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Monodisperse macroporous poly(glycidyl methacrylate) microspheres coated with silica: Design, preparation and characterization
Reactive & Functional Polymers 77 (2014) 11–17
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Monodisperse macroporous poly(glycidyl methacrylate) microspheres coated with silica: Design, preparation and characterization Silvia Grama, Zdeneˇk Plichta, Miroslava Trchová, Jana Kovárˇová, Milan Beneš, Daniel Horák ⇑ Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2, 162 06 Prague 6, Czech Republic
a r t i c l e
i n f o
Article history: Received 2 December 2013 Received in revised form 29 January 2014 Accepted 30 January 2014 Available online 8 February 2014 Keywords: Multistep swelling polymerization Poly(glycidyl methacrylate) Poly(2,3-dihydroxypropyl methacrylate) Monodisperse Macroporous Microspheres Silanization
a b s t r a c t Monosized macroporous poly(glycidyl methacrylate) (PGMA) microspheres that were 9.3 lm in size were synthesized by multistep swelling polymerization using a modified Ugelstad technique. The PGMA microspheres and their hydrolyzed analogs derived from poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) were coated by silanization with tetraethoxysilane (TEOS) and (3-aminopropyl)triethoxysilane (APTES), respectively. The particles were characterized by elemental and thermogravimetric (TGA) analysis, scanning and transmission electron microscopy (SEM and TEM) coupled with an energy dispersive X-ray analysis (EDAX) and FT-IR spectroscopy to determine the SiO2 content, morphology, particle size, polydispersity and structure. These types of particles are expected to have improved biocompatibility relative to their starting polymers. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Many techniques can be used for the preparation of microspheres. The first polymeric particles were produced at the beginning of the 20th century by suspension polymerization [1]. This technique yields large beads possessing diameters that span the low micrometer to millimeter range and that have a broad particle size distribution. Suspension polymerization is used for the commercial manufacture of many important polymers, including poly(vinyl chloride), poly(methyl methacrylate), expandable polystyrene and styrene-acrylonitrile copolymers, which are used as ion-exchangers, sorbents or adsorbents for water purification [2–4]. Precipitation and dispersion polymerization produces particles with diameters from 1 to 8 lm. The precipitation process gives larger and less regular particles than the dispersion process [5–8]. Current efforts have focused on the synthesis of monodisperse polymer particles. This synthesis along with the ability to control particle surface chemistry has led to important developments in clinical diagnostics [9,10], drug delivery [11], cell separation [12,13] and chemotherapy [14,15]. The most common production method for small-sized particles (100–600 nm) is emulsion polymerization. The technique is frequently used for production of polystyrene colloids [16,17]. Through the multistep swelling ⇑ Corresponding author. Tel.: +420 296 809 260; fax: +420 296 809 410. E-mail address: [email protected] (D. Horák). http://dx.doi.org/10.1016/j.reactfunctpolym.2014.01.010 1381-5148/Ó 2014 Elsevier Ltd. All rights reserved.
of seeds and polymerization, monodisperse microspheres are obtained [18,19]. The key issue in the design of particles for biomedical applications involves coating them with a proper shell. The importance of an appropriate shell is exemplified by the silanization of metal oxide or polymer particles to produce organic/inorganic hybrid materials that feature both biocompatibility and functionality, e.g., amino groups [20–25]. Silica can also coat metal nanoparticles, such as gold [26,27], glass and natural or synthetic polymeric particles [28–31]. Typically, silica has been applied to polystyrene [32,33] or silanization has been performed on cellulose microspheres [34] or fibers [35]. Only one report addresses silica microspheres that were obtained by the calcination of ethylenediamine-functionalized PGMA microspheres. The silica had, however, a porous or hollow structure [36]. Silanization eliminates undesirable nonspecific interactions and enables the functionalization required for biological applications. The introduced functional groups are then available for the attachment of proteins, peptides and antibodies. Silica particles are commonly synthesized by the sol–gel technique that was introduced by Stöber and Fink [37]. Primarily, this method is used for the preparation of silica nanoparticles that are < 100 nm in size. The process involves the hydrolysis of silica alkoxide precursors, such as tetraethoxysilane (TEOS), in a mixture of ethanol and aqueous ammonium hydroxide. Silicic acid is produced during hydrolysis, and when its concentration exceeds its solubility in ethanol, it nucleates homogeneously to form
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submicrometer silica particles [38]. These particles can then be functionalized with different silanization agents, usually (3-aminopropyl)triethoxysilane (APTES) [39]. This functionalization can be performed in the same step with the silanization or in a two-step process [40–42]. The silica shell on the particles facilitates a wide variety of surface reactions and allows for conjugation with biomolecules, such as proteins and DNA [13]. Silica nanoparticles are easy to prepare, nontoxic and amenable to simple surface modifications; thus, they have been widely studied and used in various applications, such as column chromatography solid support materials [43], insulating layers and silica– polymer composites in engineering and bio-imaging and drug/ gene delivery systems in nano-biotechnology [44]. Herein, we report the preparation and characterization of monodisperse macroporous poly(glycidyl methacrylate) (PGMA) and poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) microspheres and their subsequent coating with a silica shell. The PGMA microspheres were synthesized using a modified Ugelstad technique [45] based on the multistep swelling of polystyrene (PS) seeds. The PS particles were obtained by emulsifier-free emulsion polymerization. Compared to particles with broad size distributions that are subject to size classification and low product yields, monodisperse microspheres offer enhanced performance due to their uniform physical, chemical and biological properties. As far as we know, this is the first report of the direct silanization of monodisperse PGMA or PDHPMA microspheres. The well-known hydrophobicity of PGMA and polystyrene microspheres makes them highly bioincompatible; thus, the hydrophilicity imparted through silanization will improve their in vivo performance and enhance the wettability of PGMA.
(ii) A dispersion of PS latex (0.6 g corresponding to 0.3 g dry weight) in a 0.25 wt% SDS aqueous solution (5 mL) was swollen for 50 h with a dispersion of DBP (3 g) in a 0.25 wt% SDS solution (20 mL) prepared by 5-min sonication using a W-385 sonicator (Heat System – Ultrasonics; Farmingdale, NY, USA). Swelling with the DBP dispersion was performed three additional times with DBP, increasing to 4 and 5 g in the last two swellings. The final solvent-swollen PS dispersion contained 15 g of DBP. (iii) DBP-swollen PS seeds were gradually swollen with a dispersion of monomers and an initiator (192 g of GMA, 128 g of EDMA and 1.6 g of AIBN) as well as a dispersion of porogens (8 g of dodecan-1-ol and 457 g of cyclohexanol) in a 0.1 wt% SDS solution (1.65 L) for 5 h. (iv) The dispersion of monomer/porogen-swollen PS seeds in 0.1 wt% SDS solution was then transferred to a 4-L reaction vessel and a 3 wt% PVA aqueous solution (240 mL) and 6 wt% HEC aqueous solution (240 mL) were added. Polymerization proceeded at 70 °C for 16 h under a CO2 atmosphere. The macroporous PGMA microspheres were washed successively with water, methanol, toluene and methanol (three times each with 1 L) and then air-dried. Optionally, PGMA microspheres (1 g) were hydrolyzed with 0.1 M H2SO4 (30 mL) at 60 °C for 5 h under mechanical stirring. The resulting poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) microspheres were separated by centrifugation and washed 10 times with water until the effluent was neutral and vacuum-dried. 2.3. Modification of PDHPMA and PGMA microspheres with TEOS and APTES
2. Experimental 2.1. Materials Monomers, such as styrene (Synthos, Kralupy, Czech Republic), glycidyl methacrylate (GMA; Fluka; Buchs, Switzerland) and ethylene dimethacrylate (EDMA; Ugilor, France), were vacuum-distilled before use. 2,20 -Azobisisobutyronitrile (AIBN), dibutyl phthalate (DBP), sodium dodecyl sulfate (SDS) and (3-aminopropyl)triethoxysilane (APTES) were obtained from Sigma–Aldrich (St. Louis, USA). Tetraethoxysilane (TEOS) and 2-hydroxyethyl cellulose (HEC) were purchased from Fluka. Lithium persulfate, lithium hydrogen carbonate, ammonium hydroxide, sodium hydroxide and sulfuric acid were purchased from Lach-Ner (Neratovice, Czech Republic). Poly (vinyl alcohol) (PVA) was purchased from Wacker (Burghausen, Germany). Ultrapure Q-water ultra-filtered on a Milli-Q Gradient A 10 system (Millipore, Molsheim, France) was used throughout this work. All other solvents were from Lach-Ner (Neratovice, Czech Republic). 2.2. Preparation of monodisperse macroporous PGMA microspheres by multistep swelling polymerization The method consists of four steps: (i) Polystyrene (PS) latex (750 nm particles) was obtained by emulsifier-free emulsion polymerization. A 150-mL reaction vessel equipped with an anchor-type stirrer (300 rpm) was charged with styrene (10 g) and water (90 mL) containing 0.185 mM lithium persulfate (initiator) and 0.37 mM lithium hydrogen carbonate to adjust the pH to 7.5. Polymerization proceeded at 70 °C for 20 h under nitrogen atmosphere. The resulting PS latex was repeatedly washed with water.
PDHPMA microspheres (0.2 g) were dispersed in 2 mL of ethanol/water (1/1 v/v) and 0.1 M NaOH aqueous solution (0.2 mL) was added under mechanical stirring until pH 11 was reached. A solution of TEOS (0.2 mL) in ethanol (1 mL) was then added, and the reaction proceeded at 70 °C for 5 h. The resulting PDHPMA/ SiO2 microspheres were separated by centrifugation (4000 rpm), three times washed with ethanol and water, separated by centrifugation and vacuum-dried at 50 °C. Two approaches were employed for the coating of PGMA microspheres with APTES. In the one-step procedure, PGMA microspheres (0.2 g) were dispersed in methanol (2 mL), APTES (0.2 mL) in methanol (1 mL) was added under mechanical stirring and the reaction proceeded at 60 °C for 8 h. The resulting PGMA/ SiO2 microspheres were washed with methanol, separated by centrifugation and vacuum-dried at 50 °C. In the two-step procedure, PGMA microspheres were first coated with APTES in toluene and then hydrolyzed in water. PGMA microspheres (0.2 g) were dispersed in toluene (1.5 mL), and APTES (0.2 mL) in toluene (0.5 mL) was added under mechanical stirring and the reaction proceeded at 50 °C for 5 h. The particles were washed with toluene, methanol and water to remove unreacted reagents and were separated by centrifugation. The second step included the hydrolysis and condensation of alkoxides in water (2 mL) to yield silica. An ammonium hydroxide solution was added until pH 11 was reached. The reaction proceeded under mechanical stirring at 50 °C for 3 h. PGMA/SiO2 particles were separated by centrifugation, washed with water and vacuum-dried at 60 °C. 2.4. Characterization of microspheres The microspheres were observed with an Opton III light microscope (Oberkochen, Germany), a 200S Quanta scanning electron microscope (SEM; FEI, Brno, Czech Republic) equipped with an
S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
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energy dispersive X-ray analysis (EDAX) and a Tecnai Spirit G2 transmission electron microscope (TEM; FEI, Brno, Czech Republic). The particle size distribution was determined by the Atlas program (Tescan; Brno, Czech Republic), counting at least 500 particles for each experiment. The number-average diameter (Dn), weight-average (Dw) diameter and polydispersity index (PDI) were calculated by Dn = RDi/N, Dw = RD4i /RD3i and PDI = Dw/Dn, where Di is the diameter of ith particle and N is the total number of particles counted. Fourier-transform infrared (FT-IR) spectra were recorded on a Thermo Nicolet Nexus 870 FT-IR spectrometer (Madison, WI, USA) in a water-purged environment with a DTGS (deuterated triglycine sulfate) detector. The Golden GateTM single reflection ATR system (Specac Ltd., Orpington, UK) was used to measure the ATR spectra of powdered samples over a wavenumber range of 400–4000 cm1. The typical parameters were: 256 sample scans and resolution of 4 cm1, with Happ-Genzel apodization and a KBr beamsplitter. An elemental analysis was carried out on a Perkin–Elmer 2400 CHN apparatus (Norwalk, CT, USA). Thermogravimetric analyses (TGA) were performed in air (50 mL/min.) using a Perkin Elmer Pyris 1 Thermogravimetric Analyzer over a temperature interval of 30–850 °C at a heating rate of 10 °C min1. The specific surface area (SBET) of the microspheres was determined by nitrogen adsorption in liquid nitrogen (77 K) using a Gemini VII 2390 Analyzer (Micromeritics; Norcross, GA, USA).
crosslinking agent to make the particles insoluble in solvents and to introduce porosity in the microspheres. If a porogen is present in the reaction mixture in sufficient amounts, phase separation between the polymer and the porogen is ensured. An analogous porogen system was used for the preparation of chromatographic packings [46], namely, a mixture of a small amount (2 wt%) of dodecan-1-ol (a thermodynamically poor solvent of GMA) and cyclohexanol (a thermodynamically good solvent of GMA) at a conventional monomer/porogen ratio = 2/3 w/w. The advantage of porous particles is that magnetic compounds can be prospectively precipitated inside the pores, allowing for the easy manipulation of the microspheres by a magnet. Moreover, macroporosity renders the particles with increased sorption capacity to be available for interacting various target compounds, a feature beneficial in size-exclusion chromatographic applications. The oxirane group allows for a variety of subsequent modification reactions, leading to the introduction of carboxyl, amino, aldehyde and phosphonate groups. The multistep swelling polymerization method thus produced macroporous PGMA particles of regular spherical shape. Optionally, PGMA particles were hydrolyzed to yield PDHPMA microspheres. The microspheres were 9.3 lm in size and had a low polydispersity index (PDI = 1.002), documenting the monodispersity of the particles (Fig. 1a). Both TEOS and APTES were then investigated as agents for the silanization of poly(glycidyl methacrylate)-based microspheres.
3. Results and discussion
3.2. Modification of PDHPMA microspheres with TEOS
3.1. Preparation of starting monodisperse macroporous PGMA microspheres
Preliminary experiments showed that the reaction of PGMA microspheres with TEOS in an ammonia solution was inferior to that of their PDHPMA counterparts because it resulted in only amino-functionalized particles. Almost no silanization occurred, presumably because of side reactions. Therefore, PDHPMA microspheres were preferred for the reaction with TEOS in ethanol/ water. While all other reaction parameters were kept constant, the TEOS/DHPMA ratio was varied up to 1 w/w (Table 1). The morphology, size and polydispersity of PDHPMA/SiO2 microspheres
Macroporous monodisperse PGMA microspheres were obtained by a modified Ugelstad technique. The method is based on the multistep swelling of PS seeds with a hydrophobic agent (DBP) and then with a mixture of monomers and porogens, followed by conventional PVA- and HEC-stabilized suspension polymerization. The microspheres contained 60 wt% GMA and 40 wt% EDMA as a
Fig. 1. SEM (a, b, d, e) and TEM (c and f) micrographs of PDHPMA microspheres No. 1 (a–c) and PDHPMA/SiO2 microspheres No. 4 (d–f). For the specifications of the microspheres No. 1 and 4, see Table 1.
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Table 1 Properties of PDHPMA/SiO2 microspheres. No.
Solvent
TEOS/DHPMA (w/w)
Dna (lm)
PDIb
Elemental analysis (wt%) C
1g 2 3 4
– EtOH/H2O EtOH/H2O EtOH/H2O
– 0.25 0.5 1
9.3 9.3 9.4 9.5
1.002 1.005 1.003 1.003
c
55.3 54.4 54.0 53.6
SBETf (m2/g)
SiO2 from (wt%) c
Ash
C
0 1.1 1.8 2.1
0 1.6 2.4 3.1
TGA
d
EDAX
0 1.8 2.7 5.7
e
0 0 0.26 0.60
77.7 62.6 62.1 71.2
Ethanol/water = 1/1 v/v. a Number-average diameter. b Polydispersity index. c Carbon. d Thermogravimetric analysis. e Energy dispersive X-ray analysis. f Specific surface area. g Neat PDHPMA microspheres.
were observed by scanning and transmission electron microscopy (Fig. 1). It was obvious from the micrographs that while the size of PDHPMA/SiO2 No. 4 (Table 1) slightly increased (Dn = 9.5 lm; Fig. 1 d) compared with the starting PGMA microsphere No. 1 (Dn = 9.3 lm; Fig. 1 a), the polydispersity did not change. By increasing the TEOS/DHPMA ratio from 1 to 2, silica was partially formed outside the particles as well. To avoid a secondary nucleation, i.e., the formation of free silica outside the particles, the TEOS/DHPMA ratio was decreased from 1 to 0.5 and 0.25 w/w; this also resulted in slightly smaller PDHPMA/SiO2 microspheres (Table 1). An EDAX analysis determined the content of silica on the surface of the PDHPMA/SiO2 microsphere No. 4 to be 0.6 wt% (Table 1). Silica shell thickness was found to be sensitive to the amount of TEOS. With a decreasing TEOS/DHPMA ratio, the silica content was reduced. As expected, the amount of silica according to EDAX analysis was rather low because only the surface layers were measurable. By contrast, TEM images (Fig. 1c and f) revealed that the silanization smoothed the outer surface of the microspheres and that silica was also formed inside the pores of the microspheres. These results were confirmed by elemental analysis because the carbon content of the PDHPMA/SiO2 microspheres decreased compared with the starting PDHPMA particles (Table 1). Incombustible residues corresponding to silica were present in only PDHPMA/SiO2 microspheres. Both of these facts were in agreement with FT-IR spectroscopy, indicating the presence of silica both on the surface and inside the PDHPMA microspheres. Next, the thermal decomposition of the PDHPMA/SiO2 microspheres was investigated by TGA (Table 1, Fig. 2). The TGA curve
of the neat PDHPMA particles showed an approximately 3.9 wt% weight loss below 185 °C due to the evaporation of solvents. Between 200 and 600 °C, weight losses were more pronounced, and 96.1 wt% of the material was finally lost due to full decomposition of the polymer. In contrast, after heating the PDHPMA/SiO2 microspheres up to 600 °C, there were residual compounds present, the amounts of which ranged from 1.7 to 5.7 wt% depending on the DHPMA/TEOS (w/w) ratio. Moreover, silica coating also increased the thermo-oxidative stability of PDHPMA/SiO2 microspheres by 60 °C (Fig. 2). There was reasonable agreement in the analysis of silica using TGA and determination of ash. To further confirm the presence of silica on PDHPMA microspheres, FT-IR spectra were recorded (Fig. 3). The spectrum of PDHPMA/SiO2 microspheres (Table 1, No. 4 and Fig. 3, spectrum b) differed from the spectrum of the initial PDHPMA microspheres (Table 1, No. 1 and Fig. 3, spectrum a). These differences are easily detected in the corresponding differential spectrum (b) – (a) in Fig. 3, spectrum c. The enhanced absorption near 1110 cm1 in the spectrum of PDHPMA/SiO2 microspheres corresponded to the Si–O–Si asymmetric stretching vibrations. The increased absorption of the band at 3432 cm1 was due to the enhanced content of Si–OH groups. Moreover, the intensity of the peaks associated with the diol groups at 1253, 1050, 974, 852 and 752 cm1 decreased due to the presence of the silica on the surface of the microspheres. The differential spectrum (Fig. 3, spectrum c) also revealed a decrease of the carbonyl peak at 1720 cm1. This was in agreement with the TEM results regarding the formation of silica inside the pores of the microspheres.
100
Weight loss (%)
80
60
40
1150 1720 1050 1253
Absorbance (a.u.)
1 2 3 4
3432 2948 a
b
20
c
4000 0
852 974 752
100
200
300
400
500
600
Temperature (°C) Fig. 2. TGA of PDHPMA and PDHPMA/SiO2 microspheres No. 1–4 (Table 1).
3500
3000
2500
2000
1500
1000
Wavenumber (cm-1) Fig. 3. FT-IR spectra of (a) PDHPMA No. 1, (b) PDHPMA/SiO2 microspheres No. 4 and (c) differential spectrum (b) – (a). For the specifications of the microspheres No. 1 and 4, see Table 1.
S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
To establish the effect of the silica that formed on the porous properties of the PDHPMA/SiO2 microspheres, the specific surface area was measured (Table 1). The recorded isotherms were of type II. The specific surface area of the PDHPMA/SiO2 microspheres (62 m2/g) was smaller than that of the PDHPMA microspheres (75.6 m2/g). This is consistent with the results from electron microscopy and confirms the formation of silica on the surface of and inside the microspheres. 3.3. Modification of PGMA microspheres with APTES Modification of PGMA microspheres by APTES was also investigated. Reactions were performed in methanol or toluene, and the opening of the oxirane groups with APTES was followed by the hydrolysis/condensation of alkoxides under alkaline conditions. The properties of PGMA/SiO2 particles were superior to those of PDHPMA/SiO2 microspheres because the formation of undesirable silica outside of the particles was minimal. The morphology, size and polydispersity of PGMA/SiO2 microspheres were again determined from scanning and transmission electron micrographs (Fig. 4d–h). As with PDHPMA/SiO2 microspheres, silanization did not broaden the particle size distribution (PDI = 1.003). On the other hand, the PGMA/SiO2 particles were larger than the PDHPMA/SiO2 particles. The diameter increased from 9.3 to
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9.8 lm, with the APTES/GMA ratio increasing from 0 to 4 w/w (Table 2). The content of silica determined by a single particle EDAX measurement was higher for PGMA/SiO2 particles than for PDHPMA/SiO2 particles, as exemplified by the 3.2 wt% silica observed in No. 6 (Table 2). When silanization was carried out in methanol, the amount of silica on the particles increased from 3.2 to 4 wt% as the amount of APTES used in the reaction increased. A similar behavior was observed for the particles silanized in toluene and hydrolyzed in water (Table 2). Compared with silanizations using TEOS, silanization with APTES produced silica mainly on the surfaces of the microspheres, with only small amounts precipitated inside the pores, as evidenced from TEM images (Fig. 4c and f). This was due to the oxirane ring opening induced by the amine groups. Additionally, elemental analysis confirmed the formation of a silica shell around the polymer particles. As expected, the content of carbon and nitrogen in PGMA/SiO2 microspheres decreased and increased, respectively, with increasing amounts of APTES in the reaction mixture (Table 2). The presence of ash after the analysis also indicated a successful silica coating on the PGMA microspheres (Table 2). TGA of PGMA/SiO2 microspheres was in agreement with the elemental analysis results (Table 2 and Figs. 5 and 7). The TGA curve of the PGMA particles No. 5 (Fig. 5) showed 1.1 wt% weight loss below 230 °C due to the evaporation of
Fig. 4. SEM (a, b, d, e, g, h) micrographs of PGMA No. 5 (a and b), PGMA/SiO2 No. 7 (d and e) and No. 11 microspheres (g and h). TEM micrographs of the cross-section of a PGMA No. 5 (c) and PGMA/SiO2 microsphere No. 7 (f). For the specifications of the microspheres No. 5, 7 and 11, see Table 2.
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Table 2 Properties of PGMA/SiO2 microspheres. No.
Solvent
APTES/GMA (w/w)
Dna (lm)
PDIb
Elemental analysis (wt%) C
5h 6 7 8 9 10 11 12 13 a b c d e f g h
– MeOH MeOH MeOH MeOH Toluene/water Toluene/water Toluene/water Toluene/water
0 1 2 4 10 1 2 4 10
9.3 9.6 9.8 9.8 9.9 9.7 9.8 9.8 9.6
1.004 1.003 1.003 1.003 1.002 1.004 1.003 1.004 1.002
c
N
58.5 55.4 54.4 53.4 55.3 55.2 54.4 54.0 56.1
d
0 1.1 1.3 1.5 1.6 1.0 1.4 1.4 1.4
SBETg (m2/g)
SiO2 from (wt%) c
e
Ash
C
0 4.1 5.5 5.1 2 4.4 4.8 5.8 3.8
0 5.3 7.0 8.7 5.5 5.6 7.0 7.7 4.1
TGA
EDAX
0 5.2 6.2 7.7 6.5 3.5 6.7 7.2 5.6
0 3.2 2.8 3.4 4.0 1.5 2.8 3 1.9
f
85.6 99.6
98.3
Number-average diameter. Polydispersity index. Carbon. Nitrogen. Thermogravimetric analysis. Energy dispersive X-ray analysis. Specific surface area. Neat PGMA microspheres.
100 5 6 7 8 9
Absorbance (a.u.)
Weight loss (%)
80
1723
60
40
1143
1254 3435
1450
2945
1072 846 907 758
a
b
c
20 4000 100
200
300
400
500
600
700
Temperature (°C) Fig. 5. TGA of PGMA and PGMA/SiO2 microspheres No. 5–9 (Table 2).
solvents. Starting at 250 °C, a quick decomposition of organic components was observed and their full decomposition was accomplished at 550 °C. A small amount of silica remained at 750 °C. This amount increased from 5.3 to 7.8 wt% depending on the APTES/GMA ratio. It is interesting to note that the formation of a silica shell on the microspheres reduced the rate of PGMA thermo-oxidative degradation, most likely because the shell limited the amount of air accessible to the organic portion of the investigated samples. To further confirm the presence of a silica shell on the PGMA microspheres (Table 1, No. 4), the FT-IR spectra were analyzed (Fig. 6). The spectrum of PGMA (Table 1, No. 4 and Fig. 6, spectrum a) was compared with the spectrum of PGMA/SiO2 (Table 2, No. 11 and Fig. 6, spectrum b) and the corresponding differential spectrum (b) – (a) was analyzed (Fig. 6, spectrum c). The intensities of the PGMA oxirane-related peaks at 758, 846 and 907 cm1 decreased due to the ring opening induced by the amine group of APTES. The presence of Si–O–Si chains on the surface of the PGMA/SiO2 microspheres was confirmed by the appearance of a shoulder at 1072 cm1 on the band near 1143 cm1, which is a characteristic peak in the spectrum of the starting PGMA microspheres. The presence of an additional small band with a maximum near 3435 cm1 was attributed to silica–OH vibrations, confirming the presence of the Si–OH bonds. Additionally, the
3000
2500
2000
Wave number
800
1500
1000
(cm-1)
Fig. 6. FT-IR spectra of (a) PGMA No. 5, (b) PGMA/SiO2 No. 11 microspheres and (c) differential spectrum (b) – (a). For the specifications of the microspheres No. 5 and 11, see Table 2.
100 5 10 11 12 13
80
Weight (%)
0
3500
60
40
20
0
100
200
300
400
500
600
700
800
900
Temperature (°C) Fig. 7. TGA of PGMA and PGMA/SiO2 microspheres No. 5 and 10–13 (Table 2).
intensity of the carbonyl peak at 1723 cm1 decreased, suggesting the formation of silica inside the pores of the polymer bulk. The porous properties of the PGMA/SiO2 microspheres were also investigated. Due to the formation of mesoporosity, the
S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
specific surface area (SBET) of the PGMA/SiO2 microspheres increased by up to 99.6 m2/g from the SBET of 85.6 m2/g that was observed for the starting PGMA microspheres No. 5 (Table 2). This result also confirmed the formation of a silica shell around the microspheres. 4. Conclusions Monodisperse macroporous PGMA particles were obtained using a modified Ugelstad method. For first time, both the surface and pores of the microspheres were covered with silica formed by a modified Stöber procedure. The silanization of macroporous PGMA or PDHPMA particles was achieved by the hydrolysis and condensation of TEOS and APTES agents. The formation of the silica shell was confirmed by various characterization methods. As the amount of the silanization agent in the reaction mixture increased, an increase in both the microsphere diameter and the amount of the silica present was observed on SEM micrographs coupled with EDAX analysis. The PDI did not change after the silanization of the microspheres. Similar results, i.e., a higher content of silica with increasing amounts of silanization agents, were obtained from an elemental analysis and TGA measurements of PGMA/SiO2 and PDHPMA/SiO2 particles. FT-IR spectra showed a decrease in the intensity of the carbonyl band at 1723 cm1 and TEM images also showed that silica was formed inside the pores of the PGMA and PDHPMA microspheres. In the PDHPMA/SiO2 microspheres, the amount of silica was relatively small, resulting in a thin shell on the particle surface; the majority of the silica was inside the pores of the PDHPMA/SiO2 microspheres. In contrast, the use of APTES as a silanization agent provided a thicker shell on the microspheres and less silica was precipitated inside of the pores, thus, substantially increasing the PGMA/SiO2 microsphere diameter. Silica had a tendency to smooth the surface of the PDHPMA/SiO2 microspheres. In contrast, the silica formed on the PGMA surface increased the porosity of the microspheres. Due to their hydrophilic nature and potential for facile modification, the prepared silanized microspheres are suited for biomedical applications, such as drug transport, bioaffinity chromatography and immobilization of antibodies. The potential range of silanized microsphere applications can be extended if iron oxide is precipitated inside the pores of the microspheres. Acknowledgement This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grant No. EE2.3.30.0029). References [1] E. Vivaldo-Lima, P.E. Wood, A.E. Hamielec, A. Penlidis, Ind. Eng. Chem. Res. 36 (1997) 939–965. [2] B.W. Brooks, Chem. Eng. Technol. 33 (2010) 1737–1744.
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Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react
Monodisperse macroporous poly(glycidyl methacrylate) microspheres coated with silica: Design, preparation and characterization Silvia Grama, Zdeneˇk Plichta, Miroslava Trchová, Jana Kovárˇová, Milan Beneš, Daniel Horák ⇑ Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2, 162 06 Prague 6, Czech Republic
a r t i c l e
i n f o
Article history: Received 2 December 2013 Received in revised form 29 January 2014 Accepted 30 January 2014 Available online 8 February 2014 Keywords: Multistep swelling polymerization Poly(glycidyl methacrylate) Poly(2,3-dihydroxypropyl methacrylate) Monodisperse Macroporous Microspheres Silanization
a b s t r a c t Monosized macroporous poly(glycidyl methacrylate) (PGMA) microspheres that were 9.3 lm in size were synthesized by multistep swelling polymerization using a modified Ugelstad technique. The PGMA microspheres and their hydrolyzed analogs derived from poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) were coated by silanization with tetraethoxysilane (TEOS) and (3-aminopropyl)triethoxysilane (APTES), respectively. The particles were characterized by elemental and thermogravimetric (TGA) analysis, scanning and transmission electron microscopy (SEM and TEM) coupled with an energy dispersive X-ray analysis (EDAX) and FT-IR spectroscopy to determine the SiO2 content, morphology, particle size, polydispersity and structure. These types of particles are expected to have improved biocompatibility relative to their starting polymers. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Many techniques can be used for the preparation of microspheres. The first polymeric particles were produced at the beginning of the 20th century by suspension polymerization [1]. This technique yields large beads possessing diameters that span the low micrometer to millimeter range and that have a broad particle size distribution. Suspension polymerization is used for the commercial manufacture of many important polymers, including poly(vinyl chloride), poly(methyl methacrylate), expandable polystyrene and styrene-acrylonitrile copolymers, which are used as ion-exchangers, sorbents or adsorbents for water purification [2–4]. Precipitation and dispersion polymerization produces particles with diameters from 1 to 8 lm. The precipitation process gives larger and less regular particles than the dispersion process [5–8]. Current efforts have focused on the synthesis of monodisperse polymer particles. This synthesis along with the ability to control particle surface chemistry has led to important developments in clinical diagnostics [9,10], drug delivery [11], cell separation [12,13] and chemotherapy [14,15]. The most common production method for small-sized particles (100–600 nm) is emulsion polymerization. The technique is frequently used for production of polystyrene colloids [16,17]. Through the multistep swelling ⇑ Corresponding author. Tel.: +420 296 809 260; fax: +420 296 809 410. E-mail address: [email protected] (D. Horák). http://dx.doi.org/10.1016/j.reactfunctpolym.2014.01.010 1381-5148/Ó 2014 Elsevier Ltd. All rights reserved.
of seeds and polymerization, monodisperse microspheres are obtained [18,19]. The key issue in the design of particles for biomedical applications involves coating them with a proper shell. The importance of an appropriate shell is exemplified by the silanization of metal oxide or polymer particles to produce organic/inorganic hybrid materials that feature both biocompatibility and functionality, e.g., amino groups [20–25]. Silica can also coat metal nanoparticles, such as gold [26,27], glass and natural or synthetic polymeric particles [28–31]. Typically, silica has been applied to polystyrene [32,33] or silanization has been performed on cellulose microspheres [34] or fibers [35]. Only one report addresses silica microspheres that were obtained by the calcination of ethylenediamine-functionalized PGMA microspheres. The silica had, however, a porous or hollow structure [36]. Silanization eliminates undesirable nonspecific interactions and enables the functionalization required for biological applications. The introduced functional groups are then available for the attachment of proteins, peptides and antibodies. Silica particles are commonly synthesized by the sol–gel technique that was introduced by Stöber and Fink [37]. Primarily, this method is used for the preparation of silica nanoparticles that are < 100 nm in size. The process involves the hydrolysis of silica alkoxide precursors, such as tetraethoxysilane (TEOS), in a mixture of ethanol and aqueous ammonium hydroxide. Silicic acid is produced during hydrolysis, and when its concentration exceeds its solubility in ethanol, it nucleates homogeneously to form
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submicrometer silica particles [38]. These particles can then be functionalized with different silanization agents, usually (3-aminopropyl)triethoxysilane (APTES) [39]. This functionalization can be performed in the same step with the silanization or in a two-step process [40–42]. The silica shell on the particles facilitates a wide variety of surface reactions and allows for conjugation with biomolecules, such as proteins and DNA [13]. Silica nanoparticles are easy to prepare, nontoxic and amenable to simple surface modifications; thus, they have been widely studied and used in various applications, such as column chromatography solid support materials [43], insulating layers and silica– polymer composites in engineering and bio-imaging and drug/ gene delivery systems in nano-biotechnology [44]. Herein, we report the preparation and characterization of monodisperse macroporous poly(glycidyl methacrylate) (PGMA) and poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) microspheres and their subsequent coating with a silica shell. The PGMA microspheres were synthesized using a modified Ugelstad technique [45] based on the multistep swelling of polystyrene (PS) seeds. The PS particles were obtained by emulsifier-free emulsion polymerization. Compared to particles with broad size distributions that are subject to size classification and low product yields, monodisperse microspheres offer enhanced performance due to their uniform physical, chemical and biological properties. As far as we know, this is the first report of the direct silanization of monodisperse PGMA or PDHPMA microspheres. The well-known hydrophobicity of PGMA and polystyrene microspheres makes them highly bioincompatible; thus, the hydrophilicity imparted through silanization will improve their in vivo performance and enhance the wettability of PGMA.
(ii) A dispersion of PS latex (0.6 g corresponding to 0.3 g dry weight) in a 0.25 wt% SDS aqueous solution (5 mL) was swollen for 50 h with a dispersion of DBP (3 g) in a 0.25 wt% SDS solution (20 mL) prepared by 5-min sonication using a W-385 sonicator (Heat System – Ultrasonics; Farmingdale, NY, USA). Swelling with the DBP dispersion was performed three additional times with DBP, increasing to 4 and 5 g in the last two swellings. The final solvent-swollen PS dispersion contained 15 g of DBP. (iii) DBP-swollen PS seeds were gradually swollen with a dispersion of monomers and an initiator (192 g of GMA, 128 g of EDMA and 1.6 g of AIBN) as well as a dispersion of porogens (8 g of dodecan-1-ol and 457 g of cyclohexanol) in a 0.1 wt% SDS solution (1.65 L) for 5 h. (iv) The dispersion of monomer/porogen-swollen PS seeds in 0.1 wt% SDS solution was then transferred to a 4-L reaction vessel and a 3 wt% PVA aqueous solution (240 mL) and 6 wt% HEC aqueous solution (240 mL) were added. Polymerization proceeded at 70 °C for 16 h under a CO2 atmosphere. The macroporous PGMA microspheres were washed successively with water, methanol, toluene and methanol (three times each with 1 L) and then air-dried. Optionally, PGMA microspheres (1 g) were hydrolyzed with 0.1 M H2SO4 (30 mL) at 60 °C for 5 h under mechanical stirring. The resulting poly(2,3-dihydroxypropyl methacrylate) (PDHPMA) microspheres were separated by centrifugation and washed 10 times with water until the effluent was neutral and vacuum-dried. 2.3. Modification of PDHPMA and PGMA microspheres with TEOS and APTES
2. Experimental 2.1. Materials Monomers, such as styrene (Synthos, Kralupy, Czech Republic), glycidyl methacrylate (GMA; Fluka; Buchs, Switzerland) and ethylene dimethacrylate (EDMA; Ugilor, France), were vacuum-distilled before use. 2,20 -Azobisisobutyronitrile (AIBN), dibutyl phthalate (DBP), sodium dodecyl sulfate (SDS) and (3-aminopropyl)triethoxysilane (APTES) were obtained from Sigma–Aldrich (St. Louis, USA). Tetraethoxysilane (TEOS) and 2-hydroxyethyl cellulose (HEC) were purchased from Fluka. Lithium persulfate, lithium hydrogen carbonate, ammonium hydroxide, sodium hydroxide and sulfuric acid were purchased from Lach-Ner (Neratovice, Czech Republic). Poly (vinyl alcohol) (PVA) was purchased from Wacker (Burghausen, Germany). Ultrapure Q-water ultra-filtered on a Milli-Q Gradient A 10 system (Millipore, Molsheim, France) was used throughout this work. All other solvents were from Lach-Ner (Neratovice, Czech Republic). 2.2. Preparation of monodisperse macroporous PGMA microspheres by multistep swelling polymerization The method consists of four steps: (i) Polystyrene (PS) latex (750 nm particles) was obtained by emulsifier-free emulsion polymerization. A 150-mL reaction vessel equipped with an anchor-type stirrer (300 rpm) was charged with styrene (10 g) and water (90 mL) containing 0.185 mM lithium persulfate (initiator) and 0.37 mM lithium hydrogen carbonate to adjust the pH to 7.5. Polymerization proceeded at 70 °C for 20 h under nitrogen atmosphere. The resulting PS latex was repeatedly washed with water.
PDHPMA microspheres (0.2 g) were dispersed in 2 mL of ethanol/water (1/1 v/v) and 0.1 M NaOH aqueous solution (0.2 mL) was added under mechanical stirring until pH 11 was reached. A solution of TEOS (0.2 mL) in ethanol (1 mL) was then added, and the reaction proceeded at 70 °C for 5 h. The resulting PDHPMA/ SiO2 microspheres were separated by centrifugation (4000 rpm), three times washed with ethanol and water, separated by centrifugation and vacuum-dried at 50 °C. Two approaches were employed for the coating of PGMA microspheres with APTES. In the one-step procedure, PGMA microspheres (0.2 g) were dispersed in methanol (2 mL), APTES (0.2 mL) in methanol (1 mL) was added under mechanical stirring and the reaction proceeded at 60 °C for 8 h. The resulting PGMA/ SiO2 microspheres were washed with methanol, separated by centrifugation and vacuum-dried at 50 °C. In the two-step procedure, PGMA microspheres were first coated with APTES in toluene and then hydrolyzed in water. PGMA microspheres (0.2 g) were dispersed in toluene (1.5 mL), and APTES (0.2 mL) in toluene (0.5 mL) was added under mechanical stirring and the reaction proceeded at 50 °C for 5 h. The particles were washed with toluene, methanol and water to remove unreacted reagents and were separated by centrifugation. The second step included the hydrolysis and condensation of alkoxides in water (2 mL) to yield silica. An ammonium hydroxide solution was added until pH 11 was reached. The reaction proceeded under mechanical stirring at 50 °C for 3 h. PGMA/SiO2 particles were separated by centrifugation, washed with water and vacuum-dried at 60 °C. 2.4. Characterization of microspheres The microspheres were observed with an Opton III light microscope (Oberkochen, Germany), a 200S Quanta scanning electron microscope (SEM; FEI, Brno, Czech Republic) equipped with an
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13
energy dispersive X-ray analysis (EDAX) and a Tecnai Spirit G2 transmission electron microscope (TEM; FEI, Brno, Czech Republic). The particle size distribution was determined by the Atlas program (Tescan; Brno, Czech Republic), counting at least 500 particles for each experiment. The number-average diameter (Dn), weight-average (Dw) diameter and polydispersity index (PDI) were calculated by Dn = RDi/N, Dw = RD4i /RD3i and PDI = Dw/Dn, where Di is the diameter of ith particle and N is the total number of particles counted. Fourier-transform infrared (FT-IR) spectra were recorded on a Thermo Nicolet Nexus 870 FT-IR spectrometer (Madison, WI, USA) in a water-purged environment with a DTGS (deuterated triglycine sulfate) detector. The Golden GateTM single reflection ATR system (Specac Ltd., Orpington, UK) was used to measure the ATR spectra of powdered samples over a wavenumber range of 400–4000 cm1. The typical parameters were: 256 sample scans and resolution of 4 cm1, with Happ-Genzel apodization and a KBr beamsplitter. An elemental analysis was carried out on a Perkin–Elmer 2400 CHN apparatus (Norwalk, CT, USA). Thermogravimetric analyses (TGA) were performed in air (50 mL/min.) using a Perkin Elmer Pyris 1 Thermogravimetric Analyzer over a temperature interval of 30–850 °C at a heating rate of 10 °C min1. The specific surface area (SBET) of the microspheres was determined by nitrogen adsorption in liquid nitrogen (77 K) using a Gemini VII 2390 Analyzer (Micromeritics; Norcross, GA, USA).
crosslinking agent to make the particles insoluble in solvents and to introduce porosity in the microspheres. If a porogen is present in the reaction mixture in sufficient amounts, phase separation between the polymer and the porogen is ensured. An analogous porogen system was used for the preparation of chromatographic packings [46], namely, a mixture of a small amount (2 wt%) of dodecan-1-ol (a thermodynamically poor solvent of GMA) and cyclohexanol (a thermodynamically good solvent of GMA) at a conventional monomer/porogen ratio = 2/3 w/w. The advantage of porous particles is that magnetic compounds can be prospectively precipitated inside the pores, allowing for the easy manipulation of the microspheres by a magnet. Moreover, macroporosity renders the particles with increased sorption capacity to be available for interacting various target compounds, a feature beneficial in size-exclusion chromatographic applications. The oxirane group allows for a variety of subsequent modification reactions, leading to the introduction of carboxyl, amino, aldehyde and phosphonate groups. The multistep swelling polymerization method thus produced macroporous PGMA particles of regular spherical shape. Optionally, PGMA particles were hydrolyzed to yield PDHPMA microspheres. The microspheres were 9.3 lm in size and had a low polydispersity index (PDI = 1.002), documenting the monodispersity of the particles (Fig. 1a). Both TEOS and APTES were then investigated as agents for the silanization of poly(glycidyl methacrylate)-based microspheres.
3. Results and discussion
3.2. Modification of PDHPMA microspheres with TEOS
3.1. Preparation of starting monodisperse macroporous PGMA microspheres
Preliminary experiments showed that the reaction of PGMA microspheres with TEOS in an ammonia solution was inferior to that of their PDHPMA counterparts because it resulted in only amino-functionalized particles. Almost no silanization occurred, presumably because of side reactions. Therefore, PDHPMA microspheres were preferred for the reaction with TEOS in ethanol/ water. While all other reaction parameters were kept constant, the TEOS/DHPMA ratio was varied up to 1 w/w (Table 1). The morphology, size and polydispersity of PDHPMA/SiO2 microspheres
Macroporous monodisperse PGMA microspheres were obtained by a modified Ugelstad technique. The method is based on the multistep swelling of PS seeds with a hydrophobic agent (DBP) and then with a mixture of monomers and porogens, followed by conventional PVA- and HEC-stabilized suspension polymerization. The microspheres contained 60 wt% GMA and 40 wt% EDMA as a
Fig. 1. SEM (a, b, d, e) and TEM (c and f) micrographs of PDHPMA microspheres No. 1 (a–c) and PDHPMA/SiO2 microspheres No. 4 (d–f). For the specifications of the microspheres No. 1 and 4, see Table 1.
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Table 1 Properties of PDHPMA/SiO2 microspheres. No.
Solvent
TEOS/DHPMA (w/w)
Dna (lm)
PDIb
Elemental analysis (wt%) C
1g 2 3 4
– EtOH/H2O EtOH/H2O EtOH/H2O
– 0.25 0.5 1
9.3 9.3 9.4 9.5
1.002 1.005 1.003 1.003
c
55.3 54.4 54.0 53.6
SBETf (m2/g)
SiO2 from (wt%) c
Ash
C
0 1.1 1.8 2.1
0 1.6 2.4 3.1
TGA
d
EDAX
0 1.8 2.7 5.7
e
0 0 0.26 0.60
77.7 62.6 62.1 71.2
Ethanol/water = 1/1 v/v. a Number-average diameter. b Polydispersity index. c Carbon. d Thermogravimetric analysis. e Energy dispersive X-ray analysis. f Specific surface area. g Neat PDHPMA microspheres.
were observed by scanning and transmission electron microscopy (Fig. 1). It was obvious from the micrographs that while the size of PDHPMA/SiO2 No. 4 (Table 1) slightly increased (Dn = 9.5 lm; Fig. 1 d) compared with the starting PGMA microsphere No. 1 (Dn = 9.3 lm; Fig. 1 a), the polydispersity did not change. By increasing the TEOS/DHPMA ratio from 1 to 2, silica was partially formed outside the particles as well. To avoid a secondary nucleation, i.e., the formation of free silica outside the particles, the TEOS/DHPMA ratio was decreased from 1 to 0.5 and 0.25 w/w; this also resulted in slightly smaller PDHPMA/SiO2 microspheres (Table 1). An EDAX analysis determined the content of silica on the surface of the PDHPMA/SiO2 microsphere No. 4 to be 0.6 wt% (Table 1). Silica shell thickness was found to be sensitive to the amount of TEOS. With a decreasing TEOS/DHPMA ratio, the silica content was reduced. As expected, the amount of silica according to EDAX analysis was rather low because only the surface layers were measurable. By contrast, TEM images (Fig. 1c and f) revealed that the silanization smoothed the outer surface of the microspheres and that silica was also formed inside the pores of the microspheres. These results were confirmed by elemental analysis because the carbon content of the PDHPMA/SiO2 microspheres decreased compared with the starting PDHPMA particles (Table 1). Incombustible residues corresponding to silica were present in only PDHPMA/SiO2 microspheres. Both of these facts were in agreement with FT-IR spectroscopy, indicating the presence of silica both on the surface and inside the PDHPMA microspheres. Next, the thermal decomposition of the PDHPMA/SiO2 microspheres was investigated by TGA (Table 1, Fig. 2). The TGA curve
of the neat PDHPMA particles showed an approximately 3.9 wt% weight loss below 185 °C due to the evaporation of solvents. Between 200 and 600 °C, weight losses were more pronounced, and 96.1 wt% of the material was finally lost due to full decomposition of the polymer. In contrast, after heating the PDHPMA/SiO2 microspheres up to 600 °C, there were residual compounds present, the amounts of which ranged from 1.7 to 5.7 wt% depending on the DHPMA/TEOS (w/w) ratio. Moreover, silica coating also increased the thermo-oxidative stability of PDHPMA/SiO2 microspheres by 60 °C (Fig. 2). There was reasonable agreement in the analysis of silica using TGA and determination of ash. To further confirm the presence of silica on PDHPMA microspheres, FT-IR spectra were recorded (Fig. 3). The spectrum of PDHPMA/SiO2 microspheres (Table 1, No. 4 and Fig. 3, spectrum b) differed from the spectrum of the initial PDHPMA microspheres (Table 1, No. 1 and Fig. 3, spectrum a). These differences are easily detected in the corresponding differential spectrum (b) – (a) in Fig. 3, spectrum c. The enhanced absorption near 1110 cm1 in the spectrum of PDHPMA/SiO2 microspheres corresponded to the Si–O–Si asymmetric stretching vibrations. The increased absorption of the band at 3432 cm1 was due to the enhanced content of Si–OH groups. Moreover, the intensity of the peaks associated with the diol groups at 1253, 1050, 974, 852 and 752 cm1 decreased due to the presence of the silica on the surface of the microspheres. The differential spectrum (Fig. 3, spectrum c) also revealed a decrease of the carbonyl peak at 1720 cm1. This was in agreement with the TEM results regarding the formation of silica inside the pores of the microspheres.
100
Weight loss (%)
80
60
40
1150 1720 1050 1253
Absorbance (a.u.)
1 2 3 4
3432 2948 a
b
20
c
4000 0
852 974 752
100
200
300
400
500
600
Temperature (°C) Fig. 2. TGA of PDHPMA and PDHPMA/SiO2 microspheres No. 1–4 (Table 1).
3500
3000
2500
2000
1500
1000
Wavenumber (cm-1) Fig. 3. FT-IR spectra of (a) PDHPMA No. 1, (b) PDHPMA/SiO2 microspheres No. 4 and (c) differential spectrum (b) – (a). For the specifications of the microspheres No. 1 and 4, see Table 1.
S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
To establish the effect of the silica that formed on the porous properties of the PDHPMA/SiO2 microspheres, the specific surface area was measured (Table 1). The recorded isotherms were of type II. The specific surface area of the PDHPMA/SiO2 microspheres (62 m2/g) was smaller than that of the PDHPMA microspheres (75.6 m2/g). This is consistent with the results from electron microscopy and confirms the formation of silica on the surface of and inside the microspheres. 3.3. Modification of PGMA microspheres with APTES Modification of PGMA microspheres by APTES was also investigated. Reactions were performed in methanol or toluene, and the opening of the oxirane groups with APTES was followed by the hydrolysis/condensation of alkoxides under alkaline conditions. The properties of PGMA/SiO2 particles were superior to those of PDHPMA/SiO2 microspheres because the formation of undesirable silica outside of the particles was minimal. The morphology, size and polydispersity of PGMA/SiO2 microspheres were again determined from scanning and transmission electron micrographs (Fig. 4d–h). As with PDHPMA/SiO2 microspheres, silanization did not broaden the particle size distribution (PDI = 1.003). On the other hand, the PGMA/SiO2 particles were larger than the PDHPMA/SiO2 particles. The diameter increased from 9.3 to
15
9.8 lm, with the APTES/GMA ratio increasing from 0 to 4 w/w (Table 2). The content of silica determined by a single particle EDAX measurement was higher for PGMA/SiO2 particles than for PDHPMA/SiO2 particles, as exemplified by the 3.2 wt% silica observed in No. 6 (Table 2). When silanization was carried out in methanol, the amount of silica on the particles increased from 3.2 to 4 wt% as the amount of APTES used in the reaction increased. A similar behavior was observed for the particles silanized in toluene and hydrolyzed in water (Table 2). Compared with silanizations using TEOS, silanization with APTES produced silica mainly on the surfaces of the microspheres, with only small amounts precipitated inside the pores, as evidenced from TEM images (Fig. 4c and f). This was due to the oxirane ring opening induced by the amine groups. Additionally, elemental analysis confirmed the formation of a silica shell around the polymer particles. As expected, the content of carbon and nitrogen in PGMA/SiO2 microspheres decreased and increased, respectively, with increasing amounts of APTES in the reaction mixture (Table 2). The presence of ash after the analysis also indicated a successful silica coating on the PGMA microspheres (Table 2). TGA of PGMA/SiO2 microspheres was in agreement with the elemental analysis results (Table 2 and Figs. 5 and 7). The TGA curve of the PGMA particles No. 5 (Fig. 5) showed 1.1 wt% weight loss below 230 °C due to the evaporation of
Fig. 4. SEM (a, b, d, e, g, h) micrographs of PGMA No. 5 (a and b), PGMA/SiO2 No. 7 (d and e) and No. 11 microspheres (g and h). TEM micrographs of the cross-section of a PGMA No. 5 (c) and PGMA/SiO2 microsphere No. 7 (f). For the specifications of the microspheres No. 5, 7 and 11, see Table 2.
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S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
Table 2 Properties of PGMA/SiO2 microspheres. No.
Solvent
APTES/GMA (w/w)
Dna (lm)
PDIb
Elemental analysis (wt%) C
5h 6 7 8 9 10 11 12 13 a b c d e f g h
– MeOH MeOH MeOH MeOH Toluene/water Toluene/water Toluene/water Toluene/water
0 1 2 4 10 1 2 4 10
9.3 9.6 9.8 9.8 9.9 9.7 9.8 9.8 9.6
1.004 1.003 1.003 1.003 1.002 1.004 1.003 1.004 1.002
c
N
58.5 55.4 54.4 53.4 55.3 55.2 54.4 54.0 56.1
d
0 1.1 1.3 1.5 1.6 1.0 1.4 1.4 1.4
SBETg (m2/g)
SiO2 from (wt%) c
e
Ash
C
0 4.1 5.5 5.1 2 4.4 4.8 5.8 3.8
0 5.3 7.0 8.7 5.5 5.6 7.0 7.7 4.1
TGA
EDAX
0 5.2 6.2 7.7 6.5 3.5 6.7 7.2 5.6
0 3.2 2.8 3.4 4.0 1.5 2.8 3 1.9
f
85.6 99.6
98.3
Number-average diameter. Polydispersity index. Carbon. Nitrogen. Thermogravimetric analysis. Energy dispersive X-ray analysis. Specific surface area. Neat PGMA microspheres.
100 5 6 7 8 9
Absorbance (a.u.)
Weight loss (%)
80
1723
60
40
1143
1254 3435
1450
2945
1072 846 907 758
a
b
c
20 4000 100
200
300
400
500
600
700
Temperature (°C) Fig. 5. TGA of PGMA and PGMA/SiO2 microspheres No. 5–9 (Table 2).
solvents. Starting at 250 °C, a quick decomposition of organic components was observed and their full decomposition was accomplished at 550 °C. A small amount of silica remained at 750 °C. This amount increased from 5.3 to 7.8 wt% depending on the APTES/GMA ratio. It is interesting to note that the formation of a silica shell on the microspheres reduced the rate of PGMA thermo-oxidative degradation, most likely because the shell limited the amount of air accessible to the organic portion of the investigated samples. To further confirm the presence of a silica shell on the PGMA microspheres (Table 1, No. 4), the FT-IR spectra were analyzed (Fig. 6). The spectrum of PGMA (Table 1, No. 4 and Fig. 6, spectrum a) was compared with the spectrum of PGMA/SiO2 (Table 2, No. 11 and Fig. 6, spectrum b) and the corresponding differential spectrum (b) – (a) was analyzed (Fig. 6, spectrum c). The intensities of the PGMA oxirane-related peaks at 758, 846 and 907 cm1 decreased due to the ring opening induced by the amine group of APTES. The presence of Si–O–Si chains on the surface of the PGMA/SiO2 microspheres was confirmed by the appearance of a shoulder at 1072 cm1 on the band near 1143 cm1, which is a characteristic peak in the spectrum of the starting PGMA microspheres. The presence of an additional small band with a maximum near 3435 cm1 was attributed to silica–OH vibrations, confirming the presence of the Si–OH bonds. Additionally, the
3000
2500
2000
Wave number
800
1500
1000
(cm-1)
Fig. 6. FT-IR spectra of (a) PGMA No. 5, (b) PGMA/SiO2 No. 11 microspheres and (c) differential spectrum (b) – (a). For the specifications of the microspheres No. 5 and 11, see Table 2.
100 5 10 11 12 13
80
Weight (%)
0
3500
60
40
20
0
100
200
300
400
500
600
700
800
900
Temperature (°C) Fig. 7. TGA of PGMA and PGMA/SiO2 microspheres No. 5 and 10–13 (Table 2).
intensity of the carbonyl peak at 1723 cm1 decreased, suggesting the formation of silica inside the pores of the polymer bulk. The porous properties of the PGMA/SiO2 microspheres were also investigated. Due to the formation of mesoporosity, the
S. Grama et al. / Reactive & Functional Polymers 77 (2014) 11–17
specific surface area (SBET) of the PGMA/SiO2 microspheres increased by up to 99.6 m2/g from the SBET of 85.6 m2/g that was observed for the starting PGMA microspheres No. 5 (Table 2). This result also confirmed the formation of a silica shell around the microspheres. 4. Conclusions Monodisperse macroporous PGMA particles were obtained using a modified Ugelstad method. For first time, both the surface and pores of the microspheres were covered with silica formed by a modified Stöber procedure. The silanization of macroporous PGMA or PDHPMA particles was achieved by the hydrolysis and condensation of TEOS and APTES agents. The formation of the silica shell was confirmed by various characterization methods. As the amount of the silanization agent in the reaction mixture increased, an increase in both the microsphere diameter and the amount of the silica present was observed on SEM micrographs coupled with EDAX analysis. The PDI did not change after the silanization of the microspheres. Similar results, i.e., a higher content of silica with increasing amounts of silanization agents, were obtained from an elemental analysis and TGA measurements of PGMA/SiO2 and PDHPMA/SiO2 particles. FT-IR spectra showed a decrease in the intensity of the carbonyl band at 1723 cm1 and TEM images also showed that silica was formed inside the pores of the PGMA and PDHPMA microspheres. In the PDHPMA/SiO2 microspheres, the amount of silica was relatively small, resulting in a thin shell on the particle surface; the majority of the silica was inside the pores of the PDHPMA/SiO2 microspheres. In contrast, the use of APTES as a silanization agent provided a thicker shell on the microspheres and less silica was precipitated inside of the pores, thus, substantially increasing the PGMA/SiO2 microsphere diameter. Silica had a tendency to smooth the surface of the PDHPMA/SiO2 microspheres. In contrast, the silica formed on the PGMA surface increased the porosity of the microspheres. Due to their hydrophilic nature and potential for facile modification, the prepared silanized microspheres are suited for biomedical applications, such as drug transport, bioaffinity chromatography and immobilization of antibodies. The potential range of silanized microsphere applications can be extended if iron oxide is precipitated inside the pores of the microspheres. Acknowledgement This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grant No. EE2.3.30.0029). References [1] E. Vivaldo-Lima, P.E. Wood, A.E. Hamielec, A. Penlidis, Ind. Eng. Chem. Res. 36 (1997) 939–965. [2] B.W. Brooks, Chem. Eng. Technol. 33 (2010) 1737–1744.
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