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Size-Tunable and Functional Core-Shell Structured Silica Nanoparticles for Drug Release Fangli Chi,† Ya-Nan Guo,† Jun Liu,‡ Yunling Liu,† and Qisheng Huo*,† State Key Laboratory of Inorganic Synthesis and PreparatiVe Chemistry, College of Chemistry, Jilin UniVersity, Changchun 130012, China, and Pacific Northwest National Laboratory, Richland, Washington 99354 ReceiVed: NoVember 2, 2009; ReVised Manuscript ReceiVed: January 4, 2010
Size-tunable silica cross-linked micellar core-shell nanoparticles (SCMCSNs) were successfully synthesized from a Pluronic nonionic surfactant (F127) template system with organic swelling agents such as 1,3,5trimethylbenzene (TMB) and octanoic acid at room temperature. The size and morphology of SCMCSNs were directly evidenced by TEM imaging and DLS measurements (up to ∼90 nm). Pyrene and coumarin 153 (C153) were used as fluorescent probe molecules to investigate the effect and location of swelling agent molecules. Papaverine as a model drug was used to measure the loading capacity and release property of nanoparticles. The swelling agents can enlarge the nanoparticle size and improve the drug loading capacity of nanoparticles. Moreover, the carboxylic acid group of fatty acid can adjust the release behavior of the nanoparticles. Introduction Multifunctional micro/nanostructured materials, with controlled sizes and the ability to deliver large amounts of therapeutic or imaging agents, as well as the potential to encapsulate multiagents for targeted delivery, are highly desirable for biomedical applications.1-7 Polymer nanocarriers,8 microparticles,9 mesoporous silicates,10,11 nanoparticles,6,12-16 and polymeric micelles16-22 are widely investigated. Among micro/nanostructured materials, polymer micelles are especially important because they can form a hydrophobic core to solubilize and carry compounds that have poor solubility, low stability, or undesirable pharmaceutical properties.22 Recently, to improve the stability of micelles in a biological environment, scientists have tried to cross-link and lock in the micellar structures,23-26 in the core region, in the hydrophilic shell region, or on the micelle surface. We recently reported the use of block copolymer micelles based on a nonionic Pluronic block copolymer to template silica deposition. The uptake and release of various drugs and dyes from the resulting silica cross-linked micellar core-shell nanoparticles (SCMCSNs) were studied using fluorescence techniques, and the silica shells appear to retard diffusional release rates.27 The SCMCSNs are more stable than polymer micelles. Yuan et al. deposited silica on diblock copolymer micelles comprising cationic poly(2-(dimethylamino)ethyl methacrylate) coronas and poly(2-(diisopropylamino)ethyl methacrylate) hydrophobic cores from aqueous solution at pH 7.2 and 20 °C.28 The positive charge on the amine group allowed the deposition of a thick silica layer under neutral pH conditions. Schooneveld et al. improved the biocompatibility and pharmacokinetics of silica nanoparticles by means of a lipid coating.29 Niu et al. fabricated novel kinds of amino- or thiol-functionalized superparamagnetic copolymer-silica nanospheres which consist of a magnetic core and a silica cross-linked block copolymer shell.30 Liu et al. produced silica hollow nanospheres by adjusting the hydrolysis and condensation kinetics of silane * Corresponding author. Phone: +86-431-85168602. Fax: +86-43185168624. E-mail:
[email protected]. † Jilin University. ‡ Pacific Northwest National Laboratory.
precursors in the presence of F127 surfactant.31 Liu et al. reported that the hollow silica nanospheres have been successfully prepared by using the core-shell-corona structured micelles of poly(styrene-b-2-vinylpyridine-b-ethylene oxide) (PS-PVP-PEO) as a template.32 However, the studies on the tunable size and functionalization of monodisperse silica nanoparticles with high drug loading capacity have not been reported up to now. Herein, we report a simple route for the synthesis of sizetunable, functional stable silica core-shell nanoparticles by using Pluronic triblock copolymer F127 (EO106PO70EO106) as a template and 1,3,5-trimethylbenzene (TMB) or fatty acid as a swelling agent and/or functional agent in acid aqueous solution. Pyrene and coumarin 153 (C153) were used as fluorescent probe molecules to investigate the effect and location of swelling agent molecules. The size of nanoparticles can be tunable from ∼10 to ∼90 nm, and the drug loading capacity can be adjusted. Furthermore, nanoparticles functionalized by fatty acid can retard the release rate of certain drugs or chemicals. Experimental Section Chemicals. Pluronic nonionic surfactant F127, diethoxydimethylsilane (Me2Si(OEt)2, DEDMS), papaverine, pyrene, coumarin 153 (C153), and Sudan III were purchased from SigmaAldrich. Tetraethoxysilane (TEOS) was purchased from Fluka. TMB, hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid were purchased from Tianjin Guangfu Fine Chemical Research Institute of China. The dialysis bag (Mw cutoff: 8000-14 400) was purchased from Aldrich. All of the chemicals were used without further purification. Synthesis of Nanoparticles. The nanoparticles (SCMCSNs) were synthesized from the F127 template system with organic swelling agents. All of the experiments were carried out at room temperature, unless otherwise mentioned. The following is a typical synthesis example: a desired amount of TMB was added into 5 g of F127 acidic aqueous solution (HCl 0.85 M, F127 5.4 wt %), stirred for 2 days, and 0.5 g of TEOS was added to the resultant solution. The mixture was stirred for 30 min; 46 µL of DEDMS was added to control the extent of silica deposition and stabilize the silica cross-linked particle. After
10.1021/jp910460j 2010 American Chemical Society Published on Web 01/22/2010
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another 30 min of stirring, the nanoparticle solution was filtered with a Teflon filter (0.22 µm). The synthesis of particles containing octanoic acid was the same as that TMB used as a swelling agent except that the concentration of HCl was 0.57 M, and the mixture was treated with an ordinary ultrasonic bath for 1.5 h after addition of octanoic acid. Loading and Release Experiments of Drugs or Dyes. The drug or dye was added into the mixture of F127 micelles and swelling agent before the synthesis of the nanoparticles. In order to remove unloaded drug or dye, the solution was filtered with a Teflon filter (0.22 µm). The nanoparticle solution loaded with drug or dye was cleaned by dialysis against pure water and phosphate buffer solution (pH 7.4, 0.1 M) before the release experiment. The release experiment was carried out: 1 g of nanoparticle solutions containing 5 mg of papaverine were sealed in a dialysis bag, and the drug was released into 250 mL of phosphate buffer solution (pH 7.4, 0.1 M) in a beaker under stirring at room temperature. A 2 mL of solution in the beaker was taken at a predetermined time, analyzed by using a UV spectrometer at 327 nm, and then put back into the beaker after analyzing in 1 min. Characterization. Transmission electron microscopy (TEM) was carried out by using a JEOL 2100F and a JEOL JEM-3010 transmission electron microscope operated at 200 and 300 kV, respectively. The sample for TEM was prepared by directly placing a small drop of the nanoparticle solution on a TEM grid. The measurements of UV absorption of drug or dye loaded samples were done with an Ocean Optics USB4000 spectrometer. The absorbance peak intensities of samples were recorded at 515 nm for Sudan III and 327 nm for papaverine, respectively. The fluorescence emission spectra of pyrene loaded samples were measured with the USB4000 spectrometer at an excitation wavelength of 302 nm. The steady-state fluorescence of C153 in Pluronic aqueous solutions was measured using a PerkinElmer LS-55 luminescence spectrophotometer at an excitation wavelength of 370 nm. The scan speed was set at 1000 nm/ min. The dynamic light scattering (DLS) experiment was carried out with a Malvern Zetasizer Nano-Series instrument at room temperature.
Chi et al.
Figure 1. TEM picture of silica nanoparticles with different sizes prepared by using F127 polymer as a template with TMB swelling agent. (a) ∼10 nm, TMB/F127 ) 0, at 22 °C; (b) 12.3 nm, TMB/F127 ) 0.04, at 13 °C; (c) 14.7 nm, TMB/F127 ) 0.12, 22 °C; (d) 33 nm, TMB/F127 ) 0.57, at 3 °C.
Results and Discussion It is well-known that hydrophobic species may be solubilized inside the hydrophobic regions or normal (oil-in-water) micelles, causing an increase in micelle diameter.33 The solubilization efficiency and capacity depend on the relative sizes of the hydrophobic and hydrophilic blocks, the overall block copolymer molecular weight, and the polymer concentration. This phenomenon is exploited here to control the particle sizes by introducing a swelling agent into the nanoparticle synthesis system. The size and morphology of SCMCSNs were directly observed by a TEM technique. Figure 1 shows the TEM images of SCMCSNs at different ratios of TMB/F127 (by weight). In the TEM images, a uniform dark shell of silica coatings and a light interior can be observed in most cases. It is also observed that the particle size systematically increases with the TMB concentration over a wide range (Figure 1b-d), suggesting that TMB can enlarge the nanoparticles very well. We noticed that the particle sizes also depend on the temperature. A lower temperature (3 °C) (open circles in Figure 2) facilitates the solubilization of TMB in the core region, and produced a large particle size over 30 nm, which is in agreement with the result for the synthesis of large pore sized mesoporous silica with F127 as a template in the presence of TMB under low temperature reaction conditions.34 This phenomenon was
Figure 2. (A) Silica nanoparticle size based on TEM image as a function of the amount of TMB used in the synthesis system. (B) Sudan III loading on silica nanoparticle size based on UV absorption as a function of the amount of TMB used in the synthesis system (squares for 22 °C, triangles for 13 °C, and circles for 3 °C).
explained by the loose micellar structures formed at relatively low temperatures.35-37 As a result, it might be easier for the TMB molecules to enter the micellar core and produce a higher degree of swelling. On the basis of this principle, a higher temperature (22 °C) produced smaller core shell nanoparticles (solid squares in Figure 2). Notably, in this study, we demonstrate that the particle size systematically increases with the TMB concentration over a wide range. Figure 2A shows the particle sizes as a function of the
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Figure 3. TEM image of the SCMCSNs functionalized with octanoic acid: (a) octanoic acid/F127 ) 0.37; (b) octanoic acid/F127 ) 0.74.
TMB/F127 ratios. Clearly, for the experiments conducted at 13 °C, increasing the TMB/F127 ratio (by weight) from 0 to 0.4 causes an increase of the particle size from 10 to 25 nm. For the majority of the concentration range studied, the particle sizes almost increase linearly until about 0.4 (ratio of TMB/F127). However, beyond a TMB/F127 ratio of 0.4, the particle sizes level off, indicating the maximum solubilization of the swelling agent TMB in F127 micelles. We studied the cosolvent effect on the loading capacity using Sudan III as a model compound. Sudan III is a lysochrome (fatsoluble) diazo dye widely used in cosmetic products. We chose this dye in this study because of its low cost, and it can be detected easily by UV spectrum.27 Shown in Figure 2B, the loading capacity for Sudan III increases as the amount of TMB increases. This increase may be attributed to two reasons: (1) the increased particle size to provide more space and (2) the cosolubilization effect of the hydrophobic compound (TMB) inside the core. Similar to the relationship between nanoparticle size and TMB, there is an upper limit for increasing the capacity of Sudan III. The large core of the nanoparticle provides more space for solubilization of hydrophobic compounds. However, in general, the Sudan III solubilization in an enlarged silica core-shell nanoparticle with TMB can be 1 order of magnitude higher than the normal silica core shell nanoparticle without TMB (from 0.08 to 0.8 mM). The functionalization of the nanoparticles is often employed in order to enhance the drug loading capacity38 and control the drug molecule release at a proper rate.39 For example, the PEO-PLA diblock copolymers containing a small quantity of carboxylic acid in the PLA block increase dramatically the model drug papaverine loading efficiency.40 In this study, we use a simple method to functionalize the nanoparticles. We chose fatty acid as a swelling agent and functional agent because of its hydrophobic chain and carboxyl functional group. Figure 3a shows the TEM image of the functionalized SCMCSNs by octanoic acid. When the ratio of octanoic acid/F127 (by weight) is 0.37, the spherical nanoparticles are monodispersed with a size of about 17 nm which is 1.7 times of that for normal SCMCSNs; when the octanoic acid/F127 ratio increases to 0.74, the size of nanoparticles is about 37 nm (see Figure 3b). The number distribution of nanoparticles is measured by DLS, as shown in Figure 4, and the average particle size (Zav values) increases from 20 to 90 nm with increasing ratio of octanoic acid/F127 (by weight) from 0 to 1.48. The particle size from DLS measurement is larger than that from TEM (Figure 3). The reason is that the light scattering measurement includes the PEO chains stretching out into the aqueous solution which cannot be observed by TEM due to the low contrast of the PEO polymers.41 Pyrene is used successfully as a fluorescence probe in research of microenvironment changes because of its photochemical properties. The relative excitation fluorescence intensity ratio of the first peak to the third peak of pyrene is sensitive to
Figure 4. DLS size distribution of the functionalized SCMCSNs with different ratios of octanoic acid/F127 (by weight).
Figure 5. Emission spectra of pyrene (2.4 × 10-5 M, λex ) 302 nm) in the SCMCSNs with different ratios of octanoic acid/F127. For comparison, the spectrum intensities were normalized at 393 nm.
environmental polarity.42 Pyrene has an ability to form an excimer, and the excimer emission is affected by the concentration of pyrene.43,44 Here, we use pyrene as a probe to investigate the hydrophobic environment of the core of nanoparticles. The ratios of I1/I3 are between 1.31 and 1.18 for the SCMCSNs functionalized with octanoic acid, which means the polarity of the core of nanoparticles is very low and the microenvironment is hydrophobic. The formation of pyrene excimer is observed in the SCMCSNs functionalized with octanoic acid. The emission spectra of samples are shown in Figure 5. The fluorescence intensity ratio of the broadband emission measured at 490 nm for excimer to the peak measured at 393 nm for monomer is an index of pyrene excimer formation. It is observed that the ratio decreases greatly from 1.71 to 0.26 as the ratio of octanoic acid/F127 increased from 0 to 1.11. The quick decrease of excimer pyrene is due to the decrease of the pyrene concentration in the hydrophobic region,45 which results from the hydrophobic core of nanoparticle being enlarged by addition of octanoic acid. Since the log P value46 of pyrene is 5.17, we believe that pyrene is a good probe for the environment of the hydrophobic core, not sensitive to the hydrophobic-hydrophilic interface and hydrophilic shell. To understand the structure and feature of SCMCSNs with octanoic acid, we chose C153 as a new effective
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Figure 6. Steady state emission spectra of C153 (2 × 10-6 M, λex ) 370 nm) in the SCMCSNs with different ratios of octanoic acid/F127.
Figure 8. (A) Papaverine loading capacity of octanoic acid swelled SCMCSNs. (B) Release profile of papaverine from swelled nanoparticles with different swelling agents and different amounts of drug: (4) TMB swelled SCMCSNs with 25 mg papaverine; (2, 0, and 9) octanoic acid swelled SCMCSNs with 25, 35, and 75 mg of papaverine, respectively.
Figure 7. Sudan III loading based on UV absorption as a function of fatty acids used in the nanoparticle synthesis system.
probe because of its intermediate polarity (log P ) 4.53) and its large shift emission with microenvironments. C153 molecules prefer to localize in the hydrophobic core but near the PPO-PEO interface of F127 micelles.47 Our experimental results are shown in Figure 6. It is obvious that the C153 emission shifts from 502 to 522 nm with the addition of more octanoic acid. The red shift of 20 nm is attributed to an increase in polarity of microenvironments, which confirms that the carboxyl group of octanoic acid in the nanoparticle locates at the PPO-PEO interface or PEO region. Hexanoic acid, decanoic acid, and dodecanoic acid are also studied as swelling agents and functional agents. Figure 7 shows the loading capacities of Sudan III on these fatty acid functionalized nanoparticles. The loading capacity reflects the effects of swelling agents. The long carbon chain fatty acids are more hydrophobic, fill more space, and are more effective on enlargement of nanoparticles. However, the too long carbon chain is not suitable for swelling the nanoparticles; for example, dodecanoic acid is not suitable to swell the nanoparticles because the acid is solid and has low solubility. Although dodecanoic acid can be introduced into the nanoparticles by adding the cosolvent dichloromethane, the enlarged nanoparticles are only stable for 2 days because the dodecanoic acid isolates from the nanoparticles when dichloromethane molecules evaporate from the nanoparticle solution.
Papaverine was used as a basic model drug to evaluate the loading capacity and release feature of nanoparticles. The amount of drug incorporated into nanoparticles was measured by UV spectrometer and was calculated from the absorbance of papaverine at 327 nm. The drug loading capacity of nanoparticles increases with the increase of the content of the octanoic acid in the block copolymers, as is shown in Figure 8A. This result suggests that the octanoic acid swelled SCMCSNs can supply more space for drug loading. However, the loading capacity of papaverine of the octanoic acid swelled SCMCSNs is higher than that of the TMB swelled SCMCSNs, which may be attributed to the hydrogen bond interaction between octanoic acid and papaverine. The drug release behavior of nanoparticles was investigated by using a dialysis membrane against phosphate buffered (pH 7.4, 0.1 M) solution at room temperature. As is shown in Figure 8B, for TMB swelled SCMCSNs, 78% of the loaded papaverine is released within 20 h, while 55% of papaverine releases from octanoic acid swelled SCMCSNs in the same time. This result may be ascribed to the hydrogen bonding between octanoic acid and papaverine.40 The release feature is enhanced by the interaction between the nanoparticle and drug molecules. When more drug molecules are loaded, the SCMCSN sample can still release drug at a reasonable rate in long time. Conclusions We have successfully prepared stable, silica cross-linked, monodisperse nanoparticles with tunable sizes from ∼10 to ∼90 nm (based on TEM and DLS results) which is the most useful size range for the applications of nanoparticles in biomedical area.48 With the increase of nanoparticle sizes, the loading capacity of SCMCSNs is also significantly increased. The interaction between octanoic acid and drug enhances the release property of SCMCSNs. The fluorescent spectrum behaviors of probe molecules indicate that the carboxylic acid groups of fatty
Size-Tunable and Functional Silica Nanoparticles acid locate at the PPO-PEO interface region of silica crosslinked F127 micelles (SCMCSNs). Acknowledgment. We greatly acknowledge financial support from the National Nature Science Foundation of China (Grant Nos. 20788101 and 20671041), Laboratory-Directed Research and Development Program (LDRD) of the Pacific Northwest National Laboratory (PNNL) of USA, and the Office of Basic Energy Sciences (BES), U.S. Department of Energy (DOE). PNNL is a multiprogram laboratory operated by Battelle Memorial Institute for the Department of Energy under contract DE-AC05-76RL01830. References and Notes (1) Ferrari, M. Nat. ReV. Cancer 2005, 5, 161. (2) Debbage, P. Curr. Pharm. Des. 2009, 15, 153. (3) Park, K.; Lee, S.; Kang, E.; Kim, K.; Choi, K.; Kwon, I. C. AdV. Funct. Mater. 2009, 19, 1553. (4) Singh, R.; Lillard, J. W. Exp. Mol. Pathol. 2009, 86, 215. (5) Suh, W. H.; Suh, Y. H.; Stucky, G. D. Nano Today 2009, 4, 27. (6) Yong, K. T.; Roy, I.; Swihart, M. T.; Prasad, P. N. J. Mater. Chem. 2009, 19, 4655. (7) Kim, J.; Piao, Y.; Hyeon, T. Chem. Soc. ReV. 2009, 38, 372. (8) Khemtong, C.; Kessinger, C. W.; Gao, J. M. Chem. Commun. 2009, 3497. (9) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. J. Controlled Release 2004, 100, 5. (10) Trewyn, B. G.; Slowing, I. I.; Giri, S.; Chen, H. T.; Lin, V. S. Y. Acc. Chem. Res. 2007, 40, 846. (11) Slowing, I. I.; Trewyn, B. G.; Giri, S.; Lin, V. S. Y. AdV. Funct. Mater. 2007, 17, 1225. (12) Shubayev, V. I.; Pisanic, T. R.; Jin, S. H. AdV. Drug DeliVery ReV. 2009, 61, 467. (13) Veerapandian, M.; Yun, K. Dig. J. Nanomater. Biostructures 2009, 4, 243. (14) Brigger, I.; Dubernet, C.; Couvreur, P. AdV. Drug DeliVery ReV. 2002, 54, 631. (15) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969. (16) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. ReV. 2001, 53, 283. (17) Aliabadi, H. M.; Shahin, M.; Brocks, D. R.; Lavasanifiar, A. Clin. Pharmacokinet. 2008, 47, 619. (18) Alvarez-Lorenzo, C.; Concheiro, A. Mini-ReV. Med. Chem. 2008, 8, 1065. (19) Blanco, E.; Kessinger, C. W.; Sumer, B. D.; Gao, J. Exp. Biol. Med. 2009, 234, 123.
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