Preparation of Magnetic Microspheres from Water-in-Oil Emulsion

Biomacromolecules 2005, 6, 1280-1288

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Preparation of Magnetic Microspheres from Water-in-Oil Emulsion Stabilized by Block Copolymer Dispersant Guojun Liu,* Husheng Yang, and Jiayun Zhou Department of Chemistry, Queen’s University, 90 Queen’s Crescent, Kingston, Ontario, Canada K7L 3N6

Say-Jong Law,† Qingping Jiang, and Guohan Yang Bayer Healthcare, Diagnostics Division, 333 Coney Street, East Walpole, Massachusetts 02032-1597 Received October 28, 2004; Revised Manuscript Received December 31, 2004

A new method for the preparation of magnetic microspheres is reported. The preparation involved first the dispersion of an aqueous phase, containing magnetite nanoparticles and a water-soluble homopolymer, into droplets in an organic medium using an amphiphilic block copolymer as the dispersant. This was followed by water distillation at a raised temperature from the aqueous droplets to yield polymer/magnetite particles. The structure of the particles was then locked in by a reagent being added to cross-link the water-soluble copolymer block and homopolymer. Since the hydrophobic block of the copolymer consisted of a protected polyester, the removal of the protective moieties from the coronal chains yielded poly(acrylic acid) or other functional polymers to render water dispersibility to the spheres and to enable biomolecule immobilization. Introduction Water-dispersible micrometer-sized magnetic spheres with carboxyl surface groups are useful for immobilizing various biopolymers such as proteins and DNA for diagnostic applications.1 The microspheres should also be useful in controlled drug delivery,2 cell labeling and separation,3 water treatment,4 and in probing the local viscoelasticity of living cells5 or polymer chain dynamics.6 There have been different approaches to the preparation of magnetic microparticles with pros and cons to each approach. We report in this paper a new addition to the current repertoire. The reported method involves first the dispersion of an aqueous phase, consisting of magnetite nanoparticles and a water-soluble homopolymer, into droplets in an organic medium via the use of an amphiphilic block-copolymer dispersant (A f B, Scheme 1). This is followed by water distillation at a raised temperature from the aqueous droplets to yield polymer/magnetite particles stabilized by the block copolymer (B f C). The structure of the particles is then locked in by a reagent being added to cross-link the homopolymer and water-soluble copolymer block (C f D). After purification, the microspheres are made water dispersible by the protective groups being removed from the block copolymer corona chains to yield a water-soluble polymer. While many block copolymers have been tested by us over the past four years for this application, we report results in this paper obtained using only poly(t-butyl acrylate)-blockpoly(2-hydroxyethyl acrylate), PtBA-PHEA, or PtBA-PSAPHEA as the dispersant, where PSA denotes poly(solketal * Corresponding author. Phone: (613) 533-2624. Fax: (613) 533-6669. E-mail: [email protected]. † Current address: Charm Sciences, Inc., 659 Andover St., Lawrence, MA 01843.

Scheme 1

acrylate). The water-soluble homopolymer used in this case is PHEA. The PtBA block was targeted for its ready hydrolysis to yield poly(acrylic acid) to facilitate microsphere dispersion in water and biomolecule immobilization. A PSA block was incorporated for its ready hydrolysis to poly(glyceryl acrylate) or PGA, which may function as a buffering layer between biomolecules immobilized on the PAA chains and the magnetic core to minimize biomolecule adsorption by the surface of the core.

10.1021/bm049316f CCC: $30.25 © 2005 American Chemical Society Published on Web 02/17/2005

Preparation of Magnetic Microspheres from Emulsion

Known approaches to preparing composite polymer/ inorganic magnetic spheres can be classified into the following. Approach 1 is similar to ours. It involves forming an inverse emulsion from dispersing a hydrophilic polymer, magnetite nanoparticles, a surfactant, and a cross-linker in oil. Composite particles are obtained after the temperature is raised to cross-link the aqueous droplets. The hydrophilic polymers that have been used include albumin,7,8 chitosan,9 and poly(vinyl alcohol).10 The oil used includes cottonseed, mineral, and vegetable oil. The cross-linker is normally glutaraldehyde or another dialdehyde or diisocyanate. The drawback with this approach has been in the lack of a water distillation step. Since water resides in the droplets during cross-linking, the particles produced are porous and mechanically weak. Also, the surfactants used in the past are the commercially available low-molar mass surfactants or polymers such as poly(ethylene oxide)-block-poly(propylene oxide). The surfactants are not chemically grafted onto the microspheres and can be readily washed off. They are not easily derivatized to yield functional groups for biomolecule immobilization. Approach 2 is similar to approach 1 except that the hydrophilic polymer in approach 1 is replaced by a hydrophilic monomer. An inverse emulsion here is prepared from a monomer; cross-linker; magnetite, or its substitute, water; initiator; and surfactant. Microspheres are obtained after polymerization and cross-linking of the aqueous droplets.11-14 Commercial Seradyn particles are prepared from approach 3. This approach starts with uniform core particles prepared from emulsion polymerization. Magnetite particles are deposited on the core particles either by electrostatic attraction15 or by the magnetite particles being coated using a polymer as the binder.16 Further capping is possible by a polymer being applied over the magnetic layer to yield either a hydrophobic or a hydrophilic latex. A drawback with this approach is the workload required to increase magnetite loading by the multiple layer-by-layer deposition process.17 Commercial Dynal A/S beads are prepared based on approach 4.18,19 This approach starts with monodisperse porous polymer particles. The pores are pretreated to contain oxidizing surface groups and then filled with aqueous solutions of precursory salts such as FeCl2. Treatment of these particles under mild alkaline conditions causes magnetic ion oxides to precipitate as fine particles distributed evenly in the nanopores. The pores are last capped with a polymer layer to seal in magnetic particles and also to introduce functional groups to facilitate biomolecule immobilization.20 Kumacheva and co-workers recently used this approach to prepare microspheres containing not only magnetic but also metal and semiconductor nanoparticles.21 Approach 5 is a variant of approach 2.22-24 Instead of a hydrophilic monomer, a hydrophobic monomer and thus an oil-in-water emulsion are used. To facilitate the incorporation of magnetic particles into the oil monomer droplets, the

Biomacromolecules, Vol. 6, No. 3, 2005 1281

particles were coated with a surfactant to make the surface hydrophobic. More recently, Landfester and Ramirez25 prepared particles smaller than 100 nm from this approach by starting with an oil-in-water miniemulsion. Like approach 2, the spheres prepared from this approach have relatively wide size distributions. While the method reported here is new for magnetic microsphere preparation, it has been used to prepare various biodegradable polymer particles as reviewed by Nakache et al.26 More recently, it has been used to prepare diblock microspheres with regularly packed internal domains.27 As far as reactive block copolymers are concerned, they have been used mainly in the preparation of cross-linked nanostructures ranging from star polymers28 to nanospheres,28-33 hollow nanospheres,34-36 nanospheres with cross-linked shells,37-39 shaved nanospheres,40 nanospheres with molecularly imprinted cores,41 nanofibers,42 and nanotubes.43 Reports on their use as a surfactant in the preparation of chemically grafted microstructures are rare, despite the extensive use of chemically inert block copolymers as surfactants in emulsion polymerization, etc.44

Experimental Procedures Materials. Monomer solketal acrylate45 and 2-trimethylsiloxyethyl acrylate (HEA-TMS)46 were prepared following literature methods. Monomer t-butyl acrylate (98%), CuBr (98%), N,N,N′,N′,N′-pentamethyldiethylenetriamine (PMDETA, 98%), and methyl 2-bromopropionate (98%) were all products of Aldrich. CuBr2 was purchased from the Matheson Coleman and Bell Company. Acetone (EM Science) and butanone (99%, Aldrich) were of ACS grade. Polymer Characterization Techniques. Size-exclusion chromatography (SEC) was performed on a Waters system using a HT-4 column packed with ultra-Styrel-gel. The system was calibrated using monodisperse poly(methyl methacrylate) or PMMA standards. Proton NMR analysis was run in CDCl3 on a Bruker ACE-200 instrument. Synthesis and Characterization of PHEA Homopolymer. The precursor to PHEA was poly(2-trimethylsiloxyethyl acrylate) or P(HEA-TMS). P(HEA-TMS) was synthesized by atom transfer radical polymerization (ATRP). First added to a 50 mL round-bottomed flask with a glass stopcock sidearm was CuBr (17.7 mg, 0.12 mmol). The flask was evacuated and backfilled with argon, and the procedure was repeated thrice. Then added were HEA-TMS (5.0 mL, 4.65 g, 24.7 mmol, vacuum-distilled) and PMDETA (25.1 µL, 20.8 mg, 0.12 mmol, vacuum-distilled). After the addition of methyl 2-bromopropionate (In-Br, 27.6 µL, 41.2 mg, 0.25 mmol), the mixture was stirred for 15 min, and the flask was placed in an oil bath preheated to 60 °C. After 20 h, the viscous polymer was dissolved in 15 mL of THF. The mixture was filtered through a column of activated neutral aluminum oxide (Aldrich, 150 mesh). To the filtrate (∼25 mL) was added 1.0 mL of 3 M HCl to remove the TMS groups. After the mixture was stirred for 2 h, the solvent was rota-evaporated. The residual solid was redissolved in methanol and precipitated into diethyl ether to yield a solid.

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The polymer was dissolved in water for prolonged storage. For SEC, the polymer was dissolved in pyridine and reacted with excess acetic anhydride to acetate the HEA units first. Synthesis and Characterization of PtBA280-PHEA250. The diblock surfactant used had approximately 280 tBA and 250 HEA units and is denoted as PtBA280-PHEA250. The PtBA block was synthesized by ATRP following procedures similar to those described previously. The PtBA block was purified by precipitation into water/methanol (v/v ) 1/1), chilled with ice, and then dried under vacuum. To prepare the diblock, PtBA (2.39 g, 0.068 mmol) was dissolved in HEA-TMS (3.35 g, 17.8 mmol) in a 100 mL flask. CuBr (9.8 mg, 0.068 mmol) and CuBr2 (0.8 mg, 0.0034 mmol) were added. The flask was evacuated and backfilled with argon, and the procedure was repeated thrice. This was followed by the addition of butanone (3 mL) and PMDETA (14.3 µL, 11.8 mg, 0.068 mmol) that had been deoxygenated. The mixture was stirred for 30 min before the flask was placed in an oil bath preheated to 72 °C. After 20 h, 40 mL of THF was added to the flask, and the mixture was filtered through an alumina column. The filtrate was rota-evaporated to dryness to yield a soft and sticky solid, which was further dried under vacuum. SEC analysis was performed in the PtBA-P(HEA-TMS) form, and proton NMR was run in CDCl3. To hydrolyze the TMS groups, the diblock was dissolved in THF. To it was then added 1.0 mL of 1.0 M HCl. After the mixture was stirred at room temperature for 3 h, it was transferred into a dialysis tube with a molar mass cutoff of 14 000 g/mol and was dialyzed against methanol for 2 days. The methanol was changed twice daily. The resultant solution was dried by rota-evaporation, and the polymer was further dried under vacuum. PtBA145-PSA160-PHEA240. PtBA-PSA was prepared by ATRP similar to PtBA-P(HEA-TMS). The PtBA-PSA sample used consisted of 145 tBA and 160 SA units. The sample was purified by precipitation into hexanes. To prepare the triblock copolymer, PtBA-PSA (6.0 g, 0.16 mmol) was dissolved in HEA-TMS (14.8 g, 78.9 mmol) in a 100 mL flask. CuBr (23 mg, 0.16 mmol) and CuBr2 (1.8 mg, 0.008 mmol) were added. The flask was evacuated and backfilled with argon, and the procedure was repeated another two times. Then, butanone (4 mL) and PMDETA (35.5 µL, 27.7 mg, 0.16 mmol) were added. The mixture was stirred for 30 min before the flask was immersed in an oil bath preheated to 80 °C. After 20 h, 50 mL of THF was added to the flask, and the mixture was filtered through an alumina column. A portion of the filtrate was vacuum-dried for SEC and NMR characterization. To the rest of the filtrate (∼120 mL) was added 5 mL of water and 2 mL of acetic acid. The mixture was stirred for 5 h at room temperature to hydrolyze the P(HEA-TMS) block selectively. After most of the solvent was removed by rota-evaporation at room temperature, the mixture was dialyzed against methanol. The final methanol solution was rota-evaporated to dryness, and the polymer was further dried under reduced pressure. SEC analysis of the PtBA-PSA-P(HEA-TMS) sample yielded Mn ) 1.15 × 105 g/mol and Mw/Mn ) 1.67. NMR analysis yielded n/m/l ) 145:160:240.

Liu et al.

Magnetic Sol. Magnetic sol or magnetite nanoparticles were prepared following a literature method.47,48 In an example preparation, first mixed were a solution of FeCl2‚ 4H2O (4.1 g or 0.020 mol) in 25 mL of 1.0 M HCl and a solution of FeCl3‚6H2O (11.3 g or 0.042 mol) in 25 mL of water. The mixture was added to 160 mL of 1.5 M ammonia. After the resultant slurry was stirred for 24 h, the precipitate was isolated by magnetic decantation, rinsed with distilled water, and separated from the supernatant by magnetic decantation again. The precipitate was then peptized by being stirred in 2.0 M hyperchloric acid for 10 min. The hyperchloric acid was removed by magnetic decantation. This peptizing step was repeated. After the removal of the final hyperchloric acid supernatant, water was added. The aqueous magnetic sol containing undissolved precipitate was transferred into a dialysis tube and was dialyzed against distilled water changed now and then for 20 h. As the ionic strength decreased inside the tube, the dispersed magnetic sol content increased. The magnetite content was determined gravimmetrically by a sample being weighed before and after water evaporation. Samples with magnetite contents up to ∼6% were prepared using this method. Microsphere Preparation. A 1 L three-neck roundbottomed flask was immersed into an oil bath placed on a heating plate rather than a magnetic stirring heating plate to avoid its magnetic field. To one end of the stirring shaft of a mechanical stirrer was mounted a hemispherically shaped Teflon blade, which was 5.8 cm wide and 1.8 cm tall. The blade was inserted into the flask via the middle joint. Chlorobenzene (BDH, AnalaR grade, 140 mL), toluene (EM, GR grade, 140 mL), and PtBA280-PHEA250 (204 mg dissolved in 2 mL of THF and 1 mL of methanol) were added into the flask. PHEA, 224 mg in 4.0 mL of water, was mixed with 5.0 mL of the magnetic sol (34.4 mg/mL) and 51 mL of water in a beaker. The aqueous solution was then added into the organic phase at moderate stirring (e.g., at 400 rpm) over several minutes. After this, one ground joint of the reaction flask was fitted with a dropping funnel with a pressureequalization arm containing 150 mL of toluene. The other joint was fitted with the Dean-Stark trap. The stirring speed was increased to 2000 rpm to disperse the mixture and maintained thereafter until the spheres were cross-linked. After the mixture was stirred at room temperature for 1/2 h, the heater was turned on to raise the oil temperature over 1 h to 90 °C. At 90 °C, toluene and water were distilled out as an azeotrope and collected in the Dean-Stark trap. Distillation was maintained for ∼5 h until the distillate became clear (no water). During this time interval, toluene was added to maintain an approximately constant volume of the emulsion mixture. After all of the water was removed, the mixture in the flask was cooled to 80 °C. To it was added 177 mg of succinyl chloride (cross-linker, 95%, Aldrich) in 1.9 g of pyridine in two portions. The first 10% was added in one aliquot, and the rest was added dropwise over 1 h. This was followed by another 3 h of stirring to ensure complete microsphere cross-linking. After being cooled, the microspheres were cleaned repeatedly with THF by magnetic decantation and last with


Preparation of Magnetic Microspheres from Emulsion Table 1. Characteristics of the Diblock and Triblock Copolymers PtBA block

PtBA-P(HEA-TMS) or PtBA-PSA

PtBA-PSA-P(HEA-TMS)

sample

SEC Mn (g/mol)

SEC Mw/Mn

n

SEC Mn (g/mol)

SEC Mw/Mn

m

SEC Mn (g/mol)

SEC Mw/Mn

l

diblock triblock

3.6 × 104 1.8 × 104

1.24 1.21

280 145

5.9 × 104 3.8 × 104

1.42 1.33

250 160

11.5 × 104

1.67

240

dichloromethane (DCM) thrice. The spheres were suspended in trifluoroacetic acid (TFA)/DCM (1:3, v/v), and the mixture was stirred magnetically for 1.5 h. Then, more TFA was added making the final ratio of TFA/DCM equal to 1:1, and the mixture was stirred for another 1.5 h. The TFA/DMC mixture was removed at the end of the reaction, and the spheres were washed with THF and water each for six times. They were finally treated with a dilute aqueous Na2CO3 solution and rinsed with water until the supernatant was approximately neutral. The preparation of microspheres using the triblock as the dispersant was performed similarly. The PSA block was hydrolyzed partially during PtBA hydrolysis and was fully hydrolyzed only after the spheres were stirred overnight in THF containing 0.5 vol % TFA and 0.5 vol % water. Microscopy. To obtain the scanning electron microscopic (SEM) images, the microspheres were spread on a doublesided carbon adhesive tape that had been glued on a stainless steel sample holder. The spheres were then sputter-coated with a 6 nm thick Au-Pt layer and analyzed using a SEI XL30 ESEM instrument operated at 20 kV. To obtain the internal structure of the microspheres by transmission electron microscopy (TEM), the spheres were mixed with an epoxy resin (Embed 812, Electron Microscopy Science). The mixture was then used to fill the coned bottom part of a polyethylene embedded capsule and cured at 60 °C for 3 h. More resin was added on top of the cured sample until the capsule was filled, and the newly added resin was cured at 60 °C overnight before it was microtomed by an Ultracut-E Reichert-Jung instrument to yield slices that were ∼60 nm thick. The thin slices were examined by a Hitachi-700 TEM instrument operated at 75 kV. Magnetic Property Measurements. Magnetic properties of a microsphere sample in water at 10 mg/mL were measured by a vibrating sample magnetometer (ADE Technologies). The magnetite content in the microsphere sample was ∼33 wt %. Carboxyl Group and Iron Content Analysis. The number of carboxyl groups in each gram of magnetic microspheres was determined by acid-base titration. To minimize the interference of the magnetite particles, 0.5 g of a microsphere sample was dialyzed against a 0.050 M ethylenediaminetetraacetic acid disodium salt (EDTA) solution. This treatment led to the dissolution of the magnetite particles leaving a white polymer powder behind. The polymer was rinsed with a 1.0 M acetic acid solution and separated from the supernatant by centrifugation. It was then rinsed with distilled water thrice. The polymer was dried under vacuum, and a weighed amount was titrated with a standardized NaOH solution for acid content. For Fe content determination, ∼10 mg of microsphere was mixed with 2.0 mL of THF and 2.0 mL of 3 M hydrochloric acid. The mixture was equilibrated overnight to allow the

full dissolution of magnetite and then divided into two equal fractions. To each fraction was added 2.00 mL of a 10% aqueous hydroxylamine hydrochloride solution to reduce the ferric to ferrous ions and 2.00 mL of a 0.5% aqueous o-phenanthroline solution to complex with the ferrous ions. To one fraction was then added one drop of the bromophenol blue indicator. The solution was titrated with a 25% aqueous sodium citrate solution until the intermediate color of the indicator was reached at pH ≈3.4.49 The volume of sodium citrate consumed was recorded, and this amount was added to the other sample without bromophenol blue. After dilution to 100 mL, the solution was centrifuged, and absorbance at 508 nm was taken for Fe2+ content evaluation. Results and Discussion Polymer Synthesis and Characterization. The preparation of only one PHEA, one PtBA-PHEA, and one PtBAPSA-PHEA sample was described in this paper. For ATRP, the initiator (In-Br) used was methyl 2-bromopropionate, and the ligand (Lig) used was PMDETA. The mechanism for tBA polymerization by ATRP is illustrated below:50 CuBr2

was added in the preparation of block copolymers to shift the equilibrium to the left so as to minimize chain coupling during polymerization. The PtBA and PtBA-PSA samples could initiate the polymerization of other monomers because they bore the terminal Br groups. The P(HEA-TMS) sample had, based on PMMA standards, Mw/Mn ) 1.47 and Mn ) 1.73 × 104 g/mol, which corresponds to 110 HEA repeat units. Table 1 summarizes the characteristics of the block copolymers prepared. The m and l values were obtained based on the n/m and n/m/l values determined from NMR, and the Mn values of PtBA were determined from SEC. Since the Mn values of the PtBA blocks were approximate and evaluated based on PMMA standards, the m and l values were approximate as well. The polydispersity indices Mw/Mn of the block copolymers are 1.42 and 1.67, respectively. The selective hydrolysis of the P(HEA-TMS) block of PtBA-PSA-P(HEA-TMS) using acetic acid as the catalyst was demonstrated from a NMR study of the sample before and after the hydrolysis treatment. We further demonstrated by NMR spectroscopy that the selective hydrolysis of the

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Figure 1. TEM image of a magnetite sample.

PSA block over the PtBA block could be achieved using hydrochloric acid as the catalyst in THF/water. Magnetic Sol. Magnetite was produced following a literature method from reacting ferric and ferrous chloride with aqueous ammonia47-48 Fe2+(aq) + 2Fe3+(aq) + 8OH-(aq) f Fe3O4(s) + 4H2O (1) The magnetite precipitate was then rinsed with distilled water and peptized in hyperchloric acid. Figure 1 shows a TEM image of the sol sample after sample purification and aspiration from water. The primary particles, which are seen isolated from the others, are irregularly shaped with the largest dimension smaller than ∼15 nm. Magnetic Microspheres. The microspheres were prepared following procedures detailed in the Experimental Procedures or depicted in Scheme 1. The first step involved dispersing an aqueous phase consisting of magnetite sol and homopolymer PHEA in toluene and chlorobenzene. A mixture of toluene and chlorobenzene was used as the oil phase to match densities of the organic and aqueous phases. The volume ratio used between organic and aqueous phases was low at 4 mainly to save solvent cost but could be increased. We, for example, used a phase volume ratio of 8:1 for some preparations and did not, however, notice differences between the properties of the resultant spheres. An aqueous phase containing not only magnetite but also a water-soluble homopolymer was used because the homopolymer functioned as a binder for the magnetite nanoparticles and helped to increase the mechanical robustness of the final microparticles. It might have functioned incidentally also to reduce droplet coagulation by providing an osmotic pressure inside the droplets to counteract the surface tension effect.25 In all our preparations, a homopolymer that has the same structure as the water-soluble block of a block copolymer surfactant was used as the homopolymer. In the cases when PtBA-PHEA and PtBA-PSA-PHEA were used as the surfactant, the homopolymer used was PHEA. This was mainly to increase the compatibility between the binding resin (homopolymer) and the water-soluble block. While we do not know solubility parameter for PHEA, our suspicion is

Liu et al.

that we could have used other water-soluble polymers including poly(vinyl alcohol) for this purpose as well. While the use of only two block copolymers is discussed in detail in this paper, many other block copolymers have also been tested. The general guideline that we used in block copolymer synthesis was that the water-soluble block should be sufficiently long (e.g., >100 units) to ensure effective chemical grafting of the block copolymer chains. Then, the oil-soluble block should be longer than the water-soluble block, as the oil-soluble block will be eventually converted to a water-soluble block to render water dispersibility to the spheres. A longer corona block will allow better dispersibility of the microspheres. On the basis of this guideline, all of the block copolymers that we tested worked well as dispersants. The second step of preparation involved water distillation from the droplets. Water was distilled with toluene as an azeotrope. During this process, toluene was added now and then to ensure an approximately constant volume for the system. During this step, the aqueous droplets contracted, trapping probably most of the original hopolymer and magnetite inside each droplet. Evidence supporting the shrinkage of the water droplets was gathered at the very early stage of this project.51 At that time, the water-soluble block of the copolymer and the homopolymer that we used was poly(glyceryl methacrylate) or PGMA. PGMA could be cross-linked in the presence of water using glutaraldehyde (OCHCH2CH2CH2CHO, GA). We found that the diameter of the spheres prepared without water distillation was much larger (e.g., 2-3 times larger) than that of those prepared by adopting a water distillation step. Also, the spheres prepared without water distillation lacked mechanical strength probably for their porous structure. In the third step, the microspheres were cross-linked using succinyl chloride in the presence of pyridine. After the addition of one aliquot of succinyl chloride at the hydroxyl to succinyl chloride molar ratio of 10:1, the rest of the crosslinker was added dropwise so that it was the limiting reagent in the early stage to facilitate the reaction of both of its acid chloride groups.

The effectiveness of the cross-linking reaction was demonstrated by the stability of the prepared spheres in various solvents such as DMF or methanol, which solubilized both PtBA and PHEA. Furthermore, the magnetite particles were not readily extracted from the spheres by water or other solvents over the time span of years. We have used a crosslinking time of several hours at 80 °C because the condition was formerly established by us to cross-link PtBA-PHEMA micelles,52 where PHEMA denotes poly(2-hydroxyethyl methacrylate). The last step of the synthesis involved PtBA hydrolysis and also PSA hydrolysis in the case of triblock microspheres. To minimize magnetite dissolution during this process, the



Factors Affecting the Size of the Spheres. The first system that we studied involved the use of PGMA-PAMA as the surfactant, where PAMA denotes poly(allyl methacrylate). We have not submitted results from that study for publication because of the difficulty associated with the quantitative removal of the allyl protecting group from PAMA after microsphere preparation.51 Despite the deficiency in chemistry, the conclusions reached about the effect of various factors on the size of the final spheres remain valid for the current systems. These conclusions include that the diameter of the spheres prepared decreased when the copolymer amount and stirring speed were increased and the amount of homopolymer and magnetic sol was decreased. Increasing the copolymer amount initially decreased the size of the spheres, and the effect became negligible once the copolymer concentration was above ∼0.1 wt %. The most effective means to tune the size of the spheres was to vary the stirring speed. Table 3 shows the effect of changing stirring speed on the size of three batches of spheres prepared using PtBA145-PSA160-PHEA240 as the surfactant. Under otherwise essentially identical conditions, the diameter of the spheres decreased from 2.04 to 1.06 µm when the stirring speed was increased from 1200 to 2000 rpm. Properties of the Spheres. Our gravimetric analysis indicated that the final weight of the spheres was approximately equal to the total weight of magnetic sol, homopolymer, and the succinic groups. This suggests that only a small fraction of the block copolymer was grafted onto the spheres and was grafted most likely as a monolayer, as depicted in Scheme 1. The rest of the block copolymer was not grafted as it existed prior to cross-linking as block copolymer micelles or unimers in equilibrium with those chains at the interface between water droplets and the organic phase. The micelles were then cross-linked just as the polymer droplets were during the cross-linking step and later removed during the microsphere purification step. Fe2+ content analysis using a spectrophotometry method as described in the Experimental Procedures also confirmed this conclusion that a majority of block copolymer chains was wasted. When GA was used to cross-link the core of the spheres made of magnetite and PGMA,51 we also titrated the carboxyl group content of the spheres by NaOH, and the carboxyl group surface density was ∼50/ nm2, which again suggests that only one block copolymer monolayer was grafted onto the surface of the spheres. The carboxyl group content is higher for the spheres cross-linked with succinyl chloride, as some of the cross-linkers reacted only on one

Figure 2. SEM images of a diblock (top) and a triblock (bottom) microsphere sample prepared using recipes shown in Table 2.

hydrolysis media were chosen to be mainly organic based so that Fe2+ or Fe3+ had minimal solubility in them. Because of this precaution, we did not notice by SEM any significant size change of the microspheres before and after hydrolysis. Figure 2 shows the SEM images for two batches of microspheres that we prepared following the recipes shown in Table 2. Mostly spherical particles were prepared. By measuring manually the size of ∼200 particles for each sample from such SEM images, we obtained the average diameter of 0.46 ( 0.12 and 1.4 ( 0.5 µm for the two samples, respectively. The numbers after ( denote the average deviation from the mean size for the particles and offer a measure of the width in the size distribution of the particles. The distributions are relatively wide and can, in principle, be narrowed down by using techniques such as centrifugation fractionation. The major difference between the di- and triblock spheres is that the latter are larger.

Table 2. Recipes Used to Prepare the Magnetic Spheres Shown in Figure 2 surfactant

copolymer amount (mg)

homopolymer amount (mg)

magnetic soln (mg)

succinyl chloride (mg)

pyridine (g)

stirring speed (rpm)

SEM diameter (µm)

diblock triblock

258 232

318 254

399 249

490 220

2.5 2.2

1800 1600

0.46 ( 0.12 1.4 ( 0.5

Table 3. Effect of Changing Stirring Speed on Microspheres Prepared using PtBA145-PSA160-Phea240 as the Surfactant sample

triblock (mg)

PHEA (mg)

magnetic soln (mg)

succinyl chloride (mg)

pyridine (g)

stirring speed (rpm)

SEM diameter (µm)

T2 T3 T4

205 205 205

224 224 224

203 203 203

201 180 180

2.06 1.83 1.83

1200 1700 2000

2.04 ( 0.39 1.32 ( 0.30 1.06 ( 0.24

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Figure 3. Photograph showing the stability of a magnetic microsphere dispersion (left vial) and the ready capture of the microspheres by a magnet (right vial).

end with the hydroxyl groups of PHEA and the other end yielded a carboxyl group. After PtBA hydrolysis, the spheres dispersed in water especially well after treatment with Na2CO3 to produce poly(sodium acrylate). The dispersibility of the spheres in water increased as the size of the spheres decreased. The 0.46 µm diblock spheres of Table 2 remained dispersed in water for days, and the triblock 1.4 µm spheres remained dispersed for ∼10 h. Under otherwise identical conditions, the magnetic response or the rate of capturing by magnet increased with particle size. Depending on the application, the size of the magnetic particles thus needs to be optimized to ensure dispersibility and magnetic response. This need for performance optimization explains why one does not normally use polymer-coated magnetite sol directly for diagnostic applications. Since the magnetite content was high, both types of spheres of Table 2 were captured by a 0.4 T magnet within 30 s. Figure 3 shows a photograph taken of the 1.4 µm spheres in two separate vials 60 s after a magnet was placed next to one of them. While negligible particle settlement was noticed of the sample on the left, the particles in the right vial were seen essentially quantitatively captured. Distribution of Magnetite. To examine the distribution of magnetite within the microspheres, the microspheres were embedded in epoxy resin and then microtomed to yield thin sections. Figure 4 shows a TEM image of a thin section of a sample. Since the specimen was not stained, magnetite should look darker in the image. On the basis of the presence of both dark and light domains inside each microsphere, one may plausibly argue that partial segregation between magnetite and cross-linked PHEA occurred and that the PHEAor magnetite-rich domains reached the size of 10 or even 100 nanometers. This conclusion may, however, be incorrect as light domains would have been seen for voids formed due to the uneven slicing of the microsphere specimen. Uneven slicing resulting from matrix fracturing has been observed by us before for cross-linked PS.53 The cross-linked PHEA/magnetite composite might behave similarly. While we cannot conclude unambiguously about the extent of polymer and magnetite segregation, what is clear from the images is that magnetite can be found, based on the observation of many spheres, anywhere in a sphere. In regions where the magnetite nanoparticles are not agglomer-

Figure 4. TEM image of thin sections of a microsphere sample.

Figure 5. Magnetization curve of a microsphere sample dispersed in water.

ated, we found the dimension of the primary nanoparticles to be comparable with that of the original sol particles. Magnetic Properties. Figure 5 shows a magnetization curve of the microspheres dispersed in water measured at room temperature. The particles are essentially superparamagnetic possessing little hysteresis, which suggests minimal agglomeration of the magnetite nanoparticles. The saturation magnetization is 17.5 emu/g. Since the magnetite content was ∼33% in this sample, the saturation magnetization per gram of magnetite was 53 emu/g, which is lower than the bulk saturation magnetization of 92 emu/g.54 A saturation magnetization lower than that for bulk materials is, however, normal for nanoparticles. Typical reasons for this include the reaction or complexation of the surface atoms of magnetic nanoparticles with surfactant, which may create a magnetically dead layer.55 With a significant fraction of surface atoms, any crystalline disorder within the surface layer may also lead to a significant decrease in the nanoparticle saturation magnetization. Model of Microsphere Formation. We believe that the microspheres were derived directly from the aqueous droplets that were formed after the breaking up of the aqueous phase by mechanical stirring. This hypothesized model is strongly supported by the observation that a significant reduction in size was observed of the microspheres with the implementation of a water distillation step. This model also explains


the variation in the size of the microspheres prepared under different conditions. In the presence of sufficient surfactant, the aqueous phase would obviously break up into smaller droplets; thus, the final microspheres would decrease in size as the stirring speed was increased. At a given stirring speed, the particles would decrease in size initially with increasing surfactant content but increase little with further increase in surfactant concentration above a critical value. The size decreased initially as the surfactant content was the sizedetermining factor at this stage. Above a critical concentration, the size-determining factor was switched to the stirring speed. The size of the particles increased under otherwise identical conditions with magnetite or homopolymer content as the viscosity of the aqueous phase increased with the concentration of these species. As the viscosity increased, more energy was required to break up the aqueous phase, and the resultant particles were bigger for a given stirring speed. Conclusions ATRP has been used to prepare functional polymers PtBAP(HEA-TMS) and PtBA-PSA-P(HEA-TMS). In wet THF in the presence of acetic acid, the P(HEA-TMS) block was selectively hydrolyzed to yield PtBA-PHEA and PtBA-PSAPHEA. Either PtBA-PHEA or PtBA-PSA-PHEA could be used as a surfactant to disperse under vigorous stirring an aqueous phase containing PHEA and magnetite nanoparticles as droplets in toluene and chlorobenzene. After water distillation, we cross-linked the core of the microspheres using succinyl chloride to yield permanent spheres. We then made water-dispersible magnetic microspheres bearing a surface carboxyl group by hydrolyzing the PtBA or PtBAPSA surface chains under controlled conditions with minimal dissolution of the magnetite sol. The size of the spheres thus prepared could be tuned by varying the preparation conditions including the stirring speed, etc. This, in turn, allowed us to tune the water dispersibility and magnetic response of the spheres because, under otherwise identical conditions, the magnetic response decreased and water dispersibility increased for the spheres when their size was decreased. The microspheres prepared should be useful in immunoassays and other areas. Acknowledgment. BayerHealthcare, Diagnostics Division and the Collaborative Research and Development Grant Program of NSERC are cordially thanked for sponsoring this research. Mr. Matthias Haeussler and Prof. Ben Zhong Tang of the University of Science and Technology of Hong Kong are gratefully acknowledged for carrying out the magnetic property measurement. Dr. Xiaohu Yan is thanked for obtaining the cross-section TEM image of the microspheres. References and Notes (1) Hurtubise, P. E.; Bassion, S.; Gauldie, J.; Horsewood P. In Clinical ChemistrysTheory, Analysis, and Correlation, 2nd ed.; Kaplan, L. A., Pesce, A. J., Eds.; CV Mosby Company: St. Louis, 1989. (2) See, for example (a) Widder, K. J.; Morris, R. M.; Poore, G.; Howard, D. P.; Senyei, A. E. Proc. Natl. Acad. Sci. U.S.A., 1981, 78, 579. (b) Kang, J. C.; Wei, S. L. Acta Pharmaceutica Sinica 1997, 32, 536. (3) Molday, R. S.; Yen, S. P. S.; Rembaum, A. Nature 1977, 268, 437.

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BM049316F

Preparation of Magnetic Microspheres from Water-in-Oil Emulsion

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