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Targeting Colorectal Cancer Stem-Like Cells with AntiCD133 Antibody-Conjugated SN-38 Nanoparticles Sin-Tzu Ning, Shin-Yu Lee, Ming-Feng Wei, Cheng-Liang Peng, Susan Yun-Fan Lin, MingHsien Tsai, Pei-Chi Lee, Ying-Hsia Shih, Chun-Yen Lin, Tsai-Yueh Luo, and Ming-Jium Shieh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04403 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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ACS Applied Materials & Interfaces

Targeting Colorectal Cancer Stem-Like Cells with Anti-CD133 Antibody-Conjugated SN-38 Nanoparticles Sin-Tzu Ning ||,‡, Shin-Yu Lee ||,‡, Ming-Feng Wei ‡, Cheng-Liang Peng †, Susan Yun-Fan Lin ‡, Ming-Hsien Tsai ‡, Pei-Chi Lee ‡, Ying-Hsia Shih †,‡, Chun-Yen Lin,‡ Tsai-Yueh Luo,† Ming-Jium Shieh*,‡, § ‡

Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan. † Isotope Application Division, Institute of Nuclear Energy Research, Longtan, Taoyuan 325, Taiwan. § Department of Oncology, National Taiwan University Hospital and College of Medicine, Taipei 100, Taiwan.

Corresponding author: *Ming-Jium Shieh, MD, PhD, Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan. Tel: 886-2-23123456 ext 67142. Fax: 886-2-23815095. Email: [email protected]

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ABSTRACT

Cancer stem-like cells play a key role in tumor development, and these cells are relevant to the failure of conventional chemotherapy. To achieve favorable therapy for colorectal cancer, PEG-PCL-based nanoparticles, which possess good biological compatibility, were fabricated as nanocarriers for the topoisomerase inhibitor, SN-38. For cancer stem cell therapy, CD133 (prominin-1) is a theoretical cancer stem-like cell (CSLC) marker for colorectal cancer and is a proposed therapeutic target. Cells with CD133 overexpression have demonstrated enhanced tumor-initiating ability and tumor relapse probability. To resolve the problem of chemotherapy failure, SN-38-loaded nanoparticles were conjugated with anti-CD133 antibody to target CD133positive (CD133+) cells. In this study, anti-CD133 antibody-conjugated SN-38-loaded nanoparticles (CD133Ab-NPs-SN-38) efficiently bound to HCT116 cells, which overexpress CD133 glycoprotein. The cytotoxic effect of CD133Ab-NPs-SN-38 was greater than that of nontargeted nanoparticles (NPs-SN-38) in HCT116 cells. Furthermore, CD133Ab-NPs-SN-38 could target CD133+ cells and inhibit colony formation compared with NPs-SN-38. In vivo studies in an HCT116 xenograft model revealed that CD133Ab-NPs-SN-38 suppressed tumor growth and retarded recurrence. A reduction in CD133 expression in HCT116 cells treated with CD133AbNPs-SN-38 was also observed in immunohistochemistry results. Therefore, this CD133-targeting nanoparticle delivery system could eliminate CD133-positive cells and is a potential cancer stem cell targeted therapy.

KEYWORDS: colorectal cancer, cancer stem-like cells, CD133, SN-38, nanoparticles


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INTRODUCTION Cancer is the leading cause of mortality worldwide and affects over ten million people every year. However, the intravenous administration of most classical chemotherapeutic drugs still produces undesirable biodistribution. The drugs are rapidly cleared from the blood, and only a small percentage of administered drugs accumulate in the tumor site. To enhance chemotherapeutic drug accumulation in tumors, nanodelivery systems such as liposomes, micelles, nanotubes and nanogold have been developed.1–3 Through the enhanced permeation and retention (EPR) effect, these nanoparticles are considered potential nanocarriers.4 To achieve active targeting, which is also called ligand-mediated targeting, the surfaces of nanoparticles are modified with affinity ligands to increase their uptake by specific cells. Appropriate ligands are selected to target specific molecules that are overexpressed on the cell plasma membrane. The purpose of active targeting systems is to increase specific binding of nanoparticles to the cells and enhance the internalization of delivered drugs.5 In oncology, the development of targeted nanoparticles can increase the internalization of drugs in targeted tumor cells, minimize drug leakage from target sites, and protect the drug from degradation and elimination.6 To date, many nanoparticles have been designed with conjugated ligands, including antibodies, carrier proteins, and peptides.7,8 Of the many small molecules tested, antibodies have shown good targeting specificity and affinity, offering preferable therapeutic efficiency with fewer side effects for future clinical application.9 Recently, increasing evidence has demonstrated that a minor population of cancer cells possesses capacities similar to stem cells and may play an important role in resistance to cancer treatments. Stem cells are a class of undifferentiated cells, which are usually defined by some common characteristics, including the capability of self-renewal and the ability to differentiate


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into other cell types.10 Through strictly regulated self-renewal and differentiation, stem cells help to maintain normal tissues and allow them to function normally. The loss of balance in the tight regulation of self-renewal plays a critical role in the development of cancer. Tumors are organized in heterogeneous cell subpopulations, which include a rare subset of cells that have the particular biological properties for tumor initiation and maintenance.11 These cancer cells have been defined as cancer stem-like cells (CSLCs) and have self-renewal and pluripotent capabilities that influence tumor growth and metastasis.12 It has been reported that the failure of conventional chemotherapy is closely related to the existence of CSLCs, which are more resistant than the bulk of other tumor cells due to overexpression of various membrane transporter molecules (such as multi-drug resistance protein 1, MDR-1), detoxifying enzymes, and DNA repair proteins.13–15 Many markers for CSLCs in colorectal cancer have been reported, including CD133, CD44, CD90 and epithelial cell adhesion molecules (EpCAM).16 These putative markers on CSLCs are potentially attractive targets for cancer therapies.17,18 CD133 (prominin-1) is a cholesterol-interacting pentaspan-transmembrane glycoprotein that has been considered a common CSLC marker for brain, colon, prostate, breast, and liver malignancies.19–24 A previous report demonstrated that subsets of CD133+ cells have more than 200-fold the number of cancer-initiating cells compared with unsorted cancer cells.25 Several studies have also suggested that CD133 expression has high prognostic relevance for colorectal cancer progression. Kojima et al. correlated CD133 overexpression with poorer therapeutic outcomes and an increased risk of metastasis in colorectal cancer patients.26 In chemotherapy, the high expression of CD133 is related to the resistance of CSLCs to 5-FU and poorer survival.27 Targeting ligands can bind to CD133 expressed on the cell membrane and be internalized into


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the cytoplasm through receptor-mediated endocytosis, making CD133 a potential therapeutic target for cancer treatment.28,29 Camptothecin (CPT), a cytotoxic alkaloid first isolated from the bark and stem of Camptotheca acuminata, is a promising chemotherapeutic agent that induces cell death by inhibiting the nuclear enzyme topoisomerase I (Top I).30 The nuclear enzyme topoisomerase I cuts one strand of the double-stranded DNA helix, twists it around the other strand and reanneals the nicked strands during DNA replication. CPT-11 (7-ethyl-10- [4-(1-piperidino)-1piperidono] carbonyloxycamptothecin), a water-soluble derivative of CPT for clinical use, is active against various cancers.2 However, studies have shown that approximately 2–8% of CPT11

is

converted

to

its

active

form,

SN-38

(7-ethyl-10-hydroxy-camptothecin)

by

carboxylesterases in the liver and tumors, resulting in 100- to 1000-fold higher cytotoxicity.31–33 Direct administration of SN-38 could have excellent anticancer effect; however, it is infeasible because of its insolubility in all pharmaceutically acceptable solvents.34 Therefore, using nanoparticles to deliver SN-38 possesses a significant clinical advantage, which can overcome the hydrophobicity of SN-38 and the lower therapeutic efficiency of CPT-11. Here, we proposed an antibody-conjugated nanoparticle that can target cancer stem-like cells and inhibit tumor growth, thereby improving the efficacy of colorectal cancer therapy (Scheme 1). The established formulation, anti-CD133 antibody-conjugated SN-38-loaded nanoparticles (CD133Ab-NPs-SN-38), involved SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles, which were modified with anti-CD133 antibodies on the surface. We confirmed that CD133+ cells possess tumor-initiating capabilities, and then CD133Ab-NPs-SN-38 was used as an SN-38 nanovector to target CD133+ cells. Targeting efficiency and cytotoxicity were evaluated in HCT116 cells (89.08% was CD133+) and HT29 cells (1.6% was CD133+) in in vitro


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studies. The sphere-forming ability of HCT116 cells was also assessed after treatment with different concentrations of non-targeted and targeted SN-38-loaded nanoparticles. Finally, using HCT116 tumor-bearing mouse models, the tumor growth inhibition efficacy of CD133Ab-NPsSN-38 was studied in vivo, and a decrease in the CD133+ cell population after targeted treatment was observed in the immunochemical study.

EXPERIMENTAL SECTION Materials The anticancer drug 7-ethyl-10-hydroxy-camptothecin (SN-38) was purchased from ScinoPharm Taiwan Ltd (Tainan, Taiwan). R-phycoerythrin (PE)-conjugated anti-human CD133 monoclonal antibody was obtained from Miltenyi Biotec (Auburn, CA, USA). High-performance liquid chromatography (HPLC)-grade solvents, including acetonitrile and dimethyl sulfoxide (DMSO), were obtained from Tedia (Fairfield, OH, USA). Pyrene and 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO, USA).

Preparation of SN-38-loaded mPEG-PCL/mal-PEG-PCL Nanoparticles The amphiphilic block copolymer, methoxy poly(ethylene glycol)-poly(ε-caprolactone) (mPEG-PCL), was synthesized by the ring-opening polymerization of ε-caprolactone in the presence of mPEG (MW=5000) as an initiator under the catalysis of stannous octoate (Sn(Oct)2). Maleimide-terminated poly(ethylene glycol)-poly(ε-caprolactone) (maleimide-PEG-PCL) was synthesized by the same method but using maleimide-PEG (MW=5000) as the initiator. The reaction was catalyzed at 140 °C for 24 hours. Copolymer compositions were determined with


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FT-NMR at 500 MHz using chloroform-d (CDCl3) as the solvent. The molecular weights of the synthesized copolymers were determined by gel permeation chromatography as previously described.35 To determine the critical micelle concentration (CMC) of the synthesized mPEGPCL and mal-PEG-PCL, we used pyrene as a probe and the detailed procedure was described as our previously published research.35 By using the lyophilization-rehydration method, the anticancer drug SN-38 was loaded into nanoparticles. SN-38 and the amphiphilic copolymers mPEG-PCL and mal-PEG-PCL (weight ratio = 9:1) were dissolved in 1 mL DMSO with a drug/polymer weight ratio of 1/10, accompanied by sonication until the formation of a clear solution. The mixture was then freeze-dried overnight. The lyophilized cake was hydrated in 1 mL phosphate-buffered saline and then sonicated for 15 minutes using an ultrasonic cell crasher for further dispersion of the nanoparticles. The solution was filtered through a 0.22-µm filter to remove the non-incorporated drug.

Thiolation, Preparation and Observation of CD133 Antibody Conjugation For thiolation of the antibody, 5 µg of the PE-conjugated anti-human CD133 monoclonal antibody (CD133/2(293C3)-PE) and 40-times molar excess of 2-iminothilane (Traut’s reagent, SIGMA) were mixed at pH 8 in buffer containing 10 mM EDTA at room temperature for 1 hour with gentle stirring. Residual reagents were removed using a G-50 column (GE Healthcare). Conjugations of the thiolated anti-CD133 antibody (CD133-SH) to the nanoparticles were performed by mixing CD133-SH and maleimide-containing nanoparticles at 4 °C with stirring overnight. The products were then centrifuged (15,000 rpm) at 4 °C for 30 minutes and resuspended in PBS to remove unconjugated antibodies. The anti-immunoglobulin G (IgG) antibody-conjugated SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (IgGAb-NPs-SN-


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38) were synthesized through the same process but using anti-IgG antibody instead. The unconjugated probes in the supernatant were collected, and the concentration was determined by fluorescence spectroscopy. The antibody conjugation efficiency (CE, %) was then calculated using the following equation: CE (%) = [(concentration of free probe used for reaction – concentration of unconjugated probe in supernatant)/(concentration of free probe used for reaction)] × 100%.

Characterization of SN-38-loaded and Antibody-conjugated Nanoparticles The particle size, polydispersity index (PDI), and zeta potential of SN-38-loaded mPEGPCL/mal-PEG-PCL nanoparticles (NPs-SN-38) and anti-CD133 antibody-conjugated SN-38loaded mPEG-PCL/mal-PEG-PCL nanoparticles (CD133Ab-NPs-SN-38) were evaluated by a dynamic light scattering (DLS) system using the Zetasizer Nano-ZS90. The amount of SN-38 encapsulated

into

NPs-SN-38

and

CD133Ab-NPs-SN-38

was

verified

by

HPLC

(excitation/emission = 365/550 nm). Drug encapsulation efficiency (EE, %) was calculated by the following equation: EE (%) = (weight of SN-38 in the nanoparticles/total weight of SN-38 in the loading solution) × 100%. To evaluate the stability of NPs-SN-38, the prepared nanoparticle solution was stored at 4 °C, and the particle size, PDI and SN-38 encapsulation efficiency were measured at room temperature every day for 5 continuous days. The in vitro drug release profile was analyzed using a dialysis bag diffusion technique at 37 °C, and 2 mL of NPs-SN-38 was added to the dialysis bag (MWCO=3.5 kDa), which was immersed in 50 mL of PBS at different pH values (7.4 and 5.0) with continuous gentle stirring.


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At selected time points, 40 μL of solution was collected from the release medium, and the released SN-38 was detected by using a spectrofluorometer (excitation/emission = 390/427 nm).

Cell Culture Three human colorectal cancer cell lines, including HT-29, SW620 and HCT116, and the primary cultured colorectal cancer cells (CCS) were used for in vitro studies. HCT116 was maintained in McCoy’s 5A modified medium (SIGMA-ALDRICH, USA), and HT-29, SW620, and CCS were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, USA). Both media were supplemented with 10% (v/v) heat-activated fetal bovine serum (FBS, GIBCO, USA) and 1% (v/v) penicillin-streptomycin-amphotericin B antibiotic-antimycotic solutions (SIGMA-ALDRICH, USA). The McCoy’s 5A modified medium was used with the further addition of 4 mM L-glutamine (GIBCO, USA). The cells were cultured at 37 °C in the incubator in an atmosphere of 5% CO2 and were sub-cultured two to three times every week.

CD133+ Subpopulation in Different Colorectal Cancer Cell Lines The expression of CD133 in HT29, HCT116, SW620 and CCS was evaluated using a FACSCalibur apparatus (Becton Dickinson, BD FACSCaliburTM System, CA, USA). The PEconjugated anti-CD133 antibody (Miltenyi Biotec, Auburn, CA, USA) was used in the experiment. Cultured cells were trypsinized into single-cell suspensions. Then, the trypsinization was neutralized by the addition of culture medium, and cells were washed with phosphatebuffered saline (PBS). After centrifugation (1000 rpm, 5 minutes), the pellets were resuspended and incubated with PE-labeled anti-CD133 antibodies at 4 °C for 30 minutes. The cells were then analyzed on a flow cytometer after being washed with PBS.


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HT-29, HCT116, SW620, and CCS cells were also seeded onto a 6-well plate at a density of 5 × 105 cells per well and incubated overnight. After being washed with PBS, cells were fixed in 3% paraformaldehyde at 37 °C for 15 minutes and incubated with FcR blocking reagent at 37 °C for 5 minutes. Then, cells were stained with the PE-labeled anti-CD133 antibodies at 4 °C for 30 minutes; the nuclei of cells were labeled with Hoechst for 10 minutes and then imaged by fluorescence microscopy (Zeiss Axio Imager).

Binding Affinity and Cellular Uptake of CD133-Ab-NPs-SN-38 HT-29 and HCT116 cells were seeded onto 6-well plates at a density of 5 × 105 per well and incubated overnight. Cultured cells were trypsinized into single-cell suspensions and then treated with a medium containing either SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (NPs-SN-38), anti-immunoglobulin G (IgG) antibody-conjugated SN-38-loaded mPEGPCL/mal-PEG-PCL nanoparticles (IgGAb-NPs-SN-38) or anti-CD133 antibody-conjugated SN38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (CD133Ab-NPs-SN-38) at 4 °C for 1 hour. For the competition/blocking test, cells were pre-blocked with free anti-CD133 antibodies (4 °C, 30 minutes) before treatment with CD133Ab-NPs-SN-38. Then, cells were lysed by 0.1 mL lysis buffer. Half of the cell lysate was used for total protein content detection by BCA protein assay, and the remaining cell lysate was dissolved in DMSO; fluorescence intensity was measured by HPLC using excitation/emission wavelengths of 365 and 550 nm. The binding efficiency of nanoparticles was determined by the fluorescence of SN-38 normalized to total protein content. Cellular uptake was observed by using fluorescence microscopy and fluorescein isothiocyanate (FITC)-labeled nanoparticles. HCT116 and HT-29 cells were seeded onto a 6well plate at a density of 5 × 105 cells per well and incubated overnight. Subsequently, cells were


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cultured with medium containing NPs-SN-38 and CD133Ab-NPs-SN-38 at 37 °C for 3 hours, respectively; then, the nuclei of the cells were labeled with Hoechst and imaged by fluorescence microscopy.

In Vitro Cytotoxicity The in vitro cytotoxicity of the synthesized nanoparticles was measured via MTT assay. HT-29 and HCT116 cells were seeded onto a 96-well plate at a density of 8×103 and 1×104 per well, respectively, and incubated overnight. Next, different concentrations of mPEG-PCL/malPEG-PCL nanoparticles (NPs), anti-CD133 antibody-conjugated mPEG-PCL/mal-PEG-PCL nanoparticles (CD133Ab-NPs), SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (NPsSN-38), and anti-CD133 antibody-conjugated SN-38-loaded mPEG-PCL/mal PEG-PCL nanoparticles (CD133Ab-NPs-SN-38) were added into medium. After 72 hours of incubation, the medium was aspirated, and 100 μL of the MTT solution (0.5 mg/mL of MTT in serum-free medium) was added to each well and incubated for 2 hours. Then, the medium was removed, and 100 μL of DMSO was added to each well to dissolve the formazan crystals formed by the living cells. Absorbance at 570 nm was measured using an ELISA reader, with the correction wavelength set to 630 nm. The cytotoxicity of each group was calculated as a percentage relative to the non-treated control group.

Colonosphere Culture and Colony Formation Assay HCT116 and FACS sorted cells (HCT116 CD133+ and CD133- cells) were cultured in ultra-low attachment plates (Corning®) with serum-free DMEM/F12 (GIBCO, USA)


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supplemented with 20 ng/mL EGF (Pepro Tech, USA), 10 ng/mL bFGF (Pepro Tech, USA) and N-2 supplement (GIBCO, USA). For the colony formation assay, HCT116 cells were treated with NPs-SN-38 and CD133Ab-NPs-SN-38 at 4 °C for 1 hour, respectively. Treatments were then removed, and cells were incubated with growth media at 37 °C. Two thousand cells were plated per well in an ultralow attachment 96-well plate (Corning®) and cultured for 7 days. The resulting colonospheres were counted under an inverted light microscope.

In Vivo Tumor Growth Inhibition Studies The in vivo experimental protocols were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC). Female nude mice at the age of 4 weeks were purchased from the National Laboratory Animal Center, Taiwan. Each mouse was subcutaneously injected with 2×106 HCT116 cells in the hind flank region. When the tumor volume reached approximately 100 mm3, mice were randomly divided into 4 groups. The following are the treatment conditions of each group (n = 6 per group): (1) untreated control, (2) CPT-11 (10 mg/kg), (3) NPs-SN-38 (5 mg/kg), and (4) CD133Ab-NPs-SN-38 (5 mg/kg). Distinct formulations were administered via tail vein injections, and a total of five injections (one injection for every four days) were given. The tumor size and body weight of each mouse were measured twice a week. Tumor growth was monitored by measuring the tumor’s length (l) and width (w), and the tumor volume was calculated using the formula lw2/2.

Immunohistochemical Analysis


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Therapeutic efficacy was also evaluated by hematoxylin and eosin (H&E) and immunohistochemical staining. After the fifth administration, mice in different groups were sacrificed. CD133 protein expression in the tumors was analyzed by immunohistochemistry. In brief, slides containing paraffin sections of the tumor samples were deparaffinized, rehydrated and incubated in a pre-heated steamer bath at 120 °C for 3 minutes. The slides were cooled to room temperature and then subjected to the following incubation steps: 3% hydrogen peroxide for 10 minutes, 3% BSA at room temperature for 1 hour (for blocking), anti-CD133 antibody at 4 °C overnight, labeled polymer-HRP (Dako) at room temperature for 30 minutes, and DAB chromogen. After sealing with a cover slip, slides were observed and imaged under a light microscope.

Statistical Analysis All data are expressed as the mean ± standard deviation. Student’s t-test was applied to analyze the significance of the differences between groups. A P-value smaller than 0.05 was considered statistically significant; P-values less than 0.01 were considered very significant.

RESULTS AND DISCUSSION Synthesis and Characterization of SN-38-loaded Nanoparticles Both poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) polymers are approved by the US Food and Drug Administration (FDA) for many medical applications in the clinic. PEG was used as the hydrophilic segment of the copolymer because of its biocompatibility and low toxicity. PEG can help nanoparticles escape from macrophage uptake and opsonization,


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making the particles more stable in blood. PCL was used as the hydrophobic segment because it also has excellent biocompatibility and flexibility. In clinical applications, PCL has been developed as a potential material for temporary joint spacers36 and tissue-engineered skin37. It has been reported that poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) copolymers are sustained in the blood and have limited toxicity to tissues.38 Therefore, we considered a PEG-bPCL nanoparticle a good candidate for water-insoluble chemotherapeutic agents among various drug delivery systems. mPEG-PCL and mal-PEG-PCL copolymers were prepared by ring-opening polymerization of ε-caprolactone. The synthesized mPEG-PCL and mal-PEG-PCL were characterized by 1H NMR (Figure 1). 1H NMR analysis verified the maleimide protons at σ 6.7 ppm after polymerization. The molecular weights of the synthesized copolymers were evaluated by GPC (Table S1). The average molecular weight (Mn) of mPEG5k-PCL8k was 1.27 × 104 Da, similar to results we reported in a previous study,39 and that of mal-PEG5k-PCL10k was 1.43 × 104 Da. As expected, amphiphilic mPEG-PCL and mal-PEG-PCL copolymers could self-assemble into nanoparticles in a hydrophilic solution (the critical micelle concentrations are shown in Figure S2). The hydrophobic PCL blocks localize in the cores of the nanoparticles, encapsulating SN-38 in the middle, and the hydrophilic mal-PEG segments stretch outwards, forming a shell in the aqueous environment. Subsequently, thiolated anti-CD133 antibodies were conjugated to the maleimide groups, displaying targeting probes externally on the nanoparticles. The particle size, PDI, and zeta potential of the SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles were evaluated by a dynamic light scattering (DLS) system (Table 1). The average diameter of antiCD133

antibody-conjugated

SN-38-loaded

mPEG-PCL/mal-PEG-PCL

nanoparticles

(CD133Ab-NPs-SN-38) was 167.1 nm with a spherical shape and a narrow size distribution


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(Figure S1). The non-targeted SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (NPs-SN38) and the CD133Ab-NPs-SN-38 both possessed high encapsulation efficiency (EE), which was approximately 90%, and were well dispersed in PBS solution. This suggested that the synthesized nanoparticles could be readily applied to in vitro and in vivo studies in subsequent experiments. Moreover, the conjugation efficiency (CE) of the anti-CD133 antibody was determined by fluorescence of PE, and the results indicated that the CE of CD133Ab-NPs-SN-38 was approximately 70% (Table 1).

Stability and Cytotoxicity of the Synthesized Nanoparticles To evaluate the stability of SN-38-encapsulated mPEG-PCL/mal-PEG-PCL nanoparticles (NPs-SN-38), the synthesized nanoparticle solution was stored at 4 °C, and the particle size, PDI and SN-38 encapsulation efficiency were measured at room temperature every day for 5 continuous days (Table S2 and Figure 2A-B). The variation in particle size and PDI was small, with an average size of approximately 140 to 150 nm. The average encapsulation efficiency also showed limited variation and remained at approximately 85 to 90%, indicating that NPs-SN-38 were stable in aqueous solution. Before determining the cytotoxicity of the SN-38-loaded nanoparticles, we first evaluated the safety of the mPEG-PCL/mal-PEG-PCL nanoparticles (NPs) and CD133 antibody-conjugated mPEG-PCL/mal-PEG-PCL nanoparticles (CD133AbNPs) in the colorectal adenocarcinoma cell line HT-29 and the colorectal carcinoma cell line HCT116. The cell viability of HT-29 and HCT116 treated with various concentrations of nanoparticles was evaluated via MTT assay. As shown in Figure 2C, after a 72-hour incubation with the synthesized NPs and CD133Ab-NPs, there was no significant cytotoxicity in HCT116 and HT-29 cells. These data revealed that the anti-CD133 antibody-conjugated mPEG-PCL/mal-


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PEG-PCL nanoparticles have a very low cytotoxicity and can be applied as a stable and biocompatible targeted nanodelivery system.

Expression Level of CD133 Surface markers in Colorectal Cancer Cells CD133 (prominin-1) was first recognized as a surface marker of hematopoietic stem cells.40 Subsequently, it was identified in cancer stem-like cells (CSLCs) from several solid tumors.41 In the cancer stem cell hypothesis, CD133 is involved in cell survival through the regulation of autophagy, which may be necessary for cancer stem cells in the tumor microenvironment.42 It is becoming evident that CD133 surface markers can be used to identify cancer stem cells. It has also been reported that CD133high cells exhibit greater proliferation and invasion compared with CD133low cells.43 Therefore, by using flow cytometry and PE-labeled anti-CD133 antibody to identify CD133+ cells, the CD133 expression profiles of four different colorectal cancer cells were analyzed (Figure 3). The cell lines that exhibited higher CD133 expression were HCT116 (89.08%) and SW620 (40.3%), whereas HT-29 (1.6%) and primary cultured colorectal cancer cells CCS (2.94%) only possessed low levels of CD133 expression (Figure 3A). Fluorescent images of CD133 expression in HCT116, SW620, HT-29 and CCS cells are shown in Figure 3B. After incubation with PE-labeled anti-CD133 antibody for 30 minutes, the fluorescent signals of antibodies were primarily located on the plasma membrane of HCT116 cells. Consequently, in the following experiments, the HCT116 cell line was used as the CD133+ cell model because of its high CD133 expression.

The Cell Binding Affinity and Cellular Uptake of CD133-targeting nanoparticles


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Active targeting is emerging as a new strategy to make drug delivery systems more specific, allowing more drugs to be precisely delivered to the target sites, reducing unspecific drug accumulation,7 and hence, resulting in a decrease in side effects. First, we investigated the cell binding affinity of CD133Ab-NPs-SN-38 in HCT116 and HT-29 because the HCT116 cell line showed high expression of CD133 glycoprotein, whereas the HT-29 cell line showed low expression, as demonstrated above (Fig. 3). As shown in Figure 4A, CD133Ab-NPs-SN-38 enhanced nanoparticle binding affinity by approximately two-fold compared with the nontargeted NPs-SN-38 and IgG antibody-conjugated SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles (IgGAb-NPs-SN-38) in HCT116. Cells pretreated with an excess of free antiCD133 antibody before incubation with CD133Ab-NPs-SN-38 exhibited reduced binding affinity due to the competitive effect. In contrast, there were no differences in binding affinity when HT29 cells, which had low expression of CD133, were treated with NPs-SN-38 and CD133Ab-NPs-SN-38. The results indicated that CD133Ab-NPs-SN-38 could target CD133 glycoprotein on the cell plasma membrane and enhance the binding efficiency of nanoparticles. These results correlated with the previously reported finding that nanoparticles modified with anti-CD133 antibody facilitated binding towards CD133-overexpressing cells compared with non-targeted nanoparticles.44,45 The higher binding efficiency of anti-CD133 antibody-modified nanoparticles on the cell membrane could lead to higher cellular uptake through receptor-mediated endocytosis. We utilized the mPEG-PCL/FITC-PEG-PCL/mal-PEG-PCL (weight ratio = 9/1/1) nanoparticles modified with anti-CD133 antibody to quantitatively determine cellular uptake. As illustrated in Figure 4B, HCT116 cells incubated with CD133Ab-NPs showed stronger FITC fluorescence compared with that of cells treated with non-targeted NPs. However, in HT-29, there were no


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significant differences between the CD133Ab-NPs and NPs treatment. This result not only indicated that decoration with anti-CD133 antibody would enhance cellular uptake of nanoparticles but also revealed that CD133-NPs could be a potential targeted delivery system specifically recognizing cancer stem-like cells, which usually have a higher expression of CD133.

In Vitro Anticancer Effect of CD133Ab-NPs-SN-38 Against CD133high Expression Cells Biomarker identification is one of the novel strategies for cancer therapy. In previous experiments, we confirmed CD133Ab-NPs as a biocompatible nanoplatform that has potential for specific recognition of cancer stem-like cells. To further utilize CD133Ab-NPs as a drug carrier, a topoisomerase I inhibitor SN-38 was loaded into nanoparticles as a model nanomedicine. As described in the previous section, CD133Ab-NPs-SN-38 showed good encapsulation efficiency and stability. The prolonged drug release profiles of the synthesized nanoparticles under different pH values were also observed (Figure S3). We investigated the release profile under pH 7.4 and 5.0 to mimic the physiological and late endosomal or lysosomal environment (pH 4.5 to 5.5). The solubility of SN-38 increases significantly in alkaline solutions (pH ≥ 8).46 The in vitro release of SN-38 from the nanoparticles was faster at pH 7.4 than that at pH 5.0 because SN-38 has higher solubility in basic solutions, which corresponded with our previous findings.2,3,35,47 In the investigation of in vitro anticancer effect, the IC50 values of free SN-38, NPs-SN-38 and CD133Ab-NPs-SN-38 in HCT116 were 138.5, 352.0 and 176.9 ng/mL, respectively. CD133Ab-NPs-SN-38 exhibited higher cytotoxicity in HCT116, compared with non-targeting NPs-SN-38 (Figure 5). The better anticancer effect might result from the greater cellular uptake of CD133Ab-NPs-SN-38 due to its efficient targeting of CD133. Furthermore,


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our results showed that CD133Ab-NPs-SN-38 and free SN-38 had a similar degree of cytotoxicity, suggesting that the encapsulation of SN-38 into the targeted delivery system (CD133Ab-NPs) would not affect the therapeutic effect of the chemotherapeutic. Moreover, this combination could overcome the insolubility of SN-38 in biological media, increase drug accumulation in target sites, and reduce undesired side effects. On the other hand, no significant differences were observed when CD133 low-expression HT-29 cells were treated with free SN38, non-targeted NPs-SN-38 and CD133Ab-NP-SN-38 (Figure 5). Therefore, these results demonstrated that CD133Ab-NPs-SN-38 could overcome the disadvantages of free SN-38 drug and has the potential to efficiently eliminate CD133-positive cells.

CD133Ab-NPs-SN-38 Target CD133-overexpressing Cells and Inhibit Colony Formation Sphere-formation assays are widely used to identify stem cells by evaluating self-renewal capabilities.48 Additionally, the formation of 3D spheres can help to maintain the stemness of stem cells by decreasing their attachment to surfaces.49 First, to confirm the existence of stemlike cells in HCT116, we examined its sphere formation ability in vitro. In Fig. 6A, regardless of whether 2000 or 1000 cells were seeded onto ultra-low attachment wells, the average colonosphere size of CD133+ cells was markedly greater than that of CD133− cells. Similarly, the count of the colonospheres of CD133+ cells was also higher than that of CD133− cells (Fig. 6B). These data implied that HCT116 CD133+ cells possessed strong initiating capability and stemlike properties. By using anti-CD133 antibody-conjugated nanoparticles to restrain the CD133+ cells, we might be able to suppress the growth of tumors. To validate whether CD133Ab-NPs-SN-38 could target CD133-overexpressing cells, inhibit their proliferation, and thereby retard tumor growth, we investigated the colony formation


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ability of HCT116 cells pre-treated with CD133Ab-NPs-SN-38 and NPs-SN-38 at 4 °C for 1 hour. As shown in Figure 6C, when HCT116 cells were pretreated with CD133Ab-NPs-SN-38 and NPs-SN-38 with different concentrations of SN-38 (10000, 1000, and 100 ng/mL), a dosedependent inhibition of colonosphere formation was observed. When treated with nanoparticles containing 10000 ng/mL SN-38, very few spheres could be found in 3D culture because most cells were dead because of the high concentration of the drug. Noticeably, HCT116 cells pretreated with CD133Ab-NPs-SN-38 obviously inhibited colony formation compared with nontargeted NPs-SN-38 (with lower SN-38 concentrations such as 100 and 1000 ng/mL). A similar result was also observed for the cytotoxicity of NPs-SN-38 and CD133Ab-NPs-SN-38 for HCT116 spheres (Figure S4). Studies have shown that targeted nanomedicines possess the ability to enhance therapeutic effects and decrease nonspecific toxicity because the chemotherapeutic drugs are efficiently guided to the tumor sites.5,6,9 Our results demonstrated that with an actively targeting probe, CD133Ab-NPs-SN-38 could potentially inhibit tumor initiation or tumor growth even at a lower drug concentration.

In Vivo Biodistribution of CD133Ab-NPs-SN-38 and NPs-SN-38 The in vivo biodistribution of NPs-SN-38 and CD133Ab-NPs-SN-38 was evaluated by detecting the amount of SN-38 in the tumor and other organs at 24 hours after the intravenous administration (Figure 7). A major accumulation of both NPs-SN-38 and CD133Ab-NPs-SN38 was detected in liver and spleen. The fact that liver and spleen are known as parts of the reticuloendothelial system and play important roles in nanoparticle clearance might be the reason for the high accumulation of the synthesized nanoparticles in these two organs. Hepatocytes and phagocytic Kupffer cells in the liver provide clearance functions including catabolism, biliary


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excretion and elimination of foreign nanoparticles. And the high accumulation in the spleen might also result from the nonspecific uptake of nanoparticles by resident macrophages.6,50 Due to the enhanced permeability and retention (EPR) effect, some non-targeted nanoparticles could accumulate in the tumor via passive targeting. However, through the active targeting ability of the anti-CD133 antibody, CD133Ab-NPs-SN38 could achieve targeted delivery, leading to increased drug accumulation in HCT116 tumors. As a result, higher SN-38 accumulation was detected in mice treated with CD133Ab-NPs-SN38.

In vivo CD133Ab-NPs-SN-38 Inhibited Tumor Growth and Exhibited Its Potential to Reduce Tumor Recurrence A previous study reported that CD133high cells have significantly higher tumor-initiating capacity than CD133low cells, signifying that the initiating ability derived from CD133high cells is a critical factor in tumor evolution.51 In this study, we aimed to establish a cancer stem cell therapy dependent on anti-CD133 antibody-conjugated SN-38-loaded nanoparticles, which have the ability to target CD133high cells. To determine whether the targeted nanoparticles, CD133AbNPs-SN38, possess efficient targeting ability and anticancer activity, we used HCT116 tumorbearing mice as a tumor xenograft model to investigate the therapeutic outcome of the following conditions: (1) untreated control, (2) CPT-11 (10 mg/kg), (3) NPs-SN-38 (5 mg/kg of SN-38), and (4) CD133Ab-NPs-SN-38 (5 mg/kg of SN-38). A total of five injections were given intravenously during the treatment stage (one injection for every four days) (Figure 8A). As shown in Figure 8B, within 25 days after the first injection, CD133Ab-NPs-SN-38 significantly suppressed tumor growth compared with CPT-11 and NPs-SN-38. Though tumor relapse occurred in all treatment groups later during the off-therapy stage, better inhibition was still


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observed in the CD133Ab-NPs-SN-38-treated group at the end of experimental period. We assumed a complete suppression without tumor relapse might be achieved by using CD133AbNPs-SN-38 with higher SN-38 concentration. The body weight of CD133Ab-NPs-SN-38-treated mice slightly decreased during the drug treatment stage, gradually recovered to the initial weight and remained constant throughout the rest experiment period (Figure 8C). The change in body weight might be a result of better tumor growth inhibition and less adverse side effects. Pictures of treated tumor-bearing mice at the off-therapy stage showed that compared with the NPs-SN38-treated group, a smaller tumor was observed in the mouse with the best therapeutic response in the CD133Ab-NPs-SN-38-treated group. These results indicated that anti-CD133 antibodyconjugated SN-38-loaded nanoparticles have the potential to inhibit tumor growth and reduce tumor recurrence, which is consistent with previous findings that demonstrated a significant decrease in the CSLC population and improved therapeutic efficacy using CD133-targeting nanoparticles as a delivery system for paclitaxel.44 The therapeutic efficacy was also evaluated by immunohistochemical analysis. Mice were sacrificed, and the tumors were collected after five injections. As shown in Figure 9, treatments with CPT-11 and NPs-SN-38 did not completely eradicate the CD133+ cells. However, a significant reduction in CD133+ cells was observed in tumors treated with CD133Ab-NPs-SN38, suggesting that the treatment using CD133Ab-NPs-SN-38 can reduce the CD133+ subpopulation and therefore could have the potential to inhibit tumor relapse.

Conclusions In an attempt to improve the efficacy of chemotherapy, we proposed a potential cancer stem cell-targeted therapy that utilizes anti-CD133 antibody-conjugated self-assembled mPEG-


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PCL/mal-PEG-PCL nanoparticles as a drug delivery system for SN-38. The established CD133Ab-NPs exhibited excellent biocompatibility and strong CD133 binding affinity. Additionally, with the encapsulation of SN-38, CD133Ab-NPs-SN-38 can efficiently inhibit colony formation, which is usually attributed to the existence of cancer stem cells. In the in vivo studies, CD133Ab-NPs-SN-38 also exhibited the capability of selectively targeting CD133+ cells and suppressing tumor growth. The results of our study indicated that CD133 could be a superior target for drug delivery applications that specifically target cancer stem-like cells in colorectal cancer. Therefore, the application of anti-CD133 antibodyconjugated SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles might be a potential targeted therapy for future colorectal cancer treatments.

ASSOCIATED CONTENT Supporting Information The molecular weights of mPEG-PCL and mal-PEG-PCL copolymers, PDI values of 5-day stability test, size distribution and TEM images of the synthesized nanoparticles, the critical micelle concentration of synthesized copolymers, the drug release profile, and cytotoxicity of nanoparticles in HCT116 spheres. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION


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Corresponding Author * E-mail: [email protected]. Tel.: 886-2-23123456 ext 67142. Author Contributions ||

S. T. Ning and S. Y. Lee contributed equally to this work.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology, R.O.C. (MOST 102-2320-B-002-038-MY3), the Ministry of Health and Welfare, R.O.C (MOHW 103TDU-N-211-133006), and National Taiwan University Hospital (105-S3009).

REFERENCES (1)

Anders, C. K.; Adamo, B.; Karginova, O.; Deal, A. M.; Rawal, S.; Darr, D.; Schorzman, A.; Santos, C.; Bash, R.; Kafri, T.; Carey, L.; Miller, C. R.; Perou, C. M.; Sharpless, N.; Zamboni, W. C. Pharmacokinetics and Efficacy of PEGylated Liposomal Doxorubicin in an Intracranial Model of Breast Cancer. PLoS One 2013, 8 (5).

(2)

Peng, C. L.; Lai, P. S.; Lin, F. H.; Wu, S. Y.-H.; Shieh, M. J. Dual Chemotherapy and Photodynamic Therapy in an HT-29 Human Colon Cancer Xenograft Model Using SN38-Loaded Chlorin-Core Star Block Copolymer Micelles. Biomaterials 2009, 30 (21), 3614–3625.


24

Page 25 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(3)

Lee, P.-C.; Chiou, Y.-C.; Wong, J.-M.; Peng, C.-L.; Shieh, M.-J. Targeting Colorectal Cancer Cells with Single-Walled Carbon Nanotubes Conjugated to Anticancer Agent SN38 and EGFR Antibody. Biomaterials 2013, 34 (34), 8756–8765.

(4)

Greish, K. Enhanced Permeability and Retention (EPR) Effect for Anticancer Nanomedicine Drug Targeting. Methods Mol. Biol. 2010, 624, 25–37.

(5)

Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L. Active Targeting Schemes for Nanoparticle Systems in Cancer Therapeutics. Adv. Drug Deliv. Rev. 2008, 60 (15), 1615– 1626.

(6)

Lammers, T.; Hennink, W. E.; Storm, G. Tumour-Targeted Nanomedicines: Principles and Practice. Br. J. Cancer 2008, 99 (3), 392–397.

(7)

Mahon, E.; Salvati, A.; Baldelli Bombelli, F.; Lynch, I.; Dawson, K. A. Designing the Nanoparticle-Biomolecule Interface For “targeting and Therapeutic Delivery.” J. Control. Release 2012, 161 (2), 164–174.

(8)

Van Der Meel, R.; Oliveira, S.; Altintas, I.; Haselberg, R.; Van Der Veeken, J.; Roovers, R. C.; Van Bergen En Henegouwen, P. M. P.; Storm, G.; Hennink, W. E.; Schiffelers, R. M.; Kok, R. J. Tumor-Targeted Nanobullets: Anti-EGFR Nanobody-Liposomes Loaded with Anti-IGF-1R Kinase Inhibitor for Cancer Treatment. J. Control. Release 2012, 159 (2), 281–289.

(9)

Tazi, I.; Nafil, H.; Mahmal, L. Monoclonal Antibodies in Hematological Malignancies: Past, Present and Future. J. Cancer Res. Ther. 2011, 7 (4), 399–407.

(10)

Al-Hajj, M.; Becker, M. W.; Wicha, M.; Weissman, I.; Clarke, M. F. Therapeutic


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 42

Implications of Cancer Stem Cells. Curr Opin Genet Dev 2004, 14 (1), 43–47. (11)

Vermeulen, L.; Todaro, M.; de Sousa Mello, F.; Sprick, M. R.; Kemper, K.; Perez Alea, M.; Richel, D. J.; Stassi, G.; Medema, J. P. Single-Cell Cloning of Colon Cancer Stem Cells Reveals a Multi-Lineage Differentiation Capacity. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (36), 13427–13432.

(12)

Gupta, P. B.; Chaffer, C. L.; Weinberg, R. a. Cancer Stem Cells: Mirage or Reality? Nat. Med. 2009, 15 (9), 1010–1012.

(13)

Lim, M. C.; Song, Y. J.; Seo, S. S.; Yoo, C. W.; Kang, S.; Park, S. Y. Residual Cancer Stem Cells after Interval Cytoreductive Surgery Following Neoadjuvant Chemotherapy Could Result in Poor Treatment Outcomes for Ovarian Cancer. Onkologie 2010, 33 (6), 324–330.

(14)

Rich, J. N.; Bao, S. Chemotherapy and Cancer Stem Cells. Cell Stem Cell 2007, 1 (4), 353–355.

(15)

Gangemi, R.; Paleari, L.; Orengo, A. M.; Cesario, A.; Chessa, L.; Ferrini, S.; Russo, P. Cancer Stem Cells: A New Paradigm for Understanding Tumor Growth and Progression and Drug Resistance. Curr. Med. Chem. 2009, 16 (14), 1688–1703.

(16)

Fanali, C.; Lucchetti, D.; Farina, M.; Corbi, M.; Cufino, V.; Cittadini, A.; Sgambato, A. Cancer Stem Cells in Colorectal Cancer from Pathogenesis to Therapy: Controversies and Perspectives. World J. Gastroenterol. 2014, 20 (4), 923–942.

(17)

Li, S. D.; Howell, S. B. CD44-Targeted Microparticles for Delivery of Cisplatin to Peritoneal Metastases. Mol. Pharm. 2010, 7 (1), 280–290.


26

Page 27 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18)

Wang, L.; Su, W.; Liu, Z.; Zhou, M.; Chen, S.; Chen, Y.; Lu, D.; Liu, Y.; Fan, Y.; Zheng, Y.; Han, Z.; Kong, D.; Wu, J. C.; Xiang, R.; Li, Z. CD44 Antibody-Targeted Liposomal Nanoparticles for Molecular Imaging and Therapy of Hepatocellular Carcinoma. Biomaterials 2012, 33 (20), 5107–5114.

(19)

Brescia, P.; Ortensi, B.; Fornasari, L.; Levi, D.; Broggi, G.; Pelicci, G. CD133 Is Essential for Glioblastoma Stem Cell Maintenance. Stem Cells 2013, 31 (5), 857–869.

(20)

Ricci-Vitiani, L.; Lombardi, D. G.; Pilozzi, E.; Biffoni, M.; Todaro, M.; Peschle, C.; De Maria, R. Identification and Expansion of Human Colon-Cancer-Initiating Cells. Nature 2007, 445 (7123), 111–115.

(21)

Catalano, V.; Di Franco, S.; Iovino, F.; Dieli, F.; Stassi, G.; Todaro, M. CD133 as a Target for Colon Cancer. Expert Opin. Ther. Targets 2012, 16 (3), 259–267.

(22)

Miki, J.; Furusato, B.; Li, H.; Gu, Y.; Takahashi, H.; Egawa, S.; Sesterhenn, I. A.; McLeod, D. G.; Srivastava, S.; Rhim, J. S. Identification of Putative Stem Cell Markers, CD133 and CXCR4, in hTERT-Immortalized Primary Nonmalignant and Malignant Tumor-Derived Human Prostate Epithelial Cell Lines and in Prostate Cancer Specimens. Cancer Res. 2007, 67 (7), 3153–3161.

(23)

Wright, M. H.; Calcagno, A. M.; Salcido, C. D.; Carlson, M. D.; Ambudkar, S. V; Varticovski, L. Brca1 Breast Tumors Contain Distinct CD44+/CD24- and CD133+ Cells with Cancer Stem Cell Characteristics. Breast Cancer Res. 2008, 10 (1), R10.

(24)

Ma, S.; Chan, K.-W.; Hu, L.; Lee, T. K.-W.; Wo, J. Y.-H.; Ng, I. O.-L.; Zheng, B.-J.; Guan, X.-Y. Identification and Characterization of Tumorigenic Liver Cancer


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 42

Stem/progenitor Cells. Gastroenterology 2007, 132 (7), 2542–2556. (25)

O’Brien, C. A.; Pollett, A.; Gallinger, S.; Dick, J. E. A Human Colon Cancer Cell Capable of Initiating Tumour Growth in Immunodeficient Mice. Nature 2007, 445 (7123), 106– 110.

(26)

Kojima, M.; Ishii, G.; Atsumi, N.; Fujii, S.; Saito, N.; Ochiai, A. Immunohistochemical Detection of CD133 Expression in Colorectal Cancer: A Clinicopathological Study. Cancer Sci. 2008, 99 (8), 1578–1583.

(27)

Ong, C. W.; Kim, L. G.; Kong, H. H.; Low, L. Y.; Iacopetta, B.; Soong, R.; Salto-Tellez, M. CD133 Expression Predicts for Non-Response to Chemotherapy in Colorectal Cancer. Mod. Pathol. 2010, 23 (3), 450–457.

(28)

Smith, L. M.; Nesterova, A.; Ryan, M. C.; Duniho, S.; Jonas, M.; Anderson, M.; Zabinski, R. F.; Sutherland, M. K.; Gerber, H.-P.; Van Orden, K. L.; Moore, P. A.; Ruben, S. M.; Carter, P. J. CD133/prominin-1 Is a Potential Therapeutic Target for Antibody-Drug Conjugates in Hepatocellular and Gastric Cancers. Br. J. Cancer 2008, 99 (1), 100–109.

(29)

Waldron, N. N.; Kaufman, D. S.; Oh, S.; Inde, Z.; Hexum, M. K.; Ohlfest, J. R.; Vallera, D. A. Targeting Tumor-Initiating Cancer Cells with dCD133KDEL Shows Impressive Tumor Reductions in a Xenotransplant Model of Human Head and Neck Cancer. Mol. Cancer Ther. 2011, 10 (10), 1829–1838.

(30)

Jaxel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.; Pommier, Y. Structure-Activity Study of the Actions of Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for a Specific Receptor Site and a Relation to Antitumor Activity. Cancer Res. 1989, 49


28

Page 29 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6), 1465–1469. (31)

Rothenberg, M. L.; Kuhn, J. G.; Burris, H. A.; Nelson, J.; Eckardt, J. R.; Tristan-Morales, M.; Hilsenbeck, S. G.; Weiss, G. R.; Smith, L. S.; Rodriguez, G. I.; Rock, M. K.; Von Hoff, D. D. Phase I and Pharmacokinetic Trial of Weekly CPT-11. J. Clin. Oncol. 1993, 11 (11), 2194–2204.

(32)

Charasson, V.; Haaz, M. C.; Robert, J. Determination of Drug Interactions Occurring with the Metabolic Pathways of Irinotecan. Drug Metab. Dispos. 2002, 30 (6), 731–733.

(33)

Senter, P. D.; Beam, K. S.; Mixan, B.; Wahl, A. F. Identification and Activities of Human Carboxylesterases for the Activation of CPT-11, a Clinically Approved Anticancer Drug. Bioconjug. Chem. 2001, 12 (6), 1074–1080.

(34)

Chabot, G. G. Clinical Pharmacokinetics of Irinotecan. Clin. Pharmacokinet. 1997, 33 (4), 245–259.

(35)

Lee, S.-Y.; Yang, C.-Y.; Peng, C.-L.; Wei, M.-F.; Chen, K.-C.; Yao, C.-J.; Shieh, M.-J. A Theranostic Micelleplex Co-Delivering SN-38 and VEGF siRNA for Colorectal Cancer Therapy. Biomaterials 2016, 86, 92–105.

(36)

Elfick, A. P. D. Poly(epsilon-Caprolactone) as a Potential Material for a Temporary Joint Spacer. Biomaterials 2002, 23 (23), 4463–4467.

(37)

Ng, K. W.; Hutmacher, D. W.; Schantz, J.-T.; Ng, C. S.; Too, H.-P.; Lim, T. C.; Phan, T. T.; Teoh, S. H. Evaluation of Ultra-Thin Poly(ε-Caprolactone) Films for TissueEngineered Skin. Tissue Eng. 2001, 7 (4), 441–455.


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(38)

Page 30 of 42

Aliabadi, H. M.; Brocks, D. R.; Lavasanifar, A. Polymeric Micelles for the Solubilization and Delivery of Cyclosporine A: Pharmacokinetics and Biodistribution. Biomaterials 2005, 26 (35), 7251–7259.

(39)

Peng, C.-L.; Shieh, M.-J.; Tsai, M.-H.; Chang, C.-C.; Lai, P.-S. Self-Assembled StarShaped

Chlorin-Core

Poly(epsilon-Caprolactone)-Poly(ethylene

Glycol)

Diblock

Copolymer Micelles for Dual Chemo-Photodynamic Therapies. Biomaterials 2008, 29 (26), 3599–3608. (40)

Yin, A. H.; Miraglia, S.; Zanjani, E. D.; Almeida-Porada, G.; Ogawa, M.; Leary, A. G.; Olweus, J.; Kearney, J.; Buck, D. W. AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells. Blood 1997, 90 (12), 5002–5012.

(41)

Mizrak, D.; Brittan, M.; Alison, M. R. CD133: Molecule of the Moment. J. Pathol. 2008, 214 (1), 3–9.

(42)

Chen, H.; Luo, Z.; Dong, L.; Tan, Y.; Yang, J.; Feng, G.; Wu, M.; Li, Z.; Wang, H. CD133/prominin-1-Mediated Autophagy and Glucose Uptake Beneficial for Hepatoma Cell Survival. PLoS One 2013, 8 (2), e56878.

(43)

Zhang, M.; Liu, Y.; Feng, H.; Bian, X.; Zhao, W.; Yang, Z.; Gu, B.; Li, Z.; Liu, Y. CD133 Affects the Invasive Ability of HCT116 Cells by Regulating TIMP-2. Am. J. Pathol. 2013, 182 (2), 565–576.

(44)

Swaminathan, S. K.; Roger, E.; Toti, U.; Niu, L.; Ohlfest, J. R.; Panyam, J. CD133Targeted Paclitaxel Delivery Inhibits Local Tumor Recurrence in a Mouse Model of Breast Cancer. J. Control. Release 2013, 171, 280–287.


30

Page 31 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(45)

Wang, C. H.; Chiou, S. H.; Chou, C. P.; Chen, Y. C.; Huang, Y. J.; Peng, C. A. Photothermolysis of Glioblastoma Stem-like Cells Targeted by Carbon Nanotubes Conjugated with CD133 Monoclonal Antibody. Nanomedicine Nanotechnology, Biol. Med. 2011, 7 (1), 69–79.

(46)

Zhang, J. A.; Xuan, T.; Parmar, M.; Ma, L.; Ugwu, S.; Ali, S.; Ahmad, I. Development and Characterization of a Novel Liposome-Based Formulation of SN-38. Int. J. Pharm. 2004, 270 (1-2), 93–107.

(47)

Tsai, M.-H.; Peng, C.-L.; Yao, C.-J.; Shieh, M.-J. Enhanced Efficacy of Chemotherapeutic Drugs against Colorectal Cancer Using Ligand-Decorated Self-Breakable Agents. RSC Adv. 2015, 5 (112), 92361–92370.

(48)

Pastrana, E.; Silva-Vargas, V.; Doetsch, F. Eyes Wide Open: A Critical Review of Sphere-Formation as an Assay for Stem Cells. Cell Stem Cell 2011, 8 (5), 486–498.

(49)

Su, G.; Zhao, Y.; Wei, J.; Han, J.; Chen, L.; Xiao, Z.; Chen, B.; Dai, J. The Effect of Forced Growth of Cells into 3D Spheres Using Low Attachment Surfaces on the Acquisition of Stemness Properties. Biomaterials 2013, 34 (13), 3215–3222.

(50)

Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33 (9), 941–951.

(51)

Bostad, M.; Berg, K.; Høgset, A.; Skarpen, E.; Stenmark, H.; Selbo, P. K. Photochemical Internalization (PCI) of Immunotoxins Targeting CD133 Is Specific and Highly Potent at Femtomolar Levels in Cells with Cancer Stem Cell Properties. J. Control. Release 2013, 168 (3), 317–326.


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CSLCs-targeted therapy

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Tumor regression

Scheme 1. Schematic diagram of the anti-CD133 antibody-conjugated SN-38-loaded mPEGPCL/mal-PEG-PCL nanoparticles for therapy targeting cancer stem cells.


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Table 1. Characterizations of the synthesized SN-38-loaded nanoparticles. Nanoparticle formulations

D/P ratio

Size (nm)

PDI

Zeta potential (mV)

EE (%)

CE (%)

NPs-SN-38

1/10

150.1

0.152

- 1.25

91.51

-

CD133Ab-NPs-SN-38

1/10

167.1

0.189

- 0.28

90.76

68.92

EE (%) = Encapsulation efficiency (%) = (weight of SN-38 in the nanoparticles/total weight of SN-38 in the loading solution) × 100%. CE (%) = Conjugation efficiency (%) = [(concentration of free probe used for reaction – concentration of unconjugated probe in supernatant)/(concentration of free probe used for reaction)] × 100%.

A

B

Figure 1. Synthesis of mPEG-PCL and mal-PEG-PCL copolymers. (A) Illustration of amphiphilic mPEG-PCL and mal-PEG-PCL polymer synthesis. (B) 1H NMR spectra of mPEGPCL and mal-PEG-PCL polymer in CDCl3.


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A

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B

Days

Days

C

Polymer concentration (μg/mL)

Polymer concentration (μg/mL)

Figure 2. The stability and cytotoxicity of synthesized nanoparticles. (A) The size and (B) encapsulation efficiency of SN-38-loaded mPEG-PCL/mal-PEG-PCL nanoparticles were measured every day for 5 days. (C) The cell viability of HCT116 and HT29 cells after incubation with mPEG-PCL/mal-PEG-PCL nanoparticles (NPs) and anti-CD133 antibody-conjugated mPEG-PCL/mal-PEG-PCL nanoparticles (CD133Ab-NPs) for 72 hours.


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Figure 3. Expression level of CD133 surface marker in HT-29, HCT116, SW620 and CCS colorectal cancer cells. (A) The CD133 expression profiles in the four colorectal cancer cells were analyzed by flow cytometry. (B) Fluorescent images of CD133 expression in the four colorectal cancer cells (blue: Hoechst, red: PE) and the quantification of fluorescence intensity.


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A

B

Figure 4. The cell binding affinity and cellular uptake of CD133Ab-NPs-SN-38. (A) The binding affinity of CD133Ab-NPs-SN-38 and non-targeted nanoparticles in HCT116 (CD133 overexpression) and HT-29 (CD133 low-expression). Cells were treated at 4 °C for 1 hour. In competition tests, cells were pre-blocked with free anti-CD133 antibody at 4 °C for 30 minutes before the treatment. (B) Cellular uptake of FITC-labeled targeted and non-targeted


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nanoparticles in HCT116 and HT29 (37 °C for 3 hours; blue: Hoechst, green: FITC) * = P

Targeting Colorectal Cancer Stem-Like Cells with ... - ACS Publications

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