Biomaterials 61 (2015) 10e25

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Co-delivery of hydrophobic paclitaxel and hydrophilic AURKA specific siRNA by redox-sensitive micelles for effective treatment of breast cancer Tingjie Yin, Lei Wang, Lifang Yin, Jianping Zhou*, Meirong Huo* Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 January 2015 Received in revised form 8 May 2015 Accepted 14 May 2015 Available online 15 May 2015

In this study, a novel redox-sensitive micellar system constructed from a hyaluronic acid-based amphiphilic conjugate (HA-ss-(OA-g-bPEI), HSOP) was successfully developed for tumor-targeted codelivery of paclitaxel (PTX) and AURKA specific siRNA (si-AURKA). HSOP exhibited excellent loading capacities for both PTX and siRNA with adjustable dosing ratios and desirable redox-sensitivity independently verified by morphological changes of micelles alongside in vitro release of both drugs in different reducing environments. Moreover, flow cytometry and confocal microscopy analysis confirmed that HSOP micelles were capable of simultaneously delivering PTX and siRNA into MDA-MB-231 breast cancer cells via HA-receptor mediated endocytosis followed by rapid transport of cargoes into the cytosol. Successful delivery and transport amplified the synergistic effects between the drugs while leading to substantially greater antitumor efficacy when compared with single drug-loaded micelles and non-sensitive co-loaded micelles. In vivo investigation demonstrated that HSOP micelles could effectively accumulate in tumor sites and possessed the greatest antitumor efficacy over non-sensitive co-delivery control and redox-sensitive single-drug controls. These findings indicated that redox-sensitive HSOP codelivery system holds great promise for combined drug/gene treatment for targeted cancer therapy. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Co-delivery Micelles Paclitaxel Redox-sensitive siRNA Synergistic effect

1. Introduction Cancer is currently characterized as a highly heterogeneous disease that includes the presence of cells undergoing continuous indefinite growth by different mechanisms. As a result, the treatment of cancer through a single therapeutic strategy remains suboptimal as not all mechanisms of growth are targeted. However, combination therapy containing two drugs that are efficacious by different mechanisms could cooperatively inhibit the proliferation of tumor cells with synergistic or combined effects [1e4]. Furthermore, the application of nanocarrier platforms to deliver anticancer drugs offers an effective anti-tumor therapeutic strategy due to the enhanced permeability and retention (EPR) effect and tumor-specific targeting [5,6]. Recently, the co-delivery of small interfering RNA (siRNA) alongside conventional cytotoxic drugs has gained great attention

* Corresponding authors. E-mail addresses: [email protected] (J. Zhou), [email protected] (M. Huo). http://dx.doi.org/10.1016/j.biomaterials.2015.05.022 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

owing to increased anti-tumor efficacy over single regimen administrations [7,8]. Due to its high specificity and low toxicity, successful administration of siRNA has become an advent in the silencing of malignant oncogenes [9]. In particular, it was found that simultaneously delivering siRNA and cytotoxic drugs coloaded in a single nanocarrier was far more effective in treating cancers than sequential administration of two separate targeted nanocarriers with one drug in each. This finding indicates that simultaneous delivery of suitable amount of siRNA and cytotoxic drugs into the same tumor cell plays a key role in the ultimate outcome of the treatment [10]. However, it is difficult to design nanocarriers capable of encapsulating siRNA and cytotoxic drugs due to the different physicochemical properties of each agent. On one hand, siRNA exhibits a high molecular weight and polyanionic nature [11]. On the other hand, general cytotoxic drugs for cancer treatment such as paclitaxel and camptothecin are hydrophobic small molecules. To overcome this issue, researchers have specifically developed various systems for this purpose based on cationic polymeric [12,13], liposomal [14] and inorganic-based [15] nanoparticles. Among these systems, self-assembled amphiphilic conjugates

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which form cationic polymeric micelles in aqueous solution have received growing attention in the field of co-delivery nanosystems for tumor therapy [16]. Cationic polymeric micelles not only possess good solubilizing capacities for hydrophobic drugs and favorable condensing abilities for RNA based drugs, but also exhibit appealing properties such as nanoscopic dimension, distinctive core/shell structure, long-circulation property and tumor passive localization by EPR effect. Recently, some conventional cationic amphiphiles such as poly(ethylene glycol)-b-poly(ε-caprolactone)b-poly (2-aminoethyl ethylene phosphate) (mPEG-b-PCL-b-PPEE) [10], poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEGPLL-PLLeu) [13], polyethylene glycol -peptide-polyethylenimine1,2-dioleoyl-snglycero-3-phosphoethanolamine (PEG-pp-PEI-PE) [17] have been reported and exhibited good loading capacities for both chemical drugs and siRNA. However, conventional micellar drug delivery platforms often suffer from unfavorable drug release kinetics. The strong interaction between the hydrophobic drug and polymer greatly hinders drug release. Release is therefore measured in days to weeks due to the slow degradation kinetics of the polymer inside the body [18,19]. Even then, the limited amount of released drug may likely be pumped out from the intracellular compartment to the extracellular matrix, resulting in a low concentration of drug reaching its target [20]. Additionally, some studies indicated that the strong ionic interaction between siRNA and cationic segments of the delivery materials might hinder the intracellular release of siRNA, leading to a reduction in therapeutic effect [21,22]. An actively targeted micellar system capable of stimuli induced burst drug release of both hydrophobic and hydrophilic drugs at the target site is of high demand in cancer treatment. Recently, redox-sensitive micellar nanoparticle that actively delivers and releases drugs into cancer cells has emerged as a potential solution [23]. The redox gradient between the intra(approximately 2e10 mM glutathione (GSH)) and extracellular compartments (approximately 2e20 mM GSH) is used as the trigger for inducing drug release. Moreover, the cytoplasmic environment of tumor cells has a much higher reducing potential (approximately 20 mM GSH) compared to normal cells [24]. As a result, the unique intracellular redox potential promotes the design of redox-sensitive micelles which can achieve burst release of encapsulated cargoes within tumor cells. With this knowledge, we had previously developed the redox-sensitive micelles based on amphiphilic hyaluronic acid-deoxycholic acid conjugates containing cystamine as bioreducible linkages [25]. The redox-sensitive micelles exhibited excellent stability in the extracellular environment and provided a cytoplasm-selective quick release of paclitaxel (PTX), a model chemical drug, leading to enhanced efficacy. However, the codelivery of siRNA and hydrophobic anticancer drugs though a redox-sensitive delivery vehicle remains a challenge as there are only limited reports of this method [26]. In this study, redox-sensitive hyaluronic acid (HA)-based conjugates (HA-ss-(OA-g-bPEI) containing cystamine as bioreducible linkages were synthesized and developed as a micellar platform for targeted cellular co-delivery of hydrophilic siRNA and hydrophobic anticancer drugs. As a control, structurally analogous redox insensitive HA-(OA-g-bPEI) conjugates were also synthesized which lacked the disulfide bond. As shown in Fig. 1, HA was employed as the hydrophilic shell of our redox-sensitive co-delivery micelles mainly for its receptor-mediated targeting to CD44, RHAMM, HARE and LYVE-1 which were overexpressed on cell surface of many malignant tumors [27]. Octandioic acid (OA) and branched polyethyleneimine (bPEI) composed the hydrophobic core and the cationic segment for effectively encapsulation of hydrophobic anticancer drugs and condensing siRNA, respectively. It is worth noting that the cationic domain of HA-ss-(OA-g-bPEI) micelles is

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closer to the inner core compared with that of reported HA-PEI conjugates, which would provide better protection for condensed siRNA under the HA shell [28]. In addition, the induction of disulfide bonds from cystamine makes our micelles more attractive due to the redox sensitivity. Once internalized into tumor cells, HA-ss(OA-g-bPEI) micelles rapidly disassembled in response to cytoplasmic reducing reagents to achieve quick simultaneous release of both cargoes. Recent studies revealed that aurora kinase A (AURKA) was a potential therapeutic target for triple negative breast cancer (TNBC), a more aggressive and metastatic subtype of breast cancer and that pre-treatment of TNBC with AURKA inhibitors enhanced the sensitivity of cancer cells to taxanes [29e33]. Consequently, we chose PTX and AURKA specific siRNA (si-AURKA) for co-delivery and loaded both of them into redox-sensitive HA-ss-(OA-g-bPEI) micelles against MDA-MB-231 cell line which is a CD44 overexpressed TNBC. The co-loading capacities for siRNA and PTX along with the redox-sensitivity of micelles were then observed in detail. Moreover, the receptor-meditated cellular internalization, endo/lysosomal escape, reduction triggered micelle disassembly and the synergetic efficacy of the two regimens were evaluated using MDA-MB-231 cell line. Finally, in vivo biodistribution and anti-tumor efficacy of combinatory micelles in tumor-bearing nude mice were also determined. 2. Materials and methods 2.1. Materials Sodium hyaluronic acid (HA, molecular weights 20 kDa) was purchased from Freda Biochem Co., Ltd. (Shandong, China). Nontargeted control siRNA (siNonsense), Cy3-siRNA (the 50 -end of the sense strand in siNonsense was conjugated with Cy3 dye), FAMsiRNA (the 50 -end of the sense strand in siNonsense was conjugated with FAM dye), si-AURKA (sense strand: 50 -GAA GAG AGU UAU UCA UAG A dTdT-30 and anti-sense strand: 50 -UCU AUG AAU AAC UCU CUU C dTdT-30 ) were purchased from Guangzhou RiboBio Co., Ltd. (Shenzhen, China). N-hydroxysulfosuccinimide (sulfoNHS) and paclitaxel (PTX) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and Chongqing Melian Pharmaceuticals Co., Ltd. (Chongqing, China), respectively. 1-Ethyl3-(3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), octandioic acid (OA), glutathione (GSH) and branched polyethyleneimine (bPEI) with the MW of 10,000 were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Cystamine and adipic dihydrazide were purchased from TCI Development Co., Ltd. (Shanghai, China). Nile red (NR) and coumarin 6 (C6) were purchased from SigmaeAldrich (Saint Louis, Missouri, United States). The near-infrared dye DiR was obtained from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). All other chemicals and reagents were analytical grade. 2.2. Synthesis of HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) conjugates 2.2.1. Synthesis of cystamine modified HA (HA-CYS) and adipic dihydrazide modified HA (HA-ADH) HA-CYS and HA-ADH were synthesized as previously described with slight modification [25,34e36]. Briefly, EDC (0.40 mmol) and sulfo-NHS (0.40 mmol) were added to the 4 mg/mL of HA (0.50 mmol) in PBS buffer (0.01 M, pH 7.4) to activate the carboxyl groups of HA. Then, 2.24 g of cystamine dihydrochloride (10.0 mmol) was added to react for 4 h at room temperature under stirring. The resulting product HA-CYS was purified by exhaustive dialysis and obtained by lyophilization. To prepare HA-ADH, 0.20 g of HA (0.50 mmol) was dissolved in 50 mL of water followed by the

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Fig. 1. Schematic design of HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) conjugates and the illustration of intracellular trafficking pathway of redox-sensitive HA-ss-(OA-g-bPEI) codelivery micelles. The intracellular trafficking includes receptor-meditated cellular internalization (A), endo/lysosomal escape (B), reduction triggered micelle disassembly and rapid release of siRNA and PTX (C).

addition of 0.44 g of adipic dihydrazide (2.5 mmol) and 0.096 g of EDC (0.50 mmol). The pH of the reaction mixture was maintained at 4.75 by 0.1 M HCl for 1 h. Then, 0.1 M NaOH was added to terminate the reaction by adjusting the pH to 7.0. Finally, the resulting solution was dialyzed exhaustively and then lyophilized.

flocculent precipitate was collected by filtration, followed by being dissolved in 20 mL of distilled water and dialyzed (MWCO 25000) against excess amount of 1 M NaCl solution for 2 days, 25% ethanol for 1 day, and distilled water for 1 day [37,38]. Finally, the dialyzed product was lyophilized and stored at 4  C until further use.

2.2.2. Synthesis of HA-ss-OA and HA-OA conjugates HA-ss-OA and HA-OA was obtained by conjugating octandioic acid (OA) to HA-CYS and HA-ADH through amide formation, respectively. In detail, 0.16 g OA (0.9 mmol) was dissolved in 10 mL of DMF at 0  C and the carboxyl groups of OA was activated by EDC (2 mmol) and NHS (2 mmol) for 1 h. Then, HA-CYS (0.3 mmol) or HA-ADH (0.3 mmol) dissolved in 20 mL of formamide (FM) was added dropwise to the above solution and then the reaction mixture was stirred for 24 h at room temperature. Finally, HA-ss-OA or HA-OA was precipitated from the solution by adding 200 mL of previously frozen acetone, followed by being dissolved in 20 mL of distilled water. The product solution was filtered through a 0.45 mm pore-sized microporous membrane and dialyzed (MWCO 14000) against the excess amount of water/ethanol (1:1, v/v) for 1 day and distilled water for 2 days, respectively. Finally, the resulting solution was lyophilized for the further use.

2.3. Characterization of HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) conjugates

2.2.3. Synthesis of HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) conjugates Branched polyethyleneimine (bPEI) modification was achieved by coupling primary amines of bPEI to the free carboxyl groups of HA-ss-OA and HA-OA. First, HA-ss-OA (0.3 mmol) or HA-OA (0.3 mmol) was dissolved in 40 mL of DMF/FM (1:1, v/v) and activated at 0  C for 1 h using EDC (0.4 mmol) and NHS (0.4 mmol). Then, 10 mL of DMF containing 150 mg of bPEI (0.015 mmol) was added to the polymer solution dropwise while stirring. When the reaction continued for 24 h at room temperature, the mixture was precipitated into 300 mL of previously frozen acetone. The

The chemical structures of HA-ss-(OA-g-bPEI) and HA-(OA-gbPEI) conjugates were confirmed by 1H NMR spectra on a Bruker (AVACE) AV-500 spectrometer with HA-CYS, HA-ss-OA and HAADH, HA-OA as controls, respectively. HA-CYS, HA-ss-OA, HAADH, HA-OA, HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) were all dissolved in the mixed solution of D2O/CD3OD (1:1, v/v). Ellman's method was used to estimate the amount of thiol groups in the backbone of HA after the disulfide linkage in HA-ss-(OA-g-bPEI) conjugates cleaved by ten molar excess of dithiothreitol (DTT) for 24 h. The amount of free thiol groups was calculated from an according standard curve obtained by solutions with increasing concentrations (0.06e1.1 mg/mL) of L-cysteine [39]. The degree of substitution (DS) which is defined as the number of octandioic acid or polyethyleneimine molecules per 100 sugar residues of HA was analyzed by 1H NMR [28,37]. For convenience, HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) were further abbreviated as HSOP and HOP, respectively, in the following article. 2.4. Preparation and characterization of micelles 2.4.1. Preparation of blank micelles The blank redox-sensitive HSOP micelles and non-sensitive HOP micelles were prepared as following: 10 mg of HSOP conjugates or HOP conjugates were dissolved in 2 mL of ultrapurified water

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(Millipore, 18.2 MU) followed by being sonicated with a probe-type ultrasonicator (JY92-2D; Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China) at 100 W for 15 min in an ice bath, respectively. The obtained micellar solutions were filtered through a 0.45 mm filter. 2.4.2. Preparation of PTX-loaded micelles PTX-loaded micelles were prepared by a dialysis technique. Briefly, different amounts of PTX dissolved in ethanol were added dropwise to the 5 mg/mL solutions of blank micelles dissolved in ultrapurified water (Millipore, 18.2 MU) while stirring. The mixture was stirred for another 1 h at room temperature, followed by sonication for 15 min at 100 W with a probe-type ultrasonicator (JY92-2D; Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China) in an ice bath. Then the solutions were dialyzed against distilled water using a dialysis bag (MWCO 3500) for 12 h. The solution was filtered through a 0.45 mm filter to remove the unloaded PTX and then lyophilized. The entrapment efficiency (EE) and drug-loading (DL) were calculated by the following formula:

EE ð%Þ ¼

DL ð%Þ ¼

amount of PTX in micelles  100 amount of PTX dissolved in ethanol amount of PTX in micelles  100 amount of PTX­loaded micelles

The amount of PTX was determined by high performance liquid chromatography (HPLC, Shimadzu LC-2010 system, Kyoto, Japan) with UV detection at 227 nm using a Lichrosphe™ C18 column (5 mm particle size, 250 mm  4.6 mm), and the mobile phase was a 65/35 (v/v) mixture of methanol and water with a flow rate of 1.0 mL/min. 2.4.3. Preparation of siRNA micellar complexes and co-delivery micelles To prepare the siRNA micellar complexes and co-delivery micelles, desired amounts of siRNA was mixed with equal volume of blank micelle solutions or PTX-loaded micelle (the DL (w/w) was ~13.0%) solutions, and the resulting mixture was vortexed gently for 30 s followed by incubation at room temperature for 30 min. The siRNA condensed micellar complexes (HSOP/siRNA) and codelivery micells (PTXHSOPsiRNA) were confirmed by electrophoresis on a 1.0% agarose gel at 130 mV for 15 min in 0.5 TrisBorate-EDTA (TBE) buffer solution. siRNA was visualized with Goldview (Amresco, USA) staining and the gel image was taken under UV. 2.4.4. Particle size, size distribution and zeta potential of codelivery micelles The particle size and surface zeta potential of blank micelles (HSOP, HOP), PTX-loaded micelles (HSOP/PTX, HOP/PTX), siRNAcondensed micellar complexes (HSOP/siRNA, HOP/siRNA) or coloaded micelles (PTXHSOPsiRNA, PTXHOPsiRNA) were measured using dynamic light scattering (Nano-ZS90, Malvern instruments, UK) at 25  C and at a scattering angle 90 after dilution of the micelles with distilled water. All samples were equilibrated to the defined temperature for 30 min prior to the measurement. Then, siRNAloaded micelles at a charge ratio of 30 were used for the following experiments. 2.4.5. Morphological observation of co-delivery micelles Atomic force microscope (AFM, Nano Scope Ⅲa, Veeco, USA) and Transmission electron microscopy (TEM, H-600, Hitachi, Japan) were used to visualize the morphology and size distribution of codelivery micelles. AFM was operated in tapping mode and the samples to TEM analysis were negatively stained by with 0.1%

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phosphotungstic acid. 2.5. Disassembly of blank micelles triggered by GSH The disassembly of redox-sensitive HSOP micelles in response to 20 mM GSH was monitored by AFM to observe the morphology change during different time intervals. Briefly, 2 mL of HSOP micellar solution containing 20 mM GSH was placed in a shaking bed at 37  C at a rotation speed of 100 rpm for 4 h or 24 h. For comparison, HSOP micelles incubated without GSH and nonsensitive HOP micelles incubated with 20 mM GSH were designed as controls, respectively. 2.6. Serum stability assay of siRNA-loaded micelles For the serum stability assay, free siRNA (0.5 mg) and HSOP/ siRNA micellar complexes containing 0.5 mg of siRNA were incubated with 90% fetal bovine serum (FBS) at 37  C for indicated durations. As control, another group of HSOP/siRNA micellar complexes were pre-treated with 20 mM GSH for 24 h at 37  C and then followed by serum stability assay. Finally, all the results were analyzed by 1.0% agarose gel eletrophoresis. 2.7. In vitro release of PTX and siRNA in simulated extracellular and intracellular conditions 2.7.1. In vitro release of PTX The PTX release from PTXHSOPsiRNA micelles and PTXHOPsiRNA micelles triggered by different concentrations of GSH in buffer solutions was studied using a simple dialysis method. Briefly, 1 mL of PTX HSOPsiRNA micelles containing 0.5 mg PTX were placed in a clamped dialysis bag (MWCO 3500) and immersed in 0.1 M PBS (pH 7.4, pH 5.8) (150 mL) buffer solutions containing 0.1% (w/v) Tween 80. Various amounts of GSH (0 mM, 10 mM, 10 mM and 20 mM) were added to the buffer solution and the experiment was performed in an incubation shaker at 37  C at 100 rpm. At selected time intervals, 2 mL of release media was taken out for HPLC analysis as described in Section 2.4.1 and the whole media was refreshed. The drug release of PTXHOPsiRNA in the presence of 20 mM GSH was also studied as the non-sensitive control. 2.7.2. In vitro release of siRNA To investigate the release profile of siRNA from co-delivery micelles in response to the reducing environment, the PTXHSOPFAM-siRNA micelles containing 5 mg FAM-siRNA were suspended in 1.0 mL of PBS buffer (pH 7.4), with a subsequent addition of GSH (0 mM, 10 mM, 10 mM and 20 mM). The buffer solution was placed in an incubation shaker at 37  C at 100 rpm. At desired time intervals, the samples were centrifuged (20,000 rpm, 4  C) for 1 h. The concentration of the free FAM-siRNA in the supernatant was measured by HPLC (Shimadzu LC-2010 system, Kyoto, Japan) with a fluorescence detector fixed at 485 nm for excitation and 535 nm for emission. An octadecylsilane (ODS) column (5 mm particle size, 150 mm  4.6 mm) was used for FAM-siRNA concentration analysis, and the mobile phase was a 72/28 (v/v) mixture of triethylammonium acetate buffer (20 mM, pH 7.2) and acetonitrile with a flow rate of 0.5 mL/min [40,41]. The FAM-siRNA release profile from the micelles of PTXHOPFAM-siRNA in the presence of 20 mM GSH was also studied as the non-sensitive control. 2.8. Cell culture MDA-MB-231 cell line was obtained from American Type Culture Collection (ATCC) and was grown in DMEM media with 10% fetal bovine serum (FBS) at 37  C in 5% CO2 atmosphere.

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2.9. Cell uptake and intracellular trafficking of redox-sensitive codelivery micelles The cellular uptake of sensitive and non-sensitive co-delivery micelles was investigated by flow cytometric and fluorescence microscopic analyses. Firstly, hydrophobic fluorescent probe coumarin 6 (C6) and Cy3-labled siRNA was co-loaded into HSOP and HOP micelles as described in Section 2.4, apart from the change of PTX to C6 and siRNA to Cy3-siRNA. The obtained products were abbreviated as C6HSOPCy3-siRNA and C6HOPCy3-siRNA micelles. For the flow cytometric analyses, MDA-MB-231 cells (5  104 cells/well) were seeded into 24-well plates and incubated in complete DMEM to reach 80e90% confluence. The culture media were then replaced with fresh media containing C6HSOPCy3-siRNA or C6HOPCy3-siRNA micelles and cultured at 37  C for 4 h. The untreated cells were performed as a negative control. To investigate the specific uptake of micelles via HA-receptor mediated endocytosis, MDA-MB-231 cells were incubated with 25 mM of free HA polysaccharide for 2 h before the co-delivery micelles were added. The cells were rinsed with cold PBS for three times and collected by trypsinization to measure the fluorescence intensity. The fluorescence intensity of C6 and Cy3 in cell samples was analyzed using flow cytometer (BD FACS Calibur, USA). For the microscopic analyses, MDA-MB-231 cells were cultured in complete DMEM into 24-well plates for 24 h. After 4 h of incubation with different co-delivery formulations as described above, MDA-MB-231 cells were washed with PBS for three times and fixed with 4% paraformaldehyde. The cell nuclei were stained with Hoechst 33258 before the fluorescent imaging. The cellular uptake of micelles was visualized by a fluorescent microscope (Nikon TE2000, Japan). Then, confocal laser scanning microscope (CLSM) was employed to follow the internalization and endosomal release of co-delivery micelles. MDA-MB-231 cells were cultured in complete DMEM in glass bottom culture dishes for 24 h. After incubation with C6 HSOPsiRNA micelles for 2 h, 4 h and 12 h as described above, MDAMB-231 cells were washed with PBS for three times and stained with LysoTracker™ Red (SigmaeAldrich Co.) according to the manufacturer's instructions. After being washed three times with PBS, cells were fixed with 4% paraformaldehyde and observed by CLSM (Leica TCS SP5, Heidelberg, Germany). To visualize the intracellular release of cargo from co-delivery micelles, fluorescence probe nile red (NR) was loaded into codelivery micelles as described in Section 2.4, apart from the change of PTX to nile red. After cell culture in complete DMEM in glass bottom culture dishes for 24 h, the NRHSOPsiRNA or NRHOPsiRNA micelles were incubated with MDA-MB-231 cells for 4 h or 24 h, followed by being rinsed three times with cold PBS. Then, cells were fixed with 4% paraformaldehyde and cell nuclei were stained with Hoechst 33258 before the fluorescent imaging by CLSM. 2.10. In vitro gene silencing efficiency assay The gene silencing efficiency of si-AURKA at MDA-MB-231 cell was assessed by quantitative real-time PCR (qRT-PCR). MDA-MB231 cells was dispensed on 12-well plates at a density of 1  104 cells/well and incubated at 37  C for 24 h. Then, the culture medium was replaced with 1 mL of transfection medium containing HSOP/si-AURKA (200 nM, 100 nM, 50 nM) micelles, or HOP/siAURKA (100 nM) micelles. The micelles were prepared at a charge ratio (N/P) of 30. To confirm the HA receptor mediated endocytosis, HSOP/si-AURKA (200 nM) micelles were transfected with cells pretreated with 25 mM of free HA. The cell groups treated by PBS, HSOP/siNonsense and HOP/siNonsense were set as negative controls, while transfection of si-AURKA (50 nM) mediated by Lipofectamine™ 2000 according to the manufacture's protocol was

performed as a positive control. After 24 h of cell incubation, the total mRNA was isolated using RNeasy mini-kits (Qiagen, Germantown, MD) according to the manufacturer's protocol and reverse transcribed into cDNA using PrimeScript RT reagent Kit (Takara, Japan). Thereafter, 2 mL of cDNA was used to qRT-PCR analysis targeting AURKA and human 18S using an ABI Step One Plus Real-Time PCR systems (Applied Biosystems, Foster City, CA, USA) with SYBR® Premix Ex Taq™ (Takara). Primers used here for AURKA and 18S are: AURKA-forward: 50 GTCAAGTCCCCTGTCGGTTC-30 , AURKA-reverse: 50 -GTCCATGATGCCTCTAGCTGT-30 , and 18S-forward: 50 -CAGCCACCCGAGATTGAGCA-30 , 18S-reverse: 50 -TAGTAGCGACGGGCGGTGTG-30 . Amplification were performed for 40 cycles at 95  C  5 s, 60  C  34 s, 72  C  30 s and 1 cycle at 95  C  15 s, 60  C  60 s, 95  C  15 s. Human 18S was used as the endogenous reference and the data obtained were normalized before statistical analysis. 2.11. Western-blot analysis MDA-MB-231 cells was dispensed on 12-well plates at a density of 1  104 cells/well and incubated at 37  C for 24 h to reach 70% confluence. Various formulations including HOP/si-AURKA (100 nM), HOP/siNonsense (100 nM), HOP blank micelles, HSOP/ si-AURKA (100 nM), HSOP/siNonsense (100 nM), HSOP blank micelles, Lipo2000/si-AURKA (50 nM) and PBS were added and incubated with cells for 48 h for protein extraction. The free HA pretreated group (HA þ HSOP/si-AURKA (100 nM)) was also set to demonstrate the HA receptor mediated endocytosis. The cellular expression level of AURKA protein was assessed using Western blot. Protein in transfected cells were isolated by 100 mL of lysis buffer (50 mM TriseHCl, pH ¼ 8.0, 150 mM NaCl, 1% Triton X-100, 100 mg/ mL PMSF). The cell lysis was performed on ice for 30 min and the supernatant of lysates were collected after centrifugation for 10 min at 12,000 rpm. The concentration of protein was determined using the BCA protein assay method with Varioskan spectrofluorometer and spectrophotometer (Thermo) at 562 nm. Total protein was separated (at 120 V for 70 min) on 10% PAGE-SDS gels and then transferred onto the PVDF membranes (Millipore). Thereafter, the membranes were incubated with AURKA antibodies (1:1000) overnight at 4  C. After incubated with goat anti-rabbit IgG-HRP antibody for 1 h, bands were detected using ECL system (Pierce). 2.12. Cytotoxicity assay The cytotoxicity of blank micelles or PTX and siAURKA co-loaded micelles was assessed by MTT assay with MDA-MB-231 cells. In the case of biomaterial MTT assay, MDA-MB-231 cells cultured in 96well plates were incubated with blank micelles with the concentration ranging from 0.005 to 1000 mg/mL for 72 h. To perform MTT assay for drug-loaded micelles, MDA-MB-231 cells were incubated with PTXHSOPsiNonsense, PTXHOPsiNonsense, HSOP/si-AURKA or HOP/siAURKA with different concentration of PTX (0.001e10 mg/mL) or siAURKA (0.325e3.25 mg/mL) for 72 h. Meanwhile, co-delivery micelles encapsulating different combination ratios of PTX/si-AURKA (1/26, 1/13, 5/13, 1/1, 5/1 at w/w) were incubated with MDA-MB231 cells at various concentrations based on PTX (0.001e0.5 mg/ mL) for 72 h. After addition of 20 mL MTT (5 mg/mL in PBS) to each well for 4 h, 100 mL DMSO was added to dissolve the formazan crystals. Then, the absorbance was measured at 570 nm with a microplate reader (Multiskan Mk3, Thermo Labsystems, Beverly, MA, USA). All the results were normalized to the untreated group. 2.13. In vivo fluorescence imaging for tumor-targeting analysis For in vivo fluorescence imaging, the BALB/c nude mice bearing

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MDA-MB-231 tumors in the armpit region were administrated with DiR and FAM-siRNA co-loaded micelles (DiR ¼ 50 mg/kg, FAMsiRNA ¼ 2 mg/kg) via the tail vain when the tumor size reached around 500 mm3 [14]. Meanwhile, we also set free DiR (50 mg/kg) administrated group as a control. The mice were anesthetized by 10% (w/v) chloral hydrate and images were taken at different time intervals (2 h, 12 h, 24 h) on an in vivo imaging system (FX PRO, Kodak, USA). To detect the fluorescence distribution of DiR, the bandpass filter was fixed at 720 nm for excitation and 790 nm for emission. In addition, the bandpass filter fixed at 470 nm for excitation and 530 nm for emission was equipped for FAM-siRNA detection. 24 h after injection, the mice were sacrificed and different tissues including heart, liver, spleen, lung, kidney and tumor were excised for imaging. All the images were analyzed using the Kodak Molecular Imaging Software 5.X. 2.14. Antitumor efficacy of micelles in vivo BalB/c nude mice (seven weeks old, 20e25 g) bearing MDA-MB231 tumors were randomly divided into 6 groups (n ¼ 5) when the tumors grew to around 150 mm3, and received PTXHSOPsiNonsense, PTX HSOPsi-AURKA, PTXHOPsi-AURKA, HSOP/si-AURKA and Taxol® with saline as control. The doses of PTX and siRNA were fixed at 5 mg/kg and 1 mg/kg, respectively. The injections of different formulations into the tail vein were performed once every 3 days for 5 times. The tumor size and body weight of mice were recorded every 3 days for 6 times. After 18 days' observation, tumors and the normal organs (heart, lung, spleen, kidney, liver) were separated from the sacrificed mice for histological evaluation by hematoxylin and eosin (HE) staining. 2.15. Statistical analysis All quantitative data were expressed as mean ± S.D. unless otherwise noted. Statistical significance was tested using an unpaired, two-tailed Student's t-test. A value of P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Synthesis of HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) conjugates In this study, amphiphilic redox-sensitive HA-ss-(OA-g-bPEI) conjugate was successfully synthesized for the co-delivery of PTX and siRNA. In this case, the hydrophobic alkyl groups and hydrophilic cationic segment bPEI were conjugated to an HA backbone using cystamine containing a disulfide bond as a redox-sensitive connecting bridge (Fig. 1). As a control, a non-redox sensitive HA(OA-g-bPEI) conjugate was also successfully synthesized using adipic dihydrazide as connecting bridge. The chemical structures of HA-ss-(OA-g-bPEI) and HA-(OA-gbPEI) conjugates were confirmed by 1H NMR. As shown in Fig. 2A, the structure of HA-CYS was confirmed by the characteristic peaks at 2.02 ppm and 2.91e3.02 ppm of HA and cystamine, respectively. Meanwhile, the peaks at 2.23 ppm and 2.37 ppm (Fig. 2D) demonstrated successful introduction of adipic dihydrazide. The peaks at 1.32 ppm and 1.59 ppm in Fig. 2B and E were evidence for the successful conjugation of octandioic acid. Furthermore, the presence of bPEI in HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) was demonstrated by the characteristic peak appearing at 2.73e2.91 ppm (Fig. 2C and F) [42,43]. The amount of cystamine in the conjugates was quantitatively characterized from the 1H NMR (23.6% on a molar basis) and Ellman's method (24.7% on a molar basis), while the amount of adipic dihydrazide was also determined from the 1H NMR (22.1% on a

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molar basis) [25]. The degrees of octandioic acid modification in HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) were determined to be 20.7% and 19.1%, respectively, depending on the ratio of integration values of the methylene protons in octandioic acid (d ¼ 1.59 ppm, [4H, eCOeCH2eCH2e(CH2)2eCH2eCH2eCOOH]) to values corresponding to N-acetyl group in HA (d ¼ 2.02 ppm, [3H, eCOCH3]). Finally, the DS of bPEI in HA-ss-(OA-g-bPEI) and HA-(OA-g-bPEI) according to the eCH2CH2NHe protons of bPEI (2.73e2.91 ppm) was calculated to be 3.5% and 4.1%, respectively. 3.2. Preparation and characterization of co-delivery micelles The HSOP conjugates and HOP conjugates formed micellar structures in aqueous solution due to their amphipathic properties. To demonstrate the PTX-loading capacity of micelles, encapsulation efficiency (EE) and drug-loading (DL) were investigated depending on different feed ratios of PTX to conjugates at w/w [10]. As shown in Fig. 3A and B, PTX could be efficiently entrapped in the hydrophobic core of micelles with a high encapsulation efficiency (>86%) during a wide range of feed weight ratios (5/9 to 1/1200), and the drug loading content was ranged from 33.1 wt% to 0.07 wt% depending on the drug feed amount. This result indicated that the PTX loading content in micelles is adjustable, which is an important characteristic for co-delivery systems. Moreover, the linkages between HA and OA (cystamine or adipic dihydrazide) exhibited no effect on drug-loading capacities of micelles due to strong interaction and compatibility between the hydrophobic drug and hydrophobic segment of conjugates are the determinate factors for drug loading into micelles. Zeta potentials of micelles are closely related to their gene condensing ability and in vivo profiles. As shown in Fig. 3C, PTX-free and PTX-loaded micelles showed similar zeta potentials around þ14 mV, which suggested that PTX encapsulation had little effect on the siRNA-condensing capacities of micelles. The zeta potential of micelles was smaller than that of general cationic nanoparticles which are usually larger than þ20 mV [27]. The low zeta potential is because of partial shielding of cationic charge in bPEI by the negative charged HA covering the surface of micelles. Further, the ability of blank HSOP micelles and HSOP/PTX (the DL was fixed at ~13.0 wt% to simplify the experiment) micelles to condense siRNA was assessed by a gel retardation assay. As shown in Fig. 3D and E, the inhibited migration of condensed siRNA in the agarose gel was observed, indicating a complete formation of siRNA/HSOP micelles and PTXHSOPsiRNA micelles both at and above the charge ratio of 10. The particle size and zeta potential of micelles at different N/P ratio were shown in Fig. 3F and G. When the N/P ratio increased from 10 to 30, the particle size of HSOP/siRNA or PTXHSOPsiRNA micelles significantly decreased from ~220 nm to ~135 nm, which indicated that more compact micellar nanoparticles could be induced by higher electrostatic interactions. While when N/P ratio further increased up to 40, there was no significant decrease in particle size, indicating that the micelles had been compacted to the greatest extent at N/P ratio of 30. The zeta potential of nanoparticles changed from negative charges to positive charges with the increase of N/P ratio. Since nanoparticles with positive charges are prone to combine with the anionic serum components which will lead to decreased serum stability and poor tumor targeting of nanoparticles, the optimum N/P ratio for the preparation of siRNA-loaded micelles was chosen as 30. TEM and AFM were used to visualize directly the size and morphology of co-delivery micelles. The spherical morphologies of micelles are confirmed in Fig. 3H. The observed size of micelles was approximately 100 nm, which was smaller than the hydrodynamic diameter obtained from the DLS experiment. This observation was due to the collapse of micelles during drying processes of TEM and AFM samples.

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Fig. 2. 1H NMR spectra of HA-CYS conjugate (A), HA-ss-OA conjugate (B), HA-ss-(OA-g-bPEI) conjugate (C), HA-ADH conjugate (D), HA-OA conjugate (E), and HA-(OA-g-bPEI) conjugate (F).

3.3. Redox-sensitivity of blank micelles To study the redox-triggered disassembly of micelles, a visualized measurement by AFM was performed to monitor the morphological change of micelles over time in GSH-supplemented solutions (20 mM). As shown in Fig. 4A, HSOP micelles maintained structural integrity in GSH-free buffer solution and there was no significant change in particle size for 24 h. However, particles incubated in GSH solution increased in size over time. The obvious change in morphology indicated that HSOP micelles dissembled into irregular and loose particles due to the cleavage of disulfide bonds by GSH, which confirmed the sharp response of HSOP micelles to the reducing environment. As a control, the non-sensitive HOP micelles maintained structural integrity with diameter sizes around 140 nm even after incubation with 20 mM GSH for 24 h (Fig. 4Ad), which provided strong evidence that the redoxsensitivity of HSOP micelles was attribute to the disulfide bond. All results initially suggested the feasibility of HSOP micelles as a delivery system for selective burst drug release in response to the intracellular redox potential [44]. 3.4. Serum stability of siRNA-loaded micelles Both HSOP/siRNA micelles and HOP/siRNA micelles prepared at an N/P ratio of 30 had a neutral surface charge, which was significantly different from that of most siRNA-loaded cationic complexes reported in literature [45,46]. This neutral surface charge positively contributes to the enhanced serum stability of micelles due to reducing nonspecific interactions with anionic serum components. Herein, we tested the effect on siRNA degradation with 90% (vol/vol) FBS. Fig. 4B shows that naked siRNA were readily

degraded by FBS and complete degradation was observed after 36 h. In contrast, siRNA formulated in HSOP micelles remained intact even they were incubated with high concentration of FBS for 36 h. The ability of HSOP micelles in protecting siRNA from serum destruction provides a method for effective siRNA delivery in vivo. However, when HSOP/siRNA was pre-treated with 20 mM GSH for 24 h followed by incubation with FBS, degradation of naked siRNA was observed, and the band of remaining HSOP/siRNA complexes in the electrophoresis chamber became faint after 12 h of serum incubation. It can be assumed that GSH specifically accelerates the degradation of disulfide bonds in the chemical structure of HSOP, resulting in loosened HSOP/siRNA micellar complexes to facilitate the release of siRNA [21]. The hypothesis will be confirmed in the in vitro siRNA release experiment in different reducing environments. 3.5. In vitro release of PTX and siRNA in response to the reducing environment The most attractive function of PTXHSOPsiRNA micelles was their ability to quickly release both cargos in a reducing environment. Thus it is necessary to determine PTX and siRNA release behavior in different simulating physiological reducing environments. In this study, a PBS solution (pH ¼ 7.4 or 5.8, 100 mM, 1% Tween 80) containing 10 mM GSH was set corresponding to the extracellular redox condition, while PBS with 20 mM GSH was set corresponding to the intracellular redox condition in tumor cells. As shown in Fig. 4C, a burst release of PTX from PTXHSOPsiRNA micelles in the buffer solution containing 20 mM GSH was observed and about 57% of drug was released from micelles in 6 h and 91% in 36 h. In contrast, a dramatically decreased release rate of PTX from

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Fig. 3. The encapsulation efficiency (EE) (A) and drug-loading (DL) (B) at various feed ratios of PTX to conjugates (n ¼ 3). Hydrodynamic size (C) and surface charge (C) of HOP, HSOP blank micelles and HOP/PTX, HSOP/PTX micelles at different DL (wt%) (n ¼ 3). Agarose gel electrophoresis of HSOP/siRNA micellar complex formed at various N/P ratios of HSOP to siRNA (D). Agarose gel electrophoresis of PTXHSOPsiRNA co-delivery micelles formed at various N/P ratios of HSOP/PTX to siRNA (E). The drug loading content of PTX (DL) is ~13.0 wt%. Hydrodynamic size (F) and surface charge (G) of HOP/siRNA, HSOP/siRNA, PTXHOPsiRNA and PTXHSOPsiRNA with increasing N/P ratios. The drug loading content of PTX (DL) is ~13.0 wt %. (n ¼ 3). TEM image of PTXHSOPsiRNA micelles (Ha) and PTXHOPsiRNA micelles (Hb). AFM image of PTXHSOPsiRNA micelles (Hc) and PTXHOPsiRNA micelles (Hd).

PTX HSOPsiRNA micelles was observed in the absence of GSH or in the presence of 10 mM GSH (15% of PTX released in 6 h and 23% in 36 h). Furthermore, the release profile of PTX from non-sensitive co-delivery micelles in response to 20 mM GSH was similar to that from PTX HSOPsiRNA micelles in response to 10 mM GSH or no GSH. These findings indicated that the breakage of disulfide bond by reducing agents is the key to promoting disassembly of redox-sensitive micelles which allow fast release of hydrophobic drugs in the core. Meanwhile, a relatively decreased release rate of PTX from PTX HSOPsiRNA micelles was obtained in the presence of 10 mM GSH (33% of PTX released in 6 h and 51% in 36 h), suggesting a reducing reagent concentration-dependent manner of drug release. It was also found that pH of the buffer had an effect on the drug release profile. As shown in Fig. 4D, PTX-release rate was slower in PBS 5.8 than that in PBS 7.4 in spite of the same treatment with GSH (20 mM). The thiol group is the main source accounting for the reducing ability of GSH with a pKa of 8.8, while the reduction activity of the thiol group is higher in the thiolate form than in the sulfhydryl form [47]. Therefore, it is reasonable for PTXHSOPsiRNA

micelles to achieve a faster release rate of PTX at pH 7.4. Based on these results, we can conclude that the redox-sensitive HSOP micelles will be an outstanding delivery system with increased stability in blood circulation for rapid release of entrapped hydrophobic drugs upon arriving into the cytoplasm of target cells [25]. The release profiles of siRNA in different simulated physiological environments were shown in Fig. 4D. As indicated, the release pattern of siRNA was dependant on the concentration of the reducing agent as well. The percentage of siRNA released from PTX HSOPFAM-siRNA was 12% without GSH and 28% in 20 mM GSH, 36 h post treatment. The accelerated siRNA-release rate is attributed to the loosened interaction between siRNA and cationic segment of conjugates after the breakage of disulfide bonds in response to reducing environments. However, no burst release of siRNA was observed in reducing environments possibly due to the relatively high cationic charge density of bPEI which hinders conjugate disassociation with siRNA.

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Fig. 4. (A) The morphological change of HSOP and HOP micelles detected by AFM. HSOP micelles were incubated with GSH-free buffer solution for 24 h (a), and with GSHsupplemented (20 mM) buffer solution for 4 h (b), 24 h (c). HOP micelles were incubated with GSH-supplemented (20 mM) buffer solution for 24 h (d). (B) Serum stability assay of naked siRNA, HSOP/siRNA micellar complexes, and HSOP/siRNA micellar complexes pre-treated with GSH (20 mM) for 24 h by agarose gel electrophoresis. (C) GSH triggered PTX release from PTXHSOPsiRNA micelles and PTXHOPsiRNA micelles. The error bars in the graph represent standard deviations (n ¼ 3). (D) GSH triggered siRNA release from PTX HSOPFAM-siRNA micelles and PTXHOPFAM-siRNA micelles. The error bars in the graph represent standard deviations (n ¼ 3).

3.6. Cell uptake and intracellular trafficking of co-delivery micelles To demonstrate the intracellular co-delivery of PTX and siRNA by redox-sensitive HSOP micelles, a hydrophobic fluorescence probe (C6) and fluorescence labeled siRNA (Cy3-siRNA) were coloaded into micelles. The internalization of double fluorescencelabeled micelles (C6HSOPCy3-siRNA and C6HOPCy3-siRNA) in MDA-MB231 cells was first analyzed. The in vitro release experiment of C6 indicated that less than 1% of C6 was released in 24 h (data not shown), indicating that C6 detected in cells comes mostly from uptake of micelles encapsulating C6 rather than free C6. Additionally, it is known that cellular internalization of free siRNA is unfavored; therefore detected Cy3 signal in cells indicates internalization of siRNA-loaded micelles. MDA-MB-231 cells were then incubated with C6HSOPCy3-siRNA and C6HOPCy3-siRNA and fluorescence-activated cell sorting (FACS) analysis was performed. It could be clearly seen from Fig. 5A that C6 and Cy3-siRNA could be successfully co-delivered into MDA-MB-231 cells with minimal cells located outside the double-positive quadrant. To verify the function of the HA shell in facilitating the cellular internalization of co-delivery micelles via interaction with CD44 receptors, MDA-MB231 cells were pre-treated with 25 mM of free HA for 2 h prior to the incubation with C6HSOPCy3-siRNA micelles. It was found that the amount of cells located in the double-positive quadrant remarkably decreased, which demonstrated HA-dependent specific endocytic

pathway of HSOP micelles despite the partial chemical modification of HA [37]. To observe the cellular uptake of dual-labeled micelles intuitively, fluorescent microscope images were also taken. As shown in Fig. 5B, florescence of C6 and Cy3 was observed in the cytoplasm after incubating C6HSOPCy3-siRNA and C6HOPCy3-siRNA with MDA-MB-231 cells for 4 h, while little florescence was observed when the redox-sensitive co-delivery micelles were incubated with HA pre-treated tumor cells, which was consistent with results from FACS. It was also noted that redox-sensitive and non-sensitive micelles had no obvious distinction on the cell uptake, which might be attributed to the similarity of nano-structure and surface properties. The results remind us that the cellular uptake difference is not the main reason for different efficacies of the two co-delivery micelles in cell inhibition. Experiments by CLSM were further performed to determine whether HSOP micelles could effectively escape from lysosomes, in which ectogenic substances are quickly degraded. In this study, late endosome/lysosome was selectively stained with LysoTracker™ Red (exhibiting red fluorescence) after the MDA-MB-231 cells were treated with C6HSOPsiRNA micelles (exhibiting green fluorescence). The co-localization of micelles with late endosome/lysosome was indicated by yellow pixels. As shown in Fig. 6A, after 2 h of incubation, the yellow pixels in cells were obviously observed, suggesting that the micelles mostly delivered into late endosomes/ lysosomes at early stage of cellular uptake. However, yellow

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Fig. 5. Flow cytometry analysis (A) and fluorescent microscopy analysis (B) of the intercellular uptake of C6HSOPCy3-siRNA micelles, C6HOPCy3-siRNA micelles and free-HA polymer pretreated C6HSOPCy3-siRNA micelles at 4 h. For the each panel in fluorescent microscope analysis (B), images from left to right show the red fluorescence of Cy3-siRNA, the green fluorescence of C6, the blue fluorescence of Hoechst 33258 in nuclear and the merged fluorescence of Cy3-siRNA, C6 and Hoechst 33258. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

fluorescence signal was greatly diminished 4 h post incubation suggesting lysosome escape of micelles. The minimal colocalization of red and green fluorescence observed at 12 h demonstrated extremely effective lysosomal escape of C6HSOPsiRNA micelles. It was also worth noting that the red fluorescence of stained late endosomes/lysosomes weakened over time. This observation can be explained by the destruction of acidic environments in late endosomes/lysosomes by cationic materials. These materials exhibit the proton sponge effect which has been supported by previous reports [42,48]. Following endosome escape, co-delivery micelles diffused into the cytoplasm. To visualize the rapid intracellular drug release of HSOP micelles in response to cytoplasmic reducing environments, a florescence probe was used. In this study, nile red (NR) was selected as fluorescence probe and loaded into micelles to visually reflect the specific disintegration of redox-sensitive micelles in cytoplasm and quick release of cargo. As an outstanding vital stain for the detection of intracellular lipid droplets, NR has a better selectivity for cytosolic lipid droplets when viewed for yellow-gold fluorescence rather than red fluorescence. The intracellular lipid droplets stained by NR were displayed as distinct spherical fluorescent bodies distributed throughout the cytosol [49]. The intracellular distribution behaviors of NR were imaged by CLSM when

NR HSOPsiRNA and NRHOPsiRNA micelles were incubated with MDAMB-231 cells for 4 h and 24 h, respectively. Cells in Fig. 6B exhibited ample spherical yellow-gold fluorescent bodies in the cytosol 4 h post incubation with NRHSOPsiRNA micelles. Meanwhile, the total number of stained bodies increased slightly after extended incubation time of 24 h. In contrast, only a minimal number of stained bodies were observed in the non-sensitive micelle group after incubation for 4 h or 24 h. The amount of NR stained cytosolic lipid droplets exhibited in cells was significantly less in the non-sensitive micelle group than in the sensitive micelle group over the same time interval. It is worth noting that only free NR released from micelles into the cytosol can detect lipid droplets. Therefore, the ability of redox-sensitive HSOP micelles to more effectively detect intracellular lipid droplets reflects more efficient release of NR molecules over non-sensitive HOP micelles. All results indicate a sharp redox dependent response of HSOP micelles with the capacity to quickly unload NR into cytoplasm, the optimal location for inducing cytotoxicity.

3.7. In vitro AURKA transfection and analysis of AURKA expression The siRNA transfection efficiency of HSOP and HOP in MDA-MB231 cells were comparatively analyzed by gene silencing efficiency.

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Fig. 6. (A) Confocal microscopy images of MDA-MB-231 cells for intracellular delivery of C6-loaed HSOP micelles (C6HSOPsiRNA) at different times (2 h, 4 h, 12 h). Images from left to right show the green fluorescence of C6, the red fluorescence of LysoTracker™ Red and the merged fluorescence of C6 and LysoTracker™ Red. (B) Confocal microscopy images of MDA-MB-231 cells incubated with nile red-loaded HSOP micelles (NRHSOPsiRNA) and HOP micelles (NRHOPsiRNA) for 4 h and 24 h. For each panel, images from left to right show the fluorescence of nile red (yellow-gold, cytoplasma) only and the overlay of cells with fluorescence of nile red and Hoechst 33258 (blue, nuclear). (C) Expression of AURKA mRNA determined by quantitative real-time PCR. The concentration of si-AURKA with Lipofectamine 2000 (Lipo2000/si-AURKA) and siNonsense with micelles (HSOP/siNonsense, HOP/ siNonsense) were 50 and 200 nM, respectively. And the concentration of si-AURKA in group “HA þ HSOP/si-AURKA” was 200 nM. Results were expressed as means ± S.D. (n ¼ 3). *P < 0.01. (D) Expression of AURKA protein in MDA-MB-231 cells determined by western blot analysis. Lane 1: HOP/si-AURKA (100 nM); Lane 2: HOP/siNonsense (100 nM); Lane 3: HOP blank micelles; Lane 4: HA þ HSOP/si-AURKA (100 nM); Lane 5: HSOP/si-AURKA (100 nM); Lane 6: HSOP/siNonsense (100 nM); Lane 7: HSOP blank micelles; Lane 8: PBS control; Lane 9: Lipo2000/si-AURKA (50 nM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The level of AURKA mRNA expressed in MDA-MB-231 cells was evaluated by real-time PCR after 24 h transfection with si-AURKAcondensed micellar complexes. As shown in Fig. 6C, when the MDA-MB-231 cells were treated with redox-sensitive HSOP/siAURKA micellar complex at 50 mM, 37.2% knockdown of AURKA mRNA was observed. When the concentration of si-AURKA increased to 100 nM, 51.7% knockdown of AURKA mRNA was obtained. However, when the si-AURKA concentration further increased from 100 nM to 200 nM, the AURKA mRNA level decreased only slightly with no significant statistical difference (P > 0.05). Therefore, 100 nM was chosen to be the fixed concentration for further study. As compared with the non-redox sensitive HOP/si-AURKA, HSOP/si-AURKA micellar complexes exhibited much stronger gene silencing capacities at the concentration of 100 nM. The breakage of disulfide bond triggered by cytoplasmic

reducing agents resulted in loosened micellar complexes and quicker release of siRNA, which enhances gene silencing efficiency. As controls, PBS and micelles encapsulating scrambled siRNA (HSOP/siNonsense and HOP/siNonsense) showed no silencing of AURKA mRNA, whereas AURKA mRNA level was reduced by 43.7% in the Lipo2000/si-AURKA treatment group at the si-AURKA concentration of 50 nM. Though the Lipo2000/si-AURKA treatment group exhibited a slightly more effective gene silencing effect than HSOP/si-AURKA treatment at the same siRNA dosage, there is no significant statistical difference between these two groups (P > 0.05). In addition, when MDA-MB-231 cells were pre-treated with free HA, the gene silencing level decreased to 21.9% even though cells were incubated with HSOP/si-AURKA at a highest siAURKA dosage (200 nM). This result indicates the competitive binding of HA to CD44 receptors and verifies the HA receptor

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mediated specific endocytic pathway of HSOP/si-AURKA micellar complexes. In order to re-confirm gene silencing induced by siRNA formulated in different micelles, endogenous protein expressing level was further analyzed by western blot. MDA-MB-231 cells were exposed to HSOP/si-AURKA or HOP/si-AURKA micellar complexes for 48 h using blank HSOP and HOP micelles, HSOP/siNonsense and HOP/siNonsense micellar complex as controls. The western blot results were shown in Fig. 6D. As compared with HOP micelles, HSOP micelles exhibited much higher transfection efficiency, thus cells expressed a much lower level of AURKA protein when treated with HSOP/si-AURKA micellar complexes at the si-AURKA concentration of 100 nM. Moreover, when HA receptor of the cell surface was blocked, the expression of AURKA protein significantly increased, which suggested that HA receptor mediated intracellular delivery of si-AURKA by HSOP micelles can effectively downregulate the expression of AURKA protein. The result of western blot was in agreement with the result of the real-time PCR test.

3.8. Synergistic inhibition in cell proliferation in vitro It has been reported that AURKA inhibitors are capable of sensitizing MDA-MB-231 cells to taxanes [29e31]. To investigate whether the simultaneous delivery of si-AURKA and paclitaxel by redox-sensitive micelles could exhibit a much higher therapeutic efficacy by synergistic inhibition in cell proliferation, the cytotoxicity of different formulations were evaluated by MTT assay. First, no obvious cell death was observed in drug-free micelle treated groups (Fig. 7A), indicating no cytotoxicity of blank HSOP/HOP micelles. We further evaluated the cell inhibition effect of single drug loaded micelles (HSOP/si-AURKA, HOP/si-AURKA, PTXHSOPPTX HOPsiNonsense) (Fig. 7B and C). Thereafter, the cytotoxsiNonsense, icity effect of combined PTX and si-AURKA co-encapsulated in

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HSOP micelles at different weight ratios was investigated (Fig. 7D). As indicated in Fig. 7B, the IC50 of PTXHSOPsiNonsense and PTXHOPsiNonsense was 0.150 mg/mL and 0.324 mg/mL respectively. The smaller IC50 of single drug loaded HSOP micelles over corresponding HOP micelles could be attributed to the faster drug release. However, as listed in Fig. 7D, co-encapsulation of PTX and si-AURKA into single HSOP micelles at the five weight ratios (PTX/ si-AURKA at w/w) all permitted lower IC50 (based on the concentration of PTX). To confirm the synergistic effect of co-delivery HSOP micelles, the combination index (CI) was calculated using the Chou-Talalay isobologram equation [50]. As shown in Fig. 7E, HSOP micelles co-loaded with PTX/si-AURKA at all weight ratios exhibited CI value