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Controlling the Particle Size of Interpolymer Complexes through Host-Guest Interaction for Drug Delivery Yan Chen,† Yan Pang,† Jieli Wu,§ Yue Su,† Jinyao Liu,† Ruibin Wang,§ Bangshang Zhu,§ Yefeng Yao,‡ Deyue Yan,† Xinyuan Zhu,*,†,§ and Qun Chen*,‡ †
School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China, ‡ Department of Physics & Shanghai Key Laboratory of Functional Magnetic Resonance Imaging, East China Normal University, North Zhongshan Road 3663, Shanghai 200062, People’s Republic of China, and § Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China Received December 21, 2009. Revised Manuscript Received January 18, 2010 A new method to adjust the particle size of interpolymer complexes has been developed by introduction of host-guest interaction into the dilute aqueous solution of poly(acrylic acid) (PAA) and poly(ethylene glycol) (PEG). Because of the cooperative hydrogen-bonding interaction, PAA can form the interpolymer complexes with PEG. Putting β-cyclodextrin (β-CD) into dilute PAA/PEG aqueous solution, the competition between host-guest and hydrogen-bonding interactions happens. The β-CD/PAA/PEG ternary systems have been well characterized by ultraviolet-visible absorption spectroscopy (UV-vis), dynamic light scattering (DLS), transmission electron microscopy (TEM), diffusion NMR spectroscopy, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and solid-state 13C NMR spectroscopy. The results indicate that the hydrophobic cavity of β-CD is threaded by linear polymers so that the hydrophilicity of PAA/PEG interpolymer complexes is improved greatly. Adjusting the amounts of β-CD, the particle size of the interpolymer complexes can be readily controlled. The low cytotoxicity of various β-CD/PAA/PEG ternary complexes has been confirmed using the MTT assay in COS-7 cell line. Doxorubicin (DOX), an anticancer drug, has been encapsulated into the β-CD/PAA/PEG ternary complexes. The DOX-loaded β-CD/PAA/PEG ternary complexes have been analyzed by confocal laser scanning microscopy (CLSM), flow cytometry analysis, and the MTT assay against human cervical carcinoma cell (Hela). The results indicate that β-CD/PAA/PEG ternary complexes with controlled particle size could be used as safe and promising drug carriers.
1. Introduction It has been well reported that polyacids and proton-accepting polymers can form interpolymer complexes (IPCs) through hydrogen-bonding interaction.1-6 Because of their potential attractive applications in drug delivery,7-9 biomaterials,10 and membrane and separation technology,11,12 interpolymer complexes have drawn considerable attention. Mixing polyacids such as poly(acrylic acid) (PAA) and Lewis polybases such as poly(ethylene glycol) (PEG) in aqueous solution, the cooperative hydrogen-bonding interaction induces the mutual screening of hydrophilic parts of the interacting macromolecules, leading to strong hydrophobization of the resultant complexes. Therefore, the polymer complexes coil up into a compact structure, and the water-insoluble physical network or precipitate is formed. *To whom correspondence should be addressed: Tel þ86-21-34205699, Fax þ86-21-34205722, e-mail
[email protected] (X.Z.); Tel þ86-2162232274, Fax þ86-21-54344800, e-mail
[email protected] (Q.C.).
(1) Bekturov, E. A.; Bimendina, L. A. Adv. Polym. Sci. 1981, 41, 99–147. (2) Tsuchida, E.; Abe, K. Adv. Polym. Sci. 1982, 45, 1–119. (3) Philipp, B.; Dautzenberg, H.; Linow, K. J.; K€otz, J.; Dawydoff, W. Prog. Polym. Sci. 1989, 14, 91–172. (4) Bell, C. L.; Peppas, N. A. Adv. Polym. Sci. 1995, 122, 125–175. (5) Jiang, M.; Li, M.; Xiang, M.; Zhou, H. Adv. Polym. Sci. 1999, 146, 121–196. (6) Th€unemann, A. Prog. Polym. Sci. 2002, 27, 1473–1572. (7) Ozeki, T.; Yuasa, H.; Kanaya, Y. J. Controlled Release 2000, 63, 287–295. (8) Lele, B. S.; Hoffman, A. S. J. Controlled Release 2000, 69, 237–248. (9) Carelli, V.; Di Colo, G.; Nannipieri, E.; Poli, B.; Serafini, M. F. Int. J. Pharm. 2000, 202, 103–112. (10) Chun, M.-K.; Cho, C.-S.; Choi, H.-K. J. Controlled Release 2002, 81, 327–334. (11) Umana, E.; Ougizawa, T.; Inoue, T. J. Membr. Sci. 1999, 157, 85–96. (12) Bell, C. L.; Peppas, N. A. Adv. Polym. Sci. 1995, 122, 125–175.
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However, in some cases such as drug delivery, the formation of independent colloidal particles or core/shell micelles with high stability is preferred. To achieve this goal, two major strategies have been proposed: (1) the electrostatic repulsion interactions are introduced into the colloidal surface to stabilize the particles; (2) the nonionic compounds or solid particles such as PEG and Pickering system are used to provide the necessary hydrophilicity for the stabilization of polymer complexes. In both cases, the interactions between polyacids and polybases or between polymers and solvents are adjusted for reaching a suitable hydrophilic/ hydrophobic balance. Changing the intermolecular interactions, the particle size of interpolymer complexes can be well controlled. With the rapid development of supramolecular chemistry, various noncovalent interactions including the host-guest interaction have been used to design and construct nanoparticles. As one of the most popular supramolecular hosts, cyclodextrin (CD) receives intense investigation due to its ability to selectively encapsulate a wide range of guest molecules via inclusion complexation.13-30 Especially, the (13) Harada, A.; Kamachi, M. Macromolecules 1990, 23, 2821–2813. (14) Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325–327. (15) Koopmans, C.; Ritter, H. J. Am. Chem. Soc. 2007, 129, 3502–3503. (16) Fujita, H.; Ooya, T.; Yui, N. Macromolecules 1999, 32, 2534–2541. (17) Wenz, G.; Keller, B. Angew. Chem., Int. Ed. 1992, 31, 197–199. (18) Li, J.; Harada, A.; Kamachi, M. Polym. J. 1994, 26, 1019–1026. (19) He, L.; Huang, J.; Chen, Y.; Liu, L. Macromolecules 2005, 38, 3351–3355. (20) Sabadini, E.; Cosgrove, T. Langmuir 2003, 19, 9680–9683. (21) Nostro, P. L.; Lopes, J. R.; Cardelli, C. Langmuir 2001, 17, 4610–4615. (22) Jiao, H.; Goh, S. H.; Valiyaveettil, S. Macromolecules 2001, 34, 8138–8142. (23) Ritter, H.; Tabatabai, M. Prog. Polym. Sci. 2002, 27, 1713–1720. (24) Rusa, C. C.; Bridges, C.; Ha, S. W.; Tonelli, A. E. Macromolecules 2005, 38, 5640–5646.
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hydrophobic cavity of CD can be threaded by linear polymer to form the inclusion complexes. It can be imaged that putting CD into a hydrogen-bonding interpolymer complexes system, the competition between host-guest and hydrogen-bonding interactions could happen. Therefore, the cooperative hydrogenbonding interaction of interpolymer complexes might be adjusted by the amount of CD. In the present work, β-CD was introduced into a dilute PAA/PEG aqueous solution. Because of the host-guest interaction between β-CD and linear polymers, the cooperative hydrogen-bonding interaction in interpolymer complexes is weakened so that the hydrophilicity of colloidal particles is improved greatly. Adjusting the amount of supramolecular hosts, the particle size of interpolymer complexes can be readily controlled. Since β-CD, PAA, and PEG have all been used as drug delivery materials, these β-CD/PAA/PEG ternary particles can be very useful in the field of drug delivery.
2. Experimental Section 2.1. Materials. PEG with Mn = 6000 was purchased from Shanghai Sinopharm Chemical Reagent Corp. PAA (Mn = 100 000, 35 wt % in water) was acquired from Aldrich Chemical Corp. β-CD was purchased from Shanghai Sinopharm Chemical Reagent Corp. and purified once by recrystallization from deionized water before use. Doxorubicin hydrochloride was purchased from Beijing Huafeng United Technology Corp. and used as received. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma and used as received. Clear polystyrene tissue culture treated 6-well and 96-well plates were obtained from Corning Costar.
2.2. Preparation of β-CD/PAA/PEG Ternary Complexes. Different amounts of β-CD were dissolved in aqueous solutions. Then, PAA aqueous solution (7.2% w/v) and the same volume of PEG aqueous solution (4.4% w/v) were added into β-CD solutions successively. The mixtures with different molar ratio of β-CD to PEG (or PAA) repeating unit were stirred at room temperature for 24 h and then allowed to stand overnight. In order to remove free β-CD from β-CD/PAA/PEG systems, mineral ether was added into the ternary aqueous solutions to form the crystalline inclusion complexes with β-CD. The resulting precipitate was filtered, and the upper layer of mineral ether was removed from the solutions. The solid samples were collected by solution casting and further dried under vacuum at 40 °C to constant weight.
2.3. Preparation of DOX-Loaded β-CD/PAA/PEG Ternary Complexes. Certain amounts of DOX 3 HCl were
added into β-CD/PAA/PEG aqueous solutions. After stirring overnight, the obtained DOX-loaded solutions were dialyzed against distilled water for 24 h to remove free DOX. The drugloaded solutions were then analyzed by UV absorbance at 500 nm to calculate the DOX concentration. 2.4. Characterization. The turbidity of the mixing solutions was measured by a UV/vis spectrophotometer “722” (China) at wavelength λ = 540 nm at room temperature. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano-S apparatus equipped with a 4.0 mW laser (25) Chen, L.; Zhu, X.; Yan, D.; Chen, Y.; Chen, Q.; Yao, Y. Angew. Chem., Int. Ed. 2006, 45, 87–90. (26) Xue, J.; Jia, Z.; Jiang, X.; Wang, Y.; Chen, L.; Zhou, L.; He, P.; Zhu, X.; Yan, D. Macromolecules 2006, 39, 8905–8907. (27) Zhu, X.; Chen, L.; Yan, D.; Chen, Q.; Yao, Y.; Xiao, Y.; Hou, J.; Li, J. Langmuir 2004, 20, 484–490. (28) Hwang, M. J.; Bae, H. S.; Kim, S. J.; Jeong, B. Macromolecules 2004, 37, 8820–8822. (29) Chung, J. W.; Kang, T. J.; Kwak, S. Y. Macromolecules 2007, 40, 4225– 4234. (30) Wang, Y.; Zhou, L.; Sun, G.; Xue, J.; Jia, Z.; Zhu, X.; Yan, D. J. Polym. Sci., Polym. Phys. Ed. 2008, 46, 1114–1120.
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operating at λ = 633 nm and at a scattering angle of 173°. Transmission electron microscopy (TEM) measurements were carried out on a JEOL 2010 instrument operating at a voltage of 100 kV. Samples were prepared by dropping the solutions onto carbon-coated copper grids and then air-dried before measurement. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) measurements were performed on a Bruker Equinox 55 spectrometer with ATR crystal of zinc selenide (ZnSe). Each spectrum was averaged from 64 scans collected at 4 cm-1 resolution with a wavelength range from 650 to 4000 cm-1. The diffusion coefficients (D) were determined on a Bruker AVANCE III-400 M spectrometer with a maximum Z-direction gradient strength of about 50 G cm-1. Samples for NMR measurements were the same as those mentioned above except the solvent was replaced by D2O. All spectra were acquired at 300 K with 16 accumulations and a 10 s recycle delay. The D values for all samples were determined according to the following equation: I ¼ I0 exp½ -Dγ2 G2 δ2 ðΔ -δ=3Þ
ð1Þ
where I is the observed intensity; I0 is the reference intensity; G, the gradient amplitude, is increased from 2% to 95% in 16 steps; δ, the duration of the gradient, is set to 12 ms; γ is the gyromagnetic ratio of proton; and Δ, the diffusion time, is set to 200 ms. Solidstate 13C CP/MAS spectra were acquired on a Bruker DSX-300 spectrometer operated at 75.47 MHz with a spinning rate of 5 kHz. The 90° pulse width of 1H was 4 μs, and the contact time for CP was 1 ms. For MTT assay of β-CD/PAA/PEG ternary complexes, COS-7 cells were seeded into 96-well plates at a seeding density of 5000 cells/well in 200 μL medium. After 24 h incubation, the culture medium was removed and replaced with 200 μL medium containing serial dilutions of the polymers. The cells were grown for 24 h. Then, 20 μL of MTT stock solution (5 mg/mL) in PBS was added to each well. After 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL/well DMSO and measured spectrophotometrically in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm. The cytotoxicity of DOX-loaded β-CD/PAA/PEG nanoparticle solutions to Hela cell was also evaluated by the MTT method. The cellular uptake experiments were performed on flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry, COS-7 cells were seeded in 6-well plates at 3 105 per well in 1 mL complete DMEM and cultured for 24 h, followed by removing culture medium and adding DOX-loaded β-CD/PAA/PEG ternary complexes (1 mL of DMEM medium) at the DOX concentration of 10 μg/mL for 30 min, 1 h, and 3 h. Thereafter, culture medium was removed, and cells were washed with PBS three times and treated with trypsin. Then, 2 mL of PBS was added to each culture wells, and the solutions were centrifugated for 5 min (1000 rpm). After removing the supernates, the cells were resuspended in 0.5 mL of PBS. Data for 1 104 gated events were collected, and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. For CLSM studies, COS-7 cells were seeded in 6-well plates at 2 105 cells per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing culture medium and adding DOXloaded PEG/PAA/β-CD ternary complexes (1 mL of DMEM medium) at the DOX concentration of 10 μg/mL. After incubation at 37 °C for 1 h, culture medium was removed and cells were washed with PBS three times. Then, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the cells were stained with 2-(4amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min, and the slides were rinsed with PBS for three times. The slides were mounted and observed by a LSM 510META. Langmuir 2010, 26(11), 9011–9016
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Figure 1. Photographs of β-CD/PEG/PAA ternary solutions with different mole% of β-CD: 0 (a), 5% (b), 10% (c), 20% (d), and 50% (e).
Figure 3. Particle size of β-CD/PAA/PEG ternary solutions with different β-CD content determined by DLS. Table 1. β-CD/PEG/PAA Ternary Solutions composition
diffusion coefficient βEG AA av of PEG CD unit unit diam (10-11 entry (mol) (mol) (mol) (nm) PDI m2 s-1) 1 2 3 4 5 6
Figure 2. Transmittance of β-CD/PAA/PEG ternary solutions
versus β-CD content.
3. Results and Discussion Polyacids such as PAA and Lewis polybases such as PEG can form the interpolymer complexes through hydrogen-bonding interaction.31 Mixing 0.1 mol/L PAA and PEG aqueous solutions together with a volume ratio of 1:1, the system became turbid immediately, as shown in Figure 1a. Apparently, the cooperative hydrogen-bonding interaction between the carboxyl groups of PAA and ether oxygens of PEG induces the occurrence of complexation. The mutual screening of hydrophilic groups of the interacting macromolecules leads to strong hydrophobization of the resultant complexes. Therefore, the PAA/PEG complexes coil up to compact structure and then aggregate to large particles.32 β-CD, which has a unique molecular structure of hydrophobic cavity and hydrophilic outer surface, can form inclusion complexes with linear polymers through host-guest interaction. Adding β-CD into a dilute PAA/PEG aqueous solution, a competition between host-guest and hydrogen-bonding interactions happens. Correspondingly, the cooperative hydrogen-bonding interaction between PAA and PEG is weakened. Figures 1b-e give the photographs of β-CD/PAA/PEG ternary systems in an aqueous solution. With the increase of β-CD content from 5% to 50% (mole ratio of β-CD to EG or AA repeating unit), the system changes from milky solution to transparent solution. The more β-CD the mixing solution has, the less turbid it is. The transmittance of the mixing solutions was measured. Figure 2 shows the variation of transmittance of β-CD/PAA/PEG aqueous solutions versus the content of β-CD. The transmittance increases greatly (31) Bailey, F. E., Jr.; Lundberg, R. D.; Callard, R. W. J. Polym. Sci., Part A 1964, 2, 845–851. (32) Zhuunuspayev, D. E.; Mun, G. A.; Hole, P.; Khutoryanskiy, V. V. Langmuir 2008, 24, 13742–13747.
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0 0.05 0.1 0.2 0.5 0
1 1 1 1 1 1
1 1 1 1 1 0
207.5 169.3 41.0 12.2 7.0
0.23 0.24 0.27 0.37 0.38
3.13 3.31 3.43 4.66 6.83 7.39
concn of DOX loaded in the complexes (μg/mL) 21.3 23.2 21.3 27.9 36.4
with β-CD concentration, agreeing with the photographic observation very well. It can be concluded that β-CD has an important influence on the complexation of PAA and PEG. To gain insight into the structure of β-CD/PAA/PEG ternary complexes, both DLS and TEM measurements were performed. DLS results in Table 1 and Figure 3 display the effect of β-CD content on the particle size of interpolymer complexes. For the sample without any β-CD, the particle size is larger than 200 nm. After addition of 5% β-CD, the particle diameter decreases to 170 nm. Further increasing the content of β-CD leads to the rapid decrease of particle size. For example, the particle diameter is reduced to 40 and 12 nm when the β-CD content reaches 10% and 20% respectively. Finally, only small particles with a diameter of 7 nm can be observed when the content of β-CD reaches 50%. Figure 4 gives TEM images of the β-CD/PAA/PEG samples with different β-CD amount. With the increase of β-CD from 0 to 10%, the diameter of interpolymer complexes decreases from 220 to 40 nm. For samples containing 20% and 50% β-CD, no clear TEM image could be obtained because of the bad image contrast and low resolution of small particles. TEM observation further confirms the change of the particle size of β-CD/PAA/ PEG ternary complexes with different β-CD content. All these aforementioned results indicate that the particle size of interpolymer complexes can be readily adjusted by introducing different amounts of β-CD into dilute PEG/PAA aqueous solutions. The particle size change of interpolymer complexes has been further corroborated by the NMR diffusion-ordered spectroscopy (DOSY) using the pulsed gradient field technique. The relation between diffusion coefficient and particle size can be described by the Stokes-Einstein equation:33 D ¼
kT 6πηrs
ð2Þ
(33) Cameron, K. S.; Fielding, L. Magn. Reson. Chem. 2002, 40, S106–S109.
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Figure 4. TEM images for β-CD/PEG/PAA samples with different β-CD content: 0 (a), 5% (b), and 10% (c).
where D is the diffusion coefficient, k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the medium, and rs is the hydrodynamic radius. The diffusion coefficient is sensitive to the structural properties of the observed molecular species such as weight, size, and shape as well as binding phenomena, aggregation, and molecular interactions.34 Therefore, the diffusion coefficients of PEG solution, PAA/PEG mixing solution, as well as those of β-CD/PAA/PEG ternary systems were measured, and the results are summarized in Table 1. In pure PEG solution, PEG exhibits a diffusion coefficient about 7.39 10-11 m2 s-1, while in the PAA/PEG mixing solution without β-CD, the diffusion coefficient decreases to be 3.13 10-11 m2 s-1. It is clear that the decrease of the diffusion coefficient should be attributed to the increase of particle size resulting from the formation and aggregation of interpolymer complexes between PAA and PEG. When different amounts of β-CD (from 5% to 50%) are mixed with PAA/PEG solutions, the diffusion coefficient increases from 3.31 10-11 to 6.83 10-11 m2 s-1, indicating the decrease of particle size. Apparently, β-CD prevents the interpolymer complex particles from aggregating into large particles. Since the outer surface of β-CD is hydrophilic and the hydrophobic cavity can be threaded by linear polymers, it is deduced that the hydrophilic/hydrophobic balance is changed by the formation of polyrotaxane/pseudopolyrotaxane between β-CD and polymers. Thus, the interpolymer complexes are deaggregated into small particles. The existence of polyrotaxane/pseudopolyrotaxane structure can be confirmed by solid-state 13C NMR measurements.35,36 Figure 5f shows the 13C CP/MAS spectrum of pure β-CD. In a pure and crystalline state, β-CD adopts an asymmetrical conformation and is represented by multiple resolved C1 and C4 resonances of the glucose units. When the guest molecules are included into the internal cavities of β-CD, the β-CD molecules acquire a symmetrical cyclic conformation, resulting in single C1 and C4 resonances. Figures 5b-e show that for β-CD/PAA/PEG ternary complexes each carbon of the glucose units merges into a single peak. It means that β-CD molecules in the complex samples have a more symmetric conformation than in pure β-CD. In other words, β-CD host molecules are threaded by the linear polymer chains through their internal cavities. From 13C CP/MAS spectra (Figure 5a-e), we also find the existence of the resonance at 176 ppm, which corresponds to the carboxyl carbons of PAA in the complex form for all five samples,37 indicating the
(34) (35) (36) (37)
Cohen, Y.; Avram, L.; Frish, L. Angew. Chem., Int. Ed. 2005, 44, 520–554. Harada, A.; Li, J.; Kamachi, M. Macromolecules 1993, 26, 5698–5703. Gidley, M. J.; Bociek, S. M. J. Am. Chem. Soc. 1988, 110, 3820–3829. Miyoshi, T.; Takegoshi, K.; Hikchi, K. Polymer 1997, 38, 2315–2320.
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13 C CP/MAS spectra of β-CD/PEG/PAA with different β-CD content: 0 (a), 5% (b), 10% (c), 20% (d), 50% (e), and pure βCD (f).
Figure 5.
hydrogen-bonding interaction between PAA and PEG in the interpolymer complexes. FTIR spectroscopy is quite useful in characterizing the hydrogen bonding in interpolymer complexes.38 Figure 6 presents the ATR-FTIR spectra of β-CD/PAA/PEG ternary complexes. The absorption at 1955 cm-1, which is a satellite band of a hydrogen bonded hydroxyl groups, demonstrates the existence of hydrogen bonding in the interpolymer complexes with or without β-CD. Based on the aforementioned experimental results, the proposed supramolecular structure model of β-CD/PAA/PEG ternary complexes can be illustrated in Scheme 1. In PAA and PEG mixing solution, the particles of interpolymer complexes have strong hydrophobization, so they coil up and aggregate to large dimension. When β-CD hosts are added into the system, the hydrogen-bonding interaction between β-CD and PAA forms. More importantly, β-CD is threaded by the linear polymers, which partially destroy the cooperative hydrogen bonding between PAA and PEG. Therefore, the hydrophilicity of the interpolymer complexes is improved greatly, resulting in the formation of small particles. Considering that β-CD, PAA, and PEG were all used as drug delivery materials, the resulting β-CD/PAA/PEG ternary particles were evaluated for potential application as drug carriers. The cytotoxicity of β-CD/PAA/PEG ternary complexes to COS-7 cells was determined by the MTT method. Figure 7 shows the cell (38) Lu, X. Y.; Weiss, R. A. Macromolecules 1995, 28, 3022–3029.
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Figure 6. ATR-FTIR spectra of β-CD/PEG/PAA with different β-CD content: 0 (a), 5% (b), 10% (c), 20% (d), and 50% (e). Scheme 1. Influence of β-CD on the Complexation of PAA and PEG
Figure 7. Cell viability of COS-7 against β-CD/PEG/PAA ternary complexes after cultured for 24 h with different PEG and PAA concentration. β-CD content: 0 (a), 5% (b), 10% (c), 20% (d), and 50% (e).
Figure 8. Flow cytometry histogram profiles of COS-7 cells incubated with the DOX-loaded β-CD/PEG/PAA complexes.
viability after incubation for 24 h with β-CD/PAA/PEG ternary complexes at different polymer concentration (the total amount of PEG and PAA in the solution). The results demonstrate that COS-7 cells cultured with β-CD/PAA/PEG ternary complexes remain viable when the concentration of PAA and PEG is up to 1 mg/mL. This is to say, β-CD/PAA/PEG ternary nanoparticles have good biocompatibility and low cytotoxicity
to COS-7 cells. Therefore, these β-CD/PAA/PEG ternary complexes might be used as potential drug delivery carriers. Here, DOX, an antitumor drug, was chosen as a model hydrophobic drug. The concentration of DOX loaded in different β-CD/ PAA/PEG ternary complexes is listed in Table 1. It shows that varying β-CD content has only limited effect on the DOX loading content.
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Figure 9. CLSM images of COS-7 cells incubated with DOXloaded β-CD/PEG/PAA complexes (A, B, C) and DOX in free form (D, E, F) for 1 h. The red fluorescence is from DOX (A, D), and the blue is from the nuclei (cell nuclei were stained with DAPI) (B, E). The third column illustrates overlaid images (C, F).
In order to evaluate the potential intracellular application of β-CD/PAA/PEG ternary complexes, the measurement of cell internalization by COS-7 cells was conducted. First, flow cytometry analysis was performed to evaluate cellular uptake of DOX-loaded β-CD/PAA/PEG ternary complexes. DOX-loaded β-CD/PAA/PEG ternary complexes (10 μg/mL) were added to culture medium and the COS-7 cells incubated at 37 °C for the desired time. Histograms of cell-associated DOX fluorescence for COS-7 cells are shown in Figure 8. The relative geometrical mean fluorescence intensities of DOX-loaded polymeric complex pretreated cells are much higher than that of nonpretreated cells. These prominent fluorescence signals are associated with the specific uptake of the DOX-loaded β-CD/PAA/PEG ternary complexes into COS-7 cells. The mean fluorescence intensity changes with β-CD content in the DOX-loaded β-CD/PAA/ PEG ternary complexes, indicating the particle size effect on the cellular internalization progress. To further confirm the cell permeability of the DOX-loaded β-CD/PAA/PEG ternary complexes, CLSM measurements were performed. In CLSM studies, the COS-7 cells were incubated with the DOX-loaded β-CD/ PAA/PEG ternary complexes for 1 h and then stained the cell nucleus with DAPI. The pretreated cells were then observed under CLSM. Figure 9 shows that the DOX fluorescence appears in all cells but mainly in cytoplasm. These results indicate that the DOX-loaded β-CD/PAA/PEG ternary complexes are successfully internalized by COS-7 cells and mainly resided in cytoplasm. Actually, this is very important for intracellular applications of the polymer complexes, such as intracellular drug delivery. The ability of DOX-loaded β-CD/PAA/PEG ternary complexes to inhibit the proliferation of Hela cells was evaluated by the MTT assay, compared to DOX in free form. The Hela cells were treated with the DOX-loaded β-CD/PAA/PEG ternary complexes and free DOX at different DOX dose from 0.25 to 4 μg/mL. Figure 10 demonstrates that most of DOX-loaded β-CD/ PAA/PEG ternary complexes exhibit higher inhibition to Hela proliferation after 48 h culture in comparison with DOX in free form. It is conceivable that the DOX in free form across the cell
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Figure 10. Cell viability of Hela against DOX-loaded β-CD/PEG/ PAA complexes and DOX in free form after cultured for 48 h. β-CD content: 0 (a), 5% (b), 10% (c), 20% (d), 50% (e), and DOX in free form (f).
membrane directly is a less efficient means to deliver drugs into the cells because the drug molecules diffuse in and out of the cell depending on the concentration gradient. However, the cellular uptake of the DOX-loaded complexes is unidirectional. Hence, the drug is not only more efficiently transported but also retained at the site of action inside the cell. Then, the persistently intracellular release of DOX from the β-CD/PAA/PEG ternary complexes might enhance inhibition to Hela cells growth. It can be also found that samples with various β-CD display different anticancer efficacy. Among them, the sample with 10% β-CD is the most effective, which suggests the particle size has some effect on the inhibition process.
4. Conclusions A new strategy to control the particle size of interpolymer complexes based on the host-guest interaction has been developed. By introducing different amounts of β-CD into PAA/PEG aqueous solution, the cooperative hydrogen-bonding between PAA and PEG is partially replaced by the host-guest interaction between β-CD and polymers, which enhances the hydrophilicity of interpolymer complexes greatly. With the increase of β-CD content from 0 to 50%, the size of interpolymer complex particles decreases from 207 to 7 nm. The resultant β-CD/PAA/PEG ternary complexes show low cytotoxicity and can be efficiently internalized by vivid cells. Therefore, these β-CD/PAA/PEG ternary complexes with controlled particle size could be used as safe and promising drug carriers. Acknowledgment. This work is sponsored by the National Natural Science Foundation of China (50773037, 50633010, 20974062) and National Basic Research Program 2009CB930400, the Fok Ying Tung Education Foundation (111048), Shuguang Program (08SG14), and Shanghai Leading Academic Discipline Project (Project No. B202). Supporting Information Available: DLS data of β-CD/ PAA/PEG and DOX-loaded complexes and photographs of β-CD/PAA/PEG complexes with different PEG to PAA ratios and R-CD/PAA/PEG complexes. This material is available free of charge via the Internet at http://pubs.acs.org.
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