pH- and GSH-Sensitive Hyaluronic Acid-MP Conjugate Micelles for

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pH- and GSH-sensitive hyaluronic acid-MP conjugate micelles for intracellular delivery of doxorubicin to colon cancer cells and cancer stem cells Tilahun Ayane Debele, Lu-Yi Yu, Cheng-Sheng Yang, Yao-An Shen, and Chun-Liang Lo Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00856 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Biomacromolecules

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pH- and GSH-sensitive hyaluronic acid-MP

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conjugate micelles for intracellular delivery of

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doxorubicin to colon cancer cells and cancer stem

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cells

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Tilahun Ayane Debele†, Lu-Yi Yu†, Cheng-Sheng Yang†, Yao-An Shen⊥ and Chun-Liang Lo†,∥,

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‡,*

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† Department of Biomedical Engineering, National Yang-Ming University, Taipei 112, Taiwan

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∥Center for Advanced Pharmaceutics and Drug Delivery Research, National Yang-Ming

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University, Taipei 112, Taiwan

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‡

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University, Taipei 112, Taiwan

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⊥Department of Pathology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins

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Medical Institutions, Baltimore, MD 21205, USA

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[*] To whom correspondence and reprint requests should be addressed.

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Prof. C.L. Lo, E-mail: [email protected], Fax: + 886-2-2821-0847

Biomedical Engineering Research and Development Center (BERDC), National Yang-Ming

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1 ACS Paragon Plus Environment

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Abstract

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A dual-sensitive polymeric drug conjugate (HA-SS-MP) was synthesized by conjugating

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hydrophobic 6-mercaptopurine (MP) to thiolated hyaluronic acid (HA) as the carrier and ligand

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to deliver doxorubicin (Dox) to parental colon cancer and colon cancer stem cells. Due to the

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amphiphilic nature of HA-SS-MP, it was self-assembled in the aqueous media and Dox was

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physically encapsulated in the core of the micelles. The particle size and the zeta potential of the

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micelle were analyzed by dynamic light scattering (DLS), and the morphology of the micelle

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was investigated using transmission electron microscopy (TEM). Drug release study results

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revealed more drug release at pH 5.0 in the presence of GSH than that at the physiological pH

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value. The cytotoxicity of free Dox was slightly greater than that of Dox-loaded HA-SS-MP

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micelles. In vitro cytotoxicity of HA-SS-MP and Dox-loaded HA-SS-MP micelles was greater

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for cancer stem cells (HCT116-CSCs) than for parental HCT116 colon cancer cells and L929

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normal fibroblast cells. The MTT and flow cytometry results confirmed that free HA

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competitively inhibited Dox-loaded HA-SS-MP uptake. Similarly, flow cytometry result

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revealed anti-CD44 antibody competitively inhibited cellular uptake of Rhodamine B

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isothiocyanate conjugated micelles which strengthen the synthesized micelle is up taken via

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CD44 receptor. Cell cycle analysis revealed that free drugs and Dox-loaded HA-SS-MP arrested

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parental HCT116 colon cancer cells at the S phase, while cell arrest was observed at the G0G1

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phase in HCT116-CSCs. In addition, ex vivo biodistribution study showed that Dox-loaded HA-

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SS-MP micelles were accumulated more in the tumor region than in any other organ.

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Furthermore, the in vivo results revealed that Dox-loaded HA-SS-MP micelles exhibited more

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therapeutic efficacy than the free drugs in inhibiting tumor growth in BALB/C nude mice.

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Overall, the results suggested that the synthesized micelles could be promising as a stimuli 2 ACS Paragon Plus Environment

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carrier and ligand for delivering Dox to colon cancer cells and also to eradicate colon cancer

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stem cells.

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Keywords: Hyaluronic acid, Dual sensitive micelle, CD44, colon cancer stem cell

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Cancer has become one of the most dreadful public health problems, causing deaths throughout

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the world.1, 2 Among the various cancers, colon cancer is the third leading cause of cancer-related

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mortality in men and women worldwide.3 Although there are several treatment strategies, colon

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cancer is rarely cured completely due to its recurrence properties.4 Several researchers have

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demonstrated that this is due to cancer stem cells (a small population of cancer cells) that possess

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self-renewal and differentiation abilities to generate a new tumor population.5 Cancer stem cells

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(CSCs) are highly resistant to standard conventional chemotherapy due to different cellular

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processes, including rapid drug efflux, enhanced repair of damaged DNA, overexpression of

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detoxifying enzymes, and antiapoptotic proteins.6-8 Hence, several researchers have

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demonstrated that designing specific therapies targeting CSCs can enhance the survival of

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patients with cancer, primarily those with drug resistance.9,

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targeting CSCs is the use of nanocarriers that can regulate drug delivery and release the drug

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more efficiently.11 Furthermore, nanocarriers can effectively inhibit multiple types of CSCs,

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including colon cancer stem cells, by targeting specific markers (ALDH, CD44, CD90, and

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CD133) using targeting ligands on the surface of nanocarriers, which can enhance selectivity and

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internalization of drugs by CSCs.12-14 Thus, it is very essential to design an agent that can

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promote the killing of both parental cancer cells and CSCs.

Introduction

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One of the recent strategies



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In recent years, nano-sized ( 97%), 6-mercaptopurine, doxorubicin (Dox),

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glutathione (GSH, 99%, Roche), anhydrous dimethyl sulfoxide (DMSO), 1-ethyl-3-(3-

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dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and

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dialysis bag are all purchased from Sigma-Aldrich (St Louis, MO, USA). All other reagents were

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analytical grade and used without further purification. Water used in all the experiments was

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purified using an AquaMax-Ultra water purification system (Younglin Co., Anyang, Korea).

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Synthesis of HA-SS-MP conjugates

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HA-SS-MP conjugates were prepared in two separate steps. In the first step, thiolated HA were

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synthesized using L-cysteine by simple modification of our previous methods.33 Briefly, 500 mg

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of HA (1.32 mmol of carboxyl group) were dissolved in the reagent grade distilled water (20 mL,

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pH 5.3) and an excess amount of EDC/NHS (2.64 mmol each) were added to activate the

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carboxyl group of HA and stirred for 6 h at room temperature. Then, 5 mL aqueous solution of

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L-cysteine (2.64 mmol) was added to form thiolated HA. After 24 h reaction, resulting HA-Cys

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conjugates were extensively dialyzed against deionized (D.I.) water (MWCO 6-8kDa) for 48 h,

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within 6 h D.I. exchange, followed by deep freeze and lyophilization. The HA-Cys conjugates

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(sponge-like products) were stored at 4

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200 mg of HA-Cys conjugates were dissolved in 25 mL of co-solvents (reagent grade

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water/DMSO) and three times molar excess of 6-mercaptopurine (MP) (3.96 mmol) was added

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to form HA-SS-MP. Then, 5 mL of H2O2 (2mM) was added drop wise and the reactants solution

until used for the characterization. On the second step,


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were stirred at room temperature for 24 h. HA-SS-MP prodrug was acquired by extensive

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dialysis (MWCO 6-8KDa) against DMSO, water/DMSO (1:1, v/v) for 24 h and then D.I. water

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for the other 48 h followed by deep freezing and lyophilization. 1H-NMR and FTIR were used to

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confirm thiolated HA and HA-SS-MP formation. In addition, GPC analysis was performed using

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a shodex OHpack SB-803 column (8mm ID x 300mm L and 9 µm, particle size) to confirm

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thiolated HA and HA-SS-MP formation. A serious of dextrane polysaccharide (5.2, 11.6, 23.8,

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48.6 and 148kDa) was used to calculate the molecular weight of polymers. The analysis was

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carried out using acetonitrile and distilled water (20:80v/v ratio, respectively) as a mobile phase

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at a flow rate of 1 mL/min.

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Doxorubicin loading and preparation of HA-SS-MP micelles.

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Doxorubicin (Dox) loaded HA-SS-MP micelles were prepared using dialysis (solvent exchange)

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method. Briefly, 20 mg HA-SS-MP, 10mg Dox, and 30µL triethylamine were dissolved in 20

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mL of DMSO/dH2O. The solution was stirred at room temperature for 4 h and then dialyzed

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against DMSO for 24 h to remove unloaded Dox and also against D.I water for the other 48 h in

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order to remove the DMSO and to obtain the Dox-loaded HA-SS-MP micelles. The micellar

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size, zeta potential, and polydispersity index were determined by DLS measurements using a

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Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm and

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a scattering angle of 90°. All measurements were performed at 25

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from the average of three measurements. The morphology of micelles was analyzed using

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transmission electron microscopy (TEM, with a JEOLJEM-2000EX instrument at a voltage of

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200 kV). Samples were prepared by drop-casting HA-SS-MP and Dox loaded HA-SS-MP

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solutions onto carbon-coated copper grids and then air-drying at room temperature.

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Phosphotungstate (PTA) was used as the negative staining via sequential two-droplet methods.

and the data were obtained



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The critical micelle concentration (CMC) of the micelles was determined by a fluorescent

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spectroscopic method with pyrene as a fluorescence probe.

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Determination of drug loading of micelles.

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To determine the drug loading and encapsulation efficiency, 5mg of the freeze-dried micelles

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were dispersed in 5 mL of PBS (at pH 5.0 in the presence of 10mM GSH) and shook in a water

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bath at 37

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spectroscopy at 480 nm using calibration curve from 0.78125 to 400 µg/mL (0.78125, 1.5625,

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3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL). The percentage of drug loading (DL) and

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encapsulation efficiency (EE) were calculated by the following equations:

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DL ( wt %) =

for 6 h to release loaded drugs. The Dox concentration was measured by UV-VIS

weight of Dox in the micelle ×100 weight of the Dox − loaded − micelle

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GSH-Responsiveness of Dox loaded HA-SS-MP micelles

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The GSH responsive behavior of Dox loaded HA-SS-MP micelles was investigated in the

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presence of GSH using DLS measurement. In brief, an equal volume of GSH (10mM) was

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mixed with Dox loaded HA-SS-MP micelles solution at different pH values. Then, the solution

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was incubated on a shaking table at 37

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DLS at a different time of intervals and change in the morphology also investigated using TEM.

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and the size change of the micelles was measured by

In vitro release of Dox and MP from the micelles

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In vitro release behaviors of Dox and MP were investigated in PBS buffer (100 mM, pH 7.4 and

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5.0) in the presence and absence of 10 mM GSH solution. Briefly, 2 mL of Dox loaded HA-SS-

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MP solution was transferred to dialysis tubing (MWCO = 6-8 KDa). The dialysis tubing was


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immersed into 10 mL of PBS buffers (100 mM, pH 7.4 and 5.0) in the presence and absence of

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10 mM GSH solution and kept on a shaking table at 37 . At predetermined time intervals, 2 mL

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of external buffer solution was withdrawn and replaced with 2 mL of fresh PBS or PBS with 10

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mM GSH. The amount of released Dox and MP were determined by UV−VIS spectroscopy at

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480 nm and HPLC, respectively. The HPLC experiments were conducted on the HPLC, C18 (5

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µm, 150 mm × 4.6 mm i.d) instruments with UV detection at 325 nm. For 6-MP, the mobile

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phase was a mixture of acetonitrile and methanol at a volume ratio of 20:80 and flow rate of 1.0

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mL/min. 20 µL of the sample were injected and the drug was detected at 325 nm.

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In vitro stability and protein adsorption of Dox loaded HA-SS-MP micelles

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The in vitro stability and protein adsorption of Dox loaded HA-SS-MP were investigated in PBS

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and protein solutions.34, 35 Briefly, 2 mL (2 mg/mL) of Dox-loaded HA-SS-MP micelle solution

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were mixed with equal volume of each solution (i.e. 10% (v/v) FBS, 100 mM PBS at pH 7.4 and

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1wt% fibrinogen). The suspensions were constantly mixed using a sonicator at 37

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size was measured using DLS at 0, 6, 24 and 48 h.

and particle

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In vitro cytotoxicity of thiolated HA, HA-SS-MP, Free Dox, Free MP and Dox loaded

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HA-SS-MP

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The in vitro cytotoxicity of thiolated HA, HA-SS-MP, Dox loaded HA-SS-MP, free MP and free

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Dox were evaluated against parental colon cancer cells (HCT116-PC) and L929 fibroblast

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normal cells using MTT assay. Briefly, all the cells were seeded at a density of 2.5

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per well in 96-well plates and incubated for 24 h to allow cell attachment. The cells were then

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incubated in a concentration gradient at 37

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concentration of: 500, 250, 125, 62.5 and 31.25 µg/mL for each), free Dox and free MP (at Dox

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and MP concentration of 25, 12.5, 6.25, 3.125 and 1.5625µg/mL) and Dox-loaded HA-SS-MP

104 cells

with thiolated HA and HA-SS-MP (at



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(at Dox concentration of 25, 12.5, 6.25, 3.125 and 1.5625µg/mL for each). After 48 h incubation,

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the cells were washed with PBS and new medium were added with 20 µl MTT (5 mg/m1) and

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further incubated for 4 h. The medium in each well was removed and 100 µl DMSO was added

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to dissolve the internalized purple formazan crystals. The absorbance was measured at the test

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wavelength (570 nm) and reference wavelength (633 nm) using an enzyme-linked

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immunosorbent assay (ELISA) reader (Power Wave XS, BioTek, Winooski, VT). Similarly Dox

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loaded HA-SS-MP cytotoxicity was investigated for cancer stem cells (HCT116-CSCs). The

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relative cell viability (%) was calculated using the following equation: absorbance of test cells − absorbance of reference ×100 absorbance of controlled cells − absorbance of reference

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Cell viability (%) =

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Cellular uptake and competitive inhibition study

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Parental HCT116 cells and HCT116-CSCs were grown in McCoy’s 5A and DMEM modified

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medium, respectively, at 37

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were seeded and grown in a six well plate at a density of 4 × 104 cells/well and incubated at 37

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under 5% CO2 for 24 h. Cellular uptake competitive inhibition of Dox loaded HA-SS-MP and

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HA-SS-MP-Rhodamine B isothiocyanate were investigated using flow cytometry after

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incubating for 1 h at 37

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respectively. Fluorescence histograms were recorded with a BD FACS Calibur flow cytometer

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(Becton Dickinson, USA) and analyzed using Cell Quest software.

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and 5% CO2 according to reported protocol. Briefly, the cells

in the presence and absence of free HA and anti-CD44 antibody

Cell cycle analysis

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Parental cancer cells and cancer stem cells were treated with HA-SS-MP (500µg/mL), free MP,

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free Dox and Dox-MP (at the Dox and MP concentrations of 20 and 2 µg/ml, respectively) and


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Dox loaded HA-SS-MP micelles (at Dox concentration of 20µg/mL) for cell cycle analysis.

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Briefly, cells (1104 cells/well) were seeded on 6-well plates and co-cultured with HA-SS-MP,

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free MP, free Dox, Dox-MP and Dox loaded HA-SS-MP for 24 h. After incubation, cells were

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washed three times by PBS, collected by trypsinization, fixed on iced ethanol for 24 h and then

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centrifuged at 1000 rpm for 5 min to separate suspension and cell pellets. The cell pellets were

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finely washed by PBS and stained by PI/Triton X-100/ RNAase solution for 30 min at 37

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FACS Calibur flow cytometer and ModFit LT software were applied for cell cycle analysis.

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. BD

In vivo biodistribution study

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Female BALB/C nude mice (6 - 8 weeks of age, 20 g) were obtained from National Yang-Ming

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University, and kept in filter-topped cages with standard rodent chow and water available ad

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libitum, with a 12 h light/dark cycle. The experiment protocol was approved by the ethical

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committee of animal experiment of National Yang-Ming University. Briefly, 2.5 × 106 HCT116

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colon cancer cells were subcutaneous seeded by inoculation in the front armpit of BALB/C nude

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mice. After 4 weak of tumor growth (> 500 mm3), Rhodamine B isothiocyanate conjugated Dox-

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HA-SS-MP (100 µL) was intravenously (i.v.) injected into the tail vein of tumor bearing

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BALB/C nude mice. The ex vivo fluorescent scans were performed by scarifying mice after 18 h

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of post-injection using the IVIS imaging system series 50 with an excitation band filter at 563

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nm and an emission at 581 nm.

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Ex vivo anti-tumor efficacy study

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Similar to the above protocols, after 3 weeks of tumor growth (~200 mm3), Dox-loaded HA-SS-

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MP or free drugs (Dox + MP) were injected via the lateral tail vein at a dose of 5 mg/kg (for each

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Dox and MP) every 7 days for a total of 3 injections. Vernier caliper was used to measure the

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tumor sizes every 2 days and volume was measured using the formula: V = L × W ×W × 0.5, 11 ACS Paragon Plus Environment


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wherein L and W represent the tumor dimension at the longest and widest point, respectively.

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Relative tumor volumes were calculated as V/V0 (V0-volume when the treatment was initiated).

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In addition, relative body weights percent were calculated as W/W0×100 (W0-body weight when

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the treatment was initiated).

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Statistical analysis

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Data are represented as the mean ± standard deviation. All results are representative of at least

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three sets of independent experiments with samples performed in duplicate or triplicate in each

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experiment. The significances of the differences were determined using Student's t-test, one

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tailed, for each paired experiment. *p-value < 0.05 was considered statistically significant in all

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cases. *p< 0.05, ** p< 0.01, *** p< 0.001.

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Results and discussion

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Synthesis and characterization of HA-SS-MP conjugates

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Today, smart nanocarrier systems have been synthesized for targeted cancer therapy to overcome

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the adverse effects of drugs on normal cells and to enhance its therapeutic efficacy in the region

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of interest. In this study, HA-based dual-sensitive polymeric drug conjugates were designed and

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synthesized with the properties of both targeting and drug delivery against colon cancer cells and

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cancer stem cells. The HA shell permits a dual function, both as a ligand (i.e., targeting CD44 as

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a ligand to enhance receptor-mediated endocytosis to internalize nanoparticles) and allowing the

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conjugation of L-Cys via amide linkage as a spacer to conjugate the hydrophobic MP to form the

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polymer–drug conjugate. The synthesized HA-SS-MP forms a micelle by a solvent exchange

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method due to its amphiphilic nature, which in turn enables the encapsulation of Dox anticancer

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drugs in its hydrophobic core. The micelles can respond in the cancer cell cytoplasm due to

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excess GSH levels. Similarly, a micelle can respond to a low pH value due to the pKa value of

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the conjugated MP to thiolated HA. Hence, during disulfide cleavage that occurs after the cell

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internalization of the micelles into endosomes, Dox and MP are released. As shown in Scheme

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1, the activated HA carboxyl groups were covalently coupled with the amine groups of L-Cys

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through the amide linkage to form thiolated HA based on the EDC/NHS chemical approach.

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Then, the HA-SS-MP conjugates were synthesized via the disulfide linkage between thiolated

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HA and 6-MP in the presence of H2O2. The synthesis of HA-SS-MP conjugates was confirmed

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via 1H-NMR (Figure 1), FTIR (Figures S1a and S1b) spectra, and GPC (Figure S2). The 1H-

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NMR spectrum (Figure 1) demonstrated the presence of both HA and L-Cys, which indicated

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the successful formation of thiolated HA. The

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monosaccharide units of HA (H-1 from

1

H-NMR peaks of the major repeating

D-glucuronic

acid and H-1 from N-acetyl-D13

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glucosamine) were found at 4.35 and 4.55 ppm, respectively. The other protons of HA

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disaccharide units (H-2, H-3, H-4, H-5, and H-6) were found at approximately 3.2–3.9 ppm.36

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Furthermore, the proton signal of the acetyl groups of HA was found at 1.9 ppm. After thiolation

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with L-Cys, the new 1H-NMR spectrum was observed at 2.8–3.08 ppm in the HA–Cys conjugate.

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The degree of thiolation was determined by considering the ratio of the integral of the HA

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methyl proton peak (at ~1.9 ppm) to that of the protons of L-Cys (at ~2.85 ppm). The degree of

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thiolation was calculated as 95.52%. Similarly, the 1H-NMR spectrum of HA-SS-MP showed the

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successful conjugation of MP to thiolated HA via a disulfide bond. The 1H-NMR spectrum of

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HA-SS-MP showed signals at 8.3–8.41 ppm, which could be assigned to the MP moiety. The

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degree of substitution was approximately 13.33%, as calculated by comparing the integrals of

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signals at 7.3–7.41 ppm for MP and 2.85 ppm for methylene protons of L-Cys. Moreover, the

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degree of substitution was confirmed by UV-VIS measurement (about 12%) after excess GSH

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treatment at 333 nm, which was almost similar to the 1H-NMR results. Similar to previous

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reports, the percentage of MP conjugation with thiolated HA was low, which is due to the low

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reactivity and the low thiol content of MP.29, 37 The chemical structures of the thiolated HA and

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HA-SS-MP were further confirmed using FTIR (Figures S1a and S1b). All major characteristic

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peaks of HA–Cys and HA-SS-MP were observed within the range of 3600–500 cm−1. Several

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major peaks were observed at 3325 cm−1 (HA hydroxyl stretching), 2883–2915 cm−1 (C-H

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stretching), 1620 cm−1 (amide carbonyl, C=O), 1549 cm−1 (amide, N-H), 1350–1400 cm−1

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(aromatic, C-N and C=C stretching of MP), and 1027 cm−1 (C-H bending), all of which

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confirmed the formation of thiolated HA and HA-SS-MP conjugates. In addition, the synthesis

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of HA–Cys and HA-SS-MP conjugates was further confirmed by GPC analysis. The result

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(Figure S2) revealed that the retention times of HA, HA–Cys, and HA-SS-MP conjugates were


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5.69, 5.14, and 4.92 min, respectively. The decrement in the retention time after the formation of

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the HA conjugates confirmed the successful formation of thiolated HA and HA-SS-MP,

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supporting the NMR and FT-IR results.

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Preparation and characterization of HA-SS-MP micelles

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Due to their amphiphilic nature, the HA-SS-MP conjugates could self-assemble into micelles in

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an aqueous solution. The micellization behavior of HA-SS-MP was confirmed by measuring the

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CMC value using pyrene as a fluorescent probe. The CMC value of the amphiphilic HA-SS-MP

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conjugates was determined using the excitation intensity ratio of I334/I338 vs a prodrug

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concentration (Log C). As the concentration of HA-SS-MP increased and reached the CMC

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value, the hydrophobic pyrene molecules were preferentially solubilized in the core of the

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micelles, by which the intensity ratio decreased. As shown in Figure S3a, the CMC value of the

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amphiphilic HA-SS-MP conjugates was determined to be

pH- and GSH-Sensitive Hyaluronic Acid-MP Conjugate Micelles for

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