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pH-responsive composite microspheres based on magnetic mesoporous silica nanoparticle for drug delivery
European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 91–98
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Research paper
pH-responsive composite microspheres based on magnetic mesoporous silica nanoparticle for drug delivery Hao Wen, Jia Guo, Baisong Chang, Wuli Yang ⇑ State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai, China
a r t i c l e
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Article history: Received 17 February 2012 Accepted in revised form 12 November 2012 Available online 30 November 2012 Keywords: Mesoporous silica Nanoparticle pH-responsive Composite microsphere Crosslinking density Drug delivery
a b s t r a c t pH-responsive composite microspheres, consisting of a core of Fe3O4 nanoparticle, a sandwiched layer of mesoporous silica and a shell of crosslinked poly (methacrylic acid) (PMAA), were successfully synthesized via distillation precipitation polymerization. The pKa of the composite microsphere increased with the increase in the crosslinking density. Doxorubicin hydrochloride (DOX) was applied as a model drug, and the behavior of drug storage/release was investigated. The cumulative release of DOX-loaded composite microsphere in vitro showed that the drug release rate was much faster below its pKa than that of above its pKa. Because pH of most tumor tissues was lower than that of normal tissues, the pH-responsive composite microspheres are promising drug delivery system especially for cancer therapy. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction In the past few years, controlled drug delivery systems (DDSs), which can reduce the side effect of anti-cancer drugs, have attracted great interests of many research groups in cancer therapy applications. Nanoparticles are the most promising DDSs candidate for anti-cancer therapy, because they are 100- to 10,000-fold smaller than cancer cells and can pass through the cell barriers easily [1]. Due to the enhanced permeability and retention (EPR) effect, nanoparticles would easily be enriched at tumor tissues, which are called as passive targeted-drug delivery [2]. Up to date, DDSs are based on such materials as polymer, liposome, and inorganic material [3–11] After Vallet-Regi et al. firstly developed MCM-41 based DDSs in 2001 [12], mesoporous silica nanoparticles (MSNs) have been considered to be the potential candidate of DDSs, owing to their ordered pores with tunable diameters and structures, large surface areas and pore volumes, superior chemical and thermal stabilities, as well as accessible surface functionalization [13–23]. Besides, many groups reported the rapid hepatobiliary excretion [24], biodistribution [25], and low cytotoxicity of MSNs and their biodegradation products [26,27], which were quite important for DDSs.
⇑ Corresponding author. State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, No. 220, Handan Road, Shanghai 200433, China. Tel.: +86 21 6564 2385; fax: +86 21 6564 0293. E-mail address: [email protected] (W.L. Yang). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2012.11.019
In order to increase the drug delivery efficiency and decrease the side effect of most anti-cancer drugs, many kinds of stimuliresponsive MSNs, whose drug release can be triggered by external stimuli such as temperature [28,29], pH [30–33], light [34,35], and magnetic field [20,36–39], have been developed. Zink et al. did a variety of research to employ supramolecular system as pHresponsive nanovalves attached on the surface of mesoporous silica [32,33]. Jia et al. fabricated pH-responsive nanomatrix system consisted of colloidal silica and polymethylacrylate and proved that the drug could be maintained in the nanomatrix for up 1 year [40]. Hong et al. [41] also developed pH-responsive composite microspheres, which had the mesoporous silica core and the polymer outer shell, while the releasing rate of the guest molecules in these composite microspheres was faster at a higher pH than that at a lower pH. As proved previously, pH of most tumor tissues was lower (pH = 6.5–7.2) than that of human normal tissues (pH = 7.4) [4], such composite microspheres were not suitable for anti-cancer therapy. Therefore, it motivates the researchers to concentrate on the design of pH-responsive composite microspheres, which would benefit faster drug release rate at lower pH than that at higher pH. In this work, a kind of pH-responsive composite microsphere was successfully fabricated based on magnetic mesoporous silica nanoparticle (M-MSN). The inner superparamagnetic Fe3O4 core endowed the composite microsphere with magnetic response, the mesoporous silica middle-layer served as the store of drug, and the outer crosslinked poly (methacrylic acid) (PMAA) was the pH-responsive switcher to regulate the drug release behaviors at different pH values. The results indicated that the synthesized composite microspheres had tunable pH-responsive behavior by
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varying the crosslinking density of PMAA shell. Furthermore, doxorubicin (DOX), a classic anti-cancer drug, was introduced as a model drug to investigate both the loading and releasing properties of these composite microspheres. As showing a high drug-loading efficiency and a faster drug releasing rate at the pH below its pKa, the composite microsphere had a potential application in drug delivery especially for anti-cancer therapy.
The flask attached with a fractionating column, Liebig condenser, and a receiver was submerged in a heating oil bath. The temperature of the oil increased from ambient temperature to 85 °C in 15 min, and the mixture was kept at 85 °C for 2 h. The resulted milky dispersion was centrifuged and washed with deionized water for several times. 2.4. DOX loading and release
2. Materials and methods 2.1. Materials Oleic acid-capped Fe3O4 nanocrystal was prepared according to the reported procedures [42]. Cetyltrimethylammonium bromide (CTAB, AR) was purchased from Sinopharm Chemical Reagent Co., Ltd. Tetraethyl orthosilicate (TEOS, AR) was purchased from Jiangsu Qiangsheng Chemical Co., Ltd. Ammonium nitrate (AR) was purchased from Shanghai Jinghua Scientific & Technological Research Institute. 3-(Trimethoxysilyl) propyl methacrylate (MPS) was purchased from Acros Organics. N,N0 -methylene bisacrylamide (MBA) was purchased from Fluka. Methacrylic acid (MAA) was purchased from Shanghai Chemical Reagents Company, and it was distilled under the reduced pressure and nitrogen atmosphere before use. 2,20 -Azobisisobutyronitrile (AIBN) was purchased from Shanghai No. 4 Reagent & H.V. Chemical Co., Ltd., and it was purified by a recrystallization in ethanol. Doxorubicin in the form of hydrochloride salt (DOX) was purchased from Beijing Huafeng United Technology Company (Beijing, China). Phosphate-buffered saline (PBS) were obtained from Shanghai Qiangshun Chemical Reagent Company. Other chemicals were of reagent grade and were used without further purification. 2.2. Preparation of MPS-modified M-MSN Magnetic mesoporous silica nanoparticle (M-MSN) was synthesized by the modified Stöber method [43]. 7.5 mg of Fe3O4 nanocrystal (15 nm) was dispersed in 1 mL of chloroform and mixed with 12 mL of aqueous solution containing 0.25 g of CTAB by vigorously stirring. After kept at 65 °C for 30 min to vaporize chloroform, the received coffee-like Fe3O4 dispersion was diluted by 240 mL of deionized water, to which 7.5 mL of ammonia solution, 1.25 mL of TEOS and 12.5 mL of ethyl acetate were added in sequence. The mixture was kept at 40 °C for another 6 h with a stirring rate of 80 rpm. The resultant mesoporous silica nanoparticles with Fe3O4 inner cores (M-MSNs) were collected by centrifugation and washed with ethanol for five times. 100 mg of as-synthesized M-MSN and 0.1 mL of MPS were dispersed in 50 mL of ethanol, and the mixture was kept at 80 °C for 3 h. After centrifugation and ethanol washing for five times, the organic template (CTAB) was removed by refluxing in ethanol solution of ammonium nitrate (NH4NO3, 10 mg/mL) at 80 °C for 6 h. By centrifugation and washing with acetonitrile for three times, MPS-modified M-MSN was eventually obtained. 2.3. Preparation of M-MSN/PMAA composite microspheres Crosslinked poly (methylacrylic acid)-capped M-MSN (M-MSN/ PMAA) composite microspheres were generated from MPSmodified M-MSN by distillation precipitation polymerization [44]. N,N0 -methylenebisacrylamide (MBA) and AIBN were used as the crosslinker and initiator, respectively. The typical recipe for the synthesis of the composite microspheres was as follows: 50 mg of M-MSN, 200 mg of methacrylic acid, 20 mg of MBA, and 4 mg of AIBN were mixed in 20 mL of acetonitrile in a dried 50-mL single-necked flask with the aid of sonication for 1 min.
DOX was used as the model drug to assess the drug-loading and release behavior of the composite microspheres. In the loading procedure, the composite microspheres were suspended in deionized water with a concentration of 10 mg/mL and DOX was dissolved in deionized water with a concentration of 5 mg/mL. Typically, 0.2 mL of composite microspheres dispersion and 0.2 mL of DOX solution were mixed and stirred at room temperature for 1 h, then 6 mL of PBS (pH = 7.4) was added, and the mixture was stirred continuously at room temperature for another 24 h. The dispersion was centrifuged at 12,000 rpm for 5 min to collect the DOX-loaded composite microspheres. The drug-loading content and drug-loading efficiency were determined by measuring the UV absorbance of free DOX. By comparing the absorbance at 480 nm of DOX in the supernatant with the calibration curve of DOX, the loading content and drug entrapment efficiency were calculated using following equations:
Drug-loading content ð%Þ initial weight of DOX weight of DOX in supernatant solution weight of DOX-loaded composite microspheres 100% ð1Þ ¼
Drug entrapment efficiency ð%Þ initial weight of DOX weight of DOX in supernatant solution initial weight of DOX 100% ð2Þ ¼
In the release procedure, DOX-loaded composite microspheres were re-dispersed in 2 mL of PBS (pH = 7.4). The dispersion was transferred into a dialysis bag (cutoff molecular weight, 14,000 Da), and it was placed in a 200 mL of PBS solution at different pH and gently shaken. At different time intervals, 2 mL solution was collected from the dispersion and the amount of released drug was also estimated by spectrophotometer. The volume of the release medium kept constant by adding 2 mL of fresh medium after each collection. All drug release data were averaged over three measurements. 2.5. Characterization Nitrogen adsorption/desorption isotherms were obtained on a Micromeritics Tristar 3000 pore analyzer at 77 K under continuous adsorption conditions. Brunauer, Emmett, and Teller (BET) and Barrett, Joyner, and Halenda (BJH) analyses were used to calculate the surface area, pore size, and pore volume. Transmission electron microscopy (TEM) images were obtained on a JEOL-1230 transmission electron microscope, and the samples for TEM measurements were made by casting one drop of the samples on copper grids coated with carbon. The hydrodynamic diameter of the particles was determined by dynamic light scattering (Malvern Autosizer 4700) at 25 °C in 90° angle. Magnetization measurements were obtained on a vibrating-sample magnetometer (VSM, EG&G Princeton Applied Research vibrating sample magnetometer, model 155) at 25 °C. Thermogravimetric analysis (TGA) was carried on a Perkin–Elmer Pyris 1 TGA instrument, with a temperature range of 100–800 °C at a heating rate of 20 °C/min in a nitrogen flow. UV–vis absorption spectra were measured on a Lambda 35
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spectrophotometer. Powder X-ray diffraction (PXRD) patterns were recorded on a Bruker SMART APEX (II)-CCD X-ray single crystal diffractometer with Ni-filtered Cu KR radiation (40 kV, 40 mA). FTIR transmittance spectra were measured on an NEXUS 470 spectrophotometer. All measured samples were dried, and the powders were mixed with KBr and pressed to a plate for measurement.
3. Results and discussion 3.1. Characterization of M-MSN/PMAA composite microspheres Distillation precipitation polymerization is a facile and novel way to synthesize narrowly dispersed polymer microspheres without any additional surfactants or stabilizers, which is specifically applicable to the fabrication of crosslinked hydrophilic polymer microspheres [45]. In a distillation precipitation polymerization, the continuous phase is a solvent for the monomer but a nonsolvent for the resultant polymer. Besides, distillation is also essential for the synthesis of mono-dispersed polymer microspheres. As a kind of universal pH sensitive material, poly (methacrylic acid) (PMAA) is insoluble in acetonitrile, while its monomer, MAA, is soluble in acetonitrile. The schematic preparation process of M-MSN/PMAA composite microspheres is shown in Scheme 1. After MPS-modified M-MSN was obtained, distillation precipitation polymerization was introduced to obtain M-MSN/ PMAA composite microspheres. Herein, M-MSN nanoparticles were used as the seeds for the distillation precipitation polymerization of MAA onto M-MSN. Due to the limited solubility of PMAA chains or crosslinked PMAA network during the reaction, it was most likely that they were thermodynamically forced to agglomerate and/or deposit onto the seeds [46,47], resulting in the core–shell structured composite microspheres. Fig. 1a and b were the TEM images of M-MSNs and M-MSN/ PMAA-30 composite microspheres, respectively. In Fig. 1a, the black dots in the center of M-MSNs were Fe3O4 nanocrystals, while mesoporous silica shell was observable of covering the Fe3O4 nanocrystals. The average diameter of the synthesized M-MSNs observed in TEM was roughly estimated to be 175 ± 20 nm. In Fig. 1b, a grayish shell (between the two black arrows) was located out of every dark core particle, indicating that the coating of PMAA was successful in the experiment. The average diameter of the composite microspheres in TEM was 225 ± 20 nm, bigger than that of the seed particles, while the thickness of the PMAA shell was about 20–25 nm (between the two bars). FTIR was utilized to confirm the successful coating of PMAA shell on M-MSN. As shown in Fig. 2, the M-MSN/PMAA-30 composite microsphere (curve b) had a new peak at 1710 cm1, which corresponded to the stretching
Scheme 1. Schematic preparation process of M-MSN/PMAA composite microspheres. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. TEM images of the magnetic mesoporous silica nanoparticles (M-MSNs) (a) and the composite microspheres with a 30% crosslinking degree of PMAA shell (MMSN/PMAA-30) (b). The grayish PMAA shell was between the two black arrows.
vibration of carboxylic acid group [44] and was absent in the FTIR spectrum of M-MSN (curve a). The hydrodynamic diameters of M-MSN and M-MSN/PMAA at pH 4 (defined as D4) were monitored by DLS at 25 °C. The pH was achieved by adding HCl in 103 mol/L NaCl(aq), which was used to control the ionic strength (Table 1). At pH 4, the hydrodynamic diameter of M-MSN was 266 nm. For the PMAA-coated M-MSN, D4 was varied from 301 nm to 358 nm with the increase in crosslinking density from 5% to 30%. The low polydispersity index (PDI) for all composite microspheres indicated the narrow size distribution of the composite microspheres synthesized by distillation precipitation polymerization, though the PDI of M-MSN/PMAA-30 was a little larger. Because of the presence of hydrate layer in DLS measurement, both M-MSN and M-MSN/PMAA had larger size than those observed by TEM [23]. Besides, the weight loss of M-MSN and M-MSN/PMAA at 800 °C was tested by TGA (Fig. 3) and the results were listed in Table 1 as well. The weight loss of M-MSN (Fig. 3a) was 10.6%, while that of M-MSN/PMAA was increased from 50.1% to 73.7% with the increase in the crosslinking density from 5% to 30% (Fig. 3b–g). Compared with the bare M-MSN, most of the weight loss of composite microsphere was contributed by
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Fig. 2. FTIR spectra of MPS-modified M-MSN (a) and M-MSN/PMAA-30 composite microsphere (b).
Table 1 The hydrodynamic diameter at pH 4 and weight loss at 800 °C of M-MSN and M-MSN/ PMAA with different crosslinking density. Sample
Crosslinking density (%)
D4 (nm)a
Weight loss at 800 °C (%)
M-MSN
–
10.6
M-MSN/PMAA-5c M-MSN/PMAA-10 M-MSN/PMAA-15 M-MSN/PMAA-20 M-MSN/PMAA-25 M-MSN/PMAA-30
5 10 15 20 25 30
266 (0.14)b 301 (0.05) 311 (0.08) 322 (0.02) 341 (0.04) 356 (0.04) 358 (0.20)
50.1 60.6 66.0 67.4 69.9 73.7
a
The hydrodynamic diameter was tested by DLS in aqueous solution in 25 °C; D4 was the hydrodynamic diameter of M-MSN/PMAA at pH 4, which was achieved by adding HCl in 103 mol/L NaCl(aq). b The value in round brackets was the size polydispersity index. c The number in the name of the samples presented the crosslinking density of the crosslinked polymer shell. Taking M-MSN/PMAA-5 as an example, the number 5 indicated the crosslinking density was 5%.
Fig. 3. Thermogravimetric analysis (TGA) curves for MPS-modified M-MSN without CTAB (a), M-MSN/PMAA-5 (b), M-MSN/PMAA-10 (c), M-MSN/PMAA-15 (d), MMSN/PMAA-20 (e), M-MSN/PMAA-25 (f), and M-MSN/PMAA-30 (g).
the PMAA shell, and it increased with the crosslinking density increase. This was because the non-solvent acetonitrile significantly restricted the solubility of crosslinked PMAA and would enable these polymeric networks more readily to deposit onto the
Fig. 4. (a) Nitrogen absorption and desorption for M-MSN. (b) Small-angle PXRD pattern of M-MSN.
M-MSN upon the addition of more crosslinking agents [44]. Thus, a higher crosslinking density would increase the content of PMAA in the composite microspheres. This result agreed well with the variation tendency of DLS data, verifying that a higher crosslinking density would increase the PMAA-coating amount on the M-MSN. Nitrogen adsorption measurement was conducted to specify the mesopore characters of M-MSN, that is, the average pore diameter, surface area, and pore volume. Fig. 4a showed typical IV isotherm, which indicated the existence of mesopores [25]. The average pore diameter was estimated to be 2.6 nm, while the surface area and pore volume were 727 m2/g and 2.5 cm3/g, respectively. PXRD also supported the mesoporous ordering of M-MSN, showing an apparent diffraction peak that was assigned as (1 0 0) plane [13] in the pattern (Fig. 4b). For the PMAA-coated M-MSN microspheres, there was no mesopore presented even in the lowest crosslinked microspheres, therefore interpreting that the pores were most likely blocked by the outer polymer shell. As mentioned above, oleic acid-capped Fe3O4 was encapsulated in the composite microspheres to endow the DDSs with magnetic field response [38]. Vibrating-sample magnetometer (VSM) was used to monitor the magnetic properties. In Fig. 5, Fe3O4, M-MSN and composite microspheres all exhibited superparamagnetism with negligible hysteresis loops, and neither coercivity field nor remanent magnetism was observed. The saturation magnetizations (Ms) of Fe3O4 were 53.0 emu/g and Ms of M-MSN, M-MSN/ PMAA-10, and M-MSN/PMAA-30 were 3.15, 1.30, and 0.73 emu/g, respectively. The decrease in Ms for M-MSN and M-MSN/PMAA was attributed to silica and polymer covered onto Fe3O4 cores step by step. In spite of that, such structure endowed the final composite microspheres with the magnetic field response which would ensure the vehicles to be enriched in tumor tissues with the help
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Table 2 The hydrodynamic diameter of M-MSN/PMAA at pH 10 and the volume ratio at different pH. Sample M-MSN M-MSN/PMAA-5 M-MSN/PMAA-10 M-MSN/PMAA-15 M-MSN/PMAA-20 M-MSN/PMAA-25 M-MSN/PMAA-30
D10 (nm)a 263 570 524 477 474 479 468
(0.15) (0.01) (0.01) (0.07) (0.06) (0.21) (0.29)
V10/V4b c
0.97 6.8 4.8 3.2 2.7 2.4 2.2
a The hydrodynamic diameter was tested by DLS in aqueous solution in 25 °C; D10 was the hydrodynamic diameter of M-MSN/PMAA at pH 10, which was achieved by adding NH4OH in 10–3 mol/L NaCl(aq). b V10/V4 was the volume ratio of the composite microsphere’s volume at pH 10 to that at pH 4. This volume ratio was defined as the cubic value of D10/D4. c The value in round brackets was the size polydispersity index.
Fig. 5. Field-dependent magnetization curves of (a) as-prepared Fe3O4 nanoparticles, (b) M-MSN, M-MSN/PMAA-10 and M-MSN/PMAA-30 at 25 °C.
of an outer magnet [39]. Otherwise, the application of superparamagnetic iron oxide nanoparticles (IONPs) was also reported in magnetic resonance imaging [48,49] and enzyme immobilization [50], though it was beyond the scope of this article.
3.2. pH response of M-MSN/PMAA composite microspheres To demonstrate the pH sensitivity of the composite microspheres with tunable crosslinking density, the hydrodynamic diameters of the composite microsphere at pH 10 (defined as D10) were monitored by DLS (Table 2). The pH was achieved by adding NH4OH in 103 mol/L NaCl(aq), which was used to control the ionic strength. Compared with D4 in Table 1, the hydrodynamic diameter of M-MSN at pH 10 was almost the same as that at pH 4. However, M-MSN/PMAA showed different behaviors. When gradually increasing the crosslinking density from 5% to 30%, D4 of these composite microspheres increased from 301 nm to 358 nm. Correspondingly, D10 decreased from 570 nm to 468 nm, which was larger than D4 in all cases. In the other hand, the volume ratio between pH 10 and 4 (V10/V4), which was defined as the cubic value of D10/D4, decreased from 6.8 to 2.2 with the increase in crosslinking density from 5% to 30%. It appeared that a higher crosslinking density would more significantly weaken the ability of the volume transition of composite microspheres [51]. One possible explanation was that, with the increase in the crosslinking density, the segment between two nodes of PMAA networks would become shorter which lead to an extensive limitation of segment movements. Thus, it was definitely against the swelling behavior of PMAA shell upon ionization and thus resulted in the decreasing V10/V4 values when increasing the crosslinking density.
As such, M-MSN/PMAA-5, M-MSN/PMAA-10, and M-MSN/ PMAA-15 were selected to study the detailed pH-responsive behaviors due to their relatively high V10/V4 value. In Fig. 6, the hydrodynamic diameter of M-MSN/PMAA-5 increased slightly from pH 4.0 to 4.6 and then increased sharply from pH 4.6 to 6.1. After that, the size basically stayed unchanged until pH 10. Regarding to M-MSN/PMAA-10, the hydrodynamic diameter sharply increased from pH 5.8 to 7.0 and kept almost unchanged from pH 4.0 to 5.8 and from pH 7.0 to 10. And for M-MSN/PMAA-15, the hydrodynamic diameter sharply increased from pH 8.6 to 9.3 and not changed much from pH 4.0 to 8.6 and from pH 9.3 to 10. At low pH, for example, pH below 4.6 for M-MSN/PMAA-5, most of the carboxylic acid groups in composite microspheres were protonated [52]; therefore, less electrostatic repulsion occurred within the PMAA network, corresponding the small size of composite microsphere. As more and more carboxylic acid groups were deprotonated with the increasing of pH, it resulted in the stronger electrostatic repulsion [52], PMAA shell swelled and led to the larger size of composite microsphere. At high pH, taking pH over 6.1 for M-MSN/PMAA-5 as an example, as most of the carboxylic acid groups were possibly de-protonated, the electrostatic repulsion did not change much, which resulted in the platform after that. Determined as the mean value of the pH range in which hydrodynamic diameter increased sharply, pKa for M-MSN/PMAA-5, M-MSN/ PMAA-10, and M-MSN/PMAA-15 were deduced to be 5.3, 6.4, and 8.9, respectively. Interestingly, a higher crosslinking density caused a higher pKa of the composite microspheres. Dong and his co-workers grafted PMAA brushes on gold substrates, and then, they characterized the pKa of the entire polymer brushes (pKabulk) by grazing angle FTIR and that of the uppermost layer of the
Fig. 6. Hydrodynamic diameter of M-MSN/PMAA with different crosslinking density at different pH in 103 mol/L NaCl(aq).
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polymer brushes (pKasurface) by contact angle measurements [53]. They found that the acid groups further from the substrate were easy to ionize at lower pH, while the acid groups buried in the brushes near the substrate surface remained uncharged even at very high pH, which resulted in a higher pKabulk (6.9–7.0) than pKasurface (4.6 ± 0.1) [53]. They attributed the higher pKabulk to the less free volume and poor accessibility of ions for the acid groups near the substrate surface, caused by the high grafting density and carboxylic acid group density. Theoretical simulations by Szleifer and his co-workers indicated that the limited available volume was favorable of uncharged acid groups to obtain greater entropy and reduced energy cost from repulsion [54]. In our system, the higher crosslinking density of composite microspheres further limited the free volume of PMAA which would increase the fraction of uncharged carboxylic acid groups in the same condition. Thus, the pKa retarded with the crosslinking density increasing.
3.3. DOX loading and release DOX, a classic anti-cancer drug, was utilized as a model drug to evaluate the loading and release behavior of M-MSN/PMAA composite microspheres. In our experiment, M-MSN/PMAA-10 with pKa of 6.4 was selected while M-MSN/PMAA-15 with pKa of 8.9 was introduced for a comparison. For M-MSN/PMAA-10, the DOX loading content was 32.3% and the entrapment efficiency was 95.1% when R was 0.5, while the DOX loading content was 49.0% and the entrapment efficiency was 95.9% when R was 1 (Table 3). For M-MSN/PMAA-15, the DOX loading content was 30.5% and the entrapment efficiency was 91.4% when R was 0.5, while the DOX loading content was 47.3% and the entrapment efficiency was 94.7% when R was 1. Li et al. prepared PLGA coated magnetic nanoparticles by water-in-oil-in-water emulsification method as carriers for DOX, and the reported entrapment efficiency was 35.4% when the mass ratio between DOX and the composite microspheres (defined as R) was 1 [55]. It was noted that our samples afforded both high DOX loading content and high entrapment efficiency, which promised its application in drug delivery. The drug release behaviors of M-MSN/PMAA-10 and M-MSN/ PMAA-15 were evaluated at pH 5.5 (acidic condition) and 7.4 (blood circulation), and each point was performed three times to calculate an average value. For M-MSN/PMAA-10 (Fig. 7a), in the first 2 h, 19.8% of loaded DOX was released at pH 5.5 while only 5.3% was released at pH 7.4. In 24 h, the cumulative release amount was 39.0% at pH 5.5 and 15.3% at pH 7.4, respectively. The total drug release percentage at pH 5.5 was almost 2.5 times over that at pH 7.4 in 24 h. In comparison with M-MSN/PMAA-15 (Fig. 7b), the total release percentage in 24 h was 43.1% at pH 5.5 and 44.8% at pH 7.4, respectively, without showing any visible difference between them. The resulting difference between M-MSN/ PMAA-10 and M-MSN/PMAA-15 was thought to be contributed
Table 3 The loading content and the entrapment efficiency for M-MSN/PMAA-10 and M-MSN/ PMAA-15 at different R.
a
Ra
DOX loading content (%)b
Entrapment efficiency (%)c
M-MSN/PMAA10
0.5 1
32.3 49.0
95.1 95.9
M-MSN/PMAA15
0.5 1
30.5 47.3
91.4 94.7
R was defined as the mass ratio between DOX and the composite microspheres. DOX loading content was defined as the mass percentage of the loading DOX in the DOX-loaded composite microspheres. c DOX entrapment efficiency was defined as the mass percentage of the loading DOX in the total DOX. b
Fig. 7. DOX releasing curves at pH 5.5 and 7.4 PBS solution in 24 h: (a) M-MSN/ PMAA-10; (b) M-MSN/PMAA-15.
by the difference between the pKa of M-MSN/PMAA-10 and that of M-MSN/PMAA-15 and the electrostatic interaction between DOX and the composite microspheres at different pH as well. When M-MSN/PMAA-10 was dispersed at pH 5.5, which was below its pKa (6.4), PMAA was protonated and the shell was collapsed. As M-MSN did not show apparent pH response and the average diameter was 265 nm according to that in Table 1 and Table 2, one could estimate the thickness of the polymer shell to be 35 nm from the DLS data in Fig. 6 (the thickness of the polymer shell mentioned below was estimated in the same way). In such state, DOX would easily spread from the mesopores because of the shorter diffusion path (35 nm). On the other hand, the pKa of DOX was reported to be 8.6 [56], making DOX positively charged at pH 5.5. The electrostatic interaction between DOX and the neutral PMAA shell was weak, which also contributed to the fast releasing rate. By contrast, when M-MSN/PMAA-10 was dispersed at pH 7.4, which was over its pKa, PMAA was negatively charged and the shell was extended to about 125 nm because of the electrostatic repulsion. DOX was positively charged at pH 7.4 as well. In this time, the slow releasing rate was resulted from the longer diffusion path (125 nm) and the strong electrostatic interaction between the positively charged DOX and negatively charged PMAA shell. Regarding to M-MSN/PMAA-15, both pH 5.5 and 7.4 were below its pKa (8.9). The PMAA shell was collapsed due to the weak electrostatic repulsion among the protonated carboxylic acid group, which resulted in the shorter diffusion paths (40 nm at pH 5.5 and 45 nm at pH 7.4), and the electrostatic interaction between DOX and PMAA was weak, both of which contributed to drug fast releasing rate at both pH conditions. The drug release results clearly indicated that PMAA shell on the exterior surface of
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M-MSN was active in moderating the diffusion behavior of DOX from the mesoporous channels, showing a fast drug releasing rate at the pH below its pKa. For their good biocompatibility of MSN and PMAA [57,58], the pH-responsive composite microspheres could be utilized as DDS to increase the drug delivery efficiency and decrease the side effect of most anti-cancer drug. 4. Conclusion pH-responsive M-MSN/PMAA composite microspheres with magnetic mesoporous silica nanoparticle core and crosslinked poly (methacrylic acid) shell were prepared by distillation precipitation polymerization. As the diameters of the composite microspheres were larger than that of M-MSN, it supported the successful coating of the outer PMAA shell, which was also supported by the pH sensitivity of the composite microspheres. The pKa of the composite microspheres was tunable by adjusting the crosslinking density of PMAA shell. Such property promised the selection of an ideal pH sensitive polymer shell for anti-cancer drug delivery. Otherwise, when DOX was selected as the model drug, high drug-loading efficiency for both M-MSN/PMAA-10 and M-MSN/PMAA-15 was shown, and it was important for the application in drug delivery. The cumulative release of M-MSN/PMAA-10 (pKa 6.4) in vitro showed a low leakage of 15.3% at pH 7.4 while was enhanced to 39.0% at pH 5.5 in 24 h. As a contrast, M-MSN/PMAA-15 (pKa 8.9) did not show apparent 24 h cumulative release difference between pH 5.5 and 7.4. We attributed their drug-loading and releasing behavior difference to the different diffusion path and electrostatic interaction at pH 5.5 and 7.4, caused by their different pKa. The investigation of the faster drug releasing rate below its pKa demonstrates that the pH-responsive composite microspheres are intriguing candidate carriers for drug delivery in tumor therapy. Besides, our group is trying to reduce the size of the composite microspheres and make the polymer shell biodegradable for the further application of the composite microspheres. Acknowledgements We are grateful for the support of the National Science Foundation of China (Grant Nos. 20874015 and 51273047), the Shanghai Rising-Star Program (10QH1400200), and the Innovation Program of Shanghai Municipal Education Commission (09ZZ01). References [1] K. Park, S. Lee, E. Kang, K. Kim, K. Choi, I.C. Kwon, New generation of multifunctional nanoparticles for cancer imaging and therapy, Adv. Funct. Mater. 19 (2009) 1553–1566. [2] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer-chemotherapy – mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [3] S. Iwamoto, J. Watanabe, S. Ichikawa, Entrapment of some compounds into biocompatible nano-sized particles and their releasing properties, Colloid Surf., B 42 (2005) 141–146. [4] D. Schmaljohann, Thermo- and pH-responsive polymers in drug delivery, Adv. Drug Deliv. Rev. 58 (2006) 1655–1670. [5] F. Meunier, C. Pichot, A. Elaissari, Effect of thiol-containing monomer on the preparation of temperature-sensitive hydrogel microspheres, Colloid Polym. Sci. 284 (2006) 1287–1292. [6] Q. Gan, T. Wang, Chitosan nanoparticle as protein delivery carrier – systematic examination of fabrication conditions for efficient loading and release, Colloid Surf., B 59 (2007) 24–34. [7] S. Angelos, N.M. Khashab, Y.W. Yang, A. Trabolsi, H.A. Khatib, J.F. Stoddart, J.I. Zink, PH clock-operated mechanized nanoparticles, J. Am. Chem. Soc. 131 (2009) 12912–12914. [8] Z.H. Cao, R. Tong, A. Mishra, W.C. Xu, G.C.L. Wong, J.J. Cheng, Y. Lu, Reversible cell-specific drug delivery with aptamer-functionalized liposomes, Angew. Chem. Int. Ed. 48 (2009) 6494–6498. [9] L. Hosta-Rigau, R. Chandrawati, E. Saveriades, P.D. Odermatt, A. Postma, F. Ercole, K. Breheney, K.L. Wark, B. Stadler, F. Caruso, Noncovalent liposome linkage and miniaturization of capsosomes for drug delivery, Biomacromolecules 11 (2010) 3548–3555.
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Research paper
pH-responsive composite microspheres based on magnetic mesoporous silica nanoparticle for drug delivery Hao Wen, Jia Guo, Baisong Chang, Wuli Yang ⇑ State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai, China
a r t i c l e
i n f o
Article history: Received 17 February 2012 Accepted in revised form 12 November 2012 Available online 30 November 2012 Keywords: Mesoporous silica Nanoparticle pH-responsive Composite microsphere Crosslinking density Drug delivery
a b s t r a c t pH-responsive composite microspheres, consisting of a core of Fe3O4 nanoparticle, a sandwiched layer of mesoporous silica and a shell of crosslinked poly (methacrylic acid) (PMAA), were successfully synthesized via distillation precipitation polymerization. The pKa of the composite microsphere increased with the increase in the crosslinking density. Doxorubicin hydrochloride (DOX) was applied as a model drug, and the behavior of drug storage/release was investigated. The cumulative release of DOX-loaded composite microsphere in vitro showed that the drug release rate was much faster below its pKa than that of above its pKa. Because pH of most tumor tissues was lower than that of normal tissues, the pH-responsive composite microspheres are promising drug delivery system especially for cancer therapy. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction In the past few years, controlled drug delivery systems (DDSs), which can reduce the side effect of anti-cancer drugs, have attracted great interests of many research groups in cancer therapy applications. Nanoparticles are the most promising DDSs candidate for anti-cancer therapy, because they are 100- to 10,000-fold smaller than cancer cells and can pass through the cell barriers easily [1]. Due to the enhanced permeability and retention (EPR) effect, nanoparticles would easily be enriched at tumor tissues, which are called as passive targeted-drug delivery [2]. Up to date, DDSs are based on such materials as polymer, liposome, and inorganic material [3–11] After Vallet-Regi et al. firstly developed MCM-41 based DDSs in 2001 [12], mesoporous silica nanoparticles (MSNs) have been considered to be the potential candidate of DDSs, owing to their ordered pores with tunable diameters and structures, large surface areas and pore volumes, superior chemical and thermal stabilities, as well as accessible surface functionalization [13–23]. Besides, many groups reported the rapid hepatobiliary excretion [24], biodistribution [25], and low cytotoxicity of MSNs and their biodegradation products [26,27], which were quite important for DDSs.
⇑ Corresponding author. State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, No. 220, Handan Road, Shanghai 200433, China. Tel.: +86 21 6564 2385; fax: +86 21 6564 0293. E-mail address: [email protected] (W.L. Yang). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2012.11.019
In order to increase the drug delivery efficiency and decrease the side effect of most anti-cancer drugs, many kinds of stimuliresponsive MSNs, whose drug release can be triggered by external stimuli such as temperature [28,29], pH [30–33], light [34,35], and magnetic field [20,36–39], have been developed. Zink et al. did a variety of research to employ supramolecular system as pHresponsive nanovalves attached on the surface of mesoporous silica [32,33]. Jia et al. fabricated pH-responsive nanomatrix system consisted of colloidal silica and polymethylacrylate and proved that the drug could be maintained in the nanomatrix for up 1 year [40]. Hong et al. [41] also developed pH-responsive composite microspheres, which had the mesoporous silica core and the polymer outer shell, while the releasing rate of the guest molecules in these composite microspheres was faster at a higher pH than that at a lower pH. As proved previously, pH of most tumor tissues was lower (pH = 6.5–7.2) than that of human normal tissues (pH = 7.4) [4], such composite microspheres were not suitable for anti-cancer therapy. Therefore, it motivates the researchers to concentrate on the design of pH-responsive composite microspheres, which would benefit faster drug release rate at lower pH than that at higher pH. In this work, a kind of pH-responsive composite microsphere was successfully fabricated based on magnetic mesoporous silica nanoparticle (M-MSN). The inner superparamagnetic Fe3O4 core endowed the composite microsphere with magnetic response, the mesoporous silica middle-layer served as the store of drug, and the outer crosslinked poly (methacrylic acid) (PMAA) was the pH-responsive switcher to regulate the drug release behaviors at different pH values. The results indicated that the synthesized composite microspheres had tunable pH-responsive behavior by
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varying the crosslinking density of PMAA shell. Furthermore, doxorubicin (DOX), a classic anti-cancer drug, was introduced as a model drug to investigate both the loading and releasing properties of these composite microspheres. As showing a high drug-loading efficiency and a faster drug releasing rate at the pH below its pKa, the composite microsphere had a potential application in drug delivery especially for anti-cancer therapy.
The flask attached with a fractionating column, Liebig condenser, and a receiver was submerged in a heating oil bath. The temperature of the oil increased from ambient temperature to 85 °C in 15 min, and the mixture was kept at 85 °C for 2 h. The resulted milky dispersion was centrifuged and washed with deionized water for several times. 2.4. DOX loading and release
2. Materials and methods 2.1. Materials Oleic acid-capped Fe3O4 nanocrystal was prepared according to the reported procedures [42]. Cetyltrimethylammonium bromide (CTAB, AR) was purchased from Sinopharm Chemical Reagent Co., Ltd. Tetraethyl orthosilicate (TEOS, AR) was purchased from Jiangsu Qiangsheng Chemical Co., Ltd. Ammonium nitrate (AR) was purchased from Shanghai Jinghua Scientific & Technological Research Institute. 3-(Trimethoxysilyl) propyl methacrylate (MPS) was purchased from Acros Organics. N,N0 -methylene bisacrylamide (MBA) was purchased from Fluka. Methacrylic acid (MAA) was purchased from Shanghai Chemical Reagents Company, and it was distilled under the reduced pressure and nitrogen atmosphere before use. 2,20 -Azobisisobutyronitrile (AIBN) was purchased from Shanghai No. 4 Reagent & H.V. Chemical Co., Ltd., and it was purified by a recrystallization in ethanol. Doxorubicin in the form of hydrochloride salt (DOX) was purchased from Beijing Huafeng United Technology Company (Beijing, China). Phosphate-buffered saline (PBS) were obtained from Shanghai Qiangshun Chemical Reagent Company. Other chemicals were of reagent grade and were used without further purification. 2.2. Preparation of MPS-modified M-MSN Magnetic mesoporous silica nanoparticle (M-MSN) was synthesized by the modified Stöber method [43]. 7.5 mg of Fe3O4 nanocrystal (15 nm) was dispersed in 1 mL of chloroform and mixed with 12 mL of aqueous solution containing 0.25 g of CTAB by vigorously stirring. After kept at 65 °C for 30 min to vaporize chloroform, the received coffee-like Fe3O4 dispersion was diluted by 240 mL of deionized water, to which 7.5 mL of ammonia solution, 1.25 mL of TEOS and 12.5 mL of ethyl acetate were added in sequence. The mixture was kept at 40 °C for another 6 h with a stirring rate of 80 rpm. The resultant mesoporous silica nanoparticles with Fe3O4 inner cores (M-MSNs) were collected by centrifugation and washed with ethanol for five times. 100 mg of as-synthesized M-MSN and 0.1 mL of MPS were dispersed in 50 mL of ethanol, and the mixture was kept at 80 °C for 3 h. After centrifugation and ethanol washing for five times, the organic template (CTAB) was removed by refluxing in ethanol solution of ammonium nitrate (NH4NO3, 10 mg/mL) at 80 °C for 6 h. By centrifugation and washing with acetonitrile for three times, MPS-modified M-MSN was eventually obtained. 2.3. Preparation of M-MSN/PMAA composite microspheres Crosslinked poly (methylacrylic acid)-capped M-MSN (M-MSN/ PMAA) composite microspheres were generated from MPSmodified M-MSN by distillation precipitation polymerization [44]. N,N0 -methylenebisacrylamide (MBA) and AIBN were used as the crosslinker and initiator, respectively. The typical recipe for the synthesis of the composite microspheres was as follows: 50 mg of M-MSN, 200 mg of methacrylic acid, 20 mg of MBA, and 4 mg of AIBN were mixed in 20 mL of acetonitrile in a dried 50-mL single-necked flask with the aid of sonication for 1 min.
DOX was used as the model drug to assess the drug-loading and release behavior of the composite microspheres. In the loading procedure, the composite microspheres were suspended in deionized water with a concentration of 10 mg/mL and DOX was dissolved in deionized water with a concentration of 5 mg/mL. Typically, 0.2 mL of composite microspheres dispersion and 0.2 mL of DOX solution were mixed and stirred at room temperature for 1 h, then 6 mL of PBS (pH = 7.4) was added, and the mixture was stirred continuously at room temperature for another 24 h. The dispersion was centrifuged at 12,000 rpm for 5 min to collect the DOX-loaded composite microspheres. The drug-loading content and drug-loading efficiency were determined by measuring the UV absorbance of free DOX. By comparing the absorbance at 480 nm of DOX in the supernatant with the calibration curve of DOX, the loading content and drug entrapment efficiency were calculated using following equations:
Drug-loading content ð%Þ initial weight of DOX weight of DOX in supernatant solution weight of DOX-loaded composite microspheres 100% ð1Þ ¼
Drug entrapment efficiency ð%Þ initial weight of DOX weight of DOX in supernatant solution initial weight of DOX 100% ð2Þ ¼
In the release procedure, DOX-loaded composite microspheres were re-dispersed in 2 mL of PBS (pH = 7.4). The dispersion was transferred into a dialysis bag (cutoff molecular weight, 14,000 Da), and it was placed in a 200 mL of PBS solution at different pH and gently shaken. At different time intervals, 2 mL solution was collected from the dispersion and the amount of released drug was also estimated by spectrophotometer. The volume of the release medium kept constant by adding 2 mL of fresh medium after each collection. All drug release data were averaged over three measurements. 2.5. Characterization Nitrogen adsorption/desorption isotherms were obtained on a Micromeritics Tristar 3000 pore analyzer at 77 K under continuous adsorption conditions. Brunauer, Emmett, and Teller (BET) and Barrett, Joyner, and Halenda (BJH) analyses were used to calculate the surface area, pore size, and pore volume. Transmission electron microscopy (TEM) images were obtained on a JEOL-1230 transmission electron microscope, and the samples for TEM measurements were made by casting one drop of the samples on copper grids coated with carbon. The hydrodynamic diameter of the particles was determined by dynamic light scattering (Malvern Autosizer 4700) at 25 °C in 90° angle. Magnetization measurements were obtained on a vibrating-sample magnetometer (VSM, EG&G Princeton Applied Research vibrating sample magnetometer, model 155) at 25 °C. Thermogravimetric analysis (TGA) was carried on a Perkin–Elmer Pyris 1 TGA instrument, with a temperature range of 100–800 °C at a heating rate of 20 °C/min in a nitrogen flow. UV–vis absorption spectra were measured on a Lambda 35
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spectrophotometer. Powder X-ray diffraction (PXRD) patterns were recorded on a Bruker SMART APEX (II)-CCD X-ray single crystal diffractometer with Ni-filtered Cu KR radiation (40 kV, 40 mA). FTIR transmittance spectra were measured on an NEXUS 470 spectrophotometer. All measured samples were dried, and the powders were mixed with KBr and pressed to a plate for measurement.
3. Results and discussion 3.1. Characterization of M-MSN/PMAA composite microspheres Distillation precipitation polymerization is a facile and novel way to synthesize narrowly dispersed polymer microspheres without any additional surfactants or stabilizers, which is specifically applicable to the fabrication of crosslinked hydrophilic polymer microspheres [45]. In a distillation precipitation polymerization, the continuous phase is a solvent for the monomer but a nonsolvent for the resultant polymer. Besides, distillation is also essential for the synthesis of mono-dispersed polymer microspheres. As a kind of universal pH sensitive material, poly (methacrylic acid) (PMAA) is insoluble in acetonitrile, while its monomer, MAA, is soluble in acetonitrile. The schematic preparation process of M-MSN/PMAA composite microspheres is shown in Scheme 1. After MPS-modified M-MSN was obtained, distillation precipitation polymerization was introduced to obtain M-MSN/ PMAA composite microspheres. Herein, M-MSN nanoparticles were used as the seeds for the distillation precipitation polymerization of MAA onto M-MSN. Due to the limited solubility of PMAA chains or crosslinked PMAA network during the reaction, it was most likely that they were thermodynamically forced to agglomerate and/or deposit onto the seeds [46,47], resulting in the core–shell structured composite microspheres. Fig. 1a and b were the TEM images of M-MSNs and M-MSN/ PMAA-30 composite microspheres, respectively. In Fig. 1a, the black dots in the center of M-MSNs were Fe3O4 nanocrystals, while mesoporous silica shell was observable of covering the Fe3O4 nanocrystals. The average diameter of the synthesized M-MSNs observed in TEM was roughly estimated to be 175 ± 20 nm. In Fig. 1b, a grayish shell (between the two black arrows) was located out of every dark core particle, indicating that the coating of PMAA was successful in the experiment. The average diameter of the composite microspheres in TEM was 225 ± 20 nm, bigger than that of the seed particles, while the thickness of the PMAA shell was about 20–25 nm (between the two bars). FTIR was utilized to confirm the successful coating of PMAA shell on M-MSN. As shown in Fig. 2, the M-MSN/PMAA-30 composite microsphere (curve b) had a new peak at 1710 cm1, which corresponded to the stretching
Scheme 1. Schematic preparation process of M-MSN/PMAA composite microspheres. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. TEM images of the magnetic mesoporous silica nanoparticles (M-MSNs) (a) and the composite microspheres with a 30% crosslinking degree of PMAA shell (MMSN/PMAA-30) (b). The grayish PMAA shell was between the two black arrows.
vibration of carboxylic acid group [44] and was absent in the FTIR spectrum of M-MSN (curve a). The hydrodynamic diameters of M-MSN and M-MSN/PMAA at pH 4 (defined as D4) were monitored by DLS at 25 °C. The pH was achieved by adding HCl in 103 mol/L NaCl(aq), which was used to control the ionic strength (Table 1). At pH 4, the hydrodynamic diameter of M-MSN was 266 nm. For the PMAA-coated M-MSN, D4 was varied from 301 nm to 358 nm with the increase in crosslinking density from 5% to 30%. The low polydispersity index (PDI) for all composite microspheres indicated the narrow size distribution of the composite microspheres synthesized by distillation precipitation polymerization, though the PDI of M-MSN/PMAA-30 was a little larger. Because of the presence of hydrate layer in DLS measurement, both M-MSN and M-MSN/PMAA had larger size than those observed by TEM [23]. Besides, the weight loss of M-MSN and M-MSN/PMAA at 800 °C was tested by TGA (Fig. 3) and the results were listed in Table 1 as well. The weight loss of M-MSN (Fig. 3a) was 10.6%, while that of M-MSN/PMAA was increased from 50.1% to 73.7% with the increase in the crosslinking density from 5% to 30% (Fig. 3b–g). Compared with the bare M-MSN, most of the weight loss of composite microsphere was contributed by
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Fig. 2. FTIR spectra of MPS-modified M-MSN (a) and M-MSN/PMAA-30 composite microsphere (b).
Table 1 The hydrodynamic diameter at pH 4 and weight loss at 800 °C of M-MSN and M-MSN/ PMAA with different crosslinking density. Sample
Crosslinking density (%)
D4 (nm)a
Weight loss at 800 °C (%)
M-MSN
–
10.6
M-MSN/PMAA-5c M-MSN/PMAA-10 M-MSN/PMAA-15 M-MSN/PMAA-20 M-MSN/PMAA-25 M-MSN/PMAA-30
5 10 15 20 25 30
266 (0.14)b 301 (0.05) 311 (0.08) 322 (0.02) 341 (0.04) 356 (0.04) 358 (0.20)
50.1 60.6 66.0 67.4 69.9 73.7
a
The hydrodynamic diameter was tested by DLS in aqueous solution in 25 °C; D4 was the hydrodynamic diameter of M-MSN/PMAA at pH 4, which was achieved by adding HCl in 103 mol/L NaCl(aq). b The value in round brackets was the size polydispersity index. c The number in the name of the samples presented the crosslinking density of the crosslinked polymer shell. Taking M-MSN/PMAA-5 as an example, the number 5 indicated the crosslinking density was 5%.
Fig. 3. Thermogravimetric analysis (TGA) curves for MPS-modified M-MSN without CTAB (a), M-MSN/PMAA-5 (b), M-MSN/PMAA-10 (c), M-MSN/PMAA-15 (d), MMSN/PMAA-20 (e), M-MSN/PMAA-25 (f), and M-MSN/PMAA-30 (g).
the PMAA shell, and it increased with the crosslinking density increase. This was because the non-solvent acetonitrile significantly restricted the solubility of crosslinked PMAA and would enable these polymeric networks more readily to deposit onto the
Fig. 4. (a) Nitrogen absorption and desorption for M-MSN. (b) Small-angle PXRD pattern of M-MSN.
M-MSN upon the addition of more crosslinking agents [44]. Thus, a higher crosslinking density would increase the content of PMAA in the composite microspheres. This result agreed well with the variation tendency of DLS data, verifying that a higher crosslinking density would increase the PMAA-coating amount on the M-MSN. Nitrogen adsorption measurement was conducted to specify the mesopore characters of M-MSN, that is, the average pore diameter, surface area, and pore volume. Fig. 4a showed typical IV isotherm, which indicated the existence of mesopores [25]. The average pore diameter was estimated to be 2.6 nm, while the surface area and pore volume were 727 m2/g and 2.5 cm3/g, respectively. PXRD also supported the mesoporous ordering of M-MSN, showing an apparent diffraction peak that was assigned as (1 0 0) plane [13] in the pattern (Fig. 4b). For the PMAA-coated M-MSN microspheres, there was no mesopore presented even in the lowest crosslinked microspheres, therefore interpreting that the pores were most likely blocked by the outer polymer shell. As mentioned above, oleic acid-capped Fe3O4 was encapsulated in the composite microspheres to endow the DDSs with magnetic field response [38]. Vibrating-sample magnetometer (VSM) was used to monitor the magnetic properties. In Fig. 5, Fe3O4, M-MSN and composite microspheres all exhibited superparamagnetism with negligible hysteresis loops, and neither coercivity field nor remanent magnetism was observed. The saturation magnetizations (Ms) of Fe3O4 were 53.0 emu/g and Ms of M-MSN, M-MSN/ PMAA-10, and M-MSN/PMAA-30 were 3.15, 1.30, and 0.73 emu/g, respectively. The decrease in Ms for M-MSN and M-MSN/PMAA was attributed to silica and polymer covered onto Fe3O4 cores step by step. In spite of that, such structure endowed the final composite microspheres with the magnetic field response which would ensure the vehicles to be enriched in tumor tissues with the help
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Table 2 The hydrodynamic diameter of M-MSN/PMAA at pH 10 and the volume ratio at different pH. Sample M-MSN M-MSN/PMAA-5 M-MSN/PMAA-10 M-MSN/PMAA-15 M-MSN/PMAA-20 M-MSN/PMAA-25 M-MSN/PMAA-30
D10 (nm)a 263 570 524 477 474 479 468
(0.15) (0.01) (0.01) (0.07) (0.06) (0.21) (0.29)
V10/V4b c
0.97 6.8 4.8 3.2 2.7 2.4 2.2
a The hydrodynamic diameter was tested by DLS in aqueous solution in 25 °C; D10 was the hydrodynamic diameter of M-MSN/PMAA at pH 10, which was achieved by adding NH4OH in 10–3 mol/L NaCl(aq). b V10/V4 was the volume ratio of the composite microsphere’s volume at pH 10 to that at pH 4. This volume ratio was defined as the cubic value of D10/D4. c The value in round brackets was the size polydispersity index.
Fig. 5. Field-dependent magnetization curves of (a) as-prepared Fe3O4 nanoparticles, (b) M-MSN, M-MSN/PMAA-10 and M-MSN/PMAA-30 at 25 °C.
of an outer magnet [39]. Otherwise, the application of superparamagnetic iron oxide nanoparticles (IONPs) was also reported in magnetic resonance imaging [48,49] and enzyme immobilization [50], though it was beyond the scope of this article.
3.2. pH response of M-MSN/PMAA composite microspheres To demonstrate the pH sensitivity of the composite microspheres with tunable crosslinking density, the hydrodynamic diameters of the composite microsphere at pH 10 (defined as D10) were monitored by DLS (Table 2). The pH was achieved by adding NH4OH in 103 mol/L NaCl(aq), which was used to control the ionic strength. Compared with D4 in Table 1, the hydrodynamic diameter of M-MSN at pH 10 was almost the same as that at pH 4. However, M-MSN/PMAA showed different behaviors. When gradually increasing the crosslinking density from 5% to 30%, D4 of these composite microspheres increased from 301 nm to 358 nm. Correspondingly, D10 decreased from 570 nm to 468 nm, which was larger than D4 in all cases. In the other hand, the volume ratio between pH 10 and 4 (V10/V4), which was defined as the cubic value of D10/D4, decreased from 6.8 to 2.2 with the increase in crosslinking density from 5% to 30%. It appeared that a higher crosslinking density would more significantly weaken the ability of the volume transition of composite microspheres [51]. One possible explanation was that, with the increase in the crosslinking density, the segment between two nodes of PMAA networks would become shorter which lead to an extensive limitation of segment movements. Thus, it was definitely against the swelling behavior of PMAA shell upon ionization and thus resulted in the decreasing V10/V4 values when increasing the crosslinking density.
As such, M-MSN/PMAA-5, M-MSN/PMAA-10, and M-MSN/ PMAA-15 were selected to study the detailed pH-responsive behaviors due to their relatively high V10/V4 value. In Fig. 6, the hydrodynamic diameter of M-MSN/PMAA-5 increased slightly from pH 4.0 to 4.6 and then increased sharply from pH 4.6 to 6.1. After that, the size basically stayed unchanged until pH 10. Regarding to M-MSN/PMAA-10, the hydrodynamic diameter sharply increased from pH 5.8 to 7.0 and kept almost unchanged from pH 4.0 to 5.8 and from pH 7.0 to 10. And for M-MSN/PMAA-15, the hydrodynamic diameter sharply increased from pH 8.6 to 9.3 and not changed much from pH 4.0 to 8.6 and from pH 9.3 to 10. At low pH, for example, pH below 4.6 for M-MSN/PMAA-5, most of the carboxylic acid groups in composite microspheres were protonated [52]; therefore, less electrostatic repulsion occurred within the PMAA network, corresponding the small size of composite microsphere. As more and more carboxylic acid groups were deprotonated with the increasing of pH, it resulted in the stronger electrostatic repulsion [52], PMAA shell swelled and led to the larger size of composite microsphere. At high pH, taking pH over 6.1 for M-MSN/PMAA-5 as an example, as most of the carboxylic acid groups were possibly de-protonated, the electrostatic repulsion did not change much, which resulted in the platform after that. Determined as the mean value of the pH range in which hydrodynamic diameter increased sharply, pKa for M-MSN/PMAA-5, M-MSN/ PMAA-10, and M-MSN/PMAA-15 were deduced to be 5.3, 6.4, and 8.9, respectively. Interestingly, a higher crosslinking density caused a higher pKa of the composite microspheres. Dong and his co-workers grafted PMAA brushes on gold substrates, and then, they characterized the pKa of the entire polymer brushes (pKabulk) by grazing angle FTIR and that of the uppermost layer of the
Fig. 6. Hydrodynamic diameter of M-MSN/PMAA with different crosslinking density at different pH in 103 mol/L NaCl(aq).
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polymer brushes (pKasurface) by contact angle measurements [53]. They found that the acid groups further from the substrate were easy to ionize at lower pH, while the acid groups buried in the brushes near the substrate surface remained uncharged even at very high pH, which resulted in a higher pKabulk (6.9–7.0) than pKasurface (4.6 ± 0.1) [53]. They attributed the higher pKabulk to the less free volume and poor accessibility of ions for the acid groups near the substrate surface, caused by the high grafting density and carboxylic acid group density. Theoretical simulations by Szleifer and his co-workers indicated that the limited available volume was favorable of uncharged acid groups to obtain greater entropy and reduced energy cost from repulsion [54]. In our system, the higher crosslinking density of composite microspheres further limited the free volume of PMAA which would increase the fraction of uncharged carboxylic acid groups in the same condition. Thus, the pKa retarded with the crosslinking density increasing.
3.3. DOX loading and release DOX, a classic anti-cancer drug, was utilized as a model drug to evaluate the loading and release behavior of M-MSN/PMAA composite microspheres. In our experiment, M-MSN/PMAA-10 with pKa of 6.4 was selected while M-MSN/PMAA-15 with pKa of 8.9 was introduced for a comparison. For M-MSN/PMAA-10, the DOX loading content was 32.3% and the entrapment efficiency was 95.1% when R was 0.5, while the DOX loading content was 49.0% and the entrapment efficiency was 95.9% when R was 1 (Table 3). For M-MSN/PMAA-15, the DOX loading content was 30.5% and the entrapment efficiency was 91.4% when R was 0.5, while the DOX loading content was 47.3% and the entrapment efficiency was 94.7% when R was 1. Li et al. prepared PLGA coated magnetic nanoparticles by water-in-oil-in-water emulsification method as carriers for DOX, and the reported entrapment efficiency was 35.4% when the mass ratio between DOX and the composite microspheres (defined as R) was 1 [55]. It was noted that our samples afforded both high DOX loading content and high entrapment efficiency, which promised its application in drug delivery. The drug release behaviors of M-MSN/PMAA-10 and M-MSN/ PMAA-15 were evaluated at pH 5.5 (acidic condition) and 7.4 (blood circulation), and each point was performed three times to calculate an average value. For M-MSN/PMAA-10 (Fig. 7a), in the first 2 h, 19.8% of loaded DOX was released at pH 5.5 while only 5.3% was released at pH 7.4. In 24 h, the cumulative release amount was 39.0% at pH 5.5 and 15.3% at pH 7.4, respectively. The total drug release percentage at pH 5.5 was almost 2.5 times over that at pH 7.4 in 24 h. In comparison with M-MSN/PMAA-15 (Fig. 7b), the total release percentage in 24 h was 43.1% at pH 5.5 and 44.8% at pH 7.4, respectively, without showing any visible difference between them. The resulting difference between M-MSN/ PMAA-10 and M-MSN/PMAA-15 was thought to be contributed
Table 3 The loading content and the entrapment efficiency for M-MSN/PMAA-10 and M-MSN/ PMAA-15 at different R.
a
Ra
DOX loading content (%)b
Entrapment efficiency (%)c
M-MSN/PMAA10
0.5 1
32.3 49.0
95.1 95.9
M-MSN/PMAA15
0.5 1
30.5 47.3
91.4 94.7
R was defined as the mass ratio between DOX and the composite microspheres. DOX loading content was defined as the mass percentage of the loading DOX in the DOX-loaded composite microspheres. c DOX entrapment efficiency was defined as the mass percentage of the loading DOX in the total DOX. b
Fig. 7. DOX releasing curves at pH 5.5 and 7.4 PBS solution in 24 h: (a) M-MSN/ PMAA-10; (b) M-MSN/PMAA-15.
by the difference between the pKa of M-MSN/PMAA-10 and that of M-MSN/PMAA-15 and the electrostatic interaction between DOX and the composite microspheres at different pH as well. When M-MSN/PMAA-10 was dispersed at pH 5.5, which was below its pKa (6.4), PMAA was protonated and the shell was collapsed. As M-MSN did not show apparent pH response and the average diameter was 265 nm according to that in Table 1 and Table 2, one could estimate the thickness of the polymer shell to be 35 nm from the DLS data in Fig. 6 (the thickness of the polymer shell mentioned below was estimated in the same way). In such state, DOX would easily spread from the mesopores because of the shorter diffusion path (35 nm). On the other hand, the pKa of DOX was reported to be 8.6 [56], making DOX positively charged at pH 5.5. The electrostatic interaction between DOX and the neutral PMAA shell was weak, which also contributed to the fast releasing rate. By contrast, when M-MSN/PMAA-10 was dispersed at pH 7.4, which was over its pKa, PMAA was negatively charged and the shell was extended to about 125 nm because of the electrostatic repulsion. DOX was positively charged at pH 7.4 as well. In this time, the slow releasing rate was resulted from the longer diffusion path (125 nm) and the strong electrostatic interaction between the positively charged DOX and negatively charged PMAA shell. Regarding to M-MSN/PMAA-15, both pH 5.5 and 7.4 were below its pKa (8.9). The PMAA shell was collapsed due to the weak electrostatic repulsion among the protonated carboxylic acid group, which resulted in the shorter diffusion paths (40 nm at pH 5.5 and 45 nm at pH 7.4), and the electrostatic interaction between DOX and PMAA was weak, both of which contributed to drug fast releasing rate at both pH conditions. The drug release results clearly indicated that PMAA shell on the exterior surface of
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M-MSN was active in moderating the diffusion behavior of DOX from the mesoporous channels, showing a fast drug releasing rate at the pH below its pKa. For their good biocompatibility of MSN and PMAA [57,58], the pH-responsive composite microspheres could be utilized as DDS to increase the drug delivery efficiency and decrease the side effect of most anti-cancer drug. 4. Conclusion pH-responsive M-MSN/PMAA composite microspheres with magnetic mesoporous silica nanoparticle core and crosslinked poly (methacrylic acid) shell were prepared by distillation precipitation polymerization. As the diameters of the composite microspheres were larger than that of M-MSN, it supported the successful coating of the outer PMAA shell, which was also supported by the pH sensitivity of the composite microspheres. The pKa of the composite microspheres was tunable by adjusting the crosslinking density of PMAA shell. Such property promised the selection of an ideal pH sensitive polymer shell for anti-cancer drug delivery. Otherwise, when DOX was selected as the model drug, high drug-loading efficiency for both M-MSN/PMAA-10 and M-MSN/PMAA-15 was shown, and it was important for the application in drug delivery. The cumulative release of M-MSN/PMAA-10 (pKa 6.4) in vitro showed a low leakage of 15.3% at pH 7.4 while was enhanced to 39.0% at pH 5.5 in 24 h. As a contrast, M-MSN/PMAA-15 (pKa 8.9) did not show apparent 24 h cumulative release difference between pH 5.5 and 7.4. We attributed their drug-loading and releasing behavior difference to the different diffusion path and electrostatic interaction at pH 5.5 and 7.4, caused by their different pKa. The investigation of the faster drug releasing rate below its pKa demonstrates that the pH-responsive composite microspheres are intriguing candidate carriers for drug delivery in tumor therapy. Besides, our group is trying to reduce the size of the composite microspheres and make the polymer shell biodegradable for the further application of the composite microspheres. Acknowledgements We are grateful for the support of the National Science Foundation of China (Grant Nos. 20874015 and 51273047), the Shanghai Rising-Star Program (10QH1400200), and the Innovation Program of Shanghai Municipal Education Commission (09ZZ01). References [1] K. Park, S. Lee, E. Kang, K. Kim, K. Choi, I.C. Kwon, New generation of multifunctional nanoparticles for cancer imaging and therapy, Adv. Funct. Mater. 19 (2009) 1553–1566. [2] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer-chemotherapy – mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [3] S. Iwamoto, J. Watanabe, S. Ichikawa, Entrapment of some compounds into biocompatible nano-sized particles and their releasing properties, Colloid Surf., B 42 (2005) 141–146. [4] D. Schmaljohann, Thermo- and pH-responsive polymers in drug delivery, Adv. Drug Deliv. Rev. 58 (2006) 1655–1670. [5] F. Meunier, C. Pichot, A. Elaissari, Effect of thiol-containing monomer on the preparation of temperature-sensitive hydrogel microspheres, Colloid Polym. Sci. 284 (2006) 1287–1292. [6] Q. Gan, T. Wang, Chitosan nanoparticle as protein delivery carrier – systematic examination of fabrication conditions for efficient loading and release, Colloid Surf., B 59 (2007) 24–34. [7] S. Angelos, N.M. Khashab, Y.W. Yang, A. Trabolsi, H.A. Khatib, J.F. Stoddart, J.I. Zink, PH clock-operated mechanized nanoparticles, J. Am. Chem. Soc. 131 (2009) 12912–12914. [8] Z.H. Cao, R. Tong, A. Mishra, W.C. Xu, G.C.L. Wong, J.J. Cheng, Y. Lu, Reversible cell-specific drug delivery with aptamer-functionalized liposomes, Angew. Chem. Int. Ed. 48 (2009) 6494–6498. [9] L. Hosta-Rigau, R. Chandrawati, E. Saveriades, P.D. Odermatt, A. Postma, F. Ercole, K. Breheney, K.L. Wark, B. Stadler, F. Caruso, Noncovalent liposome linkage and miniaturization of capsosomes for drug delivery, Biomacromolecules 11 (2010) 3548–3555.
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