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Enhanced Cytotoxicity of Core Modified Chitosan Based Polymeric Micelles for Doxorubicin Delivery
Enhanced Cytotoxicity of Core Modified Chitosan Based Polymeric Micelles for Doxorubicin Delivery YI-QING YE,1 FENG-YING CHEN,1 QIAO-AI WU,1 FU-QIANG HU,2 YONG-ZHONG DU,2 HONG YUAN,2 HE-YONG YU1 1
Women Hospital, School of Medicine, Zhejiang University, 2 Xueshi Road, Hangzhou 310006, P.R. China
2
College of Pharmaceutical Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, P.R. China
Received 22 August 2007; revised 15 April 2008; accepted 4 May 2008 Published online 18 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21464
ABSTRACT: In this study, the cytotoxicity of doxorubicin (DOX) loaded stearic acid grafted chitosan oligosaccharide (CSO-SA) micelles and its core modified drug delivery systems were investigated in vitro. The in vitro drug release experiments using cellular culture medium, Roswell Park Memorial Institute 1640 (RPMI-1640) medium as a dissolution medium confirmed that the DOX release from CSO-SA micelles was successfully delayed by the core modification of CSO-SA micelles with stearic acid (SA). The cell viability assay against A549 cells indicated the 50% inhibition concentration (IC50) of blank CSO-SA micelles and the core modified CSO-SA micelles was 369 27 mg/mL and 234 9 mg/mL, respectively. The entrapment of DOX by CSO-SA micelles could decrease the IC50 of DOX from 3.5 to 1.9 mg/mL, and a further reduction to 0.7 mg/mL could result by the core modification of CSO-SA micelles. The fluorescence image observations of DOX and DOX concentration measurements inside A549 cells demonstrated that the DOX concentration inside cells was increased by the entrapment of CSO-SA micelles, and further enhanced by the core modification of CSO-SA micelles. The results indicated that the CSO-SA micelles with modified cores could be useful as a drug delivery vehicle for cancer chemotherapy. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:704–712, 2009
Keywords: chitosan oligosaccharide; stearic acid; micelles; doxorubicin; core modification; cytotoxicity
INTRODUCTION To overcome the poor absorption and distribution to other tissues combined with high drug toxicity of poorly soluble drugs (e.g., anti-tumor drugs), one strategy is the development of suitable drug delivery systems.1–5 The in vivo fate of the drug is no longer mainly dominated by the properties of the drug, but by the carrier system, which should permit a controlled and localized release of the Correspondence to: He-Yong YU (Telephone: 86-057187061501; fax: 86-571-87061501; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 704–712 (2009) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
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active drug according to the specific needs of the therapy. With the development of drug delivery systems, the key challenge is to look for efficient and safe vehicles to deliver cargos into the cellular interior, and intracellular drug delivery has been an important route in cancer therapy.6–9 Intracellular drug delivery could enhance the therapeutic efficiency of drugs, reduce the therapeutic dose, and minimize the toxicity of drugs.8,9 As promising drug carriers, polymeric micelles (self-assemblies of amphiphilic graft or block copolymers) have attracted significant attention for the delivery of anti-tumor drugs,10–12 due to their special architecture and nanoscale dimension. The hydrophobic core of polymeric micelles
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can provide a space for the loading of poorly watersoluble antitumor drugs, and modification of the hydrophilic shell can produce such properties as prolonged circulation and specific targeting of the micelles.13–16 The nanoscale dimensions of polymer micelles (between 5 and 100 nm) permit their efficient accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect, which termed as ‘‘passive targeting’’.17,18 For many anti-tumor drugs, the sites of action are inside tumor cells. To achieve effective tumor therapies, polymer micelles should be internalized into the tumor cells, then deliver drugs to their sites of action. Recently, many studies relating to polymeric micelles have focused on intracellular drug delivery.19–21 Great efforts have been undertaken to improve the cellular uptake efficiency of polymer micelles.15,16,22–24 One such approach is increasing the surface positive charge of delivery devices to improve cellular uptake. The positively charged delivery devices can enhance the interaction with negatively charged cellular membrane by ionic interaction. The native cationic polymer, chitosan has been widely accepted as a potential material for drug delivery carriers.25–27 The hydrophobic modified chitosan derivatives have also received great attention as drug carriers in recent years.28–31 Anti-cancer drugs, such as doxorubicin (DOX), can be physically loaded into the core of polymer micelles or chemically conjugated to the polymer molecules.4,11 Effective cellular uptake of micelles by tumor cells can increase drug concentration in the cells and enhance the pharmacological effects.15,16,22,23 In most cases, polymer micelles have loose structures and may be easily deformed or disassembled when the micelle was extremely diluted by body fluid in vivo.4,7,32 This will cause drug leakage from micelles before the drug delivery systems are internalized into the target cells.32 The loss of drug from the micelle may lead to increased toxicity as well as reduced efficacy.32 In our previous work, the amphiphilic stearic acid grafted chitosan oligosaccharide (CSO-SA) polymer was synthesized. The CSO-SA could selfaggregate to form polymer micelles in aqueous solution, which was applied to loading DNA30 and paclitaxel.31 Here, DOX was used as a model drug to incorporate into CSO-SA micelles. The core of DOX loaded CSO-SA micelles (DOX/CSO-SA) was further modified by physical solubilization with SA to reduce the initial burst release from the micelles. Using A549 cells as model tumor cells, DOI 10.1002/jps
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the in vitro cytotoxicity of core modified DOX/ CSO-SA micelles was investigated and compared in detail with that of free DOX and DOX/CSO-SA micelles.
MATERIALS AND METHODS Materials Chitosan oligosaccharide (Mw ¼ 18.7 kDa, Mw/ Mn ¼ 1.88) was prepared by enzymatic degradation of chitosan (95% deacetylated, Mw ¼ 450 kDa, Yuhuan Marine biochemistry Co., Ltd., Zhejiang, China) in our previous work.30,31 Stearic acid was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sigma (St. Louis, MO). RPMI-1640 Medium, fetal bovine serum and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from GiBco (BRL Life Technology, Grand Island, NY). Doxorubicin–hydrochlorate (DOX–HCl) was kindly donated by Zhejiang Hisun Pharmaceutical Co,. Ltd. (Zhejiang, China). Ethanol, dimethyl sulfoxide (DMSO) and other chemicals were analytical grade.
Cell Lines A549 cells, nonsmall-cell lung cancer (Alveolar type 2) cell lines derived from the respiratory epithelium, were obtained from Cell Resource Center of China Science Academe. The cells were cultured at 378C with 5% CO2 under fully humidified conditions. The culture medium was Roswell Park Memorial Institute 1640 (RPMI-1640) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin.
Synthesis and Characterization of CSO-SA The synthesis and characterization of CSOSA were reported elsewhere in detail.30,31 CSOSA was synthesized via the reaction of carboxyl groups of stearic acid with amine groups of chitosan oligosaccharide in the presence of EDC. The purified product was lyophilized, and stored under 208C for further use. The critical aggregation concentration (CAC) of CSO-SA in distilled water was estimated by fluorescence spectroscopy using pyrene as a hydrophobic JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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fluorescence probe.33 The substitutive degree of CSO-SA was determined by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method.29
Preparation of CSO-SA Micelles Blank CSO-SA micelles were prepared by dispersing CSO-SA polymers into distilled water with the probe-type ultrasonic treatment for 10 cycles (active every 1 s for 6 s duration) in an ice-bath (400 W, JY92-II, Ningbo Xinzhi scientific instrument Institute, Zhejiang). DOX–HCl (10 mg) reacted with triethylamine (two times the molar quantity of DOX) in distilled water and was extracted by chloroform. Chloroform was then evaporated, and the product was dissolved in 3 mL DMSO solution to obtain DOX solution. To prepare DOX/CSO-SA, the DOX DMSO solution was added dropwise to 50 mL CSO-SA micelle solution (2 mg/mL CSO-SA). The solution was then transferred into a dialysis bag (molecular weight cut-off 7 kDa) to dialyzed against distilled water for 24 h. The outer solution was exchanged at appropriate time intervals. After filtration through a 0.22 mm microporous membrane, the dialysis solution was lyophilized to obtain DOX/CSO-SA. The core-modified micelles were prepared by physically solubilizing stearic acid into the core of the CSO-SA micelles. Stearic acid ethanol solution (0.25 mL) with 4 mg/mL concentration was dropped into 10 mL CSO-SA micelles/or DOXloaded micelles solution (CSO-SA concentration: 1 mg/mL) under stirring at 708C for 4 h. The ethanol was then completely removed by vacuum distillation with a rotary evaporator to obtain coremodified micelles with or without DOX.
collected and the DOX content in filtrate (Cf) was measured by fluorescence spectrophotometer (F-2500, Hitachi High-Technologies Co., Tokyo, Japan). The excitation wavelength (lex) was 505 nm and the emission wavelength (lem) was 565 nm. DMSO (3.8 mL) was added into 0.2 mL DOX-loaded micelle solution to dissolve the micelles, and the drug content (Ct) was also determined. The drug content incorporated into micelles was obtained from (CtCf). The drug loading was then calculated by Eq. (1). DL ¼
Ct Cf 100% Ct Cf þ Cm
(1)
where Cm represents the amount of CSO-SA in 0.2 mL drug loaded micelle solution.
In Vitro Drug Release Studies The drug release profiles from micelles were investigated using pH 7.4 phosphate buffer solution (PBS) or RPMI-1640 medium as dissolution medium. PBS (pH 7.4) was used to simulate the extra-cellular pH environment of normal healthy tissues, and the use of RPMI-1640 Medium was used to estimate drug leakage from micelles in the cellular uptake tests. In each experiment, DOX-loaded micelles solution (1 mL) with 1 mg/mL CSO-SA concentration was dispersed into 12.5 mL phosphate buffer solution of different pH in a beaker. The beaker was then placed in a 378C water bath and stirred at 100 rpm. At certain time intervals, medium (0.2 mL) was withdraw and centrifuged by using an Ultrafilter tube with a molecular weight cutoff of 10 K for 5 min at 14000g. The DOX content in the filtrate was measured by fluorescence spectrophotometer, as described above.
Characterizations of CSO-SA Micelles CSO-SA micelle solution (2 mL) was prepared as described above (CSO-SA concentration: 1 mg/ mL). The micelle size and zeta potential were then measured by dynamic light scattering using a Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK). To determine the drug loadings (DLs) of micelles, DOX-loaded micelle solution (0.2 mL) was placed in an Ultrafilter tube with molecular weight cutoff of 10 K (Microcon YM-10, Millipore Co., Bedford, MA) and centrifuged at 14000g for 5 min (3K30, SIGMA Labrorzentrifugen GmbH, Osterode am Harz, Germany). The filtrate was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
In Vitro Cytotoxicity Assay In this study, the cytotoxicity of micelles, DOX and their drug delivery systems was evaluated by cell viability, which was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. The A549 cell was used as a model cell. After the cells were incubated with the micelles, free DOX or DOX-loaded micelles solution of different concentration for 24 or 48 h, the A549 cells were stained for 4 h with MTT reagent (final concentration 0.4 mg/mL). After the medium was carefully removed by a pipet, DMSO (500 mL) was added to dissolve the MTT formazan DOI 10.1002/jps
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crystals. Plates were shaken for 10 min and the absorbance of formazan product was measured at 570 nm in a microplate reader (BioRad, Model 680, BioRad, Hercules, CA). The cellular growth inhibition was calculated by using mock-treated cells as a control (100% survival). All the experiments were performed in triplicate.
Fluorescence Observation of DOX Internalization Into Cells After A549 cells with a density of 1 105 cells/well were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelle solution (the DOX concentration was 5 mg/mL) for different lengths of time, cells were washed three times with PBS (pH 7.4) and directly viewed under a fluorescence microscope (DMIL, Leica Microsystems Ltd., Wetzlar, Germany).
Determination of DOX Concentration in Cells After the cells were incubated with free DOX, DOX/CSO-SA micelle and core modified DOX/ CSO-SA micelle solution (the DOX concentration was 5 mg/mL) for different lengths of time, the medium was removed and the A549 cells were rinsed three times with PBS (pH 7.4). Trypsin PBS solution (50 mL) with 2.5 mg/mL concentration was then added and further incubated for 5 min. The cells were harvested by adding 950 mL DMSO solution (90%) followed by sonicate treatment (Sonic Purger CQ250, Academy of Shanghai Shipping Electric Instrument) in a water bath at room temperature for 60 s to ensure complete lysis. After the suspension was centrifuged for at 4000 rpm for 5 min, the drug content in the supernatant was measured by fluorescence spectrophotometer. The DOX concentration in cells was normalized with the protein content in cells, which was quantified using a BCA protein assay kit (Pierce, Rockford, IL).34
Statistical Analysis Data were expressed as means of the three separate experiments, and compared by analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant in all cases. DOI 10.1002/jps
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RESULTS AND DISCUSSION Characteristics of CSO-SA Micelles The synthesis of CSO-SA was reported in our previous papers.30,31 The degree of substitution and CAC of CSO-SA used in our present study was 18.5% and 0.026 mg/mL. Since only 18.5% amino groups of CSO were substituted by stearic acid, a number of primary amino groups remained in the CSO-SA molecules. As a result, the CSO-SA micelles had positive charge. The zeta-potential of CSO-SA micelles with 1 mg/mL CSO-SA concentration in distilled water was 50.7 2.9 mV. Figure 1 shows the size distributions of blank CSO-SA micelles obtained by dynamic light scattering determination. The number average hydrodynamic diameter of the CSO-SA micelles was 27.4 1.4 nm. The core modified CSO-SA micelles were prepared by physical solubilizing 1% SA into the CSO-SA micelles. The diameter of core modified CSO-SA was slightly increased to 32.1 1.3 nm ( p < 0.05, n ¼ 3). The size and distribution of drug vehicles may play important roles in transport in vivo. Drug vehicles of smaller size tend to easily to accumulate into tumor sites due to the EPR effect, and gained faster internalization into cells.17,18 The above result indicated that the core modification of CSOSA micelles by 1% SA might not significantly affect the role of size in the biomembrane transport process, because the size variation was very small. The DL of DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles was 5.51 0.01% (w/w) and 5.10 0.16% (w/w). The DL was slightly decreased after the core of DOX/CSO-SA micelle was modified by SA ( p < 0.05, n ¼ 3).
In Vitro DOX Release Behaviors In vitro drug release profiles from DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles were performed by using pH 7.4 PBS and RPMI-1640 Medium as dissolution media. As shown in Figure 2, it was confirmed that when either pH 7.4 PBS or RPMI-1640 Medium was used as dissolution medium, the drug release rate from the micelles was delayed by the core modification of the micelles with SA. The difference in released drug percentage at 24 h between DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles was above 30%. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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Figure 1. Size distribution of CSO-SA micelles (A) and core modified CSO-SA micelles with 1% SA (B) determined by dynamic light scattering.
The incorporation of SA into DOX/CSO-SA micelles could increase the hydrophobic interaction between SA and the stearate segments in CSO-SA molecules, and the ionic interaction
between DOX and SA. No obvious drug release differences were found in the runs using pH 7.4 PBS or RPMI-1640 Medium as dissolution medium.
Figure 2. In vitro DOX release profiles from DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles using pH 7.4 PBS (A) and RPMI-1640 medium (B) as dissolution mediums (n ¼ 3). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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Figure 3. Changes of cellular viability after the cells were incubated with blank CSOSA micelles and core modified CSO-SA micelles for 24 h (A) and 48 h (B). The concentration means the CSO-SA or core modified CSO-SA concentration.
In Vitro Cytotoxicity Assay The cell viabilities of blank CSO-SA micelles and core modified CSO-SA micelles against A 549 cells were evaluated using the MTT test. Figure 3 shows the cell viability changes at 24 and 48 h against CSO-SA or core modified CSO-SA concentration. It was obvious that cellular viabilities at 24 and 48 h were decreased with the increasing of CSO-SA and core modified CSO-SA concentrations, and that the cellular viability of core modified CSO-SA micelles was lower than that of CSO-SA micelles at the same concentration and incubated time. The 50% inhibition concentration (IC50) of CSO-SA micelles and core modified CSO-SA micelles was 369 27 mg/mL and 234 9 mg/mL ( p < 0.05, n ¼ 3) at 24 h, respectively. These values indicated that the CSO-SA micelles were low toxicity carriers for cancer therapy, compared with other cationic polymers like polyethylenimine and its derivatives.35 The in vitro anti-tumor activities of DOX/CSOSA micelles and core modified DOX/CSO-SA micelles against A549 cell were then evaluated and compared with that of free DOX. Figure 4 shows the cellular viabilities after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution at different drug concentrations for 48 h. It was found that the cellular viabilities were decreased when DOX was incorporated into CSO-SA micelles, and the cellular viabilities were further DOI 10.1002/jps
reduced after the core of DOX/CSO-SA micelles was modified by stearic acid. The IC50 value of free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles was 3.5, 1.9, and 0.7 mg/mL, respectively. The enhanced cytotoxicity of DOX/CSO-SA micelles was due to the faster internalization of DOX into cells mediated by the CSO-SA micelles. The further improved cytotoxicity of the core modified DOX/CSO-SA
Figure 4. Changes of cellular viability against DOX concentration after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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micelles originated from the smaller amount of drug leakage from the core modified micelles before the micelles were uptaken by cells (Fig. 2), which could enhance the drug amount internalized into cells. Since the action site of DOX was inside cells, the enhanced drug concentration in cells could increase the cellular inhibition.
Quantitative and Qualitative Analysis of Drug Content Internalized Into Cells To confirm the drug contents internalized into cells, the drug contents internalized into cells after the cells were incubated with free DOX,
DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution for different incubation times, were quantitatively and qualitatively evaluated by fluorescence image observation of DOX internalized into cells and the determination of drug content internalized into cells, respectively. Figure 5 shows the fluorescence images of DOX after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution (drug content was 5 mg/ mL) for 1, 2, 4, and 6 h, respectively. It was clear that the fluorescence intensities of DOX in different formulations were increased with the incubation time. This means that the cellular uptakes of free DOX, DOX/CSO-SA micelles and
Figure 5. Fluorescence images of DOX after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution (drug content were 5 mg/mL) for 1, 2, 4, and 6 h, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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core modified DOX/CSO-SA micelles were time dependent. Under the same incubation time frame, the fluorescence intensity of DOX was enhanced by the encapsulation of CSO-SA micelles, and further enhanced by the core modification with SA. Figure 6 shows the changes of DOX concentration in cells after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution against the incubation time. It was confirmed that the DOX concentrations in cells for different formulations were increased with the incubation time. The DOX concentration in cells was increased by the encapsulation of CSO-SA micelles, and further enhanced under the same incubation time by the core modification with SA. After 8 h incubation, for free DOX the amount of internalized DOX was approximately 4.7 3.0 mg per mg protein. Whilst the amount of internalized DOX into cells for DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles was increased to 11.7 2.7 and 19.6 3.9 mg per mg protein, respectively ( p < 0.05, n ¼ 3). The drug content in cells for DOX/CSO-SA micelle was 3.3 times higher than that for free DOX, and the drug content in cells was further roughly doubled by the core modification of DOX/CSO-SA with SA. Due to the higher cellular inhibitions that may be caused under the present drug concentrations, the incubation times were not prolonged.
Figure 6. Changes of DOX content in cells against incubation time after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution. DOI 10.1002/jps
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CONCLUSIONS The encapsulation of CSO-SA micelle could improve the cytotoxicity of DOX due to the faster cellular uptake of the CSO-SA micelles. However, the initial burst drug release from DOX/CSO-SA micelles before the micelles were uptaken by cells resulted in loss of drug, and affected the antitumor activity of DOX/CSO-SA micelles. SA was physically solubilized into DOX/CSO-SA micelles to reduce the initial burst drug release associated with DOX/CSO-SA micelles. The results indicated the cytotoxicity of DOX could be increased significantly by the use of core modified micelles due to the higher drug content internalized into cells.
ACKNOWLEDGMENTS We thank the financial supports of the National Nature Science Foundation of China under contract 30472101 and the Nature Science Foundation of Zhejiang province under contract M303817. We also thank Dr. Paul Joseph Bretz (University of Queensland, Australia) for language check of the manuscript.
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DOI 10.1002/jps
Women Hospital, School of Medicine, Zhejiang University, 2 Xueshi Road, Hangzhou 310006, P.R. China
2
College of Pharmaceutical Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, P.R. China
Received 22 August 2007; revised 15 April 2008; accepted 4 May 2008 Published online 18 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21464
ABSTRACT: In this study, the cytotoxicity of doxorubicin (DOX) loaded stearic acid grafted chitosan oligosaccharide (CSO-SA) micelles and its core modified drug delivery systems were investigated in vitro. The in vitro drug release experiments using cellular culture medium, Roswell Park Memorial Institute 1640 (RPMI-1640) medium as a dissolution medium confirmed that the DOX release from CSO-SA micelles was successfully delayed by the core modification of CSO-SA micelles with stearic acid (SA). The cell viability assay against A549 cells indicated the 50% inhibition concentration (IC50) of blank CSO-SA micelles and the core modified CSO-SA micelles was 369 27 mg/mL and 234 9 mg/mL, respectively. The entrapment of DOX by CSO-SA micelles could decrease the IC50 of DOX from 3.5 to 1.9 mg/mL, and a further reduction to 0.7 mg/mL could result by the core modification of CSO-SA micelles. The fluorescence image observations of DOX and DOX concentration measurements inside A549 cells demonstrated that the DOX concentration inside cells was increased by the entrapment of CSO-SA micelles, and further enhanced by the core modification of CSO-SA micelles. The results indicated that the CSO-SA micelles with modified cores could be useful as a drug delivery vehicle for cancer chemotherapy. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:704–712, 2009
Keywords: chitosan oligosaccharide; stearic acid; micelles; doxorubicin; core modification; cytotoxicity
INTRODUCTION To overcome the poor absorption and distribution to other tissues combined with high drug toxicity of poorly soluble drugs (e.g., anti-tumor drugs), one strategy is the development of suitable drug delivery systems.1–5 The in vivo fate of the drug is no longer mainly dominated by the properties of the drug, but by the carrier system, which should permit a controlled and localized release of the Correspondence to: He-Yong YU (Telephone: 86-057187061501; fax: 86-571-87061501; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 704–712 (2009) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
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active drug according to the specific needs of the therapy. With the development of drug delivery systems, the key challenge is to look for efficient and safe vehicles to deliver cargos into the cellular interior, and intracellular drug delivery has been an important route in cancer therapy.6–9 Intracellular drug delivery could enhance the therapeutic efficiency of drugs, reduce the therapeutic dose, and minimize the toxicity of drugs.8,9 As promising drug carriers, polymeric micelles (self-assemblies of amphiphilic graft or block copolymers) have attracted significant attention for the delivery of anti-tumor drugs,10–12 due to their special architecture and nanoscale dimension. The hydrophobic core of polymeric micelles
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can provide a space for the loading of poorly watersoluble antitumor drugs, and modification of the hydrophilic shell can produce such properties as prolonged circulation and specific targeting of the micelles.13–16 The nanoscale dimensions of polymer micelles (between 5 and 100 nm) permit their efficient accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect, which termed as ‘‘passive targeting’’.17,18 For many anti-tumor drugs, the sites of action are inside tumor cells. To achieve effective tumor therapies, polymer micelles should be internalized into the tumor cells, then deliver drugs to their sites of action. Recently, many studies relating to polymeric micelles have focused on intracellular drug delivery.19–21 Great efforts have been undertaken to improve the cellular uptake efficiency of polymer micelles.15,16,22–24 One such approach is increasing the surface positive charge of delivery devices to improve cellular uptake. The positively charged delivery devices can enhance the interaction with negatively charged cellular membrane by ionic interaction. The native cationic polymer, chitosan has been widely accepted as a potential material for drug delivery carriers.25–27 The hydrophobic modified chitosan derivatives have also received great attention as drug carriers in recent years.28–31 Anti-cancer drugs, such as doxorubicin (DOX), can be physically loaded into the core of polymer micelles or chemically conjugated to the polymer molecules.4,11 Effective cellular uptake of micelles by tumor cells can increase drug concentration in the cells and enhance the pharmacological effects.15,16,22,23 In most cases, polymer micelles have loose structures and may be easily deformed or disassembled when the micelle was extremely diluted by body fluid in vivo.4,7,32 This will cause drug leakage from micelles before the drug delivery systems are internalized into the target cells.32 The loss of drug from the micelle may lead to increased toxicity as well as reduced efficacy.32 In our previous work, the amphiphilic stearic acid grafted chitosan oligosaccharide (CSO-SA) polymer was synthesized. The CSO-SA could selfaggregate to form polymer micelles in aqueous solution, which was applied to loading DNA30 and paclitaxel.31 Here, DOX was used as a model drug to incorporate into CSO-SA micelles. The core of DOX loaded CSO-SA micelles (DOX/CSO-SA) was further modified by physical solubilization with SA to reduce the initial burst release from the micelles. Using A549 cells as model tumor cells, DOI 10.1002/jps
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the in vitro cytotoxicity of core modified DOX/ CSO-SA micelles was investigated and compared in detail with that of free DOX and DOX/CSO-SA micelles.
MATERIALS AND METHODS Materials Chitosan oligosaccharide (Mw ¼ 18.7 kDa, Mw/ Mn ¼ 1.88) was prepared by enzymatic degradation of chitosan (95% deacetylated, Mw ¼ 450 kDa, Yuhuan Marine biochemistry Co., Ltd., Zhejiang, China) in our previous work.30,31 Stearic acid was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sigma (St. Louis, MO). RPMI-1640 Medium, fetal bovine serum and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from GiBco (BRL Life Technology, Grand Island, NY). Doxorubicin–hydrochlorate (DOX–HCl) was kindly donated by Zhejiang Hisun Pharmaceutical Co,. Ltd. (Zhejiang, China). Ethanol, dimethyl sulfoxide (DMSO) and other chemicals were analytical grade.
Cell Lines A549 cells, nonsmall-cell lung cancer (Alveolar type 2) cell lines derived from the respiratory epithelium, were obtained from Cell Resource Center of China Science Academe. The cells were cultured at 378C with 5% CO2 under fully humidified conditions. The culture medium was Roswell Park Memorial Institute 1640 (RPMI-1640) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin.
Synthesis and Characterization of CSO-SA The synthesis and characterization of CSOSA were reported elsewhere in detail.30,31 CSOSA was synthesized via the reaction of carboxyl groups of stearic acid with amine groups of chitosan oligosaccharide in the presence of EDC. The purified product was lyophilized, and stored under 208C for further use. The critical aggregation concentration (CAC) of CSO-SA in distilled water was estimated by fluorescence spectroscopy using pyrene as a hydrophobic JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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fluorescence probe.33 The substitutive degree of CSO-SA was determined by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method.29
Preparation of CSO-SA Micelles Blank CSO-SA micelles were prepared by dispersing CSO-SA polymers into distilled water with the probe-type ultrasonic treatment for 10 cycles (active every 1 s for 6 s duration) in an ice-bath (400 W, JY92-II, Ningbo Xinzhi scientific instrument Institute, Zhejiang). DOX–HCl (10 mg) reacted with triethylamine (two times the molar quantity of DOX) in distilled water and was extracted by chloroform. Chloroform was then evaporated, and the product was dissolved in 3 mL DMSO solution to obtain DOX solution. To prepare DOX/CSO-SA, the DOX DMSO solution was added dropwise to 50 mL CSO-SA micelle solution (2 mg/mL CSO-SA). The solution was then transferred into a dialysis bag (molecular weight cut-off 7 kDa) to dialyzed against distilled water for 24 h. The outer solution was exchanged at appropriate time intervals. After filtration through a 0.22 mm microporous membrane, the dialysis solution was lyophilized to obtain DOX/CSO-SA. The core-modified micelles were prepared by physically solubilizing stearic acid into the core of the CSO-SA micelles. Stearic acid ethanol solution (0.25 mL) with 4 mg/mL concentration was dropped into 10 mL CSO-SA micelles/or DOXloaded micelles solution (CSO-SA concentration: 1 mg/mL) under stirring at 708C for 4 h. The ethanol was then completely removed by vacuum distillation with a rotary evaporator to obtain coremodified micelles with or without DOX.
collected and the DOX content in filtrate (Cf) was measured by fluorescence spectrophotometer (F-2500, Hitachi High-Technologies Co., Tokyo, Japan). The excitation wavelength (lex) was 505 nm and the emission wavelength (lem) was 565 nm. DMSO (3.8 mL) was added into 0.2 mL DOX-loaded micelle solution to dissolve the micelles, and the drug content (Ct) was also determined. The drug content incorporated into micelles was obtained from (CtCf). The drug loading was then calculated by Eq. (1). DL ¼
Ct Cf 100% Ct Cf þ Cm
(1)
where Cm represents the amount of CSO-SA in 0.2 mL drug loaded micelle solution.
In Vitro Drug Release Studies The drug release profiles from micelles were investigated using pH 7.4 phosphate buffer solution (PBS) or RPMI-1640 medium as dissolution medium. PBS (pH 7.4) was used to simulate the extra-cellular pH environment of normal healthy tissues, and the use of RPMI-1640 Medium was used to estimate drug leakage from micelles in the cellular uptake tests. In each experiment, DOX-loaded micelles solution (1 mL) with 1 mg/mL CSO-SA concentration was dispersed into 12.5 mL phosphate buffer solution of different pH in a beaker. The beaker was then placed in a 378C water bath and stirred at 100 rpm. At certain time intervals, medium (0.2 mL) was withdraw and centrifuged by using an Ultrafilter tube with a molecular weight cutoff of 10 K for 5 min at 14000g. The DOX content in the filtrate was measured by fluorescence spectrophotometer, as described above.
Characterizations of CSO-SA Micelles CSO-SA micelle solution (2 mL) was prepared as described above (CSO-SA concentration: 1 mg/ mL). The micelle size and zeta potential were then measured by dynamic light scattering using a Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK). To determine the drug loadings (DLs) of micelles, DOX-loaded micelle solution (0.2 mL) was placed in an Ultrafilter tube with molecular weight cutoff of 10 K (Microcon YM-10, Millipore Co., Bedford, MA) and centrifuged at 14000g for 5 min (3K30, SIGMA Labrorzentrifugen GmbH, Osterode am Harz, Germany). The filtrate was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
In Vitro Cytotoxicity Assay In this study, the cytotoxicity of micelles, DOX and their drug delivery systems was evaluated by cell viability, which was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. The A549 cell was used as a model cell. After the cells were incubated with the micelles, free DOX or DOX-loaded micelles solution of different concentration for 24 or 48 h, the A549 cells were stained for 4 h with MTT reagent (final concentration 0.4 mg/mL). After the medium was carefully removed by a pipet, DMSO (500 mL) was added to dissolve the MTT formazan DOI 10.1002/jps
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crystals. Plates were shaken for 10 min and the absorbance of formazan product was measured at 570 nm in a microplate reader (BioRad, Model 680, BioRad, Hercules, CA). The cellular growth inhibition was calculated by using mock-treated cells as a control (100% survival). All the experiments were performed in triplicate.
Fluorescence Observation of DOX Internalization Into Cells After A549 cells with a density of 1 105 cells/well were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelle solution (the DOX concentration was 5 mg/mL) for different lengths of time, cells were washed three times with PBS (pH 7.4) and directly viewed under a fluorescence microscope (DMIL, Leica Microsystems Ltd., Wetzlar, Germany).
Determination of DOX Concentration in Cells After the cells were incubated with free DOX, DOX/CSO-SA micelle and core modified DOX/ CSO-SA micelle solution (the DOX concentration was 5 mg/mL) for different lengths of time, the medium was removed and the A549 cells were rinsed three times with PBS (pH 7.4). Trypsin PBS solution (50 mL) with 2.5 mg/mL concentration was then added and further incubated for 5 min. The cells were harvested by adding 950 mL DMSO solution (90%) followed by sonicate treatment (Sonic Purger CQ250, Academy of Shanghai Shipping Electric Instrument) in a water bath at room temperature for 60 s to ensure complete lysis. After the suspension was centrifuged for at 4000 rpm for 5 min, the drug content in the supernatant was measured by fluorescence spectrophotometer. The DOX concentration in cells was normalized with the protein content in cells, which was quantified using a BCA protein assay kit (Pierce, Rockford, IL).34
Statistical Analysis Data were expressed as means of the three separate experiments, and compared by analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant in all cases. DOI 10.1002/jps
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RESULTS AND DISCUSSION Characteristics of CSO-SA Micelles The synthesis of CSO-SA was reported in our previous papers.30,31 The degree of substitution and CAC of CSO-SA used in our present study was 18.5% and 0.026 mg/mL. Since only 18.5% amino groups of CSO were substituted by stearic acid, a number of primary amino groups remained in the CSO-SA molecules. As a result, the CSO-SA micelles had positive charge. The zeta-potential of CSO-SA micelles with 1 mg/mL CSO-SA concentration in distilled water was 50.7 2.9 mV. Figure 1 shows the size distributions of blank CSO-SA micelles obtained by dynamic light scattering determination. The number average hydrodynamic diameter of the CSO-SA micelles was 27.4 1.4 nm. The core modified CSO-SA micelles were prepared by physical solubilizing 1% SA into the CSO-SA micelles. The diameter of core modified CSO-SA was slightly increased to 32.1 1.3 nm ( p < 0.05, n ¼ 3). The size and distribution of drug vehicles may play important roles in transport in vivo. Drug vehicles of smaller size tend to easily to accumulate into tumor sites due to the EPR effect, and gained faster internalization into cells.17,18 The above result indicated that the core modification of CSOSA micelles by 1% SA might not significantly affect the role of size in the biomembrane transport process, because the size variation was very small. The DL of DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles was 5.51 0.01% (w/w) and 5.10 0.16% (w/w). The DL was slightly decreased after the core of DOX/CSO-SA micelle was modified by SA ( p < 0.05, n ¼ 3).
In Vitro DOX Release Behaviors In vitro drug release profiles from DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles were performed by using pH 7.4 PBS and RPMI-1640 Medium as dissolution media. As shown in Figure 2, it was confirmed that when either pH 7.4 PBS or RPMI-1640 Medium was used as dissolution medium, the drug release rate from the micelles was delayed by the core modification of the micelles with SA. The difference in released drug percentage at 24 h between DOX/CSO-SA micelles and the core modified DOX/CSO-SA micelles was above 30%. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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Figure 1. Size distribution of CSO-SA micelles (A) and core modified CSO-SA micelles with 1% SA (B) determined by dynamic light scattering.
The incorporation of SA into DOX/CSO-SA micelles could increase the hydrophobic interaction between SA and the stearate segments in CSO-SA molecules, and the ionic interaction
between DOX and SA. No obvious drug release differences were found in the runs using pH 7.4 PBS or RPMI-1640 Medium as dissolution medium.
Figure 2. In vitro DOX release profiles from DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles using pH 7.4 PBS (A) and RPMI-1640 medium (B) as dissolution mediums (n ¼ 3). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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Figure 3. Changes of cellular viability after the cells were incubated with blank CSOSA micelles and core modified CSO-SA micelles for 24 h (A) and 48 h (B). The concentration means the CSO-SA or core modified CSO-SA concentration.
In Vitro Cytotoxicity Assay The cell viabilities of blank CSO-SA micelles and core modified CSO-SA micelles against A 549 cells were evaluated using the MTT test. Figure 3 shows the cell viability changes at 24 and 48 h against CSO-SA or core modified CSO-SA concentration. It was obvious that cellular viabilities at 24 and 48 h were decreased with the increasing of CSO-SA and core modified CSO-SA concentrations, and that the cellular viability of core modified CSO-SA micelles was lower than that of CSO-SA micelles at the same concentration and incubated time. The 50% inhibition concentration (IC50) of CSO-SA micelles and core modified CSO-SA micelles was 369 27 mg/mL and 234 9 mg/mL ( p < 0.05, n ¼ 3) at 24 h, respectively. These values indicated that the CSO-SA micelles were low toxicity carriers for cancer therapy, compared with other cationic polymers like polyethylenimine and its derivatives.35 The in vitro anti-tumor activities of DOX/CSOSA micelles and core modified DOX/CSO-SA micelles against A549 cell were then evaluated and compared with that of free DOX. Figure 4 shows the cellular viabilities after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution at different drug concentrations for 48 h. It was found that the cellular viabilities were decreased when DOX was incorporated into CSO-SA micelles, and the cellular viabilities were further DOI 10.1002/jps
reduced after the core of DOX/CSO-SA micelles was modified by stearic acid. The IC50 value of free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles was 3.5, 1.9, and 0.7 mg/mL, respectively. The enhanced cytotoxicity of DOX/CSO-SA micelles was due to the faster internalization of DOX into cells mediated by the CSO-SA micelles. The further improved cytotoxicity of the core modified DOX/CSO-SA
Figure 4. Changes of cellular viability against DOX concentration after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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micelles originated from the smaller amount of drug leakage from the core modified micelles before the micelles were uptaken by cells (Fig. 2), which could enhance the drug amount internalized into cells. Since the action site of DOX was inside cells, the enhanced drug concentration in cells could increase the cellular inhibition.
Quantitative and Qualitative Analysis of Drug Content Internalized Into Cells To confirm the drug contents internalized into cells, the drug contents internalized into cells after the cells were incubated with free DOX,
DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution for different incubation times, were quantitatively and qualitatively evaluated by fluorescence image observation of DOX internalized into cells and the determination of drug content internalized into cells, respectively. Figure 5 shows the fluorescence images of DOX after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution (drug content was 5 mg/ mL) for 1, 2, 4, and 6 h, respectively. It was clear that the fluorescence intensities of DOX in different formulations were increased with the incubation time. This means that the cellular uptakes of free DOX, DOX/CSO-SA micelles and
Figure 5. Fluorescence images of DOX after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution (drug content were 5 mg/mL) for 1, 2, 4, and 6 h, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 2, FEBRUARY 2009
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core modified DOX/CSO-SA micelles were time dependent. Under the same incubation time frame, the fluorescence intensity of DOX was enhanced by the encapsulation of CSO-SA micelles, and further enhanced by the core modification with SA. Figure 6 shows the changes of DOX concentration in cells after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/CSO-SA micelles solution against the incubation time. It was confirmed that the DOX concentrations in cells for different formulations were increased with the incubation time. The DOX concentration in cells was increased by the encapsulation of CSO-SA micelles, and further enhanced under the same incubation time by the core modification with SA. After 8 h incubation, for free DOX the amount of internalized DOX was approximately 4.7 3.0 mg per mg protein. Whilst the amount of internalized DOX into cells for DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles was increased to 11.7 2.7 and 19.6 3.9 mg per mg protein, respectively ( p < 0.05, n ¼ 3). The drug content in cells for DOX/CSO-SA micelle was 3.3 times higher than that for free DOX, and the drug content in cells was further roughly doubled by the core modification of DOX/CSO-SA with SA. Due to the higher cellular inhibitions that may be caused under the present drug concentrations, the incubation times were not prolonged.
Figure 6. Changes of DOX content in cells against incubation time after the cells were incubated with free DOX, DOX/CSO-SA micelles and core modified DOX/ CSO-SA micelles solution. DOI 10.1002/jps
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CONCLUSIONS The encapsulation of CSO-SA micelle could improve the cytotoxicity of DOX due to the faster cellular uptake of the CSO-SA micelles. However, the initial burst drug release from DOX/CSO-SA micelles before the micelles were uptaken by cells resulted in loss of drug, and affected the antitumor activity of DOX/CSO-SA micelles. SA was physically solubilized into DOX/CSO-SA micelles to reduce the initial burst drug release associated with DOX/CSO-SA micelles. The results indicated the cytotoxicity of DOX could be increased significantly by the use of core modified micelles due to the higher drug content internalized into cells.
ACKNOWLEDGMENTS We thank the financial supports of the National Nature Science Foundation of China under contract 30472101 and the Nature Science Foundation of Zhejiang province under contract M303817. We also thank Dr. Paul Joseph Bretz (University of Queensland, Australia) for language check of the manuscript.
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