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 DOI: 10.1177/0883911513491642

2013 28: 341Journal of Bioactive and Compatible PolymersDai Hai Nguyen, Jin Woo Bae, Jong Hoon Choi, Jung Seok Lee and Ki Dong Park

and folate-mediated cellular uptakeBioreducible cross-linked Pluronic micelles: pH-triggered release of doxorubicin

  

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Journal of Bioactive andCompatible Polymers

28(4) 341 –354© The Author(s) 2013

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DOI: 10.1177/0883911513491642jbc.sagepub.com

JOURNAL OF

Bioactiveand

CompatiblePolymers

Bioreducible cross-linked Pluronic micelles: pH-triggered release of doxorubicin and folate-mediated cellular uptake

Dai Hai Nguyen, Jin Woo Bae, Jong Hoon Choi, Jung Seok Lee and Ki Dong Park

AbstractBioreducible are described here, cross-linked Pluronic micelles carrying doxorubicin (DOX) for folate-mediated cancer targeting. The amine-terminated Pluronic® F-127 was functionalized by grafting acrylic acid (AA) to the hydrophobic block (AA-Pluronic-NH2). Folic acid (FA), hydrazine (H), and cystamine (C) were sequentially conjugated to AA-Pluronic-NH2, followed by DOX conjugation via an acid-labile hydrazone bond (FA-Pluronic-C/H-DOX). The DOX content was approximately 143 µg/mg of polymer. We prepared bioreducible cross-linked micelles using FA-Pluronic-C/H-DOX, which had a diameter of 156.1 nm. After incubation for 24 h with 10 mM of dithiothreitol, the micelle size decreased dramatically to 87.6 nm with a broad distribution, indicating that disulfide bonds in the micelle core were reductively cleaved. In vitro release data showed that the conjugated DOX was released slowly from the FA-Pluronic C/H-DOX micelles at pH 7.4, whereas there was a rapid DOX release at pH 5.2. Confocal images of HeLa cells showed enhanced cellular uptake of FA-Pluronic-C/H-DOX micelles as compared to nontargeted Pluronic-C/H-DOX micelles. The FA-Pluronic-C/H-DOX micelles killed more cells than the nontargeted micelles, but the cytotoxic effect was not as significant as free DOX. Additionally, micelles without DOX were not cytotoxic. On the basis of these results, pH- and redox potential–responsive FA-Pluronic-C/H-DOX micelles could potentially function as cancer-targeted and controlled DOX delivery systems.

KeywordsPluronic F-127, micelle, stimuli sensitivity, doxorubicin, folate, cancer targeting

Department of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea

Corresponding author:Ki Dong Park, Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong, Suwon 443-749, Republic of Korea. Email: kdp@ajou.ac.kr

491642 JBC28410.1177/0883911513491642Journal of Bioactive and Compatible PolymersNguyen et al.2013

Article

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342 Journal of Bioactive and Compatible Polymers 28(4)

Introduction

Stimuli-responsive polymeric micelles have attracted a rapidly growing interest as programmable drug delivery systems for targeted cancer chemotherapy. They can release payloads at a site of inter-est in response to appropriate signals for effective cancer treatment. Significant efforts have been devoted to the development of smart polymeric micelles that are responsive to various stimuli such as pH, temperature, redox potential, light, ultrasound, and magnetic fields.1–7 Among these stimuli, pH, temperature, and redox potential are particularly attractive for designing stimuli-responsive micelles as these are biologically supplied stimuli that vary with the pathological cancer state or the extra-intracellular environment.8–10 Compared with single-stimulus-responsive micelles, micelles possessing multiple-stimuli-responsive properties are likely to be more effective for cancer treat-ment because they can deliver drugs in a more controlled manner, thus maximizing therapeutic efficacy. Indeed, it has been shown in several studies that multiple-stimuli-responsive micelles are able to enhance targeting efficiency by site-specific release of drugs around/in cancer cells.11–13

Pluronics are biocompatible and bioerodible triblock copolymers with a wide range of molecu-lar weights and hydrophilic/hydrophobic block ratios. On the basis of their amphiphilic structure with modifiable hydroxyl terminal groups, micellar self-assemblies have been extensively studied as drug delivery carriers. Chemical modifications allow Pluronics to be utilized as more promising carriers by conferring additional benefits in cellular targeting and uptake.14,15 There have been several attempts to develop stimuli-responsive Pluronic micelles for site-specific drug delivery and imaging applications. Lee et al.16 described pH-responsive Pluronic micelles conjugated with dox-orubicin (DOX) via an acid-labile hydrazone linkage that could be rapidly cleaved at pH 5. Chen et al.17 used Pluronic to encapsulate an optical imaging agent and then cross-linked the micelle corona with poly(ethylenimine). The micelles showed improved fluorescence yield in response to temperature and pH.17 However, there are few reports of multiple-stimuli-responsive Pluronic micelles. In addition, the low in vivo stability of Pluronic micelles against continuous dilution in the bloodstream is an endemic problem.18

Cross-linking of the core or shell of micelles has been attempted to develop robust micelles with improved colloidal stability.19,20 Intramicellar cross-linking of the micelle core through the forma-tion of disulfide bonds is increasingly attractive as a disulfide bond that can be cleaved in response to the glutathione redox potential. Disulfide cross-linking of the micelle core is found to be stable under nonreductive physiological conditions.21,22 However, disulfides are reductively cleaved in the cytoplasm when internalized. The loosened micellar core ultimately leads to micelle dissociation, thus releasing physically entrapped drugs rapidly. Therefore, core disulfide cross-linking provides advantages for micelles in terms of improved stability and site-specific drug release. pH is used to trigger the accelerated release of drugs as described earlier since it has been demonstrated that the pH level of the interstitial space of solid tumors as well as the interior of endosomes is slightly more acidic than blood plasma.23 For prompt site-specific drug release, acid-labile linkages such as hydra-zone and acetal have been utilized for covalent linkage of amphiphilic polymers and drugs.24–26 The choice of an acid-labile linkage is dependent mainly on the chemical structures of the drugs and polymers to be linked, with consideration of the pharmacological activity of the drugs.

Surface decoration of micelles with targeting ligands is a predominant strategy used for increas-ing selectivity toward specific types of cancers. Folic acid (FA), a low-molecular-weight vitamin B9, has been extensively studied as a cancer-targeting moiety due to folate receptors (FRs) being overexpressed in various cancer cells.27 It has been well established that FA conjugation onto micellar surfaces facilitates enhanced endocytic uptake against cancer cells.28 Furthermore, the FR is an established tumor marker with elevated expression on many epithelial cancers and limited expression in normal tissues.29

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The aim of this study was the development of bioreducible cross-linked Pluronic micelles capa-ble of pH-triggered DOX release for folate-mediated cancer chemotherapy (Figure 1). First, we modified the hydrophobic block of Pluronic with acrylic acid (AA) and then conjugated FA to two terminals of the hydrophilic blocks. Thiols for disulfide bridges as well as hydrazine for DOX conjugation were introduced to the AA-modified hydrophobic block. The physicochemical proper-ties of Pluronic micelles were characterized, and in vitro release studies were performed to inves-tigate the pH-dependent release behaviors of the conjugated DOX from the micelles. Cellular uptake and cell viability of targeted micelles were evaluated using HeLa cells.

Materials and methods

Materials

Pluronic® F-127 (12,600 g/mol) was purchased from BASF (Seoul, Korea). Triethylamine (TEA), aluminum oxide, and hydrazine hydrate were purchased from Acros Organics (Morris Plains, NJ, USA). FA, DOX hydrochloride (DOX), AA, ammonium persulfate (APS), p-nitrophenyl chloro-formate (PNC), ethylenediamine (EDA), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), N-hydroxysuccinimide (NHS), cystamine dihydrochloride, dl-dithiothreitol (DTT), 2,2′-azobisisobutyronitrile (AIBN), 2,2,6,6,-tetramethyl-1-piperidinyloxy (TEMPO), 4-morpho-lineethanesulfonic acid (MES), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St Louis, MO, USA). 5,5′-dithiobis-(2-ni-trobenzoic acid) (DTNB, Ellman’s reagent) was purchased from Pierce (Rockford, IL, USA). All reagents and solvents were used as received without further purification.

Amination of Pluronic F-127

Two terminal hydroxyl groups of Pluronic F-127 were converted to primary amines as previously described.30 Briefly, a solution of PNC (1.514 g, 7.5 mmol) dissolved in 25 mL of tetrahydrofuran (THF) was added dropwise to a THF solution (250 mL) of Pluronic F-127 (31.50 g, 2.5 mmol) and

Figure 1. pH- and redox potential–responsive Pluronic micelles for cancer-targeted chemotherapy.FA: folic acid; DOX: doxorubicin.

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TEA (0.76 g, 7.5 mmol) placed in an ice bath. The reaction was carried out overnight at room tem-perature under a nitrogen atmosphere. The resulting polymer (Pluronic-PNC) was slowly added to THF solution (50 mL) containing EDA (6.1 g, 100 mmol) and stirred overnight. The residual PNC salts were removed by filtration using aluminum oxide, precipitated in cold diethyl ether, and dried under vacuum to give amine-terminated Pluronic F-127 (Pluronic-NH2). The degree of amine sub-stitution was determined to be approximately 43%, based on the nuclear magnetic resonance (NMR) integration value ratio of EDA and polypropylene oxide (PPO).

AA grafting onto amine-terminated Pluronic F-127 (AA-Pluronic-NH2)

AA was grafted onto the hydrophobic polypropylene oxide (PPO) block of Pluronic-NH2 using a dispersion/emulsion polymerization method with modifications (Figure 2).31–33 A mixture of APS

Figure 2. A synthetic route to FA-Pluronic-C/H-DOX. (i) PNC, TEA, EDA, and THF; (ii) AA, AIBN, and APS; (iii) TEMPO and MeOH; (iv) FA, EDC, NHS, and MES buffer; (v) hydrazine, cystamine dihydrochloride, EDC, NHS, and MES buffer; (vi) DTT and borate buffer; and (vii) DOX, TEA, and DMSO.FA: folic acid; DOX: doxorubicin; PNC: p-nitrophenyl chloroformate; TEA: triethylamine; EDA: ethylenediamine; THF: tetrahydrofuran; AA: acrylic acid; AIBN: 2,2′-azobisisobutyronitrile; APS: ammonium persulfate; TEMPO: 2,2,6,6,-tet-ramethyl-1-piperidinyloxy; EDC: 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; NHS: N-hydroxysuccinimide; MES: 4-morpholineethanesulfonic acid; DTT: dl-dithiothreitol; DMSO: dimethyl sulfoxide.

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and AIBN (150 µL at a concentration of 300 mg/mL for each reagent) was added to a solution of Pluronic-NH2 (8 g, 0.64 mmol) dissolved in 10 mL of AA. Dodecane (200 mL), deionized (DI) water (100 mL), and mineral oil (100 mL) were added to the mixture and sonicated for 10 min to emulsify. The emulsified solution was transferred to a 500 mL three-neck flask with a mechanical stirrer and deoxygenated by overnight exposure to nitrogen. After the bath was heated to 75°C for 2 h, the reaction was stopped by adding 500 µL of 0.1 M of methanolic solution of TEMPO. The resulting mixture was filtrated and precipitated in cold diethyl ether, followed by a membrane dialysis (molecular weight cutoff (MWCO) = 12-14 kDa) against DI water for a week. The product was lyophilized and stored at −20°C before use.

Conjugation of FA, hydrazine, and cystamine to AA-Pluronic-NH2

FA (140 mg, 0.32 mmol) dissolved in 50 mL of MES buffer (0.05 M) was activated using EDC (62 mg, 0.32 mmol) and NHS (36 mg, 0.32 mmol) for 15 min and then reacted with 2 g of AA-Pluronic-NH2 dissolved in 80 mL of MES at room temperature for 24 h. Subsequently, the carboxyl groups of Pluronic-NH2 pre-activated with excess amounts (30 fold) of EDC and NHS were reacted with cystamine (225.5 mg, 1 mmol) at room temperature for 2 h, followed by sequen-tial conjugation of hydrazine (160 mg, 5 mmol) for additional 24 h. The crude product was purified by precipitation with cold diethyl ether and dialyzed against DI water for 3 days using a dialysis membrane (MWCO = 12–14 kDa). After dialysis, 20 mL of borate buffer (0.1 M, pH 9) containing DTT (1.54 g, 10 mmol) was added to the resulting solution and stirred for 2 days, allowing for the reduction of disulfide bonds that may have formed during the synthesis. Finally, the solution was dialyzed using a dialysis membrane (MWCO = 12–14 kDa) and lyophilized for 2 days to give FA-tethered Pluronic bearing free thiols and hydrazines (FA-Pluronic-C/H). The chemical struc-tures of the modified Pluronics were analyzed by 1H-NMR (AS400; Oxford instruments, Oxford, UK) and ultraviolet/visible (UV/Vis) spectrophotometer (V-750; Jasco Co., Tokyo, Japan). The thiol content in the polymer was determined by the Ellman method.34

DOX conjugation to FA-Pluronic-C/H

To a solution of FA-Pluronic-C/H (50 mg) dissolved in 10 mL of DMSO, DOX (10 mg, 22 µmol) dissolved in 10 mL of DMSO containing TEA (6.2 µL, 22 µmol) was added and stirred for 4 h. The solution was dialyzed for 3 days to remove unbound DOX. The dialyzed solution was then treated with DTT (7.4 mg, 48 µmol) for 24 h, and residual DTT was removed by an additional dialysis step. The conjugation yield of DOX measured by UV was about 86%.

Preparation of bioreducible cross-linked micelles

For micelle preparation, 50 mg of FA-Pluronic-C/H-DOX was dissolved in 50 mL of DI water below 4°C, stirred at 37°C for 30 min, and then sonicated for 5 min. A volume of 1 mL of H2O2 (3 wt%) was added to the solution to allow complete conversion of thiols to disulfides. For the control micelles, a solution of DOX (5 mg, 1.12 mmol) dissolved in 5 mL of phosphate buffered saline (PBS) (10 mM) was added to THF (5 mg/mL) containing 25 mg of FA-Pluronic-C/H while stirring at 37°C for 30 min, followed by sonication for 5 min. The sample was dialyzed (MWCO = 6–8 kDa) and lyophilized. The DOX content in the micelles was quantitatively measured at 495 nm using a UV/Vis spectrophotometer.

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Micelle characterization

The critical micelle concentration (CMC) of the modified Pluronic was determined by fluores-cence measurement at 37°C.35 The hydrodynamic diameter and surface charge of the micelles were analyzed using dynamic light scattering (DLS) (Zetasizer Nano ZS ZEN 3600; Malvern Instruments, Malvern, UK) at a wavelength of 633 nm and at a 90° scattering angle. The sample solutions (1 mg/mL) were filtered using a 0.45-µm syringe filter, sonicated for 10 min, and then transferred to a cuvette for measurement. The change in micelle sizes in response to redox potential was also moni-tored after 24 h of incubation with 1 mL of DTT (20 mM) dissolved in PBS (50 mM, pH 7.4).

In vitro DOX release

For the in vitro release experiment, 1 mL of DOX-conjugated or -loaded micelles suspended in PBS (DOX content = 0.3 mg/mL) was transferred to a dialysis bag (MWCO = 6-8 kDa) and immersed into 14 mL of either PBS (0.01 M, pH 7.4) or acetate buffer (0.1 M, pH 5.2) at 37°C. At specific time intervals, 14 mL of the release medium was collected, and an equal volume of fresh medium was added. After lyophilization of the collected media, the released amounts of DOX were determined using a UV/Vis spectrophotometer.

Cytotoxicity assay

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C under a humidified atmos-phere containing 5% CO2. The cells were seeded into a 96-well plate (1 × 104 cells/well) and incubated overnight at 37°C. The media were then replaced with a suspension of micelles or free DOX at various concentrations, followed by incubation for 48 h. Next, 25 µL of MTT solution (2 mg/mL in PBS, pH 7.4) was added to the wells, and the cells were further incubated for 3 h. The media was removed, formazan crystals were dissolved in DMSO (130 µL/well), and the absorbance was obtained at 570 nm using a microplate reader (SpectraMax M2e®; Molecular Devices Co., Sunnyvale, CA, USA).

Intracellular uptake

Cells were seeded into a 24-well plate (5 × 104 cells/well) and cultured for 24 h. Cells were incu-bated with 1 mL of the micelle suspensions with or without FA (equivalent to DOX of 3 µg/mL) at 37°C for 1 h or 4 h. Cells were then washed three times with PBS, fixed with paraformaldehyde for 10 min, treated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min for nuclei staining, and again washed three times with PBS. Cells on a coverslip were mounted in VECTASHIELD® anti-fade mounting medium (Vector Labs, Burlingame, CA, USA) and imaged using a confocal laser scan-ning microscope (CLSM; Zeiss LSM 510; Carl Zeiss, Oberkochen, Germany). Images were ana-lyzed using image software (Carl Zeiss LSM). For flow cytometry, cells seeded into a 6-well plate (5 × 106 cells/well) were incubated with free DOX, Pluronic-C/H-DOX, or FA-Pluronic-C/H-DOX micelles at the same concentration of DOX at 37°C for 4 h. Cells were then washed three times with PBS, trypsinized at 37°C, centrifuged (1500 r/min, 5 min), and resuspended in PBS (pH 7.4, 1% bovine serum albumin (BSA)). Fluorescence histograms were recorded using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using the CellQuest™ software supplied by the manufacturer. Each histogram was generated by analyzing at least 10,000 cells.

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Results and discussion

The hydrophobic PPO block of Pluronic-NH2 was functionalized with AA to introduce bioreduci-ble and pH-responsive properties to the micelle core. We employed a one-step free radical polym-erization method to graft AA monomers onto the PPO of Pluronic.33 However, this reaction can cause intermolecular cross-linking between PPO chains, which was bridged by AA.31 To minimize this possibility and decrease the graft length of AA, the reaction time was shortened from 4 to 2 h, followed by the addition of TEMPO, which can terminate growing free radical chains of AA.31 The 1H-NMR spectrum of AA-Pluronic-NH2 showed characteristic peaks of the grafted AA at 2.25 and 1.60 ppm (Figure 3(a)). It was estimated that the degree of substitution of AA was approximately 22.7%, based on the integration value ratio of the methyl protons on each PPO block. This result indicates that AA-Pluronic-NH2 has approximately 24 carboxylic acid groups (1.921 mmol/g of polymer) in a single chain. After FA conjugation to terminal amino groups of the modified Pluronic, FA-Pluronic-C/H was prepared by sequential addition of cystamine and hydrazine. The thiol con-tent in FA-Pluronic-C/H was determined to be 0.255 ± 0.011 mmol/g of polymer. Finally, DOX was covalently bound via a pH-sensitive hydrazone linkage, and the content was 143 µg/mg of polymer.

Figure 3. 1H-NMR spectra of (a) AA-Pluronic-NH2 and (b) FA-Pluronic-C/H.NMR: nuclear magnetic resonance; AA: acrylic acid; FA: folic acid.

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Since CMC is an important parameter to predict colloidal stability of self-assembled micelles in the bloodstream, we measured the CMC of FA-Pluronic-C/H before DOX conjugation (Figure 4). The CMC value was 0.198 g/L, which was slightly higher than that of Pluronic F-127 (0.11 g/L).36 However, this CMC value was extremely low as compared with a typical CMC range (2.6–8.0 g/L) of Pluronic F-127 measured at 25°C.37 As Pluronic F-127 generally exhibits temperature-depend-ent micellization in aqueous solutions, the self-assembly process accelerates at elevated tempera-tures. Consequently, such a low CMC of FA-Pluronic-C/H was attributed to the increased temperature of 37°C. To see differences in the size of the bioreducible DOX-conjugated micelles, the FA-Pluronic-C/H-DOX micelles were incubated with 10 mM of DTT before DLS measure-ment (Figure 5). Untreated micelles were 156.1 nm in diameter. Micelle size decreased slightly after DTT treatment for 15 min, whereas 24 h of DTT treatment resulted in a dramatically decreased size as well as an increased polydispersity index (PDI). The size distribution of the micelles also became bimodal, implying that smaller micelles or aggregates were formed. This result indicates that the disulfide bonds in the micelle core were cleaved by DTT-mediated reduction, which is in agreement with reports from the literature.38

The pH-responsive release of DOX from FA-Pluronic-C/H-DOX micelles was investigated at pH 7.4 and 5.2. To compare DOX release profiles, we also prepared DOX-encapsulated Pluronic micelles (Pluronic-C/H/DOX), which also has bioreducible micelle cores but without covalent conjugation of DOX. The loading efficiency and amount of DOX in the Pluronic-C/H/DOX micelles were 73% and 122 µg/mg of micelles, respectively. DOX release from FA-Pluronic-C/H-DOX micelles was much slower at neutral pH than at acidic pH (Figure 6). A similar trend was also observed with Pluronic-C/H/DOX micelles. The accelerated release of DOX at a lower pH is prob-ably due to the basic nature of DOX with a high solubility at a low pH.39 Accordingly, the DOX release would hardly be influenced by a basic pH.40 It was expected that DOX covalently conju-gated to the PPO block of Pluronic via an acid-labile hydrazone linkage could be quickly released as compared with physically encapsulated DOX. However, FA-Pluronic-C/H-DOX micelles appeared to be stabilized by disulfide cross-linking, which could cause slower diffusion of the conjugated DOX. Although pH-triggered release profiles of DOX were clearly shown in Figure 6,

Figure 4. Intensity ratio of pyrene as a function of FA-Pluronic-C/H concentration.FA: folic acid.

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a more dramatic release of the conjugated DOX at a lower pH than expected seems to be offset by core cross-linking. It is likely that DTT treatment can facilitate a rapid release of DOX by disrupt-ing the micellar structures of FA-Pluronic-C/H-DOX, as we previously reported.27,35

The time-dependent cellular uptake of micelles analyzed by CLSM and FACS is shown in Figure 7. To evaluate the targeting ability of FA-Pluronic-C/H-DOX against FR-positive HeLa cells, Pluronic-C/H-DOX and free DOX were used as controls. Confocal images acquired after 1 h

Figure 5. Time course of changes in average diameter and size distribution of FA-Pluronic-C/H-DOX micelles after DTT treatment. The experiment was carried out in quadruplicate (n = 4) and data were expressed as mean ± standard deviation.DTT: dl-dithiothreitol; FA: folic acid; DOX: doxorubicin.

Figure 6. In vitro release behaviors of DOX from micelles at different pH values. DOX-conjugated micelles (FA-Pluronic-C/H-DOX) at pH 7.4 () and pH 5.2 (); DOX-encapsulated micelles (FA-Pluronic-C/H/DOX) at pH 7.4 (○) or at pH 5.2 (□) (n = 4).DOX: doxorubicin; FA: folic acid.

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of incubation with HeLa cells demonstrated that DOX-conjugated micelles were barely taken up by cells (Figure 7(a) and (c)), while free DOX was localized in the cells (Figure 7(e)). After 4 h of incubation, the FA-Pluronic-C/H-DOX micelles had a strong red fluorescence for the DOX distrib-uted in the cytoplasm. Nontargeted micelles were insignificantly taken up, but increased accumula-tion of free DOX in the nuclei was observed. The FACS results also supported our CLSM observations (Figure 7(g) and (h)).

The dose-dependent cytotoxicity of FA-Pluronic-C/H micelles is shown in Figure 8(a). Over 80% of cells were still viable for 2 days even at 0.5 mg/mL of micelles, indicating that the micelles

Figure 7. (a–f) Confocal microscopic images of HeLa cells treated with various DOX formulations at a concentration equivalent to 3 µg/mL of DOX. (a, b) Pluronic-C/H-DOX, (c, d) FA-Pluronic-C/H-DOX, and (e, f) free DOX were incubated with HeLa cells for 1 h (a, c, e) and 4 h (b, d, f). (g) Flow cytometry histogram and (h) fluorescence intensity of various DOX formulations internalized into HeLa cells for 4 h.FA: folic acid; DOX: doxorubicin.

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are biocompatible. Cytotoxicity of FA-Pluronic-C/H-DOX micelles was evaluated with increasing DOX content (Figure 8(b)). Viability of cells treated with FA-Pluronic-C/H-DOX micelles was significantly reduced at 1 µg/mL of DOX, and only 50% of cells were viable at 10 µg/mL of DOX. Using an equivalent amount of free DOX led to a more dramatic decrease in cell viability, and the majority of cells were killed at 1 µg/mL. Compared with Pluronic-C/H-DOX micelles, FA-Pluronic-C/H-DOX micelles appeared to be more cytotoxic, but the cytotoxic effect induced by FA was not as significant as we expected.

Cellular uptake and cytotoxicity of targeted micelles are influenced by several factors such as surface ligand density, surface charge, size, and shape.41 It is well established that conjugation of FA to amphiphilic copolymers can lead to an enhanced uptake efficiency of their micelles, resulting in increased cytotoxicity. This is most likely due to an increased endocytosis rate in FR-overexpressing cancer cells, which is mediated by FA.42 However, it is debatable whether FA conjugation always facilitates cellular internalization efficiency of drug-loaded micelles, which would result in a higher cytotoxicity than that of free drugs. Lee et al.16 reported that though FA was not conjugated, the extent of DOX-conjugated micelle uptake against MCF-7 cells was greater than that of free DOX. This increased cellular uptake of the micelles resulted in higher cytotoxicity. Park et al.43 used three separate cell lines (human fibroblast, MCF-7, and HeLa) to investigate cell type–dependent targeting effects of folate-conjugated micelles. Interestingly, no significant differ-ences in the viability of MCF-7 and HeLa cells were observed between folate-conjugated micelles and free paclitaxel. However, Tsai et al.44 found that free DOX killed more HeLa cells than FA-conjugated micelles. Recently, Jung et al.45 showed that internalization of free DOX into HeLa cells was much faster than that of folate-conjugated poly(amino acid) micelles. Additionally, free DOX was found to be more cytotoxic after 24 h of incubation. Probably the differences in FA-mediated cellular targeting efficiency are associated with different micellar properties, such as FA surface density, size, and surface charge.

To maximize the targeting efficiency of FA-conjugated micelles, several attempts have been undertaken to find an optimal number of FA molecules on micellar surfaces.46–48 However, these

Figure 8. Dose-dependent cytotoxicity of (a) FA-Pluronic-C/H micelles and (b) FA-Pluronic-C/H-DOX against HeLa cells after 48 h of incubation (n = 4). Free DOX (×), Pluronic-C/H-DOX (○), and FA-Pluronic-C/H-DOX () were incubated with cells for 48 h.

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studies were only proven to be effective in vitro, not in vivo. In this study, we showed folate-mediated cellular targeting of FA-Pluronic-C/H-DOX micelles, but no cytotoxic effect was observed. As mentioned previously, FA-Pluronic-C/H-DOX micelles were designed to respond to dual stimuli: pH and redox potential. Furthermore, Pluronic F-127 exhibits inhibitory effects on P-glycoprotein-mediated drug efflux in multi-drug-resistant cancer cells.46 Thus, we speculate that FA-Pluronic-C/H-DOX micelles may exert better therapeutic effects on multi-drug-resistant can-cer cells in vivo. In vivo experiments using FA-Pluronic-C/H-DOX micelles are ongoing, and this work will be published in the future.

Conclusion

We have developed bioreducible cross-linked Pluronic-127 micelles conjugated with DOX for effective folate-mediated cancer targeting. This micelle system containing disulfide cross-links and acid-labile hydrazone linkages was designed to respond to pH and redox potential, and FA was introduced to improve targeting efficiency. From the unique design of our micelle system, we envi-sion that the disulfide cross-linked cores can improve micelle stability during systemic circulation. The primary event after micelles reach their target area by the enhanced permeability and retention (EPR) effect is a pH-triggered release of the conjugated DOX, as the interstitial space of solid tumors is more acidic. In this case, cancer cells can be killed by a local bystander effect. The micelles can also be internalized into cancer cells via FR-mediated endocytosis, resulting in a more accelerated release of DOX due to cleavage of disulfide and hydrazone bonds in response to the glutathione redox potential and low endosomal pH. To test our hypothesis, we first synthesized and characterized FA-Pluronic-C/H-DOX. The resulting micelles showed pH and redox potential sen-sitivity. Additionally, in vitro cell studies demonstrated that the micelles can target and kill cancer cells. Further studies will be focused on investigating in vitro and in vivo therapeutic efficacies of the micelles against multi-drug-resistant cancer cells. Our results suggest that the bioreducible cross-linked Pluronic micelles carrying DOX, which are dual-stimuli responsive, have potential as a cancer-targeted and controlled DOX delivery system.

Declaration of Conflicting Interests

The authors declare that there is no conflict of interest.

Funding

This study was supported by the Pioneer Research Center Program (2013007227) and the Priority Research Centers Program (20090093826) through the National Research Foundation of Korea funded by the Ministry of Science, ICT, and Future Planning, Republic of Korea.

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