Preparation of Uniformly Sized Chitosan Nanospheres by a Premix

Ind. Eng. Chem. Res. 2009, 48, 8819–8828

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Preparation of Uniformly Sized Chitosan Nanospheres by a Premix Membrane Emulsification Technique Pi-Ping Lv,†,‡ Wei Wei,† Fang-Ling Gong,† Yue-Ling Zhang,† Hui-Ying Zhao,‡ Jian-Du Lei,† Lian-Yan Wang,*,† and Guang-Hui Ma*,† National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China, and School of Life Sciences and Technology, Beijing UniVersity of Chemical Technology, Beijing, 100029, China

Chitosan nanospheres have great potential in drug controlled release systems, because they show excellent degradability, biocompatibility, and nontoxicity. The particle size control and size distribution of nanospheres are necessary in order to improve reproducibility, bioavailability, and repeatable release behavior. In order to prepare uniformly sized and size-controllable chitosan nanospheres, in this study, a premix membrane emulsification technique was developed on the basis of direct membrane emulsification, and the uniformly sized chitosan naonospheres were successfully prepared by optimizing preparation conditions. The detail preparation process is as follows: The chitosan aqueous solution was used as a dispersed phase, and the mixture of liquid paraffin and petroleum ether containing emulsifier was used as a continuous phase. The coarse emulsions were first prepared by low-speed stator homogenization and then poured into the premix reservoir. Nanodroplets were achieved by extruding the coarse emulsions through the SPG (Shirasu porous glass) membrane with a high pressure. The nanodroplets were further cross-linked to obtain chitosan nanospheres. In this process, several factors played key roles in obtaining chitosan nanoparticles with narrow size distribution, including the amounts of emulsifier in oil phase, the composition of oil phase, the concentration of chitosan, the ratio of water to oil phase, the transmembrane pressure and number of passes, and so on. The results showed that the chitosan nanospheres from 300 nm to 1.85 µm were successfully prepared by premix membrane emulsification by changing the pore size of the membrane and the polydispersity index could be as low as 0.027 under optimized conditions, and it is a potential technique to prepare size-controllable uniform chitosan nanospheres with fast production. 1. Introduction With the rapid development of DNA-recombinant techniques and other modern biotechnology, more and more protein and peptide drugs, which are becoming a very important class of therapeutic agents, can be produced on a large scale in recent years.1–5 However, most of them are administered by veinal or parenteral injection because their bioavailabilities via orally administration are generally very low. Moreover, they are also easily degraded by enzymes in vivo, and most protein and peptide drugs pass through biological barriers poorly due to their poor diffusivity, which is unfavorable for use in lipid membrane.6,7 Consequently, the research on biodegradable polymer nanospheres has been much accounted in the field of biological medicine.8 Nanospheres are a kind of particle with diameters of less than 1 µm. As a drug delivery system, a nanoparticle has many advantages, such as prolonging the half-life time of drugs in plasma, protecting drugs from degradation by enzymes in vivo, improving the bioavailability, expanding the route of administration, and reducing drug side-effects.9,10 Moreover, better nanoformulation can also realize the controlled-release and target-oriented release for drug. In our previous study, the results showed that the smaller size of microspheres brought better bioadhesion and bioavailability when the chitosan microsphere was used as the carrier for oral administration of protein drugs.11 Furthermore, the size uniformity and controllability of nanospheres have the advantages of good reproduc* To whom correspondence should be addressed. Tel.: +86-1062557095/82627072. Fax: +86-10-82627072. E-mail: wanglianyan@ home.ipe.ac.cn (L.-Y.W.); [email protected] (G.-H.M.). † Chinese Academy of Sciences. ‡ Beijing University of Chemical Technology.

ibility and a repeatable drug release profile, the effects of nanosphere size on drug efficiency and the distribution of nanospheres with different size in vivo quantitatively being easy to study.12 Therefore, it is necessary to develop a method to prepare uniformly sized and size controllabe nanospheres. Among biodegradable polymers, chitosan, as a naturally biodegradable polysaccharide, has broad applications in the medicine field because of its excellent characteristics, such as bioadhesion, biodegradability, biocompatibility, nontoxic, and nonimmunogenicity.12 Chitosan is a unique polysaccharide with positive charge, which shows excellent bioadhesiveness with mucosa. Therefore, it has great potential in oral and mucosal administration of protein and peptide drugs. In the past few decades, chitosan nanospheres have been developed for the drug delivery system. To date, there are many methods to prepare

Figure 1. Schematic diagram of a miniature kit for premix membrane emulsification.

10.1021/ie801758e CCC: $40.75  2009 American Chemical Society Published on Web 02/11/2009

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Ind. Eng. Chem. Res., Vol. 48, No. 19, 2009

Figure 2. SEM photographs of chitosan nanospheres prepared by using different transmembrane pressures: (a) 0.50, (b) 0.65, (c) 0.80, (d) 0.95 MPa. (e) Particle size distribution of chitosan nanospheres prepared using different transmembrane pressures. (f) Viscosity of emulsion prepared using different transmembrane pressures.

chitosan nanospheres including emulsion-cross-linking, emulsion-solvent evaporation coacervation, complex coacervation, spray drying, and so on.13 Unfortunately, the size distribution of chitosan nanospheres prepared by these methods is very broad, which will result in poor targeting and low bioavailability.14 Although uniformly sized chitosan microspheres have been successfully prepared by direct membrane emulsification techniques combined with chemical cross-linking methods in our previous study, it is still difficult to prepare nanospheres.15,16 This is because the uniform water/oil (W/O) droplet is formed by utilizing high interfacial tension between the dispersed phase and the membrane pore in the direct membrane emulsification technique, the droplet size is usually as large as three times of the membrane pore size. Therefore, if we want to prepare nanodroplets, the membrane with very small pore size should be used; as a result, very high transmembrane pressure has to be applied for a long period and the production efficiency will be very lower due to the dramatically decreased emulsification rate. Therefore, it is great challenge to develop

a new method to prepare uniform and size-controllable chitosan nanosphere with high production efficiency. In present study, a novel premix membrane emulsification technique was developed on the basis of direct membrane emulsification to prepare uniformly sized and size-controllable chitosan nanospheres. The schematic diagram of preparation equipment is shown in Figure 1, where the coarse emulsion with larger droplet size was pressed through the uniform pores of the Shirasu porous glass (SPG) membrane quickly under higher nitrogen pressure to obtain uniform smaller droplets, which was further cross-linked to obtain nanospheres. By this technique, the production efficiency (emulsification rate) can attain a value as high as 104 times that of the direct membrane emulsification technique when preparing droplets 200-300 nm in diameter. In this method, several factors play key roles during preparing uniformly sized chitosan nanodroplets, including the amount of emulsifier in the oil phase, composition of the oil phase, concentration of chitosan, ratio between the water and oil phases, transmembrane pressure and number of passes, and


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Figure 3. SEM photographs of chitosan nanospheres prepared by using different transmembrane passes: (a) one, (b) three, and (c) five. (d) Particle size distribution of chitosan nanospheres prepared using different numbers of transmembrane passes.

so on. In order to obtain uniformly sized chitosan nanospheres, all the above factors are investigated in detail in this study and the membrane pore size was fixed at 1.4 µm. The results showed that the diameter of chitosan nanospheres prepared by premix membrane emulsification under optimum condition was between 200 and 300 nm, the size distribution was very narrow (polydispersity index was 0.027), and the dispersibility of nanospheres was also excellent. In addition, the chitosan nanospheres with diameters of 20-1500 nm could be prepared by selecting SPG membrane with specific pore size, and there was a linear relationship between the diameter of the chitosan nanospheres and the pore size of the SPG membrane. Therefore, the demanded diameter of chitosan nanospheres can be prepared by choosing suitable pore size of membrane. 2. Experimental Details 2.1. Reagent and Materials. Chitosan (DD 89%, Mv ) 50 000) was purchased from Zhejiang Yuhua Co., Ltd. (Zhejiang, China). The Shirasu porous glass (SPG) membrane was provied from SPG Technology Co. (Japan). The homogenizer was T18 basic from IKA WORKS of German. The ultrasonic homogenizer was S-450D from BRANSON of America. Glutaradehyde was obtained from Sigma-Aldrich Inc. (Germany). PO-500 (hexaglycerin penta ester) was purchased from Sakamoto Yakuhin Kogyo Co., Ltd. (Japan). Paraffin and petroleum ether (boiling range 60-90 °C) were purchased from Sinopharm Chemical Reagent Co., Ltd. Acetic acid was provided by Beijing Chemical Plant. All the reagent were of analytic grade. 2.2. Preparation of Chitosan Nanospheres. Chitosan nanoparticles preparation includes three steps, preparation of nanodroplets, solidification of nanodroplets, and washing of nanospheres. The process of nanodroplets prepartion was as follows: the coarse emulsions were first prepared by low-speed stator homogenization, mechanical stirring, or ultrasonic emulsification and, then, were poured into the premix reservoir, and the nanodroplets were obtained afterward by pressing the coarse

emulsions through the SPG membrane under a certain nitrogen pressure. This process is repeated 1-5 times. Subsequently, GST (glutaraldehyde saturated toluene) was slowly dropped into the emulsion to solidify chitosan nanodroplets into nanospheres. Finally, the chitosan nanoparticles were collected and washed two times with petroleum ether, acetone, and ethanol by centrifugation at 8000 rpm, separately. Finally, chitosan nanospheres were lyophilized. The effects of the following factors, the amounts of emulsifier in the oil phase, the composition of the oil phase, the concentration of chitosan, the ratio of water to the oil phase, the transmembrane pressure, and number of passes, on particle size and size distribution were investigated in detail. Unless specified, the standard formulation conditions were as follows: the pore size of the membrane was 1.4 µm, the polydispersity index value of the membrane was below 1%, the transmembrane pressure was 0.95 MPa, the concentration of chitosan solution was 0.5 wt %, the oil phase was a mixture of liquid paraffin and petroleum ether [1:2 (v/v)] containing 4 wt % PO-500 emulsifier, the volume ratio of water to the oil phase was 1:30, the amount of solidification agent was set such that the molar ratio between amino groups and aldehyde groups was 1:1, and the solidification reaction time was 10 h. 2.3. Measurement of Emulsion Viscosity. The viscosity of emulsions was measured by rheometer (L-90 Machinery and Electrical Equipment Factory of Tongji University). After the completion of L-90 rheometer installation, zero correction was carried out. Emulsions prepared by the premix membrane emulsification technique were poured into a standard rotating tank of the third unit of the L-90 rheometer, which measured the emulsions from high to low shear rate (low viscosity). A series of data (denoted Α) can be obtained from the dial plate of the L-90 rheometer at first. Then, the shear stress of measured emulsions can be calculated by the formula of τ ) 0.355Α (N/ m2), in which the coefficient 0.355 is the shear stress of standard rotating tank of the third unit of the L-90 rheometer. The

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Figure 4. SEM photographs of chitosan nanospheres prepared by using different concentrations of emulsifier in the oil phase: (a) 2, (b) 4, (c) 6, (d) 8 wt %. (e) Particle size distribution of chitosan nanospheres prepared using different concentrations of emulsifier in the oil phase.

viscosity for measured emulsions can be obtained by the formula of η ) τ/1000D. 2.4. Measurement of Size Distribution of Nanospheres. The nanospheres were dispersed into the deionized water and then sonified for a few minutes to form suspension. After that, a 2 mL suspension was taken out and added into the sample cell, and the size distribution of chitosan nanospheres was analyzed by zeta potential measurement with sub-micrometer particle size analyzer (zeta potential analyzer, Brookhaven Instruments Corporation). The uniformity and dispersity of chitosan nanospheres are presented by polydispersity index which was directly obtained by the zeta potential analyzer. The polydispersity index (PDI), mentioned above, is a measure of the distribution of nanoparticles in a given sample. The PDI is denoted as: PDI ) µ2/Γ2, where µ2 ) (D2* - D*2)q4 and Γ ) Dq2. The value of q is calculated from the equation of q ) 2Πn/λ02 sin(θ/2), where θ is the scattering angle, λ0 is the

wavelength of laser light, and n means the index of refraction of the suspending liquid. The D is the translational diffusion coefficient, which is the principle quantity measured by the quasi elastic light scattering (QELS), a preferred technique for measuring diffusion coefficients of sub-micron particles. D* is the average diffusion coefficient, the D*2 is the squared value of D*, and D2* is the mean squared value of D. The PDI is unitless, which is close to zero for a monodisperse sample, smaller for narrower distribution, and larger for a broader distribution. 2.5. Scanning Electron Microscopy Observation of Nanospheres. The morphology of chitosan nanospheres were observed by a JEM-6700F scanning electron microscope (SEM, JEOL, Japan). The chitosan nanospheres were dispersed in distilled water, and then, the dispersion was dropped on aluminum foil paper and dried at ambient atmosphere. After that, the sample was fixed with a electrically conductive


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Figure 5. SEM photographs of chitosan nanospheres prepared by using different concentration of chitosan in the water phase: (a) 0.3, (b) 0.5, (c) 1.0 wt %. (d) Particle size distribution of chitosan nanospheres prepared using different chitosan concentrations in the water phase.

adhesives on the sample stage. Subsequently, the sample was coated with platinum under vacuum by an ion sputter (JFC1600, JEOL). Finally, the sample was observed by scanning electron microscope. 3. Results and Discussion 3.1. Effect of Transmembrane Pressure on Uniformity of Nanospheres. The nanospheres were first prepared under different transmembrane pressures: 0.5, 0.65, 0.80, and 0.95 MPa. The other conditions were the same as described in section 2.2. The SEM micrographs of chitosan nanospheres by different transmembrane pressure are shown in Figure 2a-d, and the corresponding size distribution is displayed in Figure 2e. It was found that the narrowest size distribution of chitosan nanospheres was achieved when the transmembrane pressure was 0.95 MPa. The higher pressure led to stronger collisions between coarse emulsion and tortuous pore walls of SPG membrane, which accelerated the breaking of droplets. When the transmembrane pressure was lower than 0.95 MPa, it was not enough to break droplet into small and uniform emulsion and resulted in poor uniformity of chitosan nanospheres. The size of chitosan nanospheres was also confirmed with the viscosity of emulsions, the smaller size of emulsion showed higher viscosity under the same shear rate, as shown in Figure 2f. Furthermore, the poor uniformity of chitosan nanospheres resulted in poor dispersity due to the coalescence between nanoparticles, which further led to a broader size distribution of chitosan nanospheres in the measurement by the analyzer as shown in Figure 2e. 3.2. Effect of Transmembrane Number on Uniformity of Nanospheres. The chitosan nanospheres were prepared by different transmembrane numbers: one, three, and five. The other preparation conditions were the same as mentioned in section 2.2. The SEM micrographs of chitosan nanospheres prepared with different numbers of passes are shown in Figure 3a-c, and the size distribution is shown in Figure 3d. The results showed that the uniformity of chitosan nanospheres was

improved and the size became smaller with increasing numbers of passes. The larger droplets would be broken into smaller emulsions by each pass. The frequency of larger droplets being broken into smaller ones increased with increasing number of transmembrane passes, which resulted in smaller size and narrower size distribution of chitosan nanospheres. In addition, the poorer uniformity of as prepared nanospheres resulted in poorer dispersity which would further lead to broader size distribution. 3.3. Effect of Emulsifier Amount in the Oil Phase on the Uniformity of Nanospheres. The oil phase consisted of liquid paraffin-petroleum ether (1:2 v/v) and PO-500 (emulsifier). Different amounts of emulsifier, 2, 4, 6, and 8 wt %, were used to prepare chitosan nanospheres. The other preparation conditions were the same as described in section 2.2. As shown in Figure 4a-d, the chitosan nanospheres with excellent uniformity, dispersity, and narrow size distribution were obtained when the amount of emulsifier in the oil phase was 4 wt %. When the amount of emulsifier in the oil phase was 2 wt %, the interfacial tension between oil and water phase became higher because the amounts of emulsifier was not enough, which resulted in coalescence between droplets and further led to broader size distribution of nanospheres. On the other hand, when the emulsifier concentration was increased to above 4 wt %, the interfacial tension between the oil and water phases was reduced further; this would lead to the production of lots of smaller droplets during the preparation of a coarse emulsion because of the higher concentration of emulsifier. These smaller droplets would pass through pores of the SPG membrane freely without being broken to smaller ones. Furthermore, the higher emulsifier concentration, the lower the interfacial tension, which led to the larger coarse droplets being more easily broken into smaller ones and more droplets during premix membrane emulsification. Therefore, the size uniformity became poor with an increase of emulsifier concentration in the oil phase; this furthermore caused poor dispersity of chitosan nanospheres and,

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Figure 6. SEM photographs of chitosan nanospheres prepared by using different volume ratios of water to the oil phase: (a) 1:20, (b) 1:30, (c) 1:40, (d) 1:60. (e) Particle size distribution of chitosan nanospheres prepared using different ratios of water to the oil phase.

finally, led to a broader size distribution of chitosan nanospheres as shown in Figure 4e. 3.4. Effect of Chitosan Concentration on the Uniformity of Nanospheres. The chitosan nanospheres were prepared by different concentrations of chitosan: 0.3, 0.5, and 1.0 wt %. The other preparation conditions were the same as mentioned in section 2.2. The SEM micrographs of chitosan nanospheres prepared by different concentrations of chitosan are shown in Figure 5a-c, and the corresponding size distribution is displayed in Figure 5d. The chitosan nanospheres with excellent uniformity were obtained when the concentration of chitosan was 0.5 wt %. When the concentration of chitosan was 0.3 wt %, the viscosity of water phase was lower, the coarse emulsion was easy to pass through the pore of the membrane freely under higher pressure, and some of coarse droplets were not broken into smaller ones in time. Therefore, there were more and larger droplets, resulting in a broader distribution. When the concentration of chitosan was 1.0 wt %, the viscosity of the water phase

was higher, which caused more serious coagulation between droplets. Therefore, the size distribution of chitosan nanospheres became broader. 3.5. Effect of the Ratio of Water Phase/Oil Phase on the Uniformity of Nanospheres. The chitosan nanospheres were prepared by changing the water/oil phase volume ratio: 1:20, 1:30, 1:40, and 1:60. The other preparation conditions were the same as described in section 2.2. The SEM micrographs of chitosan nanospheres prepared at different ratios of the water phase to the oil phase are shown in Figure 6a-d, and the corresponding size distribution is shown in Figure 6e. It could be seen from Figure 6a-d that the chitosan nanospheres with satisfactory uniformity were obtained when the volume ratio of water/oil phase was 1:30 and 1:40. When the volume ratio was higher as 1:20, the concentration of the emulsion and the viscosity of the coarse emulsion were higher, which resulted in the easier cross-linking reaction which occurred between droplets. Consequently, poorer dispersity and


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Figure 7. SEM photographs of chitosan nanospheres prepared using different ratios of paraffin liquid to petroleum ether in the oil phase: (a) 1.5:1, (b) 1:1.5, (c) 1:2, (d) 1:3, (e) 1:11, (f) pure petroleum ether. (g) Particle size distribution of chitosan nanospheres prepared using different ratios of paraffin liquid to petroleum ether in the oil phase.

Figure 8. SEM photographs of chitosan nanospheres prepared using different cross-linking temperatures: (a) 25, (b) 40, (c) 60 °C, (d) Particle size distribution of chitosan nanospheres prepared using different cross-linking temperatures.

broader size distribution of chitosan nanospheres were revealed. When the volume ratio between the water and oil phase was

lower than 1:60, the viscosity of the coarse emulsion and the concentration of the emulsion were lower, which made it easier

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Figure 9. SEM photographs (a) and particle size distribution (b) of chitosan nanospheres prepared under optimal conditions.

Figure 10. SEM photographs of chitosan nanospheres prepared using different membrane pore sizes: (a) 2.8, (b) 5.2, (c) 7.0, and (d) 9.0 µm. (e) Particle size distribution of chitosan nanospheres prepared using different membrane pore sizes.

for the droplets to pass through the pores of the membrane and some of them were not broken in time. As a result, the size distribution of the droplets became broader. 3.6. Effect of the Viscosity of the Oil Phase on the Uniformity of Nanospheres. In this study, the oil phase was a mixture of liquid paraffin and petroleum ether. The viscosity of the oil phase varied with the volume ratio of liquid paraffin and petroleum ether, and the viscosity was reduced with an

increase of petroleum ether. Herein, different volume ratios of liquid paraffin and petroleum ether, 1.5:1, 1:1.5, 1:2, 1:3, and 1:11, and pure petroleum ether were used. The SEM micrographs of chitosan nanospheres prepared by different viscosities of the oil phase are shown in Figure 7a-f, and the corresponding size distribution is shown in Figure 7g. The results showed that the viscosity of the oil phase had little effect on the uniformity of the chitosan nanospheres;


Figure 11. Relationship between particle size and membrane pore size.

however, it played important role on the product dispersity. When the volume ratio of liquid paraffin was higher, the viscosity of the oil phase became higher, causing poorer fluidity of emulsion and further leading to coalescence between droplets. This also led to poorer dispersity and a broader size distribution of chitosan nanospheres in the measurement by the zeta potential analyzer. When the volume ratio of petroleum ether was higher, though the viscosity was lower, the poorer dispersity of chitosan nanospheres was caused by evaporation of petroleum ether during solidification of nanospheres because the solidification temperature was kept at 40 °C. Therefore, chitosan nanospheres with excellent dispersity were obtained when the volume ratios of liquid paraffin and petroleum ether were suitably chosen as 1:1.5 and 1:2. 3.7. Effect of the Solidification Temperature on the Uniformity of Nanospheres. The solidification temperature was also found to be an important factor effecting the uniformity and dispersity of chitosan nanospheres. The SEM micrographs of chitosan nanospheres with different solidification temperatures, 25, 40, and 60 °C, are shown in Figure 8a-c, and the corresponding size distribution is shown in Figure 8d. The results showed that the chitosan nanospheres with excellent uniformity and dispersity were obtained when the solidification temperature was 40 °C. When the solidification temperature was 25 °C, the reaction would be very slow, which implied that the complete cross-linking needed more time. Therefore, the coalescence of droplets would occur during longer solidification time and further lead to poor polydispersity and dispersity of chitosan nanospheres. When the solidification temperature was 60 °C, the emulsion droplets moved faster and further led to poorer stability because of coalescence between droplets. In addition, the cross-linking reaction between droplets would also occur due to the faster reaction rate and evaporation of petroleum ether. Therefore, the poorer dispersity and broader size distribution of chitosan nanospheres was examined. It can be concluded from the above results that the optimal preparation conditions of uniformly sized chitosan nanospheres with a 1.4 µm pore size membrane were as follows: the concentration of chitosan was 0.5 wt %, the oil phase was a mixture of liquid paraffin and petroleum ether (volume ratio 1:2) containing 4 wt % PO-500 (emulsifier), the volume ratio of the water and oil phases was 1:30, the transmembrane pressure was 0.95 MPa, the transmembrane number was 5, and the solidification temperature was 40 °C. The SEM image of chitosan nanospheres by optimal conditions is shown in Figure 9a, and the size distribution is shown in Figure 9b. The nanospheres showed excellent dispersity and uniformity, the

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polydispersity index was as low as 0.027, and the diameter of obtained nanospheres was 200-300 nm. 3.8. Size Control of Nanospheres. The release behavior and bioavailability of drugs from nanospheres is usually shown to be size-dependent. Therefore, it is necessary to prepare chitosan nano-microspheres with different diameters. The uniformly sized chitosan nanospheres with diameters of about 300 nm were successfully prepared using a membrane with a pore size of 1.4 µm as described above. After that, the uniformly sized chitosan microspheres were also prepared using membranes with different pore sizes of 2.8, 5.2, 7.0, and 9.0 µm, and the corresponding membrane pressures were 0.5, 0.25, 0.15, and 0.08 MPa, respectively. The SEM micrographs are shown in Figure 10a-d, and the corresponding size distribution is shown in Figure 10e. There was a linear relationship between the pore size of the membrane and the diameter of chitosan nanospheres as shown in Figure 11. The diameters of chitosan nanomicrospheres were 550, 1100, 1450, and 1850 nm when the pore sizes of the membranes were 2.8, 5.2, 7.0, and 9.0 µm, respectively. Therefore, the demanded diameter of chitosan nanospheres could be prepared by choosing a suitable pore size of membrane. 4. Conclusions The uniformly sized chitosan nano-microspheres were successfully prepared by a premix membrane emulsification technique under the optimal conditions as follows: when the pore size of the membrane was 1.4 µm and the polydispersity index value of the membrane was below 1%, the transmembrane pressure was 0.95 MPa, the concentration of the chitosan solution was 0.5 wt %, the oil phase was a mixture of liquid paraffin and petroleum ether [1:2 (v/v)] containing 4 wt % PO500 emulsifier, the volume ratio of water to the oil phase was 1:30, the amount of solidification agent was set such that the molar ratio between amino groups and aldehyde groups was 1:1, and the solidification reaction time was 10 h. The diameter of obtained chitosan nanospheres was 200-300 nm, the polydispersity was as low as 0.027, and nano-microspheres with diameters from 300 nm to 1.85 µm were obtained. The chitosan nanospheres with different diameters also could be prepared by choosing a suitable pore size of the SPG membrane. Therefore, the premix membrane emulsification technique is a potential method to prepare nano-microspheres with uniform size and excellent dispersity. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (50703043, 20536050, and 20506026) and the Chinese Academy of Sciences (KJCX2-YWM02). We also acknowledge the financial support from National Basic Research Program of China (2009CB930300). Literature Cited (1) Talmadge, J. E. The pharmaceutics and delivery of therapeutic polypeptides and proteins. AdV. Drug. DeliVery ReV. 1993, 10, 247–299. (2) Wang, L. Y.; Ma, G. H.; Su, Z. G. Preparation of uniform sized chitosan microspheres by membrane emulsification technique and application as a carrier of protein drug. J. Controlled Release 2005, 106, 62–65. (3) Shi, X. Y.; Tan, T. W. Preparation of chitosan/ethylcellulose complex microcapsule and its application in controlled release of vitamin D2. Biomaterials 2002, 23, 4469–4472. (4) Zhang, H.; Alsarra, I. A.; Neau, S. H. An in vitro evaluation of a chitosan-containing multiparticulate system for macromolecule delivery to the colon. Int. J. Pharm. 2002, 239, 197–205.

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ReceiVed for reView November 18, 2008 ReVised manuscript receiVed January 12, 2009 Accepted January 13, 2009 IE801758E

Preparation of Uniformly Sized Chitosan Nanospheres by a Premix

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