Novel Hollow Microcapsules Based on Iron−Heparin Complex

Langmuir 2008, 24, 13723-13729

13723

Novel Hollow Microcapsules Based on Iron-Heparin Complex Multilayers Lu Yu,† Yanguang Gao,† Xiuli Yue,‡ Shaoqin Liu,† and Zhifei Dai*,†,‡ Nanomedicine and Biosensor Laboratory, Bio-X Center, Harbin Institute of Technology, Harbin 150080, China, and State Key Laboratory of Urban Water Resources and EnVironment (SKLUWRE), Harbin Institute of Technology, Harbin 150090, China ReceiVed August 11, 2008. ReVised Manuscript ReceiVed September 16, 2008 Iron-polysaccharide complex have been extensively utilized in the treatment of iron deficiency anemia for parenteral administration. Herein, a novel iron-heparin complexed hollow capsules with nanoscaled wall thickness have been fabricated by means of alternating deposition of ferric ions (III) (Fe3+) and heparin (Hep) onto the surface of submicroscaled (488 nm) and microscaled (10.55 µm) polystyrene latex particles via both electrostatic interaction and chemical complexation processes, followed by dissolution of the cores using tetrahydrofuran. Confocal micrographs and atomic force microscopy (AFM) images prove that iron-heparin complexed submicroscaled hollow capsules keep spherical shapes in solution and even after drying. The activated partial thromboplastin time (APTT) assay shows that complexing with ferric ions do not compromise the catalytic capacity of heparin to promote antithrombin III-mediated thrombin inactivation. The anticoagulant activity value of (Fe3+/Hep)8 capsules is evaluated to be about 95.7 U/mg, indicating that approximately 0.55 mg heparin was in 1 mg powder of submicroscaled (Fe3+/Hep)8 hollow capsules. Compared with the same dosage of heparin, iron-heparin complexed hollow capsules display a more prolonged anticoagulant duration than heparin. All these results reveal that such submicroscaled iron-heparin complexed hollow capsules have application potential as an injectable anticoagulant vehicle.

Introduction Hollow nano- and microcapsules with nanoscaled wall thickness are of particular interest due to their potential for encapsulation of large quantities of guest molecules or largesized guests within their empty core domain.1-6 These materials could be useful in a multitude of different applications, such as confined reaction vessels, drug carriers, protective shells for cells or enzymes, transfection vectors in gene therapy, carrier systems in heterogeneous catalysis, or materials for removal of contaminated waste.7-14 Size- and shape-persistent nano- and * To whom correspondence should be addressed. (X.Y.) Nanomedicine and Biosensor Laboratory, Bio-X Center, Harbin Institute of Technology, Harbin Institute of Technology, Harbin 150080, China, Tel/Fax: 86-45186402692, Email: [email protected]. † Nanomedicine and Biosensor Laboratory. ‡ State Key Laboratory of Urban Water Resources and Environment (SKLUWRE). (1) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mohwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202–2205. (2) Leporatti, S.; Voigt, A.; Mitlohner, R.; Sukhorukov, G.; Donath, E.; Mohwald, H. Langmuir 2000, 16, 4059–4063. (3) Tao, X.; Li, J. B.; Mohwald, H. Chem.-Eur. J. 2004, 10, 3397–3403. (4) Caruso, F.; Caruso, R. A.; Mohwald, H. Science 1998, 282, 1111–1114. (5) Hah, H. J.; Kim, J. S.; Jeon, B. J.; Koo, S. M.; Lee, Y. E. Chem. Commun. 2003, 10, 1712–1713. (6) Dai, Z. F.; Dahne, L.; Mohwald, H.; Tiersch, B. Angew. Chem., Int. Ed. 2002, 41, 4019–4022. (7) Baumeister, E.; Klaeger, S.; Kaldos, A. Proc. Inst. Mech. Eng. Pt. L-J. Mater.-Design Appl. 2005, 219, 207–216. (8) Ichikawa, H.; Fukumori, Y. J. Controlled Release 2000, 63, 107–119. (9) Sah, H. K.; Toddywala, R.; Chien, Y. W. J. Controlled Release 1994, 30, 201–211. (10) Valdes-Solis, T.; Valle-Vigon, P.; Sevilla, M.; Fuertes, A. B. J. Catal. 2007, 251, 239–243. (11) Ruysschaert, T.; Germain, M.; Gomes, J. F. P. D.; Fournier, D.; Sukhorukov, G. B.; Meier, W.; Winterhalter, M. IEEE Trans. Nanobiosci. 2004, 3, 49–55. (12) Sukhorukov, G. B.; Rogach, A. L.; Zebli, B.; Liedl, T.; Skirtach, A. G.; Kohler, K.; Antipov, A. A.; Gaponik, N.; Susha, A. S.; Winterhalter, M.; Parak, W. J. Small 2005, 1, 194–200. (13) Dai, Z. F.; Heilig, A.; Zastrow, H.; Donath, E.; Mohwald, H. Chem.-Eur. J. 2004, 10, 6369–6374. (14) Okubo, M.; Konishi, Y.; Minami, H. Colloid Polym. Sci. 1998, 276, 638–642.

microcapsules can be prepared using a variety of different techniques, such as self-assembling of amphiphilic species, emulsion polymerization, and the use of dendrimers or hypobranched polymers for encapsulation.15-20 Each of them has its special advantages and disadvantages. The approach via dendrimers is quite elegant, but it is mainly of academic interest due to the tedious and costly preparation procedures. Another versatile method for the fabrication of nano- and microcapsules involves the covering a templating support with ultrathin polyelectrolyte complex film,1–6,14–22 which is fabricated using a layer-by-layer (LbL) self-assembly technique first introduced by Decher.18–20 The LbL self-assembly technique relies on the consecutive adsorption of oppositely charged polyelectrolytes via electrostatic interactions, allowing fabrication of products that are difficult or impossible to produce by conventional techniques. The core material can be removed afterward by dissolution1,2 or calcination4,22 to leave a hollow capsule. Considering the fact that multilayer hollow microcapsules have received a great deal of attention as smart drug delivery carriers intended for systemic administration, the design of novel bloodcompatible hollow capsules is clearly an actual challenge. However, there are very few reports on hollow capsule interaction with blood, although researchers have attempted to study the interaction of liposomes with blood.23,24 The design of functional (15) Kong, X. Z.; Kan, C. Y.; Li, H. H.; Yu, D. Q.; Yuan, Q. Polym. AdV. Technol. 1997, 8, 627–630. (16) Dobashi, T.; Yeh, F. J.; Ying, Q. C.; Ichikawa, K.; Chu, B. Langmuir 1995, 11, 4278–4282. (17) Dai, Z. F.; Meiser, F.; Mohwald, H. J. Colloid Interface Sci. 2005, 288, 298–300. (18) Lvov, Y.; Decher, G.; Mohwald, H. Langmuir 1993, 9, 481–486. (19) Decher, G.; Lvov, Y.; Schmitt, J. Thin Solid Films 1994, 244, 772–777. (20) Decher, G. Science 1997, 277, 1232–1237. (21) Zhang, X.; Chen, H.; Zhang, H. Y. Chem. Commun. 2007, 1395–1405. (22) Dai, Z. F.; Mohwald, H. Chem.-Eur. J. 2002, 8, 4751–4755. (23) Koziara, J. M.; Oh, J. J.; Akers, W. S.; Ferraris, S. P.; Mumper, R. J. Pharm. Res. 2005, 22, 1821–1828. (24) Pinto, L. M. A.; Pereira, R.; de Paula, E.; de Nucci, G.; Santana, M. H. A.; Donato, J. L. J. Liposome Res. 2004, 14, 51–59.

10.1021/la802611b CCC: $40.75  2008 American Chemical Society Published on Web 10/15/2008

13724 Langmuir, Vol. 24, No. 23, 2008

materials requires a much higher complexity. The multilayer capsules provide opportunities to create such advanced materials because the capsule walls can be fabricated from a variety of compounds such as charged and noncharged polymers, biopolymers, and lipids. Also, functional molecules (biotin, dyes, etc.) can be incorporated into the capsule wall by chemical conjugation to a polyelectrolyte molecule beforehand.25-27 Therefore, it is just very easy to put new combinations of molecules and thus new functionality into such layered systems to get novel functionalized capsules. Heparin is a negatively charged naturally occurring linear polysaccharide present in many living organisms and is the most complex member of the glycosaminoglycan superfamily.28 The heparin molecule is highly sulfated at both the hydroxyl and amino groups of the polymer. It is now well-established that heparin plays a pivotal role in processes such as blood coagulation, inflammatory response, antivirus, tumor cell metastasis, cell adhesion, and cell growth.29-33 Clinically, besides its original therapeutic use as an anticoagulant, other potential applications of heparin for a vast array of human diseases have been identified.34 The potentially wide-ranging clinical importance of this bioactive macromolecule warrants the building of better heparin35 and the development of better heparin delivery system.26–43 Iron is essential for most living organisms because it is required for many metabolic processes.40,41 Iron absorption can be enhanced by complexing iron with sugars.44,45 Complexation of iron with saccharides and polyols has been extensively studied because saccharides are able to stabilize iron(III) oxides to maintain a relatively high concentration of iron at physiological conditions. Iron deficiency is one of the most severe nutritional problems worldwide.46 Polysaccharide iron complex synthesized by the reaction of FeCl3 have been widely used for the treatment of iron-deficiency anemia due to its effectiveness, safety, and freedom from side effects.47-50 (25) Dai, Z.; Wilson, J. T.; Chaikof, E. L. Mater. Sci. Eng. C: Biomimetic Supramol. Syst. 2007, 27, 402–408. (26) Dai, Z. F.; Voigt, A.; Leporatti, S.; Donath, E.; Dahne, L.; Mohwald, H. AdV. Mater. 2001, 13, 1339–1342. (27) Dai, Z. F.; Dahne, L.; Donath, E.; Mohwald, H. J. Phys. Chem. B 2002, 106, 11501–11508. (28) Luppi, E.; Cesaretti, M.; Volpi, N. Biomacromolecules 2005, 6, 1672– 1678. (29) Codee, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft, H. S.; van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A. J. Am. Chem. Soc. 2005, 127, 3767–3773. (30) Suk, J. Y.; Zhang, F. M.; Balch, W. E.; Linhardt, R. J.; Kelly, J. W. Biochemistry 2006, 45, 2234–2242. (31) Jee, K. S.; Dal Park, H.; Park, K. D.; Ha Kim, Y.; Shin, J. W. Biomacromolecules 2004, 5, 1877–1881. (32) Linhardt, R. J. J. Med. Chem. 2003, 46, 2551–2564. (33) Xu, F. J.; Li, Y. L.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2005, 6, 1759–1768. (34) Lever, R.; Page, C. R. Nat. ReV. Drug DiscoVery 2002, 1, 140–148. (35) Caughey, G. H. Am. J. Respir. Cell Mol. Biol. 2003, 28, 129–132. (36) Ross, B. P.; Toth, I. Curr. Drug DeliVery 2005, 2, 277–287. (37) Pineo, G.; Hull, R.; Marder, V. Best Pract. Res. Clin. Haematol. 2004, 17, 153–160. (38) Motlekar, N. A.; Youan, B. B. C. J. Controlled Release 2006, 113, 91– 101. (39) Windsor, E.; Freeman, L. Am. J. Med. 1964, 37, 408–416. (40) Wang, H. J.; Lin, Z. X.; Liu, X. M.; Sheng, S. Y.; Wang, J. Y. J. Controlled Release 2005, 105, 120–131. (41) Thomas, A. C.; Campbell, J. H. Atherosclerosis 2004, 176, 73–81. (42) DutradeOliveira, J. E.; Freitas, M. L. S.; Ferreira, J. F.; Goncalves, A. L.; Marchini, J. S. Int. J. Vitam. Nutr. Res. 1995, 65, 272–275. (43) Spinelli, F. J.; Kiick, K. L.; Furst, E. M. Biomaterials 2008, 29, 1299– 1306. (44) Crichton, R. R. Inorganic biochemistry of iron metabolism; Ellis Horwood: West Sussex, 1991. (45) Gyurcsik, B.; Nagy, L. Coord. Chem. ReV. 2000, 203, 81–149. (46) Scrimshaw, N. S. Sci. Am. 1991, 265, 46–52. (47) Hudson, J. Q.; Comstock, T. J. Clin. Ther. 2001, 23, 1637–1671. (48) Kane, R. C. Curr. Ther. Res.-Clin. Exp. 2003, 64, 263–268.

Yu et al.

The multivalent metal ion-polyelectrolyte multilayer systems have been reported previously.22,51-53 Herein, novel anticoagulant hollow capsules depending on the template size are fabricated by alternative deposition of oppositely charged Fe3+ and heparin onto the surface of the polystyrene (PS) latex particles, followed by removal of the PS templates by dissolution. Biofunctional components, heparin and iron, could impose a variety of new functionalities and potential applications on this iron-heparin complexed multilayer hollow capsule. Such hollow architecture and very thin capsule wall could reduce the turbidity and avoid fast precipitation, enabling us to prepare a reproducible injectable formulation. To exemplify the functionality of such a new capsule, its potential anticoagulant activity is investigated in vitro and vivo.

Experimental Methods Materials. Standard heparin sodium salt (Heparin, porcine intestinal mucosa, MW: 13 500-15 000, 174 U/mg) was purchased from CalBio Chem. Ferric chloride hexahydrate (FeCl3 · 6H2O, Mw 270.3) was acquired from Tianda Chemicals, China. Polystyrene (PS, 10.55 µm, 488 nm) latex particles were purchased from Microparticles, GmbH, Berlin, Germany. APTT Lyophilized silica was purchased from Beckman-Coulter, USA. Human plasma was obtained from the Pacific Hemostasis. The deionized water used in all experiments was prepared in a three-stage Millipore Milli-Q purification system and had a resistivity higher than 18.2 MΩ. A solution of heparin (1 mg/mL) was prepared by dissolving heparin powder in the water. The ionic strength of the heparin solution was modified with sodium chloride (0.5 M). The solution of FeCl3 (5 mg/mL) was prepared by addition of FeCl3 · 6H2O to the 0.1 M hydrogen chloride aqueous solution (HCl), and the pH 3 of this solution was adjusted with 0.1 M sodium hydroxide aqueous solution (NaOH). Fabrication of Hollow Capsules of Iron-Heparin Complexed Multilayers. Fe3+ and heparin were assembled onto PS latex particles (10.55 µm and 488 nm) through the layer-by-layer self-assembly method. The added species with charge opposite that of the particle surface or last layer deposited were allowed to adsorb for 5 min. The excess Fe3+ or heparin was removed by three repeated centrifugations by washing/redispersion cycles with deionized water in each deposition step. The subsequent layers were deposited in the same manner as the oppositely charged species. After completion of the desired number of deposition cycles, hollow capsules were prepared by dissolving the PS core with tetrahydrofuran (THF) and then centrifuging at 8000 rpm for 5 min and washing with THF and water for three times, respectively. The resulting Fe3+/Hep hollow capsules were suspended in the water and kept at 4 °C until use. Fabrication of Iron-Heparin Complexed Multilayer Film on Silicon Wafer. Multilayer thin films were deposited on silicon wafers using a dip technique from ferric chloride and heparin solution at room temperature. First, the substrates were immersed in ferric chloride solution for 5 min followed by three neutral washes, of 30 s each in Millipore deionized water. Subsequently, the substrates were dipped into an aqueous heparin solution for 5 min followed by three consecutive neutral washings. Structure Characterizations of the Fe3+/Hep Microcapsules. The particle size distribution and microelectrophoretic mobility of coated PS latex particles dispersed in pure water were measured by a ZetaPALS dynamic light-scattering detector (Brookhaven Instruments, USA). (49) Sipos, P.; Stpierre, T. G.; Tombacz, E.; Webb, J. J. Inorg. Biochem. 1995, 58, 129–138. (50) Coe, E. M.; Bowen, L. H.; Speer, J. A.; Wang, Z. H.; Sayers, D. E.; Bereman, R. D. J. Inorg. Biochem. 1995, 58, 269–278. (51) Radtchenko, I. L.; Sukhorukov, G. B.; Leporatti, S.; Khomutov, G. B.; Donath, E.; Mohwald, H. J. Colloid Interface Sci. 2000, 230, 272–280. (52) Galeska, I.; Hickey, T.; Moussy, F.; Kreutzer, D.; Papadimitrakopoulos, F. Biomacromolecules 2001, 2, 1249–1255. (53) Galeska, I.; Chattopadhyay, D.; Moussy, F.; Papadimitrakopoulos, F. Biomacromolecules 2000, 1, 202–207.

Hollow Microcapsules Based on Iron-Heparin Complex

Figure 1. Zeta potential of Fe3+/Hep multilayer on 488 nm PS latex particles as a function of the number of deposition steps. The odd-layer numbers correspond to Fe3+ deposition and even-layer numbers to heparin adsorption. The first measurement (layer 0) is the surface potential of PS latex particles.

Confocal micrographs of the obtained microcapsules were taken with a confocal laser scanning microscope (CLSM, Carl Zeiss LSM 510 META, Germany), equipped with an oil immersion objective. The Fe3+/Hep multilayer film coated silicon wafers were goldsputtered before being imaged by scanning electron microscope (SEM) (HITACHI S-3400, Japan). The atomic force microscopy images (AFM) were obtained by means of a Digital Instruments Nanoscope IIIa in tapping mode. AFM images were recorded in air at room temperature, and the samples were prepared by applying a drop of the hollow capsule solution onto a freshly cleaved mica substrate. After the capsules had been allowed to settle, the substrate was dried naturally. Fourier transform infrared (FT-IR) spectra of heparin and hollow capsules of (Fe3+/Hep)8 were acquired using a Varian Resolution Fourier transform infrared spectrometer (Varian FTS 3100, USA). Samples were prepared in the forms of potassium bromide (KBr) disk. Approximately 1 mg sample and 99 mg of KBr powder was blended and triturated with agate mortar and pestle. The mixture was compacted using an IR hydraulic press at a pressure of 8 tons for 1 min. For each spectrum, a 512-scan interferogram was collected with a 4 cm-1 resolution from the 4000 to 500 cm-1 region at room temperature with nitrogen gas. Anticoagulant Activities of Fe3+/Hep Hollow Capsule in Vitro and in Vivo. The anticoagulant activities of hollow capsules of Fe3+/Hep multilayer in prevention of fibrin clot formation were determined by activated partial thromboplastin assay using Instrumentation Laboratory IL ACL 3000 Plus (Beckman-Coulte, USA). Each platelet-poor, plasma-containing heparin standards (0.1 to 0.5 U/mL, 0.2 mL) and plasma samples containing Fe3+/Hep hollow capsule were incubated with APTT reagent. The anticoagulant activity was calculated by comparing the clotting time with the heparin standard curve. The clotting time was linearly proportional to activity of heparin in the plasma. Submicroscaled Fe3+/Hep hollow capsule samples (960 U/kg) were injected into male Wistar rats (purchased from Laboratory Animal Center of Harbin Medical University) with an approximate body weight of 250 g. The clotting time measured by APTT was determined in blood plasma before and 1, 2, 3, 4, 5, 6, to 12 h after administration of Fe3+/Hep hollow capsules. The rats fasted overnight for at least 12 h with free access to water. Fe3+/Hep capsules were intravenously injected into rats via a tail vein. Serial blood samples of 450 µL were collected from heart of the rat and immediately injected into a vial containing 50 µL 3.8% sodium citrate solution. After sufficient mixing, blood samples were centrifuged at 4 °C, 2500 × g, for 5 min. The rat plasma was stored at -80 °C until analysis.

Results and Discussion Preparation and Characterization of Hollow Capsules. The layer-by-layer deposition process is easily monitored by zeta potential measurements of PS nanoparticles alternately exposed to Fe3+ and heparin, respectively (Figure 1). The uncovered PS

Langmuir, Vol. 24, No. 23, 2008 13725

Figure 2. CLSM images of microcapsules of (Fe3+/Hep)5 assemble on 10.55 µm PS latex particles with neural (pH 7) wash: before (a) and after (b) removal of PS templates by dissolution using THF.

Figure 3. SEM micrograph of (Fe3+/Hep)5 multilayer film coated silicon wafer.

particles are negatively charged and exhibited a value of -52 mV. When PS particles are exposed to Fe3+, the zeta potentials are altered to +52 mV after neutral wash. After the negatively charged heparin formed the outermost layer, zeta potentials are altered around -42 mV as measured after neutral wash. The zeta potentials alternate in a manner very similar to that reported for polyelectrolyte adsorption.51 The obvious switching of zeta potentials indicates successfully alternating deposition of oppositely charged species. After complete removal of solid core of PS particles by dissolution with THF, (Fe3+/Hep)5 hollow microcapsules are obtained. CLSM images provide direct visualization of such hollow structures (Figure 2). No closed capsules are obtained from one bilayer of Fe3+/Hep. The size and shape of the hollow capsules that have more than one bilayer are persistent. Hollow microcapsules of (Fe3+/Hep)5 maintain a spherical shape in water. The structures seen in the transmission image are due to contrast of remaining Fe3+/Hep complex layers from the original coating of templates. Such a result suggests that Fe3+/Hep microcapsules are shape-persistent in water and resist osmotic pressure during dissolution of core by THF. Thus, a multilayer is built up with heparin as one of the main structural elements on sacrificial PS templates. Because Fe3+ is absorbed on the polysaccharide surface at low pH value, Fe(OH)3 is formed on substrate surfaces after neutral water washes. The iron-free polysaccharides with random coil structures would then pack an amount of Fe(OH)3 along their flexible backbone.49 In order to verify the formation of Fe(OH)3 particles, Fe3+ and heparin are deposited alternately on a silicon wafer. In Figure 3, small particles are observed on the (Fe3+/Hep)5 multilayer film from SEM measurement, providing direct evidence for the formation of Fe(OH)3.

13726 Langmuir, Vol. 24, No. 23, 2008

Yu et al.

Figure 4. AFM images of dried hollow microcapsules of (Fe3+/Hep)n obtained by removal of cores of 10.55 um PS latex particles by dissolution with THF. (a) ) 2, (b) ) 3, (c) ) 4, (d) ) 5.

Fe3+/Hep hollow capsules typically maintain the spherical shape of the template particle in solution, while depositing on a solid substrate and air-drying induces their collapse. Figure 4 displays AFM images of dried hollow microcapsules with different numbers of Fe3+/Hep bilayer alternatively deposited onto 10.55 µm PS latex particles. A number of folds and creases are observed as a result of air-drying. As the number of Fe3+/ Hep bilayers increases, the walls of hollow capsules are found to be thicker. In order to obtain quantitative evidence for the formation of Fe3+/Hep multilayer on PS particles, the wall thickness of these capsules is further measured by using AFM from the smallest height of microcapsules. The values that are equivalent to twice the wall thickness of the microcapsule are found to increase almost linearly with the number of bilayer deposition cycles after the first few layers (Figure 5). Such behavior has often been observed, with few polyelectrolyte layers required prior to regular multilayer growth for films assembled by the layer-by-layer technique.18–20 An average growth for each additional Fe3+/Hep layer deposited is calculated to be 15.38 ( 0.28 nm from the 3-, 4-, 5-bilayer films. It is six times higher than 2.6 nm observed in earlier studies for the thickness of bilayers of PSS/PAH2 and similar to the Fe3+/Nafion capsules (15.5 nm) in earlier studies.22 Therefore, AFM measurements provide direct evidence for the creation of microcapsules from Fe3+/Hep complexes with the layer-by-layer strategy. Figure 6 shows the size distribution and representative AFM images of hollow capsules prepared from (Fe3+/Hep)8 coated 488 nm PS latex particles. Because of the uniformity of PS templates, the (Fe3+/Hep)8 capsules have a narrow size distribution

Figure 5. Wall thickness of hollow microcapsule of (Fe3+/Hep)n (n ) 2, 3, 4, 5) template on 10.55 µm PS latex particles.

according to DLS measurement. After coating 8 Fe3+/Hep bilayers, the diameter of the resulting capsules increase to 616.2 ( 10.2 nm. AFM images show that the submicroscaled hollow capsules of (Fe3+/Hep)8 are spherical, and no fold and no disruption are observed in a dry environment. It is previously reported that the wall thickness of the hollow capsule is not sufficient to maintain spherical structure after drying if the 10.55 µm PS latex particles are used as template cores instead of 488 nm PS latex particles.17 In contrast to (Fe3+/Hep)5 microcapsules that template on 10.55 µm PS latex particles, submicroscaled (Fe3+/Hep)8 hollow capsules could maintain spherical structure after drying. Interestingly, it is found that the size of the PS templates has a profound effect on the thickness of the deposited films. A relatively fast growth is observed when 10.55 µm PS

Hollow Microcapsules Based on Iron-Heparin Complex

Langmuir, Vol. 24, No. 23, 2008 13727

Figure 6. (Fe3+/Hep)8 hollow capsules template on the 488 nm PS particles: (a) Size distribution and (b) AFM image.

Figure 7. FT-IR spectra (fundamental band region): (a) heparin and (b) (Fe3+/Hep)8 hollow capsules.

particles are used as templates for the fabrication of Fe3+/Hep hollow capsules. The average growth for each Fe3+/Hep deposition cycle is evaluated to be ca. 8 and 15 nm for 488 nm and 10.55 µm PS particles, respectively. We infer that this remarkable thickness difference may result from the different surface area. The surface area of 10.55 µm PS latex particle is 467 times higher than that of 488 nm PS latex particles. The bigger Fe(OH)3 particles tend to form on the large surface, leading to the higher growth of Fe3+/Hep bilayer deposition for the larger PS templates. Spectra in infrared fundamental region (400-1750 cm-1) of heparin and submicroscaled (Fe3+/Hep)8 hollow capsules are shown in Figure 7. The quite strong vibration band appeared at 1626 and 1421 cm-1 could be assigned to the symmetric and antisymmetric stretching mode of COO- containing in heparin. The appearance of the two absorption bands at 1240 and 1032 cm-1 were due to the asymmetric and symmetric stretching vibration of -SdO- of the sulfate groups of heparin.54,55 Because heparin is the main component of hollow capsules, the spectra of (Fe3+/Hep)8 hollow capsules obtained by removal of 488 nm PS latex templates display a similar profile to the spectra of heparin. No characteristic peaks assigned to PS particles are observed in the IR spectra of (Fe3+/Hep)8 hollow capsules, suggesting complete removal of PS templates. The heparin contains potential donor groups of various types such as carboxylate, sulfonate, alcoholic hydroxyl (or hydroxylate), glycosidic and ether oxygen, carbonyl oxygen, and amide nitrogen. The participation of the amide nitrogen in binding is unlikely.49 Thus, the complexes are only oxygencoordinated. Because of their extremely high stability, the formation of chelates involving deprotonated alcoholic or, less probably, glycosidic oxygens can be suggested. Because (54) Grant, D.; Long, W. F.; Williamson, F. B. Biochem. J. 1987, 244, 143– 149. (55) Harada, N. S.; Oyama, H. T.; Bartoli, J. R.; Gouvea, D.; Cestari, I. A.; Wang, S. H. Polym. Int. 2005, 54, 209–214.

of steric reasons, the (SO4-, COO-) chelation can be ruled out. Thus, the formation of (COO-, O-) and (SO4-, O-) chelates is highly probable.49 FT-IR spectra provide insights into the interaction of Fe(III) with heparin and the nature of the complexes formed. A clue of the coordination interaction between heparin and Fe3+ is the cation dependency of frequency and intensity of hydroxyl, sulfate, and carboxylate carbonyl groups. After complexation with Fe3+, these groups on heparin are in frequency shifts of 10-20 cm-1. For example, the prominent CdO (1626 cm-l) and SdO stretching bands (1240 cm-l) in the heparin spectra shifted in the hollow capsules of Fe(III)-heparin complexes to 1610 and 1226 cm-1, respectively. In addition, these bands were found to be sharpened in the spectra of hollow capsules. These changes demonstrate that both the carboxylate and sulfonate groups are involved in complexation to the Fe(IlI). The activity of heparin is believed to depend on the number of O-sulfate groups that appear at 1240 cm-1 in infrared spectra.56,57 Therefore, the influence of the complexation with Fe3+ on the anticoagulant activity of heparin should be tested. The stability of submicroscaled (Fe3+/Hep)8 hollow capsules is also examined. The capsules are subjected to a shaken wash at 74 to 80 rpm (selected to mimic resting heart rate) in 20 mM phosphate buffered saline (PBS, pH 7.4) for a week at 37 °C. There is no free heparin detected in the aqueous solution using the reported method of toluidine blue heparin assay.58 No leakage of Fe3+ ions from the capsules is detected using a spectrophotometer too. After incubation in 10% NaOH and HCl (pH 1) in the same shaken wash model for a week, no decomposition is observed. This phenomenon indicates that (Fe3+/Hep)8 hollow capsules are stable over a wide pH range and cannot be easily depolymerized. In Vitro Characterization of Microcapsules. The particle size is one important factor that determines the fate of foreign particles in blood circulation time.59,60 The 488 nm PS latex particles are used as a template for fabrication of submicroscaled hollow capsules that might not be rapidly eliminated and sustain circulating time. Because of the predominantly negative charge of the cell membrane, several particle systems showed an increased uptake of positively charged particles compared to that of negatively charged particles.61 Heparin is used as the outermost layer to design a negatively charged capsule to reduce the uptake of cells in the blood flow. A zeta potential of -48.7 (56) Zhbankov, R. G.; Rukina, N. M.; Grinkevich, T. L. Anal. Method Prod. Control 1971, 5, 45–47. (57) Mulloy, B.; Mourao, P. A. S.; Gray, E. J. Biotechnol. 2000, 77, 123–135. (58) Park, K. D.; Piao, A. Z.; Jacobs, H.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Chem. 1991, 29, 1725–1737. (59) Barratt, G. Cell. Mol. Life Sci. 2003, 60, 21–37. (60) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. ReV. 2001, 53, 283–318. (61) Foster, K. A.; Yazdanian, M.; Audus, K. L. J. Pharm. Pharmacol. 2001, 53, 57–66.

13728 Langmuir, Vol. 24, No. 23, 2008

mV is measured for the (Fe3+/Hep)8 hollow capsules using 488 nm PS particles as templates. The negative zeta potential showed that the outermost heparin layer is not entirely neutralized with Fe3+ on the capsule wall via electrostatic interaction. Additionally, the utilization of heparin as the outermost layer may create a steric barrier around the particles to prolong circulation time. Such a steric barrier-provided density of the “brush” could reduce the opsonization so that particles could be recognized by mononuclear phagocyte system (MPS). Such structures make (Fe3+/Hep)8 capsules perform the same function as PEG in classical stealth systems, which might also enable long circulation in the bloodstream. It is also proposed that the outermost layer of heparin plays a key role in improving the hydrophilic surface that reduced protein adsorption and uptake.62,63 Heparin is conventionally monitored using APTT to measure the anticoagulant effect of prolongation. The anticoagulant activity of submicroscaled (Fe3+/Hep)8 capsules is tested by APTT assay and calculated by comparing clotting time with the heparin standard curve. Compared with standard heparin, the anticoagulant activity value of (Fe3+/Hep)8 capsules is evaluated to be about 95.7 U/mg, indicating that approximately 0.55 mg heparin is in 1 mg (Fe3+/Hep)8 hollow capsule powder. Therefore, there is about 0.45 mg Fe(OH)3 in 1 mg capsule powder. APTT reflects primarily anti-IIa activity. Its prolongation suggest that (Fe3+/Hep)8 capsules exhibited protein binding ability even though heparin molecular structure is involved in polyelectrolyte-cation interactions. In general, the layer-by-layer self-assembly is an effective method by which a heterogeneous multilayer film with unique properties can be fabricated on a variety of template particles. With the help of the layer-by-layer self-assembly technique, heparin is changed from a water-soluble anticoagulant drug into a stable three-dimensional structure that still exhibited anticoagulant activity. The mean diameter of such a capsule is made directly to submicroscale. It is important to note that regular and well-known iron polysaccharide complexes have been extensively utilized in the treatment of iron deficiency anemia for parenteral administration.64 In Vivo Evaluation of Anticoagulant Activity of Submicroscaled Hollow Capsules. A major limitation faced by foreign particles is their rapid elimination from systemic circulation by cells of the mononuclear phagocyte system within 30 min.63 This action would block foreign particles from binding to a series of thrombin inhibitors, and particles would subsequently be cleared from circulation.65 Interestingly, submicroscaled Fe3+/Hep hollow capsules are not cleared from circulation rapidly even though they have a longer anticoagulant effect than heparin in the same dose. To display the anticoagulant effect in vivo, the same significant amount of heparin and submicroscaled (Fe3+/Hep)8 hollow capsules is injected into blood flow of rats and monitored by APTT assay (Figure 8). The anticoagulant effect of an aqueous solution of heparin was lost after 8 h of intravenous injection with a dosage of 960 U/kg. Heparin is rapidly eliminated in the blood flow by binding to plasma proteins and eing altered by enzymatic degradation.66 With the same dose as heparin, the anticoagulant activity of (Fe3+/Hep)8 capsules is observed between (62) Liu, M.; Yue, X. L.; Dai, Z. F.; Xing, L.; Ma, F.; Ren, N. Q. Langmuir 2007, 23, 9378–9385. (63) Zahr, A. S.; Davis, C. A.; Pishko, M. V. Langmuir 2006, 22, 8178–8185. (64) Burns, D. L.; Mascioli, E. A.; Bistrian, B. R. Nutrition 1995, 11, 163– 168. (65) Javier, A. M.; Kreft, O.; Alberola, A. P.; Kirchner, C.; Zebli, B.; Susha, A. S.; Horn, E.; Kempter, S.; Skirtach, A. G.; Rogach, A. L.; Radler, J.; Sukhorukov, G. B.; Benoit, M.; Parak, W. J. Small 2006, 2, 394–400.

Yu et al.

Figure 8. Mean prolongation of APTT over 12 h after intravenous administration of heparin and submicroscaled (Fe3+/Hep)8 hollow capsules (960 U/kg). Data represent the mean ( ASD, n ) 6 rats.

2 and 12 h in the circulation of test animals with a value of APTT beyond 120 s from 3 to 6 h. Such results show the potential duration of anticoagulant effect of (Fe3+/Hep)8 capsules. Heparin has a short systemic half-life and is a water-soluble drug that would diffuse in organism within a short time. Nevertheless, when submicroscaled (Fe3+/Hep)8 hollow capsules enter into the blood flow, its suspension condition would prevent it from diffusing and emerging with a rapid anticoagulant effect like the heparin, causing prolongation of the anticoagulant effect from the beginning to the end after intravenous administration. The anticoagulated mechanism of heparin is very complicated, involving the following process: inhibiting transformation of the thrombinogen to thrombin, deceasing the activity of the thrombin, and preventing the aggregation and adherence of the platelets, and so forth.67,68 As we know, the basis for heparin’s anticoagulant activity in plasma is that it binds to antithrombin, which includes the rapid formation of a heparinAT III inhibitory complex that subsequently reacts with free thrombin. As mentioned above, no leakage of heparin from the capsules is detected, indicating that the anticoagulant mechanism of (Fe3+/Hep)8 hollow capsules is not dependent on the release of heparin. Therefore, we infer that, for submicroscaled hollow capsules of (Fe3+/Hep)8, the surface-bound heparin is catalytically active, and the effect is not due to the solution-phase free heparin leaking from the hollow capsules. It was demonstrated that inhibition of thrombin adsorbed on the heparin surface occurs as follows: AT III adheres to high-affinity heparin fragments on the capsule surface whereupon adsorbed thrombin migrates in the hydrophilic heparin surface of hollow capsules toward the reaction site of AT III and becomes inhibited. The inactivated thrombin-AT III complex then leaves the surface, thus enabling the process to be repeated.70

Conclusion We have described an effective method for the fabrication of anticoagulant hollow microcapsules using the colloidally templated layer-by-layer self-assembly technique with heparin and ferric ions. This technique lends iron-heparin complexed hollow capsules to a simple particle-size control in the microand submicroscale. The resulting hollow capsule is stable over a wide range of pH. Its hollow architecture and very thin (66) Hirsh, J.; Anand, S. S.; Halperin, J. L.; Fuster, V. Circulation 2001, 103, 2994–3018. (67) Olafsdottir, E. S.; Ingolfsdottir, K. Planta Med. 2001, 67, 199–208. (68) Fiore, M. M.; Kakkar, V. V. Biochem. Biophys. Res. Commun. 2003, 311, 71–76. (69) Mourao, P. A.; Giumaraes, B.; Mulloy, B.; Thomas, S.; Gray, E. Br. J. Hamaetol. 1998, 101, 647–52. (70) Pasche, B.; Kodama, K.; Larm, O.; Olsson, P.; Swedenborg, J. Thromb. Res. 1986, 44, 739–48.

Hollow Microcapsules Based on Iron-Heparin Complex

capsule wall could reduce the turbidity and avoid fast precipitation, enabling us to prepare a reproducible injectable formulation. Heparin and iron imposed a variety of new functionalities and potential applications on such iron-heparin complexed multilayer hollow capsule. The APTT assay showed that the anticoagulant activity of submicroscaled hollow capsules of (Fe3+/Hep)8 is 95.7 U/mg. Compared with the

Langmuir, Vol. 24, No. 23, 2008 13729

same dose of heparin, these hollow capsules displayed a prolonged-duration in vivo assay. Acknowledgment. This research is financially supported by National Natural Science Foundation of China (NSFC50573015 and 30740061) and Program for New Century Excellent Talents in University (NCET-05-0335). LA802611B