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Polyion Complex Micelles Encapsulating Light-Harvesting Ionic Dendrimer Zinc Porphyrins Hendrik R. Stapert,†,§ Nobuhiro Nishiyama,† Dong-Lin Jiang,‡ Takuzo Aida,‡ and Kazunori Kataoka*,† Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and PRESTO and Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8565, Japan Received March 20, 2000. In Final Form: June 19, 2000 Core-shell type micelles with a hydrodynamic diameter of 52 nm composed of 38 molecules of zinc dendrimer porphyrin with 32 carboxylate groups on the periphery and 39 poly(ethylene glycol)-poly(Llysine) (PEG-PLys) block copolymers were prepared at a stoichiometric mixing ratio. These micelles showed an unusually high stability against NaCl solutions due to the formation of hydrogen bonds at salt concentrations higher than 200 mM NaCl. In contrast, large aggregates of 250-300 nm diameter of a zinc dendrimer porphyrin were formed with 32 positively charged trimethylammonium groups and poly(ethylene glycol)-poly(aspartic acid) (PEG-PAsp) block copolymer. These aggregates lack the ability to form hydrogen bonds and almost completely dissociate at salt concentrations higher than 200 mM. Both polyion dendrimer micelle systems showed a high stability upon dilution with 150 mM NaCl without a critical association concentration being observed. Both polyion dendrimer micelle complexes have potential to be used as drug delivery systems for light-harvesting ionic zinc dendrimer porphyrin sensitizers.
Introduction Self-assembling of block copolymers with a hydrophilic and a hydrophobic block in aqueous solutions has recently gained a lot of interest because of their ability to form nicely defined nanostructures. These nanostructures may find applications as, for example, small bioreactors,1 drug and gene delivery devices,2 structure directive templates,3 or separation and purification devices.4 The formation of nanostructures from block copolymers in aqueous solutions is driven by the incompatibility between the hydrophobic block and water as well as by phase separation of the hydrophilic and hydrophobic blocks. This often leads to a core-shell architecture of condensed hydrophobic polymer chains surrounded by a palisade of hydrophilic polymer chains. Depending on the polymer’s molecular weight and the weight fraction of the blocks, many different architectures have been reported5,6 although spheres, often referred to as micelles, are the most studied systems. Recently, the groups of Kabanov and Kataoka independently introduced a new class of micelles which are formed through electrostatic interaction between two oppositely charged block or graft copolymers.7-9 These so-called polyion complex (PIC) micelles can also be formed * To whom correspondence should be addressed. † Department of Materials Science. ‡ PRESTO and Department of Chemistry and Biotechnology. § Present address: Philips Research, Polymers and Organic Chemistry, WB 7.49, Prof. Holstlaan 4, 5656 AA, Eindhoven, The Netherlands. (1) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288. (2) Mills, S. N.; Davis, S. S. In Polymers in Controlled Drug Delivery; Illum, L., Davis, S. S., Eds.; IOP Publishing: Bristol, U.K., 1987; pp 1-4. (3) Zhao, D.; Yang, P.; Melosh, N.; Feng, J.; Chmelka, B. F.; Stucky, G. D. Adv. Mater. 1998, 10, 1380. (4) Jenekhe, S. A.; Chen, X. L. Science 1999, 283, 372. (5) Won, Y.-Y.; Davis, H. T.; Bates, F. S. Science 1999, 283, 960. (6) Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6359. (7) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294. (8) Harada, A.; Kataoka, K. Science 1999, 283, 65.
by a charged block copolymer and an oppositely charged homopolymer,10 DNA,11 oligodeoxynucleotide,12-14 or enzyme.1 In work to be published elsewhere, we report15 on the use of water-soluble light-harvesting dendrimers as promising photosensitizers for treatment of cancer by photodynamic therapy.16 Dendrimers are treelike branched structures with a confined shape. The schematic structure of the ionic dendrimer zinc porphyrins is given in Figure 1. Upon irradiation with light of the appropriate wavelength, the dendrimers can transfer energy from their unstable triple state to oxygen thereby producing the highly reactive singlet oxygen which can react with many biological compounds and thus kill cells. It is well documented that a high concentration of anticancer drugs at tumors can be achieved using specially designed micellar block copolymers or conjugates due to an enhanced circulation in blood.17-18 In these systems the drugs are either chemically bound to one of the chemical blocks or physically trapped in the hydrophobic core of the micelles. In the case of ionic dendrimer porphyrins, electrostatic interaction between the groups on the periphery and oppositely charged groups of a block copolymer may be expected to induce micellar structures as well.19 (9) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797. See also ref 7. (10) Harada, A.; Kataoka, K. J. Macromol. Sci., Pure Appl. Chem. 1997, A34, 2119. (11) Katayose, S.; Kataoka, K. Bioconjugate Chem. 1997, 8, 702. (12) Kabanov, A. V.; Vinogradov, S. V.; Suzdaltseva, Y. G.; Alakhov, V. Y. Bioconjugate Chem. 1995, 6, 639. (13) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805. (14) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556. (15) Stapert, H. R.; Nishiyama, N.; Takase, D.; Liang, D.-L.; Aida, T.; Kataoka, K. Manuscript in preparation. (16) McCaughan, Jr, J. S. Drugs Aging 1999, 15, 49. (17) Torchilin, V. R. Adv. Drug Deliv. Rev. 1995, 16, No. 2, 3. (18) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119.
10.1021/la000423e CCC: $19.00 © 2000 American Chemical Society Published on Web 09/13/2000
Polyion Complex Micelles
Figure 1. Schematic structures of third-generation ionic dendrimer porphyrins.
Here, we would like to report for the first time on the formation of polyion complexes based on the complexation of ionic dendrimers and poly(ethylene glycol)-poly(R,βaspartic acid) or poly(ethylene glycol)-poly(L-lysine) block copolymers. The goal of this study is to prepare a drugdelivery system for ionic dendrimers based on the principle of PIC micelles. Experimental Section Materials. N--CBZ-L-lysine (Lys(Z)) was obtained from Sigma Co., St. Louis, MO. β-Benzyl L-aspartate (BLA), bis(trichloromethyl) carbonate (triphosgene), and HBr/AcOH 30/70 (v/v) mixture were purchased from Tokyo Kasei Kogyo Co., Ltd., Japan. Sodium chloride and sodium phosphate salts were purchased from Wako, Japan. These chemicals were used without further purification. R-Methoxy-ω-amino poly(ethylene glycol) (Mw ) 12 kg/mol) was a kind gift of Nippon Oil and Fats Co., Ltd, Japan. The polymer was precipitated in diethyl ether from chloroform, dried under reduced pressure and subsequently freeze-dried from benzene prior to use in the block copolymer synthesis (MALDITOF-MS: Mn ) 12 194 g/mol; Mw/Mn ) 1.01). Positively and negatively charged third-generation ionic dendrimer porphyrins (Figure 1) having a zinc porphyrin center and aryl ether dendrons were prepared as previously described.20-22 In the dry state the positive dendrimer is obtained as a chloride salt. Characterization showed unambiguously that both dendrimers had 32 functional groups on their periphery. In solutions used in this study these are quaternary trimethylamino groups for the positively charged dendrimer and carboxylate groups for the negatively charged dendrimer. The dendrimers are abbreviated 32(+)DPZn (MALDI-TOF-MS (3-indolacrylic acid (19) Tomalia, D. A.; Esfand, R. Chem. Ind. (London) 1997, 11, 416420. (20) Tomioka, N.; Takasu, D.; Takahashi, T.; Aida, T. Angew. Chem., Int. Ed. Engl. 1998, 37, 1531-1534. (21) Sadamoto, R.; Tomioka, N.; Aida, T. J. Am. Chem. Soc. 1996, 118, 3978-3979. (22) Tomoyose, Y.; Jiang, D.-L.; Jin, R.-H.; Aida, T.; Yamashita, T.; Horie, K.; Yashima, E.; Okamoto, Y. Macromolecules 1996, 29, 52365238.
Langmuir, Vol. 16, No. 21, 2000 8183 matrix): Mn ) 11 877 g/mol) and 32(-)DPZn (MALDI-TOF-MS: Mn ) 8030 g/mol), respectively. Tetrahydrofuran (THF), n-hexane, dimethyformamide (DMF), and n-butylamine were distilled following standard procedures. Poly(ethylene glycol)-poly(R,β-aspartic acid) (PEG-PAsp) and poly(ethylene glycol)-poly(L-lysine) (PEG-PLys) block copolymers and PLys homopolymer were prepared by previously reported methods.7,10,23,24 Preparation of Polyion Complex Dendrimer Micelles. Polyion complex micelles were made from either 32(+)DPZn and the PEG-PAsp block copolymer or 32(-)DPZn and PEG-PLys block copolymer in such a way that the ratio of positive to negative groups was exactly 1. In a typical procedure the PEG-PAsp block copolymer was dissolved in an aqueous Na2HPO4 solution (10 mM, pH ) 9.07, filtered over a 0.22 µm filter (Millex-GV, Millipore) before use) and added to an aqueous solution of 32(+)DPZn in NaH2PO4 (10 mM, pH ) 4.95) to give a solution (10 mM, pH ) 7.10) containing polyion complex micelles encapsulating ionic dendrimer zinc porphyrins. Solutions were kept for at least 24 h and filtered over a 0.45 µm filter (Dismic-13HP, Advantec) before further characterization. The procedure for the 32(-)DPZn/PEG-PLys system was similar, except that the PEG-PLys block copolymer was dissolved in an aqueous NaH2PO4 (10 mM, pH ) 4.95) solution and added to an aqueous solution of 32(-)DPZn in Na2HPO4/0.1 N NaOH (85:15 v/v) (10 mM Na2HPO4, pH ) 11.54). Addition of a little NaOH is necessary to dissolve the dendrimer completely by deprotonating carboxyl groups of 32(-)DPZn. The thus obtained micellar solution had a pH of 7.3. Gel Permeation Chromatography of Aqueous Micelle Solutions. GPC was performed on a Superose 6 HR 10/30 column (Pharmacia Biotech, Sweden) using an aqueous solution of Na2HPO4/NaH2PO4 (10 mM)/NaCl (150 mM), pH ) 7.4, as the eluent. Injection volume was 50 µL and a flow rate of 0.3 mL/min was applied. The eluted products were detected by their IR, UV (λ ) 220 nm), and fluorescence (λex ) 432, λem ) 605 nm) signals. Stability of Polyion Complex Dendrimer Micelles. The stability of both micelle dendrimer complex systems was studied as a function of sodium chloride concentration for a given micellar concentration at T ) 25.1 ( 0.2 °C and as a function of micellar concentration at T ) 37.0 ( 0.3 °C and 150 mM NaCl. The micellar concentration is defined as the sum of the weights of the dendrimer and block copolymer per unit of volume. For the study of concentration stability, all samples were characterized using dynamic and static light scattering methods. The stability was assessed by following the change in (Kc/R(0))-1 normalized to (Kc/R(0))-1 at NaCl ) 0 mM and the translational diffusion coefficient as a function of NaCl or micellar concentration. Dynamic Light Scattering Measurement.25 Dynamic light scattering (DLS) measurements were performed using a Photal dynamic laser scattering spectrometer DLS-700 (Otsuka Electronics Co., Ltd.) equipped with an argon ion laser (λ0 ) 488 nm). The general equation for the photoelectron count time correlation function is
g(2)(τ) ) 1 + β|g(1)(τ)|2
(1)
where g(2)(τ) is a normalized second-order correlation function and β is the optical constant of the instrument, g(1)(τ) is a normalized first-order correlation function and is given by formula 2. Γ is the characteristic line width. For spherical particles, Γ is
g(1)(τ) ) exp(-Γτ)
(2)
given by a function of the translational diffusion coefficient DT provided that internal motions are negligible as shown in formulas 3 and 4. where q is the magnitude of the scattering vector, θ is the scattering angle, λ is the wavelength of the laser, and n is the refractive index of the solvent. For low concentrations, (23) Yokoyama, M.; Inoue, S.; Kataoka, K.; Yui, N.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1987, 8, 431. (24) Yokoyama, M.; Inoue, S.; Kataoka, K.; Yui, N.; Sakurai, Y. Makromol. Chem. 1989, 190, 2041. (25) Xu, R.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87.
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Γ ) DTq2
(3)
q ) 4πn sin(θ/2)/λ
(4)
the dependence of the translational diffusion coefficient DT on concentration can be expressed by a linear expression:
DT ) D0(1 + kdc)
(5)
where D0 is the translational diffusion coefficient at infinite dilution, kd is the diffusion second virial coefficient, and c is the concentration. The hydrodynamic diameter (Stokes diameter) dh is given by the Stokes-Einstein equation:
dh ) kBT/(3πηD0)
(6)
where kB is the Boltzmann constant, T is absolute temperature, and η is solvent viscosity. If there is a distribution in particle size, g(1)(τ) is expressed by
g(1)(τ) )
∫G(Γ) exp(-Γτ) dΓ
(7)
where G(Γ) is the distribution function of Γ. In the cumulant method, autocorrelation functions are analyzed using an approximate equation:
g(1)(τ) ) exp[-Γ h τ + (µ2/2)τ2 - (µ3/3!)τ3 + ...]
(8)
yielding an average characteristic line width (Γ h ) and a variance h 2. In the histogram method, estimation (polydispersity index), µ2/Γ of the size distribution is carried out by a correlation function profile of the histogram analysis software, and eq 7 is replaced by
g(1)(τ) )
∑G(Γ ) exp(-Γτ)∆Γ j
(9)
G(Γj) is determined using the Marquart nonlinear least squares routine. G(Γj), which is a distribution function according to the ratio of light scattering by the particles with Γ, was then converted into the particle size distribution G(d), using eqs 3 and 6. The distribution according to the weight ratio and the number ratios was then determined from G(d). Static Light Scattering (SLS) Measurement. In general, the Rayleigh ratio (R(θ)) of diluted solutions can be expressed by
Kc/R(θ) ) 1/Mw,app[1 + q2Rg2/3] + 2A2c
(10)
K ) 4πn02/NAλ04(dn/dc)2
(11)
where Mw,app is the apparent weight-average molecular weight, A2 is the second virial coefficient, Rg2 is the mean square radius of gyration, c is the concentration of the solution, θ is the scattering angle, NA is Avogadro’s number, λ0 is the wavelength of the incident polarized light, n0 is the refractive index of the solution, and dn/dc is the refractive index increment with concentration. For low molecular weight molecules the Debye equation (12) is developed by extrapolating θ to 0.
Kc/R(0) ≈ 1/Mw,app + 2A2c
(12)
The intercept of a Debye plot provides an apparent weightaverage molecular weight (Mw,app) of the particles. The specific refractive index increment (dn/dc) of the dendrimer polyion complex solutions was determined using a DRM-1020 (Otsuka Electronics Co., Ltd.) double beam refractometer.
Results and Discussion Preparation of the Block Copolymers. PEG-PBLA and PEG-PLys(Z) block copolymers with the same PEG segment length (Mn ) 12.19 × 103 g/mol, degree of polymerization (DP) ) 275) were prepared via ringopening polymerization of BLA-NCA and Lys(Z)-NCA,
respectively, and initiated from the ω-NH2 group of PEG. Compositions of these block copolymers were determined with 1H NMR from peak intensity ratios of methylene protons of PEG (OCH2CH2: δ 3.5 ppm) and phenyl protons of β-benzyl groups of PBLA or Ζ groups of PLys(Z) (-CH2C6H5: δ 7.3 ppm). The degrees of polymerization of BLA and Lys(Z) were determined to be 28 and 31, respectively. Unimodal distributions were confirmed for PEG-PBLA (Mw/Mn ) 1.07) and PEG-PLys(Z) (Mw/Mn ) 1.09) block copolymers. The benzyl groups of PEGPBLA were removed by alkaline hydrolysis using 0.1 N NaOH to obtain PEG-PAsp (Na salt). The Z groups of PEG-PLys(Z) were removed by treatment with HBr/ AcOH (30/70 v/v) to obtain PEG-PLys (HBr salt). Complete deprotection of PBLA and PLys(Z) and no scission of the main chain were confirmed by 1H NMR measurement (in D2O, 40 °C) of the obtained copolymers. It should be noted that intramolecular isomerization and racemization of the aspartic acid units in the PAsp segment take place to form β-aspartic acid units during the deprotection process. As reported previously,7,23 the ratio of R to β units in the PAsp segment of the deprotected block copolymer was 1:3. Chain lengths of PEG-PAsp and PEG-PLys block copolymers are abbreviated as X-Y, where X stands for the molecular weight of PEG × 10-3 and Y for the polymerization degree of the Asp or Lys units. Poly(Lys(Z)) homopolymer was synthesized by ringopening polymerization of Lys(Z)-NCA using n-butylamine as the initiator. The Z groups of PEG-PLys(Z) were also removed by treatment with HBr/AcOH. The DP of the obtained PLys homopolymer was estimated to be 27 from the peak intensity ratio using 1H NMR. Preparation of Polyion Complex Dendrimer Micelles. Polyion complex micelles were prepared at a positive to negative group ratio of 1. This ratio was determined from the degree of polymerization of the amino acid segments and from the charged groups of the dendrimer peripheries assuming complete ionization in both cases. Precipitation after mixing the polymer and dendrimer solutions at 25 °C was not observed, even after a period of over 2 months. It is noteworthy that the addition of a small amount of NaOH is necessary to dissolve 32(-)DPZn completely in an aqueous Na2HPO4 solution (10 mM, pH ) 11.54). After mixing with the PEG-PLys solution (pH ) 4.95), the pH of the mixture was 7.3 (PBS, 10 mM). At this pH 32(-)DPZn is not completely dissolved without the PEG-PLys copolymer. However, no precipitate was observed indicating that complexation of 32()DPZn and PEG-PLys 12-31 occurred, resulting in a water-soluble complex. The micelle solutions were subjected to aqueous gel permeation chromatography. Due to the fluorescence of the dendrimers, it was possible to directly detect the presence of dendrimer in the micelle for the 32(-)DPZn/ PEG-PLys 12-31 system, as shown in Figure 2. Figure 3 shows the DLS histograms of “as prepared” polyion dendrimer complex systems at 25 °C, 0 mM NaCl and at 37 °C, 150 mM NaCl, a condition of special interest for possible in vivo applications. Table 1 lists some important parameters obtained from DLS analysis. The 32(-)DPZn/PEG-PLys 12-31 complex formed nicely dispersed (dw/dn < 1.15) micelles with sizes around 53 nm. No significant differences in histograms were found between 25 °C (0 mM NaCl) and 37 °C (150 mM NaCl) as shown in Figure 3A,B. The 32(+)DPZn/PEG-PAsp system was clearly bimodal at 25 °C, as shown in Figure 3C, and is indicated by the h 2 > 0.1). Relatively narrowly dispersed high variance (µ2/Γ
Polyion Complex Micelles
Figure 2. GPC elution profiles of (A) 32(-)DPZn and (B) 32(-)DPZn/PEG-PLys 12-31 dendrimer polyion complex system.
Figure 3. DLS histograms of 32(-)DPZn/PEG-Plys 12-31 (A, 25 °C, 0 mM NaCl; B, 37 °C, 150 mM NaCl) and of 32(+)DPZn/PEG-PAsp 12-28 (C, 25 °C, 0 mM NaCl; D, 37 °C, 150 mM NaCl). Detection angle 90°.
micelles with an average diameter of 26 nm were obtained as well as larger particles with an average diameter of 142 nm. However, at 37 °C and 150 mM NaCl only narrowly dispersed, relatively large micelles were formed (Figure 3D). The variance was as low as 0.013, indicating that a unimodal distribution of sizes was present. The heterogeneity of the particles in the absence of the salt may be due to the formation of some portion of nonequilibrium structures. At increasing salt concentration the rate of polyion interchange reactions increases too and the system may equilibrate forming nice particles
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with a narrow size distribution. It is important to note that the micellar sizes did not seem to alter when only the temperature was raised from 25 to 37 °C or lowered, indicating that the stabilization of larger micelles is mainly caused by an increased ionic strength. In DLS measurements, both 32(-)DPZn/PEG-PLys 12-31 and 32(+)DPZn/PEG-PAsp 12-28 systems showed almost no angular dependence (data not shown). Therefore, when rotational motion is undetectable it is reasonable to hypothesize that, under these conditions, nearly spherical particles are formed in which PEG chains form a corona surrounding complexed, encapsulated ionic dendrimer porphyrins. Stability of Polyion Complex Dendrimer Micelles in Sodium Chloride Salt Solutions. Figure 4A shows the stability of the DP32(-)/PEG-PLys 12-31 dendrimer polyion complex micelles as a function of the NaCl concentration. The behavior of the micelle solutions was assessed both by DLS and SLS. The translational diffusion coefficient (DT) as measured with DLS and a normalized (Kc/R(0))-1 parameter (normalized to Kc/R(0) of 0 mM NaCl) as obtained via SLS are presented in Figure 4A. DT can be regarded as a measure of the micellar average mobility, which relates to the hydrodynamic micellar size via the Stokes-Einstein equation, while the normalized (Kc/R(0))-1 parameter is a measure of the change in average apparent molecular weight of the micelles. Both parameters initially decreased with increasing salt concentration. It is apparent that the micelles swell a little due to increasing ionic strength up to 150 mM; however, they are also broken up and rearranged, as is clear from their decrease (of about 25%) in apparent molecular weight. When the salt concentration was increased further up to 500 mM NaCl, an increase in DT was observed. The decrease in molecular weight stopped at 55 ( 5% of its original weight at 0 mM with a salt concentration of 250 mM NaCl, and became almost independent of ionic strength at concentrations of up to 1500 mM NaCl. The DT slightly decreased in the range between 500 and 1500 mM. We explain this unique behavior by the pronounced role of hydrogen bonding in the formation of these dendrimer polyion complex micelles. At a low ionic strength the contribution of ion pairs between dendrimer and block copolymer may be significant; however, between 0 and 250 mM NaCl the ion pairs are broken by the increased ionic shielding as previously reported for other polyion complex micelles. Hydrogen bonds between carboxylic acid groups of the dendrimer and amine groups of the PLys blocks are presumably formed at the expense of ion pairs, leading to an increase in DT. At about 250 mM NaCl apparently all the ion pairs are broken and the apparent molecular weight becomes independent of ionic strength, presumably indicating that, in the micelle formation, hydrogen bonds have become the main interaction between the block copolymer and dendrimer. This hypothesis implies that ammonium groups (partly) deprotonate and carboxylic groups should become at least partly protonated. This mechanism thus assumes a complex acidbase equilibrium depending on the ionic strength. To assertain whether hydrogen bond formation had indeed occurred, urea was added to the micelle solution (NaCl ) 1500 mM). It is clear from Figure 4B that the apparent molecular weight of the micelles decreases further, indicating the disruption of hydrogen bonds and thus confirming their existence. Figure 5A presents the stability of the 32(+)DPZn/ PEG-PAsp 12-28 dendrimer polyion complex micelles as a function of the NaCl concentration. The complexes
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Table 1. Translational Diffusion Coefficient, Hydrodynamic Diameter, and Variance of 32(-)DPZn/PEG-PLys and 32(+)DPZn/PEG-PAsp PIC Micelles DT (cm2/s) 32(-)DPZn/PEG-PLys 32(+)DPZn/PEG-PAsp
25 °C, 0 mM NaCl 37 °C, 150 mM NaCl 25 °C, 0 mM NaCl 37 °C, 150 mM NaCl
10-7
1.06 × 1.28 × 10-7 3.75 × 10-8 2.80 × 10-8
dh (nm)
µ2/Γ2
46.4 57.0 130.3 235.6
0.147 0.137 0.133 0.013
Figure 4. Effect of increasing salt concentration (A, urea ) 0 mM) and urea concentration (B, NaCl ) 1500 mM) on the translational diffusion coefficients, DT (0, detection angle 90°), and normalized (Kc/R(0))-1 (O) for DP32(-)/PEG-PLys 1231. T ) 25.1 ( 0.2 °C. Micellar concentration 6.77 mg/mL.
Figure 5. Effect of increasing salt concentration on the translational diffusion coefficients, DT (0, detection angle 90°), and normalized (Kc/R(0))-1 (O) for DP32(+)/PEG-PAsp 12-28 (A, 5.48 mg/mL) and the Plys 27/PEG-PAsp 12-28 model system (B, 3.25 mg/mL). T ) 25.1 ( 0.2 °C.
initially start to swell but no disruption occurs, as can be concluded from the decrease in DT and the constant apparent molecular weight. At NaCl concentrations higher than 200 mM a steep decrease in the apparent molecular weight was observed, indicating the disruption of the ion complexes. However, the photon count of these solutions still allowed us to obtain DLS data. Above 250 mM we observed highly multimodal distributed histograms with weight averages around 5.6-6.5 (major peak), 26.0-40.0, and 155.4-468.8 nm, corresponding to free dendrimers and block copolymer, micelles, and high molecular weight agglomerates, respectively. This observation is consistent with the value of the normalized (Kc/R(0))-1 data at NaCl > 250 mM, which also indicates that some complex formation occurs at a higher ionic strength. Notably, formation of hydrogen bonds in this complex system can be excluded because of the quartenary ammonium groups
on the dendrimer periphery. This may explain the lower stability as compared to the 32(-)DPZn/PEG-Plys 1231 system. The unique complexation phenomena between charged block copolymers and micelles described above motivated us to study a model system for comparison. The salt stability of the PLys 27/PEG-PAsp 12-28 polyion complex system is shown in Figure 5B. Interestingly, at 0 mM and T ) 25 °C the hydrodynamic sizes of the particles as obtained by DLS were comparable to the 32(+)DPZn/ PEG-PAsp 12-28 dendrimer polyion complex system: w1 ) 26.6 nm, dw1/dn1 ) 1.11; w2 ) 197.1 nm, dw2/dn2 ) 1.28. However the salt stability was lower than the dendrimer polyion complexes. After a continuous decrease in DT, the complexes disrupted between 150 and 200 mM and the normalized (Kc/R(0))-1 parameter became almost
Polyion Complex Micelles
0, which is consistent with previously published data.7,26 The photon count remained too low at NaCl > 200 mM to perform further DLS analysis, indicating that all charges are completely screened and no complex formation occurs. The same behavior was also found for the “reversed” PEG-PLys/PAsp combination as previously published.27 Theoretically the PLys 27/PEG-PAsp 12-28 polyion complex system would be capable of forming hydrogen bonds, but if formed, they apparently do not stabilize these complexes at a high ionic strength. The observation that the 32(-)DPZn/PEG-Plys 12-31 dendrimer ion complex system studied here is more stable than the polyion complex model systems may be due to a lower effectieve pKa of the dendrimer carboxylic groups as compared to the carboxylic groups of PAsp, due to the vicinity of the aromatic rings. The carboxylic groups of PAsp may not become protonated under the conditions studied while the carboxylic acid groups of the dendrimer may well become protonated. The observation that both dendrimer ion complex systems studied here are more stable may also point to a specific role of the rigid dendritic architecture in the stability of the dendrimer polyion complexes. A possible explanation may be given by a simple thermodynamic comparison based on expected entropic differences in the complex formation (and dissociation) between rigid particles or flexible oligomers with the same block copolymer. The disruption of complexes is entropically driven; as the entropy gain of dendrimer polyion complexes upon disruption is likely to be smaller than polyion complexes of flexible chains, a higher stability may be expected in the former case. It should be emphasized that when (multiple) hydrogen bonds can be formed between the dendrimer and the block copolymer, a highly stable complex (essentially independent of ionic strength, as found here) can be expected to form for the same reasons. Stability of Diluted Micelle Solutions. Debye plots and the effect of concentration on the translational diffusion coefficients are presented in Figure 6A and Figure 6B for 32(-)DPZn/PEG-PLys 12-31 and 32(+)DPZn/PEG-PAsp 12-28 dendrimer polyion complex systems, respectively, to study the stability of the micelles upon dilution. It is well-known that block copolymer micelles dissociate at low concentration, corresponding to a critical association concentration (cac).28-31 However, an increment in the Kc/R(0) was not observed upon dilution for the dendrimer/block copolymer systems represented in Figure 6, suggesting that these systems might have a very low cac or may behave as nanoparticles in dilute solutions.10,30 The diffusion coefficients at infinite dilution (D0) and the diffusion second virial coefficients (kd) were obtained from the DT-micellar concentration graphs for both systems. The data were found to fit very well with a linear function as predicted by eq 5. The values obtained are D0 ) 1.3 × 10-7 cm2/s and kd ) -3.3 × 10-2 mL/mg for 32(-)DPZn/PEG-PLys 12-31 and D0 ) 2.1 × 10-8 cm2/s (26) Wolk, S. K.; Swift, G.; Paik, Y. H.; Yacom, K. M.; Smith, R. L.; Simon, E. S. Macromolecules 1994, 27, 7613. (27) Kakizawa, K.; Kataoka, K. J. Am. Chem. Soc. 1999, 121 (48), 11247-11248. (28) Tsuchida, E.; Osada, Y.; Ohno, H. J. Macromol. Sci., Phys. 1980, B17, 683. (29) Khougaz, K.; Zhong, X. F.; Eisenberg, A. Macromolecules 1996, 29, 3937. (30) Quintana, J. R.; Villacampa, M.; Katime, I. A. Macromolecules 1993, 26, 606. (31) Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1999, 15, 377.
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Figure 6. Effect of concentration on translational diffusion coefficients (0, detection angle 90°) and Debye plots (O) for DP32(-)/PEG-PLys 12-31 (A) and DP32(+)/PEG-PAsp 1228 (B). Fitting was performed using linear functions. T ) 37.0 ( 0.3 °C, 150 mM NaCl.
and kd ) 6.1 × 10-4 mL/mg for 32(+)DPZn/PEG-PAsp 12-28. The hydrodynamic radius can be calculated using D0 and the Stokes-Einstein equation (eq 6). The values obtained were Rh ) 26.4 nm for 32(-)DPZn/PEG-PLys 12-31 and Rh ) 160.7 nm for 32(+)DPZn/PEG-PAsp 1228. We explain the higher Rh of the 32(+)DPZn/PEGPAsp 12-28 system by the formation of clusters of smaller micelles (see Figure 3C) or formation of another supramolecular assembly. Determination of Association Number and Mw,app. The apparent Mw of the 32(-)DPZn/PEG-PLys 12-31 was determined to be 9.26 × 105 from a Zimm plot using a dn/dc value of 0.7313. Under charge stoichiometry conditions, about 39 PEG-PLys 12-31 block copolymers and 38 32(-)DPZn dendrimers are complexed to give a micelle. These values are close to related polyion complex data.1 The ratio of Rg/Rh as calculated from DLS and SLS data was 0.85, which is close to the Rg/Rh value of 0.776 calculated for a hard sphere.32 Judging from the threedimensional structure of a dendrimer,20-22 which is highly branched, this is not surprising. The higher value obtained (32) Douglas, J. K.; Roovers, J.; Freed, K. F. Macromolecules 1990, 23, 4168.
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as compared to the theoretical value may however reflect fluctuations in segment densities in a radial direction. Conclusions Polyion complexes of PEG-polyanion or PEG-polycation with their oppositely charged ionic zinc denrimer porphyrins were prepared through electrostatic interaction at low ionic strength. Narrowly distributed micelles consisting of about 38 32(-)DPZn dendrimers and 39 PEG-PLys 12-31 were obtained having a hydrodynamic diameter of about 50-55 nm at 37 °C and 150 mM. At higher ionic strength (NaCl < 250 mM) electrostatic interactions are broken, but hydrogen bonds are formed, rendering these micelles stable to very high ionic strengths (up to 1.5 M NaCl). In contrast, the polyion complexes of 32(+)DPZn and PEG-PAsp almost completely dissociated at NaCl > 250 mM, which is a little higher than model systems prepared
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form PLys 27 and PEG-PAsp 12-28. The 32(+)DPZn dendrimer lacks the ability to form hydrogen bonds as it has quartenary ammonium groups on its periphery. Both polyion dendrimer complex systems showed a high stability upon dilution at physiological conditions of 37 °C and 150 mM NaCl, suggesting that these systems have potential to be used as delivery systems of light-harvesting dendrimer Zn porphyrins for photodynamic therapy of cancer. Acknowledgment. The authors acknowledge Dr G. N. Phillips, MESA+ Research Institute, University of Twente, Enschede, The Netherlands, for reading the manuscript. H.R.S. is grateful to the European Union Science and Technology Fellowship program for partially sponsoring this research. LA000423E
