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Reduction-Degradable Polymeric Micelles Decorated with PArg for Improving Anticancer Drug Delivery Ef#cacy Yani Cui, Junhui Sui, Mengmeng He, Zhiyi Xu, Yong Sun, Jie Liang, Yujiang Fan, and Xingdong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10867 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 2, 2016

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ACS Applied Materials & Interfaces

Reduction-Degradable Polymeric Micelles Decorated with PArg for Improving Anticancer Drug Delivery Efficacy Yani Cui, Junhui Sui, Mengmeng He, Zhiyi Xu, Yong Sun,* Jie Liang, Yujiang Fan,* and Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China KEYWORDS:

Reduction-degradable,

Micelles,

Poly-arginine,

Doxorubicin,

Cell-penetrating ABSTRACT: In this study, five kinds of reduction-degradable polyamide amine-g-polyethylene

glycol/poly-arginine

(PAA-g-PEG/PArg)

micelles

with

different proportion of hydrophilic segment and hydrophobic segment were synthesized as novel drug delivery vehicles. Poly-arginine was not only acted as a hydrophilic segment, but also possessed a cell-penetrating function to carry out a rapid transduction into target cells. Polyamide amine-g-polyethylene glycol (PAA-g-PEG) was prepared for comparison. The characterization and antitumor effect of the DOX-incorporated PAA-g-PEG/PArg cationic polymeric micelles were investigated in vitro and in vivo. The cytotoxicity experiments demonstrated that the PAA-g-PEG/PArg

micelles

have

good

biocompatibility.

Compared

with

DOX-incorporated PAA-g-PEG micelles, the DOX-incorporated PAA-g-PEG/PArg micelles were more efficiently internalized into human hepatocellular carcinoma (HepG2) cells, and more rapidly released DOX into the cytoplasm to inhibit cell proliferation. In the 4T1-bearing nude mouse tumor models, the DOX-incorporated PAA-g-PEG/PArg micelles could efficiently accumulate in the tumor site, and had longer accumulation time and more significant aggregation concentration than that of PAA-g-PEG micelles. Meanwhile, it excellently inhibited the solid tumor growth and extended survive period of the tumor-bearing Balb/c mice. These results could be attributed to their appropriate nano-size and the cell-penetrating peculiarity of 1

ACS Paragon Plus Environment


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poly-arginine as surface layer. The PAA-g-PEG/PArg polymeric micelles as a safe and high efficiency drug delivery system was expected to be a promising delivery carrier that targeting hydrophobic chemotherapy drugs to tumor and significantly enhanced antitumor effects.

1．．INTRODUCTION Cancer treatment as a global public health problem is an urgent need for sustainable healthcare requirement. Conventional anticancer drugs often suffer from poor solubility, lack of target specificity, and serious side-effects in clinical treatment.1-3 It has been demonstrated that the drug-loaded nanoparticles are favorable for extravasation from blood stream into tumor tissues through the enhanced permeability and retention (EPR) effect, and provide significant avenues for potential therapeutic strategies, including elongated circulation half-life of the anti-cancer drugs in the blood stream, reduced side effects, improved drug bioavailability, and enhanced therapeutic efficiency.4-7 In the past decades, stimuli-responsive polymeric micelles, 8, 9

as a kind of nanoparticles, are extensively studied because chemically or physically

changeable in response to specific stimuli, such as ultrasound,10 temperature,11,

12

magnetic field,13, 14 light,15 enzyme,16 pH,17-19 and redox potential.20-23 Among these nanoparticles, the employment of reduction-sensitive micelles has showed considerable potentiality in the field of drug delivery systems. For example, in our previous work, a reduction-breakable micelle based on amphiphilic polyethylene glycol-g-polyamide amine copolymers (PAA-g-PEG) was presented. The anti-cancer drug doxorubicin (DOX) was stably encapsulated in the hydrophobic core of micelles. These drug-loaded micelles were stable in the normal physiological condition, and quickly disassembled to release the loaded drug when internalized into tumor cells, where the reducing potential was much higher than normal cells, and thus enhancing the therapy efficacy.24 However, the cell membrane forms an infaust barrier in the process that transports the nanoparticles into the cytosol.25 Therefore, it is necessary to develop a more effective drug carrier for enhancing the internalization of nanoparticles by adjusting the structure and composition of micelles. 2


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Recent studies have demonstrated that peptides and polypeptides enjoyed great popularity in the field of gene and drug delivery systems due to their acknowledged advantages of inherent bioactivity, biodegradability, and good biocompatibility.26, 27 In order to overcome the cell membrane-mediated permeability barriers, cell-penetrating peptides (CPPs) that possess an ability to go through the non-polar plasma membrane quickly was proposed, which is sometimes called protein transduction domain (PTD).28, 29 For investigating cell-penetrating mechanism, the crucial role of arginine was discovered. These studies indicated that the poly-arginine molecules showed significant ability to penetrate cell membrane when there were 5-11 arginine arranged continuously, and the R8 peptide was internalized most efficiently into cells.30,

31

Numerous recent studies have now revealed that the poly-arginine modified gene and drug vectors could strongly promote the gene transfection efficiency, enhance the internalization of drugs in the target cells and reduce side effects.32-34 O. Veiseh et al. synthesized PArg-coated magnetic nanoparticles (MNPs) to complex and deliver siRNA to cytoplasm, which showed less toxic and higher transfection efficiency by the direct membrane translocation mechanism.35 S. Biswas and his colleagues prepared octa-arginine (R8)-modified pegylated liposomal doxorubicin (R8-PLD) to treat the non-small cell lung cancer, and obtained excellent experimental results.36 H. Lee and his group’s researches showed that the histidine- and arginine-rich cell-penetrating peptides (CPPs) (HR9 peptides) combined with quantum dots were able to quickly enter into cells (within 4 min).37 The cellular uptake of HR9/QD complexes was not inhibited even F-actin polymerization and active transport was interdicted, implying that HR9 could directly transit through cell membrane. These findings suggested that arginine-rich peptides on the surface of nanoparticles could be an efficient modification method for enhancing drug delivery by the direct membrane translocation mechanism without disturbing their therapeutic activity. In this study, new CPPs-mediated stimuli-sensitive polymeric micelles were designed

as

drug

delivery

vehicles by

introducing

octa-arginine

to the

reduction-cleavable amphiphilic copolymers. The strategy were carried out by synthesizing

the

R8

grafted

polyamide

amine-g-polyethylene

3


glycol


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(PAA-g-PEG/PArg), and the amphiphilic (PAA-g-PEG) graft copolymer without R8 were prepared as comparison. The nature occurring poly-arginine moiety is expected to accelerate the internalization of drug-loaded micelles in the tumor cells, and then the drug release into the cells with the cleavage of disulfide bond under the redox-stimuli. In order to survey whether the modification of poly-arginine could safer and more effective, the characterization and antitumor effect of the Dox-incorporated PAA-g-PEG/PArg cationic polymeric micelles were investigated in vitro and in vivo. Qualitative and quantitative cellular uptake was measured by CLSM and flow cytometry. The therapeutic effect was evaluated in the 4T1 tumor-bearing mouse models.

2. MATERIALS AND METHODS 2.1. Materials Triethylamine (TEA), sodium hydroxide (NaOH), potassium hydroxide (KOH) and trifluoroacetic acide (TFA) were purchased from Furuisite Technology Development Co., Ltd. (Chengdu, China), and were used as-received. Acryloyl chloride, cystamine dihydrochloride, Boc-ethanediamine, phenethylamine and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, and were used as-received. O-[N-(3-Maleimidopropionyl) aminoethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl] polyethylene glycol (NHS-PEG-MAL, Mn 2000, ivkeyan), cysteine-modified Octa-arginine (RRRRRRRRC, GL Biochem Ltd, Shanghai, China), O, O′-bis [2-(N-succinimidyl-succinylamino)ethyl] polyethylene glycol (NHS-PEG, Mn 1000, ivkeyan) were dehydrated by azeotropic distillation from dry toluene. Doxorubicin hydrochloride (DOX·HCl, >99%, Dalian Meilun Biology Technology Co., Ltd., China) was dissolved in water (2 mg/mL) and adjust the pH to 9.6 to get the deprotonated hydrophobic DOX. Dichloromethane (DCM), 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) and toluene, were purchased from Sigma-Aldrich, and were dried by refluxing over CaH2. All other chemicals were used as-received unless otherwise specified. 2.2. Synthesis of PAA-g-PEG/PArg graft copolymer 4


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Five kinds of redox-sensitive polyamide amine-g-polyethylene glycol/poly-arginine (PAA-g-PEG/PArg)

amphiphilic graft copolymers were prepared in four steps: 1)

Cystamine bisacrylamide was synthesized according to the previously reported methods.24,38 2) Preparation of polyamide amine containing disulfide bond (PAA): cystamine biascrylamide (2.61 g, 10 mmol) and a mixture of Boc-ethanediamine and phenethylamine (mol ratio: Boc-ethanediamine/phenethylamine = 9/1, 8/2, 7/3, 6/4 and 5/5) were reacted at 125℃ under Ar atmosphere, the obtained Boc-PAA was dissolved in methanol and purified by precipitated in ethyl ether, and then deprotected via TFA to acquire the freshly PAA. 3) Preparation of amphiphilic polyamide amine-g-polyethylene glycol graft copolymer (PAA-g-PEG): the freshly obtained polyamide amine (29.34 mg, 0.1 mmol) was reacted with a mixture of NHS-PEG1K and NHS-PEG2K-MAL with different ratio as shown in Table 1 in the DMSO to acquire PAA-g-PEG-MAL. The primary amines in PAA and NHS group were allowed to reacted at room temperature for 4h under stirring. The products were purified by dialysis against phosphate buffer solution (MWCO 8000-14000, PH 7.4). 4) Conjugating

cysteine-modified

Poly-arginine

(PArg-SH)

with

functionalized

PAA-g-PEG-MAL: PArg-SH was added to the PAA-g-PEG-MAL solution at 1:1.2 molar ratios, and the thiol-reactive MAL group in PAA and sulfydryl group in cysteine-modified Poly-arginine was reacted at room temperature for 2 h in the dark under stirring. The obtained PAA-g-PEG/PArg was dialyzed against pure water and lyophilized. The PAA-g-PEG-MAL without PArg was used as control group. 2.3. Fabrication and characterization of the micelles 2.3.1. Preparation of the micelles 10 mg of the freeze-dried PAA-g-PEG/PArg or PAA-g-PEG copolymers were dissolved in 2 mL of DMSO and dialyzed in water to get the empty micelles as described previously. 24, 38 2.3.2 Preparation of the DOX-loaded micelles In dark environment, 15 mg PAA-g-PEG/PArg or PAA-g-PEG copolymers were dissolved in 0.5 mL DMSO initially. Then, 5 mg DOX in 0.5 mL DMSO solution was added. After 0.5 h stirring the mixture was dropped into 20 mL distilled water under 5



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ultrasound. The thus obtained micelle suspension was then dialyzed, filtrated and concentrated to 0.6 mL 38 2.3.3. Characterization The particle size and polydispersity index (PDI) were measured in water using dynamic light scattering (DLS, Malvern Nano-ZS). Briefly, 1 mL of polymer solution (1 mg/mL) in a glass cuvette measured at 25°C and a scattering angle of 173°. Each sample was measured three times. TEM images were obtained by a FEI TecnaiGF20S-TWIN microscope. To prepared the sample: a drop of solution was placed onto a copper grid and dried in air at room temperature, then stained with 2% phosphotungstic acid aqueous solution for 10 minutes and dried in air at room temperature. The drug loading content (DLC) was calculated from UV results as previously reported.24, 38 2.3.4. In vitro reduction-sensitive drug release from PAA-g-PEG/PArg/DOX micelles. The in vitro DOX release profile was investigated in three different kinds of buffer: PBS (10mM, PH 7.4) with 1 mM DTT, PBS (10mM, PH 7.4) with 0.1 mM DTT or PBS (10mM, PH 7.4) only. Briefly, 1 mL micelle suspension ( DLC 17.3% and containing 864 µg DOX) was transferred into a dialysis tube (MWCO 8000-14000) and dialyzed against 25 mL buffer in dark with gentle shaking at 37℃. At the predetermined time intervals, 1 mL of external buffer was removed for fluorescence measurement (excitation at 485 nm) and replaced with 1 mL of responding fresh buffer. The experiments were administrated in triplicate, and the mean value was presented. 2.4. Cell culture and biocompatibility analysis For testing the biocompatibility of the micelles, L929 fibroblast cells, HepG2 and 4T1 cancer cells was co-cultured with the micelles respectively. L929 and 4T1 were cultured in RPMI-1640 and HepG2 was cultured in DMEM at 37°C, 5% CO2, and both culture media were supplemented with 10% heat-inactivated FBS and 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin). The cytotoxicity of PAA-g-PEG/PArg micelles was assessed by MTT assay. The cells were seeded into 96-well plate at an initial density of 6 × 103 cells/well, and cultured for 12 h, and then 6


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the culture media were replaced by media containing micelles at different concentrations. After incubating for 48 h, the cell viability was determined through MTT method as described previously. 24, 38 2.5. Intracellular drug distribution The intracellular distribution of DOX was measured by confocal laser scanning microscopy (CLSM). Briefly, HepG2 cells were seeded into glass-bottomed dishes at an initial density of 2 × 105 cells/well, and maintained for 24 h at 37°C, 5% CO2. After replacing the culture media with freshly media containing DOX•HCl or DOX-loaded micelle (DOX content: 10µg/mL), the cells were cultured for another 2 and 24 h. The cells were subsequently rinsed twice with PBS to remove residual DOX or micelles. Then, the CLSM (Leica TCS SP5, Germany) was used to identify the DOX location by blue cell nucleuses stained with Hoechst 33342 (Molecular Probes) and intracellular red DOX fluorescence, which were excited at 352 and 485 nm, and emissions at 455 and 595 nm, respectively. 2.6. Evaluation of cellular uptake Flow cytometry was used to examine the quantitative cellular uptake of PAA-g-PEG/PArg/DOX nanomicelles and DOX•HCl in HepG2 cells. First, HepG2 cells were seeded into 6-well plates at cell density 4 × 105 cells/well at 37°C, 5% CO2. After 24 h, the culture medium were replaced with DOX or micelle containing medium (DOX content: 10µg/mL), and cultured for 0.5, 2 or 6 h, respectively. The culture medium was then removed and the cells were washed with PBS twice to remove residual DOX or micelles. 1 mL trypsin was added into each well, after 1 minute, the digestion was terminated by 2mL complete DMEM containing 10% FBS and 1% antibiotics, and the cells were centrifuged at 1000 rpm for 5 min, and resuspended in 3 mL of PBS. The suspensions were centrifuged again, and then the cells were suspended in 500 µL of PBS and filtered through a 40 µm nylon mesh t before measurements. The mean fluorescence intensity (MFI) of DOX in cells was analyzed by FCM (Beckman Coulter Cytomics FC-500) with an excitation wavelength of 488 nm. 2.7. Cytotoxicity of the drug-loaded micelles 7



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HepG2 and 4T1 cells were seeded into 96-well plates at an initial density of 6×103 cells/well. After 24 h, the culture media were removed. 100 µL of the DOX containing culture media (free DOX or drug-loaded micelles, DOX concentration: 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 10.0 and 100 µg/mL, respectively) were added. After incubating for 48 h, the medium was refreshed with PBS twice, and the cell viability was analyzed by MTT assay (specific experimental steps according to 2.4). For each sample, six wells of cells were used. The measurement was conducted in three replicates. 2.8. Animal experiments Nude mice and Balb/c mice in our research were provided by the Experimental Animal Center of Sichuan University, and raised in SPF-class housing of laboratory with a controlled condition (20-22 ℃, relative humidity of 50-60%, and 12 h light-dark cycles). They were fed with water and commercial rat pellet diet. All animal experiments were permitted by the Sichuan Provincial Committee for Experimental Animal Management. 2.9. In vivo tumor accumulation of DOX-incorporated micelles All animals adapted to the environment for one week before operation. Then the tumor models were prepared by subcutaneous injection of a suspension of 2×106 4T-1 cells/mouse in PBS (PH 7.4, 100µL) into the back of nude mice. When the tumor grew up to a particle like a soybean (70–100 mm3) after ten days, the DOX formulations (DOX·HCl or DOX-incorporated micelles) were intravenously injected at a dose of 10 mg/kg (DOX/body weight), and fed for another 2, 6, 12, and 24 h. At each predetermined time, the nude mice were anesthetized by chloral hydrate aqueous solution (5 wt% 7.5 mL/kg, anesthetic liquid/weight of mice), and imaged by an in vivo imaging system (Maestro Ex Pro, CRI, USA). The nude mice were sacrificed after each imaging time point, and the tumor was taken out immediately and imaged. The excitation wavelength of in vivo imaging was set in 488 nm to excite the DOX molecules. 2.10. In vivo antitumor effect of DOX-incorporated micelles against 4T1 tumor model 8


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To demonstrate the feasibility of DOX-loaded PAA-g-PEG/PArg against solid tumor in vivo, the long-term tumor inhibition efficacy was investigated using Balb/c mice as an animal model. (6 weeks of age, 20-25 g, female). All animals acclimated to the environment for one week before operation. Then the tumor models were prepared in accordance with the experiment of in vivo imaging. When the tumor grew up to a particle like a soybean (70–100 mm3) after ten days, the mice were randomly divided into

the

following

eleven

treatment

groups

(n=8):

PAA2:8-g-PEG

(A),

PAA3:7-g-PEG/PArg (B), I-PAA2:8-g-PEG/DOX (AI-DOX), II-PAA2:8-g-PEG/DOX (AII-DOX), I-PAA3:7-g-PEG/PArg/DOX (BI-DOX), II-PAA3:7-g-PEG/PArg/DOX (BII-DOX), I-PAA4:6-g-PEG/PArg/DOX (CI-DOX), II-PAA4:6-g-PEG/PArg/DOX (CII-DOX), I-DOX•HCl, II-DOX•HCl and PBS control groups. 125µL of DOX or DOX-incorporated micelles solutions were intravenously injected at 6×5 (I) or 3×10 (II) mg/kg (DOX/bodyweight) dose, and they were injected at different administration times with the same total drug content. For empty micelles, the injection amount of drug kept consistent with DOX-incorporated micelles. The tumor sizes (length and width) were measured at an interval of 2 days and the final tumor volume (mm3) was obtained by the formula: V = 0.5 × (length) × (width)2. The relative tumor volume was obtained by the formula: relative tumor volume (%) = tumor volume on that day/initial value before the first dosing) × %. The body weight (an indirect expression of physiological status), clinical status, as well as mortality of mice were recorded in detail during the experiment. After 23th day, the number of control and free DOX treated mice became not enough to carry out statistical analysis due to early death, so the tumor growth study was ended, while the life span study ended on day 65. Two mice were sacrificed for histological examination in each group at third day after finishing all of the administration. The heart, liver, spleen, lung, kidney and tumor were taken out immediately and fixed with 8% formalin for 2 days. The specimens embedded in paraffin were sectioned and were stained with hematoxylin and eosin (H&E) for further observing using optical microscope. 2.11. Systemic toxicity of DOX-incorporated micelles in normal mice 9



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For investigating the systemic toxicity, healthy Balb/c mice (6 weeks of age, 17-23 g, male) were randomly assigned into the seven experimental groups (6 mice/group): I-PAA3:7-g-PEG/PArg (BI), II-PAA3:7-g-PEG/PArg (BII), I-PAA3:7-g-PEG/PArg/DOX (BI-DOX), II-PAA3:7-g-PEG/PArg/DOX (BII-DOX), I-DOX•HCl, II-DOX•HCl and blank group. 125µL of DOX or DOX-loaded micelles solutions were intravenous injection in accordance with 2.10. The variation of mice body weight (physiological status) and clinical behavior were detailedly recorded. Histological observation of major organs was performed as described above. 2.12. Statistical analysis The statistically significant differences between groups was verified using Student’s t-Test. Differences were considered significant if P < 0.05 (*) or P

Reduction-Degradable Polymeric Micelles ... - ACS Publications

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