Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells

Biomaterials xxx (2014) 1e10

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Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells Rong Sun a, 1, Yang Liu b, 1, Shi-Yong Li b, Song Shen b, Xiao-Jiao Du b, Cong-Fei Xu a, Zhi-Ting Cao a, Yan Bao b, Yan-Hua Zhu b, Ya-Ping Li d, Xian-Zhu Yang b, Jun Wang a, b, c, * a

Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230027, China CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui 230026, China c High Magnetic Field Laboratory of CAS, University of Science and Technology of China, Hefei, Anhui 230026, China d Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 July 2014 Accepted 2 October 2014 Available online xxx

Combination treatment through simultaneous delivery of two or more drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employ a combination therapy for eliminating both the bulk tumor cells and the rare cancer stem cells (CSCs) that have a high self-renewal capacity and play a critical role in cancer treatment failure. All-trans-retinoic acid (ATRA), a powerful differentiation agent of cancer stem cells and the clinically widely used chemotherapy agent doxorubicin (DOX) are simultaneously encapsulated in the same nanoparticle by a single emulsion method. It is demonstrated that ATRA and DOX simultaneous delivery-based therapy can efficiently deliver the drugs to both non-CSCs and CSCs to differentiate and kill the cancer cells. Differentiation of CSCs into non-CSCs can reduce their self-renewal capacity and increase their sensitivity to chemotherapy; with the combined therapy, a significantly improved anti-cancer effect is demonstrated. Administration of this combinational drug delivery system can markedly augment the enrichment of drugs both in tumor tissues and cancer stem cells, prodigiously enhancing the suppression of tumor growth while reduce the incidence of CSC in a synergistic manner. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Cancer stem cell All-trans-retinoic acid Doxorubicin Combination therapy Co-delivery

1. Introduction Nanomedicine has been delivering significant benefits to the treatment of cancer over recent decades [1e3] that may overcome the limitations of therapeutic agents via prolonging the circulation half-life, improving pharmacokinetics and increasing their uptake by tumor cells [4,5]. Drug delivery systems with nanoparticles have been demonstrated to increase therapeutic efficacy against the most difficult cancer challenges, including drug resistance and tumor metastasis by incorporating targeting strategies and multifunctional capabilities [6e9]. Cancer stem cells (CSCs), also called tumor-initiating cells or stem-like cancer cells, are known to be resistant to chemotherapy and radiotherapy and are associated with tumor metastasis and recurrence after treatments [10e12]; as

* Corresponding author. School of Life Sciences and Medical Center, University of Science & Technology of China, Hefei, Anhui 230027, China. Tel.: þ86 551 63600335; fax: þ86 551 63600402. E-mail address: [email protected] (J. Wang). 1 These authors contribute equally to this work.

such, they have attracted increasing attention in recent years with regard to the development of advanced therapeutic methods. The possible therapeutic strategies that can eliminate CSCs generally include inhibiting their self-renewal pathway, differentiating the CSCs, or targeting the CSCs' niche [13e17]. In practice, delivering drugs into the rare population of CSCs in tumor tissue is still challenging. Recently, studies of nanoparticle delivery systembased approaches to tackle the CSC problem have been reported [18,19]. The superior performance of nanoparticles show some therapeutic advantages for CSCs therapy as well, such as enrichment of therapeutic agents within CSCs and delivery of more than one functional agent to CSCs [20,21]. Several studies have successfully targeted and eliminated CSCs using a nanoparticle mediated delivery system, showing good results in overcoming tumor drug resistance and relapse [19,22]. Our previous studies also demonstrated that dual pH-sensitive polymeredrug conjugate nanoparticles showed enhanced inhibition to the progression of drug-resistant SK-3rd cancer stem cells [23], and gold nanoparticles conjugated with doxorubicin via hydrazone bonds can deliver more doxorubicin (DOX) to CSCs by overcoming drug resistance through

http://dx.doi.org/10.1016/j.biomaterials.2014.10.018 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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R. Sun et al. / Biomaterials xxx (2014) 1e10

evading the efflux of P-gp, resulting in the removal of all tumor cell sub-populations and averting the potential repopulation of the tumor mass by CSCs [24]. Therefore, nanoparticle-based drug delivery systems offer a potential approach for effectively targeting CSC therapies. However, evidence exists indicating that non-CSCs in the tumor can spontaneously and stochastically turn into CSCs de novo [25,26], undermining the efficacy of therapeutic strategies that only target CSCs [27,28]. Hence, it is crucial to eliminate CSCs and nonCSCs simultaneously for effective cancer therapy. Recently, combination therapy has been developed for efficient therapy [29e31]. Co-delivery (or simultaneous delivery) is an approach that delivers two or more different functional agents in one nanoparticle. Studies have demonstrated that simultaneous delivery systems show more significant therapeutic efficiency than administration of single drug loaded nanoparticles [32e34]. More importantly, there is evidence demonstrating that co-delivery of two agents may have a synergistic effect on cancer therapy, which is not observed with a simple physical mixture of two individual drug loaded nanoparticles [35,36]. The co-delivery systems used possess some unique features, such as the similar pharmacokinetics of the two drugs and simultaneous delivery of two agents into the same cell by one nanoparticle [29,35]. The application of a co-delivery system for targeting CSC and non-CSC therapy in a synergistic manner has not been reported yet. All-trans-retinoic acid (ATRA) is a powerful differentiating agent that acts through obstructing multiple signaling pathways involved in stem cell maintenance [37e39]. However, no effective cytotoxicity and tumor inhibition can be obtained with ATRA treatment [19,40]. Therefore, combination with other therapeutics would be requisite for efficient cancer therapy, killing both CSCs and normal cancer cells. DOX has been employed as a traditional chemotherapeutic drug for multiple cancer therapies but may be resisted by CSCs in many solid tumors, which may also further enrich their CSCs after treatment, resulting in chemoresistance, tumor relapse and metastasis [41e43]. In this study, we address a promising

strategy of co-delivery of an ATRA and DOX based-therapy for both CSCs and non-CSCs (Scheme 1). Nanoparticles simultaneously encapsulating two drugs (ATRA and DOX) were prepared by a single emulsion method, and we demonstrate that drug loaded nanoparticles effectively increase drug uptake by breast CSCs. Treatment with these nanoparticles induces breast CSC differentiation by ATRA, which attenuates their tumor initiating ability and eventually enhances the cytotoxicity of DOX. Furthermore, intravenous administration of the nanoparticles effectively increases the enrichment of the drugs both in tumor tissue and cancer stem cells, and co-delivery of ATRA and DOX remarkably enhances the suppression of tumor growth while decreasing breast CSCs in the tumor in a synergistic manner. 2. Materials and methods 2.1. Materials Methoxy polyethylene glycol (MW ¼ 5000) was purchased from SigmaeAldrich (St. Louis, MO). DL-Lactide was purchased from SigmaeAldrich (St. Louis, MO). The block copolymer poly(ethylene glycol)-block-polylactide (PEG-b-PLA) was synthesized by ring-opening polymerization of D,L-lactide with methoxy polyethylene glycol as the initiator according to a previously reported method [44]. The average number of repeated polymerization units of D,L-lactide was 76, equal to a molecular weight of 11,000 for the PLA block. ATRA was purchased from SigmaeAldrich (St. Louis, MO). Doxorubicin hydrochloride was a product of Hisun Pharmaceutical Co (Hangzhou, China). The hydrophobic DOX was made according to the method previously reported [5]. The ALDEFLUOR™ KIT was purchased from STEMCELL Technologies (Vancouver, Canada). Ultra-purified water was prepared using a Milli-Q Synthesis System (Millipore, Bedford, MA). All other solvents and reagents were used as received. 2.2. Preparation of ATRA- and DOX-loaded nanoparticles DOX- and ATRA-loaded nanoparticles were prepared by a single-emulsion technique. As a typical example, a solution of ATRA (8 mg), DOX (2 mg) and PEGb-PLA (100 mg) in a mixed solvent of 2 mL of chloroform and dimethyl sulfoxide (DMSO) (3:1, v/v) was emulsified in 8 mL of ultra-purified water by probe ultrasonication at 450 W (Sonics & Materials, Newtown, CT) for 2 min over an ice bath to form an oil-in-water emulsion. The organic solvent chloroform was evaporated by using a rotary vacuum evaporator and the resulting product was further dialyzed against water for 12 h to remove DMSO with membrane dialysis tubing (molecular weight cutoff of 3.5 kDa, Spectrum Laboratories, Rancho Dominguez, CA). The free

Scheme 1. Schematic illustration of tumor suppression by eliminating both CSCs and non-CSCs by co-delivery of the ATRA and DOX nanoparticle system.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

R. Sun et al. / Biomaterials xxx (2014) 1e10 ATRA and DOX were eventually discarded by centrifugation at 1000 g for 15 min. The resulting drug-loaded nanoparticles were denoted as NPDOx, NPATRA or NPATRA/DOx, indicating nanoparticles encapsulated with DOX, or ATRA, or ATRA and DOX together, respectively. 2.3. Cell culture The human breast cancer cell MDA-MB-231 from American Type Culture Collection was cultured in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) supplied with 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China) at 37  C with 5% CO2. For mammosphere culture, MDA-MB-231 cells (1000 cells/mL) were cultured in suspension with serum-free DMEM-F12 (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen, Carlsbad, CA), 20 ng/mL hEGF (BD Biosciences, Franklin Lakes, NJ), 0.4% low-endotoxin bovine serum albumin (Sangon Biotech, Shanghai, China) and 4 mg/mL insulin (SigmaeAldrich, St. Louis, MO). To stimulate propagation in vitro, the mammospheres were collected by gentle centrifugation (800 g, 5 min), dissociated into single cells as previously described, and then cultured to generate mammospheres of the next generation. 2.4. Animals Female NOD/SCID mice were obtained from Vital River Laboratories (Beijing, China) and used at 6e8 weeks of age. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. 2.5. Cancer stem cell identification The ALDEFLUOR kit was used to analyze the acetaldehyde dehydrogenase (ALDH) activity of breast mammosphere cells or cells digested from tumor tissues following the manufacturer's protocol. The cells were acquired and analyzed using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). The proportion with ALDH was determined using FlowJo Software (TreeStar, San Carlos, CA). 2.6. Cellular uptake of ATRA and DOX 6

The MDA-MB-231 mammosphere cells were seeded at a density of 1  10 cells per well on 12-well tissue culture plates in complete DMEM/F12 culture medium. After 24 h, they were treated with equivalent doses of ATRA and DOX in different formulations for 4 h to final concentrations of 15 mg/mL and 5 mg/mL, respectively. After removing the media by centrifugation at 250 g for 5 min, cells were washed twice with cold phosphate buffered saline (PBS, 0.01 M, pH 7.4), treated with trypsin (0.05%, with 0.02% ethylene diamine tetraacetic acid, Gibco, Canada), then washed with PBS twice and stained with the ALDEFLUOR kit. The ALDHhi cells were sorted by MoFlo Astrios (Beckman Coulter). DOX fluorescence in ALDHhi was then analyzed using FlowJo Software. For quantitative determination, the ALDHhi cells were lysed with 100 mL DMSO and the drug contents were further determined by HPLC (for details see Supporting Information). The DOX fluorescence in ALDHhi was also analyzed by flow cytometry. The mammosphere cells were seeded at a density of 1  105 cells per well onto 24-well tissue culture plates in complete DMEM/F12 culture medium. After 24 h, they were treated with free DOX, NPDOx, or NPATRA/DOx, at a concentration of 5 mg/mL for 4 h. The cells were collected and stained with the ALDEFLUOR kit as described above and analyzed by flow cytometer. DOX-loaded nanoparticles (NPDOx, NPATRA/DOx) were used to investigate their intracellular distribution in MDA-MB-231 mammosphere cells with confocal laser scanning microscope. The mammosphere cells were seeded at a density of 2  104 cells per well onto 24-well tissue culture plates in complete DMEM/F12 culture medium. After 24 h, they were treated with free DOX, NPDOx, or NPATRA/DOx at a concentration of 2 mg/mL for 4 h. The cells were collected and stained as above described and imaged with a Zeiss LSM 710 confocal microscope using a 63 objective. 2.7. Determination of ALDHhi proportion after drug-loaded nanoparticle treatment in vitro MDA-MB-231 mammosphere cells were seeded at a density of 5  104 cells per well onto 24-well tissue culture plates in complete DMEM/F12 culture medium. After 24 h, they were treated with equivalent doses of ATRA and DOX in different formulations at 1.5 mg/mL and 0.1 mg/mL, respectively. After 4 days, the proportions with ALDH were acquired and analyzed by using a FACS Calibur flow cytometer as mentioned above. 2.8. The expression of stemness-associated gene MDA-MB-231 mammosphere cells were seeded at a density of 2  105 cells per well onto 12-well tissue culture plates in complete DMEM/F12 culture medium. After 24 h, they were treated with various equivalent formulations of ATRA and DOX at doses of 1.5 mg/mL and 0.1 mg/mL for 4 days, respectively. The total RNA in the cells was then collected using RNAiso Plus (TaKaRa, Dalian, China). One microgram of total RNA was transcribed into cDNA using the PrimeScript™ RT reagent Kit (TaKaRa,

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Dalian, China). To assess mRNA levels of Oct4, Nanog, and Sox2, real-time quantitative PCR (qPCR) was performed using Fast Start Universal Probe Master (Roche Applied Science, Indianapolis) with forward 50 -TGGCGTGGAGACTTTGCA-30 and reverse 50 -GAGGTTCCCTCTGAGTTGCTTTC-30 Oct 4 primers, forward 50 -GGTTGAAGACTAGCAATGGTCTGA-3 0 and reverse 50 -TGCAATGGATGCTGGGATACTC-30 Nanog primers, and forward 50 -TTGCTGCCTCTTTAAGACTAGGA-30 and reverse 50 CTGGGGCTCAAACTTCTCTC-30 Sox2 primers. The mRNA levels were normalized against the housekeeping gene GAPDH using forward 50 -ATCAAGAAGGTGGTGAAGCAGGCA-30 and reverse 50 -TGGAAGAGTGGGAGTTGCTGTTGA-30 primers. The PCR parameters consisted of 60 s of Taq activation at 95  C, followed by 40 cycles of PCR at 95  C  20 s, and 1 cycle of 95  C  15 s, 57  C  60 s and 95  C  15 s Oct4, Nanog and Sox2 mRNA levels were finally normalized to those cells with PBS treatment alone.

2.9. In vitro and in vivo limiting dilution assays (LDA) For in vitro LDA, MDA-MB-231 mammosphere cells were incubated with ATAR and DOX in various equivalent formulations of ATRA and DOX at doses of 1.5 mg/mL and 0.5 mg/mL, respectively, for 4 h. Then the mammosphere cells were collected and serially diluted from 128 to 2 cells per well in an ultralow attachment 96-well culture plate with complete mammosphere medium. Cells from each group were seeded in 10 wells. After 14 days, colonies larger than 100 mm3 were counted under the microscope. Limiting dilutions were calculated using Extreme Limiting Dilution Analysis software [45]. For in vivo LDA, MDA-MB-231 mammosphere cells were treated in the same manner as above and then transplanted into the mammary fat pads of female NOD/ SCID mice. Each mouse received 10,000 to 100 treated mammosphere cells. Tumors were monitored every 3 days by observation and palpation for up to 60 days. Cancer stem cell frequency was calculated using the L-calc program (Stem-Cell Technologies Inc.).

2.10. Pharmacokinetics studies DOX, ATRA, ATRA/DOX, NPDOx, NPATRA, NPATRA/NPDOx, and NPATRA/DOx were injected via i.v. into ICR mice at equivalent DOX and ATRA doses of 5 mg/kg and 15 mg/kg (n ¼ 3 for each group), respectively. At the predetermined time point, blood samples were collected from the retro-orbital plexus of the mouse into 1000 U/mL heparin sodium in PBS solution (10 mL). The blood was then centrifuged at 4  C (3000 g, 5 min) to collect the plasma. For analysis, the plasma was extracted with a chloroform/methanol mix (1 mL, 5:1, v/v) on a vortex-mixer for 90 s. Following centrifugation at 10,000 g for 5 min, the organic solution was collected and dried under a vacuum centrifugal concentration meter (Eppendorf, Hamburg, Germany), and the residue was dissolved in 200 mL DMSO. The concentrations of ATRA and DOX were analyzed by HPLC.

2.11. Accumulation of ATRA and DOX in tumors and CSCs DOX, ATRA, ATRA/DOX, NPDOx, NPATRA, NPATRA/NPDOx, and NPATRA/DOx were injected via i.v. into NOD/SCID mice with MDA-MB-231 breast tumors when the tumor volume was approximately 150 mm3. The DOX and ATRA doses were equivalent to 5 mg/g and 15 mg/kg body weight, respectively. After 24 h, the mice were sacrificed and tumor tissues were collected. The tissues were weighted and homogenized and the DOX and ATRA distribution in the tumor tissues were determined after extraction by HPLC as previously mentioned. For FACS analyses, the tumor tissues were transferred to a dish and cut into small pieces. The fragments were suspended in 20 mL of DMEM-F12 medium and collected by centrifugation for 5 min at 800 rpm. The pelleted materials were resuspended in 10 mL of tumor cell digestion solution (1 mg/mL collagenase Type I in PBS, Invitrogen, Carlsbad, CA) and incubated at 37  C for 3 h with persistent agitation. The tumor cells were collected by centrifuging at 1200 rpm for 6 min at room temperature and then washed twice with PBS containing 1% FBS. The tumor cells were filtered twice through a 200-mesh sieve, stained with ALDH substrate as described above, and analyzed using a FACS Calibur flow cytometer.

2.12. Orthotropic xenograft model and tumor suppression study The xenograft tumor model was generated by subcutaneous injection of MDAMB-231 cells (2  105) diluted in matrigel (3:1, BD Biosciences, Franklin Lakes, NJ) into the mammary fat pads of NOD/SCID mice. The mice were randomly divided into 9 groups when the tumor volume was approximately 70 mm3. Animals were treated with PBS, NP, free DOX, free ATRA, ATRA/DOX, NPATRA, NPDOx, NPATRA/NPDOx, and NPATRA/DOx by i.v. injection every two days. The ATRA dose per injection was equivalent to 3.0 mg/kg body weight and the DOX dose per injection was equivalent to 1.0 mg/kg body weight. Tumor growth was monitored by measuring the perpendicular diameter by caliper every other day from the 14th day to 34th day after xenograft implantation. The total doses of ATRA and DOX was 33 mg/kg and 11 mg/kg respectively. The estimated volume was calculated based on the following equation: tumor volume ¼ 1/2  length  width2.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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2.13. Analysis of the proportion of CSCs in tumors At the conclusion of the tumor suppression study, the animals were sacrificed and tumor tissues were excised. The tumor tissues were transferred to a dish and cut into small pieces. The fragments were suspended in 20 mL of DMEM/F12 medium and collected by centrifugation for 5 min at 600 rpm. The pelleted materials were resuspended in 10 mL of tumor cell digestion solution (1 mg/mL collagenase Type I in PBS, Invitrogen, Carlsbad, CA) and incubated at 37  C for 3 h with persistent agitation. The tumor cells were collected by centrifuging at 1200 rpm for 6 min at room temperature and then washed twice with PBS containing 1% FBS. The tumor cells were filtered twice through a 200-mesh sieve, stained with ALDH substrate as described above for FACS analyses. 2.14. Immunohistochemical analysis Twenty-four hours after the last treatment, tumor tissues and organs were excised. The tissues were fixed in 4% formaldehyde (SigmaeAldrich, St. Louis, MO) and embedded in paraffin for analysis. Paraffin-embedded 6 mm tumor and organ sections were prepared for immunohistochemical analysis. The proliferation of tumor cells was detected using an antibody against proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotech, Santa Cruz, CA). Deparaffinized slides were boiled for 5 min in 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker for antigen retrieval. Subsequently, slides were allowed to cool for another 5 min in the same buffer. After three rinses in PBS and pre-treatment with blocking medium for 5 min, slides were incubated with the PCNA antibody diluted to 1:200 in antibody diluent solution for 60 min at room temperature. After washing slides in Tris-buffered saline, a streptavidinebiotin system was used according to the manufacturer's instructions (BioGenex, San Ramon, CA). The slides were counterstained using Aquatex (Merck, Gernsheim, Germany). All sections were examined under a Nikon TE2000 microscope (Tokyo Prefecture, Japan). 2.15. Statistical analysis The statistical significance of treatment outcomes was assessed using Student's t-test (two-tailed); p < 0.05 was considered statistically significant in all analyses (95% confidence level). The synergism of the combined therapy was evaluated by the combination index (c.i.) method [46,47]. A c.i. of 1 indicates an effect between two agents, whereas a c.i. <1 or c.i. >1 indicates synergism or antagonism, respectively.

3. Results and discussion 3.1. Regulated preparation and characterization of ATRA and DOXloaded nanoparticles In a co-delivery system, simultaneous encapsulation of different agents in one nanoparticle is essential. High encapsulation efficiency and control over drug contents must be achieved at the same time. It has been documented in previous studies that the molar ratio can govern whether two drugs act synergistically, additively or antagonistically [29,35]. Therefore, modulation of the ratio of different agents is very important in applying suitable doses of different drugs for effective cancer treatment. In order to find an optimal proportion of ATRA and DOX in this work, we used PEG-bPLA to prepare the nanoparticles though a single emulsion method. By incorporating different ratios of ATRA and DOX in a solution of PEG-b-PLA matrix, we demonstrated that ATRA and DOX could be

simultaneously immobilized in the formed nanoparticles. As summarized in Table 1, various ratiometric drug loadings of ATRA and DOX in nanoparticles (ATRA/DOX from ~0.1 to 15, w/w) could be prepared by regulating the feeding amounts of ATRA and DOX, and the encapsulation efficiencies of ATRA and DOX were approximately 40% and 50%, respectively. The prepared nanoparticles (NPDOx, NPATRA and NPATRA/DOx) exhibited similar diameters (~140 nm) and almost neutral zeta potential with a spherical shape as shown in Fig. S1A. In addition, the release of drugs from nanoparticles might be a crucial element for synergetic therapy. The in vitro release profiles of ATRA and DOX from the NPs were investigated by HPLC. In Fig. S1B and C, the results show that ATRA and DOX each presented a steady sustained release from NPs over 8 days (nearly 55% of drug released ATRA or DOX from NPATRA or NPDOx). However, ATRA and DOX both presented slower release speeds when they were simultaneously encapsulated in nanoparticles (nearly 40% and 36% drug release of ATRA and DOX, respectively, from NPATRA/DOx). 3.2. Simultaneous delivery of DOX and ATRA to breast CSCs by drug-loaded nanoparticles ATRA and DOX should be simultaneously delivered to CSCs in order to induce CSC differentiation by ATRA and subsequently kill them by DOX. To investigate whether the nanoparticles could deliver the drugs to CSCs, we first enriched CSCs in mammosphere cell cultures of MDA-MB-231 cells in serum-free medium [13,47]. Breast cancer cells with ALDHhi phenotypes have been demonstrated to have stem cell-like tumor-initiating and invasive features [48,49]. For instance, flow cytometric analysis of MDA-MB-231 mammosphere cells demonstrated a 9.9-fold increase in the ALDHhi population compared with adherent cells (Fig. S2A, proportions of ALDHhi cells were 33.6% and 3.4% in mammosphere cells and adherent cells, respectively). Moreover, it has demonstrated that Nanog, Sox2, Oct4 and ALDH are highly expressed in breast CSCs [50e52]. The qRT-PCR analysis indicated an increased expression of stemness-associated genes in mammosphere cells compared with adherent cells (Fig. S2B). Furthermore, the mammosphere cells were more resistant to DOX than adherent cells (Fig. S2C). In addition, the result of in vivo LDA also demonstrated that the oncogenicity of mammosphere cells was greater than that of adherent cells (Fig. S2D). These results indicated that CSCs could be enriched in non-adherent mammosphere cell cultures. To evaluate the properties of nanoparticle delivery to CSCs, we incubated mammosphere cells with drug-loaded nanoparticles or free drugs for 4 h. Then, CSCs (ALDHhi population) were sorted from the incubated cells. Quantitative analysis of intracellular total ATRA and DOX contents by high-performance liquid chromatography

Table 1 The size, zeta potential, drug loading content and drug loading efficiency of nanoparticles with different ratios of ATRA and DOX. Feeding ratio of ATRA/DOX (w/w)

Resultant ratio of ATRA/DOX (w/w)

Diametera (nm)

1:0 0:1 18:1 10:1 4:1 2:1 1:2 1:5 1:10

1:0 0:1 15.02 8.52 3.00 1.39 0.38 0.15 0.07

150.10 137.11 145.14 148.12 146.50 151.60 141.42 143.32 140.16

a b c d

± ± ± ± ± ± ± ± ±

2.04 2.16 1.12 1.13 3.47 1.83 1.98 3.06 2.18

Zeta potentialb (mV)

DLC (%)c ATRA

DOX

ATRA

DOX

1.14 1.44 1.01 0.12 0.17 1.80 1.25 2.60 3.11

4.32 0.00 6.34 3.76 3.03 3.05 1.45 0.83 0.33

0.00 4.67 0.42 0.45 1.01 2.19 3.83 5.56 4.76

45.21 0.00 37.90 39.54 39.45 40.01 38.56 44.43 35.32

0.00 48.95 45.46 46.41 52.60 57.57 50.74 59.24 50.46

± ± ± ± ± ± ± ± ±

0.18 0.20 0.14 0.11 0.13 0.11 0.07 0.39 0.15

DLE (%)d

Z-average diameter determined by dynamic light scattering (DLS). Zeta potential determined by DLS. Drug-loading content (DLC) determined by high-performance liquid chromatography (HPLC). Drug-loading efficiency (DLE) determined by HPLC.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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(HPLC) showed that the nanoparticles (NPATRA, NPDOx, mixed delivery system NPATRA/NPDOx, and co-delivery system NPATRA/DOx) could more effectively deliver ATRA and DOX to breast CSCs in comparison to culturing with free DOX and DOX (Fig. 1A). Notably, the ratio of intracellular ATRA and DOX delivered by nanoparticles was consistent with the ratio of the two drugs added (the

Fig. 1. Nanoparticles significantly increase cellular uptake of ATRA and DOX by CSCs in vitro. (A) HPLC analyses of intracellular ATRA and DOX concentrations in ALDHhi MDA-MB-231 mammosphere cells after a 4 h treatment with different formulations. (B) Flow cytometric analyses of DOX fluorescence in ALDHhi MDA-MB-231 mammosphere cells after a 4 h treatment with different formulations. MFI: mean fluorescence intensity. (C) Confocal laser scanning microscopy images of MDA-MB-231 mammosphere cells incubated with different formulations for 4 h. The CSCs were labeled with ALDEFLUOR™ (green). Data are shown as means ± s.d. (n ¼ 3). *p < 0.05, **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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intracellular ratio of ATRA and DOX was 2.9 and the added ratio was 3.0), but it was less than the added drug ratio when they were combinationally treated with free ATRA and DOX (the intracellular ratio of ATRA and DOX was 2.2 while the added ratio was 3.0), indicating reduced uptake of free ATRA. Moreover, the uptake of NPATRA/DOx was similar to that of NPATRA/NPDOx, the results demonstrating that NPATRA/DOx could simultaneously deliver ATRA and DOX to CSCs. In addition, we also demonstrated that the nanoparticles (NPATRA, NPDOx, NPATRA/NPDOx, NPATRA/DOx) could deliver drugs to bulk cancer cells (Fig. S3). The adherent MDA-MB231 cells were incubated with free drugs and drug-loaded nanoparticles. Quantitative analyses of intracellular total DOX and ATRA contents following treatment showed that the drug-loaded nanoparticles could be internalized by the adherent cells. Furthermore, the fluorescence of DOX in ALDHhi cells analyzed by flow cytometer showed a similar uptake tendency as the results of the quantitative analysis (Fig. 1B). The intracellular DOX fluorescence after treatment with nanoparticles was approximately 2.5fold stronger when compared with free DOX treatment, and no difference was observed between the NPDOx, NPATRA/DOx and NPATRA/ NPDOx treatments. In addition, confocal microscopy corroborated the same result. The mammosphere cells were incubated with free

Fig. 2. NPATRA/DOx significantly induces CSC differentiation and affects the stemness of the CSCs. (A) The proportion of ALDHhi cells in MDA-MB-231 mammosphere cells after 4 days of treatment with different formulations. (B) In vitro limiting dilution assays performed with MDA-MB-231 mammosphere cells on day 14 after pretreatment with different formulations for 4 h. The CSC frequency was calculated using Extreme Limiting Dilution Analysis software. Data are shown as means ± s.d. (n ¼ 3).

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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DOX and DOX-loaded nanoparticles for 4 h. The CSCs were labeled with the ALDEFLUOR™ kit, showing green fluorescence. As depicted in Fig. 1C, the disappearance of green fluorescence upon blocking ALDH with diethylaminobenzaldehyde (DEAB) indicated successful labeling of CSCs, and the percentage of green fluorescence-labeled cells in mammosphere cells was about 30%, which was consistent with the FACS analysis. After incubation with DOX-loaded NPs (NPDOx and NPATRA/DOx), obvious DOX fluorescence was observed in both ALDH-negative cells and ALDHhi cells, whereas DOX fluorescence was only observed in ALDH-negative non-CSCs but not in ALDHhi cells when treated with free DOX. The above results demonstrated that the co-delivery nanoparticle system could simultaneously deliver more ATRA and DOX to CSCs than utilizing the free drugs, which might lead to better therapeutic efficiency.

Fig. 3. The expression of stemness-associated genes (Nanog, Sox2 and Oct4) in MDAMB-231 mammosphere cells after 4 days of treatment with different formulations. Data are shown as means ± s.d. (n ¼ 3).

3.3. Simultaneous delivery of ATRA and DOX in nanoparticles significantly inhibits cancer initiating activity of CSCs To evaluate whether ATRA could differentiate CSCs in vitro, flow cytometry was used to analyze the ALDHhi population of mammosphere cells in different concentrations of ATRA. The results showed that ATRA reduced the ALDHhi population in mammosphere cells in a dose-dependent manner (Fig. S4A, ALDHhi cell ratios decreased from 20.6% to 5.4% as ATRA was increased from 100 nM to 10 mM, respectively). Moreover, the MTT assay indicated that very weak cytotoxicity was observed with ATRA in the 0.3 mg/ mL to 1.5 mg/mL range (Fig. S4B). After verifying the differentiating capacity of ATRA on CSCs, we sought to examine whether simultaneous delivery of ATRA and DOX via nanoparticles can inhibit cancer initiation. On account of the enhanced cytotoxicity of NPATRA/DOx with various ATRA and DOX concentrations, we chose a suitable ATRA dose of 1.5 mg/mL that exerted low cytotoxicity (Fig. S4B) and high differentiation ability. Meanwhile, we chose to use DOX at 0.1 mg/mL with low cytotoxicity to MDA-MB-231 mammosphere cells. MDA-MB-231 mammospheres cells were incubated with NPATRA/DOx simultaneous delivery nanoparticles or various other formulations at equivalent DOX (0.1 mg/mL) and ATRA doses (1.5 mg/mL) for 4 days. As indicated in Fig. 2A, CSCs (identified as the ALDHhi population) were enriched by treatment with free

Fig. 4. In vivo limiting dilution assays. The tumorigenic ability (A) and CSC frequency (B) of MDA-MB-231 mammosphere cells serially transplanted into NOD/SCID mice after pretreatment with different formulations for 4 h. Tumors were monitored every 3 days by observation and palpation for up to 60 days.

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DOX, which could be reduced when combined with ATRA. The proportion of ALDHhi cells in mammosphere cells was 38.1% when treated with DOX, which was reduced to 11.7% when combinationally treated with ATRA. With NPDOx treatment, CSCs were not enriched (ALDHhi cell proportion 27.0%), and could be reduced by NPATRA (ALDHhi cell proportion 7.6%). The drug loaded nanoparticles (NPATRA/NPDOx or NPATRA/DOx) showed a more effective decrease in CSCs than free ATRA/DOX (the proportion of ALDHhi cells in mammospheres was 7.4% or 6.3% when treated with NPATRA/ NPDOx or NPATRA/DOx, respectively), which might be attributed to the increased uptake of drugs in nanoparticles by CSCs. In order to prove that the simultaneous delivery of ATRA and DOX nanoparticles could inhibit cancer initiation, in vitro and in vivo LDA were performed in both the tumor-initiating and nontumor-initiating fractions. As shown in Fig. 2B, in vitro LDA showed that the CSCs frequency of MDA-MB-231 mammosphere cells could be decreased by ATRA treatment, and the drug loaded NPs (NPATRA, NPDOx, NPATRA/NPDOx, NPATRA/DOx) could reduce the frequency of CSCs compared with the respective free drugs. Importantly, treatment with NPATRA/DOx showed a significant decrease of the CSCs frequency compared with NPATRA/NPDOx treatment. In addition, the expression of stemness-associated genes Nanog, Sox2 and Oct4, three of the most common genes overexpressed in breast CSCs, was determined. The mammosphere cells were treated as described above and analyzed by qRT-PCR. We found increased expression of the three genes after treatment with free DOX and decreased expression upon differentiation of the CSCs

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by combining DOX with ATRA (Fig. 3). Moreover, drug loaded nanoparticles also revealed greater suppression of expression of stemness-associated genes. Meanwhile, nanoparticles alone had no effect on the genes' expression and a slight increase in the geness expression was evident after treating with NPDOx, while a significant decrease in the genes' expression on mammosphere cells was shown by NPATRA treatment. Importantly, NPATRA/NPDOx or NPATRA/ DOx treatment showed more effective suppression of expression of the three genes than the treatment with free ATRA/DOX, supporting the results shown in Fig. 2A. Furthermore, we determined the number of cells required to generate tumors and the CSC frequency in vivo. MDA-MB-231 mammosphere cells were treated with various formulations for 4 h. Equal cell numbers from each of the treatment groups were then injected into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (n ¼ 5) at three different cell numbers, and the tumor formation rate was assessed after 60 days. As shown in Fig. 4A and B, the treatment with ATRA/DOX significantly reduced the tumor forming ability and CSC frequency of the cells compared to either DOX or ATRA, which indicated better inhibition of tumor initiation by the combination differentiation therapy and chemotherapy. Moreover, there was an increased inhibition of tumor forming ability and CSC frequency in mammosphere cells following treatment with NPDOx or NPATRA compared to either free DOX or free ATRA. In addition, the cells treated with either NPATRA/NPDOx or NPATRA/DOx showed reduced tumor forming ability and CSC frequency than either NPDOx or NPATRA and, importantly, treatment with NPATRA/DOx was more

Fig. 5. Plasma ATRA (A) and DOX (B) concentration versus time after intravenous administration of different formulations for 24 h at an equivalent dose of 15 mg ATRA and 5 mg DOX per kg of mice body. (C) HPLC analyses of the ATRA and DOX concentrations in MDA-MB-231 xenograft tumors 24 h after i.v. administration of different formulations. (D) Flow cytometric analyses of DOX fluorescence in ALDHhi MDA-MB-231 xenograft tumor cells after i.v. administration of different formulations. MFI: mean fluorescence intensity. Data are shown as means ± s.d. (n ¼ 3), **p < 0.01.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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efficient than NPATRA/NPDOx in inhibiting the tumor forming ability of mammosphere cells in a synergistic manner. 3.4. Simultaneous delivery of ATRA and DOX with nanoparticles significantly increases the accumulation of DOX and ATRA in tumor and CSCs in vivo Nanoparticle-mediated therapeutics have been demonstrated to enhance anticancer effects compared with small-molecule therapeutic agents, a feature attributed to their overcoming the limitations of the therapeutic agents via their longer circulation half-lives, improved pharmacokinetics and highly specific tumor site enrichment [4,5]. Herein, we investigated whether ATRA and DOX loaded nanoparticles could prolong circulation half-lives and enhance the delivery of DOX and ATRA into engrafted tumors and CSCs in vivo. The ATRA and DOX concentrations in plasma versus time after intravenous administration of free drugs or drug-loaded nanoparticles are shown in Fig. 5A and B. The results were consistent with previous reports that free DOX or ATRA is rapidly eliminated following injection [53,54]. However, drug loaded nanoparticles showed significantly slower clearance than free drugs. More importantly, ATRA and DOX in nanoparticles (NPATRA, NPDOx, NPATRA/NPDOx, NPATRA/DOx) had similar pharmacokinetics. To further verify whether the nanoparticles could facilitate the accumulation of ATRA and DOX in tumors via their enhanced permeability and retention effects (EPR effect) [55], the MDA-MB231 tumor-bearing mice were treated with different formulations via intravenous injection at equivalent DOX and ATRA doses of 5 mg/kg and 15 mg/kg, respectively. We analyzed the amounts of DOX and ATRA in the tumors at 24 h post injection. As shown in Fig. 5C, the DOX and ATRA levels in the tumor tissue were 0.55e0.59 mg/g and 1.45e2.01 mg/g for mice receiving a single injection of drug-loaded NPs, whilst the corresponding levels for mice treated with free DOX or ATRA alone were only 0.11e0.14 mg/g and 0.40e0.44 mg/g, respectively. This demonstrated that the drug loaded nanoparticles could enhance the accumulation of DOX or ATRA in breast tumors due to the EPR effect of the nanoparticles. Afterwards, we determined whether drug-loaded nanoparticles could enhance DOX accumulation in CSCs in tumors (Fig. 5D). The intracellular DOX fluorescence of CSC defined by ALDHhi was determined by flow cytometric analysis. The relative mean fluorescence intensity of DOX in CSCs after treatment with a single injection at 24 h of DOX-loaded NPs (NPDOx, NPATRA/NPDOx, NPATR/ DOx) was 3.01 ± 0.38 when compared with PBS treatment (set as 1.00). However, negligible levels of DOX were detected in the CSCs after the same administration of DOX (1.20 ± 0.02) or ATRA/DOX (1.23 ± 0.15). 3.5. Simultaneous delivery of ATRA and DOX with nanoparticles significantly inhibits tumor growth and synergistically reduces the proportion of CSCs in vivo To further verify whether the simultaneous delivery system (NPATRA/DOx) could effectively inhibit tumor growth and induce CSC differentiation following systemic administration, we treated MDAMB-231 orthotropic xenograft-bearing mice with NPATRA/DOx or various other formulations through i.v. injection every two days from the 14th day after xenograft implantation. As indicated in Fig. 6A, free DOX and ATRA inhibited tumor growth only slightly, while combining ATRA with DOX treatment only slightly increased the inhibition at the same doses. Moreover, drug-loaded nanoparticles at the same dose of DOX or ATRA could better suppress tumor growth than treatment with free DOX or ATRA. Additionally, the co-delivery system NPATRA/DOx exhibited particularly significant inhibition of tumor growth compared with free ATRA/DOX, NPDOx

Fig. 6. (A) Inhibition of tumor growth by NPATRA/DOx in MDA-MB-231 xenograft NOD/ SCID mice in comparison with other formulations. Data are shown as means ± s.d. (n ¼ 5). (B) Weights of MDA-MB-231 xenograft tumors at the end of the treatment. Data are shown as means ± s.d. (n ¼ 5) *p < 0.05. (C) The CSCs proportion within tumors after the tumor suppression study. Data are shown as means ± s.d. (n ¼ 3) *p < 0.05.

or NPATRA (p < 0.01). More importantly, a synergistic inhibitory effect of the two therapeutic agents delivered by nanoparticles on tumor growth was demonstrated (c.i. < 1). In contrast, separate combinatorial delivery of ATRA and DOX by NPATRA/NPDOx showed less suppression of tumor growth than NPATRA/DOx. At the end of treatment, the tumor weight and images was analyzed (Fig. 6B and Fig. S5), which supported the results of tumor suppression. Moreover, it is worth noting that none of the drug-loaded nanoparticle treatments led to significant body weight loss (Fig. S6), suggesting

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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Fig. 7. H&E and PCNA analyses of tumor tissues after treatment with various formulations. The PCNA-positive proliferating cells are stained brown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

that these formulations of drug-loaded nanoparticles would be potentially safe for breast cancer therapy. Subsequently, to investigate the differentiation effect of ATRA and the anti-proliferative effect of simultaneous delivery of DOX by the NPATRA/DOx system on cancer stem cells in vivo, the population of cancer stem cells (ALDHhi) recovered from the tumors at the end of the treatments was examined. As shown in Fig. 6C, the percentage of cancer stem cells in tumor cells treated with ATRA (the proportion of ALDHhi cells in tumor cancer cells was 5.8%) was moderately lower than in the PBS control (the proportion of ALDHhi cells was 7.8%). In contrast, free DOX treatment induced enrichment of CSCs in tumor cells (the proportion of ALDHhi cells was raised to 16.3%), which was consistent with findings reported by others [24,56]. This result also suggested the necessity of using a drug that can target cancer stem cells in combination with DOX. When treated with ATRA/DOX, the percentage of CSCs (the proportion of ALDHhi cells was 8.9%) was less than with DOX treatment, but more than after ATRA treatment (the proportion of ALDHhi cells was 5.6%). In addition, the nanoparticle delivery system could promote the suppression of CSCs which might attribute to enhanced drug accumulation by nanoparticles in CSCs. Notably, the co-delivery system (NPATRA/DOx) showed a particularly significant decrease in CSCs (the proportion of ALDHhi cells was 1.7%) compared with NPDOx (the proportion of ALDHhi cells was 8.1%) and NPATRA (the proportion of ALDHhi cells was 2.8%) treatment (p < 0.05). Moreover, a synergistic inhibitory effect on CSCs of the two therapeutic agents loaded into nanoparticles was demonstrated (c.i. < 1). In contrast, combinatorial delivery of separate ATRA and DOX by NPATRA/NPDOx showed less inhibition of CSCs than NPATRA/DOx and no synergistic effect was observed, primarily due to the separate internalization of the two nanoparticles by CSCS. Therefore, our findings suggest that co-delivery of the CSC differentiation regulator ATRA together with the chemotherapeutic drug DOX by nanoparticles could simultaneously eliminate CSCs and non-CSCs in tumors and achieve a synergistic effect in CSC inhibition, resulting in effective cancer therapy. Additionally, in Fig. 7, compared with free DOX or ATRA, administration of ATRA/DOX moderately reduced the percentage of proliferating PCNA-positive tumor cells, indicating the enhanced efficiency of treatment in inhibiting proliferation in tumor cells. The nanoparticle delivery system promoted the inhibition of cell proliferation, and the codelivery system (NPATRA/DOx) showed a particularly significant

decrease of proliferating PCNA-positive tumor cells. Moreover, in Fig. S7, the H&E analyses of heart, liver and kidney tissues showed no obvious toxicity by treatment with the formulations. 4. Conclusions In this study, we proved that the co-delivery system NPATRA/DOx can simultaneously deliver a CSC differentiation agent and a chemotherapeutic drug to CSCs and non-CSCs. The co-delivery system shows great superiority in delivering the two payloads into the same CSC, revealing effective tumor suppression by inducing CSC differentiation into non-CSCs and inhibiting all of the tumor cells without triggering CSC enrichment subsequent to treatment. By this treatment, ATRA induced the differentiation of CSCs, which reduced their tumor initiating ability and increased their sensitivity to DOX. In addition, the prolonged circulation halflives, improved pharmacokinetics and increased uptake by tumors of drug-loaded nanoparticles carrying ATRA and DOX also makes them more effective for tumor inhibition. Additionally, CSCs could be more efficiently suppressed by the NPATRA/DOx co-delivery system than by NPDOx, NPATRA and NPATRA/NPDOx, both in vitro and in vivo; in particular a synergistic inhibitory effect was noted in vivo. Therefore, combination therapy of ATRA and DOX with simultaneous delivery can be a potential strategy for inhibiting tumor growth and relapse by targeting both CSCs and non-CSCs in cancer therapy. Acknowledgments This work was supported by the National Basic Research Program of China (2010CB934001, 2013CB933900 and 2012CB932500), the National Natural Science Foundation of China (31470965, 51125012, 51390482), and the open project of State Key Laboratory of Supramolecular Structure and Materials (sklssm201413). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.10.018.

Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018

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Please cite this article in press as: Sun R, et al., Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.018