at room temperature according to the manufacturer's instructions (LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells).30 Briefly, the MDAMB-231 and A549 cells after treatment with the drug for different times were incubated with 2 μM calcein acetoxymethyl ester (Calcein AM, 0.05%) and 0.5 μM ethidium homodimer-1 (EthD-1, 0.2%) for 10 mins in a dark place. Viable cells were fluorescently stained green by Calcein AM; however, dead cells were fluorescently stained red by EthD-1 as a result of membranolysis. Fluorescent images were captured using the Nikon Eclipse 80i epifluorescence microscope. The integrity of the cell membrane in the fluorescent images was evaluated by quantifying the percentage of green pixels out of the total colored pixels.

2.8. SEM analysis of orlistat-loaded PHC and free orlistat-treated cells. MCF7 and MDA-MB-231 cells (2.0×105 cells/well) were seeded on collagen-coated silicon wafers (1.0 × 1.0 cm) for different times (6/18 h), respectively. Cells seeded on silicon wafers were triple rinsed with PBS, then exposed to 50 µg/mL orlistat-loaded PHC and free orlistat as a control for 120 mins. Afterward, cells were soaked with 4% paraformaldehyde (PFA) for 30 mins, and then triple rinsed with PBS. After fixation, 10

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Molecular Pharmaceutics

cells were incubated in ethanol with gradually increased concentrations (50%, 70%, 80%, 90%, and 100%). Then, the cells were coated with gold and imaged using SEM.

2.9. Cellular uptake. A549 cells were incubated with nile red-loaded PHC (equivalent orlistat concentration of 25 µg/mL) for different times (0, 0.5, 1, 2 and 4 h) at 37 ℃, rinsed with cold PBS (pH=7.4) for three times, and soaked in 4% PFA for 20 mins. After fixation, the cells were rinsed with PBS, stained with 4’,6-diamidino-2phenylindole (DAPI) for 20 mins. Afterward, the cells were washed with PBS three times again and examined with a Nikon Eclipse 80i epifluorescence microscope. Additionally, cellular uptake efficiency was evaluated on PC3, VCaP, MDA-MB-231, MCF7, A549, and Hela cells. Briefly, cells’ suspension (2.0×104 cells/well) were added in 24-well plates and cultured at 37 °C. The cells were incubated until 80-90% confluence was observed prior to use, and they were exposed to free nile red and nile red-loaded PHC with the same orlistat concentration of 12.5 µg/mL for 2 h. The medium was then removed and the cells were washed three times with cold PBS (pH=7.4). The addition of 1 mL DMSO to each well and 10 mins incubation resulted in the extraction of intracellular nile red. A homogenized solution was obtained after centrifugation at 10,000 rpm for 10 mins. The intracellular nile red intensity was quantitatively measured at Emission wavelength of 635 nm and Excitation wavelength of 545 nm. The regression parameters obtained from the calibration curve (Figure S2B) were used to find out the concentration of nile red in each sample.

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3. Results and Discussion 3.1. Synthesis and characterization of orlistat-loaded PHC. The orlistat-loaded PHC was fabricated by using a quick sonication method based on octane/water emulsion in which orlistat was packaged into the oil droplet (Scheme 1). Then, PDA was selected to modify the surface of oil droplets containing orlistat. Under weak basic condition (pH=8.0), the dopamine catechol was oxidized to quinone in the presence of oxygen as an oxidizing agent, which reacted with other catechols and quinones to form PDA. A layer of PDA then wrapped tightly on the surface of the oil droplet.31 When dopamine was added, the suspension gradually turned light dark, indicating successful polymerization of dopamine. Afterward, 0.1 M HCL was gradually added into the solution to change the pH back to 7.4, and terminated the polymerization process. The particle size and surface potential of the nanoparticles played important roles in cellular uptake, drug release, and in vivo pharmacokinetics.32, 33 As shown in Table S1, the obtained orlistat-loaded PHC had a mean particle size of 246.2±6.7 nm and polydispersity index (PDI) value of 0.126 at pH 7.4 condition. A small PDI value (