© Copyright 2002 American Chemical Society
JUNE 11, 2002 VOLUME 18, NUMBER 12
Letters Block Copolymer Self-Assembly into Monodispersive Nanoparticles with Hybrid Core of Antisense DNA and Calcium Phosphate Yoshinori Kakizawa† and Kazunori Kataoka* Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received November 29, 2001. In Final Form: April 22, 2002 Self-assembled particles comprised of calcium phosphate, oligonucleotide, and block copolymers of poly(ethylene glycol)-block-poly(aspartic acid) (PEG-PAA) were prepared by the simple mixing of calcium/DNA and phosphate/PEG-PAA solutions. It was shown that the block copolymerization of PEG and PAA segments is essential to prevent precipitation of calcium phosphate crystals and to allow nanoparticles to form. With dynamic light scattering measurements, the diameters of the particles were determined to be around 100 nm with a significantly narrow size distribution. The particles have the ability to incorporate DNA in the core with sufficient efficiency as determined by gel permeation chromatography and fluorescence measurements. Further, the cytotoxicity of the particles assessed by MTT (3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide) assay was significantly low. The organic-inorganic hybrid nanoparticles containing DNA molecules thus prepared are expected to be utilized as DNA delivery systems for gene and antisense therapy.
The synthesis of functional particles with nanometer dimensions has attracted growing interest from both fundamental and applied fields of science.1 One of the most promising strategies to synthesize such particles is to make use of the self-association process of amphiphilic block copolymers.2 Block copolymers aligned at the interface between two phases can prevent aggregation of colloidal dispersants, often resulting in the formation of * To whom correspondence should be addressed. Phone: +813-5841-7138 Fax: +81-3-5841-8653. † Present address: Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tukuba-shi, Ibaraki 305-0044, Japan. (1) (a) Caruso, F.; Caruso R. A.; Mohwald, H. Science 1998, 282, 1111-1114. (b) Li, M.; Schnablegger, H.; Mann, S. Nature 1999, 402, 393-395. (c) von Werne, T.; Patten, T. E. J. Am. Chem. Soc. 1999, 121, 7409-7410. (d) Elder, S. H.; Cot, F. M.; Su, Y.; Heald, S. M.; Tyryshkin, A. M.; Bowman, M. K.; Gao, Y.; Joly, A. G.; Balmer, M. L.; Kolwaite, A. C. Magrini, K. A.; Blake, D. M. J. Am. Chem. Soc. 2000, 122, 51385146. (e) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Mater. Today 2001, 47, May/June, 30. (f) Colfen, H. Macromol. Rapid. Commun. 2001, 22, 219252.
spherical particles with a separated phase core coated with a polymer layer having a steric repulsion effect. Especially, nanoparticles of polymeric micelles formed from amphiphilic block copolymers in water have been well studied and are regarded as vehicle systems used for drug delivery because the micellar core segregated from the aqueous phase can serve as a reservoir of hydrophobic drugs including cytotoxic reagents.3 Recent studies showed that the inner core of the block copolymer micelles can be a polyion complex comprised of oppositely charged polymers4 or a metal-polymer complex,5 which precipitates as insoluble aggregates unless in the form of block (2) (a) Webber, S. E., Munk, P., Tuzar, Z., Ed. Solvents and Selforganization of Polymers; Kluwer Academic Publishers: Dordrecht, Netherlands, 1996. (b) Zhang, L.; Eisenberg, A. Science 1995, 268, 17281731. (3) (a) Kataoka, K.; Yokoyamam, M.; Kwon, G. S.; Okano, T.; Sakurai, Y. J. Contolled Release 1993, 24, 119-132. (b) Kwon, G. S.; Kataoka, K. Adv. Drug Deliv. Rev. 1995, 16, 295-309. (c) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug. Deliv. Rev. 2001, 47, 113-131. (d) Kakizawa, Y.; Kataoka, K. Adv. Drug. Deliv. Rev. 2002, 54, 203-222.
10.1021/la011736s CCC: $22.00 © 2002 American Chemical Society Published on Web 05/16/2002
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Figure 1. Schematic illustration of the spontaneous formation of the CaP/PEG-PAA/DNA particles.
copolymers. Utilizing this strategy, we herein report the formation of monodispersive nanoparticles of calcium phosphate (CaP) crystals surrounded by hydrophilic polymer palisades through complexation with block copolymers and also the capability of such nanoparticles to incorporate DNA molecules (Figure 1). It should be noted that the control of the growth and size of the CaP/DNA hybrid is of great importance in the field of developing DNA delivery systems. Most of the synthetic nonviral DNA delivery systems already reported are composed of positively charged organic molecules such as cationic lipids and cationic polymers because of their ability to form a complex with negatively charged DNA molecules.6 Among the few exceptions, the CaP coprecipitation method has been used to introduce exogenous DNA including plasmid DNA7a-b and oligonucleotides7c into cells for decades. When calcium and phosphate solutions are mixed in the presence of DNA, DNA coprecipitates with a CaP crystal, followed by uptake into cells. However, the major limitation of this method is the uncontrollable rapid growth of CaP after mixing of the solutions, which results in a heterogeneous size distribution of CaP/DNA hybrids that induces a large deviation in the transfection efficiency, because the size of the CaP/ DNA crucially affects the transfection process.7b Thus, it is essential to develop the methodology to control the size of CaP in order to obtain the maximum efficiency. To this end, we first evaluated the effect of the homopolymer of poly(aspartic acid) (PAA; degree of polymerization (DP) 51), which is known to adsorb on CaP crystals,8 on the growth of the CaP-DNA composite. CaP crystals were formed by the simple mixing of calcium and phosphate solutions under the conditions employed in typical protocols used to introduce DNA into cells with slight variations. In this study, 16-mer oligonucleotide with the complementary sequence to insulin-like growth factor II was purchased from Takara Shuzo Co., Ltd., Japan, and used as a model DNA.9 One volume of the 2× calcium/DNA solution10 was added to an equal volume of (4) (a) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 52945299. (b) Harada, A.; Kataoka, K. Science 1999, 283, 65-67. (c) Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem. Soc. 1999, 121, 11247-11248. (5) (a) Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1999, 15, 377-383. (b) Nishiyama, N.; Kato, Y.; Sugiyama, Y.; Kataoka, K. Pharm. Res. 2001, 18, 1035-1041. (6) (a) Kabanov, A. V., Felgner, P., Seymour, L. W., Ed. Selfassembling Complexes for Gene Delivery; John Willey & Sons: Chichester, England, 1998. (b) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556-58557. (c) Lim, Y.; Kim, C.; Kim, K.; Kim, S. W.; Park, J. J. Am. Chem. Soc. 2000, 122, 6524-6525. (7) (a) Chen, C.; Okayama, H. Mol. Cell. Biol. 1987, 7, 2745-2752. (b) Jordan, M.; Schallhorn, A.; Wurm, F. M. Nucleic Acids Res. 1996, 24, 596-601. (c) Tolou, H. Anal. Biochem. 1993, 215, 156-158. (8) Tsortos, A.; Nancollas, G. H. J. Colloid Interface Sci. 1999, 209, 109-115. (9) Lin, S., Hsieh, S., Hsu, H., Lai, M., Kan, L., Au, L. J. Biochem. 1997, 122, 717-722. (10) 250 mM CaCl2, DNA 70 µg/mL. 1 mM Tris-HCl, 0.1 mM EDTA, pH 7.6.
Figure 2. The effect of PAA and PEG-PAA on the growth of CaP crystals evaluated by turbidity measurements. (a) Change in turbidity accompanied with crystallization in the presence of the PAA homopolymers (polymer concentration: (0) 14 µg/ mL, (]) 29 µg/mL, (O) 43 µg/mL, (4) 57 µg/mL). (b) Turbidity at 3 min after the mixing of solutions containing (0) the PAA homopolymer, (O) the PEG and PAA homopolymers, (4) the PEG-PAA block copolymer. Concentrations are normalized as the PAA contents. Total polymer concentrations of four CaP/ PEG-PAA solutions are 70, 140, 210, and 280 µg/mL, respectively.
the 2× phosphate/PAA solution11 (final concentrations: DNA 35 µg/mL, calcium ion 125 mM, phosphate ion 0.75 mM). The effects of PAA on the growth of the crystals were evaluated by the turbidity measurement.12 The increase in the turbidity of the solution accompanied with the growth of CaP was monitored by the change in the transmittance at 350 nm. At this wavelength, no components of the solutions showed absorption. Calcium/DNA and phosphate/PAA solutions preincubated at 37 °C were directly mixed in a polystyrene cuvette with continuous stirring at 1000 rpm. Figure 2a shows the time course of the change in transmittance during the initial 3 min at various PAA concentrations. The transmittances of the solutions were rapidly decreased after mixing the solutions (11) 140 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.1 (12) Turbidity measurements were performed using a V-550 instrument (Jasco, Japan).
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and reached constant values within 30 s. These plateau values decreased as the concentrations of PAA increased. These results suggest that PAA is involved in the early event of the crystallization, possibly, the nucleation process, and the enhancement of the crystal growth rather than inhibition occurs in the presence of the PAA homopolymer. In an effort to control the crystal growth, we synthesized the diblock copolymers in which the end of the interacting PAA segments (DP 24) are covalently linked to the end of the poly(ethylene glycol) (PEG; Mw 12000), the noninteracting segment, and investigated their role in the crystal growth using turbidity measurements (Figure 2b). A detailed synthetic procedure of PEG-PAA was previously reported.5a Briefly, the polymerization of the N-carboxy anhydride of β-benzyl L-aspartate was initiated by the terminal amino group of R-methoxy-ω-aminoPEG. The deprotection of the benzyl group was conducted by alkaline hydrolysis in 0.5 N NaOH to obtain the block copolymer. In the presence of the block copolymers over the concentration range of 70-280 µg/mL, which corresponds to a 14-57 µg/mL PAA segment as indicated on the x axis of Figure 2b, the turbidity after the mixing of the calcium/ DNA and phosphate/PEG-PAA solutions was not apparent, maintaining 97-99% of the initial transparency. Furthermore, the transparency of the block copolymer/ CaP solutions remained for at least a month without any precipitate or turbidity formation. On the other hand, a rapid increase in the turbidity of the solutions was observed for the CaP solution with the simple mixture of the PAA and PEG homopolymer as in the case of the PAA/ CaP system. The transmittance measured at 3 min after mixing showed no significant difference between the solutions containing PAA only and those containing both the PEG and PAA homopolymers, supporting the noninfluencing nature of the PEG homopolymer on CaP crystallization. The difference between DP of the PAA homopolymer and that of the PAA segment of the block copolymer is not likely the cause of inhibition of precipitation because solutions containing the block copolymer with DP of PAA 67 showed 92-98% in transmittance at polymer concentrations ranging from 70 to 280 µg/mL (data not shown). These results clearly show the essential importance of the covalent linkage between the PEG and PAA segments to form a block copolymer in order to inhibit the overgrowth of the CaP crystals. To elucidate the sizes of the CaP crystals in the presence of the block copolymer, PEG-PAA, dynamic light scattering (DLS) measurements were performed.13 The CaP crystals were prepared by mixing of the calcium/DNA and phosphate/PEG-PAA solutions, followed by vigorous stirring using a vortex mixer and incubation at 37 °C overnight before the DLS measurements. The data at a detection angle of 90° were analyzed by the cumulant method and the obtained diffusion coefficient of the particles was converted to hydrodynamic diameters using the StokesEinstein equation.14 Figure 3 shows the diameters of the particles plotted versus the polymer concentration. The diameters ranged from 91 to 125 nm with the minimum value at the polymer concentration of 140 µg/mL. The most likely explanation for the observed minimum is that the sizes of the particles are modulated in the balance between two conflicting factors: (1) the particle size (13) DLS measurements were carried out using a DLS-7000 instrument (Otsuka Electronics Co., Ltd., Japan). Vertically polarized light of 488 nm wavelength from an argon ion laser was used as the incident beam. All measurements were carried out at 25 °C. (14) Elias, H. G. In Light Scattering from Polymer Solutions; Huglin, M. B., Ed.; Academic Press: New York, 1972; Chapter 9.
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Figure 3. Diameters of CaP particles determined by DLS measurements (n ) 3, (standard deviation).
reduction due to the increase in the surface stabilizing effect with an increased concentration of the block copolymer, and (2) the enhanced rate of crystal growth due to an increase in the concentration of the PAA segment. The polydispersity indices of the particles, which are equivalent to the normalized z-average variance of the distribution of the diffusion coefficient of the particles, were as low as 0.05-0.09, suggesting the relatively narrow size distribution and the defined structure of the particles. The particle sizes around 100 nm should be appropriate from the viewpoint for application as the DNA carrier, considering the size of the endocytic vesicles (e150 nm in diameter) for cellular uptake through the endocytosis.15 We then investigated the amount of DNA incorporated into the particles, which is of primary importance with respect to DNA delivery systems. To separate the particles and free DNA, gel permeation chromatography (GPC) was carried out using Superose 6HR columns with the HEPES buffer containing calcium ion as the eluent. The calcium concentration in the eluent was adjusted to the same as in the CaP solution (125 mM) to suppress the dissolution of the CaP particles during the course of the analysis. Note that because the CaP crystals have the composition of Ca10(OH)2(PO4)6 and the molar concentration of the calcium ion is 100-fold excess with respect to the phosphate ion, the calcium ions that participated in the CaP crystal should be less than 1% of the total amount. Figure 4a shows the chromatograms of free DNA (upper figure) and the CaP particle formed at the polymer concentration of 70 µg/mL (lower figure). The peaks corresponding to the particles and free DNA were observed at the retention times of 12 and 31 min, respectively. The amount of the incorporated DNA was calculated as the percentage of the peak area of the particles to the total area (summation of areas of the particles and free DNA) and plotted versus the polymer concentration in Figure 4b. The incorporated amount was 45% of the total DNA at the polymer concentration of 70 µg/mL and decreased with the increment of the polymer concentration to 14% at 280 µg/mL. This is probably because the PAA segment of PEG-PAA and DNA, both of which can interact with CaP crystals, are competitively bound to the crystal, resulting in the exclusion of DNA from the particles in the presence of an excess amount of PEG-PAA. The particle with the minimal size formed at the polymer concentration of 140 µg/mL retained 30% DNA. A GPC analysis was also performed using the eluent containing calcium and phosphate ions at concentrations adjusted to those in a typical culture (15) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D., Ed.; Molecular Biology of the Cell, 3rd ed.; Garland Publishing: New York, 1994; Chapter 13.
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Figure 5. The modified Stern-Volmer plot for free DNA and CaP particles: (0) free DNA; (O) CaP particles.
(585 nm) were measured with excitation at 560 nm at 37 °C.18 The quantum yield was assumed to be proportional to the peak fluorescence, because the spectral shape did not change significantly. Obtained data were analyzed according to the method described by Lehrer.19 Assuming independent and equally absorbing fluorophors in different environments, the fluoresence quantum yields in the absence and presence of quencher, F0 and F, respectively, are given by
∑F0i/n F ) ∑Fi/n ) (1/n)∑F0i/(1 + KQi[I]) F0 )
Figure 4. The amount of DNA incorporated in the CaP particles evaluated by GPC analysis: (a) chromatograms of free DNA (upper figure) and CaP particles (lower figure); (b) the amount of DNA in the particles plotted versus the block copolymer concentration. GPC analysis was performed using Superose 6HR column with the eluent solution of 125 mM CaCl2, 25 mM HEPES, 70 mM NaCl, pH 7.1 (O) or CaCl2 200 mg/L, NaH2PO4‚H2O 125 mg/L, NaCl 6400 mg/L, HEPES 5958 mg/L, pH 7.4 (4). Flow rate was 0.6 mL/min, and DNA was detected by absorbance at 260 nm.
medium.16 The incorporated amount obtained under such conditions suggests that the particles formed at the lower polymer concentrations slowly dissociate, while those formed at a high concentration are sufficiently stable at physiological calcium and phosphate concentrations. As for the structure of the CaP particles reported here, the core-shell architecture with a PEG shell and the core of the CaP/PAA hybrid is the most suitable candidate, indicated by the high colloidal stability presumably due to the steric repulsion of the PEG shell. To gain insight into the location of DNA in the particle, fluorescence quenching experiments were performed using 5′-rhodamine-labeled DNA and iodide ion as a quencher. If DNA is segregated into the core of the particles, it is expected that the quencher cannot access to the fluorophor attached to DNA, resulting in the repression of quenching. For the purpose, CaP particles were prepared using the labeled DNA mixed with the nonlabeled DNA at the mixing ratio of 1:5 (DNA 35 µg/mL, PEG-PAA 87.5 µg/mL). The concentrations of iodide ion (0.10-0.47 M) were adjusted by addition of 4 M stock solution of iodide to 380 µL of the sample solutions of free DNA17 or CaP particles. Fluorescence intensities at the wavelength of peak fluorescence (16) CaCl2 200 mg/L (calcium 1.8 mM), NaH2PO4‚H2O 125 mg/L (phosphate 0.9 mM), NaCl 6400 mg/L, HEPES 5958 mg/L, pH 7.4. The concentrations of calcium and phosphate in plasma or extracellular fluids are similar to these values. See a textbook on physiology, for example: Guyton, A. C. Human Physiology and Mechanisms of Disease, 5th ed.; W. B. Saunders Company: Philadelphia, PA, 1992; Chapter 4.
where the sums are taken over the n fluorophors in the different situation, [I] is the concentration of iodide, and KQi is the quenching constant. On the basis of the SternVolmer law, the following equation is derived at low concentration of iodide
F0/∆F ) 1/([I]
∑fiKQi) + ΣKQi/ΣfiKQi
where ∆F ) F0 - F and fi ) F0i/∑F0i. In this modified Stern-Volmer equation, 1/intercept can be considered as an effective fractional maximum accessible flourescence (feff) and intercept/slope as an effective quenching constant (KQeff). The modified Stern-Volmer plots for free DNA and CaP particles are shown in Figure 5. The linearity was seen in the plots for both samples. The feff value for free DNA was 0.97, suggesting that all the fluorophors are subject to a similar degree of fluorescence quenching. The feff value for CaP particles ()0.58), as well as the similar KQeff values for free DNA ()2.7 M-1) and CaP particles ()2.5 M-1), indicates that about 58% of the fluorophor attached to DNA in the particle solution is free state and accessible for quenching by iodide. The other 42% of DNA should be in the region where the quencher is not accessible, most likely the core of the particles. Note that the value is well consistent with the percentage of the incorporated DNA determined by the GPC analysis (40%). The application of the delivery systems in vivo as well as in vitro has been hampered by the toxicity, which is a particular concern for those composed of organic compounds with a positive charge. We hypothesized that CaP (17) DNA 35 µg/mL, 250 mM CaCl2, 0.5 mM Tris-HCl, 0.05 mM EDTA, 70 mM NaCl, 25 mM HEPES, pH 7.1 (18) Fluorescence measurements were performed using a FP-777 spectrofluorometer (Jasco, Japan). (19) Lehrer, S. S. Biochemistry 1971, 10, 3254-3263.
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Thereafter, the surviving cells were quantified by MTT assay20 (Figure 6). No remarkable decrease in the viability of the cells incubated with CaP particles was observed as compared to the untreated cells within experimental error in the polymer concentration range required to inhibit precipitation of the crystals. The result implies the essentially nontoxic nature of the particles, which is obviously advantageous as a delivery system. In conclusion, we found that the terplex particles composed of CaP, PEG-PAA, and DNA were formed by simple mixing of the calcium/DNA and phosphate/PEGPAA solutions under mild condition. The self-assembled particles had diameters around 100 nm and high colloidal stability with the capability to incorporate DNA in the core and the reduced cyotoxicity. To our knowledge, this is the first example of the organic-inorganic hybrid nanoparticles intended to be utilized as synthetic DNA carriers. Figure 6. Cytotoxicity test. The viable cells were expressed as the percentage of the surviving cells, relative to untreated HeLa cells (100%) (mean ( standard deviation, n ) 4).
particles reported here would be low toxic, since the particles have no component with positive charges except for the naturally occurring calcium ions. As an initial cytotoxicity screening, MTT assay was carried out to assess the viability of HeLa cells incubated with CaP particles incorporating DNA with the random sequence.9 HeLa cells were plated into 96-well plates at 5 × 103 cells/well and maintained for 2 days in DMEM containing 10% serum at 37 °C. After the addition of CaP particle solution (10 µL) to the medium (90 µL), cells were incubated for 24 h.
Acknowledgment. The authors acknowledge a Grandin-Aid for Scientific Research on Priority Area (A) (Molecular Synchronization for Design of New Materials System) from Ministry of Education, Science, Sports, and Culture, Japan (MEXT), the Special Coordination Funds for Promoting Science and Technology from MEXT, and Core Research for Evolutional Science and Technology (CREST) from Japan Science and Technology Corporation (JST). Y.K. acknowledges financial support from Japan Society for the Promotion of Science. LA011736S (20) Hansen, M. B.; Neilson, S. E.; Berg, K. J. Immunol. Methods 1989, 119, 203-210.
