Tumor cells-specific targeting delivery achieved by A54 peptide functionalized polymeric micelles

Biomaterials 33 (2012) 8858e8867

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Tumor cells-specific targeting delivery achieved by A54 peptide functionalized polymeric micelles Yong-Zhong Du*, Li-Li Cai, Ping Liu, Jian You, Hong Yuan, Fu-Qiang Hu* College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 June 2012 Accepted 19 August 2012 Available online 6 September 2012

The delivery of all of administrated chemotherapeutics into tumor cells is an extreme object for tumor targeting therapy to enhance the curative effect and eliminate the side effect. However, until now, the targeting delivery has only partial been realized by passive targeting, which was called “enhanced permeability and retention” effect, and only few targeting delivery system was commercialized. Here, we designed and synthesized a hepatocarcinoma-binding peptide (A54 peptide, which was identified from a phage-display random peptide library) functionalized and PEGylated stearic acid grafted chitosan (A54ePEGeCSeSA) micelles for targeting therapy of doxorubicin. The A54ePEGeCSeSA micelles presented special internalization ability into human hepatoma cells (BEL-7402) when the cells were co-incubated with normal liver cells in vitro, and high distribution ability to liver and hepatoma tissue in vivo. In vitro and in vivo anti-tumor activity results showed that A54ePEGeCSeSA micelles loading doxorubicin treatments suppressed tumor growth more effectively and reduced toxicity compared with commercial adriamycin injection. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Stearic acid grafted chitosan Poly(ethylene glycol) Homing peptide Active targeting Anti-tumor activity

1. Introduction Hepatocarcinoma is one of the deadliest malignancies in the world [1]. Nowadays, anthracyclines anti-tumor drug doxorubicin is widely utilized in the clinical field for patients with the liver cancer. However, the current therapy efficacy of doxorubicin for hepatocarcinoma is still far from satisfactory because of its severe side effects, especially the dose-limited cardiotoxicity and myelosuppression [2]. Therefore, an attractive strategy to enhance the therapeutic index of anti-tumor drugs is to specifically deliver these agents to tumor cells thereby keeping them away from nonmalignant cells sensitive to the toxic effects of the drug. This would allow for more effective treatments achieved with doses that are better tolerated [3]. It has successfully been demonstrated that polymers can form effective delivery systems for drugs, proteins and genes [4e7]. However, the human body recognizes drug delivery systems as foreign intruders, and hence, they become easily opsonized and removed from the circulation long prior to completion of their function. Therefore, much effort has been spent on the design of long circulating drug carriers which can slowly accumulate in pathological sites with leaky vasculature and poor lymphatic * Corresponding authors. Tel./fax: þ86 571 88208439. E-mail addresses: [email protected] (Y.-Z. (F.-Q. Hu).

Du),

[email protected]

0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.08.043

drainage via the enhanced permeability and retention (EPR) effect, also termed “passive targeting” [8,9], or can even achieve a better effect of targeted drug delivery systems due to a longer interaction time with their target. Poly(ethylene glycol) (PEG), a nontoxic and nonirritant hydrophilic polymer, is the mostly used polymer to enhance the circulation time of such drug carriers in the blood stream [10,11] by a sterical hindrance of their interaction with blood components as very nicely shown for proteins [12], drugs [13], liposomes [14,15] and nanoparticles [16]. Similarly, PEGylated polymers may improve the stability of the drug delivery system in the blood by preventing protein absorption and reticuloendothelial systems (RES) uptake [17,18]. Furthermore, the intracellular delivery is one key step in drug delivery. However, the lipophilic nature of the cellular membranes prevents the delivery of PEGylated nanoparticles. Therefore, the next step is to develop the property of specific target recognition, in order to selective drug delivery to certain cell types with the aid of ligands that are specific to cell surface characteristic structures. In some cases, such drug delivery systems can exploit the mechanism of receptor-mediated endocytosis and accomplished their cellular entry via plasma membrane receptors or other cell surface antigens, which are unique to certain cells or related to certain diseases. Upon their binding to cell surface receptors, nanoparticle- and polymer-based drug delivery systems are internalized by a process called endocytosis/phagocytosis and end up in small vesicles called endosomes [19]. Tumor-specific binding peptide is a peptide that

Y.-Z. Du et al. / Biomaterials 33 (2012) 8858e8867

specifically binds to tumor, while the tumor homing peptide home to tumors through the circulation. Homing peptides [20,21], as excellent targeting agents for human tumor, are important in the development of tumor therapeutics. The advantages of homing peptides include relatively small molecular weights, being easily synthesized, having relatively low cytotoxicity and immunogenicity, and degrading in vivo to naturally occurring compounds [22]. Herein, a peptide combined the abilities of hepatocarcinomahoming and specific binding, named A54 peptide, has been obtained by in vivo phage-display technology [1]. After three rounds of panning, only the potential motif Pro-Ser was found in 80 sequenced phage clones. Phage A54 (sequence AGKGTPSLETTP) was shown to be the most effective and specific to the liver cancer cells via the cell surface marker-mediated endocytosis [1], especially human hepatoma cell line BEL-7402 by cell-based ELISA in all 130 tested clones. We have developed stearic acid grafted chitosan (CSeSA) micelles with excellent cellular uptake ability for the delivery of anti-tumor agent [23]. The CSeSA micelle delivery system presented higher cytotoxicity than drug itself, however it indicated the same high cytotoxicity in normal cells and faster elimination in vivo. The PEGylated CSeSA (PEGeCSeSA) micelles could significantly reduce the uptake by macrophage [24], while the antitumor activity was lowered. Herein, the active targeting of PEGe CSeSA micelles was further designed. A hepatocarcinomahoming and specific binding peptide, AGKGTPSLETTP peptide (A54), was used as an active-targeting ligand to functionalize the PEGeCSeSA micelles. 2. Materials and methods 2.1. Materials Chitosan (CS) with about 5.0 kDa average molecular weight was obtained by enzymatic degradation of 95% deacetylated chitosan (Mw ¼ 45.0 kDa) was supplied by Yuhuan Marine Biochemistry Co., Ltd, China. Stearic acid (SA) was purchased from Shanghai Chemical Reagent Co., Ltd, China. AGKGTPSLETTP peptide (A54) was synthesized by Guangzhou Sinoasis Pharmaceuticals Inc., China. mPEG2000 with aldehyde side group and NH2ePEGeNH2 were purchased from SigmaeAldrich Inc., USA. N,N0 -Disuccinimidyl Carbonate (DSC) was purchased from BIO BASIC Inc., USA. Di-tert-butyl dicarbonate ((Boc)2O) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Shanghai Medpep Co., Ltd, China. Pyrene was purchased from Aldrich Chemical Co., USA. 2,4,6-trinitrobenzene sulfonic acid (TNBS), Fluorescein isothiocyanate (FITC) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co., St., USA. Doxorubicin hydrochlorate (DOX$HCl) was gifted from Hisun Pharm Co., Ltd, China. Chitosanase was purchased from Dyadic International, Inc., USA. Trypsin, RPMI 1640 Medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL, USA. Fetal bovine serum (FBS) was purchased from Sijiqing Biologic, China. All other chemicals were analytical or chromatographic grade. 2.2. Synthesis of A54 functionalized PEGylated CSeSA (A54ePEGeCSeSA) The CSeSA conjugate was synthesized via the coupling reaction between carboxyl group of SA and amine group of CS in the presence of EDC [25]. Briefly, 1.68 g SA and 11.2 g EDC were firstly dissolved in 70 mL ethanoleacetone mixed solvent (ethanol:acetone ¼ 2:5, v/v) under stirring for 1 h at 60  C, and then added into 133 mL DI water containing 2.0 g CS under stirring for another 24 h. The reaction solution was dialyzed against DI water using a dialysis membrane (MWCO: 3.5 kDa, Spectrum Laboratories, Laguna Hills, CA) for 2 days with frequent exchange of fresh DI water to remove water-soluble by-products, and then lyophilized. After that, the lyophilized product was further washed thrice with ethanol to remove un-reacted stearic acid. The solution was then lyophilized. For synthesis of A54ePEGeCSeSA, 5.16 mL (Boc)2O was added into 20 mL anhydrous DMF containing 20 mg A54 at ice-bath, followed by stirring with light protection at room temperature for 12 h. Then 33.4 mg EDC in DMF was added drop wise into the reaction system. After stirred for 1.5 h at room temperature, 35.2 mg NH2ePEGeNH2 in DMF was added for another 24 h to produce t-BoceA54ePEGe NH2. And then 4.42 mg DSC (DSC:t-BoceA54ePEGeNH2 ¼ 1:1, mol/mol) in DMF was added and stirred for 9 h at room temperature to obtain succinimidyl t-Boce A54ePEGeNH2 solution. To obtain A54ePEGeCSeSA, 111.3 mg CSeSA was dissolved in 20 mL DI water and subjected to probe-type ultrasonic treatment (400 W,

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30 cycles with 2 s activee3 s duration, JY92-II, Scientz Biotechnology Co., Ltd., China). The CSeSA solution was added into the succinimidyl t-BoceA54ePEGeNH2 solution in drops and stirred for 24 h at room temperature. The t-Boc (tert-butoxycarbonyl) group was removed by treatment with 2 M HCl to give A54ePEGeCSeSA. The resulting product was purified by dialysis (MWCO: 7 kDa) against DI water for 2 days, followed by lyophilization. For PEGylation of CSeSA as a control, mPEG2000 with aldehyde side group (PEG:CSeSA ¼ 1: 1, mol/mol) was added into 10 mg mL1 CSeSA solution under stirring overnight at room temperature. Then the mixture solution was dialyzed (MWCO: 7 kDa) against DI water for 24 h and lyophilized. 2.3. Preparation of DOX-loaded CSeSA, PEGeCSeSA and A54ePEGeCSeSA micelles Doxorubicin base (DOX), used for the preparation of DOX-loaded micelles, was obtained by the reaction between DOX$HCl and double mole triethylamine in DMSO for 24 h. Briefly, 50 mg polymers were dissolved in 10 mL DI water with probe-type ultrasonic treatment for 30 times (active every 2 s for a 3 s duration) in ice-bath. Then 1 mg mL1 DOX DMSO solution was added into polymeric micelle solution (DOX: polymers ¼ 5%, w/w). After that, the mixture solution was dialyzed against DI water (MWCO ¼ 3.5 kDa) for 24 h followed by probe-type ultrasonic treatment for 30 times. After dialyzed products were centrifuged at 3000 rpm for 8 min to remove precipitated drug during dialysis process, the DOX-loaded micelles solution was obtained. 2.4. Physicochemical characteristics of polymers and micelles The 1H NMR spectra of chemicals were obtained by a NMR spectrometer (AC-80, Bruker Biospin, Germany). SA was dissolved in dimethylsulfoxide-D6; A54 peptide, CS, mPEG2000, NH2ePEGeNH2, CSeSA, PEGeCSeSA and A54ePEGeCSeSA were dissolved in D2O at the concentration of 20 mg mL1. The degrees of amino-substitution (SD %) for CSeSA and PEGeCSeSA were assayed using 2,4,6-trinitrobenzene sulfonic acid (TNBS) test. Briefly, 2 mL 4% NaHCO3 and 2 mL 0.1% TNBS solution were added into 2 mL 100 mg mL1 polymers solution, and then incubated for 2 h at 37  C, followed by the addition of 2 mL 2 M HCl. The absorbed intensity of the mixture solution at 344 nm was detected by a UV spectrophotometer (TU-1800PC, Beijing Purkinje General Instrument Co., Ltd., China). The critical micelle concentration (CMC) of CSeSA, PEGeCSeSA and A54ePEGe CSeSA was determined by fluorescence measurement using pyrene as a probe [26]. Pyrene was firstly dissolved in acetone for quantitation. After the acetone was evaporated under 50  C, 10 mL polymers solution with different concentrations ranging from 5.0  104 to 1.0 mg mL1 were added into pyrene. The concentration of pyrene was controlled at 5.94  107 M. After the solution was treated by waterbath ultrasonication for 30 min, the fluorescence spectra of solution were recorded on a fluorometer (F-2500, Hitachi Co., Japan) at room temperature. The excitation wavelength was 336 nm and the slit openings were set at 10 nm (excitation) and 2.5 nm (emission). The pyrene emission was monitored at a wavelength range of 360e450 nm. From the pyrene emission spectra, the intensity ratio of the first peak (I1, 374 nm) to the third peak (I3, 384 nm) was analyzed for calculation of CMC. The hydrodynamic diameters of blank and DOX-loaded micelles in DI water solution at the concentration of 1.0 mg mL1 were determined by dynamic light scattering using a Zetasizer (3000 HS, Malvern Instruments Ltd., UK). The surface zeta potential at the same concentration in DI water was also detected by the Zetasizer. The morphological examinations of the blank and DOX-loaded micelles were performed by a transmission electron microscopy (TEM) (JEOL JEM-1230, Japan). The samples were stained with 2% (w/v) phosphotungstic acid and placed on copper grids with films for viewing. 2.5. Determination of drug encapsulation efficiency and drug loading To determine DOX content, a fluorescence spectrophotometer was utilized. The excitation wavelength was set at 505 nm, emission wavelength at 565 nm, and slit openings at 5 nm. The drug encapsulation efficiency and drug loading were measured by centrifugal-ultrafiltration method. Briefly, 0.4 mL DOX-loaded micelle solution was added into a centrifugal-ultrafiltration tube (Microcon YM-10, MWCO 3000, Millipore Co., USA) and centrifuged at 10 000 rpm for 20 min. The DOX concentration (Cu, mg mL1) in ultrafiltrate was measured. Another 0.4 mL DOXloaded micelle solution was diluted 100-fold by DMSO aqueous solution (DMSO/ H2O ¼ 9:1, v/v) and dissociated. The DOX concentration (C0, mg mL1) in diluted solution was measured, and was considered as the total drug amount in 0.4 mL DOXloaded micelle solution. The drug encapsulation efficiency (EE %) and drug loading (DL %) of the DOX-loaded micelles could be calculated by the following equations: EE% ¼ ðC0  100  Cu Þ  V =Md  100%;

(1)

DL% ¼ ðC0  100  Cu Þ  V =½Mm þ ðC0  100  Cu Þ  V   100%;

(2)

where Cu is the DOX concentration in ultrafiltrate; C0 is the DOX concentration in the DOX-loaded micelle solution; Md is the charged amount of DOX and the unit is mg;

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Mm is the amount of polymer micelles used, which is 20 000 mg in this experiment; V is the total volume of the DOX-loaded micelle solution and the unit is mL. 2.6. In vitro DOX release from DOX-loaded micelles For in vitro drug release tests, pH 7.4 Phosphate-buffered saline (PBS) was used as the dissolution medium. 1 mL DOX-loaded micelle solution was put into a dialysis membrane (MWCO: 3.5 kDa) and added into a plastic tube containing 15 mL PBS solution. The plastic tube was then placed in an incubator shaker (HZ-8812S, Scientific and Educational Equipment plant, Tai Cang, China), which was maintained at 37  C and shaken horizontally at 60 rpm. At predetermined time intervals, all medium out of the dialysis membrane was withdrawn and changed with the fresh medium. The drug concentration was determined by a fluorescence spectrophotometer. All drug release tests were performed thrice. 2.7. Cell culture BEL-7402 (human hepatocellular carcinoma cell line), HepG2 (human liver tumor cell line) and BRL-3A (immortalized rat liver cell line) cells were donated from the second Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China). BEL-7402, HepG2 and BRL-3A cells were maintained in DMEM at 37  C in a humidified atmosphere containing 5% CO2. All the mediums were supplemented with 10% (v/v) FBS (fetal bovine serum) and penicillin/streptomycin (100 U mL1, 100 U mL1). Cells were sub-cultured regularly using trypsin/EDTA. 2.8. Cellular competitive uptake Before incubation with polymer micelles, the HepG2 or BRL-3A cells were stained with PKH67 Fluorescent Cell Linker (SigmaeAldrich; St. Louis, MO USA), which incorporates into cell membrane with no modification of biological activity [27], following to the protocol with some arrangements. Briefly, cells were re-suspended in 200 mL Diluent C, then 200 mL of PHK67 dye (4 mM) was added and the cells were incubated for 10 min at room temperature. To stop the staining reaction, 1 mL of serum was added and incubated for 2 min, following by centrifuged at 400g for 10 min. The cell pellet was washed 2 more times with 10 mL of complete medium to ensure removal of unbound dye and re-suspended to desired concentration. The PKH67 labeled HepG2 or BRL-3A cells were then respectively co-cultured with BEL-7402 cells in the same well of a 24-well plate and incubated for 24 h to attach. And then the cells were incubated with Rhodamine B Isothiocyanate labeled polymer micelles (RITC: polymer ¼ 2:1, mol/mol) in growth medium for 1 h. Final concentrations of polymers will be 20 mg mL1. After washing the cells with PBS three times, the cellular uptake was observed by Confocal laser scanning microscopy (Carl Zeiss LSM 510, Germany). 2.9. Quantification of cellular uptake BEL-7402, HepG2, and BRL-3A cells were respectively seeded at 1.0  105 cells mL1 per well in a 24-well plate (Nalge Nunc International, Naperville, IL, USA) and allowed to attach for 24 h, respectively. Twenty mg RITC labeled CSeSA, PEGe CSeSA or A54ePEGeCSeSA was added into each well, followed by further incubation for 1, 12, and 24 h, respectively. After the cells were washed with PBS for three times, 70 mL of WIP lysis buffer (Tissue and Cell lysis solution for Western Blot and Immunoprecipitation; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) was added into each well to lysis. And then 630 mL DMSO was added into each well, and the cells were harvested. The harvested cells were further fragmented through repeated freezing and thawing. Finally, the cell lysate was centrifuged at 10 000g for 5 min, and then subjected to fluorescence assay using fluorometer (F-2500, HITACHI Co., Japan) (excitation wavelength: 544 nm; emission wavelength: 593 nm). The cellular uptake contents of micelles were corrected to per mg intracellular protein. Using BCA protein assay kit, the intracellular protein content was measured. Briefly, 20 mL cell lysate was added into each well, and 200 mL BCA working reagent was then added, followed by incubation at 37  C for 30 min. UV absorbance at 570 nm of cell lysate was measured by a microplate reader (Bio-Rad, Model 680, USA). The protein concentration was calculated from calibration curve obtained from bovine serum albumin (BSA). The cellular uptake percentage of micelles was calculated from the following equation: Pt ð%Þ ¼ Ft =F0  100%

(3)

where Pt represents the cellular uptake percentage of the micelles at t time; Ft and F0 are the fluorescence absorbance corrected by intracellular protein concentration at t time and 0 time, respectively.

concentrations of blank and DOX-loaded CSeSA, PEGeCSeSA or A54ePEGeCSeSA micelles for another 48 h. After that, cells were incubated with 20 mL MTT solution (5 mg$mL1) each well for further 4 h at 37  C. At the end of incubation, 100 mL DMSO was added into each well to replace the culture medium and dissolve the insoluble formazan-containing crystals. Finally, the plate was shaken for 30 min, and optical density was determined at 570 nm using an automatic reader (Bio-Rad, Model 680, USA). Cell viability was calculated in reference to cells incubated with culture medium alone. All the experiments were repeated thrice. 2.11. Specific internalization of DOX-loaded micelles The internalization of CSeSA/DOX, PEGeCSeSA/DOX and A54ePEGeCSeSA/ DOX micelles was studied using BEL-7402 and HepG2 cells. Cells were seeded in a 24-well plate (Nalge Nunc International, Naperville, IL, USA) at a seeding density of 2.0  104 cells per well and allowed to attach for 24 h, respectively. And then the cells were incubated with FITC-labeled CSeSA/DOX, PEGeCSeSA/DOX or A54ePEGe CSeSA/DOX micelles (final drug content was 5 mg mL1) in growth medium for 1, 2, 4, 6, 10, and 24 h, respectively. After washing the cells with PBS three times, the cellular uptake was observed by a fluorescence microscopy (Leica, Germany). 2.12. In vivo and ex vivo imaging All animal procedures were performed according to national regulations and approved by the local animal experiments ethical committee. The xenografted tumor models were established by subcutaneous injection of BEL-7402 cells (w5  106 cells in 100 mL of serum-free DMEM) into the flank of male BALB/C þ nu/F1 nude mice, respectively. The mice were subjected to imaging studies when the tumor reached the acceptable sizes. After the tail vein injection of DiRloaded micelles (CSeSA/DiR, PEGeCSeSA/DiR and A54ePEGeCSeSA/DiR; 0.5 mg polymers and 4.9 mg DiR in 200 mL PBS buffer) respectively, the mice were anesthetized and imaged at multiple time points (e.g., 1 h, 3 h, and 6 h) using the Maestro In Vivo Imaging System (CRI Inc., Woburn, MA). The tunable filter was automatically stepped in 10-nm increments from 580 to 700 nm while the camera captured images at each wavelength with constant exposure. Overall acquisition time was about 0.5 s. For ex vivo imaging, after the tumors and hearts were harvested, the tissues were subjected to fluorescence imaging using the Spectral Imaging System immediately. 2.13. In vivo anti-tumor activity To investigate the in vivo anti-tumor activity of DOX-loaded micelles, BEL-7402 cells were transplanted into BALB/C þ nu/F1 nude mice about 6e8 weeks old subcutaneously. When the tumor volume reached approximately 100 mm3, drug injection via tail vein was started. Mice were divided randomly into eight groups. The negative control group was injected with 0.2 mL 0.9% saline solution and the positive control group with 0.2 mL adriamycin (commercial doxorubicin hydrochloride injection, 2.0 mg kg1 body weight). The CSeSA/DOX or PEGeCSeSA/DOX group was injected with 0.2 mL CSeSA/DOX or PEGeCSeSA/DOX micelles (2.0 mg kg1 body weight). Another four groups were injected with A54ePEGeCSe SA/DOX micelles (4 mg kg1, 2 mg kg1, 1 mg kg1 and 0.5 mg kg1). All the groups were dosed for seven days consecutively [28], except that the treatment with A54e PEGeCSeSA/DOX micelles (4 mg kg1) was four days. The size of tumor and the body weight of each mouse were monitored every five days thereafter. After 25 days, the nude mice were sacrificed, and the tumor weights were measured. Tumor volume was calculated using the formula, a2  b/2, where a is the smallest and b is the largest diameter. The inhibition of tumor growth was calculated using the formula as follows: The inhibition of tumor growthð%Þ ¼ ðWc  Wt Þ=Wc  100%

(4)

where, Wc is the average tumor weight of controlled group, Wt is the average tumor weight of treated group. The treatment is considered effective if the value of the inhibition of tumor growth is higher than 60%. 2.14. Statistical analysis Data were expressed as means of three separate experiments. Differences between groups were assessed using unpaired two-tailed Student’s t-test, and a pvalue <0.05 was considered statistically significant in all cases.

3. Results and discussion

2.10. In vitro anti-tumor activity

3.1. Synthesis and characteristics of A54ePEGeCSeSA

The cytotoxicity tests against BEL-7402 and HepG2 cells were used to evaluate in vitro anti-tumor activity of the DOX-loaded micelles. Briefly, 1.0  104 cells per well were placed in a 96-well microtiter plate (Nalge Nunc International, Naperville, IL, USA). After cultured at 37  C for 24 h, the cells were exposed to a series of

The stearic acid grafted chitosan (CSeSA) was firstly synthesized by coupling reaction between the amino groups of CS and carboxyl group of SA in the presence of water-soluble 1-Ethyl-3-(3-

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dimethylaminopropyl) carbodiimide (EDC), and the PEGylation of stearic acid grafted chitosan (PEGeCSeSA) was conducted by the reaction between the remaining amino groups in CSeSA and aldehyde group of mPEG. The degrees of amino-substitution (SD %) of CSeSA and PEGeCSeSA were measured as 5.03  0.31% and 6.69  0.004%, respectively. Fig. 1a presented the synthesis scheme of A54ePEGeCSeSA. The t-BoceA54ePEGeNH2 was then prepared by the chemical reaction between A54 (its amino terminus pre-protected by (Boc)2O) and NH2ePEGeNH2 in the presence of EDC. EDC, a coupling crosslinker, can react with the carboxyl group of the t-Boc protected A54 peptide and form an active intermediate-O0 -acylisourea derivation [29], which can react with the amine group of NH2ePEGeNH2 to form an amide bond. And N,N0 -Disuccinimidyl Carbonate (DSC) has been recognized as a versatile reagent for active ester synthesis [30]. The t-BoceA54ePEGeCSeSA was synthesized by conjugating the remaining amino groups of NH2ePEGeNH2 and the remaining primary amino groups in CSeSA in the presence of DSC. The chemical structures of obtained CSeSA, PEGeCSeSA and A54e PEGeCSeSA were confirmed by 1H NMR spectra (Fig. 1b). The peaks at about 0.95 ppm and 1.08 ppm were attributed to the eCH3 and eCH2e for SA of CSeSA, respectively. Compared with CSeSA, the 1H NMR spectrum of the A54ePEGeCSeSA showed a double signal at 0.75e0.81 ppm, which were the chemical shifts of the proton of eCH3 for Leucine of peptide A54. And the peaks at about 3.58 ppm were attributed to eCH2CH2Oe of NH2ePEGeNH2. These results indicated that A54ePEGeCSeSA was synthesized successfully. Due to the molecular weight of used CS was 5 kDa, which easily dissolved in water, there were found the synthesized CSeSA, PEGe CSeSA and A54ePEGeCSeSA could form polymeric micelles by self-aggregation in aqueous phase with low critical micelles concentration (CMC), which was much lower than those of low molecular weight surfactants in water [31]. As shown in Fig. 1c, the value of I1/I3 kept around 1.7 until the A54ePEGeCSeSA concentration reached up to CMC. The CMC values of CSeSA, PEGeCSeSA and A54ePEGeCSeSA were determined as 249.35  5.87, 229.85  1.77, and 191.70  3.11 mg mL1, respectively. The TEM images of formed micelles, the distribution of size and surface potential was presented in Fig. 2a and b. As we known, in hypervascular tumors with a highly permeable structure, sub100 nm micellar nanomedicines showed no size-dependent restrictions on extravasation and penetration in tumors [32]. Moreover, the carrier system with smaller size (<200 nm) and hydrophilic surface can avoid clearance by macrophages [33]. The CSeSA micelle size was only 24.02  0.41 nm. After PEGylation and further A54 modification, the micelle size increased to 67.17  14.36 nm and 77.85  8.31 nm, shown in Fig. 2a. These might result from the stretch of PEG chains and hydrophilous peptide A54 on the surface of A54ePEGeCSeSA micelle toward water phase. When hydrophobic drug was further entrapped into the hydrophobic core, the micelle size was always larger than blank micelles [34]. However, A54ePEGeCSeSA micelles and its drugloaded micelles were small enough (<100 nm) for efficient binding to cell surface receptor. The zeta potential of PEGeCSeSA and A54ePEGeCSeSA micelles was lower than that of CSeSA micelles, which might be caused from the barrier shield effect of PEG chain on the micelles [35] and the reduced amino groups after the PEGylation. However, the zeta potential of A54ePEGeCSeSA micelles was a little higher than that of PEGeCSeSA micelles, which might resulted from the protonation of peptide A54. The zeta potential of drug-loaded micelles remained positive in DI water varied from 33.2  5.8 to 36.2  1.8 mV, shown in Fig. 2b. It is a general rule of thumb that an absolute value of zeta potential above 30 mV yields good stability [36].

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3.2. Preparation and characteristics of DOX-loaded micelles DOX-loaded micelles were then prepared successfully by dialysis method. After loading DOX, the micelle size was bigger than that of blank one (Fig. 2a and b), while the size still smaller than 100 nm. When the drug feeding amount was 5.00%, the Drug Loading (DL %) of DOX-loaded micelles was ranged from 3.44% to 4.00%. Considering the drug loss in preparation process, the Drug Encapsulation Efficiency (EE %) of DOX-loaded micelles was above 65%. In vitro drug release from DOX-loaded micelles was carried out using pH 7.4 PBS as dissolution medium. As shown in Fig. 2c, the release profiles of DOX from CSeSA/DOX, PEGeCSeSA/DOX and A54ePEGeCSeSA/DOX micelles were mostly similar, which implied that the modification of PEG and A54 had no obvious influences on drug release behaviors. The curves in Fig. 2c exhibit a typical biphasic pattern consisting of an initial fast release in 12 h followed by sustained release for a prolonged time. There were about 70% drugs released after 72 h. 3.3. Cellular internalization ability of the micelles To investigate the targeting ability of A54ePEGeCSeSA micelles toward BEL-7402 cells, cellular competitive uptake of RITC labeled micelles on BEL-7402/HepG2 cells co-cultured and BEL-7402/BRL3A cells co-cultured systems were observed by a Confocal laser scanning microscopy. Obviously, Line 3 and 4 in Fig. 3 indicated that there was significant difference in the cellular uptake of A54ePEGe CSeSA on the cell co-cultured systems (BEL-7402/HepG2 cocultured and BEL-7402/BRL-3A co-cultured systems). The cellular uptake of A54ePEGeCSeSA micelles on BEL-7402 cells was more efficient compared with HepG2 and BRL-3A cells co-cultured, due to the A54 homing peptide-mediated endocytosis. However, CSeSA and PEGeCSeSA micelles showed approximate uptake on the cell co-cultured systems, either the BEL-7402/HepG2 or BEL-7402/BRL3A co-cultured systems (Line 1 and 2 in Fig. 3). The cellular competitive uptake data confirmed strong and specific binding of the A54ePEGeCSeSA micelles to BEL-7402 cells, due to the presence of an abundant cell surface marker that may be highly expressed on BEL-7402 cells [1]. These results established that the A54ePEGeCSeSA micelles retain their A54 peptide binding activity and specificity, and may be employed as a promising activetargeting carrier via A54 peptide mediation [37e39]. The results of quantitative cellular uptake for RITC labeled CSe SA, PEGeCSeSA and A54ePEGeCSeSA micelles were presented in Fig. 4 after the micelles were incubated with BEL-7402, HepG2 and BRL-3A cells, respectively. The A54ePEGeCSeSA micelles had great internalization ability in BEL-7402 cells. About 50% A54e PEGeCSeSA micelles could be uptaked by BEL-7402 cells in 24 h. However, the uptaked amounts of the micelles by HepG2 or BRL-3A cells were lower than 20% in 24 h. Fig. 5a showed the fluorescence images after the A54ePEGeCSe SA/DOX micelles was incubated with BEL-7402 cells for 1 h, 6 h and 24 h, respectively. It was found the anti-tumor agent, DOX, could be internalized into tumor cells mediated by the micelles. And the uptake of the A54ePEGeCSeSA/DOX micelles by BEL-7402 cells was time dependent. Fig. 5b revealed that the best DOX accumulation in BEL-7402 cells was reached by A54ePEGeCSeSA micelles in 24 h, while there were no significant differences among the DOXloaded micelles in HepG2. 3.4. In vitro anti-tumor activity of the DOX-loaded micelles In vitro anti-tumor activities of DOX-loaded micelles were evaluated by the determination of cytotoxicities of DOX-loaded micelles using BEL-7402 and HepG2 as model tumor cells, and

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DOX$HCl solution as a control. Using the MTT method, the 50% cellular growth inhibitions (IC50) of the DOX$HCl solution, blank micelles and DOX-loaded micelles against BEL-7402, HepG2 and BRL-3A cells were determined. As shown in Fig. 5c, the IC50 values

O

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of the blank micelles ranged from 380 to 680 mg mL1, which indicated the relatively high biocompatibility of blank micelles. After loading DOX (the Drug Loading was ranged from 3.44% to 4.00%), the cytotoxicities of micelles increased obviously, especially

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Fig. 1. Synthesis and characteristics of A54ePEGeCSeSA. (a) Synthetic scheme of A54ePEGeCSeSA. (b) 1H NMR spectra of peptide A54, NH2ePEGeNH2, CSeSA and A54ePEGeCSeSA. The important peaks were pointed out. (c) Variation of fluorescence intensity ratio for I1/I3 against logarithm of A54ePEGeCSeSA concentration. The unit of concentration was mg mL1.

Y.-Z. Du et al. / Biomaterials 33 (2012) 8858e8867

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showed similar cytotoxicities against HepG2 cells. This can be explained by the specific interaction (target-based binding) between the BEL-7402 cells and the A54ePEGeCSeSA micelles and the non-specific interaction between the micelles with the cells.

that of A54ePEGeCSeSA/DOX against BEL-7402 cells. The IC50 value was about 2.0 mg mL1, which increased the cytotoxicity by approximately 3.2e3.4-folded compared with CSeSA/DOX and PEGeCSeSA/DOX micelles. However, the DOX-loaded micelles

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Fig. 2. Characteristics of blank micelles and DOX-loaded micelles. (a) TEM images of CSeSA, PEGeCSeSA, A54ePEGeCSeSA micelles and CSeSA/DOX, PEGeCSeSA/DOX, A54ePEGe CSeSA/DOX micelles. (b) Size distribution and Zeta potential of A54ePEGeCSeSA micelles (upper) and A54ePEGeCSeSA/DOX micelles (lower). (c) In vitro drug release profiles of CSeSA/DOX, PEGeCSeSA/DOX and A54ePEGeCSeSA/DOX micelles.

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Fig. 3. Confocal microscopy images of cellular competitive uptake of RITC labeled micelles for 1 h. BRL-3A cells (the cytoplasmic membrane labeled with PKH67 fluorescent linker, Green) co-cultured with BEL-7402 cells were incubated with RITCeCSeSA, RITCePEGeCSeSA and RITCeA54ePEGeCSeSA micelles (Red). HepG2 cells (PKH67 labeled, Green) cocultured with BEL-7402 cells were incubated with RITCeA54ePEGeCSeSA (Red). The cells were all stained with Hoechst 33342. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

This effect was also observed by other researchers [37,40]. All these results suggested that A54ePEGeCSeSA had a high targeting (binding) ability to the BEL-7402 cells (shown in Fig. 5b) because of the attached small peptide A54, which could significantly increase the cellular cytotoxicity of the DOX-loaded micelles (CSeSA/DOX and PEGeCSeSA/DOX). In a word, PEGeCSeSA coupled with homing peptides was endowed the specific affinity to corresponding tumor cells in vitro. 3.5. In vivo targeting imaging of the micelles Far-red and near-infrared light, in the spectral range 650e 900 nm, provides a “clear” window for in vivo optical imaging

because it is separated from the major absorption peaks of blood and water [41]. Based on optical calculations, we estimated that the use of near-infrared-emitting DiR (DiIC18(7), Invitrogen, Staley Rd. Grand Island, NY USA) should improve the tumor imaging sensitivity by at least 10-fold. Fig. 6a compared the in vivo fluorescence images of BEL-7402 human hepatoma bearing nude mice resulted from three types of drug delivery systems: CSeSA/DiR, PEGeCSe SA/DiR and A54ePEGeCSeSA/DiR micelles. As seen from the characteristic fluorescence of DiR (spectral unmixing algorithms resolved), the uptake and retention of CSeSA/DiR micelles took place primarily in the liver, with little accumulation in tumor. For PEGeCSeSA micelles, the length of blood circulation was improved, leading to slow accumulation of the nanoparticles in the tumors.

Y.-Z. Du et al. / Biomaterials 33 (2012) 8858e8867

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Fig. 4. Cellular uptake percentages of CSeSA, PEGeCSeSA and A54ePEGeCSeSA micelles in different cell lines: (a) BEL-7402, (b) HepG2 and (c) BRL-3A for 1, 12, and 24 h, respectively. Data represent the mean  standard deviation (n ¼ 3). *, P < 0.05 and **, P < 0.01.

Furthermore, with the favor of homing peptide A54, the A54ePEGe CSeSA micelles were delivered and retained by the tumor xenografts more efficient with heart uptake reduced (Fig. 6a). This comparison provided further evidence for the conclusion that active tumor targeting by using a tumor-specific ligand is much faster and more efficient than passive targeting based on tumor permeation, uptake and retention [42]. Therefore, A54ePEGeCSe SA micelles can be delivered to tumors by both passive and active-targeting mechanisms under in vivo conditions.

3.6. In vivo anti-tumor activity of the DOX-loaded micelles In vivo anti-tumor activity of adriamycin (a commercial doxorubicin hydrochloride injection) and the DOX-loaded micelles were studied using BEL-7402 human hepatoma bearing nude mice. The changes in the tumor volume were plotted as shown in Fig. 6b. It was evident that adriamycin and the DOX-loaded micelles treatments effectively suppressed the tumor growth: e.g., 5 days after i.v. injection, tumor volumes of nude mice treated with adriamycin

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Fig. 5. In vitro cellular uptake and cytotoxicity of DOX-loaded micelles in different cell lines. (a) Fluorescence images of DOX drug after BEL-7402 cells incubated with A54ePEGe CSeSA/DOX micelles (drug content were 5 mg mL1) for 1, 6, and 24 h, respectively. (b) Fluorescence images of DOX drug after BEL-7402 and HepG2 cells incubated with CSeSA/DOX, PEGeCSeSA/DOX and A54ePEGeCSeSA/DOX micelles (drug content were 5 mg mL1) for 24 h. (c) Cytotoxicity of blabk micelles, drug-loaded micelles, and DOX$HCl against BEL7402 (left) and HepG2 cells (right). Data represent the mean  standard deviation (n ¼ 3).

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and the DOX-loaded micelles were significantly smaller than those treated with saline (p < 0.01), and after 10 days, p < 0.001. At the 15th day, the tumor volumes of nude mice treated with A54ePEGe CSeSA/DOX (2 mg kg1) were significantly smaller than the positive control group. What is more, the tumor volumes of nude mice injected with A54ePEGeCSeSA/DOX micelles (4 mg kg1, i.v. 4qd) were significantly smaller than all the other groups since the 5th day (p < 0.001). At the 10th day, the tumor volumes of nude mice treated with CSeSA/DOX micelles were significantly smaller than those treated with adriamycin (p < 0.05), while not at the 25th day. Compared with the adriamycin treated group (2 mg kg1), the tumor volumes of nude mice treated with A54ePEGeCSeSA/DOX (1 mg kg1) didn’t decrease significantly (p > 0.05) until the 25th day (p < 0.05) as well as the PEGeCSeSA/DOX (2 mg kg1). Fig. 6c also showed that the body weight of drug treated and un-treated mice was continuously increased. It indicated that the doses of these eight groups were within the safe range.

The tumor inhibition rate of different treatments was 73.57% treated with adriamycin (2 mg kg1), 73.38% with CSeSA/DOX (2 mg kg1), 72.92% with PEGeCSeSA/DOX (2 mg kg1), 85.91% with A54ePEGeCSeSA/DOX (4 mg kg1), 80.32% with A54ePEGe CSeSA/DOX (2 mg kg1) and 73.83% with A54ePEGeCSeSA/DOX (1 mg kg1), respectively. All the tumor inhibition values were larger than 60%, which were considered to be effective treatment. There were no significant differences of the tumor inhibition rate between the groups treated with A54ePEGeCSeSA/DOX with 7 mg kg1 total drug dose and adriamycin with 14 mg kg1 total drug dose (p > 0.05). The results powerfully confirmed that A54e PEGeCSeSA/DOX micelles could target to the tumor tissue more efficiently, and even realize the same anti-tumor efficiency with adriamycin while halved the drug dose. Furthermore, with increasing the drug dose of A54ePEGeCSeSA/DOX micelles, the tumor inhibition rate was enhanced, while given the same dose of adriamycin all of the animals were dead. The reduced toxicity of

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Fig. 6. In vivo bio-distribution of DiR-loaded micelles and anti-tumor activities of drug-loaded micelles after i.v. injection in the tail of tumor-bearing nude mice. (a) The fluorescence images of tumor-bearing nude mice using DiR probes with three different micelles: CSeSA/DiR (left), PEGeCSeSA/DiR (middle) and A54ePEGeCSeSA/DiR (right). The amount of micelles and probes DiR was 500 mg and 4.9 mg, respectively. The images were spectrally resolved and the tumor sites were emphasized by red circles. (b, c) In vivo antitumor activities of adriamycin and DOX-loaded micelles in tumor-bearing nude mice: mice tumor volume (b) and mice body weight (c) changed within 25 days. Data represent the mean  standard deviation (n ¼ 6).

Y.-Z. Du et al. / Biomaterials 33 (2012) 8858e8867

A54ePEGeCSeSA/DOX might originate from the high targeting efficiency and reduction of heart distribution (Fig. 6a). It was assumed that A54ePEGeCSeSA/DOX could elevate the maximum tolerated dose in comparison with the usual clinical dose of adriamycin. 4. Conclusions Above all, it could be attributed to triple function of rapid uptake, long circulation times and biomolecular targeting of A54e PEGeCSeSA. Long circulation times will allow effective transport of micelles to the tumor tissue through the EPR effect, and the targeting molecule can increase endocytosis of the micelles. The triple functional A54ePEGeCSeSA successfully inherit the advantages of CSeSA, PEG chains and biomolecular targeting peptide A54, so that the micelles can be targeted to the tumor tissue with more efficiency than the other two micelles. All the results suggested that the A54ePEGeCSeSA micelles were a potential candidate for active-targeting drug delivery. Acknowledgments We are grateful for financial support from the National Basic Research Program of China (973 Program) under Contract 2009CB930300, the National HighTech Research and Development Program (863) of China (2007AA03Z318), and the National Nature Science Foundation of China under Contract 81072583. References [1] Du B, Han H, Wang Z, Kuang L, Wang L, Yu L, et al. Targeted drug delivery to hepatocarcinoma in vivo by phage-displayed specific binding peptide. Mol Cancer Res 2010;8:135e44. [2] Watts RG, George MK, Johnson Jr WH. Pretreatment and routine echocardiogram monitoring during chemotherapy for anthracycline-induced cardiotoxicity rarely identifies significant cardiac dysfunction or alters treatment decisions. Cancer 2011;18:1919e24. [3] Mastrobattista E, Koning GA, Storm G. Immunoliposomes for the targeted delivery of antitumor drugs. Adv Drug Deliv Rev 1999;40:103e27. [4] Duncan R. Polymer conjugates as anticancer nanomedicines. Nat Rev Cancer 2006;6:688e701. [5] Scott AW, Tyler BM, Masi BC, Upadhyay UM, Patta YR, Grossman R, et al. Intracranial microcapsule drug delivery device for the treatment of an experimental gliosarcoma model. Biomaterials 2011;32:2532e9. [6] Vila A, Sanchez A, Tobi’O M, Calvo P, Alonso MJ. Design of biodegradable particles for protein delivery. J Control Release 2002;78:15e24. [7] Liu Y, Huang R, Han L, Ke W, Shao K, Ye L, et al. Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles. Biomaterials 2009;30:4195e202. [8] Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 2000;65:271e84. [9] Danquah MK, Zhang XA, Mahato RI. Extravasation of polymeric nanomedicines across tumor vasculature. Adv Drug Deliv Rev 2010;63:623e39. [10] Greenwald RB. PEG drugs: an overview. J Control Release 2001;74:159e71. [11] Torchilin VP, Trubetskoy VS. Which polymers can make nanoparticulate drug carriers long-circulating? Adv Drug Deliv Rev 1995;16:141e55. [12] Inada Y, Furukawa M, Sasaki H, Kodera Y, Hiroto M, Nishimura H, et al. Biomedical and biotechnological applications of PEG-and PM-modified proteins. Trends Biotechnol 1995;13:86e91. [13] Shibata H, Yoshioka Y, Ikemizu S, Kobayashi K, Yamamoto Y, Mukai Y, et al. Functionalization of tumor necrosis factor-a using phage display technique and PEGylation improves its antitumor therapeutic window. Clin Cancer Res 2004;10:8293e300. [14] Terada T, Mizobata M, Kawakami S, Yamashita F, Hashida M. Optimization of tumor-selective targeting by basic fibroblast growth factor-binding peptide grafted PEGylated liposomes. J Control Release 2007;119:262e70.

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