Colloids and Surfaces B: Biointerfaces 131 (2015) 191–201
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Polymeric complex micelles with double drug-loading strategies for folate-mediated paclitaxel delivery Min Li 1 , Yongjun Liu 1 , Lixia Feng, Fengxi Liu, Li Zhang, Na Zhang ∗ School of Pharmaceutical Science, Shandong University, Ji’nan 250012, China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Received in revised form 15 April 2015 Accepted 27 April 2015 Available online 5 May 2015 Keywords: Polymeric complex micelles Pluronic P123 Paclitaxel Double drug-loading
a b s t r a c t Drug loading is a key procedure in the preparation of drug-loaded nano-carriers. In this study, the paclitaxel (PTX)-loaded polymeric complex micelles (FA-P123-PTX/PTX micelles) with double drug-loading strategies were designed and prepared to improve the drug loading percentage of carriers and its antitumor efficiency. PTX was simultaneously conjugated to pluronic P123 (P123) polymer and encapsulated inside the P123 complex micelle. Folate (FA) was linked to the surface of micelles for the active target delivery of micelles to tumor cells. The FA-P123-PTX/PTX micelles showed spherical shaped with high drug loading of 18.08 ± 0.64%. The results of cellular uptake studies suggested that FA could promote the internalization of micelles into the FR positive cells. FA-P123-PTX/PTX micelles showed significant higher anti-tumor activity against FR positive tumor cells compared to Taxol® (p < 0.05). Moreover, the FA-P123-PTX/PTX micelles exhibited higher anti-tumor efficacy in B16 bearing mice with better safety property compared with Taxol® . These results suggested that FA-P123-PTX/PTX micelles with double drug-loading strategies showed great potential for targeted delivery of anti-cancer drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past decades, drug-loaded nanoparticles were widely studied to improve the poor biodistribution and pharmacokinetics properties of free drug molecules in cancer drug delivery. Drug encapsulation and chemical conjugation were the common methods for drug loading. For chemical conjugation method, polymer–drug conjugates have been widely investigated as novel drug delivery systems [1]. It is formed by conjugation of drugs to hydrophilic or amphiphilicity polymers through chemical bonds. Due to the amphiphilicity, the polymer–drug conjugates selfassemble into micelles or micelle-like nanoassemblies in aqueous media. Drug loading by chemical conjugation method have many advantages such as high drug loading capacity, simplified preparation process, accurate loading efficiency [2]. However, this method also suffers from the possibility of decrease drug activity by the linkage of drugs and materials, the difficulty of controlled drug release from the drug conjugates. Drug encapsulation is another common method for drug loading which could solve these problems of
∗ Corresponding author at: Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel.: +86 0531 88382015; fax: +86 0531 88382548. E-mail address: [email protected] (N. Zhang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.04.057 0927-7765/© 2015 Elsevier B.V. All rights reserved.
chemical conjugation. However, its drug loading capacity needs to be improved [3,4] and the preparation formulation/process needs to be carefully adjusted to reach the highest drug loading and encapsulation efficiency [5]. Base on these problems, the paclitaxel (PTX)-loaded pluronic P123 (P123) complex micelles (FA-P123PTX/PTX micelles) combined with drug encapsulation and chemical conjugation drug-loading strategies were designed and prepared. The double drug-loading strategies in one carrier could increase the drug loading efficiency, improve the release profile, and eventually increase the anti-tumor efficiency. Pluronic block copolymer (PEO-b-PPO-b-PEO), which is amphiphilic synthetic polymers containing hydrophilic poly(ethylene oxide) (PEO) blocks and hydrophobic poly(propylene oxide) (PPO) blocks, has been commonly used for solubilization of hydrophobic drugs [6]. The hydrophobic PPO segments can comprise a hydrophobic core as a microenvironment for the incorporation of hydrophobic drugs [7]. The hydrophilic PEO corona can prevent aggregation, protein adsorption, and recognition by the reticuloendothelial system (RES) [7]. Currently, a micellar formulation consisting of DOX and two Pluronic copolymers (Pluronic L61 and Pluronic F127) has been already in clinical trials [8]. Among different kinds of Pluronic copolymers, Pluronic P123 (PEO20 -b-PPO69 -b-PEO20 ) is a hydrophobic block copolymer with a relatively low critical micelle concentration (CMC, 4.4 × 10−6 mol/L) [9]. Besides, Pluronic P123 presented the advantages of cheap, non-toxic, non-immunogenic and biocompatibility
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Scheme 1. Schematic diagram illustrating for P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles bearing paclitaxel with chemical conjugation and encapsulation.
[10]. Thus, P123 was selected as the polymer for the linkage and encapsulation of PTX at the same time. It was expected that when the double loading micelles (P123-PTX/PTX micelles) were delivered to the tumor tissue, the encapsulated PTX could be released quickly to reach effective therapeutic drug concentrations. Then the conjugated PTX was released continuously to maintain the therapeutic concentration of PTX. Therefore, the therapeutic efficiency could be greatly increased. Tumor active target delivery is a promising approach to increase the accumulation of drugs at tumor area. Folate receptor (FR) was known to be vastly over-expressed on malignant cells [11]. Therefore, folate (FA) has been employed as a targeting moiety of various anti-cancer agents to reduce their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells via receptor-mediated endocytosis [12]. In addition, FA possesses some advantages such as low immunogenicity, ease of modification, and low cost. The small size of FA also allows for good tissue penetration and rapid clearance from receptor negative tissues [13]. Hence, FA-based targeting systems were widely used to selectively delivering therapeutic agents to tumors. In this study, FA-mediated double drug-loading delivery system (FA-P123-PTX/PTX micelles) was prepared (Scheme 1). The in vitro PTX release of FA-P123-PTX/PTX micelles was performed in different pH values to evaluate the pH sensitive of ester bond [14]. The cellular uptake was investigated to verify the active-targeting of FA-P123-PTX/PTX micelles. In vitro anti-tumor activity of PTX loaded micelles against human lung adenocarcinoma A549 cells, human breast adenocarcinoma MCF-7 cells and mouse malignant melanoma B16 cells was assessed by the MTT method and annexin V-FITC/PI double labeling. In vivo therapeutic effect and the maximum tolerated dose were investigated in Kunming mice.
2. Materials and methods 2.1. Materials Paclitaxel was provided by Chenxin Pharmaceutical Co Ltd. (China). Pluronic P123 (P123) was purchased from BASF China Co., Ltd. (Shanghai, China). FA was purchased from Sigma–Aldrich Shanghai Trading Co, Ltd (Shanghai, China). 4-Dimethylaminopyridine (DMAP), succinic anhydride, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), N-hydroxysulfosuccinimide (NHS) and MTT were all obtained from Sigma–Aldrich (China). Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Fetal bovine serum (FBS) was obtained from Sijiqing Co., Ltd. Annexin V-FITC apoptosis kit was provided by Bestbio (Shanghai, China). All other chemicals
and reagents used were of analytical grade or higher and obtained commercially. 2.2. Cells and animals Human lung adenocarcinoma cells (A549), human breast carcinoma cell lines (MCF-7) and murine malignant melanoma cell lines (B16) were kindly provided by Institute of Immunopharmacology and Immunotherapy of Shandong University (Ji’nan, China) and cultured in DMEM medium supplemented with 10% FBS, streptomycin at 100 g/mL and penicillin at 100 U/mL. All cells were cultured in a 37 ◦ C incubator with 5% CO2 . Female Kunming mice (18–22 g) were supplied by Laboratory Animals Center of Shandong University (Ji’nan, China). All animal procedures were performed according to the guidelines of the Ethical Committee for Animal Experiments of Shandong University. 2.3. Synthesis of P123-PTX conjugates 2.3.1. Synthesis of succinyl-PTX PTX (300 mg), succinic anhydride (48 mg) and DMAP (5.9 mg) were dissolved in 10 mL of anhydrous dichloromethane and stirred for 24 h at room temperature. Then the dichloromethane was removed by rotary evaporation. After the residue was dissolved in 50 mL of ethyl acetate, 50 mL of dilute hydrochloric acid (0.1 M) was added and stirred for 15 min. The organic phase was then separated and dried with anhydrous magnesium overnight. The succinylPTX was purified by silica gel column chromatography eluted with petroleum ether–ethyl acetate mixture (1:2) and used directly in the next step. The structure of succinyl-PTX was confirmed by 1 H NMR (300 MHz, CDCl3 ) and mass spectrometry. 2.3.2. Synthesis of P123-PTX conjugates Succinyl-PTX (100 mg), DMAP (10.9 mg), EDC (34 mg) were dissolved in anhydrous dichloromethane and then cooled down to 0 ◦ C by ice bath. 500 mg of P123 was dissolved in 2 mL of anhydrous dichloromethane and added dropwise into the reaction solution and then stirred for 48 h at room temperature. The dichloromethane was removed by rotary evaporation. Then residue was dissolved in tetrahydrofuran and dialyzed with a 3500 Da molecular weight cutoff dialysis bag against 1000 mL deionized water for 72 h. The structure of P123-PTX conjugates was confirmed by 1 H NMR (300 MHz, CDCl3 ). 2.4. Synthesis of FA-P123-PTX conjugates 30 mg of folic acid was dissolved in 10 mL of anhydrous DMSO. NHS (32 mg), EDC (53 mg), and triethylamine (20 L) were
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successively added. 400 mg of P123-PTX conjugates was dissolved in 2 mL of anhydrous DMSO and added dropwise into the reaction solution. After the completion of the reaction (about 48 h at room temperature), the resultant solution was dialyzed against 50 mL saline for 24 h and against distilled water for 48 h using a dialysis membrane (MWCO: 3500 Da). The structure of FA-P123-PTX conjugates was also confirmed by 1 H NMR (300 MHz, DMSO). 2.5. Quantitation of PTX content of P123-PTX conjugates The conjugated PTX content of the P123-PTX conjugates was determined using an UV-Visible spectrophotometer (UV-2102PCS; UNICO [SHANG-HAI] Instruments Co., Ltd, Shanghai, China). P123PTX conjugates were dissolved in acetonitrile and the content of conjugated PTX was estimated from the UV–visible spectroscopy. 2.6. Preparation of PTX loaded FA-P123-PTX (FA-P123-PTX/PTX) micelles FA-P123-PTX/PTX micelles and P123-PTX/PTX micelles were prepared by a dialysis method with 6 mg of PTX and 100 mg of FAP123-PTX and P123-PTX, respectively. They were dissolved in 5 mL of tetrahydrofuran and dialyzed against deionized water at room temperature for 48 h using a 3500 Da molecular weight cutoff dialysis bag. P123-PTX micelles and FA-P123-PTX micelles were also prepared with 100 mg of P123-PTX and FA-P123-PTX conjugates, respectively. The three micelles were used as the experimental control and all prepared using the method the same as FA-P123PTX/PTX micelles. 2.7. Preparation of FITC labeled P123-PTX and FA-P123-PTX micelles Briefly, 100 mg of P123-PTX and FA-P123-PTX conjugates were pre-dissolved in 10 mL of dimethyl sulfoxide (DMSO), respectively. Then 1.36 mg of FITC was added into the reaction solution. After stirring for 48 h at room temperature, the reaction mixture was dialyzed with a 3500 Da molecular weight cut off dialysis bag against PBS (pH 7.4) to remove free FITC. Finally, FITC labeled P123-PTX and FA-P123-PTX micelles were obtained. 2.8. Determination of encapsulation efficiency and drug loading The encapsulated PTX content was detected by HPLC. The HPLC conditions were as follows: Venusil XBP C-18 (4.6 mm × 50 mm, pore size 5 m, Agela); measured wavelength, 227 nm; the mobile phase, acetonitrile–water (55:45, v/v); and flow rate, 1.0 mL/min. The calibration curve of peak area (A) against concentration of PTX (C) was A = 22507C + 31580 (r = 0.9992) under the concentration of PTX 5–100 g/mL; the limit of detection was 0.1 ng/mL. The encapsulation efficiency (EE) and drug loading (DL) were calculated from the following equations: DL% =
We + Wc × 100% Wtotal
EE% =
We × 100% Wd
We is the weight of encapsulated drug, Wc the weight of conjugated drug, Wtotal the weight of the feeding polymer and drug in the micelles, Wd the weight of the feeding drug. 2.9. Particle size, -potential and morphology measurement The morphology of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles were investigated using transmission electron
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microscopy (JEM-1200EX, Japan) and the method was shown as follows: a sample of the P123-PTX/PTX micelles and FA-P123-PTX/PTX dispersion (dispersed in PBS, pH 7.4) was dropped onto the surface of the copper grid; then, they were negatively stained with phosphotungstic acid solution (2%, w/v) for 30 s and air-dried at room temperature before observation. Particle size and -potential were measured by Zetasizer 3000 (Malvern Instruments Ltd., UK). 2.10. In vitro drug release study The P123-PTX micelles, P123-PTX/PTX micelles, FA-P123-PTX micelles, FA-P123-PTX/PTX micelles were dispersed in 6 mL of PBS at pH 7.4 and 5.0, and the dispersion was divided into three equal aliquots. Each 2 mL aliquot sample was then transferred into a dialysis bag (MWCO: 3500 Da), which were dialyzed against 20 mL of the corresponding PBS (pH 7.4 and 5.0) containing 1 M of sodium salicylate at 37 ± 0.5 ◦ C under oscillation at 100 rpm, respectively. At the predetermined time intervals, 1 mL of solution was removed, and fresh medium was added back to the reservoir to replace the remaining release medium. The concentration of PTX in the samples was then determined by the HPLC method described above. 2.11. In vitro cellular uptake studies Human lung adenocarcinoma cells (A549), human breast carcinoma cell lines (MCF-7) and murine malignant melanoma cell lines (B16) were selected to investigate the cellular uptake of FITC labeled P123-PTX and FA-P123-PTX micelles. The cells were seeded in a 12-well plate cells at 1.0 × 105 cells/well and incubated for 24 h. At a confluence level of 70–80%, FITC loaded P123-PTX micelles and FA-P123-PTX micelles (the concentration of PTX was 10 g/mL) were added, respectively. The cells were then incubated for 0.5, 2.0, 4.0 h at 37 ◦ C. After that, all cells were harvested for trypsinization and washed in cold PBS three times. The cell-associated fluorescence was quantitatively determined by FACSCalibur flow cytometry (BD Biosciences, USA) by counting 10,000 events. All experiments were performed in triplicate. 2.12. In vitro cytotoxicity studies A549, MCF-7 and B16 cells were seeded in a 96-well plate at a density of 4000 viable cells/well and incubated for 24 h to allow cell to adhere. Cells were exposed to a series of doses of Taxol® , blank P123 micelles, P123-PTX micelles, P123-PTX/PTX micelles, and FAP123-PTX/PTX micelles, respectively. The range of concentrations of PTX used was 0.002, 0.02, 0.2, 1 and 2 M. After 72 h of incubation at 37 ◦ C, 20 L of MTT (5 mg/mL) was added to each well. The plate was incubated for an additional 4 h and DMSO (200 L per well) was added to dissolve purple blue formazan crystals in the plate. The absorbance of each well was measured by a microplate reader (FL600TM ; BioTek Instruments, Winooski, VT) at a test wavelength of 570 and 630 nm. All experiments were repeated thrice. 2.13. Annexin V-FITC/PI double staining The apoptosis induced by PTX-loaded micelles was assessed by the Annexin V-FITC and PI kit. A549, MCF-7 and B16 cells were treated with 1 mM of Taxol® , blank P123 micelles, P123-PTX micelles, P123-PTX/PTX micelles, and FA-P123-PTX/PTX micelles, respectively. After incubation for 24 h, cells with different drug treatments were harvested and washed with 4 ◦ C pre-cold PBS. Then 200 L of calcium-containing binding buffer containing 5 L Annexin V-FITC was used to resuspend cells. The cells were incubated in 4 ◦ C for 15 min and 10 L of PI was added and incubated
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with cells for 5 min. Finally, the samples were measured by FACSCalibur flow cytometry (BD Biosciences, USA). 2.14. In vivo anti-tumor efficacy Mice implanted with B16 cell were used to qualify the efficacy of PTX-loaded micelles administrated by intravenous injection. The mice were subcutaneously injected at the right axillary space with 0.1 mL of cell suspension containing 105 B16 cells. After 8–10 days of implantation, the mice with tumor volume of ∼100 mm3 were selected and randomly divided into six groups (n = 8 mice/group) as follows: (A) physiological saline (NS) as a control group; (B) blank P123 micelles group; (C) Taxol® group (dosage of 20 mg/kg); (D) P123-PTX micelles group (dosage of 20 mg/kg); (E) P123-PTX/PTX micelles group (dosage of 20 mg/kg); (F) FA-P123-PTX/PTX micelles group (dosage of 20 mg/kg). Each group of mice was treated once a week by tail vein injection with above formulations. After administration, the tumor diameter was measured with calipers, and the body weight of the mice was also recorded every other day during the period of study. Three weeks later, the mice were sacrificed, and the tumors were excised and weighed. The tumor volume was calculated using the equation of (1/2)L × W2 , where L is the tumor dimension at the longest point and W is the tumor dimension at the widest point, respectively. 2.15. In vivo MTD studies The maximum tolerated dose (MTD) for P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was administered intravenously female Kunming mice. The mice were randomly assigned to groups (three mice per group) and were administered intravenously with a single dose of Taxol® (20, 30, 40, 50, 60 mg PTX/kg body weight), P123-PTX/PTX micelles (50, 100, 150, 200, 250, 300 mg PTX/kg body weight) or FA-P123-PTX/PTX (50, 100, 150, 200, 250, 300 mg PTX/kg body weight), and NS as a control, respectively. The body weight of the mice was also recorded every other day for 1 week following drug administration. The MTD was determined to be the dose that induced ∼10% loss in body weight with no lethality and no notable behavior differences were noted [15]. The animals showing weight loss exceeding 20% were sacrificed, as changes of this magnitude often indicate lethal toxicity [16]. 3. Results and discussion 3.1. Synthesis of P123-PTX conjugates The synthesis route of the P123-PTX conjugates was shown in Fig. 1. Succinic anhydride was chosen as the linker to covalently link PTX to P123 via ester bond which was a degradable bond under
physiological conditions. The mass spectrum of PTX and succinylPTX was shown in Fig. S1. The molecular ion peak of succinyl-PTX was 952.8 (m/z), which fitted the molecular weight of succinylPTX (953.99, C51 H55 NO17 ). This result indicated the successfully synthesis of succinyl-PTX. The 1 H NMR of succinyl-PTX and P123PTX conjugates in CDCl3 was shown in Fig. S2. The peaks of PTX were ı (ppm): 4.2–4.4, 4.9, 5.6–6.3, 7.0–8.0, 8.1 (Fig. S2b). The peaks at 3.0–4.0 ppm (O CH2 CH2 O) were assigned to the protons of P123 block (Fig. S2a). The characteristic PTX and P123 peaks, which both appeared in the 1 H NMR spectrum of P123-PTX (Fig. S2c), which verified the successful synthesis of P123-PTX. Furthermore, the 1 H NMR analysis showed that the molar ratio of PTX and P123 in the P123-PTX conjugate was about 1.2:1 (PTX loading rate: 14.7%, wt.%). And the PTX content was determined to be (14.00 ± 0.36)% (wt.%) by the UV–visible spectrophotometry. These two results were unanimously confirmed the high loading of PTX by chemical conjugation. 3.2. Synthesis of FA-P123-PTX conjugates FA is used as the tumor-targeting ligand in drug-delivery systems, which could lead nanoparticles accumulated into cancer cells by FR-mediated endocytosis [17]. Fig. 2 showed the synthesis route of the FA-P123-PTX conjugates and Fig. S3 showed the 1 H NMR of FA-P123-PTX conjugates in DMSO. Compared with the 1 H NMR of P123-PTX, the appearance of an additional signal at ı8.67 (s, 1H, C7 H) indicated the successful conjugation of FA. And the FA-modified rate of FA-P123-PTX conjugates was 27.6% by the UV–visible spectrophotometry. 3.3. Characterization of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles The morphology of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles was shown in Fig. 3. P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed spherical shapes with good dispersity. The characterization parameters of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were summarized in Table 1. High drug loading (around 18%) of P123-PTX/PTX or FAP123-PTX/PTX micelles which consisted of conjugated PTX and encapsulated PTX was observed. The particle size of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was 102.63 ± 7.36 nm and 147.57 ± 2.82 nm, respectively. It is reported that limiting the size of nanoparticles to less than 200 nm could promote extravasation from microvessels as well as interstitial transport in tumor issue due to EPR effect [18]. Therefore, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed great potential for tumor targeted drug delivery. The Zeta potential of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was (−11.13 ± 0.62) mV and (−9.31 ± 1.07) mV, respectively.
Fig. 1. Synthesis route of the P123-PTX conjugates.
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Fig. 2. Synthesis of the FA-P123-PTX conjugates.
Fig. 3. TEM images of (a) P123-PTX/PTX micelles and (b) FA-P123-PTX/PTX micelles; Particle size distribution of (c) P123-PTX/PTX micelles and (d) FA-P123-PTX/PTX micelles.
3.4. In vitro drug release The PTX release of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles were investigated in PBS (containing 1 M of sodium salicylate) with different pH 7.4 (physiological pH) and pH
5.0 (lysosomal pH). Because PTX was a water-insoluble drug, 1 M of sodium salicylate was added to PBS to increase its solubility to achieve sink conditions. The in vitro release behavior presented as the accumulative percentage release is shown in Fig. 4. The cumulative release percentage of PTX from P123-PTX micelles and FA-P123-PTX micelles at pH 7.4 in 48 h were 1.8% and 6.7%, respectively. The possible reason of the slow release rate was that the cleavage of ester bond between PTX and P123 at pH 7.4 was a slow procedure. The incremental release of PTX from FA-P123-PTX micelles may cause by minor changes in the structure of micelles after the introduction of FA in the micelles and resulted in a slight faster PTX release. In contrast, the cumulative release percentage of PTX from the P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles at pH 7.4 in 48 h were 16.3% and 26.8%, respectively. The incremental release of PTX indicated that encapsulated PTX can be released more easily from micelles by diffusion [19]. Because ester bonds were more easily cleaved in acidic pH 5.0 than neutral pH 7.4, the PTX release profile from FA-P123-PTX/PTX micelles was expected to exhibit pH-sensitive PTX release when delivered to lysosome. As showed in Fig. 4, the release of PTX from FA-P123-PTX/PTX micelles at pH 5.0 was significantly faster than at pH 7.4 (p < 0.05). The reason was that PTX was linked to P123 by ester bonds. Ester bonds were more easily cleaved in acid environment compared to neutral environment. This pH-sensitivity of ester bonds resulted to the fast release of PTX in acidic environments. Thus, it could be speculated that when the P123-PTX/PTX micelles or FA-P123-PTX/PTX micelles were in acidic environments of tumor tissue or cells, the encapsulated PTX would be released quickly to inhibit the tumor growth. Then, the conjugated PTX could release continuously to maintain stable drug concentration at the site of tumor. 3.5. In vitro cellular uptake studies To evaluate the targeting properties of FA, the cellular uptake of FA-P123-PTX micelles were performed on FR positive (FR+ ) cells (MCF-7 [20] and B16 [21]) and FR negative cells (FR− ) (A549 [22]). FITC-labeled P123-PTX micelles and FITC-labeled FA-P123PTX micelles were incubated with MCF-7, B16 and A549 cells for 0.5 h, 2.0 h, 4.0 h, respectively. The results were shown in Fig. 5.
Table 1 The characterization parameters of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles. Types of micelles
Drug loading (%)
Particle size (nm)
P123-PTX/PTX micelles FA-P123-PTX/PTX micelles
18.69 ± 0.52 18.08 ± 0.64
102.63 ± 7.36 147.57 ± 2.82
Zeta potential (mV) −11.13 ± 0.62 −9.31 ± 1.07
Polydispersity index 0.212 0.203
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Fig. 4. In vitro drug release profiles of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles upon incubation in PBS (pH 5.0 and 7.4).
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Fig. 5. Cellular uptake of P123-PTX micelles and FA-P123-PTX micelles for 0.5 h, 2.0 h, 4.0 h in MCF-7, B16 and A549 cells, respectively: (a) MCF-7 cells; (b) B16 cells; and (c) A549 cells.
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Table 2 IC50 of A549, MCF-7 and B16 incubated with different formulations (n = 3). Cell line
Taxol®
P123-PTX micelles
P123-PTX/PTX micelles
FA-P123-PTX/PTX micelles
A549 MCF-7 B16
0.309 ± 0.081 (7.78 ± 1.94) × 10−3 0.378 ± 0.075
0.713 ± 0.233* , # 0.133 ± 0.024* , # 0.794 ± 0.079* , #
0.204 ± 0.004* (21.3 ± 6.5) × 10−3 * 0.476 ± 0.087*
0.178 ± 0.026* (2.38 ± 1.45) × 10−3 * , # 0.282 ± 0.011* , #
* #
p < 0.05 versus the Taxol group. p < 0.05 versus the P123-PTX/PTX micelles group.
In FR+ cells, the uptake rate of FITC labeled FA-P123-PTX micelles was significantly higher than FITC labeled P123-PTX micelles at the same incubation time (p < 0.05). In MCF-7 cells, although the uptake rate closed to 100% after 4 h, the fluorescence intensity of cells incubated with FITC-labeled FA-P123-PTX micelles was significantly higher than FITC-labeled P123-PTX micelles (p < 0.05). Nevertheless, there were no differences in uptake ratio and fluorescence intensity detected in cells treated with FITC-labeled P123-PTX micelles and FA-P123-PTX micelles in A549 cells. The increased uptake ratio and the fluorescence intensity both suggested that the modification of FA could enhanced the cellular uptake of FA-P123-PTX micelles in FR+ cells through the FA and FR recognition. 3.6. In vitro cytotoxicity study The IC50 values of Taxol® , P123-PTX micelles, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were summarized in Table 2. Blank P123 micelles had no effect on the cell viability at equivalent excipient concentration in the micelles. The IC50 values of P123-PTX micelles in three experimental cell lines were significantly higher compared to Taxol® (p < 0.05), which might be caused by the slow drug release rate of P123-PTX micelles in vitro. Compare with P123-PTX micelles and Taxol® , the IC50 values of P123-PTX/PTX micelles were significantly lower in three experimental cell lines (p < 0.05). The possible reason was that the encapsulated PTX could fast release from P123-PTX/PTX micelles and increase the therapeutic effect. These results indicated that the cytotoxicity of the micelles prepared by chemical conjugation and encapsulation was better than the micelles only prepared by chemical conjugation. In MCF-7 and B16 cells (FR+ cells), FA-P123-PTX/PTX micelles exhibited higher cytotoxicity than P123-PTX/PTX micelles (p < 0.05). In contrast, compare with P123-PTX/PTX micelles, no therapeutic advantageous effect on cytotoxicity was induced by FA-P123-PTX/PTX micelles against the FR− A549 cell. These results implied that FA-P123-PTX/PTX micelles had greater sensitivity to FR+ cells compared to P123-PTX/PTX micelles and could effectively inhibit the proliferation of FR+ cells. 3.7. Detection of apoptosis by FCM By staining cells with the combination of Annexin V-FITC and PI, non-apoptotic cells (Annexin V-FITC negative/PI negative), early apoptotic cells (Annexin V-FITC positive/PI negative), late apoptotic cells (Annexin V-FITC positive/PI positive) were possible distinguished and quantitatively analyzed [23]. As shown in Fig. 6, the induced apoptosis of A549, MCF-7 and B16 cells by P123-PTX/PTX micelles were 23.24%, 35.33% and 61.48%, respectively, which was significantly higher than apoptosis induced by P123-PTX micelles which were 15.12%, 12.12% and 24.18% (p < 0.05). These results might cause by the faster drug release from the P123-PTX/PTX micelles compared to P123-PTX micelles.
There is no significant difference between the apoptosis induced by P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles against A549 cells (p > 0.05). However, the apoptosis induced by FA-P123PTX/PTX micelles against MCF-7 and B16 cells was significantly higher than P123-PTX/PTX micelles (p < 0.05). The apoptosis detection results suggested that FA-P123-PTX/PTX micelles were more effective in inducing apoptosis of FR+ MCF-7 and B16 cells than P123-PTX/PTX micelles. The results of both cytotoxicity and apoptosis studies indicated that the in vitro anti-tumor effect of P123-PTX/PTX micelles was better than P123-PTX micelles. And the in vitro anti-tumor effect of FA-P123-PTX/PTX micelles was superior to P123-PTX/PTX micelles in the FR+ MCF-7 and B16 cells. According to cellular uptake results and previous studies [24,25], it could be speculated that a possible mechanism that enhanced therapeutic efficacy of FA-P123-PTX/PTX micelles against FR+ tumor cells was by FA-mediated internalization and intracellular drug accumulation. 3.8. In vivo anti-tumor effect The anti-tumor effects of FA-P123-PTX/PTX micelles, P123PTX/PTX micelles, P123-PTX micelles, blank micelles and normal saline (NS) on B16 tumor-bearing mice were shown in Fig. 7a and b. Compared with NS group, no anti-tumor effect was observed in blank micelles group (p > 0.05). Taxol® , P123-PTX micelles, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed significant tumor growth suppression compared to blank micelles and NS. Although the in vitro cytotoxicity of P123-PTX micelles was not as good as Taxol® , the in vivo anti-tumor effect of P123PTX micelles was similar to Taxol® . The possible reason was that P123-PTX micelles could reach the tumor site by EPR effect and PTX could be released in the acidic environment of tumor interstitial space or lysosomal of cancer cells. More importantly, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were found to be more effective in the prohibition of the tumor growth than Taxol® (p < 0.05) and the mice treated with FA-P123-PTX/PTX micelles showed better anti-tumor effect compared to P123PTX/PTX micelles group (p < 0.05). It was speculated that the high anti-tumor activity of FA-P123-PTX/PTX micelles was achieved by the following mechanisms. First, FA-P123-PTX/PTX micelles could accumulate in tumor tissue by EPR effect. Then, FA-P123-PTX/PTX micelles could link to the tumor cells and be internalized inside the tumor cells via FR-mediated endocytosis [26]. After the FAP123-PTX/PTX micelles enter the lysosomal, the encapsulated PTX was released quickly and reach the therapeutic concentration of PTX. The conjugated PTX was sustained released by the cleavage of ester bonds of PTX-P123 to maintain the therapeutic concentration of PTX, which resulted in the increase of anti-tumor activity [27]. The body weight variations could be used to reflect the adverse effects of the different therapy regiments [27]. As shown Fig. 7c, the body weight variations of blank micelles group were similar to NS group, which indicated that the blank micelles were safe carrier to mice body. While the other three micelle groups and Taxol® group showed body weight loss compared with NS
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Fig. 6. Induction of apoptosis on MCF-7 cells, B16 cells and A549 cells: (a) control; (b) blank micelles; (c) Taxol® ; (d) P123-PTX micelles; (e) P123-PTX/PTX micelles; and (f) FA-P123-PTX/PTX micelles.
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Fig. 7. Anti-tumor effects of FA-P123-PTX/PTX micelles, P123-PTX/PTX micelles, P123-PTX micelles and blank micelles and NS on B16 tumor-bearing mice after intravenous administration. Data represent mean ± standard deviation (n = 8). (a) Tumor volume; (b) tumor weight; (c) body weight change. Notes: *p < 0.05 versus the Taxol group; **p < 0.01 versus the Taxol group; # p < 0.05 versus the P123-PTX/PTX micelles group; ## p < 0.01 versus the P123-PTX/PTX micelles group.
group. In contrast, the mice treated with Taxol® showed obvious body weight loss compared with the other three micelle groups (p < 0.01). These results suggested that the three micelles generated less toxicity and fewer side effects to mice than Taxol® after administered intravenously under the present experimental conditions. 3.9. In vivo MTD studies The maximum tolerated dose for a single i.v. administration of Taxol® , P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was shown in Table 3. Owing to ethanol and polyoxyethylene castor oil, the mice showed apathy, catatonia, prostration after
administration of Taxol® at a single dose of 30 mg/kg. Increasing the PTX dosage to 60 mg/kg resulted in the death of one mouse among the three treated mice and there was more than 10% body weight loss. For the mice treated with P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles, there was less than 10% weight loss and no noticeable changes in normal activity at a PTX dosage as high as 150 mg/kg and 200 mg/kg, respectively. The MTD of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were 150 mg/kg and 200 mg/kg, respectively, which were 3.0-fold and 4.0-fold higher than that of Taxol® (50 mg/kg). These results indicated that the FA-P123-PTX/PTX micelles could reduce the toxicity of the drug delivery system and improve the safety for clinical application.
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Table 3 Determination of MTD after i.v. injection of Taxol, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles. Formulations
Dose (mg/kg)
Lethality
Performance after injection
Weight loss
MTD
20 30 40 50 60
0/3 0/3 0/3 0/3 1/3
Normal Apathy Apathy Apathy Apathy, dead
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Polymeric complex micelles with double drug-loading strategies for folate-mediated paclitaxel delivery Min Li 1 , Yongjun Liu 1 , Lixia Feng, Fengxi Liu, Li Zhang, Na Zhang ∗ School of Pharmaceutical Science, Shandong University, Ji’nan 250012, China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Received in revised form 15 April 2015 Accepted 27 April 2015 Available online 5 May 2015 Keywords: Polymeric complex micelles Pluronic P123 Paclitaxel Double drug-loading
a b s t r a c t Drug loading is a key procedure in the preparation of drug-loaded nano-carriers. In this study, the paclitaxel (PTX)-loaded polymeric complex micelles (FA-P123-PTX/PTX micelles) with double drug-loading strategies were designed and prepared to improve the drug loading percentage of carriers and its antitumor efficiency. PTX was simultaneously conjugated to pluronic P123 (P123) polymer and encapsulated inside the P123 complex micelle. Folate (FA) was linked to the surface of micelles for the active target delivery of micelles to tumor cells. The FA-P123-PTX/PTX micelles showed spherical shaped with high drug loading of 18.08 ± 0.64%. The results of cellular uptake studies suggested that FA could promote the internalization of micelles into the FR positive cells. FA-P123-PTX/PTX micelles showed significant higher anti-tumor activity against FR positive tumor cells compared to Taxol® (p < 0.05). Moreover, the FA-P123-PTX/PTX micelles exhibited higher anti-tumor efficacy in B16 bearing mice with better safety property compared with Taxol® . These results suggested that FA-P123-PTX/PTX micelles with double drug-loading strategies showed great potential for targeted delivery of anti-cancer drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past decades, drug-loaded nanoparticles were widely studied to improve the poor biodistribution and pharmacokinetics properties of free drug molecules in cancer drug delivery. Drug encapsulation and chemical conjugation were the common methods for drug loading. For chemical conjugation method, polymer–drug conjugates have been widely investigated as novel drug delivery systems [1]. It is formed by conjugation of drugs to hydrophilic or amphiphilicity polymers through chemical bonds. Due to the amphiphilicity, the polymer–drug conjugates selfassemble into micelles or micelle-like nanoassemblies in aqueous media. Drug loading by chemical conjugation method have many advantages such as high drug loading capacity, simplified preparation process, accurate loading efficiency [2]. However, this method also suffers from the possibility of decrease drug activity by the linkage of drugs and materials, the difficulty of controlled drug release from the drug conjugates. Drug encapsulation is another common method for drug loading which could solve these problems of
∗ Corresponding author at: Department of Pharmaceutics, College of Pharmacy, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel.: +86 0531 88382015; fax: +86 0531 88382548. E-mail address: [email protected] (N. Zhang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.04.057 0927-7765/© 2015 Elsevier B.V. All rights reserved.
chemical conjugation. However, its drug loading capacity needs to be improved [3,4] and the preparation formulation/process needs to be carefully adjusted to reach the highest drug loading and encapsulation efficiency [5]. Base on these problems, the paclitaxel (PTX)-loaded pluronic P123 (P123) complex micelles (FA-P123PTX/PTX micelles) combined with drug encapsulation and chemical conjugation drug-loading strategies were designed and prepared. The double drug-loading strategies in one carrier could increase the drug loading efficiency, improve the release profile, and eventually increase the anti-tumor efficiency. Pluronic block copolymer (PEO-b-PPO-b-PEO), which is amphiphilic synthetic polymers containing hydrophilic poly(ethylene oxide) (PEO) blocks and hydrophobic poly(propylene oxide) (PPO) blocks, has been commonly used for solubilization of hydrophobic drugs [6]. The hydrophobic PPO segments can comprise a hydrophobic core as a microenvironment for the incorporation of hydrophobic drugs [7]. The hydrophilic PEO corona can prevent aggregation, protein adsorption, and recognition by the reticuloendothelial system (RES) [7]. Currently, a micellar formulation consisting of DOX and two Pluronic copolymers (Pluronic L61 and Pluronic F127) has been already in clinical trials [8]. Among different kinds of Pluronic copolymers, Pluronic P123 (PEO20 -b-PPO69 -b-PEO20 ) is a hydrophobic block copolymer with a relatively low critical micelle concentration (CMC, 4.4 × 10−6 mol/L) [9]. Besides, Pluronic P123 presented the advantages of cheap, non-toxic, non-immunogenic and biocompatibility
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Scheme 1. Schematic diagram illustrating for P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles bearing paclitaxel with chemical conjugation and encapsulation.
[10]. Thus, P123 was selected as the polymer for the linkage and encapsulation of PTX at the same time. It was expected that when the double loading micelles (P123-PTX/PTX micelles) were delivered to the tumor tissue, the encapsulated PTX could be released quickly to reach effective therapeutic drug concentrations. Then the conjugated PTX was released continuously to maintain the therapeutic concentration of PTX. Therefore, the therapeutic efficiency could be greatly increased. Tumor active target delivery is a promising approach to increase the accumulation of drugs at tumor area. Folate receptor (FR) was known to be vastly over-expressed on malignant cells [11]. Therefore, folate (FA) has been employed as a targeting moiety of various anti-cancer agents to reduce their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells via receptor-mediated endocytosis [12]. In addition, FA possesses some advantages such as low immunogenicity, ease of modification, and low cost. The small size of FA also allows for good tissue penetration and rapid clearance from receptor negative tissues [13]. Hence, FA-based targeting systems were widely used to selectively delivering therapeutic agents to tumors. In this study, FA-mediated double drug-loading delivery system (FA-P123-PTX/PTX micelles) was prepared (Scheme 1). The in vitro PTX release of FA-P123-PTX/PTX micelles was performed in different pH values to evaluate the pH sensitive of ester bond [14]. The cellular uptake was investigated to verify the active-targeting of FA-P123-PTX/PTX micelles. In vitro anti-tumor activity of PTX loaded micelles against human lung adenocarcinoma A549 cells, human breast adenocarcinoma MCF-7 cells and mouse malignant melanoma B16 cells was assessed by the MTT method and annexin V-FITC/PI double labeling. In vivo therapeutic effect and the maximum tolerated dose were investigated in Kunming mice.
2. Materials and methods 2.1. Materials Paclitaxel was provided by Chenxin Pharmaceutical Co Ltd. (China). Pluronic P123 (P123) was purchased from BASF China Co., Ltd. (Shanghai, China). FA was purchased from Sigma–Aldrich Shanghai Trading Co, Ltd (Shanghai, China). 4-Dimethylaminopyridine (DMAP), succinic anhydride, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), N-hydroxysulfosuccinimide (NHS) and MTT were all obtained from Sigma–Aldrich (China). Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech Co., Ltd (Shanghai, China). Fetal bovine serum (FBS) was obtained from Sijiqing Co., Ltd. Annexin V-FITC apoptosis kit was provided by Bestbio (Shanghai, China). All other chemicals
and reagents used were of analytical grade or higher and obtained commercially. 2.2. Cells and animals Human lung adenocarcinoma cells (A549), human breast carcinoma cell lines (MCF-7) and murine malignant melanoma cell lines (B16) were kindly provided by Institute of Immunopharmacology and Immunotherapy of Shandong University (Ji’nan, China) and cultured in DMEM medium supplemented with 10% FBS, streptomycin at 100 g/mL and penicillin at 100 U/mL. All cells were cultured in a 37 ◦ C incubator with 5% CO2 . Female Kunming mice (18–22 g) were supplied by Laboratory Animals Center of Shandong University (Ji’nan, China). All animal procedures were performed according to the guidelines of the Ethical Committee for Animal Experiments of Shandong University. 2.3. Synthesis of P123-PTX conjugates 2.3.1. Synthesis of succinyl-PTX PTX (300 mg), succinic anhydride (48 mg) and DMAP (5.9 mg) were dissolved in 10 mL of anhydrous dichloromethane and stirred for 24 h at room temperature. Then the dichloromethane was removed by rotary evaporation. After the residue was dissolved in 50 mL of ethyl acetate, 50 mL of dilute hydrochloric acid (0.1 M) was added and stirred for 15 min. The organic phase was then separated and dried with anhydrous magnesium overnight. The succinylPTX was purified by silica gel column chromatography eluted with petroleum ether–ethyl acetate mixture (1:2) and used directly in the next step. The structure of succinyl-PTX was confirmed by 1 H NMR (300 MHz, CDCl3 ) and mass spectrometry. 2.3.2. Synthesis of P123-PTX conjugates Succinyl-PTX (100 mg), DMAP (10.9 mg), EDC (34 mg) were dissolved in anhydrous dichloromethane and then cooled down to 0 ◦ C by ice bath. 500 mg of P123 was dissolved in 2 mL of anhydrous dichloromethane and added dropwise into the reaction solution and then stirred for 48 h at room temperature. The dichloromethane was removed by rotary evaporation. Then residue was dissolved in tetrahydrofuran and dialyzed with a 3500 Da molecular weight cutoff dialysis bag against 1000 mL deionized water for 72 h. The structure of P123-PTX conjugates was confirmed by 1 H NMR (300 MHz, CDCl3 ). 2.4. Synthesis of FA-P123-PTX conjugates 30 mg of folic acid was dissolved in 10 mL of anhydrous DMSO. NHS (32 mg), EDC (53 mg), and triethylamine (20 L) were
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successively added. 400 mg of P123-PTX conjugates was dissolved in 2 mL of anhydrous DMSO and added dropwise into the reaction solution. After the completion of the reaction (about 48 h at room temperature), the resultant solution was dialyzed against 50 mL saline for 24 h and against distilled water for 48 h using a dialysis membrane (MWCO: 3500 Da). The structure of FA-P123-PTX conjugates was also confirmed by 1 H NMR (300 MHz, DMSO). 2.5. Quantitation of PTX content of P123-PTX conjugates The conjugated PTX content of the P123-PTX conjugates was determined using an UV-Visible spectrophotometer (UV-2102PCS; UNICO [SHANG-HAI] Instruments Co., Ltd, Shanghai, China). P123PTX conjugates were dissolved in acetonitrile and the content of conjugated PTX was estimated from the UV–visible spectroscopy. 2.6. Preparation of PTX loaded FA-P123-PTX (FA-P123-PTX/PTX) micelles FA-P123-PTX/PTX micelles and P123-PTX/PTX micelles were prepared by a dialysis method with 6 mg of PTX and 100 mg of FAP123-PTX and P123-PTX, respectively. They were dissolved in 5 mL of tetrahydrofuran and dialyzed against deionized water at room temperature for 48 h using a 3500 Da molecular weight cutoff dialysis bag. P123-PTX micelles and FA-P123-PTX micelles were also prepared with 100 mg of P123-PTX and FA-P123-PTX conjugates, respectively. The three micelles were used as the experimental control and all prepared using the method the same as FA-P123PTX/PTX micelles. 2.7. Preparation of FITC labeled P123-PTX and FA-P123-PTX micelles Briefly, 100 mg of P123-PTX and FA-P123-PTX conjugates were pre-dissolved in 10 mL of dimethyl sulfoxide (DMSO), respectively. Then 1.36 mg of FITC was added into the reaction solution. After stirring for 48 h at room temperature, the reaction mixture was dialyzed with a 3500 Da molecular weight cut off dialysis bag against PBS (pH 7.4) to remove free FITC. Finally, FITC labeled P123-PTX and FA-P123-PTX micelles were obtained. 2.8. Determination of encapsulation efficiency and drug loading The encapsulated PTX content was detected by HPLC. The HPLC conditions were as follows: Venusil XBP C-18 (4.6 mm × 50 mm, pore size 5 m, Agela); measured wavelength, 227 nm; the mobile phase, acetonitrile–water (55:45, v/v); and flow rate, 1.0 mL/min. The calibration curve of peak area (A) against concentration of PTX (C) was A = 22507C + 31580 (r = 0.9992) under the concentration of PTX 5–100 g/mL; the limit of detection was 0.1 ng/mL. The encapsulation efficiency (EE) and drug loading (DL) were calculated from the following equations: DL% =
We + Wc × 100% Wtotal
EE% =
We × 100% Wd
We is the weight of encapsulated drug, Wc the weight of conjugated drug, Wtotal the weight of the feeding polymer and drug in the micelles, Wd the weight of the feeding drug. 2.9. Particle size, -potential and morphology measurement The morphology of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles were investigated using transmission electron
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microscopy (JEM-1200EX, Japan) and the method was shown as follows: a sample of the P123-PTX/PTX micelles and FA-P123-PTX/PTX dispersion (dispersed in PBS, pH 7.4) was dropped onto the surface of the copper grid; then, they were negatively stained with phosphotungstic acid solution (2%, w/v) for 30 s and air-dried at room temperature before observation. Particle size and -potential were measured by Zetasizer 3000 (Malvern Instruments Ltd., UK). 2.10. In vitro drug release study The P123-PTX micelles, P123-PTX/PTX micelles, FA-P123-PTX micelles, FA-P123-PTX/PTX micelles were dispersed in 6 mL of PBS at pH 7.4 and 5.0, and the dispersion was divided into three equal aliquots. Each 2 mL aliquot sample was then transferred into a dialysis bag (MWCO: 3500 Da), which were dialyzed against 20 mL of the corresponding PBS (pH 7.4 and 5.0) containing 1 M of sodium salicylate at 37 ± 0.5 ◦ C under oscillation at 100 rpm, respectively. At the predetermined time intervals, 1 mL of solution was removed, and fresh medium was added back to the reservoir to replace the remaining release medium. The concentration of PTX in the samples was then determined by the HPLC method described above. 2.11. In vitro cellular uptake studies Human lung adenocarcinoma cells (A549), human breast carcinoma cell lines (MCF-7) and murine malignant melanoma cell lines (B16) were selected to investigate the cellular uptake of FITC labeled P123-PTX and FA-P123-PTX micelles. The cells were seeded in a 12-well plate cells at 1.0 × 105 cells/well and incubated for 24 h. At a confluence level of 70–80%, FITC loaded P123-PTX micelles and FA-P123-PTX micelles (the concentration of PTX was 10 g/mL) were added, respectively. The cells were then incubated for 0.5, 2.0, 4.0 h at 37 ◦ C. After that, all cells were harvested for trypsinization and washed in cold PBS three times. The cell-associated fluorescence was quantitatively determined by FACSCalibur flow cytometry (BD Biosciences, USA) by counting 10,000 events. All experiments were performed in triplicate. 2.12. In vitro cytotoxicity studies A549, MCF-7 and B16 cells were seeded in a 96-well plate at a density of 4000 viable cells/well and incubated for 24 h to allow cell to adhere. Cells were exposed to a series of doses of Taxol® , blank P123 micelles, P123-PTX micelles, P123-PTX/PTX micelles, and FAP123-PTX/PTX micelles, respectively. The range of concentrations of PTX used was 0.002, 0.02, 0.2, 1 and 2 M. After 72 h of incubation at 37 ◦ C, 20 L of MTT (5 mg/mL) was added to each well. The plate was incubated for an additional 4 h and DMSO (200 L per well) was added to dissolve purple blue formazan crystals in the plate. The absorbance of each well was measured by a microplate reader (FL600TM ; BioTek Instruments, Winooski, VT) at a test wavelength of 570 and 630 nm. All experiments were repeated thrice. 2.13. Annexin V-FITC/PI double staining The apoptosis induced by PTX-loaded micelles was assessed by the Annexin V-FITC and PI kit. A549, MCF-7 and B16 cells were treated with 1 mM of Taxol® , blank P123 micelles, P123-PTX micelles, P123-PTX/PTX micelles, and FA-P123-PTX/PTX micelles, respectively. After incubation for 24 h, cells with different drug treatments were harvested and washed with 4 ◦ C pre-cold PBS. Then 200 L of calcium-containing binding buffer containing 5 L Annexin V-FITC was used to resuspend cells. The cells were incubated in 4 ◦ C for 15 min and 10 L of PI was added and incubated
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with cells for 5 min. Finally, the samples were measured by FACSCalibur flow cytometry (BD Biosciences, USA). 2.14. In vivo anti-tumor efficacy Mice implanted with B16 cell were used to qualify the efficacy of PTX-loaded micelles administrated by intravenous injection. The mice were subcutaneously injected at the right axillary space with 0.1 mL of cell suspension containing 105 B16 cells. After 8–10 days of implantation, the mice with tumor volume of ∼100 mm3 were selected and randomly divided into six groups (n = 8 mice/group) as follows: (A) physiological saline (NS) as a control group; (B) blank P123 micelles group; (C) Taxol® group (dosage of 20 mg/kg); (D) P123-PTX micelles group (dosage of 20 mg/kg); (E) P123-PTX/PTX micelles group (dosage of 20 mg/kg); (F) FA-P123-PTX/PTX micelles group (dosage of 20 mg/kg). Each group of mice was treated once a week by tail vein injection with above formulations. After administration, the tumor diameter was measured with calipers, and the body weight of the mice was also recorded every other day during the period of study. Three weeks later, the mice were sacrificed, and the tumors were excised and weighed. The tumor volume was calculated using the equation of (1/2)L × W2 , where L is the tumor dimension at the longest point and W is the tumor dimension at the widest point, respectively. 2.15. In vivo MTD studies The maximum tolerated dose (MTD) for P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was administered intravenously female Kunming mice. The mice were randomly assigned to groups (three mice per group) and were administered intravenously with a single dose of Taxol® (20, 30, 40, 50, 60 mg PTX/kg body weight), P123-PTX/PTX micelles (50, 100, 150, 200, 250, 300 mg PTX/kg body weight) or FA-P123-PTX/PTX (50, 100, 150, 200, 250, 300 mg PTX/kg body weight), and NS as a control, respectively. The body weight of the mice was also recorded every other day for 1 week following drug administration. The MTD was determined to be the dose that induced ∼10% loss in body weight with no lethality and no notable behavior differences were noted [15]. The animals showing weight loss exceeding 20% were sacrificed, as changes of this magnitude often indicate lethal toxicity [16]. 3. Results and discussion 3.1. Synthesis of P123-PTX conjugates The synthesis route of the P123-PTX conjugates was shown in Fig. 1. Succinic anhydride was chosen as the linker to covalently link PTX to P123 via ester bond which was a degradable bond under
physiological conditions. The mass spectrum of PTX and succinylPTX was shown in Fig. S1. The molecular ion peak of succinyl-PTX was 952.8 (m/z), which fitted the molecular weight of succinylPTX (953.99, C51 H55 NO17 ). This result indicated the successfully synthesis of succinyl-PTX. The 1 H NMR of succinyl-PTX and P123PTX conjugates in CDCl3 was shown in Fig. S2. The peaks of PTX were ı (ppm): 4.2–4.4, 4.9, 5.6–6.3, 7.0–8.0, 8.1 (Fig. S2b). The peaks at 3.0–4.0 ppm (O CH2 CH2 O) were assigned to the protons of P123 block (Fig. S2a). The characteristic PTX and P123 peaks, which both appeared in the 1 H NMR spectrum of P123-PTX (Fig. S2c), which verified the successful synthesis of P123-PTX. Furthermore, the 1 H NMR analysis showed that the molar ratio of PTX and P123 in the P123-PTX conjugate was about 1.2:1 (PTX loading rate: 14.7%, wt.%). And the PTX content was determined to be (14.00 ± 0.36)% (wt.%) by the UV–visible spectrophotometry. These two results were unanimously confirmed the high loading of PTX by chemical conjugation. 3.2. Synthesis of FA-P123-PTX conjugates FA is used as the tumor-targeting ligand in drug-delivery systems, which could lead nanoparticles accumulated into cancer cells by FR-mediated endocytosis [17]. Fig. 2 showed the synthesis route of the FA-P123-PTX conjugates and Fig. S3 showed the 1 H NMR of FA-P123-PTX conjugates in DMSO. Compared with the 1 H NMR of P123-PTX, the appearance of an additional signal at ı8.67 (s, 1H, C7 H) indicated the successful conjugation of FA. And the FA-modified rate of FA-P123-PTX conjugates was 27.6% by the UV–visible spectrophotometry. 3.3. Characterization of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles The morphology of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles was shown in Fig. 3. P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed spherical shapes with good dispersity. The characterization parameters of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were summarized in Table 1. High drug loading (around 18%) of P123-PTX/PTX or FAP123-PTX/PTX micelles which consisted of conjugated PTX and encapsulated PTX was observed. The particle size of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was 102.63 ± 7.36 nm and 147.57 ± 2.82 nm, respectively. It is reported that limiting the size of nanoparticles to less than 200 nm could promote extravasation from microvessels as well as interstitial transport in tumor issue due to EPR effect [18]. Therefore, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed great potential for tumor targeted drug delivery. The Zeta potential of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was (−11.13 ± 0.62) mV and (−9.31 ± 1.07) mV, respectively.
Fig. 1. Synthesis route of the P123-PTX conjugates.
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Fig. 2. Synthesis of the FA-P123-PTX conjugates.
Fig. 3. TEM images of (a) P123-PTX/PTX micelles and (b) FA-P123-PTX/PTX micelles; Particle size distribution of (c) P123-PTX/PTX micelles and (d) FA-P123-PTX/PTX micelles.
3.4. In vitro drug release The PTX release of P123-PTX/PTX micelles and FA-P123PTX/PTX micelles were investigated in PBS (containing 1 M of sodium salicylate) with different pH 7.4 (physiological pH) and pH
5.0 (lysosomal pH). Because PTX was a water-insoluble drug, 1 M of sodium salicylate was added to PBS to increase its solubility to achieve sink conditions. The in vitro release behavior presented as the accumulative percentage release is shown in Fig. 4. The cumulative release percentage of PTX from P123-PTX micelles and FA-P123-PTX micelles at pH 7.4 in 48 h were 1.8% and 6.7%, respectively. The possible reason of the slow release rate was that the cleavage of ester bond between PTX and P123 at pH 7.4 was a slow procedure. The incremental release of PTX from FA-P123-PTX micelles may cause by minor changes in the structure of micelles after the introduction of FA in the micelles and resulted in a slight faster PTX release. In contrast, the cumulative release percentage of PTX from the P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles at pH 7.4 in 48 h were 16.3% and 26.8%, respectively. The incremental release of PTX indicated that encapsulated PTX can be released more easily from micelles by diffusion [19]. Because ester bonds were more easily cleaved in acidic pH 5.0 than neutral pH 7.4, the PTX release profile from FA-P123-PTX/PTX micelles was expected to exhibit pH-sensitive PTX release when delivered to lysosome. As showed in Fig. 4, the release of PTX from FA-P123-PTX/PTX micelles at pH 5.0 was significantly faster than at pH 7.4 (p < 0.05). The reason was that PTX was linked to P123 by ester bonds. Ester bonds were more easily cleaved in acid environment compared to neutral environment. This pH-sensitivity of ester bonds resulted to the fast release of PTX in acidic environments. Thus, it could be speculated that when the P123-PTX/PTX micelles or FA-P123-PTX/PTX micelles were in acidic environments of tumor tissue or cells, the encapsulated PTX would be released quickly to inhibit the tumor growth. Then, the conjugated PTX could release continuously to maintain stable drug concentration at the site of tumor. 3.5. In vitro cellular uptake studies To evaluate the targeting properties of FA, the cellular uptake of FA-P123-PTX micelles were performed on FR positive (FR+ ) cells (MCF-7 [20] and B16 [21]) and FR negative cells (FR− ) (A549 [22]). FITC-labeled P123-PTX micelles and FITC-labeled FA-P123PTX micelles were incubated with MCF-7, B16 and A549 cells for 0.5 h, 2.0 h, 4.0 h, respectively. The results were shown in Fig. 5.
Table 1 The characterization parameters of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles. Types of micelles
Drug loading (%)
Particle size (nm)
P123-PTX/PTX micelles FA-P123-PTX/PTX micelles
18.69 ± 0.52 18.08 ± 0.64
102.63 ± 7.36 147.57 ± 2.82
Zeta potential (mV) −11.13 ± 0.62 −9.31 ± 1.07
Polydispersity index 0.212 0.203
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Fig. 4. In vitro drug release profiles of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles upon incubation in PBS (pH 5.0 and 7.4).
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Fig. 5. Cellular uptake of P123-PTX micelles and FA-P123-PTX micelles for 0.5 h, 2.0 h, 4.0 h in MCF-7, B16 and A549 cells, respectively: (a) MCF-7 cells; (b) B16 cells; and (c) A549 cells.
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Table 2 IC50 of A549, MCF-7 and B16 incubated with different formulations (n = 3). Cell line
Taxol®
P123-PTX micelles
P123-PTX/PTX micelles
FA-P123-PTX/PTX micelles
A549 MCF-7 B16
0.309 ± 0.081 (7.78 ± 1.94) × 10−3 0.378 ± 0.075
0.713 ± 0.233* , # 0.133 ± 0.024* , # 0.794 ± 0.079* , #
0.204 ± 0.004* (21.3 ± 6.5) × 10−3 * 0.476 ± 0.087*
0.178 ± 0.026* (2.38 ± 1.45) × 10−3 * , # 0.282 ± 0.011* , #
* #
p < 0.05 versus the Taxol group. p < 0.05 versus the P123-PTX/PTX micelles group.
In FR+ cells, the uptake rate of FITC labeled FA-P123-PTX micelles was significantly higher than FITC labeled P123-PTX micelles at the same incubation time (p < 0.05). In MCF-7 cells, although the uptake rate closed to 100% after 4 h, the fluorescence intensity of cells incubated with FITC-labeled FA-P123-PTX micelles was significantly higher than FITC-labeled P123-PTX micelles (p < 0.05). Nevertheless, there were no differences in uptake ratio and fluorescence intensity detected in cells treated with FITC-labeled P123-PTX micelles and FA-P123-PTX micelles in A549 cells. The increased uptake ratio and the fluorescence intensity both suggested that the modification of FA could enhanced the cellular uptake of FA-P123-PTX micelles in FR+ cells through the FA and FR recognition. 3.6. In vitro cytotoxicity study The IC50 values of Taxol® , P123-PTX micelles, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were summarized in Table 2. Blank P123 micelles had no effect on the cell viability at equivalent excipient concentration in the micelles. The IC50 values of P123-PTX micelles in three experimental cell lines were significantly higher compared to Taxol® (p < 0.05), which might be caused by the slow drug release rate of P123-PTX micelles in vitro. Compare with P123-PTX micelles and Taxol® , the IC50 values of P123-PTX/PTX micelles were significantly lower in three experimental cell lines (p < 0.05). The possible reason was that the encapsulated PTX could fast release from P123-PTX/PTX micelles and increase the therapeutic effect. These results indicated that the cytotoxicity of the micelles prepared by chemical conjugation and encapsulation was better than the micelles only prepared by chemical conjugation. In MCF-7 and B16 cells (FR+ cells), FA-P123-PTX/PTX micelles exhibited higher cytotoxicity than P123-PTX/PTX micelles (p < 0.05). In contrast, compare with P123-PTX/PTX micelles, no therapeutic advantageous effect on cytotoxicity was induced by FA-P123-PTX/PTX micelles against the FR− A549 cell. These results implied that FA-P123-PTX/PTX micelles had greater sensitivity to FR+ cells compared to P123-PTX/PTX micelles and could effectively inhibit the proliferation of FR+ cells. 3.7. Detection of apoptosis by FCM By staining cells with the combination of Annexin V-FITC and PI, non-apoptotic cells (Annexin V-FITC negative/PI negative), early apoptotic cells (Annexin V-FITC positive/PI negative), late apoptotic cells (Annexin V-FITC positive/PI positive) were possible distinguished and quantitatively analyzed [23]. As shown in Fig. 6, the induced apoptosis of A549, MCF-7 and B16 cells by P123-PTX/PTX micelles were 23.24%, 35.33% and 61.48%, respectively, which was significantly higher than apoptosis induced by P123-PTX micelles which were 15.12%, 12.12% and 24.18% (p < 0.05). These results might cause by the faster drug release from the P123-PTX/PTX micelles compared to P123-PTX micelles.
There is no significant difference between the apoptosis induced by P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles against A549 cells (p > 0.05). However, the apoptosis induced by FA-P123PTX/PTX micelles against MCF-7 and B16 cells was significantly higher than P123-PTX/PTX micelles (p < 0.05). The apoptosis detection results suggested that FA-P123-PTX/PTX micelles were more effective in inducing apoptosis of FR+ MCF-7 and B16 cells than P123-PTX/PTX micelles. The results of both cytotoxicity and apoptosis studies indicated that the in vitro anti-tumor effect of P123-PTX/PTX micelles was better than P123-PTX micelles. And the in vitro anti-tumor effect of FA-P123-PTX/PTX micelles was superior to P123-PTX/PTX micelles in the FR+ MCF-7 and B16 cells. According to cellular uptake results and previous studies [24,25], it could be speculated that a possible mechanism that enhanced therapeutic efficacy of FA-P123-PTX/PTX micelles against FR+ tumor cells was by FA-mediated internalization and intracellular drug accumulation. 3.8. In vivo anti-tumor effect The anti-tumor effects of FA-P123-PTX/PTX micelles, P123PTX/PTX micelles, P123-PTX micelles, blank micelles and normal saline (NS) on B16 tumor-bearing mice were shown in Fig. 7a and b. Compared with NS group, no anti-tumor effect was observed in blank micelles group (p > 0.05). Taxol® , P123-PTX micelles, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles showed significant tumor growth suppression compared to blank micelles and NS. Although the in vitro cytotoxicity of P123-PTX micelles was not as good as Taxol® , the in vivo anti-tumor effect of P123PTX micelles was similar to Taxol® . The possible reason was that P123-PTX micelles could reach the tumor site by EPR effect and PTX could be released in the acidic environment of tumor interstitial space or lysosomal of cancer cells. More importantly, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were found to be more effective in the prohibition of the tumor growth than Taxol® (p < 0.05) and the mice treated with FA-P123-PTX/PTX micelles showed better anti-tumor effect compared to P123PTX/PTX micelles group (p < 0.05). It was speculated that the high anti-tumor activity of FA-P123-PTX/PTX micelles was achieved by the following mechanisms. First, FA-P123-PTX/PTX micelles could accumulate in tumor tissue by EPR effect. Then, FA-P123-PTX/PTX micelles could link to the tumor cells and be internalized inside the tumor cells via FR-mediated endocytosis [26]. After the FAP123-PTX/PTX micelles enter the lysosomal, the encapsulated PTX was released quickly and reach the therapeutic concentration of PTX. The conjugated PTX was sustained released by the cleavage of ester bonds of PTX-P123 to maintain the therapeutic concentration of PTX, which resulted in the increase of anti-tumor activity [27]. The body weight variations could be used to reflect the adverse effects of the different therapy regiments [27]. As shown Fig. 7c, the body weight variations of blank micelles group were similar to NS group, which indicated that the blank micelles were safe carrier to mice body. While the other three micelle groups and Taxol® group showed body weight loss compared with NS
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Fig. 6. Induction of apoptosis on MCF-7 cells, B16 cells and A549 cells: (a) control; (b) blank micelles; (c) Taxol® ; (d) P123-PTX micelles; (e) P123-PTX/PTX micelles; and (f) FA-P123-PTX/PTX micelles.
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Fig. 7. Anti-tumor effects of FA-P123-PTX/PTX micelles, P123-PTX/PTX micelles, P123-PTX micelles and blank micelles and NS on B16 tumor-bearing mice after intravenous administration. Data represent mean ± standard deviation (n = 8). (a) Tumor volume; (b) tumor weight; (c) body weight change. Notes: *p < 0.05 versus the Taxol group; **p < 0.01 versus the Taxol group; # p < 0.05 versus the P123-PTX/PTX micelles group; ## p < 0.01 versus the P123-PTX/PTX micelles group.
group. In contrast, the mice treated with Taxol® showed obvious body weight loss compared with the other three micelle groups (p < 0.01). These results suggested that the three micelles generated less toxicity and fewer side effects to mice than Taxol® after administered intravenously under the present experimental conditions. 3.9. In vivo MTD studies The maximum tolerated dose for a single i.v. administration of Taxol® , P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles was shown in Table 3. Owing to ethanol and polyoxyethylene castor oil, the mice showed apathy, catatonia, prostration after
administration of Taxol® at a single dose of 30 mg/kg. Increasing the PTX dosage to 60 mg/kg resulted in the death of one mouse among the three treated mice and there was more than 10% body weight loss. For the mice treated with P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles, there was less than 10% weight loss and no noticeable changes in normal activity at a PTX dosage as high as 150 mg/kg and 200 mg/kg, respectively. The MTD of P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles were 150 mg/kg and 200 mg/kg, respectively, which were 3.0-fold and 4.0-fold higher than that of Taxol® (50 mg/kg). These results indicated that the FA-P123-PTX/PTX micelles could reduce the toxicity of the drug delivery system and improve the safety for clinical application.
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Table 3 Determination of MTD after i.v. injection of Taxol, P123-PTX/PTX micelles and FA-P123-PTX/PTX micelles. Formulations
Dose (mg/kg)
Lethality
Performance after injection
Weight loss
MTD
20 30 40 50 60
0/3 0/3 0/3 0/3 1/3
Normal Apathy Apathy Apathy Apathy, dead
