Novel, Fluorescent, Magnetic, Polysaccharide-Based Microsphere for

Institute of Life Science and Technology, Inner Mongolia Normal University,. Huhhhot, 010020, People's Republic of China. Received November 3, 2004; ...
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Biomacromolecules 2005, 6, 1041-1047

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Novel, Fluorescent, Magnetic, Polysaccharide-Based Microsphere for Orientation, Tracing, and Anticoagulation: Preparation and Characterization Guang-Ming Qiu,† You-Yi Xu,*,† Bao-Ku Zhu,† and Guang-Liang Qiu‡ Department of Polymer Science & Engineering, Zhejiang University, Hangzhou, 310027, P. R. China, and Institute of Life Science and Technology, Inner Mongolia Normal University, Huhhhot, 010020, People’s Republic of China Received November 3, 2004; Revised Manuscript Received December 31, 2004

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 µm, contain 586-1013 µg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54 212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence. 1. Introduction There is an increasing interest for the synthesis of the socalled fluorescent, magnetic, polysaccharide-based microspheres, mainly for their potent orientating and tracing action and a wide range of physicochemical properties, modulated by the structural units of the polysaccharide.1-4 The fluorescent, magnetic, polysaccharide-based microspheres have a magnetite core containing a rare-earth complex and polymer shell bearing sugar residues. The microspheres have a potent fluorescence and magnetic susceptibility and reactive polymer surfaces for conjugation with heparin, galactose, glucose, lactose, etc. They can be moved to a given site with a powerful magnetic field, and also traced by any fluorescent scanning technique. Thus, they show an extensive biomedical application prospect in cell culture and separation, tumor diagnosis, antithrombosis, target pharmaceutical drug design, detection and trapping of viruses, and so on.3-7 Rare-earth phthalate complexes can be used as fluorescence tracers for their luminescence in near-ultraviolet light.8,9 Nanosized magnetite particles have been applied to the synthesis of magnetic composite polymer microspheres due * To whom correspondence should be addressed. Phone: 0086-571-87953011. Fax: 0086-571-87953011. E-mail: [email protected]. † Zhejiang University. ‡ Inner Mongolia Normal University.

to their powerful magnetic susceptibility.10-13 In the presence of a mixture of rare-earth complexes and magnetite, copolymerization of vinyl monomers will produce the core-shell composite polymer microspheres with fluorescence and magnetic susceptibility. The surface property of the polymer shell can be modulated by comonomers and functional molecules bound on the surface of the composite microspheres. Poly(styrene-co-maleic anhydride) (SMA) is a synthetic copolymer with interesting features from both the chemical and the biological points of view.14-19 From the chemical point of view, SMA copolymers are readily subjected to modification. The anhydride groups can easily react with saccharides and peptides containing hydroxyl and amino groups. From the biological point of view, SMA has been used for conjugation with the antitumor protein neocarzinostatin (NCS) and Laminin peptide YIGSR, resulting in an increase of the plasma half-life of NCS and the antimetastatic effect of YIGSR and a decrease of the toxicity of NCS.16,17 Therefore, SMA-glycoconjugates have been identified as a group of biocompatible polymer derivatives with interesting biofunctional capability. Hence, in the present paper we report the synthesis and characterization of magnetite-europium phthalate/SMAheparin glycoconjugate core-shell composite microspheres. The magnetite powder (MP) and europium phthalate complex

10.1021/bm049302+ CCC: $30.25 © 2005 American Chemical Society Published on Web 02/11/2005

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Table 1. Synthesis of FMCPs/SMA Composite Microspheresa

no.

FMCP (g)

KPS soln (mL)

St (g)

MAn (g)

DVB (mL)

EtCOMe (mL)

H 2O (mL)

δb (cal0.5 cm-1.5)

diameter (µm)

DPc

1 2 3 4 5

1.0 0.6 0.9 0.8 1.2

100 100 100 100 100

13.5 19.8 15.2 15.2 17.5

2.5 7.5 3.4 10.2 9.0

0.2 0.2 0.2 0.2 0.2

45.0 85.0 50.0 35.0 40.0

60 35 70 85 80

17.2 13.4 17.4 19.1 18.6

0.36 0.75 0.15 0.21 0.18

0.120 0.292 0.108 0.036 0.075

a 70 °C, N , 10 h. FMCP, fluorescent, magnetic colloid particles, St, styrene; MAn, maleic anhydride; DVB, divinylbenzene; EtCOMe, butanone; KPS 2 soln, aqueous solution of saturated potassium peroxydisulfate. b Solubility parameter of dispersion medium. c Dispersion parameter of microsphere size: DP ) [∑in)1(di - d h )2/n - 1]1/2, where di is the diameter of a microsphere and d h is the average diameter of the microspheres.

(EPC) were blended and dispersed in a poly(ethylene glycol) (PEG) solution to obtain the fluorescent magnetite colloid particles (FMCPs). Copolymerization of styrene and maleic anhydride in the presence of FMCPs seeds led to magnetiteeuropium phthalate/poly(styrene-co-maleic anhydride) (FMCPs/SMA) core-shell composite microspheres. Heparin was then conjugated with the surface anhydride. The surface anhydrides conjugated with heparin to form FMCPs/SMAheparin glycoconjugate core-shell composite microspheres. Heparin, a polysaccharide, exhibits antithrombogenic properties.20-23 Heparin is most effective when free and mobile, but it is also desired to retain it for a long period by immobilizing heparin onto the microsphere surface. The fluorescent, magnetic, polysaccharide-based microspheres, which combined the specific properties of FMCPs/SMA microspheres with those of carbohydrate ligands recognized by receptors on cell surfaces, have been designed to be used for antithrombus and targeted drugs. Such a system would also serve as a biofunctional matrix for cell cultures and separation and as a bioreactor to synthesize important proteins. The new fluorescent, magnetic, polysaccharidebased microspheres reported here were comparatively tested for orientation, tracing, and anticoagulation in a preliminary biological study. The research work on the targeted drugs using the microspheres as carriers will be reported elsewhere. 2. Materials and Methods 2.1. Materials. Magnetite powders (φ ∼ 10 nm, Sigma) were dried before use. Europium phthalate complexes were synthesized via Eu2O3 and potassium phthalate.9 Styrene (St), divinylbenzene, and maleic anhydride (MAn) were purified by vacuum distillation before use. Analytical grade poly(ethylene glycol) (MW ) 3000), potassium peroxosulfate (KPS), and butanone were used without further purification. Heparin sodium (MW ) 12 000; 198 U/mg; Seikagaku Co., Ltd. Jokyo, Japan) and thromboplastin (Pathromtin SL) were purchased from Dade Behring Inc. Citrated fresh human blood plasma was supplied by the Blood Center of Inner Mongolia, China. D-Glucose, sodium citrate dehydrate, and citric acid monohydrate were purchased from Sigma. All other chemical reagents were of analytical grade. The ACD solution consisting of 100 mL of H2O, 2.45 g of D-glucose, 2.20 g of sodium citrate dihydrate, and 0.08 g of citric acid. 2.2. Preparation of Fluorescent, Magnetic Colloid Particles (FMCPs). Magnetite powders and europium phthalate complexes were blended in an aqueous solution

of PEG with stirring and nitrogen purging. The resulting FMCPs were wrapped by PEG with an average particle size of ∼30 nm.24 They were then dialyzed for 1 week and used as the polymerization seeds. The fluorescence intensity was strongly influenced by the magnetite/europium phthalate ratio, which is the highest with a magnetite/europium phthalate ratio of 1:5 (w/w). It might be possible that the structure of europium phthalate complexes was affected by magnetite. 2.3. FMCPs/SMA Fluorescent, Magnetic Composite Microspheres. FMCPs were first immersed in an aqueous solution saturated by KPS to absorb the initiator (KPS). After magnetic separation, they were then swelled by the butanone solution with the monomers (St, MAn, DVB) for 24 h.The mixture was poured into a 250 mL round-bottomed fournecked flask with an incubating dispersion medium of butanone/water. MAn monomer was added to the solution to copolymerize with St on the surface of MPs at 70 °C. The reaction mixture was stirred at 400 rpm for 10 h with the reaction temperature at 70 °C and constant N2 purge. The fluorescent, magnetic latex microspheres (FMCPs/SMA) were separated by a given magnetic field (0.5 Wb/m2) and then washed repeatedly with butanone. The synthesis conditions are given in Table 1. 2.4. Conjugation of Heparin with Fluorescent, Magnetic Composite Microspheres. Heparin contains amino and hydroxyl groups, which can react with the anhydride on the surface of FMCPs/SMA composite microspheres. However, to prevent destruction of its anticoagulant properties, heparin must be attached under mild conditions and should preferably retain some flexibility even when conjugated. The FMCPs/ SMA composite microspheres were purified and dispersed by ultrasound in a phosphate-buffered solution (PBS; 0.05 M, pH 7.5) for 5 h. They were separated in a 0.5 Wb/m2 magnetic field and added into 5 mL of 0.1 M heparin solution incubated with 1 M NaHCO3 (pH 9.0), in which the heparin was purified with anion-exchange resin. The mixture was oscillated at 40 °C for 24 h. The fluorescent, magnetic, polysaccharide-based microspheres (FMCPs/SMA-heparin), conjugating with heparin on the surface of the microspheres, were separated from the mixture by a 0.5 Wb/m2 magnetic field and washed by 0.1 M NaHCO3, followed by 1 M NaCl solution and distilled water. They were washed repeatedly until no heparin was detected in the washed water by spectrophotometry. The reaction mixture was dialyzed exhaustively against MilliQ water. FMCPs/SMA-heparin

Fluorescent, Magnetic, Polysaccharide-Based Microsphere

was then separated by the magnetic field and then lyophilized in a vacuum. 2.5. Characterization of Composite Microspheres. The average diameter of the composite microspheres was estimated from the micrographs using a JEM-100CX electron microscope (TEM; JEOL, Tokyo, Japan). The magnetite content of the FMCPs/SMA composite microspheres was determined from atomic absorption spectrum. The surface maleic anhydride contents were measured by conductometric titration. An excess amount of 0.1 M aqueous hydrochloride was added into the suspension and stirred at room temperature for 0.5 h to change surface anhydride groups into carboxyl groups, which were titrated by a conductometer with 0.01 N NaOH aqueous solution used as titrant. Assuming complete conversion, the amount of anhydride groups equals to that of the carboxyl groups.10 The content of conjugated heparin was analyzed by the toluidine blue colorimetric method.25 Heparin solution (0.1 mL, in 0.2% NaCl) was added to 0.1 mL of toluidine blue solution (0.05 g of toluidine blue was dissolved in 1 L of 0.01 N HCl containing 0.2% NaCl) and then followed by 1.0 mL of hexane. The solution was vortexed for 30 s and the phases were allowed to separate. The 631-nm absorbance of the aqueous layers was determined by a UV spectrophotometer. The structure and properties of the FMCPs/SMA-heparin and the FMCPs/SMA composite microspheres were characterized by a Fourier transform infrared spectrophotometer (FT-IR, Nicolet, Magma-IR 750) and a spectrofluorimeter (Hitchi, F-3010). The ESCA spectra for the determination of surface chemical composition was recorded using a PHI 5000C XPS spectrometer (Perkin-Elmer Instruments), with a monochromatic Al KR X-ray radiation source at a taking off angle of 45°. 2.6. Quantitative Analysis of FMCPs/SMA-Heparin Composite Microspheres. The content of FMCPs/SMAheparin composite microspheres was analyzed by the spectrofluorimeter. A 10 mg/mL FMCPs/SMA-heparin solution was obtained by supersonic dispersion of FMCPs/SMAheparin composite microspheres in ACD solution, which was diluted into four 10-mL aliquots of FMCPs/SMA-heparin standard solution with various concentrations (0.1-2 mg/ mL in ACD). The fluorescence intensity of the solution was determined at 618 nm on a spectrofluorimeter with nearUV excitation at 365 nm. 2.7. In Vitro Experiments for Magnetic Orientation and Fluorescence Tracer. A 0.1 g portion of FMCPs/SMAheparin composite microspheres was dispersed by ultrasound in an ACD solution to obtain a 10 mg/mL solution of FMCPs/SMA-heparin composite microspheres in ACD, incubated at 37 °C for 2 h. FMCPs/SMA-heparin (1 mL of 10 mg/mL) was injected into the artery of a tested rabbit’s kidney, and the rabbit’s kidney was placed at 37 °C in the 0.5 Wb/m2 magnetic field. After 1 h, 1 µL samples were drawn by syringe from kidney, artery, and vein, respectively, and then observed at 365 nm using a fluorescence microscope (Olympus BX51TR-32FB3E01), and then the quantitative analysis was performed by spectrofluorimeter.

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2.8. Anticoagulant Activity of FMCPs/SMA-Heparin. The anticoagulant activity of FMCPs/SMA-heparin was determined by means of an activated partial thromboplastin time (APTT) assay.26 In conjunction with incubated plasma, thromboplastin (Pathromtin SL) enables the individual factors of the intrinsic coagulation system to be quantified and permits diagnosis of hemophilia. Calcium ions of calcium chloride trigger the coagulation process; the time to formation of a fibrin clot is measured. The FMCPs/SMA and FMCPs/SMA-heparin composite microspheres (2 mg) were added into the test tube. Citrated standard plasma (100µL) and Pathromtin SL (100 µL) were then pipetted into a test tube prewarmed to 37 °C, and the mixture was incubated for 3 min at 37 °C. The recording of clotting time started when the calcium chloride solution (100 µL, 0.025 M) was added at 37 °C. A calibration curve of clotting time was obtained by measuring the clotting time of heparin solution with various concentrations (0.05-3 µg/ mL). A standard curve of clotting time was made using heparin as a reference. The anticoagulant activity of FMCPs/ SMA and FMCPs/SMA-heparin was calculated by the standard curve, which is compared with the activities of FMCPs/SMA and free heparin. 3. Results and Discussion 3.1. Synthesis and Characterization of FMCPs/SMA Composite Microspheres. Copolymerization of MA and St was performed on the surface of FMPs to make the FMCPs/ SMA core-shell composite micrspheres with reactive anhydride groups on the microsphere surface. In this copolymerization, it is vital that polymerization seeds are pretreated with PEG having suitable chain length. First, the particles of magnetite and europium phthalate complexes were wrapped with poly(ethylene glycol) to improve the affinity between FMCPs and initiators and monomers for enhancing the adsorption of initiators and monomers, and second, the flexible PEG chains on the surface of “incipient latex particles” may hinder agglomeration of latex particles. In addition, a butanone/water mixture was used as the dispersion medium for the polymerization reaction not only to control the interface tension of the composite microspheres and improve the dispersion stability of them but also to change the comonomer distribution between the latex particles and dispersion medium in favor of polymerization on the surface of FMPs. As a result, the St/Man ratio of the polymer shell changed from 9:1 to 2:1. The FMCPs/SMA composite micrspheres have a coreshell multiphase structure and narrow size distribution, as illustrated in Figure 1, with an average diameter between 0.15 and 0.7 µm. The comoposite microspheres are composed of a magnetite/europium phthalate core and a poly(styrenemaleic anhydride) shell. The infrared spectrum of the composite microspheres (Figure 2) indicated that the maleic anhydride had been copolymerized with styrene and introduced into the composite microspheres (maleic anhydride, 1860,1790 cm-1; benzene ring, 1600,1065, and 698 cm-1). The amount of anhydride groups on the surface of composite microspheres was 0.5-16 mmol/g of mirospheres, deter-

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Figure 1. TEM photograph of FMCPs/SMA core-shell composite microsphere. Figure 3. The fluorescence emission spectrum of FMCPs/SMA composite microspheres with excitation at 365 nm near-UV. Scheme 1. Schematic Diagram of the Conjugation of Heparin to FMCPs/SMA Composite Microspheres

Figure 2. FT-IR spectra of FMCPs/SMA and FMCPs/SMA-heparin composite microspheres.

mined by the titration method. The infrared absorption band corresponding to Fe3O4 at 580 cm-1 (Figure 2) showed that the magnetite particles were entrapped into the composite polymer microspheres. The content of Fe3O4 is 586.5-1013.6 µg/g obtained by atomic absorption spectrum of FMCPs/ SMA composite microspheres, which show potent magnetic susceptibility in a 0.5 Wb/m2 magnetic field. In Figure 2, the infrared absorption bands corresponding to symmetrical and asymmetric stretching of phthalate (1384-1426 and 1533-1583 cm-1) indicated that phthalate was involved in complexation with europium. As was seen in the fluorescence emission, bands related to europium phthalate complex peak at 593 and 618 nm (Figure 3). Moreover the FMCPs/SMA composite micrspheres have a strong pink absorption at 365 nm long ultraviolet radiation. 3.2. Conjugation of Heparin with FMCPs/SMA Composite Microspheres. Heparin, a naturally occurring sulfated linear polysaccharide, accelerates the antithrombin inhibition of the coagulation proteinases by several-hundred-fold,20,21 which forms the basis for polysaccharide’s clinical use as an anticoagulant. Yet, it is the cause of many undesirable pathophysiological consequences, including hemorrhage, thrombocytopenia, osteoporosis, and inconsistent patient response. Conjugation of heparin with FMCPs/SMA composite microspheres leads to the fluorescent, magnetic,

polysaccharide-based composite micrspheres (FMCPs/SMAheparin) containing a coating of heparin on the surface of the microspheres. The FMCPs/SMA-heparin composite microspheres, combining the biological activities of heparin with the fluorescence and magnetic susceptibility of FMCPs/ SMA composite microspheres, may be used as a targeted drug for increasing the drug concentration at the focus and decreasing the side effects. They can be promptly delivered to the focus area by means of a magnetic field and traced by virtue of their fluorescence. Coupling of heparin onto FMCPs/SMA surfaces is mainly based upon the formation of amido bonds between the amino groups of the heparin and the anhydrides on the surface of the composite microspheres, as shown in Scheme 1. The covalent linkages are strong and do not release the heparin into solution in the presence of a substrate or in solution with a high ionic strength.27 The microspheres combining with heparin were a little large and surface-rough compared with virginal microspheres, but they still had a narrow size distribution. FT-IR spectroscopy confirmed that heparin conjugated with FMCPs/SMA composite microspheres, according to reaction Scheme 1. As an example, in Figure 2 the FT-IR spectrum of the FMCPs/SMA is compared with that of the FMCPs/SMA-heparin. The symmetric CdO stretching absorption band (1790 cm-1) and the asymmetric one (1860 cm-1) of the anhydride monomers disappeared in the spectra of FMCPs/SMA-heparin, where the signals corresponding to the carboxylate CdO stretching vibrations (∼1560 cm-1) and the amide group CdO stretching (∼1670 cm-1) developed instead. As can be seen in the ESCA spectrum of Figure 4, heparin was indeed grafted onto the

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Fluorescent, Magnetic, Polysaccharide-Based Microsphere

Table 2. Effect of the Properties of the Carrier Microspheres on Conjugation between Heparin and FMCPs/SMAa microsphere properties

Figure 4. The ESCA spectra of FMCPs/SMA, FMCPs/SMA-heparin, and heparin.

Figure 5. Surface-bound heparin increase by the conjugation of heparin to FMCPs/SMA microspheres as functions of heparin, measured at 50 mg of FMCPs/SMA microspheres with a diameter of 0.236 µm and 4.2 mmol of surface anhydride groups/g of microsphere (a, the amount of the heparin bound/gram of microspheres; b, the amount of the heparin conjugating/millimole of surface anhydride).

microsphere surface with the conjugation. After the reaction with heparin, ESCA measurements on the FMCPs/SMAheparin showed much higher amounts of oxygen, and peaks from sodium, nitrogen, and traces of sulfur, which were not present before the reaction, were also detected. The content of conjugated heparin is 60-600 mg/g of microsphere, measured by the toluidine blue colorimetric method after magnetic separation. The conjugation with heparin was performed under mild conditions in order to decrease the changes of heparin conformation, which play pivotal roles in the inhibition of thrombin and some other serine proteases of the blood coagulation cascade.28,29 The effects of the heparin/microsphere ratio, microsphere diameter, and surface anhydride on the conjugation with heparin were investigated. Figure 5 indicates that the heparin-binding capacity of the fluorescent, magnetic, polysaccharide-based composite microspheres, increased distinctly with increase in the ratio of heparin to microspheres till the amount of the conjugated heparin reached 350 mg/g of microsphere. When the amount of the conjugated heparin exceeded 350 mg/g of microspheres, the heparin-binding capacity approached saturation. It could be

heparin-binding capacity of FMCPs/SMA-heparin

diameter (µm)

surface anhydride (mmol/g)

conjugated heparin/microsphere (mg/g)b

conjugated heparin/anhydride (mg/mmol)c

0.152 0.235 0.451 0.672 0.209 0.226 0.218

4.5 4.6 4.3 4.7 3.2 5.1 7.3

399.0 383.0 287.6 269.4 259.8 409.5 607.1

88.7 83.3 66.9 57.3 81.2 80.3 83.2

a Heparin, 125 mg; FMCPs/SMA composite microspheres, 50 mg; 1 M NaHCO3. b The amount of the heparin bound on 1 g of FMCPs/SMA composite microspheres. c The amount of the heparin conjugating with 1 mmol of anhydride on the surface of FMCPs/SMA composite microspheres.

Figure 6. Standard fluorescence emission curve for quantitative analysis of FMCPs/SMA-heparin composite microspheres measured at 618 nm.

reasonable that the binding sites on the surface of the magnetic composite microspheres were limited. The specific area of the magnetic composite microspheres increased with the decrease in their size, so they could bind more heparin, as demonstrated in Table 2. The data showed that as the amount of the surface anhydride increased, the amount of the conjugated heparin increased remarkably while the amount of heparin bound with per millimole of surface anhydride hardly changed. 3.3. Quantitative Analysis of FMCPs/SMA-Heparin Composite Microspheres by Fluorescence. Direct quantitative measurement of FMCPs/SMA-heparin composite microspheres were performed by a spectrofluorimeter. Figure 6 shows the standard fluorescence emission curve of FMCPs/ SMA-heparin (0.05-0.3 mg/mL), which shows a good linear relationship within the measurement range. The FMCPs/SMA-heparin composite microspheres were separated by a magnetic field from a blood sample, dispersed by ultrasound in ACD, and then measured at 618 nm on a spectrofluorimeter with an excitation wavelength of 365 nm. 3.4. In Vitro Experiments for Magnetic Orientation and Fluorescence Trace. The FMCPs/SMA-heparin composite microspheres have a core composed of magnetite and europium phthalate. Consequently, they show potent magnetic susceptibility in a 0.5 Wb/m2 magnetic field and a sharp pink fluorescence emission with 365 nm UV, which can be

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Table 3. In Vitro Experiments on Rabbit Kidney for Magnetic Orientation and Fluorescence Tracea artery timeb (min) 0 20 40 60

colorc deep pink light pink

concnd (µg/mL) 180.2 3.05 0.01 0

kidney color

concn (µg/mL)

light pink pink deep pink

0 41.6 159.4 159.8

Table 4. Anticoagulant Activity of FMCPs/SMA-Heparin Composite Microspheres by an APTT Assay

vein color

concn (µg/mL) 0 0 0 0

a

FMCPs/SMA-heparin: diameter, 0.209 µm; bound heparin, 259.8 mg/g of microspheres; 1 mL of 10 mg/mL was injected into the artery of a tested rabbit’s kidney and placed in the 0.5 Wb/m2 field at 37 °C. b Time intervals for taking samples from artery, kidney, and vein. c The fluorescence color of the samples. d The FMCPs/SMA-heparin concentration in the samples.

Figure 7. Increase in time for appearance of pink fluorescence in rabbit kidney by embedding magnetite into FMCPs/SMA-heparin microspheres as functions of magnetite in the composite microspheres, measured at 0.5 Wb/m2 magnetic field.

used for magnetic orientation and fluorescence tracing, as was confirmed in the experiments in rabbit kidney. A 1-mL portion of FMCPs/SMA-heparin solution was injected into the artery of the rabbit kidney placed in 0.5 Wb/m2 magnetic field, and at different time intervals, the blood samples were drawn by syringe from kidney, artery, and vein respectively, which were observed at 365 nm on a fluorescence microscope and quantitatively measured using a spectrofluorimeter. The shape and size of the microspheres from vein did not changed compared with those of the microspheres from kidney, which means that the microspheres are stable for UV irradiation or in magnetic field, possibly due to the mutual link between the polymer chains by divinylbenzene. The fluorescence color of the blood samples and the FMCPs/ SMA-heparin amount in the artery, kidney, and vein at 0, 20, 40, and 60 min is shown in Table 3. Figure 7 indicates that the magnetic susceptibility of FMCPs/SMA-heparin composite microspheres increases with the magnetite content of the composite microspheres. 3.5. Anticoagulant Activity of FMCPs/SMA-Heparin Composite Microspheres. Heparin’s in vitro bioactivity was determined by an APTT assay. In Table 4, the clotting time of FMCPs/SMA-heparin composite microspheres was measured by APTT and then compared with that of FMCPs/ SMA to give the clotting over time ratio. The conjugation with heparin remarkably improved the anticoagulant activity of the composite microsphere, and the clotting time of FMCPs/SMA-heparin was extended by 99.7% compared

microsphere properties

APPT assay

bound heparina diameter clotting extended (mg/g) (µm) time (s) timeb (%) 0 259.8 409.5 607.1 287.6 269.4

0.212 0.209 0.226 0.218 0.451 0.672

38.2 55.1 68.5 96.8 50.7 46.2

44.24 79.32 153.40 32.72 20.94

activity of conjugated heparin activityc (U/g)

activity retentiond (%)

22016.5 40702.7 54212.8 22948.8 17869.3

42.8 50.2 45.1 40.3 33.5

a The content of the heparin bound on 1 g of FMCPs/SMA-heparin composite microspheres. b The extended clotting time of the FMCPs/ SMA-heparin compared with FMCPs/SMA. c The biological activity of per gram of FMCPs/SMA-heparin. d The activity retention of the conjugated heparin on the FMCPs/SMA-heparin composite microspheres compared with that of free heparin (198 U/mg).

with that of FMCPs/SMA, as shown in Table 4. The activity retention of conjugated heparin was calculated to be 50.2% compared to free heparin (198.6 U/mg). Table 4 indicates that the clotting time of FMCPs/SMA-heparin increases with the increase of the conjugated heparin and decrease of the microsphere diameter. The unique anticoagulant property of heparin is contributed to its combination with antithrombin III (AT-III) present in the blood plasma,30,31 the major inhibitor of the coagulation cascade; thereby, the complex formed readily reacts with thrombin to inhibit the formation of a fibrin network, which is an important step for the thrombosis.32 The conjugated heparin might inhibit the intrinsic coagulation factors, thus leading to a suppression of thrombin activity. Thus, it is possible that the FMCPs/SMA-heparin surface may bind AT-III from the plasma and is thus able to neutralize the activity of clotting factors generated in the blood. The more conjugated heparin on the surface of the composite microspheres and the less the microsphere diameter, the more easily the reaction with AT-III and clotting factors. So, the FMCPs/SMA-heparin composite microspheres have the higher anticoagulant activity. Yet, the excessively dense heparin on the surface of the composite microspheres may limit combining with AT-III, which decreases the anticoagulant activity of the FMCPs/SMA-heparin composite microspheres. 4. Conclusions Nanosized core-shell composite spheres with magnetic inductivity, fluorescence, and active anhydride surface groups were prepared. Heparin was covalently bound onto the surface of the spheres to give fluorescent, magnetic, polysaccharide-based microspheres with blood compatibility and related biocompatibility. In vitro studies indicate that the bound heparin has remarkable anticoagulant activity. The bound heparin can be moved to a given position in a magnetic field and show a sharp pink fluorescence at 365 nm near-UV. Therefore, The fluorescent, magnetic, polysaccharide-based microspheres could be used as a potent targeted drug for thrombus therapy and so on.

Fluorescent, Magnetic, Polysaccharide-Based Microsphere

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