Langmuir 2006, 22, 2323-2328
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Complexes of Poly(ethylene oxide)-block-Poly(L-glutamate) and Diminazene Andreas F. Thu¨nemann,*,† Dagmar Schu¨tt,‡ Robert Sachse,§ Helmut Schlaad,| and Helmuth Mo¨hwald| Federal Institute for Material Research and Testing, Richard-Willsta¨tter-Straβe 11, 12489 Berlin, Germany, Technical UniVersity Berlin, Institut fu¨r Werkstoffwissenschaften, Englische Straβe 20, 10587 Berlin, Germany, Fraunhofer Institute for Applied Polymer Research, Geiselbergstraβe 69, 14476 Potsdam, Germany, and Max Planck Institute of Colloids and Interfaces, Am Mu¨hlenberg 1, 14476 Potsdam, Germany ReceiVed August 2, 2005. In Final Form: December 20, 2005 Nanoparticles with a mean hydrodynamic radius of 16 nm and low polydispersity (P.I. ) 0.1) were spontaneously formed by the complexation of poly(ethylene oxide)-block-poly(L-glutamate) (PEO-b-PLGlu) with diminazene. Only one of two possible binding sites of each diminazene molecule was involved in complexation. As determined by UV-vis difference spectra measurements, the complex binding constant is on the order of 1-2 × 104 M-1. Circular dichroism measurements showed that the highly water-soluble diminazene can induce and stabilize the R-helical secondary structure of a PLGlu block.
Introduction Block copolymer micelles are interesting colloidal objects for fundamental research as well as for advanced applications in nanotechnology like drug and gene delivery.1-3 Using block copolymers made of poly(ethylene oxide) (PEO) and poly(amino acid) blocks is advantageous because they may undergo hydrolysis and enzymatic degradation into biocompatible fragments (for a review see Kwon et al.4). For instance, Kataoka et al. reported on conjugated PEO-b-PAsp (PAsp ) poly(R,β-aspartic acid)), in which the drug is covalently bound to aspartic acid.5 Thu¨nemann et al. described the noncovalent binding (complexation) of all-trans-retinoic acid to the poly(L-lysine) block of PEO-b-PLLys, by which core-shell particles with a diameter of 60 nm are formed.6,7 Also, the all-trans-retinoic acid, which is an amphiphile, can stabilize the R-helical conformation of the PLLys block when the pH value changes. Self-assembled polymer-metal complex micelles from cis-dichlorodiammine platinum(II) and PEO-b-PAsp, in which the drug were linked to the polymer via the metal ion, were also reported.8 In all of these examples, the presence of a shell of hydrophilic PEO chains stops the growing of aggregates on a nanometer scale in aqueous solution. Otherwise macroscopic precipitation occurs. For the complexes of amphiphiles (noncovalent systems), a * Corresponding author. E-mail:
[email protected]. † Federal Institute for Material Research and Testing. ‡ Technical University Berlin. § Fraunhofer Institute for Applied Polymer Research. | Max Planck Institute of Colloids and Interfaces. (1) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf. B: Biointerfaces 1999, 16, 3-27. (2) Lavasanifar, A.; Samuel, J.; Kwon, G. S. AdV. Drug DeliVery ReV. 2002, 54, 169-190. (3) Kwon, G. S.; Kataoka, K. AdV. Drug DeliVery ReV. 1995, 16, 295-309. (4) Lavasanifar, A.; Samuel, J.; Kwon, G. S. AdV. Drug DeliVery ReV. 2002, 54, 169-190. (5) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113-131. (6) Thu¨nemann, A. F.; Beyermann, J.; Kukula, H. Macromolecules 2000, 33, 5906-5911. (7) Thu¨nemann, A. F.; M. Mu¨ller, Dautzenberg, H.; J.-F. Joanny, Lo¨wen, H.; in AdVances in Polymer Science: Polyelectrolytes with Defined Molecular Architecture II; Schmidt, M., Ed.; Springer-Verlag: Berlin, 2004, Vol. 166, pp 113-171. (8) Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1999, 15, 377-383.
diversity of different mesomorphous solid-state structures has been observed.9 In contrast to complexes of amphiphilic molecules,10 only a few studies deal with complexes of highly water-soluble molecules and poly(amino acid)s. An interesting exception is diminazene, a water-soluble positively charged drug of low molecular weight. Diminazene is used to treat trypanosomiasis (sleeping sickness) in animals, predominantly for cattle, but the mechanism of its action in vivo is not exactly known.11 Aggregation of the trypanosomes in the presence of diminazene seems to be important.12 Stolnik et al. used diminazene as a model drug to study its complex formation with PAsp13 and PEO-b-PAsp.14 The PEO-b-PAsp/diminazene complex forms nanoparticles with sizes in the range of 22-60 nm. The aim of the present study was to improve the basic understanding of the binding properties of diminazene to an anionic poly(amino acid) block. PEO-b-PLGlu (LGlu ) Lglutamic acid) was chosen as a simple polymeric model system to study binding and self-assembly. Diminazene has two amidino groups which are both possible binding sites for complexation (cf. Chart 1). For example, strong indications are found that both amidino groups participate in a positively charged state in the complex of diminazene and DNA.15 Here, we investigate whether the water-soluble diminazene binds with one or two of its possible binding sites to PEO-PLGlu and how large are the binding constants when the length of the PLGlu block varies. In addition, we wanted to see if there is an influence of diminazene on the pH-stability of the R-helical secondary structure of the PLGlu block, comparable to the one observed for surfactant molecules. (9) Thu¨nemann, A. F. Prog. Polym. Sci. 2002, 27, 1473-1572. (10) Thu¨nemann, A. F.; Mu¨ller, M.; Dautzenberg, H.; Joanny, J.-F.; Lo¨wen H. AdV. Polym. Sci., Vol. 168: Polyelectrolytes with Defined Molecular Architechture II; Springer: Berlin, 2004; pp 113-171. (11) Gummow, B.; Swan, G. E.; Du Preez, J. L. J. Vet. Res. 1994, 61, 317326. (12) Peregrine, A. S.; Mamman, M Acta Trop. 1993, 54, 185-203. (13) Ehtezazi, T. Govender, T.; Stolnik, S. Pharm. Res. 2000, 17, 871-878. (14) Govender, T.; Stolnik, S.; Xiong, C.; Zhang, S.; Illum, L.; Davis, S. S. J. Controlled Release 2001, 75, 249-258. (15) Pilch, D. S.; Kirolos, M. A.; Liu, X. Y.; Plum, G. E.; Breslauer, K. J. Biochemistry 1995, 34, 9962-9976.
10.1021/la0521138 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/26/2006
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Chart 1. Proposed Chemical Structure of the Complex Formed between PEO-b-PLGlu and Diminazenea
a Therein diminazene is bound with one of two amidino groups to the carboxylate groups of LGlu.
Figure 1. Sketch of the experimental setup for the measurement of UV-difference spectra. The UV beam was split and one beam was guided through a double chain cuvette where diminazene and polymer are separated in the two chambers. The second beam was guided through an equal cuvette but with diminazene and polymer mixed to form a complex. The difference spectra were calculated from the light transmitted through both cuvettes.
Experimental Section Materials. Diminazene aceturate and 2-amino-2-hydroxymethyl1,3-propanediol hydrochloride (TrisHCl) were purchased from Aldrich and used as received. The PEO110-b-PLGlu6-25 copolymers were prepared by the polymerization of γ-benzyl-L-glutamate N-carboxyanhydride using R-methoxy-ω-amino-PEO110 (subscript denotes the number of repeating units, MW ) 5000 g/mol) as a macroinitiator;16 benzyl protecting groups were subsequently removed by hydrogenation.17 Five samples with different length of the PLGlu were prepared: PEO110-b-PLGlu6, PEO110-b-PLGlu10, PEO110-b-PLGlu16, PEO110-b-PLGlu20, and PEO110-b-PLGlu25. Details of the polymer synthesis and characterization by 1H NMR spectroscopy and size exclusion chromatography have been described elsewhere by Kasparova et al.18 Methods and Instrumentation. UV-difference spectra were measured with a UV-vis spectrophotometer model Cary 1 UV from Varian using double chamber cuvettes, type Suprasil from Helma, with a 2 × 4.375 mm light pathway. The spectra were measured at 25 ( 1 °C with a Lauda thermoregulator connected to the sample compartment. For the measurement of an UV-difference spectrum, both the sample cuvette and the reference cell were filled with a solution of diminazene in the first and a solution of PEO-b-PLGlu in the second of the separated compartments of each cuvette. The spectrum measured with this configuration was used as a baseline. Then, the content of the compartments of the sample cell were mixed rigorously, thereby allowing the complexation of diminazene and PEO-b-PLGlu. The UV-difference spectrum was then measured over the wavelength range from 200 to 500 nm and corrected by the baseline. A sketch of the experimental setup is shown in Figure 1. Circular dichroism (CD) spectra were detected by a spectropolarimeter J715 from Jasco to determine the ellipticity, θ, of the PEO-b-PLGlu when complexed with diminazene. The measurements were performed in the far-UV range (λ ) 190-260 nm). Dynamic light scattering (DLS) measurements were carried out with a particle sizer, model HPPS 3.3-ET from Malvern Instruments, at a temperature of 25 °C. Complex Formation. The solubility of PEO-b-PLGlu in aqueous solution decreases with increasing length of the PLGlu segment. Turbid solutions were formed at pH 7 when the PLGlu comprises more than 10 units. We therefore used a 25 mM TrisHCl buffer (pH ) 7.4) in which all the block copolymers were soluble and were (16) Yotzoyama, M.; Inue, S.; Kataoka, K.; Yui, N.; Sakurai, Y. Makromol. Chem., Rapid. Comm. 1987, 8, 431. (17) Yang, J.-Z.; Autoun, S.; Ottenbrite, R. M.; Milstein, S. J. BioactiVe Compatible Polym. 1996, 11, 219. (18) Kasparova, P.; Antonietti, M.; Co¨lfen, H. Colloids Surf. A: Physicochem. Surf. 2004, 250, 153-162.
Figure 2. Absorption spectra of PEO110-b-PLGlu16 (solid line; concentration: 0.8 mM with respect to LGlu), diminazene (dashed line; 0.025 mM), and TrisHCl buffer (dotted line; 25 mM).
Figure 3. UV-vis difference spectra of the complex of PEO110b-PLGlu16 and diminazene with LGlu:diminazene ) 8.0, 2.0, 1.0, and 0.5; concentration of diminazene: 0.05 mM. producing clear solutions. Note that Stolnik et al. used the same buffer to study the complexation of PEO-b-PAsp and diminazene.14 Diminazene shows a high light absorption with a maximum at λ ) 368 nm (cf. Figure 2, dashed line). We determined its extinction coefficient in distilled water to be ) (26 500 ( 2300) M-1 cm-1 at a wavelength of 368 nm. The average extinction coefficient is the same in physiological sodium chloride solution (cNaCl ) 0.15 mol L-1) and 25 mM TrisHCl buffer within the limit of experimental error. The high absorption of light of diminazene limits the upper diminazene concentration for complexation when monitored by UVdifference measurements. We chose concentrations of 0.10 mM diminazene to fill the first compartment of the cuvette, which resulted in 0.05 mM after mixing it with PEO-b-PLGlu for complexation in the sample cuvette. The concentration of the PEO-b-PLGlu samples
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binding constant (K) can be determined by combining the laws of mass action and of Lambert-Beer. The law of mass action is given by
K ) cDM/(cDcnM)
(1)
where cDM, cD, and cM are the concentrations of the complex, of diminazene, and of LGlu units, respectively. Lambert-Beer’s law, written in the form of the difference in absorbance, is
∆A ) ∆c∆λd ) cDM∆λd Figure 4. Concentration dependency of the ∆Absorbance at 426 nm in the UV-vis difference spectra of the complexes of PEO110b-PLGlun: n ) 6 (squares), 10 (circles), 16 (triangles), 20 (crosses), and 25 (diamonds). The straight lines are produced on the basis of eq 4 used for the determination of binding constants (cf. Table 1). Concentration of diminazene: 0.05 mM. was varied to produce LGlu-to-diminazene ratios of nLGlu:ndiminazene ) 8.0, 4.0, 2.0, 1.0, and 0.5. The mean extinction coefficient at λ ) 206 nm of the PEO-bPLGlu copolymers in 25 mM TrisHCl was found to be ) (2210 ( 140) M-1 cm-1 (per LGlu unit). It can be seen in Figure 2 that there is no overlap of the absorbance band of PEO-b-PLGlu and diminazene and that the absorbance of TrisHCl is negligible in the region of the maximum absorbance of diminazene.
Results and Discussion Formation of Complexes of PEO-b-PLGlu and Diminazene. The mixing of diminazene and PEO-b-PLGlu should lead to the formation of complexes that are stabilized by ionic and hydrogen bonds as illustrated in Chart 1. The pKa value of the carboxylic acid is 4.38,19 and we can therefore assume that PLGlu is deprotonated when complexation is done at pH 7.4. The UV spectra of complexes should be different from those of the noncomplexed compounds (see Figure 2), which indeed was observed in the UV-vis difference spectra (Figure 3) of the mixtures of diminazene and PEO-b-PLGlu (in cuvette b) and the pure compounds (in cuvette a). We found that the difference in absorbance increases with increasing concentration of PEO-bPLGlu. Further, the shape of the spectra is constant immediately after mixing diminazene and PEO-PLGlu. Three isosbestic points at λ ) 260, 308, and 407 nm are present in the difference spectra, suggesting the presence of a two state system, i.e., diminazene in a defined complexed and in a noncomplexed state (however, one cannot exclude the existence of further species with the same absorbance spectrum). The maximum positive difference in the UV-vis absorbance is seen at λ ) 426 nm. We therefore used the UV-vis difference at 426 nm to monitor the complexation reaction. From the large values of the differences in absorbance, as shown in Figure 3, we concluded that PEO-bPLGlu/diminazene complexes are spontaneously formed when the diminazene is mixed with polymer. The higher the concentration of PEO-b-PLGlu the higher is the amount of diminazene bound to the polymer. On the basis of its chemical structure shown in Chart 1, diminazene may bind with one or two of its amidino groups to the carboxylic acid groups of PEO-b-PLGlu. To determine the number of binding sites, we assumed a simple complexation reaction: Diminazene (D) and LGlu units (M) form a complex DMn according to D + nM T DMn, where n is the number of LGlu units that bind one diminazene molecule. The complex (19) Nilsson, S.; Zhang, W. Macromolecules 1990, 23, 5234-5239.
(2)
∆λ is the change of the absorbance coefficient at a given wavelength (here: λ ) 426 nm). In case that only one binding site (one amidino group) of diminazene is involved in complex formation, the concentration of free diminazene is given as cD ) cD,t)0 - cDM, that of bound LGlu units is cM,b ) cDM, and that of free LGlu is cM ) cM,t)0 - cM,b. The binding constant is therefore
K)
cDM
(3)
(cD,t)0 - cDM)(cM,t)0 - cDM)
Together with the difference in absorbance, which is correlated to the concentration of the complex by ∆A ) cDM∆λd ) cDM∆extλ, the fit function for the determination of K is given as
∆A ) (∆extλ/2)(cM,t)0 + cD,t)0 + 1/K -
x(cM,t)0 + cD,t)0 + 1/K)2 - 4cM,t)0cD,t)0)
(4)
In the case of two binding sites (both amidino groups of diminazene), it is cD ) cD,t)0 - cDM, cM,b ) 2cDM, and cM ) cM,t)0 - cM,b, and thus
K)
{
cDM
(5)
(cD,t)0 - cDM)(cM,t)0 - 2cDM)2
}
The fit function for the determination of K is then given by
∆A )
2 ‚21/3(a2 - 3b)
2a -
∆extλ 6
with
(- 2a2 + 9ab - 27c +
x(2a2 - 9ab + 27c)2 - 4(a2 - 3b)3)1/3 -22/3(- 2a2 + 9ab - 27c +
x(2a2 - 9ab + 27c)2 - 4(a2 - 3b)3)1/3
(6)
a ) cM,t)0 + cD,t)0 b)
(cM,t)0)2 1 + cM,t)0cD,t)0 + 4 4K
and c)
(cM,t)0)2cD,t)0 4
The quality of a fit to experimental data is reflected in the value of the coefficient R2; the closer the value of R2 to unity the better the fit. We found that the ∆A data obtained for the complexes of diminazene with PEO110-b-PLGlu6,25 are better described by
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Table 1. Molecular Characteristics, Complex Binding Constants (K), and Amount of Diminazenea Bound to Glutamate Units amount of bound diminazeneb
polymer
K (103 M-1)
LGlu: diminazene 1:1 (%)
LGlu: adiminazene 8:1 (%)
PEO110-b-PLGlu6 PEO110-b-PLGlu10 PEO110-b-PLGlu16 PEO110-b-PLGlu20 PEO110-b-PLGlu25
0.8 ( 0.1 19.8 ( 1.3 23.8 ( 1.6 19.0 ( 1.2 11.6 ( 0.7
7 50 53 49 41
43 95 96 95 91
a
Concentration of diminazene: 0.05 mM.
eq 4 than by eq 6. Averaging the coefficients of determination, R2, of the five polymers results in a much higher value for eq 4 (R2 ) 0.996) than for eq 6. (R2 ) 0.636). Accordingly, only one binding site of diminazene is involved in the complex with the PEO-b-PLGlu. It might be speculated that the second binding site is not involved in complexation due to steric restrictions. It must be mentioned that Stolnik et al. suggested, on the basis of calorimetric data, that each diminazene molecule interacts with more than one monomer unit (1.35-1.88) of PAsp and PEOb-PAsp.13,14 The reason for the different numbers of binding sites of diminazene in the complexes with PAsp and PLGlu is not known yet. The measured ∆A values are a function of cM,t)0 for the different lengths of the PLGlu and were fitted with the equations for one and two binding sites (eqs 4 and 6, respectively) using (∆λ d) ) 1762 M-1 to access binding constants (see Table 1). Except for the sample exhibiting the shortest PLGlu block (K ) 0.8 × 103 M-1), the value of K is in the order of 1-2 × 104 M-1 and thus very similar to the ones reported earlier for diminazene/ PAsp complexes (K > 1 × 104 M-1)13 or other host-guest complexes.20-22 The binding of diminazene to PEO-b-PLGlu can therefore be considered as strong. The conversion of diminazene, i.e., the amount of diminazene molecules bound to the polymer, was calculated according to UD ) 1 - (cD/cD,t)0) ) 1 KcD,t)0 - KcM,t)0 - 1 + x(KcD,t)0 - KcM,t)0 - 1)2 + 4KcD,t)0 2KcD,t)0
(7) UD was exemplarily determined for LGlu-to-diminazene ratios of 1:1 and 8:1 at very low concentrations of diminazene (0.05 mM); results are provided in Table 1. It can be seen that the amount of diminazene bound to PEO110-b-PLGlu10-25 is 4153% at 1:1 ratio and >90% for 8:1 ratio. Much lower amounts of diminazene (7% and 43%, respectively) bind to PEO110-bPGlu6. Hence, diminazene can be bound quantitatively to PEOb-PLGlu when the PLGlu block has 10 or more units and the polymer is used in excess. Stabilization of the r-Helix. It is known that conformational transitions of PLGlu from a random coil to an R-helix can be stimulated by selected surfactants.23,24 Such behavior, which might be considered as a special case of molecular recognition, has not been reported for nonsurface active, highly water-soluble molecules such as diminazene. It seemed reasonable to assume (20) Ravoo, B. J.; Jacquier, J.-C. Macromolecules 2002, 35, 6412-6416. (21) Funasaki, N.; Sumiyoshi, T.; Ishikawa, S.; Neya, S. Mol. Pharm. 2004, 1, 166-172. (22) D’Amico, M. L.; Paiotta, V.; Secco, F.; Venturini, M. J. Phys. Chem. B 2002, 106, 12635-12641. (23) Liu, J.; Takisawa, N.; Kodama, H.; Shirahama, K. Langmuir 1998, 14, 4489-4494. (24) Wang, Y.; Chang, C. Macromolecules 2003, 36, 6503-6510.
Figure 5. CD spectra of PEO110-b-PLGlu25 in the absence of diminazene at different pH. The arrow indicates the increase in pH from 3.4 (solid line), 5.1 (dotted line), 5.6 (dashed line), to 7.4 (dash-dotted line). The inset shows the development of the ellipticity at λ ) 222 nm.
Figure 6. CD spectra of the PEO110-b-PLGlu25/diminazene complex at different pH. The arrow indicates the increase in pH from 7.4 (solid line), 9.3 (dotted line), 11.5 (dashed line), to 12.4 (dashdotted line). The inset shows the development of the ellipticity at λ ) 222 nm.
that diminazene can alter the secondary structure of the PLGlu blocks in the PEO-b-PLGlu/diminazene complexes. It is wellknown that about 20 amino acid units are needed to form a stable R-helix.25,26 Therefore, we used the polymer with the longest PLGlu block to compare its secondary structure in its noncomplexed and complexed states as a function of the pH value. Figure 5 shows a number of CD spectra of PEO110-b-PGlu25 at different pH values. The typical spectrum of an R-helix can be seen at pH 3.4 with two minima at λ ) 208 and 222 nm and one maximum at ∼192 nm (see Figure 5). The ellipticity at λ ) 222 nm, θ222, is a measure of the R-helix content. Its value is negative and increases with increasing pH (between pH 5.1 and 5.6) and finally reaches positive values at pH 6. The shape of the spectrum at pH larger than 6 with a minimum at 197 nm and a maximum at 218 nm is typical for a random coil. As can be seen from the inset in Figure 5, the transition clearly takes place between pH 5 and 6, with the inflection point at pH 5.3. A collection of CD spectra of the PEO110-b-PLGlu25/diminazene complex is shown in Figure 6. The poor quality of the spectra at λ < 200 is supposedly due to a strong absorption of the diminazene (cf. Figure 2). Surprisingly, we found two minima at λ ) 206 and 224 nm that are characteristic for an R-helix in a very broad pH range from 3.4 to 11.5. It is remarkable that the intensity of the first minimum is amplified but not that of the second one; a similar effect has been observed for complexes of PLGlu and (25) Harada, A.; Cammas, S.; Kataoka, K. Macromolecules 1996, 29, 61836188. (26) Rinaudo, M.; Domard, A. J. Am. Chem. Soc. 1976, 98, 6360-6364.
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Figure 7. Comparison of the helix-to-coil transition induced by pH for PEO110-b-PLGlu25 in the absence (squares) and in the presence of diminazene (circles). Hydrodynamic radius (triangles) of nanoparticles of the PEO110-b-PLGlu25/diminazene complex as a function of pH. Lines are to guide the eye.
Figure 8. CD spectra of (a) PEO-b-PLGlu25 (b) and PLGlu26 at pH 7.4 (25 mM Tris HCl buffer) in the presence of diminazene at 0 (curve 1), 0.19 (2), 0.29 (3), 0.49 (4), and 0.97 mM (5). The concentration of LGlu in monomer units was 1.34 mM.
dodecylammonium.23 As seen from the development of the value of θ222 (see inset in Figure 6), the R-helix to random coil transition occurred between pH 11.0 and 12.5 with the inflection point at pH 12.0. The θ222 value varies only slightly between -1.9 and -2.1 × 104 degrees cm2 dmol-1 in the pH interval from 3.4 to 11.0 indicating that the content of the R-helix is constant within this interval. The R-helix fraction of PLGlu, fH, can be calculated24 by
fH )
θC - θ222 θC - θH
(8)
θH denotes the ellipticity for the PLGlu R-helix at pH 3.4 and θC that for the PLGlu random coil at pH 12.0 (λ ) 222 nm). Figure 7 shows fH as a function of pH of the solutions of PEO110b-PLGlu25 and its complex with diminazene. The strong shift of the helix-to-coil transition from pH 5 to 12 due to complexation can be clearly seen. It seems obvious that the interaction with diminazene tremendously increases the stability of a PLGlu R-helix. DLS measurements showed that the PEO110-b-PLGlu25/ diminazene complex assembles into discrete nanoparticles with a hydrodynamic radius of 16 nm and low polydispersity index27,28 (P.I. ) 0.1) at pH 7.4. The noncomplexed PEO110-b-PLGlu25 displays only scattering entities smaller than 5 nm in the TrisHCl buffer, which most likely are individual polymer chains. As can be seen in Figure 7, the size of nanoparticles remains the same (27) Finsy, R. AdV. Colloid Interface Sci. 1994, 52, 79-143. (28) Gun’ko, V. M.; Klyueva, A. V.; Levchuk, Y. N.; Leboda, R. AdV. Colloid Interface Sci. 2003, 105, 201-328.
Figure 9. Molar ellipticity of PEO-b-PLGlu25 (circles).and PLGlu26.(squares) at 222 nm as a function of diminazene concentration. The ellipticity is given with respect to LGlu monomers (LGlu concentration was 1.34 mM).
up to pH 11.5. Above pH 12, the radius increases rapidly to about 100 nm and the P. I. to about 0.5. It is known that the amino acid residue expands to over 0.15 nm in an R-helix.29 In the case of PEO110-b-PLGlu25, the helical end-to-end distance was calculated to be 3.75 nm. Several reports are available regarding the end-to-end distance of PEO in their fully extended, meander, and random coil conformation,30 which for PEO110 should be 40, 20, and 2.5 nm, respectively. Combining the geometrical characteristics with the experimental data it is safe to assume that the R-helical PLGlu segment is complexed with diminazene and surrounded by PEO to form a spherical micelle-like structure with core-shell symmetry. The coreshell micelles of PEO110-b-PLGlu25 are stable within the range of pH 6.5 to 11.5 (cf. Figure 7). The complex disintegrates at higher pH values as a result of a deprotonation of diminazene molecules followed by a breaking of the ionic bonds between the LGlu units and diminazene. The scattering at the higher pH values larger than 11.5 may be the result of poorly defined clusters in which diminazene is connected weakly to the PEO and PLGlu segments by dipole-dipole and van der Waals forces. Our finding that both the particles stay stable and the helical conformation is stabilized until a high pH is reached let us assume that the particle formation contributes significantly to the stability of the R-helices. We earlier reported core-shell micelles for complexes of retinoic acid and PEO-PLLys,6 which showed larger hydrodynamic diameters of 50-60 nm. There, the R-helix of the PLLys block was also stable as long as the bonding between retinoic acid and the PLLys was intact. Complexation was done at pH 7.4 when the PLGlu segment of PEO-b-PLGlu25 is in a random coil and not an R-helix conformation. Therefore, complexation with diminazene induced a disorder to order transition from random coil to R-helix. The change of the CD signal of the PEO-b-PLGlu25 was measured at pH 7.4 as a function of the diminazene concentration to verify this; data are shown in Figure 8a. We performed the same experiment with a PLGlu26 homopolymer in order to see the influence of the PEO segment (Figure 8b). In both cases, the shape of the curves obtained in the absence of diminazene is typical for a random coil conformation, as expected at pH 7.4.23 Upon the addition of diminazene, the shape of the curves changes and becomes typical for the PLGlu R-helix. The only difference between the spectra of PEO-b-PLGlu25 and PLGlu26 is the absolute value of the ellipticity around 200 nm. It is lower for the block (29) Richardson, J. S.; Richardson, D. C. Prediction of Protein Structure and the Principles of Protein Conformation; Plenum Press: New York, 1989; pp 1-98. (30) Tanford, C.; Nozaki, Y.; Rohde, M. F. J. Phys. Chem. 1977, 81, 15551560.
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copolymer, probably as a matter of some steric hindrance of the complexation of diminazene by the PEO block. At a defined threshold (diminazene:LGlu > 0.3), however, the PLGlu26/ diminazene complex starts to precipitate, which is not observed for the PEO-b-PLGlu25 complex (see Figure 9). The different stability of nanoparticles can be explained by a steric stabilization of the nanoparticles by PEO chains.
Conclusion In this study, we have found that complexes in the form of nanoparticles (16 nm) were produced by the self-assembly of an
Thu¨nemann et al.
anionic PEO-b-PLGlu block copolymer and the cationic drug diminazene. Diminazene binds only with one of two possible sites to the LGlu units with binding constants K ∼ 1-2 × 104 M-1. The helix-to-coil transition of the PLGlu blocks is remarkably shifted from pH 5 to 12. This effective stabilization of the R-helix structure seems to be due to the formation of a protective coating of diminazene and a shell of PEO. One may speculate that diminazene can also stabilize R-helical rich protein structures against pH-induced denaturation. LA0521138
