Formation and Morphologies of Novel Self-Assembled Micelles from

Sep 18, 2007 - We successfully synthesized N-phthaloyl-carboxymethylchitosan (CMPhCh) from chitosan. CMPhCh could be self-assembled to form various ...
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Langmuir 2007, 23, 10493-10498

10493

Formation and Morphologies of Novel Self-Assembled Micelles from Chitosan Derivatives Xianghong Peng†,‡ and Lina Zhang*,† Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China and College of Chemistry and EnVironmental Engineering, Jianghan UniVersity, Wuhan 430056, China ReceiVed May 9, 2007. In Final Form: July 30, 2007 We successfully synthesized N-phthaloyl-carboxymethylchitosan (CMPhCh) from chitosan. CMPhCh could be self-assembled to form various morphologies of crew-cut micelle-like aggregates using a mixed solution of water and N,N-dimethylformamide (DMF). The results of scanning electron microscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS) revealed that the morphologies of the aggregates exhibited vesicles, vesicle-encapsulating vesicles, onion-like vesicles, and large compound micelles (LCM). Their morphologies were changed with varying the concentration of CMPhCh, the ratio of DMF in the mixture, and the self-assembly process. In relatively high CMPhCh concentration and content of DMF, CMPhCh was self-assembled to form the onion-like vesicles with a thin wall and 80-240 nm diameter. For the first time, TEM observation provided straightforward evidence of the onion-like vesicles prepared from natural polymer.

Introduction Recently, block copolymers which can be self-assembled into various morphologies of crew-cut aggregates in a selective solvent have attracted much attention.1-4 Depending on the structure of the block copolymers there are two types of micelles, including star and crew-cut aggregates. Crew-cut aggregates exhibit various morphologies such as spheres, rods, lamellae, vesicles, large compound micelles, etc.5-9 The morphologies of crew-cut aggregates can be adjusted by tuning the block weight fraction and the composition of select solvent.10-12 Crew-cut aggregates have great potential for encapsulation of a large amount of guest molecules.13 Among them, vesicles have been demonstrated to be particularly interesting for application of encapsulation agents.14-16 Polymeric vesicles not only have superior stability and toughness but also offer numerous possibilities of tailoring the physical, chemical, and biological properties by variation of * To whom correspondence should be addressed. Phone: +86-2787219274. Fax: +86-27-68754067. E-mail: [email protected]. † Wuhan University. ‡ Jianghan University. (1) Shen, H.; Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1999, 121, 27282740. (2) Determan, M. D.; Guo, L.; Thiyagarajan, P.; Mallapragada, S. K. Langmuir 2006, 22, 1469-1743. (3) Won, Y. Y.; Brannan, A. K.; Davis, H. T.; Bates, F. S. J. Phys. Chem. B 2002, 106, 3354-3364. (4) Zhao, Y.; Liang, H.; Wang, S.; Wu, C. J. Phys. Chem. B 2001, 105, 848851. (5) Borisov, O. V.; Zhulina, E. B. Langmuir 2005, 21, 3229-3231. (6) Yu, K.; Zhang, L.; Eisenberg, A. Langmuir 1996, 12, 5980-5984. (7) Maskos, M.; Harris, J. R. Macromol. Rapid. Commun. 2001, 22, 271-273. (8) Lei, L.; Gohy, J. F.; Willet, N.; Zhang, J.; Varshney, S. Macromolecules 2004, 37, 1089-1094. (9) Liu, X.; Kim, J.; Wu, J.; Eisenberg, A. Macromolecules 2005, 38, 67496751. (10) Zhang, W.; Shi, L.; An, Y.; Gao, L.; Wu, K.; Ma, R. Macromolecules 2004, 37, 2551-2555. (11) Quarti, N.; Viville, P.; Lazzaroni, R.; Minatti, E.; Schappacher, M.; Deffieux, A.; Borsali, R. Langmuir 2005, 21, 1180-1186. (12) Adams, D. J.; Butler, M. F.; Weaver, A. C. Langmuir 2006, 22, 45344540. (13) Dou, H.; Jiang, M.; Peng, H.; Chen, D.; Hong, Y. Angew. Chem., Int. Ed. 2003, 42, 1516-1519. (14) Lim Soo, P.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923-938. (15) Che´cot, F.; Bruˆlet, A.; Oberdisse, J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux, S. Langmuir 2005, 21, 4308-4315. (16) Vial, F.; Rabhi, S.; Tribet, C. Langmuir 2005, 21, 853-862.

block lengths, chemical structure, and conjugation with biomolecules.17,18 Therefore, they have great potential application for encapsulation and stabilization of active ingredients in the cosmetic, pharmaceutical, and environmental fields.19-21 In the past two decades, onion-like vesicle, a special vesicle, has been investigated in small molecules surfactant system, pluronic system, and block-copolymer solutions.22-26 Most studies of crewcut aggregates have focused on the diblock, triblock, and graft copolymers because they are easily synthesized. However, the multiple morphologies of crew-cut aggregates prepared from natural polymers and their derivatives have scarcely been reported because of their complex structure. To date, onion-like vesicles from natural polysaccharides such as chitin have never been published. In the 21st century science and technology have moved toward renewable raw materials and more environmentally friendly and sustainable resources and processes.27 Wide application of chitin and chitosan from annually renewable resources have been found in the field of biomaterials, especially as drug and gene carriers because of their biocompatibility and nontoxicity.28,29 It has been reported that chitosan derivatives or a chitosan-surfactant mixture can be self-assembled to form spherical nanoparticles in aqueous solution.30-33 In our previous work hollow microspheres were successfully prepared using the cyclohexane droplets as the (17) Antoniett, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323-1333. (18) Harris, J. K.; Rose, G. D.; Bruening, M. L. Langmuir 2002, 18, 53375342. (19) Choucair, A.; Lim Soo, P.; Eisenberg, A. Langmuir 2005, 21, 93089313. (20) Nguyen, P. M.; Hammond, P. T. Langmuir 2006, 22, 7825-7832. (21) Rodrı´gurz-Herna´ndez, J.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026-2027. (22) Sein, A.; Engberts, J. B. F. N. Langmuir 1993, 9, 1714-1720. (23) Danino, D.; Talmon, Y.; Zana, R. J. Colloid Interface Sci. 1997, 185, 84-93. (24) Evans, C. C.; Zasadzinski, J. Langmuir 2003, 19, 3109-3113. (25) Tsitsilianis, C.; Voulgaris, D. Langmuir 2000, 16, 6868-6876. (26) Shen, H.; Eisenberg, A. Angew. Chem., Int. Ed. 2002, 39, 3310-3312. (27) Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A. Angew. Chem., Int. Ed. 2005, 44, 3358-3393. (28) Zhu, C.; Lee, J.; Raghavan, S. R.; Payne, G. F. Langmuir 2006, 22, 2951-2955. (29) Strand, S. P.; Danielsen, S.; Christensen, B. E.; Vårum, K. M. Biomacromolecules 2005, 6, 3357-3366. (30) Peng, X.; Zhang, L. Langmuir 2005, 21, 1091-1095. (31) Grant, J.; Cho, J.; Allen, C. Langmuir 2006, 22, 4327-4335.

10.1021/la701325v CCC: $37.00 © 2007 American Chemical Society Published on Web 09/18/2007

10494 Langmuir, Vol. 23, No. 21, 2007 Scheme 1. Synthetic Route for Preparation of N-Phthaloyl-carboxymethylchitosan (CMPhCh)a

a (A) Phthalic anhydride, DMF, 130 °C. (B) Chloroacetic acid, isopropyl alcohol 60 °C.

template and N-methylated chitosan cross-linked with gultaraldehyde as the shell.30 Thus, it motivated us to investigate the unique morphologies of crew-cut aggregates prepared by chitosan. In present paper, we focus on preparation of N-phthaloylcarboxymethylchitosan (CMPhCh) and its self-assembly in a mixture solution of water and N,N-dimethylformamide (DMF). Their morphologies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). This work may contribute some meaningful information to understand the self-assembly behavior in the fields of colloid and surface chemistry for the complex polysaccharides, which are naturally occurring polymers and possess safeness, biocompatibilities, and some bioactivities. Experimental Section Sample Preparation. All of the chemical reagents were commercially obtained from China and of analytical grade. Chitosan (Ch) having a weight-average molecular weight (Mw) of 13.44 × 104 and 82.4% deacetylation degree was purchased from Yuhuan Ocean Biochemistry Co. Ltd. in Zhejiang, China. The degree of deacetylation of chitosan was determined by the C/N value from elemental analysis. N-Phthaloylchitosan was prepared according to the procedure described by Holappa et al.34 A 10 g amount of chitosan was dispersed in 200 mL of N,N-dimethylformamide (DMF) and stirred for 24 h. To the resultant solution, phthalic anhydride (20 g) was added, and the mixture was stirred at 130 °C for 10 h. The reacted mixture was poured into ice water, centrifuged, and washed with ethanol to obtain N-phthaloylchitosan, coded as PhCh. A 2 g amount of PhCh was mixed with 40 wt % aqueous NaOH and then kept overnight at -18 °C. The mixture was dissolved in 40 mL of isopropyl alcohol, and then 5.5 g of chloroacetic acid was added into the mixture solution and kept at room temperature for 2 h and at 60 °C for 2 h. The resultant solution was neutralized with 0.1 M HCl at 40 °C, yielding a precipitate. The precipitate was dissolved in water, dialyzed in distilled water for 3 days, and centrifuged to obtain a clear solution, which was evaporated and precipitated with acetone to obtain N-phthaloyl-carboxymethylchitosan after vacuum drying, coded as CMPhCh. The synthesis route for preparation of CMPhCh is shown in Scheme 1. CMPhCh was dissolved in water and dialyzed in DMF solvent for 2-5 days to obtain self-assembly aggregates in a mixture solution. The ratio of DMF in the mixture solution was changed from 67 to 90 wt %. By changing the concentration of CMPhCh and the ratio of DMF in the mixture solution we obtained a series of crew-cut aggregates. Their conditions and the sample code are listed in Table 1. Characterization. Fourier transform infrared spectroscopy (FTIR) was recorded on a Nicolet 5700 spectrometer (Nicolet, Minnesota) in a range from 4000 to 400 cm-1 using a KBr-pellet method. 1H (32) Liu, C.; Desai, K. G. H.; Chen, X.; Park, H. J. J. Agric. Food Chem. 2005, 53, 437-441. (33) Bodnar, M.; Hartmann, J. F.; Borbely, J. Biomacromolecules 2005, 6, 2521-2527. (34) Holappa, J.; Nevalainen, T.; Savolainen, J.; Soininen, P.; Elomaa, M.; Safin, R.; Suvanto, S.; Pakkanen, T.; Loftsson, T. M. M.; Ja¨rvinen, T. Macromolecules 2004, 37, 2784-2789.

Peng and Zhang NMR and 13C NMR spectra for CMPhCh in D2O were recorded on an INOVA-300 MHz and INOVA-600 MHz (Varian Inc., Palo, Alto, CA) at 25 °C. TEM observation of the crew-cut aggregates was carried out with a transmittance electron microscope [JEM2010 FEF (UHR), JEOL, Tokyo, Japan] at an accelerating voltage of 200 kV and TEM (H-8100, Hitachi, Japan) at an accelerating voltage of 70 kV. A droplet of the sample solution was supported on a carbon-coated copper grid and dried in the air at room temperature for TEM measurement. In addition, the morphologies of the aggregates were observed by a scanning electron microscope (Sirion FEG-SEM’FEI Co., Holland). The samples were coated with gold for SEM observation. The Mw value of CMPhCh was determined by size-exclusion chromatography (SEC) combined with laser light scatting (LLS, DAWN-DSP, Wyatt Technology Co., Santa Barbara, CA). SECLLS measurements were carried out on size-exclusion chromatograph combined with the multiangle laser photometer mentioned above. A P100 pump (Thermo Separation Products, San Jose, CA) equipped with G4000PWXL (MicroPak, TSK) and G6000PWXL (MicroPak, TSK) columns was used as the SEC instrument. A differential refractive index detector (RI-150, USA) was simultaneously connected. The carrier solution was 0.1 M aqueous NaCl. The sample was dissolved in 0.1 M aqueous NaCl. The solvents and polysaccharide solutions were purified by a 0.2 µm filter and degassed before use. The injection volume was 200 µL with a concentration from 0.05 to 0.16 mg/mL for the sample, and the flow rate was 1.0 mL/min in 0.1 M aqueous NaCl at 25 °C. The refractive index increment (dn/dc) of CMPhCh in 0.1 M NaCl aqueous was measured with a double-beam differential refractometer (DRM-1020, Otsuka Electronics Co., Tokyo, Japan) at 633 nm to be 0.168 mL g-1. Astra software (version 4.90.07) was utilized for data acquisition and analysis. The hydrodynamic diameter (Dh) and its distribution of CMPhCh in the mixture solution of H2O-DMF at 25 °C were measured on a Zetasizer Nano particle analyzer (Malvern Instruments Ltd., U.K.) using dynamic light scattering (DLS).

Results and Discussion Structure of CMPhCh. FT-IR spectra of chitosan, PhCh, and CMPhCh are shown in Figure 1. The characteristic peaks at 1649 and 1599 cm-1 are assigned to the amide I and II bands of chitosan, respectively. It is worth noting that the peak at 1599 cm-1 for primary amine N-H bending almost disappears in PhCh as a result of acylation at C-2 of the glucosamine unit. The peaks at 1777 and 1713 cm-1 indicate that a CdO imide group exists in PhCh. In addition, the peaks at 1392 and 722 cm-1 of PhCh are assigned to CdC of phthaloyl groups. There are the peaks at 1642 and 1587 cm-1 in the FT-IR spectrum of CMPhCh, assigned to carboxyl groups and acetyl groups, respectively. The results from FT-IR indicate that a carboxymethylated reaction occurred between PhCh and chloroacetic acid in the isopropyl alcohol solution. Figure 2 shows the 1H NMR and 13C NMR spectra of CMPhCh in D2O. The peaks at 7.26, 7.30, 7.35, and 7.69 ppm in the 1H NMR spectrum are assigned to the phthaloyl groups. The peaks at 1.67 and 1.96 ppm indicate that there are the methyl groups in CMPhCh. The peaks at 127.1, 128.2, 129.5, 130.9, 133.4, and 138.0 ppm in 13C NMR are assigned to the phthalimide protons.34 The peaks at 176.4, 177.5, and 172.8 ppm are assigned to the CdO of the acetyl and carboxyl groups, respectively.35 In view the results from FT-IR and NMR, it is considered that chitosan has been reacted with phthalic anhydride to yield PhCh and then with chloroacetic acid to produce water-soluble CMPhCh. According to the C/N value from elemental analysis and the 1H NMR spectrum the molecular structure of CMPhCh was calculated to be (C14H13NO6)0.345(C16H15NO8)0.368(C8H13NO5)0.085(C10H15NO7)0.091(C8H13NO6)0.054(C10H15NO8)0.057: C, 43.61; H, (35) Baumann, H.; Faust, V. Carbohydr. Res. 2001, 331, 43-57.

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Table 1. Concentration (c) and Ratio of DMF in Mixture Solution as Well as the Hydrodynamic Diameter (Dh) during Formation of CMPhCh Aggregates CMPhChI

a

CMPhChII

samples

CMPhChI-1

CMPhChI-2

CMPhChII-1

CMPhChII-2

CMPhChII-3

c (mg/mL) DMF (wt %) Dh (nm)

0.2 67 61 ( 6; 486 ( 53

0.2 86 73 ( 8; 503 ( 58

22.2 67 576 ( 69

22.2 75 518 ( 48

22.2 90 a

Dh of CMPhChII-3 not determined.

Figure 3. Hydrodynamic diameter (Dh) distribution of CMPhChI (a) and CMPhChII (b) in a mixture solution containing different amounts of DMF. Figure 1. FT-IR spectra of chitosan, PhCh, and CMPhCh.

Figure 2. 1H NMR and 13C NMR spectra of CMPhCh in D2O at 25 °C.

3.83; N, 3.82. Found: C, 43.61; H, 5.13; N, 3.82. Chitosan was phthaloylated to yield N-phthaloylchitosan (PhCh), as shown in Scheme 1. The phthaloylation took place at the amino groups at the reaction conditions, as pointed out by Holappa et al.34 The degree of substitution of the phthaloyl group was 0.713. Then part of the hydroxyl groups of PhCh was replaced by carboxymethyl groups to yield N-phthaloyl-6-O-carboxymethylchitosan (CMPhCh); the degree of substitution of carboxymethyl groups was 0.516. The values of Mw, the square radius of gyration (〈S2〉z1/2), and the second virial coefficient (A2) were determined

by SEC-LLS from a Zimm plot (not shown here) to be 2.01 × 105, 49.0 nm, and 9.24 × 10-3 mol‚cm3‚g-2, respectively. The A2 value is positive, suggesting that 0.1 M NaCl is a good solvent for CMPhCh. According the molecular structure of chitosan, the calculated Mw value of CMPhCh is 2.48 × 105, close to that measured by SEC-LLS. It is believed that CMPhCh exists as a single chain in water and 0.1 M NaCl aqueous solution rather than an aggregate at the lower concentration of 0.05-0.16 mg/ mL. Formation of Aggregates and Vesicles. The morphologies of the macromolecular aggregates depend on the balance of the attractive and repulsive forces, including the stretching of the core-forming segments, the intercoronal interactions, and the free energy of the interface between the solvent and the micelle.36 It is noted that CMPhCh is easily dissolved in water and difficult to dissolve in DMF solvent. In addition, the phthaloylchitosan (PhCh) could be dissolved or swollen in DMF because of the hydrophobic segments.37 Water is a good solvent for both hydrophilic and hydrophobic segments of CMPhCh, whereas DMF is a good solvent only for the hydrophobic segments and a precipitant for the hydrophilic segments of CMPhCh. Thus, the mixture of water and DMF was used as a selective solvent for CMPhCh in the self-assembly progress. The effects of the concentration of CMPhCh, the ratio of DMF in a mixture solution, and the self-assembly process on the morphologies of aggregates have been studied with DLS, TEM, and SEM. Figure 3 shows the hydrodynamic diameter distribution (Dh) of CMPhChI and CMPhChII determined by DLS. At relatively high CMPhCh (36) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1999, 118, 3168-3181. (37) Nishimura, S. I.; Kohgo, O.; Kurita, K. Macromolecules 1991, 24, 47454748.

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Figure 4. TEM image of CMPhChI-2 vesicle (a), and SEM image of CMPhChI-2 (b).

concentration, the Dh value of CMPhChII-1 is larger than that of CMPhChI-1. It is noted that the Dh patterns of CMPhChI exhibit two peaks: one corresponds to aggregates with large Dh and the other to a single chain. The distribution of Dh of CMPhChI is broader than that of CMPhChII. This suggests that a single chain and aggregates coexist in the mixture solution at low CMPhCh concentration. The results prove that with an increase of the CMPhCh concentration and DMF content the CMPhCh chains aggregate gradually and the size and content of the aggregates increase. As CMPhCh concentration reaches 22.2 mg/mL, only aggregates have been found in solution, as shown in Figure 3b. For a given polymer solution the ratio of the geometric to hydrodynamic radius (F ) 〈S2〉z1/2/Rh, Dh ) 2Rh) is related to the chain architecture and conformation. Many experimental results have verified the F value in the range of 1.5-1.7 for flexible linear polymers in good solvent,38 whereas that for a homogeneous sphere is 0.78. An approximated F value of the ratio of 〈S2〉z1/2 to Rh was estimated from the data to be 1.6 for CMPhCh I-1 and 1.4 for CMPhCh I-2. The F values are close to that of the flexible polymers, suggesting a pliable chain structure of CMPhCh. It implies that CMPhCh would form coil or sphere aggregates in aqueous solution. Figure 4 presents the morphologies of CMPhChI-2 as vesicles. Amphiphilic block copolymers have been proven to be useful precursors for preparation of vesicles,39 so CMPhCh, a kind of amphiphilic polymer, has formed the vesicles. The results indicate that at the relatively low concentration of CMPhCh (0.2 mg/ mL), with an increase of the ratio of DMF from 67 to 86 wt %, CMPhCh could be self-assembled to form vesicles. During (38) Park, S.; Chang, T.; Park, I. H. Macromolecules 1991, 24, 5729-5731. (39) Hu, Z.; Jonas, A. M.; Varshney, S. K.; Gohy, J. F. J. Am. Chem. Soc. 2005, 127, 6526-6527.

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dialysis DMF gradually diffuses into the dialysis bag, and the quality of the solvent for the hydrophilic segments gradually became poor. Therefore, the hydrophobic segments formed the corona of the aggregates and the hydrophilic segments were involved into the core of the aggregates. The CMPhCh chains in the solution system would continue to aggregate in the interface of the vesicles, leading to multilayer vesicles, as shown in Figure 4a. The diameter of vesicles lies in the range from 70 to 200 nm, and the thickness of the wall is about 20 nm. The hydrodynamic diameter of the vesicles determined by DLS is in the range from 73 to 503 nm, as shown in Table 1. It is noted that Dh of vesicles is larger than the diameter observed by TEM because the vesicles have been swollen in the mixture solution whereas TEM has been performed at the dry state. Further, the spherical shape of vesicles has also been proved by the SEM photographs, as shown in Figure 4b. The size of aggregates in the solid state measured with SEM is smaller than that from TEM. This can be explained by the further constriction of aggregates during coating with gold at the vacuum state. Onion-like Vesicles. The probability or chance of the molecules chain colliding together in the system would be increased at a high concentration of amphiphilic block polymer. Therefore, polymer with relatively high concentration can be more easily self-assembled to form crew-cut aggregates in the selective solvent, including large vesicle encapsulating small vesicles, onion-like vesicles, and large compound micelles, etc.6,26 As a result of the amphiphilic chitosan derivative, CMPhCh could form onion-like vesicles. Figure 5 shows the TEM photographs of aggregates from CMPhChII-1 having a relatively high concentration (22.2 mg/mL). The image in Figure 5a exhibits large vesicle encapsulating small vesicles, similar to the pregnant vesicles.40 In the relatively high polymer concentrations with an increase of the ratio of DMF in solution, large vesicles have occurred in solution as a result of the incompatibility between the core-forming segments and DMF and the balance of forces operating in solution self-assembly.26 Formation of large vesicle encapsulating small vesicles can be described as follows. When water was removed and DMF diffused into large vesicles, smaller vesicles were formed inside the large vesicle by self-assembly of the entrapped polymer. The process is similar to formation of the outermost large vesicle.23 Broken vesicles are also observed (arrowhead in Figure 5b). Usually, encapsulation of small vesicles or colloidal particles within a phospholipid bilayer affords multicompartment structures. Such interesting structure has also been exploited in targeted drug delivery, imaging, and separations, especially in the field of efficient delivery for drug release.31 Figure 6 shows the TEM photographs of onion-like vesicles of CMPhChII-2. DMF is a good solvent only for the hydrophobic segments and a precipitant for the hydrophilic segments of CMPhCh. Further increasing the ratio of DMF the hydrophobic segments tend outside to form the corona and the hydrophilic segments are involved inside to form the core of the aggregates, leading to formation of the vesicles compound. During this process the small vesicles encapsulated in vesicles have been aggregated to form the multilamellar vesicles compound. This can be explained as follows: CMPhCh chains in the mixture solution could be self-assembled at the interface of the vesicle shell, leading to multilamellar vesicles. Therefore, the aggregation process would continue to form the onion-like vesicles, even their aggregates. The number of layers depends on the polymer concentration and size of the outermost vesicles. This process is similar to onion-like vesicles formed from small(40) Yu, K.; Eisenberg, A. Macromolecules 1998, 31, 3509-3518.

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Figure 6. TEM images of onion-like vesicles of CMPhChII-2 (a); the vesicle at higher magnification (b).

Figure 5. TEM images of CMPhChII-1: large vesicle encapsulating small vesicles (a) and the broken large vesicles (b).

molecule amphiphilic systems and block copolymer.41 The aggregates of CMPhChII-2 have a compact multilamella structure and onion-like spherical shape with 80-240 nm overall diameter. The onion-like vesicle consists of more than five layers and a wall thickness from 2 to 4 nm, as shown in Figure 6b. Comparing with the onion-like vesicles prepared from PS132-b-PAA20 with 500-1200 nm diameter and 22 nm wall thickness,26 onion-like vesicles formed by CMPhCh have a small diameter and thin wall. Such results can be explained due to the molecule weight of CMPhCh being higher than the block copolymer, so the onionlike vesicles from CMPhCh become stronger and more stable in solution, leading to easy formation of the small multilamellar vesicles having a thin wall. The hydrodynamic diameter Dh of the onion-like vesicles self-assembled from CMPhChII-2 is 518 ( 48 nm with a narrow distribution, which is larger than that of CMPhChI-2 at low concentration. The onion-like vesicles formed with block copolymer are spherical shape with fairly smooth internal layers and more rugged outer layers.26 It is noted that the morphologies of onion-like vesicles from chitosan derivatives are similar to that from block copolymer, but there is a lack of a clear interface because of the complex structure for the N-phthaloyl-carboxymethylchitosan. The unique onionlike vesicles are considered for potential control release delivery because the drug has to have gradual diffusion through the walls and be broken with erosion of the layers during the progressive (41) Bergmeier, M.; Gradzielski, M.; Hoffmann, H.; Mortensen, K. J. Phys. Chem. B 1999, 103, 1605-1617.

release. Most of the onion-like vesicles are composed of block copolymer and small molecules surfactant. For the first time, onion-like vesicles self-assembled by chitosan derivatives that are safe have been reported here. On the basis of the results from DLS, SEM, and TEM, a schematic illustration of formation of the onion-like vesicles is presented in Figure 7. CMPhCh exists as a flexible chain in aqueous solution, as shown in Figure 7a. In the mixture solution of water and DMF, as water diffuses out and DMF diffuses into the dialysis bag, the CMPhCh chains have been self-assembled to form the outside large vesicles (Figure 7b). The CMPhCh chains inside the vesicles gradually aggregate to form the small vesicles, leading to the large vesicle encapsulating small vesicles, corresponding to the TEM image in Figure 5a and the DLS results in Figure 3a. With increasing CMPhCh concentration, DMF content, and time, the small vesicles could be further aggregated to form vesicles aggregates in which CMPhCh chains in the solution were gradually aggregated at the interface of the compound of the vesicles. The process continues to form the onion-like vesicles, as shown in Figure 7c, corresponding to the DLS results in Figure 3b. TEM images in Figure 6 provide straightforward evidence for the onion-like vesicles. Therefore, the onion-like vesicles from natural polymers such as chitosan as raw material were observed directly with TEM. The onionlike vesicles are a kind of biocompatible and safe vesicle and could find broad application as time-release devices in biomedicine. Moreover, the morphologies of crew-cut aggregates can be controlled easily by adjusting the concentration of CMPhCh, the ratio of DMF in the solution, and the dialysis time. Compound Micelles. Figure 8 shows the TEM photograph of CMPhCh II-3 as large compound micelles (LCM). As the content of DMF reaches 90 wt % and the dialysis time was 5 days, secondary aggregation of the spherical micelles of CMPhCh

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Figure 7. Schematic formation process of the onion-like vesicles: CMPhCh as flexible chains in aqueous solution (a), as a single chain and aggregates of a large vesicle encapsulating small vesicles in a mixture solution of H2O-DMF (b), and as onion-like vesicles in H2O-DMF system with increasing time and polymer concentration (c).

Figure 8. TEM image of large compound micelles (CMPhChII-3) with a dialysis time of 5 days and observed by H-8000 TEM, stained with 2% phosphotungstic acid.

occurred to form LCM. The diameter of LCM observed by TEM is from 1 to 4 µm. The Dh value of LCM could not be determined by DLS because of the high viscosity of LCM. It can be explained that the attractive force driving aggregation increases at a high ratio of DMF in the solution having a high concentration of CMPhCh. In addition, more and more micelles collide together to form LCM to reduce the interface energy of the system as the dialysis time increases. The LCM from CMPhCh is larger than that of PS200-b-PAA4,36 suggesting that LCM could encapsulate more drugs inside as delivery.

Conclusion N-Phthaloyl-carboxymethylchitosan (CMPhCh) was successfully prepared by reacting N-phthaloylchitosan with chloroacetic

acid in isopropyl alcohol. Dynamic light scattering revealed that CMPhCh existed as a flexible chain in the aqueous solution and aggregated gradually to form the sphere aggregates in the mixture solution of H2O-DMF. TEM showed that CMPhCh could be self-assembled to form various morphologies of crew-cut aggregates including vesicles, vesicle encapsulating vesicles, onion-like vesicles, and large compound micelles in the mixture system. At low CMPhCh concentration (0.2 mg/mL) and a ratio of DMF from 67 to 86 wt %, part of CMPhCh was self-assembled to form the vesicles. At relatively high CMPhCh concentration (22.2 mg/mL) and a content of DMF of 75 wt %, CMPhCh was self-assembled to form onion-like vesicles as a result of the selfassembly at the interface of the vesicle shell. For the first time, onion-like vesicles self-assembled from natural polymer derivative were observed with TEM. As the ratio of DMF reached 90 wt % and the dialysis time was 5 days, large compound micelles with a size of 1-4 µm were formed. The crew-cut aggregates with various morphologies and size could be controlled by the concentration of CMPhCh, the ratio of DMF in the mixture solution, and the self-assembly process. Acknowledgment. This work was financially supported by a major grant from the National Natural Science Foundation of China (59933070 and 30530850) and National Natural Science Foundation of China (20474048 and 20404010). The authors thank the Center for Electron Microscopy of Wuhan University for TEM measurements. Supporting Information Available: N-Phthaloyl-carboxymethylchitosan (CMPhCh) was self-assembled to form crew-cut micellelike aggregates in a mixture of water and N,N-dimethylformamide; TEM images of the onion-like vesicles with 80-240 nm diameter and thin wall (Figure S1); TEM image of the large compound micelles with size 1-4 µm (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. LA701325V