Sequential Release of Autophagy Inhibitor and ... - ACS Publications

Mar 25, 2014 - Department of Oral and Maxillofacial Surgery, School of Dentistry, Harbin Medical University, 141 Yiman Street, Nangang District, Harbi...
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Sequential Release of Autophagy Inhibitor and Chemotherapeutic Drug with Polymeric Delivery System for Oral Squamous Cell Carcinoma Therapy Wuliji Saiyin,†,‡ Dali Wang,‡,§ Lili Li,† Lijuan Zhu,§ Bing Liu,† Lijian Sheng,∥ Yanwu Li,† Bangshang Zhu,§ Limin Mao,*,† Guolin Li,*,† and Xinyuan Zhu*,§ †

Department of Oral and Maxillofacial Surgery, School of Dentistry, Harbin Medical University, 141 Yiman Street, Nangang District, Harbin 150001, People’s Republic of China § School of Chemistry and Chemical Engineering, Shanghai Key Lab of Electrical Insulation and Thermal Aging, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ∥ Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Nangang District, Harbin 150001, People’s Republic of China S Supporting Information *

ABSTRACT: Autophagy inhibition is emerging as a new paradigm for efficient cancer therapy by overcoming multidrug resistance (MDR). Here, we developed an effective chemotherapeutic system for oral squamous cell carcinoma (OSCC) based on polymeric nanomicelles for codelivery of the anticancer drug doxorubicin (DOX) and the autophagy inhibitor LY294002 (LY). The hydrophobic DOX was conjugated onto a hydrophilic and pHresponsive hyperbranched polyacylhydrazone (HPAH), forming the DOXconjugated HPAH (HPAH−DOX). Due to its amphiphilicity, HPAH−DOX self-assembled into nanomicelles in an aqueous solution and the autophagy inhibitor LY could be loaded into the HPAH−DOX micelles. The release of DOX and LY from the LY-loaded HPAH−DOX micelles was pH-dependent, whereas LY was released significantly faster than DOX at a mildly acidic condition. The in vitro evaluation demonstrated that the LY-loaded HPAH− DOX micelles could rapidly enter cancer cells and then release LY and DOX in response to an intracellular acidic environment. Compared to the HPAH−DOX micelles and the physical mixture of HPAH− DOX and LY, the LY-loaded HPAH−DOX micelles induced a higher proliferation inhibition of tumor cells, illustrating a synergistic effect of LY and DOX. The preferentially released LY inhibited the autophagy of tumor cells and made them more sensitive to the subsequent liberation of DOX. The polymeric codelivery system for programmable release of the chemotherapy drug and the autophagy inhibitor provides a new platform for combination of traditional chemotherapy and autophagy inhibition. KEYWORDS: combination therapy, autophagy inhibition, chemotherapy, polymeric micelles, drug delivery

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INTRODUCTION

repression of autophagy has demonstrated an increased efficacy of chemotherapeutics, both in vitro and in vivo.10−13 However, most of the previous work in this research area focused on the combination of small molecular weight chemotherapy drugs and autophagy inhibitors. Generally, these chemotherapy drugs and autophagy inhibitors exhibit poor stability, limited water solubility, undesirable toxicity, and relatively short half-life, which prevent their widespread clinical application. Moreover, the simple combination of chemotherapy drugs and autophagy inhibitors does not provide a sequential release of these components in a controlled manner that may have a better

Carcinomas of the oral cavity, particularly oral squamous cell carcinoma (OSCC), have become a critical healthcare problem worldwide.1 Traditional chemotherapy is one of the most effective treatments, but it could not meet requirements in clinical therapy since the tumor does not respond to the current chemotherapeutic agents due to the multidrug resistance (MDR) of tumor cells.2,3 Recently, the elegant concept of autophagy inhibition provides a new strategy to overcome MDR for efficient cancer therapy.4−7 In general, when the cells respond to limited nutrition and growth factors, autophagy is activated and contributes to maintaining homeostasis through degradation of impaired or unnecessary macromolecules and organelles, thereby providing energy to cancer cells. 8 Autophagy usually serves as a protective mechanism for tumor cells exposed to anticancer drugs.9 In preclinical trials, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1662

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phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against cleaved caspase-3, cleaved PARP (Poly ADP-ribose polymerase), Beclin-1, SQSTM1/p62, and LC3B were supplied by Cell Signaling Inc. (Massachusetts, USA). The Annexin V-FITC/PI Apoptosis Detection Kit was purchased from BD PharMingen (San Diego, CA, USA). The FITC-conjugated secondary antibody for indirect immunofluorescence, anti-β-actin antibody, and the fluorescent marker Rhodamine 123 (Rh123) were obtained from Beyotime Institute of Biotechnology (Beijing, China). All other chemicals were high performance liquid chromatography (HPLC) or analytical grade and used without further purification. Dialysis tubes (molecular weight cutoff, MWCO = 1 kDa) were purchased from Shanghai Lvniao Technology Corp. Clear polystyrene tissue culture treated 6-well and 96-well plates were obtained from SangonBiotech (Shanghai, China). Ultrapure water (18.2 MΩ/cm) was used in all experiments. Measurements. All nuclear magnetic resonance (NMR) spectra were carried out on Varian Mercury plus 400 NMR spectrometer (400 MHz, 298 K) with dimethyl sulfoxide-d6 (DMSO-d6) or deuterated chloroform (CDCl3) as solvents. Quantitative 13C NMR spectra were measured by the method of inverse gated 1H decoupling. Fourier transform infrared (FTIR) spectra were performed on a Paragon 1000 instrument using the potassium bromide (KBr) method. The molecular weights of the synthesized samples were measured by the sizeexclusion chromatography multiangle laser light scattering (SEC-MALLS) system, which consisted of a Waters 2690D Alliance liquid chromatography system, a Wyatt Optilab DSP differential refractometer detector, and a Wyatt MALLS detector. Two PL mix-D columns (Styragel HR3, HR4) were used in series. DMF containing 0.5 M LiBr was used as the mobile phase at a flow rate of 1 mL/min at 30 °C. The data were analyzed by Astra software (Wyatt Technology). Transmission electron microscopy (TEM) studies were performed to detect the morphology and size of micelles with a JEM2010HT microscope at an accelerating voltage of 200 kV. A drop of the micelle solution (1 mg/mL) was spread onto an amorphous holey-carbon film supported by a copper grid, then lyophilized by a freeze-dryer for observation. The cell samples were examined with a TEM (Hitachi-7650). Dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples were measured at a scattering angle of 173°. Synthesis of HPAH and HPAH−DOX. HPAH and HPAH−DOX conjugate under study were prepared as previously described, and the detailed characterizations were given in our previous report.26 Briefly, hyperbranched polymer HPAH was synthesized by a simple polycondensation of BD and 1-(2-aminoethyl)piperazine tripropionylhydrazine (AEPNHNH2). Then HPAH−DOX was prepared by conjugation of DOX onto the surface of HPAH through further condensation of ketone and acylhydrazine groups. The synthesis details and characterization data of HPAH and HPAH−DOX are also described in the Supporting Information. Preparation of HPAH−DOX Micelles. In brief, 10 mg of HPAH was dissolved in 2 mL of DMF and stirred at room temperature for 1 h. Then, the solution was slowly added to 5 mL of deionized water and stirred for 0.5 h. Subsequently, the solution was dialyzed against deionized water for 24 h (MWCO = 1 kDa), and the deionized water was exchanged every 4 h.

synergistic effect for cancer therapy. Hence, it would be significant to construct a highly efficient codelivery system of chemotherapy drugs and autophagy inhibitors with controlled release ability for OSCC therapy. As one emerging smart material, stimulus-responsive polymeric micelles derived from amphiphilic polymers have attracted increasing interest as delivery vehicles for anticancer drugs.14−18 The previous studies have demonstrated that these stimulus-responsive nanocarriers could release the loaded drugs by exerting an appropriate stimulus, minimize side effects, and greatly enhance therapeutic efficacy.19,20 In particular, the pHsensitive nanocarriers have gained significant attention since the tumor environment is more acidic than healthy tissues.21−25 Therefore, it can be imagined that if the chemotherapy drugs and autophagy inhibitors are encapsulated into the pHresponsive nanomicelles in different binding forms, a nanodelivery system with programmable release ability can be easily obtained. Ascribed to the perfect combination of nanomicelles and combination therapy, an efficient drug delivery system with a synergistic effect can be expected. In our previous work, a backbone-degradable hyperbranched polyacylhydrazone (HPAH) with pH-responsive properties was successfully synthesized by polycondensation of diketone and trihydrazine.26 HPAH possessed a large number of acylhydrazine terminals for further conjugation of drugs and exhibited good water solubility and low cytotoxicity, making it an excellent carrier for drug delivery. In the present work, we constructed pH-responsive nanomicelles based on HPAH for codelivery of the chemotherapy drug doxorubicin (DOX) and the autophagy inhibitor LY294002 (LY) for OSCC therapy. The anticancer drug DOX was readily conjugated onto HPAH via acylhydrazone linkages, and the autophagy inhibitor LY was encapsulated into the core of self-assembled HPAH−DOX micelles. The release of the conjugated DOX and encapsulated LY was pH-dependent, and the release of LY was faster due to the physical encapsulation. The disruption of these nanomicelles inside tumor cells would result in a rapid deployment of the autophagy inhibitor, leading to early inhibition of protective autophagy of tumor cells. The subsequent release of the chemotherapy drug from the micelles killed the tumor cells. Indeed, here we demonstrate that treatment with the LY-loaded HPAH−DOX micelles results in a superior anticancer outcome as compared to the HPAH−DOX micelles and the physical mixture of HPAH−DOX and LY. This work highlights the potential of stimulus-responsive nanodelivery system as combination therapeutic platforms of chemotherapy and autophagy inhibition for enhanced efficacy against aggressive OSCC.

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EXPERIMENTAL SECTION Materials. 2,3-Butanedione (99%, BD), methyl acrylate (MA), and 1-(2-aminoethyl)piperazine (AEP, 99%) were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOX·HCl) and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, LY) were purchased from Beijing Huafeng United Technology Corp. and used as received. Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF) were supplied by Sinopharm Chemical Reagent Co. (Shanghai, China). MA, ethanol, DMF, and hydrazine hydrate were purified according to standard procedure. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamino-21663

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micelles, and the cells were incubated at 37 °C for predetermined time intervals. Then the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, and the slides were rinsed with cold PBS three times. Finally, the cells were stained with DAPI for 5 min, and the slides were mounted and observed with a fluorescence microscope (Olympus Bx60). In Vitro Cytotoxicity Studies. Drug-induced cytotoxic effects were measured in vitro by the MTT assay. HN-6 cells or CAL-27 cells were seeded into 96-well plates with a density of 6 × 103 cells per well in 200 μL of medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of a medium containing serial dilutions of the LYloaded HPAH−DOX micelles, the HPAH−DOX micelles, the mixture of HPAH−DOX and LY, and HPAH at 37 °C for predetermined time intervals. After treatment, the wells were quickly washed 3 times with PBS. Then, 20 μL of 5 mg/mL MTT assay stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL per well DMSO, and the absorbance was measured in a BioTek SynergyH4 at a wavelength of 490 nm. Apoptosis Analyses with Flow Cytometry. Cells were exposed to HPAH−DOX, the LY-loaded HPAH−DOX micelles and the mixture of HPAH−DOX and LY at equivalent DOX and LY doses for 48 h. After that, both floating and attached cells were collected, washed twice with ice-cold PBS, and incubated at 37 °C for 15 min with propodium iodide (PI) and Annexin V-FITC to determine cell apoptosis. The samples were analyzed with a FACScan flow cytometer. Apoptosis and Autophagy Analyses with Western Blot. HN-6 cells or CAL-27 cells were seeded in 6-well plates at 5 × 105 cells per well in 2 mL of complete DMEM and allowed to adhere at 37 °C for 24 h, followed by removing culture medium and adding micelles in 2 mL of DMEM medium at a final DOX concentration of 1.6 μg/mL or LY concentration of 1.4 μg/mL. Then the cells were cultured at 37 °C for 48 h. The protein levels of cleaved caspase-3, cleaved PARP, LC3B I/LC3B II, Beclin-1, and p62 were analyzed by Western blotting. Briefly, cell lysates were prepared and electrotransferred, and 40 μg of protein from each sample was resolved on polyacrylamide sodium dodecyl sulfate (SDS) gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with primary antibody, followed by incubation with an alkaline phosphataseconjugated secondary antibody. The membranes were then developed using the enhanced chemiluminescence system (Pierce). Blots were stained with an anti-β-actin antibody to confirm that each lane contained a similar amount of protein. To determine whether HPAH induced autophagy, cells were allowed to adhere and serial dilutions of HPAH were added. After incubation for 48 h, the autophagy-related proteins LC3B I/LC3B II, Beclin-1, and p62 were measured by Western blot in the same way as described above. Autophagy Analyses with Immunofluorescence and TEM. Microtubule-associated protein light chain 3 (LC3) was examined by indirect immunofluorescence as previously reported. 27−29 The treated cells were fixed with 4% paraformaldehyde for 30 min, blocked with BSA, and incubated with the primary antibody (anti-LC3B, used at a 1:200 dilution) overnight at 4 °C. Then the slides were incubated

The appearance of turbidity in the aqueous solution indicated the formation of aggregation. Preparation of LY-Loaded HPAH−DOX Micelles and Rh123-Loaded HPAH−DOX Micelles. For preparing the LY-loaded micelles, HPAH−DOX (10.0 mg) and a predetermined amount of LY were dissolved in an aliquot of DMF and stirred at room temperature for 4 h. Then, 10× volumes of deionized water were added dropwise to the solution under stirring. The mixture was stirred overnight at room temperature. The solution was then placed in a dialysis bag with a MWCO of 1 kDa and dialyzed for 24 h to eliminate any residual solvent and free LY. To determine the total loading of drug, the LY-loaded HPAH−DOX micelle solution was lyophilized. The Rh123-loaded HPAH−DOX micelles were prepared using the same method. Drug loading content (DLC) and drug loading efficiency (DLE) of DOX and LY in the LY-loaded HPAH−DOX micelles were measured by HPLC. The acylhydrazone linkage of HPAH−DOX was cleaved by addition of 0.1 N HCl solution and stirred at room temperature for 48 h. After removal of HCl solution, the sample was redissolved in acetate buffer solution. It was assayed on a Shimadzu LC-10AD (Shimadzu, Japan) HPLC system equipped with a Shimadzu UV detector and an Agilent RP-HPLC analytical column (C-18, 5 μm, 200 mm × 4.6 mm). Calibration curves were generated using known concentrations of DOX or LY. DLC and DLE of HPAH−DOX and LY-loaded HPAH−DOX micelles were calculated using the following equations: DLC (wt %) = (weight of loaded drug/weight of polymer) × 100% DLE (%) = (weight of loaded drug/weight of drug in feed) × 100%

In Vitro DOX and LY Release. Drug-release studies were performed at 37 °C in pH 7.4 PBS solution and pH 5.0 acetate buffer medium to mimic physiological and lysosomal conditions, respectively. A total of 20 mg of lyophilized LYloaded HPAH−DOX micelles was dissolved in 2 mL of the appropriate release medium and placed in a dialysis bag with an MWCO of 1 kDa. The dialysis bag was then immersed in 50 mL of the release medium and stirred at a constant temperature. At specified time points, 2 mL samples were withdrawn from the surrounding release medium, and an equal volume of fresh PBS or acetate buffer was immediately added for keeping the sink condition. The amount of released DOX and LY was measured by HPLC system and plotted as a function of time. Cell Cultures. The human OSCC cell lines HN-6 and CAL27 were cultured in DMEM supplied with 10% FBS and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Cellular Uptake of LY-Loaded HPAH−DOX Micelles. LY was substituted with Rh123, which was used as a fluorescent marker to assess the efficiency of LY-loaded HPAH−DOX micelles taken up by tumor cells. HN-6 cells or CAL-27 cells were seeded in 6-well plates at 2 × 105 cells per well containing 2 mL of DMEM supplemented with 10% FBS and allowed to adhere at 37 °C for 24 h prior to the assay. The medium was then replaced with fresh DMEM culture medium containing free DOX, free Rh123, or Rh123-loaded HPAH−DOX 1664

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Scheme 1. Synthesis Route of HPAH−DOX and Construction of the LY-Loaded HPAH−DOX Micellea

a I, HPAH was synthesized by a simple polycondensation of BD and AEP-NHNH2; II, HPAH−DOX was obtained through conjugation reaction between DOX and HPAH.

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with FITC-conjugated secondary antibody (1:200 dilution) for 1 h, stained with DAPI for 5 min, and washed three times with PBS. After mounting, the cells were observed using an Olympus Bx60 fluorescence microscope with a FITC filter. TEM was used to detect autophagic vesicles with double membrane structure, also referred to as autophagosomes. The presence of these vesicles is morphological evidence of autophagy.30 The treated cells were harvested by trypsinization, washed twice with PBS, and fixed with ice-cold glutaraldehyde (3% in 0.1 M phosphate buffer, pH 7.4) for 90 min at room temperature. After washing in PBS, the cells were postfixed in 2% OsO4 for 2 h at 4 °C. After several washes with PBS, the samples were dehydrated in graded alcohol, transferred into toluene, and embedded in Ep on 812 resin. The resin was allowed to polymerize in a dry oven at 60 °C for 24 h. Ultrathin sections were prepared and stained with uranyl acetate−lead citrate. The sections were then observed at 80 kV using a TEM (Hitachi-7650).

H NMR and FTIR analyses confirmed the successful formation of HPAH. The degree of branching of HPAH was 0.60 according to quantitative 13C NMR analysis. The weightaverage molecular weight (Mw) and its polydipersity index of HPAH determined by SEC-MALLS were 4 kDa and 1.60, respectively. As a novel kind of biodegradable materials, HPAH combines high stability and stimulus-responsive property together. It can be degraded into low molecular weight products due to the existence of acid-sensitive acylhydrazone bonds in the backbone of highly branched polymer, illustrating a low cytotoxicity. The pH-sensitive biodegradability and low cytotoxicity make HPAH a promising biomaterial for biomedical applications. In particular, HPAH can be used as an ideal carrier for hydrophobic drugs because of its good hydrophilicity and a large number of functional acylhydrazine terminals. The anticancer drug DOX, which has a ketone group, was chemically conjugated with acylhydrazine terminals of HPAH to form pH-sensitive acylhydrazone bonds. The degree of conjugation was determined to be around 6.5% based on the HPLC measurement. The synthetic route of HPAH and HPAH−DOX is described in the Supporting Information, and for detailed characterization data and properties of HPAH and HPAH−DOX, refer to our previous publication.26 Fabrication of HPAH−DOX Micelles and LY-Loaded HPAH−DOX Micelles. HPAH−DOX’s amphiphilic nature provides an opportunity for it to self-assemble into nanometer

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RESULTS AND DISCUSSION Synthesis and Characterization of HPAH and HPAH− DOX. HPAH and HPAH−DOX conjugate were prepared as described in our previous report.26 As shown in Scheme 1, the backbone-cleavable and pH-sensitive HPAH was synthesized through A 2 + B 3 polycondensation of diketone and trihydrazine. With the feeding ratio of BD/AEP-NHNH2 at 1:1, HPAH with plentiful acylhydrazine terminals was obtained. 1665

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Figure 1. DLS plots of (A) the HPAH−DOX micelles and (C) the LY-loaded HPAH−DOX micelles and representative TEM images of (B) the HPAH−DOX micelles and (D) the LY-loaded HPAH−DOX micelles (the scale bar is 200 nm).

Figure 2. (A) Size of the LY-loaded HPAH−DOX micelles at different time intervals in PBS (pH = 7.4) determined by DLS. Error bars represent the standard deviation (n = 3). (B) In vitro release profiles of LY and DOX from the LY-loaded HPAH−DOX micelles at different pH values (7.4 and 5.0) at 37 °C.

aggregates in an aqueous solution. After being dissolved in DMF and dialyzed against water, HPAH−DOX was able to spontaneously self-assemble into micelles with hydrophobic DOX core and hydrophilic HPAH outer shell. The size of HPAH−DOX micelles was determined by DLS measurement. The size distribution histogram of the micelles in Figure 1A displays a monomodal distribution with an average hydrodynamic diameter of 27 nm and a PDI of 0.26. On the other hand, the size and morphology of HPAH−DOX micelles were also measured and observed by TEM. The TEM image in Figure 1B shows that HPAH−DOX can aggregate into approximate spherical micelles in an aqueous solution, and

the average diameter of them is close to the result of DLS measurement. LY is a potent cell permeable inhibitor of phosphoinositide 3-kinase (PI3K).31,32 Inhibition of PI3K with LY can inhibit autophagic sequestration.33 Therefore, LY has been well studied as autophagy inhibitor to exhibit excellent antitumor activity with chemotherapy drugs in a number of in vitro and in vivo studies.34,35 However, it suffers from poor pharmacokinetics, low water solubility, and undesirable toxicity, which limits its application in clinical treatment.36,37 In the present work, hydrophobic LY was encapsulated into the core of the HPAH−DOX micelles (Scheme 1), which would overcome the 1666

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Figure 3. Fluorescence images of CAL-27 and HN-6 cells incubated with (a1, a2) free DOX and free Rh123 for 5 min; (b1, b2) Rh123-loaded HPAH−DOX for 5 min; (c1, c2) free DOX and free Rh123 for 1 h; and (d1, d2) Rh123-loaded HPAH−DOX for 1 h at 37 °C (DOX concentration: 10 μg/mL, Rh123 concentration: 10 μg/mL).

conjugated DOX at an acidic condition, which is beneficial for inhibiting autophagy before DOX induction and makes the tumor cells more sensitive to DOX. Moreover, the fast cleavage of DOX in lower pH leads to the disassembly of LY-loaded HPAH−DOX micelles and the subsequent release of the loaded LY, which may result in continuous autophagy inhibition that leads to more cell death induced by DOX. Cell Internalization. To determine whether the LY-loaded HPAH−DOX micelles could effectively deliver DOX and LY into cells, the cellular uptake and intracellular release behavior of the LY-loaded HPAH−DOX micelles by CAL-27 and HN-6 cells was monitored by fluorescence microscopy. The hydrophobic fluorescent probe Rh123 was loaded into micelles in place of LY because LY has no obvious fluorescence and it is difficult to be detected by fluorescence microscopy. Both CAL27 and HN-6 cells were incubated with free DOX, free Rh123, and Rh123-loaded HPAH−DOX at 37 °C for 5 min and 1 h, respectively, and then fixed with paraformaldehyde. The cell nucleus was stained by DAPI and the pretreated cells were observed directly by fluorescence microscopy. As shown in Figures 3a1 and 3a2, cells exposed to free DOX and free Rh123 show very low fluorescence signals in both the cytoplasm and the nucleus after 5 min, while cells incubated with Rh123loaded HPAH−DOX exhibit relatively high fluorescence intensity of DOX and Rh123 in the cytoplasm but no fluorescence in the nucleus (Figures 3b1 and 3b2). These differences occur because the positively charged HPAH enables it to penetrate the cells more easily. After 1 h incubation, free DOX enters the nucleus and free Rh123 still exhibits a weak fluorescence signal (Figures 3c1 and 3c2). The Rh123-loaded HPAH−DOX micelles remain in the cytoplasm (Figures 3d1 and 3d2) and exhibit strong fluorescence intensity of DOX and Rh123, indicating that high cellular uptake of the micelles and efficient release of DOX and Rh123 take place. It has been reported that free DOX molecules can enter the nucleus rapidly via passive diffusion.38,39 Free Rh123 has low solubility in water and limits the cellular uptake. After having been encapsulated in the HPAH−DOX micelles, Rh123 increases its solubility and enhances its delivery to CAL-27 cells and HN-6 cells. These results suggest that the LY-loaded HPAH−DOX micelles can

aforementioned limitations and provide for a desirable inhibitory profile. For the preparation of the LY-loaded micelles, LY and HPAH−DOX were dissolved in DMF together and then dropped slowly into deionized water under stirring. After the formation of the LY-loaded micelles, the solution was ultrafiltered and dialyzed against deionized water using a dialysis tube (MWCO = 1 kDa) until the absence of LY signals in dialysate by UV measurements. When the feed ratio of HPAH−DOX to LY was 10:1, the DLC in the HPAH−DOX micelles was 5.7%, and the corresponding DLE was 53.2%. As shown in Figure 1C, the size and size distribution of the LYloaded HPAH−DOX micelles measured by DLS show that the diameters of these LY-loaded micelles are larger than their parent micelles without LY. The average hydrodynamic diameter of the LY-loaded micelles is 40 nm with PDI of 0.23. The increase of micelle size after LY loading is also revealed by the TEM measurement in Figure 1D, which shows that the average diameter of the LY-loaded HPAH−DOX micelles increases to about 38 nm. The stability of LY-loaded HPAH−DOX micelles was evaluated by DLS technique. As shown in Figure 2A, no apparent change in micelle size is observed after 120 h in PBS (pH = 7.4). Therefore, the high stability of LY-loaded HPAH−DOX micelles at physiological condition offers the possibility for the potential of therapeutic delivery. In Vitro DOX and LY Release. The in vitro release behavior of the LY-loaded HPAH−DOX micelles was investigated under a simulated physiological condition (PBS, pH 7.4) and in an acidic environment (acetate buffer, pH 5.0) at 37 °C. The release profiles of DOX and LY from the micelles at different pH values are presented in Figure 2B. The LY-loaded HPAH− DOX micelles exhibit high stability in pH = 7.4 PBS solution with less than 30% release of each therapeutic component over a period of 106 h, while the two components show staggered release in pH = 5.0 acetate buffer medium within 40 h with a faster release rate of LY from the micelles, comparing to DOX from the HPAH−DOX micelles. These results indicate that the acylhydrazone linkages are stable under physiological conditions but readily cleavable in an acidic condition. It is worth noting that the encapsulated LY is released faster than 1667

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Figure 4. Cell viability of (A) CAL-27 cells and (B) HN-6 cells after 48 h incubated with the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY (HPAH−DOX+LY), and the LY-loaded HPAH−DOX micelles (HPAH−DOX/LY) at various concentrations. Cell viability of (C) CAL-27 cells and (D) HN-6 cells incubated with the above three drug formulations at equivalent dose of DOX (1.6 μg/mL) and LY (1.4 μg/ mL) for various time periods. Values represent mean ± SD (n = 3).

effectively deliver both DOX and LY into cells. The cellular uptake of the LY-loaded HPAH−DOX micelles may be based on an endocytosis mechanism, not the simple passive diffusion of small molecules between the extracellular and intracellular milieu. Moreover, the release of DOX and LY from the LYloaded HPAH−DOX micelles is dependent on a pH-triggered, sustained release rather than a burst one, which is in accordance with the in vitro release measurements. In Vitro Anticancer Effect of LY-Loaded HPAH−DOX Micelles. After incubation for 48 h, HPAH displayed very little toxic effects in both CAL-27 cells and HN-6 cells, with inhibition rates of less than 10%, even at high concentration of 1 mg/mL (Figure S1 in the Supporting Information). The antitumor activity of the LY-loaded HPAH−DOX micelles was evaluated by MTT assay against CAL-27 cells and HN-6 cells. The HPAH−DOX micelles and the physical mixture of HPAH−DOX and LY were also evaluated under identical conditions as the control. As depicted in Figures 4A and 4B, the cytotoxicity of all three formulations is concentration-dependent after 48 h incubation. Both the LY-loaded HPAH−DOX micelles and the physical mixture of HPAH−DOX and LY exhibit higher cytotoxicity than the HPAH−DOX micelles in CAL-27 cells and HN-6 cells, suggesting the additive or synergistic anticancer effect of DOX and LY. This is because administration of LY can inhibit autophagy, thus making tumor cells more sensitive to DOX treatment. Moreover, the LYloaded HPAH−DOX micelles exhibit higher cytotoxicity than the physical mixtures of HPAH−DOX and LY. It may be related to the fact that the LY-loaded HPAH−DOX micelles improve the solubility of hydrophobic LY and release the LY and DOX in a controlled programmable manner. The preferential release of LY from LY-loaded HPAH−DOX micelles leads to autophagy inhibition and improves the sensitivity of tumor cells to DOX, thus resulting in more cell death. These results demonstrate that the HPAH−DOX

micelles could effectively encapsulate LY and subsequently release it in tumor cells, enhancing drug bioavailability and exhibiting better antitumor effect. The in vitro cytotoxicity of the LY-loaded HPAH−DOX micelles, the HPAH−DOX micelles, and the physical mixture of HPAH−DOX and LY was further evaluated at equivalent dose of DOX (1.6 μg/mL) and LY (1.4 μg/mL) for different time periods. Typically, the tumor cells (CAL-27 cells and HN6 cells) were first cultured overnight for adherence and then incubated with three formulations for 12, 24, and 48 h, respectively. Figures 4C and 4D show that the LY-loaded HPAH−DOX micelles inhibit the proliferation of cancer cells in a time-dependent fashion. After 12 h incubation, all of them exhibit low abilities of proliferation inhibition in both CAL-27 cells and HN-6 cells. After 24 h incubation, the anticancer effect of the LY-loaded HPAH−DOX micelles and the physical mixture of HPAH−DOX and LY improves apparently, which is much better than that of the HPAH−DOX micelles, indicating the synergistic effect of the two drugs. With the increase of incubation time to 48 h, the LY-loaded HPAH−DOX micelles display markedly improved abilities of proliferation inhibition in the cancer cells compared to the HPAH−DOX micelles and the physical mixture of HPAH−DOX and LY. These results confirm that the program release of LY and DOX from the LYloaded HPAH−DOX micelles improves the synergistic effect of two drugs and thus exhibits enhanced inhibition of the OSCC proliferation. In Vitro Apoptosis-Inducing Effect of LY-Loaded HPAH−DOX Micelles. DOX-induced cancer cell death is mainly apoptotic (programmed cell death).40,41 The FITCAnnexin V/PI method was used to verify whether the cancer cell death caused by the LY-loaded HPAH−DOX micelles was associated with apoptosis. First, cells were incubated with the LY-loaded HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY, and the blank HPAH−DOX micelles, at 1668

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Figure 5. (A) Representative flow cytometric analysis results of cell apoptosis of CAL-27 cells and HN-6 cells treated with the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY (HPAH−DOX+LY), and the LY-loaded HPAH−DOX micelles (HPAH−DOX/LY) at equivalent DOX concentrations (1.6 μg/mL) and LY concentrations (1.4 μg/mL) for 48 h. Lower left, living cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area. (B) Ratio of apoptotic CAL-27 cells based on the results of flow cytometry measurements. (C) Ratio of apoptotic HN-6 cells based on the results of flow cytometry measurements. Values represent mean ± SD (n = 3). A significant increase in the apoptosis rate compared with the control is denoted by “*” (P < 0.01), a significant increase compared with HPAH−DOX-treated cells is denoted by “§” (P < 0.01), and a significant increase compared with HPAH−DOX-treated cells and HPAH−DOX+LY-treated cells is denoted by “#” (P < 0.01).

equivalent dose of DOX (1.6 μg/mL) and LY (1.4 μg/mL) for 48 h and then stained with FITC-Annexin V and PI. The apoptosis-inducing effect was evaluated by counting the early apoptotic percentage plus the late apoptotic percentage. As shown in Figure 5, after applying the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY, and the LYloaded HPAH−DOX micelles, the induced apoptotic percentages in CAL-27 cells are 13.46%, 32.16%, and 52.4%; and those in HN-6 cells are 13.99%, 28.69%, and 54.26%, respectively. LY-loaded HPAH−DOX micelles result in 16.52% early apoptotic cells (positive for FITC-Annexin V only) and 35.88% late apoptotic cells (double positive for

FITC-Annexin and PI), indicating that polymer-conjugated DOX induces similar apoptotic progression in CAL-27 cells as free DOX and the biological function of DOX is retained following conjugation to the polymer (Figure 5A). As compared to 11.04% of cells in early apoptosis and 2.42% in late apoptosis after treatment by HPAH−DOX micelles, the enhanced apoptosis induced by LY-loaded HPAH−DOX micelles is likely due to the fact that the preferential release of LY leads to autophagy inhibition of tumor cells and makes them more sensitive to the subsequent liberation of DOX. Interestingly, the LY-loaded HPAH−DOX micelles induce much more apoptotic cells than the physical mixture of 1669

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Figure 6. Cells treated with the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY (HPAH−DOX+LY), and the LY-loaded HPAH−DOX micelles (HPAH−DOX/LY) at equivalent DOX concentrations (1.6 μg/mL) and LY concentrations (1.4 μg/mL) for 48 h. Then the apoptosis related proteins were detected by Western blot. (A) Western blot analysis of cleaved caspase-3 and cleaved PARP in CAL-27 and HN-6 cells. (B) Semiquantitative analysis of cleaved caspase-3 and cleaved PARP in CAL-27 cells. (C) Semiquantitative analysis of cleaved caspase-3 and cleaved PARP in HN-6 cells. Values represent mean ± SD (n = 3). A significant increase compared with the control is denoted by “*” (P < 0.01), a significant increase compared with HPAH−DOX-treated cells is denoted by “§” (P < 0.01), and a significant increase compared with HPAH−DOXtreated cells and HPAH−DOX+LY-treated cells is denoted by “#” (P < 0.01).

HPAH−DOX and LY, indicating an effective delivery of LY by the HPAH−DOX micelles with enhanced anticancer efficacy. Similar results are also observed in HN-6 cells. Apparently, little apoptosis or cell death is observed in controls in which CAL-27 or HN-6 cells are grown in media alone. Therefore, flow cytometry data confirm that the LY-loaded HPAH−DOX micelles can significantly induce apoptosis of OSCC, which is in accordance with MTT analysis. LY-Loaded HPAH−DOX Micelles Regulate ApoptosisRelated Protein Expression. Caspase-3 plays a central role in the execution of the apoptotic program and is primarily responsible for the cleavage of PARP during cell death. Cleaved caspase-3 indicates the activity of caspase-3, while PARP is a well-known substrate of caspase-3 and cleaved PARP indicates the extent of apoptosis.42 The expression levels of cleaved caspase-3 and cleaved PARP were detected to further analyze the apoptosis of the tumor cells response to the treatment. CAL-27 and HN-6 cells were treated with the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY, and the LY-loaded HPAH−DOX micelles at equivalent dose of DOX (1.6 μg/mL) and LY (1.4 μg/mL) for 48 h, and the expression levels of cleaved caspase-3 and cleaved PARP were detected by Western blot analysis. As shown in Figure 6, the expression levels of cleaved caspase-3 and cleaved PARP in response to the formulations are significantly upregulated compared to the control. It is found that cells treated with the physical mixture of HPAH−DOX and LY and the LY-loaded HPAH−DOX micelles exhibit higher expression levels of cleaved caspase-3

and cleaved PARP compared to the cells treated with the HPAH−DOX micelles. It indicates that the coadministration of DOX and LY has a better apoptosis-inducing effect than the single agent of DOX. Among the cells treated with these formulations, the cells treated with the LY-loaded HPAH− DOX micelles exhibit the highest expression levels of the apoptosis-related proteins, revealing that the LY-loaded HPAH−DOX micelles can remarkably improve the apoptosis of OSCC and further lead them to death. LY-Loaded HPAH−DOX Micelles Enhance Apoptosis through Preferential Autophagy Inhibition. Recently, it has been recognized that the anticancer efficiency of drugs can be enhanced by autophagy inhibition. Here, we introduced the well-known autophagy inhibitor LY with the anticancer agent DOX into a nanodelivery system for combination treatment, as shown in Scheme 2. To judge whether the LY-loaded HPAH− DOX micelles had a better effect of autophagy inhibition, indirect immunofluorescence, TEM, and Western blot analysis were performed. For indirect immunofluorescence, CAL-27 and HN-6 cells were treated with HPAH (25 μg/mL, the amount that equal to the content in other formulations), the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY, and the LYloaded HPAH−DOX micelles at an equivalent dose of DOX (1.6 μg/mL) and LY (1.4 μg/mL) for 48 h. After that, the autophagy induced by these formulations was detected by fluorescence microscope. As shown in Figure 7, the cells treated with HPAH exhibit low fluorescence intensity and have no 1670

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Scheme 2. Schematic Representation for Proposed Mechanism of Cellular Uptake of the LY-Loaded HPAH−DOX Micelles and Intracellular Drug Releasea

a

The strategy of nanomicelle-based LY/DOX delivery can effectively lead to high cell death. The encapsulated LY was preferentially released to induce autophagy inhibiton, which could efficiently enhance the cytotoxicity of DOX and lead to cell death.

Figure 7. Fluorescence images of CAL-27 and HN-6 cells exposed to HPAH (25 μg/mL), the HPAH−DOX micelles, the physical mixture of HPAH−DOX and LY (HPAH−DOX+LY), and the LY-loaded HPAH−DOX micelles (HPAH−DOX/LY) at equivalent DOX concentrations (1.6 μg/mL) and LY concentrations (1.4 μg/mL) for 48 h and stained with LC3B antibody.

significant difference with the control, indicating a low amount of autophagy induction. The fluorescence intensity in HPAH− DOX-treated cells is much higher than that of the physical mixture of HPAH−DOX and LY, and the LY-loaded HPAH− DOX-treated ones. It indicates that the introduction of LY can effectively inhibit the autophagy induced by the anticancer drug DOX. Among the cells treated with all these three formulations, the LY-loaded HPAH−DOX-treated cells exhibit the lowest amount of fluorescence intensity, indicating the least amount of autophagy. It is consistent with the hypothesis that the LYloaded HPAH−DOX micelles may have a better effect of

autophagy inhibition due to the preferentially sustained release of LY, which provides the basis for an efficient cancer therapy. Considering that immunofluorescence is an indirect measurement of autophagosome formation, the immunofluorescence results partly reflect the autophagy inhibition effect of the LYloaded HPAH−DOX micelles. Therefore, we used TEM to directly observe the formation of autophagosomes caused by the drugs. As shown in Figure 8, HPAH induces low amounts of autophagosomes in CAL-27 and HN-6 cells, which has no significant differences with the control. However, the cells incubated with the HPAH−DOX micelles induce a large 1671

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Figure 8. (A) TEM images of CAL-27 and HN-6 cells exposed to HPAH (25 μg/mL), the HPAH−DOX micelles, the physical mixture of HPAH− DOX and LY (HPAH−DOX+LY), and the LY-loaded HPAH−DOX micelles (HPAH−DOX/LY) at equivalent DOX concentrations (1.6 μg/mL) and LY concentrations (1.4 μg/mL) for 48 h. (B) The average number of autophagic vesicles (AVs) per CAL-27 cell. (C) The average number of AVs per HN-6 cell. AVs, defined as double-membrane structures, such as the ones marked by black arrows, were counted and calculated by two independent investigators. At least 200 cells from each group were counted. A significant increase compared with the control is denoted by “*” (P < 0.01), a significant increase compared with HPAH−DOX/LY-treated cells is denoted by “§” (P < 0.01), and a significant increase compared with HPAH−DOX/LY-treated cells and HPAH−DOX+LY-treated cells is denoted by “#” (P < 0.01).

we found that the ratios of LC3 II/LC3 I and the expression levels of Beclin-1 and p62 were not significantly changed in HPAH-treated cells compared to the control, even the concentration was up to 100 μg/mL (Figure S2 in the Supporting Information). It testifies that HPAH induces no obvious autophagosome formulation, even at a high concentration. As shown in Figure 9, the tumor cells treated with the HPAH−DOX micelles exhibit higher expression levels of Beclin-1 and conversion rate of LC3 I/II, and lower levels of p62 compared to the cells treated with the physical mixture of HPAH−DOX and LY, and the LY-loaded HPAH−DOX micelles. It is because LY inhibits the early stage of autophagy effectively which is induced by DOX. In particular, the cells incubated with the LY-loaded HPAH−DOX micelles exhibit the lowest expression level of Beclin-1 and conversion rates of LC3 I/II, and the highest level of p62. These data indicate that the LY-loaded HPAH−DOX micelles inhibit the autophagy effectively. Similar to the results of indirect immunofluorescence and TEM observation, the Western blot analysis further confirms that the LY-loaded HPAH−DOX micelles have excellent ability of autophagy inhibition.

amount of autophagosomes, which can be attributed to the introduction of the anticancer drug DOX. In contrast, the cells treated with the LY-loaded HPAH−DOX micelles possess much lower amounts of autophagosomes than those incubated with the physical mixture of HPAH−DOX and LY, and the HPAH−DOX micelles. These data suggest that the programmable release of LY and DOX from the LY-loaded HPAH− DOX micelles successfully inhibits the autophagy of CAL-27 and HN-6 cells, which is consistent with the results of indirect immunofluorescence. To further explore the mechanism of the LY-loaded HPAH− DOX micelles in autophagy inhibition, the expression levels of autophagy-related proteins LC3, Beclin-1, and p62 were measured by Western blot analysis. LC3, the microtubuleassociated protein light chain 3, exists in cytosolic form (LC3-I) and membrane-bound form (LC3-II). The ratio of conversion from LC3-I to LC3-II is closely correlated with the extent of autophagosome formation.43 Beclin 1, a mammalian homologue of yeast Atg6, plays a proximal role in stimulation of autophagy by recruiting the proteins from cytoplasm for formation of autophagosomes and triggering the signaling pathway for induction of autophagy.44 p62, which is also known as SQSTM1, is a ubiquitin-binding protein that is involved in lysosome- or proteasome-dependent degradation of proteins. It incorporates into the autophagosome via direct interaction with LC3-II and degrades in the process of autophagy. Inhibition of autophagy would lead to increased levels of p62.45 In our study,

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CONCLUSION

This study has demonstrated that pH-responsive HPAH-based nanomicelles for codelivery of autophagy inhibitor LY and chemotherapy drug DOX have been successfully constructed for oral squamous cell carcinoma therapy. Nanomicelles not 1672

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Figure 9. (A) Western blot analysis of LC3 I/II, Beclin-1, and p62 in CAL-27 and HN-6 cells. (B) The ratios of LC3 II/I in CAL-27 and HN-6 cells. (C) Semiquantitative analysis of Beclin-1 in CAL-27 and HN-6 cells. A significant increase compared with the control is denoted by “*” (P < 0.01), a significant increase compared with HPAH−DOX/LY-treated cells is denoted by “§” (P < 0.01), and a significant increase compared with HPAH− DOX/LY-treated cells and HPAH−DOX+LY-treated cells is denoted by “#” (P < 0.01). (D) Semiquantitative analysis of p62 in CAL-27 and HN-6 cells. A significant decrease compared with the control is denoted by “*” (P < 0.01), a significant decrease compared with HPAH−DOX/LY-treated cells is denoted by “§” (P < 0.01), and a significant decrease compared with HPAH−DOX/LY-treated cells and HPAH−DOX+LY-treated cells is denoted by “#” (P < 0.01). Values represent mean ± SD (n = 3).

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only are especially useful to compatibilize the lipophilic LY and DOX but also release the LY and DOX in a controlled programmable manner. In vitro evaluations demonstrate that, compared to the HPAH−DOX micelles or the physical mixture of HPAH−DOX and LY, the LY-loaded HPAH−DOX micelles result in synergistic inhibition of tumor cell proliferation. Furthermore, in vitro cell experiments confirm that the preferential release of LY leads to autophagy inhibition of tumor cells and makes them more sensitive to the subsequent liberation of DOX, thus resulting in significantly high antitumor efficacy. We believe that this stimulus-responsive polymer nanomicelle system for programmable release of chemotherapy drugs and autophagy inhibitors will open up new perspectives in clinically applicable combination therapy.

ASSOCIATED CONTENT

S Supporting Information *

The synthesis details and characterizations of HPAH and HPAH−DOX, cell viability of CAL-27 and HN-6 cells against HPAH, and Western blot analysis of autophagy-related proteins in CAL-27 and HN-6 cells treated with different concentrations of HPAH. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]; iamwuliji@ 163.com. Tel: +86-21-34203400. Fax: +86-21-54741297. Notes

The authors declare no competing financial interest. 1673

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‡

These authors are joint first authors.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81272466, 51373099), Provincial Youth Science Fund of Heilongjiang (QC2011C037), and Provincial Education Department of Science and Technology Research Fund of Heilongjiang (11551188), the Open Project of State Key Laboratory of Chemical Engineering (SKL-ChE12C04), China National Funds for Distinguished Young Scientists (21025417).

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