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Poly(ε-caprolactone)/Poly(ethylene glycol)/Poly(ε-caprolactone) Nanoparticles: Preparation, Characterization, and Application in Doxorubicin Delivery MaLing Gou,† XiuLing Zheng,† Ke Men, Juan Zhang, Lan Zheng, XiuHong Wang, Feng Luo, YinLan Zhao, Xia Zhao, YuQuan Wei, and ZhiYong Qian* State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan UniVersity, Chengdu, 610041, P. R. China ReceiVed: June 19, 2009; ReVised Manuscript ReceiVed: August 10, 2009
Biodegradable poly(ε-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer nanoparticles showed potential application in drug delivery systems. In this article, monodisperse poly(ε-caprolactone)/poly(ethylene glycol)/ poly(ε-caprolactone) (PCL/PEG/PCL, PCEC) nanoparticles, ∼40 nm, were prepared by solvent extraction method using acetone as the organic solvent. These PCL/PEG/PCL nanoparticles did not induce hemolysis in vitro and did not show toxicity in vitro or in vivo. The prepared PCL/PEG/PCL nanoparticles were employed to load doxorubicin by a pH-induced self-assembly method. In vitro release study indicated that doxorubicin release from nanoparticles at pH 5.5 was faster than that at pH 7.0. The encapsulation of doxorubicin in PCL/PEG/PCL nanoparticles enhanced the cytotoxicity of doxorubicin on a C-26 cell line in vitro. Meanwhile, compared with free doxorubicin, doxorubicin in nanoparticles could more efficiently treat mice bearing subcutaneous C-26 tumors. The doxorubicin-loaded PCL/PEG/PCL nanoparticles might be a novel doxorubicin formulation for cancer therapy. 1. Introduction Cancer is one of the major causes of mortality, and the worldwide incidence of cancer continues to increase. The discovery of cytotoxic agents was revolutionary for cancer treatment in the last century, improving survival rates and the quality of life of patients with different types of cancers. However, the development of agents that combine efficacy, safety, and convenience remains a great challenge due to the narrow therapeutic index of some drugs, the fact that they may damage not only cancer cells but also healthy and normal tissue, and the occurrence of resistance. Previous development in nanotechnology provides researchers with new tools for cancer therapy.1-4 Biodegradable polymeric nanoparticles are highlighted as an anticancer drug delivery system to improve the anticancer effect and safety of the cargo.5-7 Many previous reports have indicated that nanoparticles could passively target agents that are cytotoxic to tumors by the enhanced permeability and retention (EPR) effect8,9 could release drugs in an extended period in vivo to improve the pharmacokinetics of anticancer drugs,10 could quickly release drugs to tumors rather than to normal tissue,11 could actively target tumor cells,12 and could overcome multidrug resistance.13 In past decades, drug-loaded nanoparticles were paid extensive attention. Some innovative drugs based on nanovectors have been the subject of clinical research.1,14 It might be attractive to develop innovative anticancer drugs based on anticancer agents-loaded polymeric nanoparticles.15,16 Poly(ε-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymers are biodegradable, biocompatible, and amphiphilic, which makes PCL/PEG nanoparticles good candidates for an advanced drug delivery system.17-19 The PCL/PEG/PCL is one kind of triblock PCL/PEG copolymers that can be easily * To whom correspondence should be corresponded. Phone: +86-2885164063. Fax: +86-28-85164060. E-mail:
[email protected]. † These authors contributed equally to this work.
Figure 1. Synthesis scheme of PCL/PEG/PCL copolymer (a) and the molecular structure of doxorubicin (b).
synthesized by ring-opening polymerization of ε-caprolactone initiated by PEG as shown in Figure 1a.20 In the past decades, PCL/PEG/PCL nanoparticles have been studied as drug delivery systems.21,22 Previously, we also successfully prepared PCL/ PEG/PCL nanoparticles (>100 nm) to deliver hydrophobic honokiol,23,24 pDNA,25 and bFGF protein antigen.26,27 In this paper, we report the preparation of PCL/PEG/PCL nanoparticles by a solvent extraction method to load doxorubicin, whose molecular structure was shown in Figure 1b. Doxorubicin is a potent anticancer agent, but it can induce severe toxicity in normal tissue, so many advanced drug delivery systems have been developed for doxorubicin to improve the anticancer effect and safety of doxorubicin by others groups.28,29 The C-26 colon carcinoma cell line is relatively sensitive to free doxorubicin in cell culture but not in vivo, a finding that has been attributed to the inability of the drug to attain sufficient intratumor concentrations.30 In this paper, the prepared PCL/PEG/PCL nanoparticles were safe as an intravenous drug delivery system. The encapsulation of doxorubicin in PCL/PEG/PCL nanoparticles efficiently improved the anticancer activity of doxorubicin on the C-26 cell line in vitro and in vivo. The doxorubicin-loaded PCL/ PEG/PCL nanoparticle might be a novel anticancer agent. 2. Experimental Methods 2.1. Materials. Materials used included poly(ethylene glycol) (PEG, Mn ) 4000, Aldrich, St. Louis, MO), ε-caprolactone (ε-
10.1021/jp905781g CCC: $40.75 2009 American Chemical Society Published on Web 09/09/2009
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CL, Alfa Aesar, Ward Hill, MA), stannous octoate (Sn(Oct)2, Sigma, St. Louis, MO), Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT, Sigma, St. Louis, MO), acetonitrile (AN, Fisher Scientific, Loughborough, U.K.), dimethyl sulfoxide (DMSO, KeLong Chemicals, Chengdu, China), dichloromethane (DCM, KeLong Chemicals, Chengdu, China), petroleum ether (KeLong Chemicals, Chengdu, China), and doxorubicin chloride (doxorubicin, Dox, Zhejiang Hisun Pharmaceutical Company, Zhejiang, China). Sprague-Dawley (SD) rats, at weight of 200 ( 20 g, were used for the acute toxicity study. The animals were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, China). The animals were housed at a temperature of 20-22 °C, relative humidity of 50-60%, and 12 h light-dark cycles. Free access to food and water was allowed. All the animals were in quarantine for a week before treatment. All animal care and experimental procedures were conducted according to Institutional Animal Care and Use guidelines. 2.2. Preparation of Empty or Doxorubicin-Loaded PCL/ PEG/PCL Nanoparticles. The PCL/PEG/PCL triblock copolymer with the designed molecular weight of 20 000 was synthesized by ring-opening polymerization of ε-caprolactone initiated by PEG4000.20,24 The number molecular weight of the synthesized PCL/PEG/PCL was ∼17 500 calculated from 1H NMR spectrum (data not shown here). Blank PCL/PEG/PCL nanoparticles were prepared by a solvent extraction method. Briefly, PCL/PEG/PCL polymer in acetone solution (80 mg/mL) was added into water under moderate stirring. With the diffusion of acetone into water, PCL/ PEG/PCL self-assembled into nanoparticles. Then, the acetone was evaporated in a fuming cupboard. Finally, the obtained nanoparticles were dialyzed against distilled water for 72 h, and the concentration of the PCL/PEG/PCL nanoparticles was adjusted to 80 mg/mL. Doxorubicin-loaded PCL/PEG/PCL nanoparticles were prepared by a pH-induced self-assembly method. Briefly, 0.1 mL of PBS (10×, pH ) 7.4) was added into 0.7 mL of nanoparticle slurry (30 mg/mL), then 0.2 mL of doxorubicin aqueous solution (5 mg/mL) was dropped into the above PCL/PEG/PCL nanoparticles under moderate stirring. Because of the low solubility of doxorubicin in PBS at a pH 7.4, doxorubicin self-assembled into the hydrophobic core of PCL/PEG/PCL nanoparticles. Twenty minutes later, doxorubicin-loaded PCL/PEG/PCL (Dox-PCEC) nanoparticles were obtained. 2.3. Evaluation of the Drug Loading and Encapsulation Efficiency. The drug loading and encapsulation efficiency of Dox-PCEC were determined by a subtraction method. Briefly, 0.2 mL of Dox-PCEC was centrifuged through a filter with a molecular mass cutoff of 10 KDa. Although the free doxorubicin could pass through the filter, the doxorubicin-encapsulated PCEC nanoparticles could not pass through the filter. The unincorporated doxorubicin was quantified by determining the absorbance at 485 nm using a spectrophotometer (Spectramax M5, Molecular Devices Corp., Sunnyvale, CA). The drug loading (DL) and encapsulation efficiency (EE) was calculated according to the following equations: DL ) concentration of (total drug - free drug) × 100% concentration of (polymer + total drug - free drug)
(1)
EE )
concentration of (total drug - free drug) × 100% concentration of total drug (2)
2.4. Morphology Study. The morphology of empty or doxorubicin-loaded PCL/PEG/PCL nanoparticles was observed under a transmission electron microscope (TEM) (H-6009IV, Hitachi, Japan). Nanoparticles were placed on a copper grid covered with nitrocellulose. The samples were negatively stained with phosphotungstic acid and dried at room temperature. 2.5. Cytotoxicity of PCL/PEG/PCL Nanoparticles. The cytotoxicity of the PCL/PEG/PCL nanoparticles was evaluated by a cell viability assay on the HEK293 cell line. Briefly, HEK293 cells were plated at a density of 5 × 103 cells per well in 100 µL of DMEM medium in 96-well plates and grown for 24 h. The cells were then exposed to a series of PCL/PEG/ PCL nanoparticles at different concentrations for 48 h, and the viability of cells was measured using the MTT method. Results were the mean of 6 test runs. 2.6. Hemolytic Test in Vitro. The hemolytic study was performed on empty PCL/PEG/PCL nanoparticles in vitro. Briefly, 0.5 mL of PCL/PEG/PCL micelles (80 mg/mL) in normal saline was diluted to 2.5 mL by normal saline and added into 2.5 mL of rabbit erythrocyte suspension (2%) in normal saline at 37 °C. Distilled water and normal saline were employed as the positive and negative controls, respectively. Three hours later, the erythrocyte suspension was centrifuged, and the color of the supernatant was compared with the controls. If the supernatant solution was absolutely achromatic, it was implied that the prepared PCL/PEG/PCL nanoparticles would not induce hemolysis. In contrast, the prepared PCL/PEG/PCL nanoparticles would induce hemolysis if the supernatant solution was red. 2.7. Acute Toxicity of PCL/PEG/PCL Nanoparticles in Vivo. The acute toxicity of PCL/PEG/PCL nanoparticles was evaluated on SD rats. Briefly, PCL/PEG/PCL nanoparticles (80 mg/mL) in normal saline were intravenously injected into SD rats at a dosage of 1.6 g/kg, and normal saline was used as the control (12 rats per group, six female and six male). The following week, the adverse effects were observed. On day 7, the rats were sacrificed. The blood was withdrawn from the caudal vena cava. The heart, liver, spleen, lung, and kidney were subjected to histological evaluation. 2.8. Release Study in Vitro. To determine the release kinetics of doxorubicin from nanoparticles, the prepared 0.5 mL of doxorubicin-loaded PCL/PEG/PCL nanoparticles was placed in a dialysis bag (molecular mass cutoff, 8-14 kDa). The dialysis bags were incubated in 30 mL of phosphate buffer (pH ) 7.0 or 5.5) containing Tween80 (0.5%) at 37 °C with gentle shaking, and the incubation medium was replaced with fresh incubation medium at predetermined time points. The released drug was quantified by determining the absorbance at 485 nm using a spectrophotometer (M5, Molecular Corporation), and the cumulative release profile with time was demonstrated. This study was repeated three times, and the result was expressed as mean value ( sd. 2.9. Cytotoxicity of Doxorubicin in Nanoparticles on the C-26 Cell Line in Vitro. The cytotoxicity of doxorubicin in PCL/PEG/PCL nanoparticles, free doxorubicin, and empty PCL/ PEG/PCL nanoparticles on the C-26 cell line was evaluated by a cell viability assay. Briefly, C-26 cells were plated at a density of 5 × 103 cells per well in 100 µL of RPMI medium 1640 containing 10% FBS in 96-well plates and grown for 24 h. The cells were then exposed to a series of empty PCL/PEG/PCL nanoparticles, free doxorubicin, or doxorubicin in PCL/PEG/
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Figure 2. Preparation scheme of doxorubicin-loaded PCL/PEG/PCL (PCEC) nanoparticles.
Figure 3. Characterization of empty PCL/PEG/PCL nanoparticles. (a) Morphology of PCL/PEG/PCL nanoparticles determined by TEM (the bar is 100 nm); (b) cytotoxicity of PCL/PEG/PCL nanoparticles on HEK293 cells; and (c) hemolytic study of PCL/PEG/PCL nanoparticles in vitro (1, PCL/PEG/PCL nanoparticles; 2, normal saline as negative control; and 3, distilled water as a positive control).
PCL nanoparticles at different concentrations for 48 h. The viability of the cells was measured using the MTT method. Results are the mean of six test runs. 2.10. The Anticancer Effect of Doxorubicin in Nanoparticles on Subcutaneous C-26 Tumor. Female BALB/c mice (6-8 weeks old) were inoculated subcutaneously with 5 × 105 C-26 cells on day 0. On day 6, the tumors were palpable. Then, the mice were randomized into four groups (6 mice per group) and numbered. The four groups were injected intravenously with three dosages of normal saline (control), empty PCL/PEG/PCL nanoparticles (100 mg/kg) (PCEC), 5 mg/kg of free doxorubicin (Dox), and 5 mg/kg of doxorubicin in PCL/PEG/PCL nanoparticles (Dox-PCEC) on days 6, 9, and 12. The tumor size was measured externally daily using calipers during the experimental period. The tumor volume was approximated by using the equation vol ) (a × b2)/2, where vol is volume, a is the length of the major axis, and b is the length of the minor axis. The results were further analyzed statistically by the OneWay ANOVA test using SPSS software. 3. Results In this article, the PCL/PEG/PCL nanoparticles were prepared and employed to load doxorubicin as shown in Figure 2. First, empty PCL/PEG/PCL nanoparticles were prepared by a solvent extraction method. The acetone is a water-soluble organic solvent, whereas the PCL/PEG/PCL is water-insoluble amphiphilic copolymer. When PCL/PEG/PCL organic solution in acetone was added into water under stirring, solvent extraction occurred, and the PCL/PEG/PCL copolymer self-assembled into core-corona structured micelle-like nanoparticles due to the amphiphilic property of the PCL/PEG/PCL. Then, doxorubicin was loaded into the PCL/PEG/PCL nanoparticles by a pHinduced self-assembly method. It is known that the solubility of doxorubicin in water is pH-dependent. Doxorubicin could be well-dissolved in distilled water (pH ) 5-6), but the solubility of doxorubicin in PBS at pH 7.4 is low. Meanwhile, amphiphilic polymer micelle always has a loose structure in
water. After doxorubicin aqueous solution was dropped into the PCL/PEG/PCL nanoparticles in PBS (pH ) 7.4) under stirring, doxorubicin became hydrophobic and would self-assemble into the hydrophobic core of PCL/PEG/PCL nanoparticles. 3.1. Characterization of Empty PCL/PEG/PCL Nanoparticles. The prepared empty PCL/PEG/PCL nanoparticles were characterized as shown in Figure 3. First, the particle size and morphology of the PCL/PEG/PCL was determined by TEM image. As presented in Figure 3a, it could be observed that the prepared PCL/PEG/PCL nanoparticles are spherical, monodisperse, and have a mean particle size of ∼40 nm. Then, the cytotoxicity of PCL/PEG/PCL nanoparticles on the HEK293 cell line was evaluated by the MTT method in vitro, as shown in Figure 3b. It was suggested that the PCL/PEG/PCL nanoparticles (e1 mg/mL) did not suppress the proliferation of HEK293 cells in vitro. Meanwhile, the hemolytic study was performed on the prepared PCL/PEG/PCL nanoparticles, and the result indicated that PCL/PEG/PCL nanoparticles (80 mg/ mL) did not induce hemolysis in vitro, as shown in Figure 3c. The acute toxic study of PCL/PEG/PCL nanoparticles indicated that intravenous administration of PCL/PEG/PCL nanoparticles at a dosage of 1.6 g/kg did not cause mortality in the rat model. Meanwhile, there were no signs of abnormal behavioral reactions. The condition of the oral cavity, teeth, eyes, and eye slits were normal. There was no difference in food consumption between the groups throughout the study. In Figure 4, it could be found that intravenous administration of PCL/PEG/PCL nanoparticles at a dosage of 1.6 g/kg did not induce any histological damage to the heart, liver, spleen, lung, or kidney in the rat model. 3.2. Characterization of Doxorubicin-Loaded PCL/PEG/ PCL Nanoparticles. Doxorubicin was loaded into PCL/PEG/ PCL nanoparticles by a pH-induced self-assembly method. When the in-feed mass ratio of doxorubicin /PCL/PEG/PCL was 5/100, the nanoparticles had an encapsulation efficiency of 91.7% and drug loading of 4.2%. The TEM image of doxorubicin-loaded PCL/PEG/PCL nanoparticles is shown in Figure
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Figure 5. Characterization of doxorubicin-loaded PCL/PEG/PCL nanoparticles (Dox-PCEC). (a) Morphology of Dox-PCEC determined by TEM and (b) release profile of doxorubicin from Dox-PCEC at pH 5.0 or pH 7.0.
Figure 6. Anticancer effect of doxorubicin-loaded PCL/PEG/PCL nanoparticles (Dox-PCEC) on a C-26 colon carcinoma cell line ex and in vivo. (a) Cytotoxicity of doxorubicin (Dox) and Dox-PCEC on C-26 cells in vitro; and (b) the antitumor effect of Dox and Dox-PCEC on subcutaneous C-26 tumor. Normal saline was used as the negative control.
Figure 4. Histological staining of the organs of rats treated with normal saline or PCL/PEG/PCL nanoparticles at a dosage of 1.6 g/kg. Panels a, b, c, d, and e were the heart, liver, spleen, lung, and kidney in the control group, respectively. Panels f, g, h, i, and j were heart, liver, spleen, lung and kidney in the treated group (PCL/PEG/PCL, 1.6 g/kg), respectively.
5a. It could be found that the doxorubicin-loaded PCL/PEG/ PCL nanoparticles were spherical and had a mean particle size
of ∼45 nm. Previously, Chan et al. found that nanoparticlemediated cellular response was size-dependent, and mammalian cells could most efficiently uptake nanoparticles with a size between 40 and 50 nm.31,32 The obtained doxorubicin-loaded PCL/PEG/PCL nanoparticles with a particle size of ∼45 nm might be expected when we design nanoparticles as a drug delivery system. The release profile of doxorubicin from the PCL/PEG/PCL nanoparticles was studied using a dialysis method. As shown in Figure 5b, it was implied that doxorubicin could be released from the nanoparticles over an extended period. Meanwhile, it could be observed that doxorubicin release from Dox-PCEC was faster at pH 5.5 than at pH 7.0. This pH-dependent releasing behavior might be due to the reprotonation of the amino group of doxorubicin and faster degradation of PCL/PEG/PCL nanoparticles at lower pH.33 This pHdependent releasing behavior was very interesting in achieving the tumor-targeted doxorubicin delivery with nanoparticles. It was expected that doxorubicin could be very slowly released in the plasma under normal physiological conditions (pH ) 7.4), but quickly released at the solid tumor site (pH ) 5.5). In addition, nanoparticles are usually internalized inside the cells by endocytosis. So, a further accelerated release inside the endosome/lysosome (pH 5-5.5) of tumor cells might occur due to the low pH values. 3.3. Anticancer Effect in Vitro and Vivo. We compared the antitumor effect of Dox-PCEC with that of free doxorubicin in the C-26 cell line in vitro and in vivo. To compare the cytotoxicity of the encapsulated and free doxorubicin, C-26 cells were exposed to a series of empty PCL/PEG/PCL nanoparticles and equivalent concentrations of free doxorubicin or Dox-PCEC for 48 h, and the percentage of viable cells was quantified using the MTT method. The concentration of doxorubicin in the nanoparticles that caused 50% killing was lower than that of
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Figure 7. Schematic illustration of the proposed mechanism of the antitumor effect of doxorubicin-loaded PCL/PEG/PCL nanoparticles in the subcutaneous C-26 tumor model. In the left panel of the figure, the EPR effect is demonstrated. In the right panel of the figure, it indicates that in normal tissues, the drug release is slow, whereas in tumor tissues, the drug release is fast.
free doxorubicin (0.382 µg versus 0.767 µg), as shown in Figure 6a, whereas empty PCL/PEG/PCL nanoparticles (e1 mg/mL) did not suppress C-26 cell proliferation (data not shown here). This result implied that the encapsulation of doxorubicin in a PCL/PEG/PCL nanoparticle enhances the cytotoxicity of Dox. We also compared the antitumor effect of Dox-PCEC with that of free doxorubicin in the subcutaneous C-26 model, as shown in Figure 6b. When empty PCEC nanoparticles did not show an anticancer effect on the subcutaneous C-26 tumor, both free doxorubicin and PCEC nanoparticles encapsulating doxorubicin could suppress subcutaneous a C-26 tumor in vivo (P < 0.05). Meanwhile, compared with free doxorubicin, doxorubicin in PCL/PEG/PCL nanoparticles could more efficiently treat subcutaneous C-26 tumor (P < 0.05). In summary, biodegradable and biocompatible PCL/PEG/PCL nanoparticles were successfully prepared and employed to load doxorubicin. Encapsulation of doxorubicin in PCL/PEG/PCL nanoparticles enhanced the anticancer effect of doxorubicin on the C-26 cell line in vitro and in vivo. 4. Discussion To improve the anticancer effect and safety of cytotoxic agent, targeted drug delivery systems were employed to deliver the cargo to tumors. Biodegradable nanoparticles were highlighted as the intravenously injected targeted drug delivery system. Taking advantage of the inherent size of nanoparticles, the unique properties of tumor vasculature and the tumor microenvironment, passive targeting anticancer to tumor tissues could be achieved by nanoparticles.14 Angiogenesis is crucial to tumor progression. Angiogenic blood vessels in tumor tissues, rather than those in normal tissues, have gaps as large as 600 to 800 nm between adjacent endothelial cells. The defective vascular architecture coupled with poor lymphatic drainage induces the EPR effect, which allows nanoparticles to extravasate through these gaps into extravascular spaces and accumulate inside tumor tissues.8,9 Meanwhile, compared with normal tissues, interstitial fluid in tumors is known to have a lower pH value.34 In addition, nanoparticles are usually internalized inside the cells by endocytosis and end up in the acidic environment of the endosome/lysosome (pH 5-5.5) of tumor cells.35 So the passive tumor targeting also could be achieved by nanoparticles that release the drug in a pH-dependent manner (drug could be slowly released in the plasma (pH ) 7.4), but quickly released in the acidic solid tumor site). When poly(lactic acid) (PLA)/PEG and poly(lactide-coglycolide) (PLGA) nanoparticles were widely studied as an anticancer drug delivery system, PCL/PEG nanoparticles also were paid extensive attention.12,36,37 The commonly used PCL/ PEG copolymers included PCL/PEG/PCL, PEG/PCL/PEG and MPEG/PCL. In this article, PCL/PEG/PCL nanoparticles were
successfully prepared and employed to load doxorubicin. Previously, PCL/PEG/PCL nanoparticles were prepared by a solvent extraction method using DMSO as the organic solvent, and PCL/PEG/PCL nanoparticles with a particle size of ∼80 nm were obtained.24 When DMSO was replaced by acetone in this work, the particle size of the PCL/PEG/PCL decreased from 80 nm 40 nm. It was indicated that when PCL/PEG/PCL nanoparticles were prepared by the solvent extraction method, using acetone as the organic solvent would result in smaller PCL/PEG/PCL nanoparticles than that using DMSO as organic solvent. When we design a nanoparticle as a targeted drug delivery system, extended-time circulation of nanoparticles in vivo should be first considered.19 PEGylated nonionic polymeric nanoparticles with small size (
