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In situ magnetization technique for synthesis of magnetic polymer microspheres
Powder Technology 235 (2013) 1017–1024
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Powder Technology journal homepage: www.elsevier.com/locate/powtec
In situ magnetization technique for synthesis of magnetic polymer microspheres Lianyan Wang ⁎, Tingyuan Yang, Jian Yang, Qiang Li, Dongxia Hao, Juanjuan Yang, Guanghui Ma ⁎⁎ State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190, China
a r t i c l e
i n f o
Article history: Received 28 November 2011 Received in revised form 6 December 2012 Accepted 6 December 2012 Available online 16 December 2012 Keywords: In situ magnetization Magnetic polymer microspheres Ethyl acetate (EA) Double emulsion
a b s t r a c t A new process was developed for synthesis of magnetic polymer microspheres which can be used as hyperthermia carrier for cancer therapy. Polymer microspheres containing magnetic nanoparticles were successfully prepared by in situ formation technique of magnetite combination with double emulsion method. Poly (styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA) dissolved into ethyl acetate (EA) was employed as oil phase (O). Ferrous chloride (Fe 2 +) and ferric chloride (Fe 3 +) aqueous solution was used as an internal water phase (W1), which was dispersed into polymer solution to form primary emulsion of W1/O. Then, this emulsion was further dispersed into external water phase (W2) to obtain double emulsion of W1/O/W2. Finally, the alkaline solution of ammonia was added into this system, and the ammonia continuously diffused into the internal water phase (W1) to react with Fe2+ and Fe3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase. Various process parameters including Fe2+/Fe3+ concentrations in internal water phase, PS-HEMA concentrations, Span85 amounts in oil phase and volume fractions of pre-solidification solution affected stability of primary emulsion, loading efficiency of magnetic nanoparticles, morphology and magnetization of microspheres, which were systematically investigated in this study. Compared with the traditional method, the magnetic polymer microspheres prepared by in situ magnetization technique demonstrated excellent encapsulation efficiency (26.1%) and high magnetization (12.2 emu/g). Therefore, the as-prepared magnetic polymer microspheres show good potential for cancer therapy. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Magnetic polymer microspheres have been widely used in the fields of biotechnology and biomedical engineering, such as cell isolation, protein immobilization, enzyme immobilization, immunoassay, RNA and DNA purification, biochemical assays, drug targeting and delivery, hyperthermia for cancer therapy and so on [1–5]. Until now, various methods have been developed for preparation of magnetic polymer microspheres with inorganic magnetic core and polymer shell. For these methods, the iron oxide nanoparticles are prepared at first, and then encapsulated or adsorbed inside the polymer microspheres [6–11]. However, in these processes, it is time-consuming to separately prepare the iron oxide nanoparticles and polymer microspheres. Moreover, the inorganic magnetic nanoparticles are difficult to be encapsulated or adsorbed inside the polymer microspheres due to weak affinity, which resulted in lower encapsulation efficiency and magnetization. To overcome these problems, a novel process of in situ formation technique of magnetite combined with double emulsion method was developed in this study. In details, poly(styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA) dissolved into ethyl acetate (EA) was employed ⁎ Corresponding author. Tel./fax: +86 10 82544931. ⁎⁎ Corresponding author. Tel./fax: +86 10 82627072. E-mail addresses: [email protected] (L. Wang), [email protected] (G. Ma). 0032-5910/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.powtec.2012.12.014
as oil phase (O). Ferrous chloride (Fe2+) and ferric chloride (Fe3+) aqueous solution was used as an internal water phase (W1), which was dispersed into polymer solution to form primary emulsion of W1/O. Then, this emulsion was further dispersed into external water phase (W2) to obtain double emulsion of W1/O/W2. Finally, the alkaline solution of ammonia was added into the system, and the ammonia continuously diffused into the internal water phase (W1) to react with Fe2+ and Fe3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase of microspheres. Both formation and encapsulation of iron oxide nanoparticles were simply accomplished in a single step. Furthermore, the process avoided directly encapsulating nanoparticles into the polymer matrix [12,13]. Therefore, this novel method developed in this study is expected to significantly improve the encapsulation efficiency and magnetic properties of microspheres. Solvent for dissolving polymers is very important for improving encapsulation efficiency and magnetization of magnetic polymer microspheres. It affects not only the stability of primary emulsion, but also alkaline diffusion into internal water phase and the following yield of Fe3O4 magnetic nanoparticles. As we know, dichloromethane (DCM) is a commonly used solvent of lipophilic polymers for microsphere preparation because it is convenient to be removed by evaporation due to low boiling point [14]. However, it is time-consuming to solidify microspheres, which will lead to diffusion and leakage of the internal water phase. This will result in lower encapsulation efficiency of Fe 2+/Fe 3 + salt, which will further lead to lower yield of Fe3O4 nanoparticles in microspheres. Moreover, the larger aggregates of
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the formed Fe3O4 nanoparticles will easily leak out from microspheres, which will also lead to lower encapsulation of magnetic nanoparticles. In order to overcome disadvantages above, ethyl acetate (EA) was chosen as organic solvent in this study. EA has relatively higher solubility in water (8.7%, w/v) than DCM, which can accelerate EA to diffuse from oil phase into external water phase, and further rapidly solidify W1/O/W2 droplets into microspheres. The rapid solidification will be undoubtedly favorable for retaining the Fe3O4 nanoparticles in the internal water phase and lead to improvement of encapsulation efficiency. Although rapid solidification can improve encapsulation efficiency, the fast diffusion of EA will lead to rapid precipitation of polymer from oil phase, which will further result in formation of coagulation rather than microspheres. In order to overcome this disadvantage, we employed pre-solidification at first by pouring the double emulsion into a small volume fraction of aqueous solution. In this step, the precipitation of polymer from oil droplets was slowed down and permitted partial solidification of the droplet. Then the complete solidification was carried out by transferring this system into an excess aqueous solution. This process effectively retarded the diffusion or coalescence of internal phase to external water phase, which greatly improved encapsulation of magnetic nanoparticles and magnetic properties of magnetic polymer microspheres [15,16].
Company (China). All other regents were of reagent grade and used as received. 2.2. Preparation of magnetic PS-HEMA microspheres The schematic illustration for preparation of magnetic PS-HEMA microspheres was shown in Fig. 1. Ferrous chloride and ferric chloride were dissolved into deionized water as an internal water phase (W1). PS-HEMA was dissolved into EA containing emulsifier (Span85) as an oil phase (O). These two phases were mixed and emulsified to form primary emulsion of W1/O. Then, the W1/O emulsion was further dispersed into external water phase (W2) containing PVA and Na2SO4 to obtain double emulsion of W1/O/W2. After that, the double emulsion was transferred into a small volume fraction of external water phase for pre-solidification. Afterward, the alkaline solution of ammonia was added into the system, and it diffused into the internal water phase (W1) to react with Fe 2+ and Fe 3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase, and the double emulsion of W1/O/W2 was changed into S/O/W2. Finally, the S/O/W2 was poured into a large volume of external water phase (W2) to completely remove EA by solvent diffusion, and the magnetic polymer microspheres were obtained. 2.3. Measurement of primary emulsion stability
2. Materials and methods 2.1. Materials Styrene (St, reagent grade) was distilled under vacuum and stored in a refrigerator until use. Hydroxyethylmethacrylate (HEMA, reagent grade) was used without further treatment. Poly (styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA, 95:5, Mw=1.69×105 kDa) was synthesized by soap-free emulsion polymerization in our lab. Poly (vinyl alcohol) (PVA-217, average degree of polymerization 1700, degree of hydrolysis 88.5%) was provided by Kuraray (Tokyo, Japan) and used as a stabilizer in the external water phase. Span85 (Sorbitantrioleat, Beijing Chemical Reagents Company, China) of biochemical grade was selected as oil emulsifier. FeCl3∙6H2O, FeCl2∙4H2O, ammonia solution (25%), and EA (ethyl acetate) of analytical grade were purchased from Beijing Chemical Reagents
Definite amounts of PS-HEMA were dissolved into 5.0 mL EA containing emulsifier of Span85 as oil phase (O). 1.0 mL ferric solution (W1) was emulsified into oil phase to form primary emulsion of W1/O. Then, the primary emulsion was poured into a test tube and kept still to observe the occurrence of phase separation. The stability was expressed by the elapsed time until the phase separation was observed. The longer elapsed time meant the better stability of primary emulsion. 2.4. Characterization of magnetic PS-HEMA microspheres 2.4.1. Morphology and microstructure of microspheres The surface morphology of magnetic polymer microspheres was observed by a scanning electron microscope (SEM) (JSM-6700F, JEOL, Japan). The specimens for SEM observation were prepared by mounting
Fig. 1. Schematic illustration of the preparation of magnetic polymer microspheres.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
2.4.2. Measurement of size and size distribution The volume-average diameter and the size distribution of the magnetic polymer microspheres were measured by a dynamic light scattering analysis (DLS) (Malvern 2000E apparatus, USA). The term “SPAN” was used to present the width of the particle size distribution, which was defined as: SPAN ¼
D90−D10 D50
The values of D10, D50 and D90 were the diameters corresponding to 10%, 50% and 90% in volume on a relative cumulative droplet size distribution curve, respectively. 2.4.3. Measurement of Fe content in microspheres and encapsulation efficiency A certain weight of magnetic polymer microspheres (W0) was put into a ceramic crucible, and then it was incinerated in a Muffle furnace under 800 °C for 6 h. The wall material of microspheres as PS-HEMA was burnt into carbon dioxide and steam, and the magnetic nanoparticles of Fe3O4 were changed into Fe2O3. The weight of residual substance (Fe2O3) was measured and expressed as W1. The molecular weight of Fe and Fe2O3 were 56 and 160 g/mol, respectively. The Fe content of magnetic polymer microspheres was calculated by the following formula: Fe contentð%Þ ¼
W1 56 2 100 W0 160
The encapsulation efficiency was obtained according to the following calculation: Encapsulation efficiencyð%Þ ¼
Total amount of Fe in microspheres 100 Total amount of Fe added
2.4.4. Magnetic properties of microspheres The magnetite type of microspheres was analyzed by X-ray diffractometer (D/max 2200 PC, Rigaku Co., Japan). The magnetization of magnetic polymer microspheres was measured by vibrating sample magnetometer (VSM) (model-155, Digital measurement System, Inc.). The maximum applied field was 8 kOe, and the measurement temperature was kept at 290 K. 3. Results and discussion 3.1. Stability of primary emulsion Stability of the primary emulsion is important for improving encapsulation efficiency of microspheres prepared from double emulsion system. The primary emulsion is usually prepared by dispersing water phase into oil phase. As usual, there exists a density difference between water and oil phase, which will lead to poor stability of
primary emulsion. The amounts of polymer and surfactant in oil phase usually have significant influence on stability of primary emulsion. 3.1.1. Effect of PS-HEMA concentration on stability PS-HEMA with different concentrations (0.7 wt.%, 1.0 wt.%, 1.3 wt.%, 1.6 wt.% and 1.9 wt.%) was used to prepare primary emulsion, and the elapsed time before phase separation was recorded. The result of Fig. 2 showed that the phase separation for primary emulsion was retarded with the increase of PS-HEMA concentrations. The primary emulsion could maintain stability for more than 2 h when PS-HEMA concentrations were 1.3 wt.% and 1.6 wt.%. This suggested that higher polymer concentrations were favorable for stability of primary emulsion. The possible reason was that the viscosity of oil phase increased with enhancement of polymer concentrations, and the oil film with higher viscosity could effectively prevent coalescence of droplets, which further led to excellent stability of primary emulsion. When PS-HEMA concentrations reached to 1.9 wt.%, it was difficult to obtain primary emulsion due to much higher viscosity of oil phase. The above results suggested that the suitable polymer concentrations for obtaining stable primary emulsion were 1.3 wt.% and 1.6 wt.%. In addition, the polymer concentrations would also affect the subsequent solidification of microspheres. The lower concentrations could slow down the precipitation of polymer from oil phase with extraction of EA and favor for formation of microspheres. Therefore, PS-HEMA concentrations of 1.3 wt.% were chosen for the further study. 3.1.2. Effect of Span85 amounts on stability Different amounts of Span85 (0.00 wt.%, 0.25 wt.%, 0.50 wt.%, 0.75 wt.% and 1.00 wt.%) were dissolved into EA to be employed as oil phase for preparation of primary emulsion, and the phase separation of primary emulsion was observed. The corresponding result was demonstrated in Fig. 3, which exhibited that the stability of primary emulsion was enhanced with increase of Span85 concentrations. When the concentrations of Span85 were more than 0.75 wt.%, the amounts of emulsifier had little influence on stability. As we know, emulsifier can form protective layer around surface of droplet, which can effectively suppress the coalescence of droplets. The emulsifier-assembled layer could not completely cover the surface of droplet when the Span85 concentrations were less than 0.75 wt.%, which led to poor stability of primary emulsion. Therefore, the amounts of Span85 in oil phase were chosen as 0.75 wt.% in the subsequent study. 3.2. Investigation of preparation conditions for PS-HEMA microspheres In double emulsion system which employed EA containing PS-HEMA as oil phase, the volume fractions of external water phase and pre-
Elapsed time before phase separation (h)
sample on metal stubs with double-sided conductive adhesive tape. Then, the samples were coated with a thin gold film (approx. 60 nm in thickness) under a reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan). Finally, the samples were put into SEM to observe the morphology of microspheres. Transmission electron microscope (TEM) (H-600, Hitachi, Japan) was used to observe the microstructure of microspheres and the distribution of magnetic nanoparticles in microspheres. The specimens for TEM observation were prepared by dropping the alcohol-diluted sample onto ultra thin carbon film supported by a copper mesh, and then the copper mesh grid was dried under light. Then, the sample was put into the sample cell for observation.
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2.5 2.0 1.5 1.0 0.5 0.0 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
PS-HEMA concentration (wt.%) Fig. 2. Effect of PS-HEMA concentrations on stability of primary emulsion.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
Elapsed time before phase separation (h)
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7 6 5 4 3 2 1 0 0.0
0.2
0.4
0.6
0.8
1.0
Concentration of Span 85 (wt.%) Fig. 3. Effect of Span85 concentrations in oil phase on stability of primary emulsion.
solidification aqueous solution have important influence on morphology of microspheres due to larger solubility of EA in water (8.7%, w/v). This will further affect encapsulation and magnetization of magnetic polymer microspheres. Therefore, the effects of the volume fractions of external water phase and pre-solidification aqueous solution on morphology, encapsulation efficiency and magnetization of microspheres have been systematically investigated in this study.
3.2.1. Effect of volume ratio between external water phase and oil phase 1.0 mL ferric salt solution as internal water phase, 5.0 mL EA containing 1.3 wt.% PS-HEMA and 0.75 wt.% Span85 as oil phase, different volumes of external water phase (20, 30, 40 and 50 mL) containing 1.0 wt.% PVA and 2.0 wt.% Na2SO4 were used to prepare magnetic microspheres, and the volume ratios between external water phase and oil phase were 4:1, 6:1, 8:1 and 10:1, respectively. The morphology of
magnetic microspheres was shown in Fig. 4, and the results of encapsulation efficiency, volume-mean size and magnetization of microspheres were represented in Table 1. Fig. 4 suggested that the volume ratios between external water phase and oil phase had significant influence on morphology of microspheres. With the increase of volume fractions of external water phase, the amounts of EA extracted into external water phase were also increased, which further affected the precipitation of polymer from oil droplets and subsequent formation of microspheres. For the system with low volume fractions of external water phase (volume ratios between oil phase and external water phase as 4:1 and 6:1), there were small amounts of EA to diffuse into external water phase during double emulsion processes. Mostly no polymer was precipitated from the oil droplets, which led to the increase of viscosity of oil droplet and further facilitated adhesion and coagulation between oil droplets. Moreover, the concentrations of emulsion were also increased with the decrease of volume fraction of external water phase, which resulted in a crowded environment and increased collision frequency between oil droplets. Both the increased viscosity of oil droplet and the crowded environment promoted the formation of coagulation as shown in Fig. 4a and b. The coagulation between oil droplets also caused leakage of internal water phase with final lower encapsulation and magnetization results. While the volume fraction of external water phase was increased (volume ratios between external water phase and oil phase as 8:1 and 10:1), large amounts of EA could diffused into external water phase, and the droplets became quasi-solid during double emulsion process. Droplets coagulation was effectively inhibited, and microspheres showed an excellent morphology. As a result, higher encapsulation efficiency and magnetization were obtained as shown in Table 1. Therefore, the higher volume fractions of external water phase were favorable for formation of microspheres with excellent morphology and higher encapsulation efficiency and magnetization. 3.2.2. Effect of volume ratio between pre-solidification aqueous solution and oil phase The magnetic polymer microspheres were prepared with various volume ratios between pre-solidification aqueous solution and oil phase as 40:1, 30:1, 20:1 and 10:1. The morphology of microspheres
a
b
c
d
Fig. 4. SEM micrographs of microspheres prepared by different volume of external water phase (a, 20 mL b, 30 mL c, 40 mL d, 50 mL).
L. Wang et al. / Powder Technology 235 (2013) 1017–1024 Table 1 The characteristics of magnetic polymer microspheres prepared by different volume of external water phase. Volume of external water phase (mL)
Encapsulation efficiency (%)
Volume-mean size of microspheres (μm)
Magnetization (emu/g)
20 30 40 50
15.61 17.96 22.35 25.78
30.2 32.4 35.8 37.5
8.42 10.0 12.2 13.8
as shown in Fig. 5 suggested that the volume ratio between presolidification aqueous solution and oil phase had important influence on the morphology of microspheres. The microspheres showed better spherical shape with a few pores on the surface under the conditions with lower volume ratios as 20:1 and 10:1. With the increase of volume fraction of pre-solidification aqueous solution as 30:1 and 40:1, microspheres appeared more and large surface pores, and especially some microspheres collapsed to form coagulations as shown in Fig. 5a and b. For the large fractions of pre-solidification aqueous solution, faster diffusion of EA led to rapid precipitation of polymer, and the deposited polymer could not shrink into the dense surface in time, resulting in the formation of lager pores. If the pores were large enough, the coalescence between internal and external water phase would occur, which further resulted in the collapse of microspheres. While in the systems with lower fractions of pre-solidification aqueous solution, the above phenomenon could be avoided to some extent due to slow extraction of EA and precipitation of polymer. Therefore, lower fractions of pre-solidification aqueous solution were much favorable for formation of microspheres with better morphological characters.
3.3. Encapsulation efficiency and magnetization of microspheres After obtaining spherical microspheres, the effects of total concentration of Fe 2+/Fe 3+ solution, PS-HEMA concentration and Na2SO4
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concentration in external water phase on encapsulation and magnetization microspheres were investigated thoroughly.
3.3.1. Effect of total concentrations of Fe 2+/Fe 3+ The Fe 2+ and Fe 3+ concentrations in internal water phase would cause difference of osmotic pressure between internal and external water phase, which would further influence various encapsulation efficiency and magnetization of polymer microspheres. Here, the different concentrations of Fe 2+ and Fe 3+ solutions as 0.72, 1.08, 1.44, 1.80 and 2.16 mol/L with a fixed Fe2+/Fe3+ molar ratio of 1:2 were employed to prepare magnetic microspheres, and their effects on the encapsulation and magnetization of magnetic polymer microspheres were shown in Figs. 6 and 7. Fig. 6 showed that the encapsulation efficiency declined with the increase of total concentrations of Fe2+/Fe3+. The difference of osmotic pressure between internal and external water phase increased with increase of the total concentrations of Fe 2 +/Fe 3 +. The external water phase would enter into oil droplets under the higher osmotic pressure of internal water phase, and this further resulted in gradually thinned till broken of oil film, which finally decreased the encapsulation efficiency of magnetic polymer microspheres. Comparatively, Fig. 7 showed that the magnetization increased at first, and then tend to decline with increase of the total concentrations of Fe 2+/Fe 3+. This tendency resulted from two controlling factors, the dynamic reaction rate for formation of Fe3O4 nanoparticles and the encapsulation efficiency of microspheres. When the total concentrations of Fe 2+/Fe 3+ were less than 1.8 mol/L, the magnetization of microspheres was mainly affected by reaction rate for formation of Fe3O4 nanoparticles. The yield of Fe3O4 nanoparticles increased with the increase of total concentrations of Fe2+/Fe3+, which resulted in the increase of magnetization. The higher concentrations of Fe2+/Fe3+ in internal water phase would lead to the higher osmotic pressure, which drove more alkali diffuse into internal water phase from external water phase to react with Fe 2+ and Fe3+ solutions, and resulted in the higher yield of Fe3O4 nanoparticles. However, when the concentrations of Fe 2+/Fe 3+ were over 1.8 mol/L, the encapsulation efficiency became
a
b
c
d
Fig. 5. SEM micrographs of microspheres prepared by different volume of pre-solidification solution (a, 200 mL b, 150 mL c, 100 mL d, 50 mL).
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28
34
Encapsulation efficiency (%)
Encapsulation efficiency (%)
32 30 28 26 24 22 20 18
26
24
22
16 20 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
0
2.6
2
4
6
8
10
HEMA content in the polymer (wt%)
Concentration of Fe2+/Fe3+ (moL/L) Fig. 6. Effect of total concentrations of Fe2+/Fe3+ on encapsulation efficiency of magnetic polymer microspheres.
Fig. 8. Effect of HEMA content in polymer on encapsulation efficiency of magnetic nanoparticles.
the main controlling factor. The rapid decrease of encapsulation efficiency of microspheres caused the decrease of magnetization.
layer and emulsion, and eventually led to lower encapsulation efficiency. Therefore, the optimum content of HEMA in polymer was 2.0 wt.%.
3.3.2. Effect of HEMA content in PS-HEMA The PS-HEMA with different HEMA amounts of 0.0, 2.0, 5.0, 8.0 and 10.0 wt.% was employed to prepare magnetic microspheres, and their effect on encapsulation efficiency was investigated. The corresponding result as shown in Fig. 8 demonstrated that suitable amounts of HEMA in polymer were necessary for the improvement of encapsulation efficiency of magnetic nanoparticles. The encapsulation efficiency reached the highest of 26.1% when HEMA content in PS-HEMA was 2.0 wt.%. The hydrophilic hydroxyl group on PS-HEMA could promote HEMA units to distribute interface between internal water phase and oil phase, which preferred to form chelates with Fe2+/Fe3+ ion and favored for increase of encapsulation efficiency. Furthermore, PS-HEMA was amphiphilic, and played a role of emulsifier during formation of W/O and W/O/W. This was helpful for the stability improvement of primary and double emulsion, which was also favorable for increase of encapsulation efficiency. Therefore, the encapsulation efficiency was raised with the increase of HEMA content in polymer when HEMA was less than 2.0 wt.%. When the content of HEMA increased to more than 2.0 wt.%, steric repulsion of HEMA became dominant due to limited space of internal water phase. This would greatly reduce the stability of polymer
3.3.3. Effect of salt concentrations in outer water phase The salt of Na2SO4 was added into external water phase to adjust osmotic pressure between internal and external water phase. Different salt concentrations of 0.5, 1.0, 1.5 and 2.0 wt.% were used to prepare magnetic microspheres, and the corresponding encapsulation efficiency was measured as shown in Fig. 9. The result suggested that the encapsulation efficiency was raised with the increase of salt concentrations. In this system, internal water phase had higher ionic strength due to addition of Fe2+/Fe3+. When concentrations of Na2SO4 were low, the ionic strength of external water phase was lower than that of internal water phase. A part of external water phase entered into internal water phase by virtue of osmotic pressure, which led to volume increase of internal water phase. This further resulted in coalescence and leakage of internal water phase. On the contrary, the ionic strength of water phase was raised by increasing Na2SO4 concentrations to achieve higher osmotic pressure of external water phase, which effectively inhibited diffusion of external water phase into internal water phase and improved the stability of double emulsion. This was promising for the subsequent improvement of encapsulation efficiency. The Na2SO4 concentration was chosen as 2.0 wt.%, because
13 26
Encapsulation efficiency (%)
Magnetization (emu/g)
12 11 10 9 8 7
25 24 23 22 21 20 19
6 0.6
0.8
1.0
1.2
1.4
Concentration of
1.6
1.8
Fe2+/Fe3+
2.0
2.2
2.4
2.6
(moL/L)
Fig. 7. Effect of total concentrations of Fe2+/Fe3+ on magnetization of magnetic polymer microspheres.
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Concentration of Na2SO4 (wt%) Fig. 9. Effect of Na2SO4 concentrations in external water phase on the encapsulation efficiency of magnetic nanoparticles.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
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50
Intensity (cps)
40 30 20 10 0 0
20
30
40
50
60
70
80
90
Fig. 12. XRD of magnetic polymer microspheres prepared under optimal conditions.
10 µm Fig. 10. TEM microphotographs of magnetic polymer microspheres prepared under optimal conditions.
PVA flocculation appeared if the concentrations were higher than 2.0 wt.%. 3.4. Magnetic PS-HEMA microspheres under optimal preparation conditions Under the optimized conditions, the magnetic polymer microsphere was successfully prepared. The Fe 2 +/Fe 3 + aqueous solution with concentration of 1.8 mol/L was internal water phase (W1), the EA containing 1.3 wt.% PS-HEMA with 2.0 wt.% HEMA and 0.75 wt.% Span85 was oil phase (O), the aqueous solution containing 1.0 wt.% PVA and 2.0 wt.% Na2SO4 was external water phase (W2), and the volume ratio among internal water phase, oil phase and external water phase was fixed at 1:5:10. After preparation of double emulsion, it was added into pre-solidification aqueous solution to partially solidify the oil droplets, and the volume ratio between pre-solidification aqueous solution and oil phase was 20:1. Then, the ammonia was dropped into the system for in situ magnetization of Fe2+/Fe3+, and the volume ratio between solidification aqueous solution and oil phase was fixed at 160:1. Fig. 10 demonstrated a TEM image of obtained magnetic microspheres, in which magnetite nanoparticles appeared darker and welldispersed in the whole matrix of microspheres. This meant that the in situ formation of magnetic nanoparticle was homogeneous, and there
15 10
Magnetization (emu/g)
10
5 0 -5 -10
was no agglomeration of the magnetite nanoparticles. A magnetization curve of the microspheres in Fig. 11 showed that the magnetic microspheres were super-paramagnetic, and no hysteresis was found, and the magnetization reached to 12.2 emu/g, which was much higher than those of microspheres prepared by the conventional methods. The crystalline structures of nanoparticles in microspheres were further characterized by X-ray diffraction (XRD) patterns. As shown in Fig. 12, the patterns of in situ formed nanoparticles could be easily indexed to standard Fe3O4 crystal with its six diffraction peaks: {2 2 0}, {3 1 1}, {4 0 0}, {4 2 2}, {5 1 1} and {4 4 0}, and no other peaks of impurities were detected. Therefore, we concluded that the nanoparticles dispersed in the PS-HEMA microspheres matrix were mainly composed of super-paramagnetic Fe3O4 nanoparticles.
4. Conclusion The magnetic PS-HEMA microspheres were successfully fabricated by in situ magnetization techniques in double emulsion system. We first prepared W1/O/W2 emulsion with Fe 2 +/Fe 3 + solutions as internal water phase (W1), EA dissolved with polymer of PS-HEMA and surfactant of Span85 as oil phase (O), and deionized water dissolved with PVA-217 and Na2SO4 as external water phase (W2). After presolidification, the in situ co-precipitation reaction of Fe 2+ and Fe3+ into magnetic nanoparticles was performed in the internal water phase by diffusing of alkali solution from external to internal water phase. Finally, the obtained S/O/W2 droplet was further solidified into microspheres by further solvent diffusion. In this novel preparation process, the critical factors affecting the structure and magnetic properties of final microspheres included PS-HEMA concentration, HEMA content in PS-HEMA, the type of emulsifier in oil phase, concentrations of Fe2+/Fe3+ solutions in internal water phase, concentrations of Na2SO4 added in external water phase, volume fractions of external water phase and pre-solidification aqueous solution. The final microspheres with better morphological characters and higher magnetization of 12.2 emu/g were obtained under the optimal conditions. The magnetization of microspheres prepared by this novel process was much higher than that of microspheres prepared by conventional methods. This technique can also be utilized in preparation of other inorganic-polymer composite microspheres when the inorganic particles can be synthesized by chemical co-precipitation reaction. Therefore, this is a potential method for preparation of polymer microspheres with higher loading efficiency of inorganic nanoparticles.
-15 -10
-8
-6
-4
-2
0
2
4
6
8
10
Applied magnetic field (KOe) Fig. 11. Magnetization curves of magnetic polymer microspheres prepared under optimal conditions.
Acknowledgement The authors are thankful to Natural Science Foundation of China for financial support to this work (grant no. 20376082, no. 20820102036).
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Powder Technology journal homepage: www.elsevier.com/locate/powtec
In situ magnetization technique for synthesis of magnetic polymer microspheres Lianyan Wang ⁎, Tingyuan Yang, Jian Yang, Qiang Li, Dongxia Hao, Juanjuan Yang, Guanghui Ma ⁎⁎ State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190, China
a r t i c l e
i n f o
Article history: Received 28 November 2011 Received in revised form 6 December 2012 Accepted 6 December 2012 Available online 16 December 2012 Keywords: In situ magnetization Magnetic polymer microspheres Ethyl acetate (EA) Double emulsion
a b s t r a c t A new process was developed for synthesis of magnetic polymer microspheres which can be used as hyperthermia carrier for cancer therapy. Polymer microspheres containing magnetic nanoparticles were successfully prepared by in situ formation technique of magnetite combination with double emulsion method. Poly (styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA) dissolved into ethyl acetate (EA) was employed as oil phase (O). Ferrous chloride (Fe 2 +) and ferric chloride (Fe 3 +) aqueous solution was used as an internal water phase (W1), which was dispersed into polymer solution to form primary emulsion of W1/O. Then, this emulsion was further dispersed into external water phase (W2) to obtain double emulsion of W1/O/W2. Finally, the alkaline solution of ammonia was added into this system, and the ammonia continuously diffused into the internal water phase (W1) to react with Fe2+ and Fe3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase. Various process parameters including Fe2+/Fe3+ concentrations in internal water phase, PS-HEMA concentrations, Span85 amounts in oil phase and volume fractions of pre-solidification solution affected stability of primary emulsion, loading efficiency of magnetic nanoparticles, morphology and magnetization of microspheres, which were systematically investigated in this study. Compared with the traditional method, the magnetic polymer microspheres prepared by in situ magnetization technique demonstrated excellent encapsulation efficiency (26.1%) and high magnetization (12.2 emu/g). Therefore, the as-prepared magnetic polymer microspheres show good potential for cancer therapy. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Magnetic polymer microspheres have been widely used in the fields of biotechnology and biomedical engineering, such as cell isolation, protein immobilization, enzyme immobilization, immunoassay, RNA and DNA purification, biochemical assays, drug targeting and delivery, hyperthermia for cancer therapy and so on [1–5]. Until now, various methods have been developed for preparation of magnetic polymer microspheres with inorganic magnetic core and polymer shell. For these methods, the iron oxide nanoparticles are prepared at first, and then encapsulated or adsorbed inside the polymer microspheres [6–11]. However, in these processes, it is time-consuming to separately prepare the iron oxide nanoparticles and polymer microspheres. Moreover, the inorganic magnetic nanoparticles are difficult to be encapsulated or adsorbed inside the polymer microspheres due to weak affinity, which resulted in lower encapsulation efficiency and magnetization. To overcome these problems, a novel process of in situ formation technique of magnetite combined with double emulsion method was developed in this study. In details, poly(styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA) dissolved into ethyl acetate (EA) was employed ⁎ Corresponding author. Tel./fax: +86 10 82544931. ⁎⁎ Corresponding author. Tel./fax: +86 10 82627072. E-mail addresses: [email protected] (L. Wang), [email protected] (G. Ma). 0032-5910/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.powtec.2012.12.014
as oil phase (O). Ferrous chloride (Fe2+) and ferric chloride (Fe3+) aqueous solution was used as an internal water phase (W1), which was dispersed into polymer solution to form primary emulsion of W1/O. Then, this emulsion was further dispersed into external water phase (W2) to obtain double emulsion of W1/O/W2. Finally, the alkaline solution of ammonia was added into the system, and the ammonia continuously diffused into the internal water phase (W1) to react with Fe2+ and Fe3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase of microspheres. Both formation and encapsulation of iron oxide nanoparticles were simply accomplished in a single step. Furthermore, the process avoided directly encapsulating nanoparticles into the polymer matrix [12,13]. Therefore, this novel method developed in this study is expected to significantly improve the encapsulation efficiency and magnetic properties of microspheres. Solvent for dissolving polymers is very important for improving encapsulation efficiency and magnetization of magnetic polymer microspheres. It affects not only the stability of primary emulsion, but also alkaline diffusion into internal water phase and the following yield of Fe3O4 magnetic nanoparticles. As we know, dichloromethane (DCM) is a commonly used solvent of lipophilic polymers for microsphere preparation because it is convenient to be removed by evaporation due to low boiling point [14]. However, it is time-consuming to solidify microspheres, which will lead to diffusion and leakage of the internal water phase. This will result in lower encapsulation efficiency of Fe 2+/Fe 3 + salt, which will further lead to lower yield of Fe3O4 nanoparticles in microspheres. Moreover, the larger aggregates of
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the formed Fe3O4 nanoparticles will easily leak out from microspheres, which will also lead to lower encapsulation of magnetic nanoparticles. In order to overcome disadvantages above, ethyl acetate (EA) was chosen as organic solvent in this study. EA has relatively higher solubility in water (8.7%, w/v) than DCM, which can accelerate EA to diffuse from oil phase into external water phase, and further rapidly solidify W1/O/W2 droplets into microspheres. The rapid solidification will be undoubtedly favorable for retaining the Fe3O4 nanoparticles in the internal water phase and lead to improvement of encapsulation efficiency. Although rapid solidification can improve encapsulation efficiency, the fast diffusion of EA will lead to rapid precipitation of polymer from oil phase, which will further result in formation of coagulation rather than microspheres. In order to overcome this disadvantage, we employed pre-solidification at first by pouring the double emulsion into a small volume fraction of aqueous solution. In this step, the precipitation of polymer from oil droplets was slowed down and permitted partial solidification of the droplet. Then the complete solidification was carried out by transferring this system into an excess aqueous solution. This process effectively retarded the diffusion or coalescence of internal phase to external water phase, which greatly improved encapsulation of magnetic nanoparticles and magnetic properties of magnetic polymer microspheres [15,16].
Company (China). All other regents were of reagent grade and used as received. 2.2. Preparation of magnetic PS-HEMA microspheres The schematic illustration for preparation of magnetic PS-HEMA microspheres was shown in Fig. 1. Ferrous chloride and ferric chloride were dissolved into deionized water as an internal water phase (W1). PS-HEMA was dissolved into EA containing emulsifier (Span85) as an oil phase (O). These two phases were mixed and emulsified to form primary emulsion of W1/O. Then, the W1/O emulsion was further dispersed into external water phase (W2) containing PVA and Na2SO4 to obtain double emulsion of W1/O/W2. After that, the double emulsion was transferred into a small volume fraction of external water phase for pre-solidification. Afterward, the alkaline solution of ammonia was added into the system, and it diffused into the internal water phase (W1) to react with Fe 2+ and Fe 3+. As a result, Fe3O4 magnetic nanoparticles were formed in situ in the internal water phase, and the double emulsion of W1/O/W2 was changed into S/O/W2. Finally, the S/O/W2 was poured into a large volume of external water phase (W2) to completely remove EA by solvent diffusion, and the magnetic polymer microspheres were obtained. 2.3. Measurement of primary emulsion stability
2. Materials and methods 2.1. Materials Styrene (St, reagent grade) was distilled under vacuum and stored in a refrigerator until use. Hydroxyethylmethacrylate (HEMA, reagent grade) was used without further treatment. Poly (styrene-co-2-hydroxyethyl methacrylate) (PS-HEMA, 95:5, Mw=1.69×105 kDa) was synthesized by soap-free emulsion polymerization in our lab. Poly (vinyl alcohol) (PVA-217, average degree of polymerization 1700, degree of hydrolysis 88.5%) was provided by Kuraray (Tokyo, Japan) and used as a stabilizer in the external water phase. Span85 (Sorbitantrioleat, Beijing Chemical Reagents Company, China) of biochemical grade was selected as oil emulsifier. FeCl3∙6H2O, FeCl2∙4H2O, ammonia solution (25%), and EA (ethyl acetate) of analytical grade were purchased from Beijing Chemical Reagents
Definite amounts of PS-HEMA were dissolved into 5.0 mL EA containing emulsifier of Span85 as oil phase (O). 1.0 mL ferric solution (W1) was emulsified into oil phase to form primary emulsion of W1/O. Then, the primary emulsion was poured into a test tube and kept still to observe the occurrence of phase separation. The stability was expressed by the elapsed time until the phase separation was observed. The longer elapsed time meant the better stability of primary emulsion. 2.4. Characterization of magnetic PS-HEMA microspheres 2.4.1. Morphology and microstructure of microspheres The surface morphology of magnetic polymer microspheres was observed by a scanning electron microscope (SEM) (JSM-6700F, JEOL, Japan). The specimens for SEM observation were prepared by mounting
Fig. 1. Schematic illustration of the preparation of magnetic polymer microspheres.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
2.4.2. Measurement of size and size distribution The volume-average diameter and the size distribution of the magnetic polymer microspheres were measured by a dynamic light scattering analysis (DLS) (Malvern 2000E apparatus, USA). The term “SPAN” was used to present the width of the particle size distribution, which was defined as: SPAN ¼
D90−D10 D50
The values of D10, D50 and D90 were the diameters corresponding to 10%, 50% and 90% in volume on a relative cumulative droplet size distribution curve, respectively. 2.4.3. Measurement of Fe content in microspheres and encapsulation efficiency A certain weight of magnetic polymer microspheres (W0) was put into a ceramic crucible, and then it was incinerated in a Muffle furnace under 800 °C for 6 h. The wall material of microspheres as PS-HEMA was burnt into carbon dioxide and steam, and the magnetic nanoparticles of Fe3O4 were changed into Fe2O3. The weight of residual substance (Fe2O3) was measured and expressed as W1. The molecular weight of Fe and Fe2O3 were 56 and 160 g/mol, respectively. The Fe content of magnetic polymer microspheres was calculated by the following formula: Fe contentð%Þ ¼
W1 56 2 100 W0 160
The encapsulation efficiency was obtained according to the following calculation: Encapsulation efficiencyð%Þ ¼
Total amount of Fe in microspheres 100 Total amount of Fe added
2.4.4. Magnetic properties of microspheres The magnetite type of microspheres was analyzed by X-ray diffractometer (D/max 2200 PC, Rigaku Co., Japan). The magnetization of magnetic polymer microspheres was measured by vibrating sample magnetometer (VSM) (model-155, Digital measurement System, Inc.). The maximum applied field was 8 kOe, and the measurement temperature was kept at 290 K. 3. Results and discussion 3.1. Stability of primary emulsion Stability of the primary emulsion is important for improving encapsulation efficiency of microspheres prepared from double emulsion system. The primary emulsion is usually prepared by dispersing water phase into oil phase. As usual, there exists a density difference between water and oil phase, which will lead to poor stability of
primary emulsion. The amounts of polymer and surfactant in oil phase usually have significant influence on stability of primary emulsion. 3.1.1. Effect of PS-HEMA concentration on stability PS-HEMA with different concentrations (0.7 wt.%, 1.0 wt.%, 1.3 wt.%, 1.6 wt.% and 1.9 wt.%) was used to prepare primary emulsion, and the elapsed time before phase separation was recorded. The result of Fig. 2 showed that the phase separation for primary emulsion was retarded with the increase of PS-HEMA concentrations. The primary emulsion could maintain stability for more than 2 h when PS-HEMA concentrations were 1.3 wt.% and 1.6 wt.%. This suggested that higher polymer concentrations were favorable for stability of primary emulsion. The possible reason was that the viscosity of oil phase increased with enhancement of polymer concentrations, and the oil film with higher viscosity could effectively prevent coalescence of droplets, which further led to excellent stability of primary emulsion. When PS-HEMA concentrations reached to 1.9 wt.%, it was difficult to obtain primary emulsion due to much higher viscosity of oil phase. The above results suggested that the suitable polymer concentrations for obtaining stable primary emulsion were 1.3 wt.% and 1.6 wt.%. In addition, the polymer concentrations would also affect the subsequent solidification of microspheres. The lower concentrations could slow down the precipitation of polymer from oil phase with extraction of EA and favor for formation of microspheres. Therefore, PS-HEMA concentrations of 1.3 wt.% were chosen for the further study. 3.1.2. Effect of Span85 amounts on stability Different amounts of Span85 (0.00 wt.%, 0.25 wt.%, 0.50 wt.%, 0.75 wt.% and 1.00 wt.%) were dissolved into EA to be employed as oil phase for preparation of primary emulsion, and the phase separation of primary emulsion was observed. The corresponding result was demonstrated in Fig. 3, which exhibited that the stability of primary emulsion was enhanced with increase of Span85 concentrations. When the concentrations of Span85 were more than 0.75 wt.%, the amounts of emulsifier had little influence on stability. As we know, emulsifier can form protective layer around surface of droplet, which can effectively suppress the coalescence of droplets. The emulsifier-assembled layer could not completely cover the surface of droplet when the Span85 concentrations were less than 0.75 wt.%, which led to poor stability of primary emulsion. Therefore, the amounts of Span85 in oil phase were chosen as 0.75 wt.% in the subsequent study. 3.2. Investigation of preparation conditions for PS-HEMA microspheres In double emulsion system which employed EA containing PS-HEMA as oil phase, the volume fractions of external water phase and pre-
Elapsed time before phase separation (h)
sample on metal stubs with double-sided conductive adhesive tape. Then, the samples were coated with a thin gold film (approx. 60 nm in thickness) under a reduced pressure below 5 Pa with a JFC-1600 fine coater (JEOL, Japan). Finally, the samples were put into SEM to observe the morphology of microspheres. Transmission electron microscope (TEM) (H-600, Hitachi, Japan) was used to observe the microstructure of microspheres and the distribution of magnetic nanoparticles in microspheres. The specimens for TEM observation were prepared by dropping the alcohol-diluted sample onto ultra thin carbon film supported by a copper mesh, and then the copper mesh grid was dried under light. Then, the sample was put into the sample cell for observation.
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2.5 2.0 1.5 1.0 0.5 0.0 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
PS-HEMA concentration (wt.%) Fig. 2. Effect of PS-HEMA concentrations on stability of primary emulsion.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
Elapsed time before phase separation (h)
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7 6 5 4 3 2 1 0 0.0
0.2
0.4
0.6
0.8
1.0
Concentration of Span 85 (wt.%) Fig. 3. Effect of Span85 concentrations in oil phase on stability of primary emulsion.
solidification aqueous solution have important influence on morphology of microspheres due to larger solubility of EA in water (8.7%, w/v). This will further affect encapsulation and magnetization of magnetic polymer microspheres. Therefore, the effects of the volume fractions of external water phase and pre-solidification aqueous solution on morphology, encapsulation efficiency and magnetization of microspheres have been systematically investigated in this study.
3.2.1. Effect of volume ratio between external water phase and oil phase 1.0 mL ferric salt solution as internal water phase, 5.0 mL EA containing 1.3 wt.% PS-HEMA and 0.75 wt.% Span85 as oil phase, different volumes of external water phase (20, 30, 40 and 50 mL) containing 1.0 wt.% PVA and 2.0 wt.% Na2SO4 were used to prepare magnetic microspheres, and the volume ratios between external water phase and oil phase were 4:1, 6:1, 8:1 and 10:1, respectively. The morphology of
magnetic microspheres was shown in Fig. 4, and the results of encapsulation efficiency, volume-mean size and magnetization of microspheres were represented in Table 1. Fig. 4 suggested that the volume ratios between external water phase and oil phase had significant influence on morphology of microspheres. With the increase of volume fractions of external water phase, the amounts of EA extracted into external water phase were also increased, which further affected the precipitation of polymer from oil droplets and subsequent formation of microspheres. For the system with low volume fractions of external water phase (volume ratios between oil phase and external water phase as 4:1 and 6:1), there were small amounts of EA to diffuse into external water phase during double emulsion processes. Mostly no polymer was precipitated from the oil droplets, which led to the increase of viscosity of oil droplet and further facilitated adhesion and coagulation between oil droplets. Moreover, the concentrations of emulsion were also increased with the decrease of volume fraction of external water phase, which resulted in a crowded environment and increased collision frequency between oil droplets. Both the increased viscosity of oil droplet and the crowded environment promoted the formation of coagulation as shown in Fig. 4a and b. The coagulation between oil droplets also caused leakage of internal water phase with final lower encapsulation and magnetization results. While the volume fraction of external water phase was increased (volume ratios between external water phase and oil phase as 8:1 and 10:1), large amounts of EA could diffused into external water phase, and the droplets became quasi-solid during double emulsion process. Droplets coagulation was effectively inhibited, and microspheres showed an excellent morphology. As a result, higher encapsulation efficiency and magnetization were obtained as shown in Table 1. Therefore, the higher volume fractions of external water phase were favorable for formation of microspheres with excellent morphology and higher encapsulation efficiency and magnetization. 3.2.2. Effect of volume ratio between pre-solidification aqueous solution and oil phase The magnetic polymer microspheres were prepared with various volume ratios between pre-solidification aqueous solution and oil phase as 40:1, 30:1, 20:1 and 10:1. The morphology of microspheres
a
b
c
d
Fig. 4. SEM micrographs of microspheres prepared by different volume of external water phase (a, 20 mL b, 30 mL c, 40 mL d, 50 mL).
L. Wang et al. / Powder Technology 235 (2013) 1017–1024 Table 1 The characteristics of magnetic polymer microspheres prepared by different volume of external water phase. Volume of external water phase (mL)
Encapsulation efficiency (%)
Volume-mean size of microspheres (μm)
Magnetization (emu/g)
20 30 40 50
15.61 17.96 22.35 25.78
30.2 32.4 35.8 37.5
8.42 10.0 12.2 13.8
as shown in Fig. 5 suggested that the volume ratio between presolidification aqueous solution and oil phase had important influence on the morphology of microspheres. The microspheres showed better spherical shape with a few pores on the surface under the conditions with lower volume ratios as 20:1 and 10:1. With the increase of volume fraction of pre-solidification aqueous solution as 30:1 and 40:1, microspheres appeared more and large surface pores, and especially some microspheres collapsed to form coagulations as shown in Fig. 5a and b. For the large fractions of pre-solidification aqueous solution, faster diffusion of EA led to rapid precipitation of polymer, and the deposited polymer could not shrink into the dense surface in time, resulting in the formation of lager pores. If the pores were large enough, the coalescence between internal and external water phase would occur, which further resulted in the collapse of microspheres. While in the systems with lower fractions of pre-solidification aqueous solution, the above phenomenon could be avoided to some extent due to slow extraction of EA and precipitation of polymer. Therefore, lower fractions of pre-solidification aqueous solution were much favorable for formation of microspheres with better morphological characters.
3.3. Encapsulation efficiency and magnetization of microspheres After obtaining spherical microspheres, the effects of total concentration of Fe 2+/Fe 3+ solution, PS-HEMA concentration and Na2SO4
1021
concentration in external water phase on encapsulation and magnetization microspheres were investigated thoroughly.
3.3.1. Effect of total concentrations of Fe 2+/Fe 3+ The Fe 2+ and Fe 3+ concentrations in internal water phase would cause difference of osmotic pressure between internal and external water phase, which would further influence various encapsulation efficiency and magnetization of polymer microspheres. Here, the different concentrations of Fe 2+ and Fe 3+ solutions as 0.72, 1.08, 1.44, 1.80 and 2.16 mol/L with a fixed Fe2+/Fe3+ molar ratio of 1:2 were employed to prepare magnetic microspheres, and their effects on the encapsulation and magnetization of magnetic polymer microspheres were shown in Figs. 6 and 7. Fig. 6 showed that the encapsulation efficiency declined with the increase of total concentrations of Fe2+/Fe3+. The difference of osmotic pressure between internal and external water phase increased with increase of the total concentrations of Fe 2 +/Fe 3 +. The external water phase would enter into oil droplets under the higher osmotic pressure of internal water phase, and this further resulted in gradually thinned till broken of oil film, which finally decreased the encapsulation efficiency of magnetic polymer microspheres. Comparatively, Fig. 7 showed that the magnetization increased at first, and then tend to decline with increase of the total concentrations of Fe 2+/Fe 3+. This tendency resulted from two controlling factors, the dynamic reaction rate for formation of Fe3O4 nanoparticles and the encapsulation efficiency of microspheres. When the total concentrations of Fe 2+/Fe 3+ were less than 1.8 mol/L, the magnetization of microspheres was mainly affected by reaction rate for formation of Fe3O4 nanoparticles. The yield of Fe3O4 nanoparticles increased with the increase of total concentrations of Fe2+/Fe3+, which resulted in the increase of magnetization. The higher concentrations of Fe2+/Fe3+ in internal water phase would lead to the higher osmotic pressure, which drove more alkali diffuse into internal water phase from external water phase to react with Fe 2+ and Fe3+ solutions, and resulted in the higher yield of Fe3O4 nanoparticles. However, when the concentrations of Fe 2+/Fe 3+ were over 1.8 mol/L, the encapsulation efficiency became
a
b
c
d
Fig. 5. SEM micrographs of microspheres prepared by different volume of pre-solidification solution (a, 200 mL b, 150 mL c, 100 mL d, 50 mL).
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28
34
Encapsulation efficiency (%)
Encapsulation efficiency (%)
32 30 28 26 24 22 20 18
26
24
22
16 20 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
0
2.6
2
4
6
8
10
HEMA content in the polymer (wt%)
Concentration of Fe2+/Fe3+ (moL/L) Fig. 6. Effect of total concentrations of Fe2+/Fe3+ on encapsulation efficiency of magnetic polymer microspheres.
Fig. 8. Effect of HEMA content in polymer on encapsulation efficiency of magnetic nanoparticles.
the main controlling factor. The rapid decrease of encapsulation efficiency of microspheres caused the decrease of magnetization.
layer and emulsion, and eventually led to lower encapsulation efficiency. Therefore, the optimum content of HEMA in polymer was 2.0 wt.%.
3.3.2. Effect of HEMA content in PS-HEMA The PS-HEMA with different HEMA amounts of 0.0, 2.0, 5.0, 8.0 and 10.0 wt.% was employed to prepare magnetic microspheres, and their effect on encapsulation efficiency was investigated. The corresponding result as shown in Fig. 8 demonstrated that suitable amounts of HEMA in polymer were necessary for the improvement of encapsulation efficiency of magnetic nanoparticles. The encapsulation efficiency reached the highest of 26.1% when HEMA content in PS-HEMA was 2.0 wt.%. The hydrophilic hydroxyl group on PS-HEMA could promote HEMA units to distribute interface between internal water phase and oil phase, which preferred to form chelates with Fe2+/Fe3+ ion and favored for increase of encapsulation efficiency. Furthermore, PS-HEMA was amphiphilic, and played a role of emulsifier during formation of W/O and W/O/W. This was helpful for the stability improvement of primary and double emulsion, which was also favorable for increase of encapsulation efficiency. Therefore, the encapsulation efficiency was raised with the increase of HEMA content in polymer when HEMA was less than 2.0 wt.%. When the content of HEMA increased to more than 2.0 wt.%, steric repulsion of HEMA became dominant due to limited space of internal water phase. This would greatly reduce the stability of polymer
3.3.3. Effect of salt concentrations in outer water phase The salt of Na2SO4 was added into external water phase to adjust osmotic pressure between internal and external water phase. Different salt concentrations of 0.5, 1.0, 1.5 and 2.0 wt.% were used to prepare magnetic microspheres, and the corresponding encapsulation efficiency was measured as shown in Fig. 9. The result suggested that the encapsulation efficiency was raised with the increase of salt concentrations. In this system, internal water phase had higher ionic strength due to addition of Fe2+/Fe3+. When concentrations of Na2SO4 were low, the ionic strength of external water phase was lower than that of internal water phase. A part of external water phase entered into internal water phase by virtue of osmotic pressure, which led to volume increase of internal water phase. This further resulted in coalescence and leakage of internal water phase. On the contrary, the ionic strength of water phase was raised by increasing Na2SO4 concentrations to achieve higher osmotic pressure of external water phase, which effectively inhibited diffusion of external water phase into internal water phase and improved the stability of double emulsion. This was promising for the subsequent improvement of encapsulation efficiency. The Na2SO4 concentration was chosen as 2.0 wt.%, because
13 26
Encapsulation efficiency (%)
Magnetization (emu/g)
12 11 10 9 8 7
25 24 23 22 21 20 19
6 0.6
0.8
1.0
1.2
1.4
Concentration of
1.6
1.8
Fe2+/Fe3+
2.0
2.2
2.4
2.6
(moL/L)
Fig. 7. Effect of total concentrations of Fe2+/Fe3+ on magnetization of magnetic polymer microspheres.
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Concentration of Na2SO4 (wt%) Fig. 9. Effect of Na2SO4 concentrations in external water phase on the encapsulation efficiency of magnetic nanoparticles.
L. Wang et al. / Powder Technology 235 (2013) 1017–1024
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50
Intensity (cps)
40 30 20 10 0 0
20
30
40
50
60
70
80
90
Fig. 12. XRD of magnetic polymer microspheres prepared under optimal conditions.
10 µm Fig. 10. TEM microphotographs of magnetic polymer microspheres prepared under optimal conditions.
PVA flocculation appeared if the concentrations were higher than 2.0 wt.%. 3.4. Magnetic PS-HEMA microspheres under optimal preparation conditions Under the optimized conditions, the magnetic polymer microsphere was successfully prepared. The Fe 2 +/Fe 3 + aqueous solution with concentration of 1.8 mol/L was internal water phase (W1), the EA containing 1.3 wt.% PS-HEMA with 2.0 wt.% HEMA and 0.75 wt.% Span85 was oil phase (O), the aqueous solution containing 1.0 wt.% PVA and 2.0 wt.% Na2SO4 was external water phase (W2), and the volume ratio among internal water phase, oil phase and external water phase was fixed at 1:5:10. After preparation of double emulsion, it was added into pre-solidification aqueous solution to partially solidify the oil droplets, and the volume ratio between pre-solidification aqueous solution and oil phase was 20:1. Then, the ammonia was dropped into the system for in situ magnetization of Fe2+/Fe3+, and the volume ratio between solidification aqueous solution and oil phase was fixed at 160:1. Fig. 10 demonstrated a TEM image of obtained magnetic microspheres, in which magnetite nanoparticles appeared darker and welldispersed in the whole matrix of microspheres. This meant that the in situ formation of magnetic nanoparticle was homogeneous, and there
15 10
Magnetization (emu/g)
10
5 0 -5 -10
was no agglomeration of the magnetite nanoparticles. A magnetization curve of the microspheres in Fig. 11 showed that the magnetic microspheres were super-paramagnetic, and no hysteresis was found, and the magnetization reached to 12.2 emu/g, which was much higher than those of microspheres prepared by the conventional methods. The crystalline structures of nanoparticles in microspheres were further characterized by X-ray diffraction (XRD) patterns. As shown in Fig. 12, the patterns of in situ formed nanoparticles could be easily indexed to standard Fe3O4 crystal with its six diffraction peaks: {2 2 0}, {3 1 1}, {4 0 0}, {4 2 2}, {5 1 1} and {4 4 0}, and no other peaks of impurities were detected. Therefore, we concluded that the nanoparticles dispersed in the PS-HEMA microspheres matrix were mainly composed of super-paramagnetic Fe3O4 nanoparticles.
4. Conclusion The magnetic PS-HEMA microspheres were successfully fabricated by in situ magnetization techniques in double emulsion system. We first prepared W1/O/W2 emulsion with Fe 2 +/Fe 3 + solutions as internal water phase (W1), EA dissolved with polymer of PS-HEMA and surfactant of Span85 as oil phase (O), and deionized water dissolved with PVA-217 and Na2SO4 as external water phase (W2). After presolidification, the in situ co-precipitation reaction of Fe 2+ and Fe3+ into magnetic nanoparticles was performed in the internal water phase by diffusing of alkali solution from external to internal water phase. Finally, the obtained S/O/W2 droplet was further solidified into microspheres by further solvent diffusion. In this novel preparation process, the critical factors affecting the structure and magnetic properties of final microspheres included PS-HEMA concentration, HEMA content in PS-HEMA, the type of emulsifier in oil phase, concentrations of Fe2+/Fe3+ solutions in internal water phase, concentrations of Na2SO4 added in external water phase, volume fractions of external water phase and pre-solidification aqueous solution. The final microspheres with better morphological characters and higher magnetization of 12.2 emu/g were obtained under the optimal conditions. The magnetization of microspheres prepared by this novel process was much higher than that of microspheres prepared by conventional methods. This technique can also be utilized in preparation of other inorganic-polymer composite microspheres when the inorganic particles can be synthesized by chemical co-precipitation reaction. Therefore, this is a potential method for preparation of polymer microspheres with higher loading efficiency of inorganic nanoparticles.
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Applied magnetic field (KOe) Fig. 11. Magnetization curves of magnetic polymer microspheres prepared under optimal conditions.
Acknowledgement The authors are thankful to Natural Science Foundation of China for financial support to this work (grant no. 20376082, no. 20820102036).
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