Synthesis and Characterization of Amphiphilic Glycidol−Chitosan

Oct 28, 2010 - The critical aggregation concentration decreased from 0.043 mg/mL to 0.013 mg/mL with the increase of degree of substitution (DS) of DC...
1 downloads 12 Views 3MB Size
Download PDF
3480

Biomacromolecules 2010, 11, 3480–3486

Synthesis and Characterization of Amphiphilic Glycidol-Chitosan-Deoxycholic Acid Nanoparticles as a Drug Carrier for Doxorubicin Huofei Zhou,†,‡ Weiting Yu,† Xin Guo,† Xiudong Liu,*,§ Nan Li,† Ying Zhang,† and Xiaojun Ma*,† Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, People’s Republic of China, Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China, and College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian, 116622, People’s Republic of China Received August 24, 2010; Revised Manuscript Received September 28, 2010

Novel amphiphilic chitosan derivatives (glycidol-chitosan-deoxycholic acid, G-CS-DCA) were synthesized by grafting hydrophobic moieties, deoxycholic acid (DCA), and hydrophilic moieties, glycidol, with the purpose of preparing carriers for poorly soluble drugs. Based on self-assembly, G-CS-DCA can form nanoparticles with size ranging from 160 to 210 nm, and G-CS-DCA nanoparticles maintained stable structure for about 3 months when stored in PBS (pH 7.4) at room temperature. The critical aggregation concentration decreased from 0.043 mg/mL to 0.013 mg/mL with the increase of degree of substitution (DS) of DCA. Doxorubicin (DOX) could be easily encapsulated into G-CS-DCA nanoparticles and keep a sustained release manner without burst release when exposed to PBS (pH 7.4) at 37 °C. Antitumor efficacy results showed that DOX-G-CS-DCA have significant antitumor activity when MCF-7 cells were incubated with different concentration of DOX-G-CS-DCA nanoparticles. The fluorescence imaging results indicated DOX-G-CS-DCA nanoparticles could easily be uptaken by MCF-7 cells. These results suggested that G-CS-DCA nanoparticles may be a promising carrier for DOX delivery in cancer therapy.

Introduction Nanoparticles have been paid more and more attention as drug delivery vehicles in the last two decades1,2 due to advantages such as improving drug solubility and stability in serum, extending drug circulation time, enhancing its bioavailability, and reducing the drug toxicity and side effects. As the development of nanoparticle drug delivery system, biocompatible, and biodegradable properties of carrier should be taken in account. Therefore, a lot of nontoxic and biodegradable polymers have been used as carrier materials including polyhydroxyalkanoates,3 poly(L-lysine),4 poly(L-aspartic acid),5,6 poly(γ-glutamic acid),7,8 poly(aminoester),9,10 heparin,11 PLA,12 PLGA,13,14 chitosan,15 dextran,16 pullulan,17 and so on. Chitosan (CS), a naturally cationic polysaccharide composed of glucosamine and N-acetyl-glucosamine, has been widely used for delivery of anticancer drugs,18 peptide,19 protein,20 DNA,21 and siRNA.22 Up to date, different hydrophobic molecules, such as, octaldehyde,23 palmitic acid,24 stearic acid,25 cholanic acid,26 and cholesterol27 have been grafted onto chitosan to get amphiphilic derivatives so as to form micelles or micelle-like self-aggregates as drug carriers. However, because the pKa value of chitosan is around 6.5, it is insoluble in biological solution or neutral solution. Undoubtedly, grafting hydrophobic molecules onto chitosan further decreased its solubility in physiological solution, which limited its widely biomedical applica* To whom correspondence should be addressed. E-mail: [email protected] (X.L.); [email protected] (X.M.). † Dalian Institute of Chemical Physics, Chinese Academy of Sciences. ‡ Graduate School of the Chinese Academy of Sciences. § College of Environment and Chemical Engineering, Dalian University.

tion. Therefore, improvement of the solubility of the amphiphilic chitosan derivatives has been pursued recently by chemical modification with different hydrophilic groups. The watersoluble amphiphilic chitosan derivatives, for instance, cholic acid chitosan-g-mPEG,28 N-cholesterol-O-carboxymethyl chitosan,27 N-alkyl-O-sulfate chitosan,29 N-alkyl-N-trimethyl chiotsan,23 and glycol chitosan,26 bearing cholanic acid have been reported to form self-aggregates under neutral condition. In this study, hydrophobic molecule of deoxycholic acid with a rigid cyclopentenophenanthrene nucleus structure was chosen to modify CS to get amphiphilic chitosan-deoxycholic acid (CSDCA). A small hydrophilic molecule glycidol was then grafted onto CS backbone by nucleophilic addition to produce a novel chitosan derivative, G-CS-DCA. The physicochemical properties of G-CS-DCA were characterized by Fourier transform infrared (FTIR) spectroscopy, elemental analysis, and 1H NMR. G-CSDCA nanoparticles formed by self-assembly in water solutions with sonication were characterized by fluorescence spectroscopy with pyrene molecular probe, dynamic light scattering (DLS), laser Doppler velocimetry (LDV), and TEM. The poor soluble anticancer drug, doxorubicin (DOX), was loaded into G-CSDCA nanoparticles. Both drug loading and encapsulation efficiency of DOX in G-CS-DCA nanoparticles were determined. The release profile of DOX in G-CS-DCA nanoparticles and antitumor efficacy were also studied.

Experimental Section Materials. Chitosan (Mw ) 230 kDa, degree of deacetylation (DD) ) 97%) was supplied from Yuhuan Ocean Biochemical Co., Ltd. (Zhejiang, China), and depolymerized by our lab. 1-Ethyl-3-(3-

10.1021/bm100989x  2010 American Chemical Society Published on Web 10/28/2010

Glycidol-Chitosan-Deoxycholic Acid Nanoparticles dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccimide (NHS), and pyrene (purity 98%) were obtained from Acros Organics (U.S.A.). Deoxycholic acid (DCA) and glycidol were purchased from Sigma Co. (U.S.A.). Doxorubicin · HCl (DOX · HCl) was kindly donated by Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). Other reagents and solvents were bought from Damao Chemical Reagent Co., Ltd. (Tianjin, China) and used as received. Synthesis of Amphiphilic Chitosan Derivatives. CS (Mw ) 230 kDa) was first depolymerized by NaNO2 as before.30 Briefly, CS was dissolved overnight at 0.1% (w/V) in 500 mL of acetic acid solution under magnetic stirring. After being treated with specific amounts of sodium nitrite for 5 h at room temperature, the reaction was stopped by sodium hydroxide with an adjustment of pH above 8. The resulting precipitate was collected and washed with water and ethanol, followed by drying in vacuum. The Mw of CS was 24 kDa (PDI ) 1.45) detected with GPC. Amphiphilic chitosan derivatives (CS-DCA) were synthesized by grafting hydrophobic molecule DCA with catalysis of EDC and NHS. Briefly, 4 g 24 kDa CS was dissolved in 200 mL of acidic water with pH of 5.6. DCA (at feed ratio of deoxycholic acid to aminoglucose from 10 to 30%), EDC, and NHS (EDC/DCA ) 1.2/1) were dissolved in 100 mL of DMSO and prereacted for 30 min so as to activate DCA. Subsequently, 100 mL of activated DCA solution was added drop by drop into CS in 60 min. After reaction was undergone for 24 h, the resulting reaction solution was poured into 300 mL methanol/NaOH solution (pH 8). The precipitate was recovered by centrifugation, washed with methanol, water, and ethanol more than five times, respectively, followed by drying in vacuum at room temperature. To improve the solubility of amphiphilic chitosan derivatives (CSDCA) in wide pH range, water-soluble amphiphilic chitosan derivatives G-CS-DCA were synthesized as follows. CS-DCA (1.0 g) was dispersed in 50 mL of deionized water, and then 0.5 mL of acetic acid was added to the suspension as a catalyst. The CS-DCA and acetic acid mixture was stirred for 6 h prior to the dropwise addition of glycidol with continuous stirring. The mole ratio of glycidol to CS-DCA was 4:1. The reaction mixture was stirred at 1000 rpm at 50 °C for 24 h. Then, the pH of solution was adjusted to around 8, followed by centrifugation of mixture at 8000 rpm for 30 min at room temperature so as to remove undissolved polymer. For purification, the supernatant was dialyzed against water for more than 3 days, followed by lyophilization. G-CSDCA with different DS of DCA and different DS of glycidol was obtained using the same method and was denoted as G(DS2)-CSDCA(DS1) in the paper. Characterization of G-CS-DCA Derivatives. Dried CS-DCA powder and lyophilized G-CS-DCA samples were pressed with KBr and scanned from 4000 to 400 cm-1 with a resolution of 4 cm-1. Infrared spectra were recorded using a Bruker spectrometer (VECTOR22, Switzerland). The DS of DCA in different CS-DCA was calculated based on the different C, H, N ratios between CS and CS-DCA derivatives through elemental analysis (Elemental Analyzer Vario EL III, Germany). 1 H NMR analysis was performed by using a Bruker Avance 500 MHz spectrometer (Switzerland) with samples of CS, G-CS-DCA with different DS of DCA in deuterated water (D2O) at room temperature. Preparation of G-CS-DCA Nanoparticles. G-CS-DCA nanoparticles were prepared by a sonication method. G-CS-DCA (20 mg) was dissolved in 10 mL of PBS (0.02 M) at desired pH, followed by probe sonication using a JY92-II probe sonicator (Zhejiang, China) with the output set at 60 W for 2 min each, in which the pulse was turned off for 1 s with the interval of 5 s to prevent the increase in temperature. The resulting solution was passed through membrane filter (pore size: 0.45 µm, Millipore), therefore, the G-CS-DCA nanoparticles solution was obtained and stored at room temperature. CAC Determination of G-CS-DCA Nanoparticles. The critical aggregation concentration (CAC) of G-CS-DCA samples in PBS (0.02 M, pH 7.4) buffer was determined by fluorescence spectroscopy using pyrene as a probe. The aliquots of pyrene stock solution (6.0 × 10-5

Biomacromolecules, Vol. 11, No. 12, 2010

3481

M in acetone, 50 µL) were added to 5 mL volumetric flasks, and acetone was allowed to evaporate. A total of 5 mL of G-CS-DCA derivatives (DS of glycidol was 52, but the DS of DCA was 2.3, 3.2, and 4.2, respectively) with different concentrations (2 × 10-4 mg/mL to 2.0 mg/mL) in PBS were then added to the volumetric flasks containing the pyrene residues with the final concentration of 6.0 × 10-7 M. The solutions were kept on a shaker at 37 °C for 24 h to reach the solubilization equilibrium of pyrene between water phase and nanoaggregates. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer, U.S.A.) at room temperature. The emission spectra were scanned at speed 120 nm/min from 350 to 450 nm with the excitation wavelength of 336 nm. The excitation and emission slit widths were set at 7.5 and 2.5 nm, respectively. The intensity ratio (I1/I3) of the first band (373 nm, I1) to the third band (383 nm, I3) from the emission spectra was analyzed as a function of polymer concentration. Furthermore, the first point of inflection of intensity ratio (I1/I3) against polymer concentration was taken as the CAC.31 Size and Zeta Potential Measurements of G-CS-DCA Nanoparticles. The size of G-CS-DCA nanoparticles was determined by dynamic light scattering (DLS) and zeta potential by laser Doppler velocimetry (LDV) at 25 °C using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, U.K.). The size and zeta potential of nanoparticles (2 mg/ mL) were plotted as the mean values of three measurements ( SD (standard deviation). Morphology Observation of G-CS-DCA Nanoparticles. A drop of sample with the concentration of 2 mg/mL was placed on the copper grids coated with carbon. The grids were dried and imaged at 120 kV. The morphological examination of G-CS-DCA nanoparticles was characterized by TEM (JEM-2000 EX). Drug Loading and In Vitro Release of DOX from Nanoparticles. DOX-loaded G-CS-DCA nanoparticles were prepared by a membrane dialysis method. Briefly, G-CS-DCA (20 mg) was dissolved in 4 mL of DMSO, while DOX was neutralized with 5 moles of excess triethylamine in 1 mL of DMSO. The DOX solution was added into the G-CS-DCA solution and mixed by stirring overnight. The mixture was dialyzed against deionized water at room temperature for 24 h using a dialysis membrane with MWCO of 3500 Da. After dialysis, the solution in the dialysis bag was filtered through 0.8 µm syringe filter and lyophilized for two days. To determine the drug loading (DL) and encapsulation efficiency (EE) of DOX in G-CS-DCA, a certain amount of DOX-loaded nanoparticles was dissolved in DMSO. The concentration of DOX was determined by using the UV-vis spectrophotometer at 481 nm. Furthermore, both DL and EE were calculated based on the standard curve obtained from DOX in DMSO. In vitro release of DOX from G-CS-DCA nanoparticles was investigated in PBS (0.02 M, pH 7.4). Briefly, DOX-G-CS-DCA nanoparticles (3 mg) were dispersed in 3 mL of PBS. The bulk solution (3 mL) was placed into a dialysis tube. The dialysis tube was placed in 30 mL of PBS buffer and gently shaken at 37 °C at 100 rpm. A total of 3 mL of solution was taken from the medium outside of the dialysis tube at predetermined times with replenishing of the same volume fresh medium, and DOX concentrations were determined by calculation from the standard curve obtained from DOX in PBS. Cytotoxicity Assay of DOX-G-CS-DCA Nanoparticles. The cytotoxicity of CS derivatives and DOX-loaded nanoparticles was assessed by MTT test. Both DOX · HCl and DOX-G-CS-DCA nanoparticles were dispersed in PBS (pH 7.4) and then serially diluted to get different drug concentration. MCF-7 cells were seeded in 96-well plate at a density of 10000 cells/well and cultured in 200 µL of RPMI 1640 containing 10% FBS in a humidified 37 °C environment with 5% CO2 overnight. Then, the culture medium was replaced by mixture of 180 µL fresh culture medium and 20 µL of DOX · HCl or DOX-G-CS-DCA nanoparticles with different concentration. Different concentrations (0-1000 µg/mL) of G-CS-DCA dissolved in culture medium without drug was also added to test the cytotoxicity. The plates were then returned to the incubator and maintained in 5% CO2 at 37 °C for 48 h. Fresh culture medium (180 µL) and 20 µL aliquots of MTT solutions

3482

Biomacromolecules, Vol. 11, No. 12, 2010

Zhou et al.

Scheme 1. Synthesis Route of Glycidol-Chtiosan-Deoxycholic Acid (G(DS2)-CS-DCA(DS1)) and Encapsulation of Doxorubicin in G-CS-DCA Nanoparticles Based on Self-Assembly for Intracellular Release of DOX

(5 mg/mL) were used to replace the mixture in each well after 48 h. After being incubated for 4 h, the culture medium was removed and 200 µL of DMSO was then added to each well to dissolve the internalized purple formazan crystals. Plates were vigorously shaken before measuring the relative color intensity using a microplate reader (Well Scan MK3, Labsystems Dragon, Finland). A test wavelength of 550 nm and a reference wavelength of 630 nm were used. The intensity of each well was then given by (absorbance550 nm - absorbance630 nm). The relative cell viability was calculated as cell viability (%) ) (ODsample/ODcontrol) × 100, where ODcontrol was obtained in the absence of polymers, and ODsample was obtained in the presence of polymers. Results were expressed as mean ( SD for six replicates. Uptake Process of G-CS-DCA Nanoparticles Observed by Confocal Laser Scanning Microscopy (CLSM). Uptake of the fluorescent G-CS-DCA nanoparticles by MCF-7 cells was visualized by CLSM (Leica, Germany). Doxorubicin (DOX) was loaded in G52-CS-DCA3.2 (3.2 is DS of DCA (DS1), 52 is DS of glycidol (DS2)) nanoparticles as a fluorescence marker. MCF-7 cells were cultured with culture media containing 10% FBS in the plates at density of 2 × 104 cells/well for 24 h. Then the culture media were replaced by the DOX (5 µg/mL) encapsulated in G52-CS-DCA3.2. After 5 h, the MCF-7 cells were washed three times with PBS and detected by CLSM with excitation and emission wavelength of 485 and 595 nm, respectively.

of the N-H bending vibration of the primary amino group at 1600 cm-1 (Figure 1b-d). It also showed that with increasing of the feed ratio of DCA to chitosan (from 10 to 30%), the amide I peak at 1652 cm-1 became sharper, which indirectly indicated the increase of DS of DCA. The DS of DCA was calculated from elemental analysis results based on the changes of C/N between CS and CS-DCA and was tuned by adjusting the feed ratio of reagents. With the changed ratio of deoxycholic acid to aminoglucose units from 10 to 30%, the DS of DCA was changed from 2.3 to 4.2. Second, following the synthesis route of grafting glycidol to CS-DCA shown in Scheme 1, G-CS-DCA derivatives were successfully obtained as characterized with FTIR and 1H NMR spectra. Figure 2 shows the FTIR spectra of CS, CS-DCA3.2 (DS of DCA is 3.2), and G52-CS-DCA3.2 (DS of glycidol is 52). By comparing the FTIR spectra of CS-DCA3.2 (Figure 2b) with G52-CS-DCA3.2 (Figure 2c), the disappearance of 1600 cm-1 peak ascribing to the N-H bending vibration of the primary

Results and Discussion Synthesis and Characterization of Amphiphilic Chitosan Derivatives. Hydrophobic deoxycholic acid with a rigid cyclopentenophenanthrene nucleus structure was selected to graft onto chitosan. The synthesis procedure of water-soluble amphiphilic chitosan derivatives was illustrated in Scheme 1. First, a small fatty acid molecule of DCA was covalently grafted onto chitosan in the presence of EDC and NHS, which produced amphiphilic chitosan-deoxycholic acid (CS-DCA). The formation of amide linkage between DCA and chitosan was evidenced in the FTIR spectra (Figure 1). In comparison with the FTIR spectrum of CS (Figure 1a), the peak of amide I band at 1652 cm-1 was clearly intensified in CS-DCA accompanied by the weakening

Figure 1. FTIR spectra of chitosan and chitosan-deoxycholic acid (CS-DCA) derivatives: (a) CS (DD ) 97%); (b) CS-DCA2.3; (c) CSDCA3.2; (d) CS-DCA4.2; 4.2 is the degree of substitution of DCA.

Glycidol-Chitosan-Deoxycholic Acid Nanoparticles

Biomacromolecules, Vol. 11, No. 12, 2010

DS(%) )

Figure 2. FTIR spectra of chitosan and its derivatives: (a) CS (DD ) 97%); (b) CS-DCA3.2; (c) G52-CS-DCA3.2; 3.2 is the degree of substitution of DCA (DS1) and 52 is the degree of substitution of glycidol (DS2).

amino group indicated hydrogens of the primary amino group were further substituted by glycidol molecules. Figure 3 shows the 1H NMR spectra of chitosan and its derivatives. Both peaks at 2.9 and 2.5 ppm represented the -N-CH2- group in glycidol, while the peaks at 2.60-2.72 ppm ascribed to C-2 in saccharide unit for both G-CS and G52-CS-DCA3.2 (Figure 3b-e). The DS values of glycidol in G-CS and G-CS-DCA were calculated according to eq 1 shown as follows. Based on the comparison of 1H NMR spectra of G-CS (Figure 3b) with G52-CS-DCA3.2 (Figure 3d), it can be concluded that the small hydrophobic molecule DCA was successfully grafted onto chitosan due to the appearance of new peaks from 0.7 to 1.9 ppm (Figure 3d). For example, the peak at 0.7 ppm was affiliated to 21-CH3 in DCA moiety.

[(

∫ H(-N-CH2-) ∫ H(C-2)

) ] ×

1 × 100 2

3483

(1)

CAC Determination of G-CS-DCA Nanoparticles. The CAC value of G52-CS-DCA is not only strong evidence to prove formation of micellar nanoaggregates by self-assembly, but is also an important parameter to evaluate the stability of the nanoaggregates in the blood circulation system post-administration. Pyrene is molecular fluorescence probe frequently used to determine the CAC of amphiphilic polymers. With the formation of G52-CS-DCA nanoaggregates, the microenvironment of pyrene changed from hydrophilic to hydrophobic. Correspondingly, the ratio of pyrene fluorescence intensities between peak 1 (373 nm) and peak 3 (383 nm), that is I1/I3, would decrease gradually. The influence of DS of DCA on the CAC of G52CS-DCA is shown in Table 1 and Figure 4. It was indicated that CAC of G52-CS-DCA decreased with the increasing DS of a hydrophobic molecule. It can be explained that the higher DS, the higher hydrophobicity, which gave rise to stronger selfaggregation ability. The CAC values of G52-CS-DCA were in the ranges of 0.013 to 0.043 mg/mL with the decrease of DS of DCA from 4.2 to 2.3. These results demonstrated that G52CS-DCA can form very stable nanoaggregates at low concentration in aqueous medium. The morphology and size distribution of G52-CS-DCA nanoparticles characterized by TEM and dynamic light scattering are shown in Figure 5. It indicated that the nanoaggregates were spherical nanoparticles and with a narrow size distribution. Size and Zeta Potential Measurements of G-CS-DCA Nanoparticles. For the purpose of achieving longevity during systemic circulation, the nanoparticles must be small enough to evade

Figure 3. 1H NMR spectra of chitosan and its derivatives: (a) CS (D2O/CD3COOD, 70 °C); (b) G-CS (D2O, 25 °C); (c) G52-CS-DCA2.3 (D2O, 25 °C); (d) G52-CS-DCA3.2 (D2O, 25 °C); (e) G52-CS-DCA4.2 (D2O, 25 °C).

3484

Biomacromolecules, Vol. 11, No. 12, 2010

Zhou et al.

Table 1. CAC, Size, Zeta Potential of G52-CS-DCA Nanoparticles with a Different DS of DCA samplea

CAC (mg/mL)

size (nm)b

zeta potential (mV)

G52-CS-DCA2.3 G52-CS-DCA3.2 G52-CS-DCA4.2

0.043 0.020 0.013

166.9 ( 2.9(0.156) 201.0 ( 3.0(0.151) 212.5 ( 2.6(0.120)

0.4 ( 0.2 0.7 ( 0.1 2.1 ( 0.6

a G52-CS-DCA3.2 means that the DS of glycidol and the DS of deoxycholic acid are 52 and 3.2, respectively. b Values in parentheses represent the polydispersity index (PDI).

Figure 6. Size of G52-CS-DCA4.2 (2 mg/mL) nanoparticles in PBS at room temperature as a function of time.

Figure 4. Fluorescence intensity ratio I1/I3 of pyrene as a function of G-CS-DCA concentration: (a) G52-CS-DCA2.3; (b) G52-CS-DCA3.2; (c) G52-CS-DCA4.2.

Figure 7. In vitro release profile of DOX from G52-CS-DCA nanoparticles incubated in 0.02 M PBS (pH 7.4) at 37 °C: (a) DOX-G52-CSDCA2.3; (b) DOX-G52-CS-DCA3.2; (c) DOX-G52-CS-DCA4.2.

Figure 5. Size distribution and transmission electron micrograph (TEM) image of G52-CS-DCA3.2 nanoparticles at the concentration of 2 mg/mL.

detection and destruction by the reticulo-endothelial system.32 Thus, the size and size distribution of self-aggregated G52-CSDCA nanoparticles in PBS buffer at a pH of 7.4 were determined using dynamic light scattering and were illustrated in Table 1. The size of G52-CS-DCA nanoparticles increased from 160 to 210 nm with increasing of DS of DCA. This phenomenon may be ascribed to that higher DS of DCA would cause much more chains to aggregate together. Correspondingly, more chains aggregated together also brought about more repulsion force between inter- and intrachains of chitosan derivatives resulting in the increase of size. Furthermore, the size of G52-CS-DCA nanoparticles were kept constant when stored at room temperature for almost 3 months, suggesting that they were very stable (Figure 6). Table 1 showed that the zeta potential of these chitosan derivatives nanoparticles almost near to zero, thus, it seems that these nanoparticles might avoid the unspecific conjugation with proteins or enzymes by electrostatic interaction and keep long circulation time in vivo.

Drug Loading and In Vitro Release of DOX from Nanoparticles. CAC determination not only supplied the information of the minimum concentration for forming nanoparticles, but it also suggested that hydrophobic molecules could be encapsulated into G52-CS-DCA nanoparticles. Fluorescence anticancer drug DOX, one of the most potent anticancer drugs widely used in the treatment of different types of solid malignant tumors, was chosen as the drug model. A delivery system based on G52CS-DCA nanoparticles was developed for enhancing cancer chemotherapy of DOX. DOX was encapsulated into nanoparticles by dialysis of a DOX/polymer solution in DMSO against water. The results showed that DOX could be easily loaded in G52-CS-DCA nanoparticles, and the drug loading level increased from 5.6 to 7.7% with increasing of DS of DCA while encapsulation efficiency was up to 70%. Furthermore, the size of nanoparticles increased about 70 nm while PDI remained low after drug encapsulation. In vitro release evaluation showed that DOX was released slowly from DOX-G52-CS-DCA nanoparticles without burst release phenomenon in the first 6 h (Figure 7). Meanwhile, it was also noticed that the higher DS of DCA, the slower release rate of drugs. The accumulative amount of DOX released from G52-CS-DCA2.3, G52-CS-DCA3.2, and G52-CS-DCA4.2 nanoparticles was 46.7, 43.8, and 31.2%, respectively, within 48 h. The slow release rate of DOX from G-CS-DCA nanoparticles may be attributed to the strong interaction between hydrophobic DCA and DOX.

Glycidol-Chitosan-Deoxycholic Acid Nanoparticles

Figure 8. Effect of G52-CS-DCA derivatives on the MCF-7 cell viability at different concentration after incubation for 48 h: (a) G52-CS-DCA2.3; (b) G52-CS-DCA3.2; (c) G52-CS-DCA4.2. The results represent the means ( SD (n ) 6).

Biomacromolecules, Vol. 11, No. 12, 2010

3485

concentration. However, when DOX was loaded in G52-CSDCA nanoparticles, the cell viability decreased from almost 100% to less than 30% as the DOX concentration increased from 1 to 50 µg/mL (Figure 9), while the cell viability decreased to about 10% when free DOX with the same concentration was used in cells for 48 h. The possible reason is that DOX loaded in G52-CS-DCA nanoparticles has to be released from the carrier when delivered into tumor cells and then enters into the nucleus to take effect through interaction with DNA by intercalation and inhibition of macromolecular biosynthesis. DOX-loaded G52-CS-DCA nanoparticles with lower DS of DCA showed better antitumor efficiency than that with higher DS of DCA, which may be also the results that drug release speed increased with the decrease of DS of DCA and that DOX-loaded G52CS-DCA nanoparticles with lower DS of DCA have a smaller size that is beneficial for entering into cells. As shown in Figure 10, stronger fluorescence was observed in the cytoplasm than that in nucleus when cells were incubated with DOX-loaded G52-CS-DCA3.2 nanoparticles. It may indicate that DOX-G52CS-DCA3.2 nanoparticles were easily internalized by the cells through endocytosis pathway within 5 h and then escaped from the endosome and the lysosome to enter the cytoplasm. Then the DOX may be released from the nanoparticles and entered into nucleus.

Conclusion

Figure 9. Viability of MCF-7 cells after incubation with DOX- G52CS-DCA2.3 nanoparticles (a), DOX- G52-CS-DCA3.2 nanoparticles (b), DOX-G52-CS-DCA4.2 nanoparticles (c), and DOX · HCl (d) for 48 h with different concentration. The results represent the means ( SD (n ) 6).

Cytotoxicity Assay of DOX-G-CS-DCA Nanoparticles. The cytotoxicity of DOX loaded G52-CS-DCA nanoparticles was compared with that of free DOX · HCl dissolved in PBS. To ensure that the cytotoxicity was caused by DOX itself and not by G52-CS-DCA, cells were also incubated with empty G52CS-DCA nanoparticles alone. As shown in Figure 8, G52-CSDCA nanoparticles did not show cytotoxicity even at high

Water-soluble amphiphilic chitosan derivatives G52-CS-DCA was successfully synthesized and used to fabricate nanoparticles for delivery of the antitumor cell drug DOX. The nanoparticles were spherical morphology, confirmed by TEM, and the size ranged from 160 to 210 nm, but the zeta potential was almost zero. The CAC of G52-CS-DCA nanoparticles ranged from 0.043 to 0.013 mg/mL according to the increasing of DS of DCA from 2.3 to 4.2. DOX was easily encapsulated into G52-CS-DCA nanoparticles with size less than 300 nm and was released slowly from G52-CS-DCA nanoparticles. Furthermore, the release could be controlled by adjusting the DS of DCA. DOX-G52-CS-DCA nanoparticles were easily internalized into tumor cells and showed a significant antitumor activity in vitro. The DOX-G52CS-DCA may have promising potential as a carrier of poorly soluble antitumor drugs. Acknowledgment. The authors are thankful for the financial support from the National Natural Science Foundation of China (No. 20876018, No. 20736006), Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.M02 and

Figure 10. Confocal microscopic images of MCF-7 cells incubated with DOX-G52-CS-DCA3.2 nanoparticles at a concentration of 5 µg/mL of DOX and cultured for 5 h.

3486

Biomacromolecules, Vol. 11, No. 12, 2010

KJCX2-YW-210-02), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

References and Notes (1) Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2 (12), 751–760. (2) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263 (5153), 1600–1603. (3) Kim, H. N.; Lee, J.; Kim, H. Y.; Kim, Y. R. Chem. Commun. 2009, (46), 7104–7106. (4) Abbasi, M.; Uludag, H.; Incani, V.; Olson, C.; Lin, X. Y.; Clements, B. A.; Rutkowski, D.; Ghahary, A.; Weinfeld, M. Biomacromolecules 2007, 8 (4), 1059–1063. (5) Lee, Y.; Ishii, T.; Cabral, H.; Kim, H. J.; Seo, J. H.; Nishiyama, N.; Oshima, H.; Osada, K.; Kataoka, K. Angew. Chem., Int. Ed. 2009, 48 (29), 5309–5312. (6) Cheng, H.; Li, Y. Y.; Zeng, X.; Sun, Y. X.; Zhang, X. Z.; Zhuo, R. X. Biomaterials 2009, 30 (6), 1246–1253. (7) Lin, Y. H.; Mi, F. L.; Chen, C. T.; Chang, W. C.; Peng, S. F.; Liang, H. F.; Sung, H. W. Biomacromolecules 2007, 8 (1), 146–152. (8) Luo, K.; Yin, J. B.; Song, Z. J.; Cui, L.; Cao, B.; Chen, X. S. Biomacromolecules 2008, 9 (10), 2653–2661. (9) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122 (44), 10761– 10768. (10) Huynh, D. P.; Nguyen, M. K.; Pi, B. S.; Kim, M. S.; Chae, S. Y.; Kang, C. L.; Bong, S. K.; Kim, S. W.; Lee, D. S. Biomaterials 2008, 29 (16), 2527–2534. (11) Wang, X.; Li, J.; Wang, Y. Q.; Cho, K. J.; Kim, G.; Gjyrezi, A.; Koenig, L.; Giannakakou, P.; Shin, H. J. C.; Tighiouart, M.; Nie, S. M.; Chen, Z.; Shin, D. M. ACS Nano 2009, 3 (10), 3165–3174. (12) Jain, J. P.; Kumar, N. Biomacromolecules , 11 (4), 1027–1035. (13) Yoo, H. S.; Park, T. G. J. Controlled Release 2001, 70 (1-2), 63–70. (14) Dong, Y.; Feng, S. S. Int. J. Pharm. 2007, 342, 208–214. (15) Kumar, M.; Muzzarelli, R. A. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A. J. Chem. ReV. 2004, 104 (12), 6017–6084. (16) Li, Y. L.; Zhu, L.; Liu, Z. Z.; Cheng, R.; Meng, F. H.; Cui, J. H.; Ji, S. J.; Zhong, Z. Y. Angew. Chem., Int. Ed. 2009, 48 (52), 9914–9918.

Zhou et al. (17) Na, K.; Lee, E. S.; Bae, Y. H. Bioconjugate Chem. 2007, 18, 1568– 1574. (18) Zhao, Z. M.; He, M.; Yin, L. C.; Bao, J. M.; Shi, L. L.; Wang, B. Q.; Tang, C.; Yin, C. H. Biomacromolecules 2009, 10 (3), 565–572. (19) Mi, F. L.; Wu, Y. Y.; Lin, Y. H.; Sonaje, K.; Ho, Y. C.; Chen, C. T.; Juang, J. H.; Sung, H. W. Bioconjugate Chem. 2008, 19 (6), 1248– 1255. (20) Sonaje, K.; Chen, Y. J.; Chen, H. L.; Wey, S. P.; Juang, J. H.; Nguyen, H. N.; Hsu, C. W.; Lin, K. J.; Sung, H. W. Biomaterials , 31 (12), 3384–3394. (21) Liu, W. G.; Sun, S. J.; Cao, Z. Q.; Xin, Z.; Yao, K. D.; Lu, W. W.; Luk, K. D. K. Biomaterials 2005, 26 (15), 2705–2711. (22) Liu, X. D.; Howard, K. A.; Dong, M. D.; Andersen, M. O.; Rahbek, U. L.; Johnsen, M. G.; Hansen, O. C.; Besenbacher, F.; Kjems, J. Biomaterials 2007, 28 (6), 1280–1288. (23) Zhang, C.; Ding, Y.; Yu, L. L.; Ping, Q. N. Colloids Surf., B 2007, 55 (2), 192–199. (24) Wang, W.; McConaghy, A. M.; Tetley, L.; Uchegbu, I. F. Langmuir 2001, 17 (3), 631–636. (25) Hu, F. Q.; Liu, L. N.; Du, Y. Z.; Yuan, H. Biomaterials 2009, 30 (36), 6955–6963. (26) Hwang, H. Y.; Kim, I. S.; Kwon, I. C.; Kim, Y. H. J. Controlled Release 2008, 128 (1), 23–31. (27) Wang, Y. S.; Liu, L. R.; Weng, J.; Zhang, Q. Q. Carbohydr. Polym. 2007, 69 (3), 597–606. (28) Ngawhirunpat, T.; Wonglertnirant, N.; Opanasopit, P.; Ruktanonchai, U.; Yoksan, R.; Wasanasuk, K.; Chirachanchai, S. Colloids Surf., B 2009, 74 (1), 253–259. (29) Zhang, C.; Qu, G. W.; Sun, Y. J.; Wu, X. J.; Yao, Z. L.; Guo, Q. L.; Ding, Q. O.; Yuan, S. T.; Shen, Z. L.; Ping, Q. E.; Zhou, H. P. Biomaterials 2008, 29 (9), 1233–1241. (30) Zhou, H. F.; Liu, X. D.; Zhang, Y.; Ma, X. J. J. Nanosci. Nanotechnol. 2010, 10 (4), 2304–2313. (31) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99 (7), 2039–2044. (32) Seow, W. Y.; Xue, J. M.; Yang, Y. Y. Biomaterials 2007, 28 (9), 1730–1740.

BM100989X