Chinese Chemical Letters  2014, Vol.25 Issue (11):1435-1440   PDF    
Preparation and characterization of mPEG grafted chitosan micelles as 5-fluorouracil carriers for effective anti-tumor activity
Dong-Jun Fua, Yu Jina, Mu-Qing Xiea, Ya-Jing Yea, Dong-Dong Qina, Kai-Yan Loua,b,c, Yan-Zuo Chena, Feng Gaoa,b,c     
a Department of Pharmaceutics, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China;
b Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China;
c Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai 200237, China
Abstract: The objective of this study was to investigate the potential of methoxy polyethylene glycol (mPEG) grafted chitosan (mPEG-g-CS) to be used as a drug carrier. mPEG-g-CS was successfully synthesized by one-step method with formaldehyde. The substitution degree of mPEG on chitosan was calculated by elemental analysis and was found to be (3.23±0.25)%. mPEG-g-CS self-assembled micelles were prepared by ultrasonic method with the controlled size of 178.5-195.1 nm and spherical morphology. Stable dispersion of the micelles was formed with the zeta potential of 2.3-30.2 mV. 5-Fluorouracil (5-FU), an anticancer chemotherapy drug, was used as a model drug to evaluate the efficiency of the new drug delivery carrier. The loading efficiency of 5-FU was (4.01±0.03)%, and the drug-loaded mPEG-g-CS self-assembled micelle showed a controlled-release effect. In summary, the mPEG-g-CS self-assembled micelle is proved to be a promising carrier with controlled particle size and controlled-release effect. Therefore, it has great potential for the application as 5-FU carriers for effective anti-tumor activity.
Key words: Self-assembled micelles     Methoxy polyethylene glycol grafted     chitosan     5-Fluorouracil     Controlled-release    
1. Introduction Nanoparticle drug delivery systems (NDDS) have been extensively studied because of their numerous advantages,such as protecting sensitive drug molecules from degradation,controlledrelease properties,and increasing the bioavailability of poorly water-soluble drugs [1]. Self-assembled polymeric micelle is one kind of the NDDS and has proven to be useful in drug delivery. Amphiphilic block or graft copolymers spontaneously assemble into polymeric micelles which have a unique core-shell structure above the critical micelle concentration (CMC) [2]. The hydrophilic outer shell which acts as inter-face could avoid biological incompatibilities and the hydrophobic inner core serves as a reservoir for hydrophobic drugs. Many efforts have been made to develop novel self-assembled polymeric micelles from amphiphilic copolymers for drug delivery,especially for anticancer drug delivery. They offer routes to improved water solubility of hydrophobic anticancer drugs,enhanced drug accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect,prolonged blood circulation time and decreased toxicity to normal cells [3]. Numerous biodegradable and biocompatible copolymers have been synthesized for the construction of selfassembled polymeric micelles [4].

Chitosan,the only natural cationic polysaccharide,is an aminopolysaccharide in a deacetylated form of chitin. Chitosan and its derivatives have gained considerable attention in pharmaceutical and biomedical field owing to their favorable biological properties,such as low-toxicity,good biocompatibility and good biodegradability. It can be used to fabricate particles easily [5]. Several research groups have reported the use of chitosan micro- or nanoparticles for the delivery of poorly watersoluble drugs [6, 7, 8] as well as water-soluble protein [9, 10]. However,chitosan has an apparent pKa of 6.5,which means it is only soluble in few dilute acid solutions such as acetic acid and hydrochloric acid,but cannot form micelles in water [11]. As a result,a considerable number of different chemical agents for chitosan modification have been employed. Grafting hydrophilic polymers onto chitosan chain cannot only improve the water solubility of chitosan but also form nano-micelles and maintain the stability in dilute conditions. Among these modified chitosans,N,N,N-trimethyl chitosan,N-lauryl-carboxymethyl chitosan,N-octyl-O-sulfate chitosan andN-methylene phosphonic chitosan have been reported for the preparation of polymeric micelles [12, 13, 14]. However,these applications might involve toxicity issues.

To overcome these issues,polyethylene glycol (PEG) is a good choice to modify chitosan. Due to its favorable hydrophilicity and biocompatibility,PEG has been extensively adopted as a soluble polymeric modifier in organic synthesis [15]. At present,PEGylation of chitosan derivatives with PEG of different molecular weights has been reported by several research groups in order to increase the aqueous solubility of chitosan [16]. With the properties of biodegradability,biocompatibility,low toxicity, low immunogenicity,and hydrophilic flexibility,PEG conjugated chitosan can be prepared into microspheres or nanoparticles to enhance the stability of incorporated active agents,prolong their half-life,alter their pharmacokinetic behavior,tissue distribution and pharmacological properties [17, 18].

In this study,we synthesized methoxy polyethylene glycol (mPEG) grafted chitosan (mPEG-g-CS) by one-step method and prepared polymeric micelles by self-assembly using ultrasonic method. The influence of pH on core-shell integrity of mPEG-g-CS micelles was tested. 5-Fluorouracil (5-FU),one of the best anticancer chemotherapy drugs,was used as the model drug to evaluate the potential of mPEG-g-CS self-assembled micelle for drug loading. The release behavior of 5-FU-loaded mPEG-g-CS micelles was also investigated. 2. Experimental

The mPEG-g-CS was prepared according to literature [18]. Briefly,the purified chitosan (MW = 110,000 Da,100 mg) was dissolved in formic acid (4 mL) and then diluted with dimethyl sulfoxide (DMSO,45 mL). After 0.48 g of mPEG was added and stirred for 15 min,a certain amount of formaldehyde was added and mixed for 1 h to obtain the mPEG-g-CS solution. Then,the solution was dialyzed and the solution pH was adjusted to 14. The precipitate was filtered off and washed several times with absolute ethyl alcohol. Finally,mPEG-g-CS was obtained after dialysis and lyophilization. The polymer was characterized by 1HNMR spectra (Bruker AVANCE400 NMR),FT-IR (Nicolet-6700 spectrometer) and elemental analysis (Euro EA3000 analyser). The solubility of the prepared mPEG-g-CS in aqueous solution was determined by measuring their optical transmittances. A weighed amount of materials was placed in large screw-cap tubes (50 mL) containing ultrapure water. The solution pH was adjusted to 4 with 1.0 mol/L HCl and left overnight at a final concentration of 4 mg/mL. Then,the pH was readjusted with 0.1 mol/L NaOH to a pH gradient (4-9). After adjusting the pH value,the solutions were mixed by vortex for 30 s. The absorbance (A) of solutions was measured at 500 nm using a UV-visible spectrometer (Jasco V-530). The transmittance (T) was calculated as:

mPEG-g-CS self-assembled micelles were obtainedviaultrasonic method. Briefly,a certain amount of mPEG-g-CS was dissolved in 0,20,30 and 40 mmol/L phosphate buffered saline or cosolvent (water:dimethyl formamide (DMF) = 10:1) to obtain final concentrations of 0.5,1,2.0,2.5 and 3.0 mg/mL. The solution pHwasadjustedto3,4,5,6,6.4,6.8,7and7.4,respectively.Then, the solution was sonicated using a probe-type sonicator (BILON92-11DL ultrasonic cell disruptor) at 100 W for 2 min in cycles of 1 s followed by 0.5 s of pauses. The particle size and zeta potential of the assemblies in aqueous solution were determined using dynamic light scattering (DLS,NanoZS4700 nanoseries). Furthermore,the solutions were freeze-dried to obtain dry micelles for transmission electron microscope (TEM,JEM-2010JEOL) observation.

To measure the CMC value of mPEG-g-CS polymer,a hydrophobic fluorescence probe,pyrene,was used as described previously [19]. A stock solution of pyrene (6 mg) dissolved in methanol was stored at a final concentration of 297×10 -6 mol/L, then 100mL of the solution was transferred to centrifuge tube and evaporated under nitrogen gas. The methanol-free pyrene was mixed with mPEG-g-CS solution (1.0×10 -4 -4.0 mg/mL,6 mL) to give a final pyrene concentration of 4.94×10 -6 mol/L. The solution was sonicated for 2 min in cycles of 1 s followed by 0.5 s of pauses. After placing at room temperature for 12 h,the fluorescence spectra were recorded on a fluorescence spectrophotometer (Thermo Lumina) from 350 nm to 400 nm with an excitation wavelength of 334 nm. The intensity ratio (I3/I1, I3: the third band at 385 nm; I1: the first band at 373 nm) was plottedversusthe log mPEG-g-CS concentration to calculate the CMC. To investigate the influence of pH on core-shell integrity of the self-assembled micelles,mPEG-g-CS solution (0.4 mg/mL, 6 mL) at various pH values (pH 1.5,3.0,4.0,5.0,6.0 or 7.0) were sonicated to prepare micelles and the fluorescence spectra were recorded as said above. The intensity ratio (I3/I1, I3: the third band at 385 nm;I1: the first band at 373 nm) was plotted versusthe solution pH.

In order to study the stability of the mPEG-g-CS micelles,mPEGg-CS solution (1 mg/mL) was adjusted to pH 7.0. Then,the solution was sonicated and stored at 4℃. Samples were collected at time points of day 0,1,2,3,5,7,10,15,20 and 25. The size distribution of the micelles was measured by DLS.

The incorporation of 5-FU into mPEG-g-CS self-assembled micelles was also achieved by ultrasonic method. Briefly,5-FU (1.2,2.4,3 and 4 mg) was dissolved in DMF,(1 mL). Then,5-FU solution (0.5 mL) in DMF was added to mPEG-g-CS solution (1.2 mg/mL,5 mL). The mixed solution was vortexed for 1 min and adjusted to pH 7. The resulting mixture was then sonicated at 100 W for 2 min in cycles of 1 s followed by 0.5 s of pauses. The resultant solutions were subjected to particle size and zeta potential analysis. The entrapment efficiency (EE) and the loading efficiency (LE) of 5-FU-loaded self-assembled micelles were calculated as:

In vitro release profiles of 5-FU from drug-loaded mPEG-g-CS micelles were investigated for 12 h in the PBS solution (pH 7.4). The micelles (20 mg) and 2 mL of release medium were put into a dialysis tube (MWCO: 14,000 Da). The dialysis tube was placed in 48 mL of release medium at 37℃ and stirred continuously at 500 rpm. At specific time intervals,3 mL of solution was withdrawn from the release medium and replaced with fresh PBS (3 mL). The concentration of the released 5-FU was determined by UV spectrophotometer at 266 nm. The analysis was performed in triplicate for each sample. 3. Results and discussion

The synthetic route of mPEG-g-CS was depicted in Fig. 1. The chitosan and mPEG-g-CS polymer were characterized by 1HNMR, FT-IR and elemental analysis. 1HNMR spectra are presented in Fig. 2. In the spectrum of chitosan (Fig. 2A),the solvent peak of D2O was found at 4.7 ppm. Typical peaks at 3.4-3.8 ppm (H-c,H-d,H-e and H-f) were assigned to methyne protons of chitosan saccharide units. Peaks at 1.82 ppm (H-g) and 2.94 ppm (H-b) were attributed to -COCH3and -CHNH2from chitosan. In the spectrum of mPEG-gCS (Fig. 2B),the peaks at 3.10 ppm and 3.6 ppm (H-2,H-3,H-4) appeared,which were assigned to -OCH3 from mPEG and methylene protons of repeat units in PEG. The IR spectra of chitosan and mPEG-g-CS polymer are presented in Fig. 3. The chitosan spectrum showed characteristic bands of amide I (1644 cm-1 ) and amide II (1587 cm-1 ). The double peaks at 3356 and 3293 cm-1 in chitosan scaffolds corresponded to -NH2 stretching bands. For mPEG-g-CS (Fig. 3B),the chitosan amide peaks slightly shifted to 1632 cm-1 and 1525 cm-1 ,respectively. The shifts were possibly due to hydrogen bonding between amide carbonyl with PEG hydroxyl [18]. Furthermore,the increased intensity of the peaks at around 2884 and 1100 cm-1 indicated the CH2groups and C-O-C stretch of mPEG. These results show that the amino groups of chitosan were successfully substituted by mPEG groups. The substitution degree of mPEG on chitosan was calculated using the obtained elemental analysis values. The compositions of mPEG-g-CS are listed in Table 1. The results showed that the substitution degree of mPEG on chitosan was (3.23±0.25)%.

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Fig. 1. Synthetic pathway of mPEG g-CS polymer.

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Fig. 2. 1 H NMR spectra of (A) chitosan,(B) mPEG-g-CS.

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Fig. 3. FT-IR spectra of (A) chitosan,(B) mPEG-g-CS.

Table 1
Elemental analysis of mPEG-g-CS.
The solubility of the prepared mPEG-g-CS in aqueous solution was determined by measuring their optical transmittances. Higher transmittance represents better solubility. As shown in Fig. 4,the solubility of the prepared polymer was affected by the solution pH value. After chemically linked with mPEG,the solubility of chitosan derivative in aqueous solution at a pH range of 4-5 increased slightly. At pH 5.0,chitosan and the polymer studied showed an optical transmittance of>98%,indicating a good solubility in aqueous solution due to protonation of the amino groups on chitosan. At pH 6.1,chitosan formed a milky solution,which was an indication of large size aggregates suspended in the solution. However,due to mPEG substitution,the polymer solution began to aggregate at a higher pH value (pH 6.7).
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Fig. 4. The influence of pH on water solubility of chitosan and mPEG-g-CS (n= 3).
The effects of solution pH value,concentration of mPEG-g-CS, ionic strength and water/organic cosolvent on the characteristics of mPEG-g-CS micelles were investigated in this study. As seen from Tables 2 and 3,particle size and zeta potential of mPEG-g-CS micelles largely depended on these critical conditions. When the pH value was more than 7 or less than 4,micelles failed to form. At pH 7,the particle size of micelles formed was smaller than that of those formed under other solution pH. As the pH value decreased, the particle size increased. This was because of the protonation of amino groups on chitosan at low acidic pH,which could cause electrostatic repulsion between the entangled polymeric segments. This idea of protonation was also supported by the results that zeta potential of micelles increased with decreasing solution pH value. In addition,the ideal micelles were obtained with the concentration of mPEG-g-CS at 1 mg/mL (Table 2). Furthermore, when adding organic solvent into mPEG-g-CS solution,ideal micelles could also be obtained at pH 7 (Table 3). The morphology of the micelles was investigated by TEM. As shown in Fig. 5, micelles had spherical morphology and maintained non-aggregation state. The particle size ranged from 150 nm to 200 nm and was distributed uniformly.
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Fig. 5. TEM microgragh of mPEG-g-CS self-assembled micelles.

Table 2
Physicochemical properties of mPEG-g-CS self-assembled micelles.

Table 3
The influence of pH on mPEG-g-CS self-assembled micelles in cosolvent. a
CMC value of the self-assembled micelles was measured using a hydrophobic fluorescence probe,pyrene. The threshold concentration of micelle formation via intra- and/or inter-molecular entanglements of mPEG-g-CS was determined from the crossover point at the lower concentration range. As shown in Fig. 6A,the ratios of I3/I1 were nearly unchanged at low concentrations of mPEG-g-CS,whereas at higher concentrations,the ratios decreased,indicating self-aggregation of mPEG-g-CS. The calculated value of CMC of mPEG-g-CS micelles was about 264.8mg/mL in aqueous solution (pH 6.57),a relatively high value as compared to the literature [17],which was due to the low substitution degree of mPEG.
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Fig. 6. (A) Critical micelle concentration of mPEG-g-CS. (B) The influence of pH on core-shell integrity of 0.4 mg/mL mPEG-g-CS self-aseembled micelles (n= 3).
The influence of pH on core-shell integrity of the selfassembled micelles was investigated by the change of CMC because the property of pyrene can be utilized to study core/shell micelle formation and deformation. At concentrations above the CMC,the polymers self-assemble into core/shell micelles. The sharp change of intensity ratio (I3/I1) suggests the destruction of the micelle integrity. As shown in Fig. 6B,when pH increased from 1.5 to 4.0,the sharp change ofI3/I1compared with the intensity ratio under pH 6.57 was caused by the destruction of micelles. However,as the pH increasing from 4.0 to 7.0,the intensity ratio (I3/I1) changed slightly,which meant the integrity of micelles. Therefore,the self-assembled micelles should be prepared at a pH value higher than 4 and lower than 7. The blank mPEG-g-CS micelles were stored in the deionized water at 4℃ for 25 days. The change of micelle size is shown in Fig. 7. The results showed that the particle size of micelles changed inconspicuously from (198.6±20.0) nm to (240.5±15.1) nm within 25 days. In short,under a neutral condition,the micelles could maintain a stable micellar system.
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Fig. 7. Micelle size change of mPEG-g-CS micelles (n= 3).
The physiochemical properties of 5-FU-loaded micelles are summarized in Table 4. Loading efficiency (LE) increased with the increasing concentration of 5-FU. When the weight ratio of 5-FU to mPEG-g-CS was 5/10,the LE of the obtained micelles was (4.01±0.03)%. Moreover,the particle sizes of 5-FU-loaded micelles were larger than blank micelles. The size of mPEG-g-CS selfassembled micelles increased from (336.0±14.5) nm to (769.0±7.8) nm when the concentration of 5-FU increased,which might be due to the encapsulation of more 5-FU.
Table 4
Physicochemical properties of 5-FU-loaded mPEG-g-CS micelles. a
The in vitro cumulative release of 5-FU from self-assembled micelles was carried out in PBS (pH 7.4) at 37℃ and the results are shown in Fig. 8. It was characterized by an initial fast release phase followed by a delayed release. The burst release at 0.5 h of the 5-FU-laoded micelles was 34.7%. This phase (within the first 0.5 h) was mainly caused by desorption of the surface-bound or adsorbed drug. And the second phase was a relatively slow release up to 12 h of 76.9%,which attributed to the drug diffusion through the matrix and matrix erosion or degradation.
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Fig. 8. In vitro release of 5-FU from mPEG-g-CS micelles in the PBS (pH 7.4,n= 3).
4. Conclusion

In this study,amphiphilic polymer mPEG-g-CS was successfully synthesized by one-step method as a new carrier material for drug delivery. The chemical structure of mPEG-g-CS was characterized by 1HNMR,FT-IR and elemental analysis. After chemically linked with mPEG,the pH range for solubilizing chitosan derivative in aqueous solution became wider. The results of TEM photograph and DLS analysis indicated that the synthesized mPEG-g-CS could self-assemble into stable micelles with controlled size by ultrasonic method. Systematic design and modulation of the surface charge,particle size of mPEG-g-CS micelles could be achieved with the right control of critical processing parameters. Moreover,in vitro release study indicated that 5-FU was released from the micelles in a controlled-release manner. Therefore,we believe that mPEG-g-CS could be a potential carrier for controlled release of 5-FU for effective anti-tumor activity. Having laid the groundwork of the mPEG-g-CS micelles as a general efficient delivery system,we will direct our efforts toward applying it for other poorly water-soluble anti-tumor drugs to demonstrate their practical application potential. Acknowledgments

The authors acknowledge the financial support from the Fundamental Research Funds for the Central Universities (No. WY1213013ECUST). This work was also supported by Science and Technology Commission of Shanghai Municipality (STCSM,contract Nos.11DZ2260600 and 10DZ2220500).

References
[1] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS Nano 3 (2009) 16-20.
[2] T. Ngawhirunpat, N. Wonglertnirant, P. Opanasopit, et al., Incorporation methods for cholic acid chitosan-g-mPEG self-assembly micellar system containing camptothecin, Colloids Surf. B: Biointerfaces 74 (2009) 253-259.
[3] X.H. Peng, L. Zhang, Self-assembled micelles of N-phthaloyl-carboxymethy chitosan for drug delivery, Colloids Surf. A: Physicochem. Eng. Asp. 337 (2009) 21-25.
[4] G. Gaucher, M.H. Dufresne, V.P. Sant, et al., Block copolymer micelles: preparation, characterization and application in drug delivery, J. Control. Release 109 (2005) 169-188.
[5] A.A. Sunil, N.M. Nadagouda, M.A. Tejraj, Recent advances on chitosan-based micro- and nanoparticles in drug delivery, J. Control. Release 100 (2004) 5-28.
[6] Z.T. Yuan, Y.J. Ye, F. Gao, et al., Chitosan-graft-β-cyclodextrin nanoparticles as a carrier for controlled drug release, Int. J. Pharm. 446 (2013) 191-198.
[7] S.S. Gao, J. Sun, F. Gao, et al., Preparation, characterization and pharmacokinetic studies of tacrolimus-dimethyl-β-cyclodextrin inclusion complex-loaded albumin nanoparticles, Int. J. Pharm. 427 (2012) 410-416.
[8] Y. Sun, L. Gu, Y. Gao, Preparation and characterization of 5-fluorouracil loaded chitosan microspheres by a two-step solidification method, Chem. Pharm. Bull. 58 (2010) 891-895.
[9] Q. Gan, T. Wang, Chitosan nanoparticle as protein delivery carrier - systematic examination of fabrication conditions for efficient loading and release, Colloids Surf. B: Biointerfaces 59 (2007) 24-34.
[10] A.W. Wu, B.B. Wu, J.M. Wu, et al., Chitosan nanoparticles crosslinked by glycidoxypropyltrimethoxysilane for pH triggered release of protein, Chin. Chem. Lett. 20 (2009) 79-83.
[11] X.Y. Kong, X.Y. Li, X.H. Wang, et al., Synthesis and characterization of a novel mPEG-chitosan diblock copolymer and self-assembly of nanoparticles, Carbohydr. Polym. 79 (2010) 170-175.
[12] A. Miwa, A. Ishibe, M. Nakano, et al., Development of novel chitosan derivatives as micellar carriers of taxol, Pharm. Res. 15 (1998) 1844-1850.
[13] C. Zhang, P. Qineng, H.J. Zhang, Self-assembly and characterization of paclitaxelloaded N-octyl-O-sulfate chitosan micellar system, Colloids Surf. B: Biointerfaces 39 (2004) 69-75.
[14] D.W. Zhu, J.G. Bo, K.D. Yao, et al., Synthesis of N-methylene phosphonic chitosan (NMPCS) and its potential as gene carrier, Chin. Chem. Lett. 18 (2007) 1407-1410.
[15] P. Chan, M. Kurisawa, J.E. Chung, et al., Synthesis and characterization of chitosang- poly (ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery, Biomaterials 28 (2007) 540-549.
[16] A.J. Dong, M.H. Feng, H.Y. Qi, et al., Synthesis and properties of O-carboxymethyl chitosan/methoxy poly (ethylene glycol) graft copolymers, J. Mater. Sci. Mater. Med. 19 (2008) 869-876.
[17] S.Y. Zhu, F. Qian, Y. Zhang, et al., Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticle, Eur. Polym. J. 43 (2007) 2244-2253.
[18] A.R. Kulkarni, Y.H. Lin, H.F. Liang, et al., A novel method for the preparation of nanoaggregates of methoxy polyethyleneglycol linked chitosan, J. Nanosci. Nanotechnol. 6 (2006) 2867-2873.
[19] T. Peng, Y. Li, D. Ahn, et al., Synthesis and characterization of pH-responsive poly (2-hydroxyethyl aspartamide)-g-poly (b-amino ester) graft copolymer micelles as potential drug carriers, Macromol. Res. 21 (2013) 400-405..