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
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. |
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Fig. 4. The influence of pH on water solubility of chitosan and mPEG-g-CS (n= 3). |
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Fig. 5. TEM microgragh of mPEG-g-CS self-assembled micelles. |
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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). |
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Fig. 7. Micelle size change of mPEG-g-CS micelles (n= 3). |
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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). |
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).
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