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alginate multilayers-coated CaCO3 microparticles
Colloids and Surfaces A: Physicochem. Eng. Aspects 353 (2010) 132–139
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Sustained delivery of doxorubicin by porous CaCO3 and chitosan/alginate multilayers-coated CaCO3 microparticles Caiyu Peng, Qinghe Zhao, Changyou Gao ∗ MOE of Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
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Article history: Received 2 July 2009 Received in revised form 24 September 2009 Accepted 4 November 2009 Available online 10 November 2009 Keywords: CaCO3 microparticles Doxorubicin Drug delivery Multilayers
a b s t r a c t Carboxymethyl cellulose (CMC)-doped CaCO3 microparticles with an average diameter of 5 m were prepared and coated by chitosan and alginate multilayers. The prepared CaCO3 microparticles had a dominant phase of vaterite and a spherical morphology with nanopores on their surface. After LbL assembly of chitosan and alginate, the CaCO3 microparticles were significantly smoothened. Treatment of the multilayers-coated particles yielded hollow microcapsules. These particles could spontaneously load positively charged doxorubicin (DOX) molecules, whose amount was 475 and 482 mg DOX/g CaCO3 for the CaCO3 (CMC) microparticles and the (chitosan/alginate)5 coated CaCO3 (CMC) microparticles, respectively. Brunauer–Emmett–Teller (BET) method was used to analyze the specific surface area and the pore size distribution of the CaCO3 (CMC) microparticles before and after DOX loading. After DOX loading, SBET and pore volume were reduced obviously, and the volume of smaller pores decreased significantly, whereas that of larger pores were increased. The increase of the volume of larger pores was explained by an electric charge screening effect. DOX release from the CaCO3 microparticles in pH 5 was relatively slow within the first 15 h, and could be sustained to more than 150 h. The release amount at lower pH was larger at the same time. Coating of the CaCO3 (CMC) microparticles with the chitosan/alginate multilayers could obviously assuage the initial burst release and reduce the release rate. © 2009 Elsevier B.V. All rights reserved.
1. Introduction During the past decades, the controlled drug release techniques have attracted much attention in modern pharmaceutical and medication area. Unlike conventional forms of dosage, controlled drug delivery undoubtedly has many advantages. As a result of reducing the drug release rate and prolonging the release time, the controlled release technologies can minimize poisonous side effects and increase drug efficiency [1,2]. So far various delivery systems such as micro/nano-spheres [3,4], lipid vesicles [5], micelles [6], hydrogels [7], and microparticles [8,9] have been formulated from many types of materials including polymers, amphiphilic molecules, and other organic and inorganic substances. During fabrication of these systems, organic solvents, additives (crosslinking agents and emulsifiers) and temperature or pH changes are generally necessary, which may bring side effects on the bioactive substances. In contrast, porous inorganic materials such as porous calcium carbonate micro/nanoparticles can load drugs in very mild conditions, either during the preparation [10] or by post-adsorption into the vast number of nanopores [11]. The loaded drugs or
∗ Corresponding author. Tel.: +86 571 87951108; fax: +86 571 87951948. E-mail address: [email protected] (C. Gao). 0927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2009.11.004
biomacromolecules can preserve their native properties to a great extent. Furthermore, the surface of the inorganic particles can be easily modified by methods such as the layer-by-layer (LbL) selfassembly. Indeed, the LbL technique has achieved great progress in fabricating two-dimensional multilayer films [12] and threedimensional microparticles [13,14]. It has also been utilized in encapsulating the drug-loaded microparticles to reduce the release rate and suppress the initial burst release [11,15]. In this work, carboxymethyl cellulose (CMC), a negatively charged polysaccharide, was used to mediate formulation of CaCO3 microparticles, which was simultaneously incorporated (Fig. 1). This negatively charged polymer can then provide additional attractive forces for positively charged drugs such as doxorubicin (DOX). In fact, most anti-cancer drugs are positively charged at physiological conditions. Moreover, the particles can be alternatingly coated with chitosan and alginate multilayers to tune the release kinetics of the loaded drugs. It is also known that the kinetics of drug release can be greatly influenced and controlled by the pore size or steric hindrance [2]. Generally, the drug release rate decreases with the decrease of the pore size [16,17]. The CaCO3 microparticles prepared by this method have a large number of nanopores, which provide a capillary force to load substances regardless of their charging properties or hydrophilicity. For example, Wang et al. [11] reported that ibuprofen, a lipophilic
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Fig. 1. Schematic illustration to show synthesis of CaCO3 microparticles mediated by the carboxymethyl cellulose (CMC), assembly of biocompatible polysaccharides and adsorption of doxorubicin into the CaCO3 microparticles.
drug, was loaded into the polystyrene sulfonate (PSS)-doped CaCO3 microparticles. Due to the charge repulsion, the loading amount of ibuprofen was not very large. Moreover, the additive of PSS has some uncertainties for biomedical applications. We shall show in this work that our strategy is very effective to delivery DOX, an anti-cancer drug. In this system, CMC will powerfully attract DOX into the nanopores due to the “spontaneous deposition” and the “charge-controlled attraction” effects [18–22]. These micron-sized particles can be directly injected into tumor tissues as demonstrated before by Zhao et al. [8], in which the encapsulated DOX was more effective to restrict tumor growth than free drug. Compared with the hollow microcapsules, the CaCO3 (CMC) microparticles cannot only load more drugs but also
be able to reduce the toxicity of drugs to normal tissues, since they are only released at acidic conditions. 2. Materials and methods 2.1. Materials The materials used in this work included carboxymethyl cellulose sodium salt (CMC, viscosity 30–80 cps for a 1 mg/ml solution in 0.5 M NaCl at pH 5.5, China Curative and Medicine Corp., Shanghai), doxorubicin (DOX, Zhejiang Haizheng Pharmaceutics Corp., China), chitosan (viscosity 115 cps for a 0.5 mg/ml solution in 0.5 M NaCl at pH 5.5, Haidebei Halobios, Ji’nan, China), and sodium alginate
Fig. 2. (a and b) SEM images and (c) XRD pattern of CaCO3 (CMC) microparticles. In (c), V and C denote vaterite and calcite, respectively.
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Fig. 3. (a) -Potential of CaCO3 (CMC) microparticles coated with chitosan/alginate multilayers as a function of layer number. The odd and even numbers represent chitosan and alginate, respectively, except for number 0, CaCO3 (CMC) microparticles. (b and c) SEM images of CaCO3 (CMC)–(chitosan/alginate)5 microparticles. (d) CLSM image of CaCO3 (CMC)–(chitosan/alginate)4 –rodamin–chitosan microcapsules.
(viscosity 250 cps for a 1 mg/ml solution in 0.5 M NaCl at pH 5.5, Sigma–Aldrich, St. Louis, MO). The water used in all experiments was triple-distilled. Other chemicals were all analytical reagents and used as received.
assembled with alginate as the outermost layer. The core–shell CaCO3 (CMC) particles were then treated with 1% glutaraldehyde (GA) solution for 12 h at room temperature to crosslink the chitosan component. Then the particles were rinsed with distilled water and freeze-dried.
2.2. Methods 2.2.1. Preparation of porous CaCO3 (CMC) microparticles and chitosan/alginate polyelectrolyte multilayers coating The CaCO3 microparticles doped with CMC, abbreviated as CaCO3 (CMC), were synthesized by mineralization of Ca(NO3 )2 and Na2 CO3 solutions in the existence of CMC [9]. Briefly, 100 ml 0.025 M Ca(NO3 )2 solution was mixed with 2 ml 5% CMC, into which 100 ml Na2 CO3 solution was rapidly poured under ultrasonication. After 10–20 min the CaCO3 microparticles were filtered off, washed with triple-distilled water and dried in air. The prepared CaCO3 (CMC) microparticles were encapsulated by polycation chitosan and polyanion alginate through LbL selfassembly. Solutions of chitosan (1 mg/ml, in 0.5 M NaCl, pH 5.0) and alginate (1 mg/ml, in 0.5 M NaCl, pH 5.0) were obtained by dissolving chitosan and alginate in 0.5 M NaCl solution, respectively. The CaCO3 (CMC) particles were washed with 0.5 M NaCl solution (pH 5.0) 3 times before assembly. In a typical fabrication process, the CaCO3 (CMC) particles were incubated in the chitosan or alginate solution for 15 min. The multilayers were deposited onto the CaCO3 (CMC) particles by consecutive adsorption of chitosan and alginate using a centrifugation protocol [13]. The suspension was then centrifuged at 2000 rpm for 5 min and the supernatant was carefully removed. Three washes in 0.2 M NaCl solution (pH 5.0) at each interval were conducted before the next adsorption. The adsorption was repeated until 5 bilayers of polysaccharides were
2.2.2. Deposition of DOX Before DOX loading, the CaCO3 (CMC) microparticles were dried in a oven at 50 ◦ C. 20 mg CaCO3 (CMC) microparticles or CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles were immersed in 5 ml DOX solution (2 mg/ml). The suspension was brought to equilibrium under gentle agitation at room temperature for 24 h, followed by centrifugation at 4000 rpm for 5 min. After centrifugation, the DOX concentration of the supernatant was determined with the absorbance at 479 nm by a UV–visible spectrophotometer (UV-2550; Shimadazu, Japan). A calibration curve was obtained using the pure DOX. The amount of incorporated DOX was then obtained and expressed as milligram DOX/g CaCO3 (CMC). The DOX-loaded microparticles were freezedried before Brunauer–Emmett–Teller (BET) analysis and release experiment. For the study of loading kinetics, 60 mg CaCO3 (CMC) microparticles was immersed in 2 ml DOX solution (2 mg/ml), and the supernatant concentration was measured as a function of time. 2.2.3. In vitro release The DOX-loaded CaCO3 (CMC) microparticles (20 mg, = 37.5 mg DOX/g CaCO3 from UV, ca. 0.75 mg DOX in total) and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles (20 mg, = 67 mg DOX/g CaCO3 from UV, ca. 1.34 mg DOX in total) were put in dialysis bags (cut-off MW 8000) with 5 ml release medium separately, which were immersed in 195 ml
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Fig. 4. (a) Loading kinetics of DOX into CaCO3 (CMC) microparticles. (b and c) CLSM images of DOX-loaded CaCO3 (CMC) microparticles.
release medium. The in vitro release was performed with continuous agitation at 37 ◦ C. Two different solutions of pH 2.0 and pH 5.0 were used as the release medium (0.1 M sodium citrate-HCl buffer). 1 ml solution was withdrawn from the release medium and then supplemented with 1 ml fresh release medium of the same pH at desired time interval. The DOX concentration in the release medium was then determined with UV spectroscopy. The cumulative released amount of DOX was integrated from each measurement.
2.2.4. Characterization For scanning electron microscopy (SEM, SIRION-100, FEI) observation, the CaCO3 (CMC) microparticles suspension was dropped on a glass slide, air dried and then sputtered with gold. The acceleration voltage was set at 15 kV. The DOX-loaded CaCO3 (CMC) microparticles and hollow microcapsules were observed by confocal laser scanning microscopy (CLSM, Zeiss LSM510). The specific surface area and the pore size distribution of the CaCO3 (CMC) microparticles were measured by the Brunauer–Emmett–Teller
Fig. 5. N2 adsorption–desorption isotherms of CaCO3 (CMC) microparticles before (a) and after DOX loading (b).
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method of N2 adsorption–desorption at −196 ◦ C with an ASAP 2010 surface area analyzer (Micrometrics BET, USA). -Potential of the CaCO3 (CMC) microparticles after each adsorption was measured in 0.2 M NaCl solution with a pH value of 5.0 by a Zeta-sizer (Mastersizer 2000, UK). Each datum was averaged from 3 measurements. 3. Results and discussion 3.1. Preparation of porous CaCO3 (CMC) microparticles and multilayers coating To obtain spherical CaCO3 microparticles, CMC, a negatively charged polysaccharide, was added into the reaction mixture during the preparation to control the crystallization and growth behavior of the CaCO3 particles. Fig. 2 displays SEM images of the CaCO3 (CMC) microparticles with two different magnifications. On the surface of the CaCO3 (CMC) microparticles there existed a lot of carbonate nanoparticles and many tiny channel pores with a size of 20–50 nm. As a biocompatible macromolecule, the added CMC plays an important role. It can not only help to get spherical microparticles with narrow size distribution through its control over the crystallization process, but also be beneficial to maintain the biocompatibility of the microparticles. The XRD pattern (Fig. 2c) shows that the CaCO3 (CMC) microparticles had a dominant phase of vaterite, although a trace amount of calcite was present also. The broadening diffraction peaks suggest that the produced vaterite microspheres were composed of tiny nanoparticles. Generally, spherical CaCO3 microparticles will change to rhombohedral calcite microcrystals in water for several days [23,24]. Recrystallization of the porous CaCO3 (CMC) microparticles composed of carbonate nanoparticles was blocked by the CMC adsorbed on the surface of the carbonate nanoparticles. Thus, the CaCO3 (CMC) microparticles were much more stable than those without additives. To decrease release rate of the loaded DOX from the microparticles, the CaCO3 (CMC) microparticles were further coated with chitosan and alginate polyelectrolyte multilayers before DOX loading. The LbL self-assembly process was monitored by -potential as shown in Fig. 3. The optimal pH value of the polyelectrolyte solutions was set to 5.0 because at this pH both chitosan and alginate are highly charged [25–27]. Since CMC is negatively charged, the -potential of the CaCO3 (CMC) microparticles was about −20 to −30 mV. The -potential of the microparticles deposited with the first chitosan layer still remained a negative value, however, increased about 10 mV as compared with the bare microparticles. After the second cycle of the assembly process, regular charge reversal of -potential indicated adsorption of polycation chitosan and polyanion alginate on the CaCO3 (CMC) microparticles alternatingly. After 4 bilayers of chitosan/alginate were assembled, the CaCO3 (CMC) microparticles became smoother and the original pores disappeared (Fig. 3b and c), which are distinctive with the pristine particles (Fig. 2a and b). Moreover, removal of the template particles yielded hollow microcapsules (Fig. 3d). All these results confirm the successful coating of the CaCO3 (CMC) microparticles with the chitosan/alginate multilayers.
Table 1 BET characterization of porous CaCO3 (CMC) microparticles before and after DOX loading.
Before loading After loading
BET surface area (m2 /g)
Adsorption average diameter (nm)
Total volume of pores at P/P0 0.976 (cm3 /g)
77.60 5.32
3.53 20.87
0.068 0.028
images of the DOX-loaded CaCO3 (CMC) microparticles. Strong fluorescence emitted from the particles, which could be only attributed to DOX and thereby qualitatively confirmed the successful loading. The loaded amount ( ) of DOX was quantified with UV–vis spectroscopy by subtracting the supernatant amount from the feeding amount. The values were found to be 475 mg and 480 mg DOX/g CaCO3 for the CaCO3 (CMC) microparticles and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles, respectively. The value is about an order of magnitude larger than that of ibuprofen ( = 30 ∼ 80 mg ibuprofen/g CaCO3 ) loaded in CaCO3 (PSS) microparticles [11]. Under the loading conditions, DOX is positively charged, and CMC, PSS and ibuprofen are all negatively charged. Therefore, the reason for larger loading of DOX in the CaCO3 (CMC) microparticles is attributed to attraction between CMC and DOX. By contrary, the repulsion between PSS and ibuprofen is unfavorable for ibuprofen loading. Besides, the drugs may be also filled into the nanopores of the CaCO3 (CMC) microparticles, as confirmed by the BET analysis. Although the chitosan/alginate multilayers may slow down the loading rate, the pore size of the multilayers is typically around tens of nanometer, and thereby the eventual loading should not be influenced significantly as evidenced by the same amount of DOX. To prove that the DOX molecules could penetrate into the nanopores of the CaCO3 (CMC) microparticles, BET analysis was also carried out. N2 adsorption–desorption isotherm of CaCO3 (CMC) microparticles before and after DOX loading are depicted in Fig. 5a and b, respectively. Fig. 5a shows that at higher pressures the pores of the CaCO3 (CMC) microparticles were filled with adsorbed or condensed N2 leading to the plateau, indicating that the multilayers-adsorbed N2 was saturated because of limited pore volume. The hysteresis loop of desorption isotherm in Fig. 5a belongs to Type E classified by de Boer [28], conveying that the pores possess a narrow neck and wide body shape. The hysteresis loop of desorption isotherm of the microparticles loaded with DOX in Fig. 5b suggests the pores have a inter-slanting slices shape, which is possibly formed by filling of the nanopores by the complexes of the CMC molecules and DOX molecules.
3.2. DOX loading By simple incubation of the CaCO3 (CMC) microparticles or the CaCO3 (CMC) microparticles coated with chitosan/alginate multilayers in DOX solution, the positively charged anti-cancer drug DOX was spontaneously loaded into the microparticles (Fig. 4a). The loading was almost completed within 5 h, with a final encapsulation efficiency of >95%. Therefore, our loading time of 24 h was long enough to reach an equilibrium state. Fig. 4b and c display the CLSM
Fig. 6. Pore size distribution of CaCO3 (CMC) microparticles before and after DOX loading.
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Fig. 7. Schematic demonstration of the change of pore size distribution of CaCO3 (CMC) microparticles before and after DOX loading.
The change of specific surface area, pore size and pore volume distribution of the microparticles before and after DOX loading are summarized in Table 1. The BET surface area SBET of CaCO3 (CMC) microparticles was 77.6 m2 /g, which is much higher than that of CaCO3 microparticles without dispersants (8.8 m2 /g) [23,24]. The microcrystal carbonates were loosely packed due to the intertwining of the dispersant CMC molecules adsorbed on surface of the carbonate nanocrystals, and thus possessed larger specific surface area. After DOX loading, the BET surface area of the CaCO3 (CMC) microparticles decreased to 5.32 m2 /g and the pore volume was reduced from 0.068 to 0.028 cm3 /g (Table 1) as well, indicating penetration of the DOX molecules into the nanopores. A simple calculation found that theoretical density of DOX was as high as 12 g/cm3 , which is apparently unreasonable. As shall be demonstrated by the pore size distribution (Fig. 6), many DOX molecules were filled into the smaller pores, and those smaller than 2 nm could not be measured by the BET method. Moreover, the CMC macromolecules occupied some internal volume of the particles. By charge attraction, the DOX molecules bind with the CMC macromolecules and thereby make the CMC more compact without change of volume. Another reason should be that some DOX molecules adsorbed on the particle surface, and contributed considerably to the loading amount, as illustrated by the burst release in Fig. 8. Pore size distribution of the microparticles before and after DOX loading is shown in Fig. 6. After DOX loading, the volume of small pores less than 9 nm decreased greatly while that of larger ones increased to some extent. The smaller pores possess high surface free energy and thus are easily to be filled by the DOX molecules. The increased volume of larger pores could probably be explained by electric charge screening effect. The proposed mechanism of the change of pore size distribution is presented in Fig. 7. Being soaked in DOX solution, the CMC macromolecules doped in the CaCO3 (CMC) microparticles are highly swollen and effectively charged. The positively charged DOX molecules preferentially adsorb into the smaller pores and effectively screen the electric repulsion among the negatively charged CMC macro-
molecules. Consequently, the gaps preferentially occupied by DOX between the CaCO3 nanocrystals became smaller, and the space between neighboring nanocrystals which form the larger pores became larger. This can explain why after loading the adsorption average diameter increased from 3.53 to 20.87 nm (Table 1). This is different from the result of adsorption of ibuprofen by CaCO3 microparticles doped with PSS [11]. In that case, the electric repulsion among the negatively charged PSS molecules adsorbed on the CaCO3 nanocrystals would be enhanced by loading of the ibuprofen molecules, leading to decrease of volume of both smaller and larger pores. 3.3. In vitro drug release It is well known that the local pH of tumor tissue is lower than that of normal tissue in human body, i.e. the pH of normal tissue is about 7.4, and the pH of tumor tissue may be as low as 5 or 6. Therefore, we chose a pH 5 buffer for the release experiment to simulate the pH of tumor tissue. The release was also carried out at pH 2 to expedite the behavior since the release kinetic is mainly controlled by dissolution of the CaCO3 particles at low pH. It should be pointed out that the release amount at physiological condition (pH 7.4) of DOX from the CaCO3 (CMC) microparticles was very low and could hardly be detected by UV spectroscopy. This would mean that this drug delivery system should have smaller toxicity to normal tissues (pH 7.4), illustrating the advantages of this “safer” delivery system. The DOX release profiles from the CaCO3 (CMC) microparticles and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles are shown in Fig. 8 (pH 5 buffer and pH 2 buffer). Fig. 8a shows that the release rate was relatively slow after the initial burst release within the first 15 h, and uncommonly the release process can be sustained to more than 150 h. This is rather promising for the application of in vivo drug delivery. Coating of the microparticles by chitosan and alginate multilayers could obviously assuage the initial burst release and control the release rate during the whole process. The long release duration also supports the fact that the
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Fig. 8. Release profiles of DOX from CaCO3 (CMC) microparticles and CaCO3 (CMC)–(chitosan/alginate)5 microparticles in (a) pH 5.0 and (b) pH 2.0 solution (0.1 M sodium citrate-HCl buffer).
DOX molecules are mainly adsorbed in the inner pores other than on the outer surface of the CaCO3 microparticles, because DOX release from the (chitosan/alginate)5 microcapsules was rather fast and could hardly be sustained over 20 h [8]. When the medium pH was set at 2 (Fig. 8b), the release rate was increased obviously, which should be attributed to the faster dissolution of the CaCO3 microparticles at lower pH. Again the multilayer coating could still lower down the release amount of the loaded DOX within the same incubation time. Unlike the 100% release of ibuprofen from the PSS-doped CaCO3 microparticles [11], the DOX release amount could not reach 100% regardless of the pH values investigated so far. This behavior is consistent with the structure of the CaCO3 (CMC) microparticles due to the strong charge attraction. Moreover, the CMC macromolecules become hydrophobic at acidic conditions as a result of protonation of the carboxyl groups, and thereby can block the further dissolution of the inner CaCO3 microparticles. 4. Conclusions The CMC-doped CaCO3 microparticles were prepared and coated by chitosan and alginate polyelectrolyte multilayers. These particles showed a strong ability to load DOX (475 mg DOX/g CaCO3 ), which should be mainly attributed to the charge attraction between negatively charged CMC and positively charged DOX. The large number of nanopores in the CaCO3 (CMC) microparticles provided additional capillary force for the loading. SBET of the CaCO3 (CMC) microparticles was much higher than that without dispersants. After DOX loading, the SBET and pore volume were reduced drastically. The volume of small pores also decreased significantly, whereas that of larger ones increased, indicating the penetration of DOX molecules into the nanopores. The increase of the volume of larger pores and average adsorption diameter may be explained by an electric charge screening effect. DOX release from the CaCO3 microparticles could be sustained to more than 150 h. Moreover, the multilayer coating could lower down the release amount of the loaded DOX within the same incubation time. Thus, this system has great potential for the application of in vivo drug delivery. Acknowledgements This study is financially supported by the Natural Science Foundation of China (20774084, 50873087) and the Major State Basic Research Program of China (2005CB623902). The author is also grateful for H. T. Yang’s help of drawing the schematic illustration figure in this paper.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Sustained delivery of doxorubicin by porous CaCO3 and chitosan/alginate multilayers-coated CaCO3 microparticles Caiyu Peng, Qinghe Zhao, Changyou Gao ∗ MOE of Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
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Article history: Received 2 July 2009 Received in revised form 24 September 2009 Accepted 4 November 2009 Available online 10 November 2009 Keywords: CaCO3 microparticles Doxorubicin Drug delivery Multilayers
a b s t r a c t Carboxymethyl cellulose (CMC)-doped CaCO3 microparticles with an average diameter of 5 m were prepared and coated by chitosan and alginate multilayers. The prepared CaCO3 microparticles had a dominant phase of vaterite and a spherical morphology with nanopores on their surface. After LbL assembly of chitosan and alginate, the CaCO3 microparticles were significantly smoothened. Treatment of the multilayers-coated particles yielded hollow microcapsules. These particles could spontaneously load positively charged doxorubicin (DOX) molecules, whose amount was 475 and 482 mg DOX/g CaCO3 for the CaCO3 (CMC) microparticles and the (chitosan/alginate)5 coated CaCO3 (CMC) microparticles, respectively. Brunauer–Emmett–Teller (BET) method was used to analyze the specific surface area and the pore size distribution of the CaCO3 (CMC) microparticles before and after DOX loading. After DOX loading, SBET and pore volume were reduced obviously, and the volume of smaller pores decreased significantly, whereas that of larger pores were increased. The increase of the volume of larger pores was explained by an electric charge screening effect. DOX release from the CaCO3 microparticles in pH 5 was relatively slow within the first 15 h, and could be sustained to more than 150 h. The release amount at lower pH was larger at the same time. Coating of the CaCO3 (CMC) microparticles with the chitosan/alginate multilayers could obviously assuage the initial burst release and reduce the release rate. © 2009 Elsevier B.V. All rights reserved.
1. Introduction During the past decades, the controlled drug release techniques have attracted much attention in modern pharmaceutical and medication area. Unlike conventional forms of dosage, controlled drug delivery undoubtedly has many advantages. As a result of reducing the drug release rate and prolonging the release time, the controlled release technologies can minimize poisonous side effects and increase drug efficiency [1,2]. So far various delivery systems such as micro/nano-spheres [3,4], lipid vesicles [5], micelles [6], hydrogels [7], and microparticles [8,9] have been formulated from many types of materials including polymers, amphiphilic molecules, and other organic and inorganic substances. During fabrication of these systems, organic solvents, additives (crosslinking agents and emulsifiers) and temperature or pH changes are generally necessary, which may bring side effects on the bioactive substances. In contrast, porous inorganic materials such as porous calcium carbonate micro/nanoparticles can load drugs in very mild conditions, either during the preparation [10] or by post-adsorption into the vast number of nanopores [11]. The loaded drugs or
∗ Corresponding author. Tel.: +86 571 87951108; fax: +86 571 87951948. E-mail address: [email protected] (C. Gao). 0927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2009.11.004
biomacromolecules can preserve their native properties to a great extent. Furthermore, the surface of the inorganic particles can be easily modified by methods such as the layer-by-layer (LbL) selfassembly. Indeed, the LbL technique has achieved great progress in fabricating two-dimensional multilayer films [12] and threedimensional microparticles [13,14]. It has also been utilized in encapsulating the drug-loaded microparticles to reduce the release rate and suppress the initial burst release [11,15]. In this work, carboxymethyl cellulose (CMC), a negatively charged polysaccharide, was used to mediate formulation of CaCO3 microparticles, which was simultaneously incorporated (Fig. 1). This negatively charged polymer can then provide additional attractive forces for positively charged drugs such as doxorubicin (DOX). In fact, most anti-cancer drugs are positively charged at physiological conditions. Moreover, the particles can be alternatingly coated with chitosan and alginate multilayers to tune the release kinetics of the loaded drugs. It is also known that the kinetics of drug release can be greatly influenced and controlled by the pore size or steric hindrance [2]. Generally, the drug release rate decreases with the decrease of the pore size [16,17]. The CaCO3 microparticles prepared by this method have a large number of nanopores, which provide a capillary force to load substances regardless of their charging properties or hydrophilicity. For example, Wang et al. [11] reported that ibuprofen, a lipophilic
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Fig. 1. Schematic illustration to show synthesis of CaCO3 microparticles mediated by the carboxymethyl cellulose (CMC), assembly of biocompatible polysaccharides and adsorption of doxorubicin into the CaCO3 microparticles.
drug, was loaded into the polystyrene sulfonate (PSS)-doped CaCO3 microparticles. Due to the charge repulsion, the loading amount of ibuprofen was not very large. Moreover, the additive of PSS has some uncertainties for biomedical applications. We shall show in this work that our strategy is very effective to delivery DOX, an anti-cancer drug. In this system, CMC will powerfully attract DOX into the nanopores due to the “spontaneous deposition” and the “charge-controlled attraction” effects [18–22]. These micron-sized particles can be directly injected into tumor tissues as demonstrated before by Zhao et al. [8], in which the encapsulated DOX was more effective to restrict tumor growth than free drug. Compared with the hollow microcapsules, the CaCO3 (CMC) microparticles cannot only load more drugs but also
be able to reduce the toxicity of drugs to normal tissues, since they are only released at acidic conditions. 2. Materials and methods 2.1. Materials The materials used in this work included carboxymethyl cellulose sodium salt (CMC, viscosity 30–80 cps for a 1 mg/ml solution in 0.5 M NaCl at pH 5.5, China Curative and Medicine Corp., Shanghai), doxorubicin (DOX, Zhejiang Haizheng Pharmaceutics Corp., China), chitosan (viscosity 115 cps for a 0.5 mg/ml solution in 0.5 M NaCl at pH 5.5, Haidebei Halobios, Ji’nan, China), and sodium alginate
Fig. 2. (a and b) SEM images and (c) XRD pattern of CaCO3 (CMC) microparticles. In (c), V and C denote vaterite and calcite, respectively.
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Fig. 3. (a) -Potential of CaCO3 (CMC) microparticles coated with chitosan/alginate multilayers as a function of layer number. The odd and even numbers represent chitosan and alginate, respectively, except for number 0, CaCO3 (CMC) microparticles. (b and c) SEM images of CaCO3 (CMC)–(chitosan/alginate)5 microparticles. (d) CLSM image of CaCO3 (CMC)–(chitosan/alginate)4 –rodamin–chitosan microcapsules.
(viscosity 250 cps for a 1 mg/ml solution in 0.5 M NaCl at pH 5.5, Sigma–Aldrich, St. Louis, MO). The water used in all experiments was triple-distilled. Other chemicals were all analytical reagents and used as received.
assembled with alginate as the outermost layer. The core–shell CaCO3 (CMC) particles were then treated with 1% glutaraldehyde (GA) solution for 12 h at room temperature to crosslink the chitosan component. Then the particles were rinsed with distilled water and freeze-dried.
2.2. Methods 2.2.1. Preparation of porous CaCO3 (CMC) microparticles and chitosan/alginate polyelectrolyte multilayers coating The CaCO3 microparticles doped with CMC, abbreviated as CaCO3 (CMC), were synthesized by mineralization of Ca(NO3 )2 and Na2 CO3 solutions in the existence of CMC [9]. Briefly, 100 ml 0.025 M Ca(NO3 )2 solution was mixed with 2 ml 5% CMC, into which 100 ml Na2 CO3 solution was rapidly poured under ultrasonication. After 10–20 min the CaCO3 microparticles were filtered off, washed with triple-distilled water and dried in air. The prepared CaCO3 (CMC) microparticles were encapsulated by polycation chitosan and polyanion alginate through LbL selfassembly. Solutions of chitosan (1 mg/ml, in 0.5 M NaCl, pH 5.0) and alginate (1 mg/ml, in 0.5 M NaCl, pH 5.0) were obtained by dissolving chitosan and alginate in 0.5 M NaCl solution, respectively. The CaCO3 (CMC) particles were washed with 0.5 M NaCl solution (pH 5.0) 3 times before assembly. In a typical fabrication process, the CaCO3 (CMC) particles were incubated in the chitosan or alginate solution for 15 min. The multilayers were deposited onto the CaCO3 (CMC) particles by consecutive adsorption of chitosan and alginate using a centrifugation protocol [13]. The suspension was then centrifuged at 2000 rpm for 5 min and the supernatant was carefully removed. Three washes in 0.2 M NaCl solution (pH 5.0) at each interval were conducted before the next adsorption. The adsorption was repeated until 5 bilayers of polysaccharides were
2.2.2. Deposition of DOX Before DOX loading, the CaCO3 (CMC) microparticles were dried in a oven at 50 ◦ C. 20 mg CaCO3 (CMC) microparticles or CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles were immersed in 5 ml DOX solution (2 mg/ml). The suspension was brought to equilibrium under gentle agitation at room temperature for 24 h, followed by centrifugation at 4000 rpm for 5 min. After centrifugation, the DOX concentration of the supernatant was determined with the absorbance at 479 nm by a UV–visible spectrophotometer (UV-2550; Shimadazu, Japan). A calibration curve was obtained using the pure DOX. The amount of incorporated DOX was then obtained and expressed as milligram DOX/g CaCO3 (CMC). The DOX-loaded microparticles were freezedried before Brunauer–Emmett–Teller (BET) analysis and release experiment. For the study of loading kinetics, 60 mg CaCO3 (CMC) microparticles was immersed in 2 ml DOX solution (2 mg/ml), and the supernatant concentration was measured as a function of time. 2.2.3. In vitro release The DOX-loaded CaCO3 (CMC) microparticles (20 mg, = 37.5 mg DOX/g CaCO3 from UV, ca. 0.75 mg DOX in total) and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles (20 mg, = 67 mg DOX/g CaCO3 from UV, ca. 1.34 mg DOX in total) were put in dialysis bags (cut-off MW 8000) with 5 ml release medium separately, which were immersed in 195 ml
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Fig. 4. (a) Loading kinetics of DOX into CaCO3 (CMC) microparticles. (b and c) CLSM images of DOX-loaded CaCO3 (CMC) microparticles.
release medium. The in vitro release was performed with continuous agitation at 37 ◦ C. Two different solutions of pH 2.0 and pH 5.0 were used as the release medium (0.1 M sodium citrate-HCl buffer). 1 ml solution was withdrawn from the release medium and then supplemented with 1 ml fresh release medium of the same pH at desired time interval. The DOX concentration in the release medium was then determined with UV spectroscopy. The cumulative released amount of DOX was integrated from each measurement.
2.2.4. Characterization For scanning electron microscopy (SEM, SIRION-100, FEI) observation, the CaCO3 (CMC) microparticles suspension was dropped on a glass slide, air dried and then sputtered with gold. The acceleration voltage was set at 15 kV. The DOX-loaded CaCO3 (CMC) microparticles and hollow microcapsules were observed by confocal laser scanning microscopy (CLSM, Zeiss LSM510). The specific surface area and the pore size distribution of the CaCO3 (CMC) microparticles were measured by the Brunauer–Emmett–Teller
Fig. 5. N2 adsorption–desorption isotherms of CaCO3 (CMC) microparticles before (a) and after DOX loading (b).
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method of N2 adsorption–desorption at −196 ◦ C with an ASAP 2010 surface area analyzer (Micrometrics BET, USA). -Potential of the CaCO3 (CMC) microparticles after each adsorption was measured in 0.2 M NaCl solution with a pH value of 5.0 by a Zeta-sizer (Mastersizer 2000, UK). Each datum was averaged from 3 measurements. 3. Results and discussion 3.1. Preparation of porous CaCO3 (CMC) microparticles and multilayers coating To obtain spherical CaCO3 microparticles, CMC, a negatively charged polysaccharide, was added into the reaction mixture during the preparation to control the crystallization and growth behavior of the CaCO3 particles. Fig. 2 displays SEM images of the CaCO3 (CMC) microparticles with two different magnifications. On the surface of the CaCO3 (CMC) microparticles there existed a lot of carbonate nanoparticles and many tiny channel pores with a size of 20–50 nm. As a biocompatible macromolecule, the added CMC plays an important role. It can not only help to get spherical microparticles with narrow size distribution through its control over the crystallization process, but also be beneficial to maintain the biocompatibility of the microparticles. The XRD pattern (Fig. 2c) shows that the CaCO3 (CMC) microparticles had a dominant phase of vaterite, although a trace amount of calcite was present also. The broadening diffraction peaks suggest that the produced vaterite microspheres were composed of tiny nanoparticles. Generally, spherical CaCO3 microparticles will change to rhombohedral calcite microcrystals in water for several days [23,24]. Recrystallization of the porous CaCO3 (CMC) microparticles composed of carbonate nanoparticles was blocked by the CMC adsorbed on the surface of the carbonate nanoparticles. Thus, the CaCO3 (CMC) microparticles were much more stable than those without additives. To decrease release rate of the loaded DOX from the microparticles, the CaCO3 (CMC) microparticles were further coated with chitosan and alginate polyelectrolyte multilayers before DOX loading. The LbL self-assembly process was monitored by -potential as shown in Fig. 3. The optimal pH value of the polyelectrolyte solutions was set to 5.0 because at this pH both chitosan and alginate are highly charged [25–27]. Since CMC is negatively charged, the -potential of the CaCO3 (CMC) microparticles was about −20 to −30 mV. The -potential of the microparticles deposited with the first chitosan layer still remained a negative value, however, increased about 10 mV as compared with the bare microparticles. After the second cycle of the assembly process, regular charge reversal of -potential indicated adsorption of polycation chitosan and polyanion alginate on the CaCO3 (CMC) microparticles alternatingly. After 4 bilayers of chitosan/alginate were assembled, the CaCO3 (CMC) microparticles became smoother and the original pores disappeared (Fig. 3b and c), which are distinctive with the pristine particles (Fig. 2a and b). Moreover, removal of the template particles yielded hollow microcapsules (Fig. 3d). All these results confirm the successful coating of the CaCO3 (CMC) microparticles with the chitosan/alginate multilayers.
Table 1 BET characterization of porous CaCO3 (CMC) microparticles before and after DOX loading.
Before loading After loading
BET surface area (m2 /g)
Adsorption average diameter (nm)
Total volume of pores at P/P0 0.976 (cm3 /g)
77.60 5.32
3.53 20.87
0.068 0.028
images of the DOX-loaded CaCO3 (CMC) microparticles. Strong fluorescence emitted from the particles, which could be only attributed to DOX and thereby qualitatively confirmed the successful loading. The loaded amount ( ) of DOX was quantified with UV–vis spectroscopy by subtracting the supernatant amount from the feeding amount. The values were found to be 475 mg and 480 mg DOX/g CaCO3 for the CaCO3 (CMC) microparticles and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles, respectively. The value is about an order of magnitude larger than that of ibuprofen ( = 30 ∼ 80 mg ibuprofen/g CaCO3 ) loaded in CaCO3 (PSS) microparticles [11]. Under the loading conditions, DOX is positively charged, and CMC, PSS and ibuprofen are all negatively charged. Therefore, the reason for larger loading of DOX in the CaCO3 (CMC) microparticles is attributed to attraction between CMC and DOX. By contrary, the repulsion between PSS and ibuprofen is unfavorable for ibuprofen loading. Besides, the drugs may be also filled into the nanopores of the CaCO3 (CMC) microparticles, as confirmed by the BET analysis. Although the chitosan/alginate multilayers may slow down the loading rate, the pore size of the multilayers is typically around tens of nanometer, and thereby the eventual loading should not be influenced significantly as evidenced by the same amount of DOX. To prove that the DOX molecules could penetrate into the nanopores of the CaCO3 (CMC) microparticles, BET analysis was also carried out. N2 adsorption–desorption isotherm of CaCO3 (CMC) microparticles before and after DOX loading are depicted in Fig. 5a and b, respectively. Fig. 5a shows that at higher pressures the pores of the CaCO3 (CMC) microparticles were filled with adsorbed or condensed N2 leading to the plateau, indicating that the multilayers-adsorbed N2 was saturated because of limited pore volume. The hysteresis loop of desorption isotherm in Fig. 5a belongs to Type E classified by de Boer [28], conveying that the pores possess a narrow neck and wide body shape. The hysteresis loop of desorption isotherm of the microparticles loaded with DOX in Fig. 5b suggests the pores have a inter-slanting slices shape, which is possibly formed by filling of the nanopores by the complexes of the CMC molecules and DOX molecules.
3.2. DOX loading By simple incubation of the CaCO3 (CMC) microparticles or the CaCO3 (CMC) microparticles coated with chitosan/alginate multilayers in DOX solution, the positively charged anti-cancer drug DOX was spontaneously loaded into the microparticles (Fig. 4a). The loading was almost completed within 5 h, with a final encapsulation efficiency of >95%. Therefore, our loading time of 24 h was long enough to reach an equilibrium state. Fig. 4b and c display the CLSM
Fig. 6. Pore size distribution of CaCO3 (CMC) microparticles before and after DOX loading.
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Fig. 7. Schematic demonstration of the change of pore size distribution of CaCO3 (CMC) microparticles before and after DOX loading.
The change of specific surface area, pore size and pore volume distribution of the microparticles before and after DOX loading are summarized in Table 1. The BET surface area SBET of CaCO3 (CMC) microparticles was 77.6 m2 /g, which is much higher than that of CaCO3 microparticles without dispersants (8.8 m2 /g) [23,24]. The microcrystal carbonates were loosely packed due to the intertwining of the dispersant CMC molecules adsorbed on surface of the carbonate nanocrystals, and thus possessed larger specific surface area. After DOX loading, the BET surface area of the CaCO3 (CMC) microparticles decreased to 5.32 m2 /g and the pore volume was reduced from 0.068 to 0.028 cm3 /g (Table 1) as well, indicating penetration of the DOX molecules into the nanopores. A simple calculation found that theoretical density of DOX was as high as 12 g/cm3 , which is apparently unreasonable. As shall be demonstrated by the pore size distribution (Fig. 6), many DOX molecules were filled into the smaller pores, and those smaller than 2 nm could not be measured by the BET method. Moreover, the CMC macromolecules occupied some internal volume of the particles. By charge attraction, the DOX molecules bind with the CMC macromolecules and thereby make the CMC more compact without change of volume. Another reason should be that some DOX molecules adsorbed on the particle surface, and contributed considerably to the loading amount, as illustrated by the burst release in Fig. 8. Pore size distribution of the microparticles before and after DOX loading is shown in Fig. 6. After DOX loading, the volume of small pores less than 9 nm decreased greatly while that of larger ones increased to some extent. The smaller pores possess high surface free energy and thus are easily to be filled by the DOX molecules. The increased volume of larger pores could probably be explained by electric charge screening effect. The proposed mechanism of the change of pore size distribution is presented in Fig. 7. Being soaked in DOX solution, the CMC macromolecules doped in the CaCO3 (CMC) microparticles are highly swollen and effectively charged. The positively charged DOX molecules preferentially adsorb into the smaller pores and effectively screen the electric repulsion among the negatively charged CMC macro-
molecules. Consequently, the gaps preferentially occupied by DOX between the CaCO3 nanocrystals became smaller, and the space between neighboring nanocrystals which form the larger pores became larger. This can explain why after loading the adsorption average diameter increased from 3.53 to 20.87 nm (Table 1). This is different from the result of adsorption of ibuprofen by CaCO3 microparticles doped with PSS [11]. In that case, the electric repulsion among the negatively charged PSS molecules adsorbed on the CaCO3 nanocrystals would be enhanced by loading of the ibuprofen molecules, leading to decrease of volume of both smaller and larger pores. 3.3. In vitro drug release It is well known that the local pH of tumor tissue is lower than that of normal tissue in human body, i.e. the pH of normal tissue is about 7.4, and the pH of tumor tissue may be as low as 5 or 6. Therefore, we chose a pH 5 buffer for the release experiment to simulate the pH of tumor tissue. The release was also carried out at pH 2 to expedite the behavior since the release kinetic is mainly controlled by dissolution of the CaCO3 particles at low pH. It should be pointed out that the release amount at physiological condition (pH 7.4) of DOX from the CaCO3 (CMC) microparticles was very low and could hardly be detected by UV spectroscopy. This would mean that this drug delivery system should have smaller toxicity to normal tissues (pH 7.4), illustrating the advantages of this “safer” delivery system. The DOX release profiles from the CaCO3 (CMC) microparticles and the CaCO3 (CMC)–(chitosan/alginate)5 core–shell microparticles are shown in Fig. 8 (pH 5 buffer and pH 2 buffer). Fig. 8a shows that the release rate was relatively slow after the initial burst release within the first 15 h, and uncommonly the release process can be sustained to more than 150 h. This is rather promising for the application of in vivo drug delivery. Coating of the microparticles by chitosan and alginate multilayers could obviously assuage the initial burst release and control the release rate during the whole process. The long release duration also supports the fact that the
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Fig. 8. Release profiles of DOX from CaCO3 (CMC) microparticles and CaCO3 (CMC)–(chitosan/alginate)5 microparticles in (a) pH 5.0 and (b) pH 2.0 solution (0.1 M sodium citrate-HCl buffer).
DOX molecules are mainly adsorbed in the inner pores other than on the outer surface of the CaCO3 microparticles, because DOX release from the (chitosan/alginate)5 microcapsules was rather fast and could hardly be sustained over 20 h [8]. When the medium pH was set at 2 (Fig. 8b), the release rate was increased obviously, which should be attributed to the faster dissolution of the CaCO3 microparticles at lower pH. Again the multilayer coating could still lower down the release amount of the loaded DOX within the same incubation time. Unlike the 100% release of ibuprofen from the PSS-doped CaCO3 microparticles [11], the DOX release amount could not reach 100% regardless of the pH values investigated so far. This behavior is consistent with the structure of the CaCO3 (CMC) microparticles due to the strong charge attraction. Moreover, the CMC macromolecules become hydrophobic at acidic conditions as a result of protonation of the carboxyl groups, and thereby can block the further dissolution of the inner CaCO3 microparticles. 4. Conclusions The CMC-doped CaCO3 microparticles were prepared and coated by chitosan and alginate polyelectrolyte multilayers. These particles showed a strong ability to load DOX (475 mg DOX/g CaCO3 ), which should be mainly attributed to the charge attraction between negatively charged CMC and positively charged DOX. The large number of nanopores in the CaCO3 (CMC) microparticles provided additional capillary force for the loading. SBET of the CaCO3 (CMC) microparticles was much higher than that without dispersants. After DOX loading, the SBET and pore volume were reduced drastically. The volume of small pores also decreased significantly, whereas that of larger ones increased, indicating the penetration of DOX molecules into the nanopores. The increase of the volume of larger pores and average adsorption diameter may be explained by an electric charge screening effect. DOX release from the CaCO3 microparticles could be sustained to more than 150 h. Moreover, the multilayer coating could lower down the release amount of the loaded DOX within the same incubation time. Thus, this system has great potential for the application of in vivo drug delivery. Acknowledgements This study is financially supported by the Natural Science Foundation of China (20774084, 50873087) and the Major State Basic Research Program of China (2005CB623902). The author is also grateful for H. T. Yang’s help of drawing the schematic illustration figure in this paper.
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