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Characterization of Methylprednisolone Esters of Hyaluronan in Aqueous Solution: Conformation and Aggregation Behavior Anna Taglienti,† Massimiliano Valentini,‡,§ Paolo Sequi,‡ and Vittorio Crescenzi*,† Department of Chemistry, University “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, Experimental Institute for Plant Nutrition, Via della Navicella 2-4, 00184 Rome, Italy, and Institute for Biomedical Engineering and Department of Materials, Swiss Federal Institute of Technology and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland Received December 9, 2004; Revised Manuscript Received February 9, 2005
Methylprednisolone steroid esters of hyaluronan differing in degree of functionalization and molecular weight were investigated in aqueous solution. Conformation and aggregation phenomena were elucidated by means of circular dichroism, viscometry, rheology, and nuclear magnetic resonance, mainly by 1H pulsed field gradient (PFG) NMR, which allows the determination of the diffusion coefficient of the species under investigation. The functionalization of hyaluronan with the steroid induces a reduction of the molecular volume, as a consequence of intramolecular hydrophobic interactions. For concentrated samples we have observed the coexistence of unimolecular collapsed chains and of aggregates, the latter disappearing upon dilution. The methylprednisolone ester of lower molecular weight hyaluronan has a larger molecular volume than its higher molecular weight analogue, even though still smaller than the underivatized polymer. This effect can be explained with the reduced flexibility of the polymer backbone probably impairing intramolecular interactions. Introduction glycosaminoglycan1,2
Hyaluronan (HA) is a with important biological functions.3 It is one of the major components of the extracellular matrix and participates in several relevant biological processes, such as cell motility,4 cell differentiation,5 wound healing,6 and cancer metastasis.7 As a consequence of its high degree of biocompatibility, biodegradability, nonimmunogenicity, and viscoelastic properties, HA is currently used in viscosurgery,8 ophthalmic surgery,9 and osteoarthritis therapy.10,11 The biological relevance and the versatility of HA structure make this molecule an attractive building block for the preparation of polymers with potential biomedical applications.12 Many chemical modifications of the native HA structure13 have been obtained; for example, partial or total esterification of the carboxylic groups produces biomaterials with interesting rheological properties and with high processability for preparing films and microspheres.14 The covalent binding of drugs15 to the carboxylic functions has received considerable attention since this procedure gives a polymeric prodrug as product. One of the most interesting and promising polymeric prodrugs derived from HA is obtained from the proper combination of hyaluronan with the steroid methylprednisolone.16 The latter was covalently bound to HA by esterification of the hydroxyl groups in C-21 position of the steroid with the carboxyl groups of the glucuronic acid moieties of hyaluronan.17 * To whom correspondence should be addressed: tel +39 6 49913630; e-mail
[email protected]. † University “La Sapienza”. ‡ Experimental Institute for Plant Nutrition. § Swiss Federal Institute of Technology and University of Zurich.
Prior to any in vivo investigation of the hyaluronan/ methylprednisolone system, a structural characterization in solution of such derivative was necessary since it is known that physical and chemical properties can often influence drastically the efficiency of polymeric drug carriers in terms of pharmacodynamics, pharmacokinetics, drug delivery, and biodegradability. In this paper we report a structural study of the hyaluronan/ methylprednisolone derivative for which the influence of some key parameters such as degree of functionalization, polymer concentration, and molecular weight has been investigated and elucidated. Experimental Section Materials. D2O was purchased from Acros Organics and was used without further purification. Sodium hyaluronate of high (about 600 000, fraction Hyalectin) and medium (about 200 000, fraction Hyalastine) molecular weight, and hyaluronan methylprednisolone esters of high and medium molecular weight, HYC141 and HYC41, respectively, were supplied by Fidia Farmaceutici S.p.A. (Abano Terme, PD, Italy) and used without further purification (Figure 1). HYC141 derivatives have 60%, 45%, or 16% of the carboxylic groups of glucuronic acid functionalized with the steroid, denoted HYC141p60, HYC141p45, and HYC141p16, respectively, while the HYC41 derivative has 45% of carboxyl functions esterified, denoted HYC41p45. The remaining carboxylic groups are in the sodium salt form. It should be noted that quite naturally the charge density decreases as the degree of functionalization increases.
10.1021/bm049221d CCC: $30.25 © 2005 American Chemical Society Published on Web 03/17/2005
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where A(τ1 + τ2) and A(0)/2 are the amplitudes of the echo with and without gradients, respectively; T1 and T2 are the longitudinal and transverse relaxation times, respectively; D is the diffusion coefficient of the species under investigation; γ is the gyromagnetic ratio of the observed nucleus; and the other parameters are defined in Figure 2. For nuclei having relatively long relaxation times, eq 1 can be rewritten as A(τ1 + τ2) )
Figure 1. Structure of hyaluronan salt with X ) Na+ and methylprednisolone hyaluronic acid ester (HYC) with X ) methylprednisolone (% derivatization) and having the remaining carboxyl groups as sodium salt. Methylprednisolone is covalently linked through the OH group at position 21 by ester bonds to the carboxyl group of the glucuronic acid moiety.
A(0) δ exp - (γGδ)2D ∆ 2 3
[
ln
()
( )
I δ ) -(γGδ)2D ∆ - ) -kD I0 3
A(τ1 + τ2) )
[
)] (1)
τ2 - τ1 2τ1 A(0) δ - (γGδ)2D ∆ exp 2 T1 T2 3
(
(2)
(3)
where I and I0 are the intensities of the NMR peaks of the spectra measured with and without the gradients, respectively. The diffusion coefficient D is generally determined as the slope of the linear plot of ln (I/I0) vs k, where k is equal to (γGδ)2[∆ - (δ/3)]. In the case of spherical objects it is finally possible to correlate the diffusion coefficient with the hydrodynamic radius rH via the Stokes-Einstein equation:
1H
These products were presented as freeze-dried powders and were dissolved in distilled water at stock concentration of 1.5% (w/v) before use. NMR Experiments. NMR measurements were performed on a Bruker Avance 400 MHz spectrometer equipped with a microprocessor-controlled gradient unit and a multinuclear probe with an actively shielded Z-gradient coil. All samples were measured in H2O, used for the reconstitution of the powder, with the addition of variable amounts of D2O to obtain the desired concentration. The 1H pulsed field gradient (PFG) NMR measurements18-20 were performed at 298 K without spinning and with the threepulse stimulated-echo sequence21 shown in Figure 2. The minimum time required for eddy current effects to decay was found to be ca. 200 µs, so that delays between the first π/2 pulse and the first gradient and between the last gradient and the acquisition were set longer than 200 µs. The shapes of the gradients were rectangular and their length was set equal to 10 ms. The diffusion time, ∆, was held constant so that the attenuation resulting from spinlattice relaxation (T1) would not vary; ∆ value was equal to 150 ms. Gradient strengths between 0.054 and 0.558 T m-1 were used. For a single diffusing species, the free induction decay (FID) amplitude is given by
)]
After Fourier transformation, the echo amplitudes are proportional to the signals’ intensities and one obtains
rH ) Figure 2. Stimulated Echo sequence used in PFG-NMR experiments. Solid rectangles represent gradient pulses with length and amplitude equal to δ and to G, respectively. The diffusion time ∆ is the interval between the midpoints of the gradients.
(
kBT 6πηD
(4)
where kB is the Boltzmann constant, T is the temperature expressed in kelvins, and η is the viscosity of the solution. Data Treatment. In the most general case of a polydisperse sample, the diffusion coefficient distribution P(D) is related to the intensities of the NMR peaks through the following relationship: I ) I0
∫0∞ P(D) exp(-kD) dD
(5)
which is obtained from eq 3 with the assumption of NMR intensity additiveness. In this mathematical treatment, analogous to that usually applied to dynamic light scattering data, eq 5 presents the problem of a physically interesting function, such as the diffusion coefficient distribution, that is to be obtained from noisy experimental data through the numerical inversion of an integral equation. In the most common approach to this problem, a priori information about the unknown distribution is applied to the experimental data. The most common regularization adopted is that due to Tikhonov,22 which, among the distributions fitting the experimental data, favors the smoothest. In this work, the inversion of eq 5 was performed with CONTIN,23,24 a Fortran IV software package that implements a Tikhonov regularization coupled with nonnegativity constraints in the framework of least-squares programming. Circular Dichroic Analysis. Circular dichroic spectra were recorded at 25 °C with a Jasco J715-A dichrograph equipped with quartz cuvettes 0.1 cm thick, in the wavelength range 190-300 nm according to the following setup: bandwidth 1 nm; time constant 2 s; scan rate 50 nm/min; sensitivity 5 mdeg; four spectra corrected with background were averaged for each sample.
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Viscometry. The viscosity at 20 °C of HYC141p60 and HYC141p45 in distilled water have been measured on a Shott-Gerate automatic dilution viscometer (AVS) with concentration ranging from 0.005% to 0.015% (w/v); intrinsic viscosity values have been calculated by a double extrapolation of the relevant Huggins-Kraemer plots.25 Rheological Measurements. Steady-shear viscosity experiments were performed in a stress/rate-controlled TA Instruments AR1000 rheometer that uses a cone-and-plate geometry with a cone diameter of 40 mm and a cone angle of 2°. The rotor position is controlled by airflow, ensuring the application of a virtually friction-free torque to the rotating unit; the measuring device is equipped with a temperature unit (Peltier plate) that ensures very good temperature control ((0.1 °C) over an extended period of time. A constant volume of sample, equal to 0.59 mL, was used for all measurements. The viscosity measurements were performed in rate-controlled mode over an extended shear rate range (covering both the nonlinear and, if present, the linear regions) and the shear rate dependence of viscosity was usually monitored as a function of increasing shear rate, as no significant hysteresis effects were detected in the preliminary measurements.
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Figure 3. CD signal at 238 nm of aqueous solutions of HYC141p45 at different concentrations; the monoexponential function best fitting the points indicates strong deviation from linearity starting from 0.2% (w/v) concentration.
Results and Discussion Self-Assembly Properties. The CD spectrum of HYC shows a negative peak at 210 nm corresponding to the free carboxylic groups and a positive signal at 238 nm due to the steroid moieties. This assignment is consistent with the CD spectra of HA, which shows the negative signal at 210 nm,26 and of aqueous solutions of methylprednisolone hemisuccinate, where the positive peak at 238 nm is present. CD spectra of all HYC141 samples measured at polymer concentrations ranging from 0.1% to 0.45% (w/v) in water show a nonlinear dependence of the signal at 238 nm from the polymer concentration, suggesting the offspring of intermolecular aggregates at concentrations higher than 0.2% (w/v). Figure 3 reports the CD signal at 238 nm of HYC141p45 vs concentration and indicates that the experimental data can be fitted with a single-exponential function. To elucidate the nature of the proposed aggregation, and in particular of the inter- and/or intramolecular interactions, investigations with other analytical techniques were performed. Molecular Conformation in Dilute Solution. Intrinsic viscosities and Huggins’ coefficients kH for HYC141p45 and HYC141p60 are reported in Table 1. The kH value is almost the same for both derivatives and agrees with the usual values observed for polymers in good solvents. We have found that the intrinsic viscosity, [η], decreases significantly with increasing degree of functionalization. Considering that the molar mass is constant for both biopolymers and that the quantity [η]M is proportional to hydrodynamic radius (rH), the [η] data suggest that the average hydrodynamic radius of the samples decreases with increasing degree of functionalization. This implies that these solutions may contain either collapsed unimolecular systems or compact aggregates formed for intra- and intermolecular
Figure 4. Steady shear viscosity at 20 °C of 0.5% (w/v) aqueous solutions of HA, HYC141p45, and HYC141p60. Table 1. Intrinsic Viscosity and Huggin’s Coefficient of HYC141p45 and HYC141p60 in Water sample
[η] (dL/g)
kH
HYC141p45 HYC141p60
79 ( 1 47 ( 1
0.43 ( 0.01 0.41 ( 0.01
interactions. The former explanation seems more realistic, also because the kH values indicate water as a good solvent for these biopolymers. Rheological Properties. Figure 4 shows the steady-shear viscosity of HA and HYC141 samples as a function of the shear rate. A qualitative consideration of the shapes of the curves indicates that there is not any newtonian linear region in HYC samples, different from what is found for HA. This behavior, observed also at very low shear rates, suggests the existence of extremely deformable aggregates.27 Furthermore, we have observed that both functionalized biopolymers are less viscous than HA, thus suggesting the breakage of the hydrogen-bond network, which is responsible for the stiffness of the HA molecule according to the proposed model.28,29
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Figure 5.
1H
NMR spectrum of HYCp45 in D2O.
The viscosity values for the two HYC141 systems considered are quite similar, albeit the viscosity seems to decrease by increasing the degree of functionalization. Nature of the Aggregates. The peculiar properties of HYC141 in solution, highlighted by the experimental data discussed above, suggest large differences in the macromolecular structure between HA and the HA-steroid systems. To elucidate the aggregation phenomenon, 1H PFG-NMR experiments were carried out. Other sizing techniques commonly used for macromolecular systems, for example, laser light scattering, gel-permeation chromatography, and osmometry, were not taken into account because of experimental dfficulties: basically the HYC141 molecule, although highly soluble in water and isotonic buffers, cannot be filtered properly by the use of both hydrophilic and hydrophobic membranes, probably as a consequence of its amphiphilic nature. The 1H PFG-NMR method fulfils perfectly our experimental needs since one can analyze unfiltered samples and the measurements can be performed in the desired physiological and/or applicative conditions in terms of temperature, polymer concentration, pH, etc. Moreover this technique, which allows direct measurement of the diffusion coefficient of the species under investigation, gives excellent results also in the case of opaque solutions or suspensions, even though this was not the case, and no pretreatment or purification of the sample is required.30 The 1H NMR spectra of HA and HYC (the latter is in Figure 5) show few sharp and intense peaks, and we have chosen to monitor during the 1H PFG-NMR experiments the intensity of the signal at δ ) 1.95 ppm, belonging to the polysaccharide’s acetamido group. Its sharpness and intensity gives excellent results without requiring long acquisition times. For some HYC samples also the peaks at δ ) 0.81, 1.35, 6.02, and 6.25 ppm were considered in order to ensure the absence of misleading effects on the resonance at δ ) 1.95 ppm, but due to the low signal/noise ratio we report below only the data obtained by monitoring the signal at δ ) 1.95 ppm. For the HA samples only the frequency at δ ) 1.95 ppm is considered since it is the only one present in the 1H NMR spectrum. 1 H PFG-NMR data of HYC141 and of HA (fraction Hyalectin) at a concentration equal to 0.5% (w/v) are shown in Figure 6, which reports the plot of the echo attenuation,
Figure 6. Plot of the echo attenuation, ln (I/I0), vs k, where k is equal to (γ∆δ)2[∆ - (δ/3)], of HA fraction Hyalectin and HYC141 having different degrees of functionalization in 0.5% (w/v) aqueous solution. The resonance at 1.95 ppm is monitored. The slopes of the fitting lines, equal to the diffusion coefficients, are reported in parentheses for each sample. Table 2. Measured Diffusion Coefficient Values entry
sample
D × 1012 (m2 s-1)
1 2 3 4 5 6 7 8
HA Hyalectin 0.5% HYC141p16 0.5% HYC141p45 0.5% HYC141p60 0.5% HYC141p45 0.05% HYC141p60 0.05% HA Hyalastine 0.5% HYC41p45 0.5%
0.82 1.6 2.0 2.2 3.1 5.4 3.2 3.8
ln (I/I0), vs k for HA, HYC141p16, HYC141p45, and HYC141p60. The experimental points for all the measured samples in the plot of ln (I/I0) vs k can be fitted with linear functions having different slopes, and thus different diffusion coefficients, depending on the degree of functionalization: 8.2 × 10-13 m2 s-1 for HA, 1.6 × 10-12 m2 s-1 for HYC141p16, 2.0 × 10-12 m2 s-1 for HYC141p45, and 2.2 × 10-12 m2 s-1 for HYC141p60; see Table 2. These values can be correlated to the hydrodynamic radii via the Stokes-Einstein equation (eq 4), keeping clearly in mind that this analysis is semiquantitative since the drastic simplification of a spherical geometry is assumed. The calculation for HA with the Stokes-Einstein equation is not reported since its geometry differs greatly from a spherical one and gives unreliable data. One obtains sizes equal to 9, 7, and 6 nm for HYC141p16, HYC141p45, and HYC141p60, respectively, which implies that a higher degree of functionalization with the steroid of hyaluronan induces a reduction in the macromolecular sizes of the objects. Since no significant degradation of the backbone occurs during the synthetic preparation, one can explain these data by the existence of strong intramolecular associations that induce a collapse of the structure. These interactions are likely to be hydrophobic due to the large amount of steroidic groups present, which can be segregated in the core, while the hydrophilic backbone of the polysaccharide are exposed to the solvent for stabilizing the structure.
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Figure 7. Plot of ln (I/I0) vs k of HYC141p45 and HYC141p60 in 0.05% (w/v) aqueous solution.
The diffusion coefficient data agree with the intrinsic viscosity values; in fact, both measurements indicate a decrease of the hydrodynamic radius with increasing degree of functionalization. Moreover, the CD data clearly account for intermolecular interactions at concentrations higher than 0.2% (w/v), so we suppose that few multimolecular aggregates coexist with the major part of isolated and collapsed chains. Effect of Dilution. To further ascertain that the aggregates are mainly formed by intramolecular interactions, we have repeated the 1H PFG-NMR experiments on samples diluted 10-fold, that is, at a concentration of 0.05% (w/v). The plot of the echo attenuation, ln (I/I0), vs k for the HYC141p60 and HYC141p45 samples is shown in Figure 7. Due to the low signal-to-noise ratio, as a consequence of the dilution, the quality of the measurements is not as excellent as the one discussed in Figure 5, but nevertheless we can observe increasing diffusion coefficients, and thus a reduction of the size with dilution, for both biopolymers: 3.1 × 10-12 m2 s-1 for HYC141p45 and 5.4 × 10-12 m2 s-1 for HYC141p60. These data support the idea of the presence of a small amount of multimolecular aggregates formed for intermolecular interactions, which are weakened by dilution. The effect is particularly large for the most heavily functionalized polymer, HYC141p60, whose diffusion coefficient is increased by a factor of about 1.5 upon dilution. Effect of Molecular Weight. We have also elucidated the influence of the molecular weight of the HA-steroid system in the macromolecular organization. In fact, modification of the molar mass can produce a noticeable change in conformation, so that we have considered low molecular weight HYC41p45, prepared staring from HA fraction Hyalastine, that is, MW 200 000, and the nonfunctionalized analogue, and compared the 1H PFG-NMR data. The latter are reported in Figure 8, which shows the plot of the echo attenuation, ln (I/I0), vs k. The observed trend is similar to that found for high molecular weight analogues, the HYC41p45 having a larger diffusion coefficient, and thus a smaller size, 3.8 × 10-12 m2 s-1, than HA, 3.2 × 10-12 m2 s-1. The reduction of the D values is smaller with respect to the higher molecular weight analogues, for which the functionalization induces
Taglienti et al.
Figure 8. 1H PFG-NMR data for HA fraction Hyalastine and HYC41p45 in 0.5% (w/v) aqueous solution.
Figure 9. Size distribution plot obtained by 1H PFG-NMR experiments for HYC41p45 in 0.5% (w/v) aqueous solution.
an increase of about 60% of the diffusion coefficient value, while in low molecular weight systems the difference is only 17%. The reason is that lower masses impair intramolecular interactions, because of the decrease of both the number of hydrophobic groups per macromolecule and of intrinsic flexibility. Furthermore it was possible to obtain the size distribution of the species in solution by the use of the CONTIN algorithm based on the Laplace inverse transformation: Figure 9 shows the hydrodynamic radius distribution for HYC41p45 as an example. The abundance peak at ca. 7 nm is consistent, at least in the order of magnitude, with the D value obtained with the Stokes-Einstein equation, ca. 4 nm. The larger component, in terms of size, of the distribution is probably due to the intermolecular aggregates previously discussed on the basis of CD data and the effect of dilution on diffusion coefficients. Conclusions The conformation and aggregation in aqueous solution of hyaluronan and hyaluronan functionalized with the steroid
Methylprednisolone Esters of Hyaluronan
methylprednisolone have been elucidated by means of CD, viscometry, rheology, and 1H PFG-NMR. The functionalization gives rise to very compact species in solution, and competition between intramolecular and intermolecular interactions has been observed, depending mainly on the amount of steroid subsistent and on polymeric concentration. Samples with different molecular weights have highlighted that competitive forces exist between intramolecular interactions and internal stress according to the intrinsic flexibility of the polymer backbone. Moreover, samples with different concentrations have revealed the competition between intermolecular interactions and probability of different polymer chains to overlap. These results provide a clear insight in the behavior of the steroid/hyaluronan biopolymer in aqueous solution, useful for developing the intended biomedical application as drug carrier. Acknowledgment. We gratefully thank Fidia Farmaceutici S.p.A. for providing the samples and for financial support. We warmly thank Professor Nicola Tirelli, University of Manchester, for useful discussions and advice. M.V. thanks Gebert Ru¨f Stiftung (Basel, Switzerland) for the fundamental financial support. References and Notes (1) Meyer, K.; Palmer, J. J. Biol. Chem. 1934, 107, 629-634. (2) Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Chem. ReV. 1998, 98, 2663-2684. (3) Laurent, T. C. The chemistry, biology and medical applications of hyaluronan and its deriVatiVes; Portland Press: London, 1998. (4) Collis, L.; Hall, C.; Lange, L.; Ziebell, M.; Prestwich, R.; Turley, E. A. FEBS Lett. 1998, 440, 444-449. (5) Entwistle, J.; Hall, C. L.; Turley, E. A. J. Cell. Biochem. 1996, 61, 569-577. (6) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharm. Biopharm. 2000, 50, 27-46.
Biomacromolecules, Vol. 6, No. 3, 2005 1653 (7) Hall, C. L.; Yang, B. H.; Yang, X. W.; Zhang, S. W.; Turley, M.; Samuel, S.; Lange, L. A.; Wang, C.; Curpen, G. D.; Savani, R. C.; Greenberg, A. H.; Turley, E. A. Cell 1995, 82, 19-28. (8) Shah, M. V. Patent WO 03059391, 2003. (9) Arshinoff, S. A. In 12th International Cellucon Conference: Wrexham, 2002; pp 119-128. (10) Day, R.; Brooks, P.; Conaghan, P. G.; Petersen, M. J. Rheumatol. 2004, 31, 775-782. (11) Weiss, C. In New Frontiers in Medical Sciences: Redefining Hyaluronan; Abatangelo, G., Weigel, P. H., Eds.: Padua, Italy, 1999; pp 89-103. (12) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487-492. (13) Bulpitt, P.; Aeschlimann, D. J. Biomed. Mater. Res. 1999, 47, 152169. (14) Crescenzi, V.; Francescangeli, A.; Renier, D.; Bellini, D. Biopolymers 2002, 64, 86-94. (15) Luo, Y.; Prestwich, G. D. Bioconjugate Chem. 1999, 10, 755-763. (16) Payan, E.; Jouzeau, J. Y.; Lapicque, F.; Bordji, K.; Simon, G.; Gillet, P.; Oregan, M.; Netter, P. J. Controlled Release 1995, 34, 145153. (17) Romeo, A.; Della Valle, F. (Fidia Farmaceutici S.p.A.). U.S. Patent 4851521, 1989. (18) Price, W. S. Concepts Magn. Resonance 1997, 9, 299-336. (19) Price, W. S. Concepts Magn. Resonance 1998, 10, 197-237. (20) Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1-45. (21) Tanner, J. E. J. Chem. Phys. 1970, 52, 2523-2525. (22) Groetsch, C. W. The theory of TikhonoV regularization for Fredholm equations of the first kind; Pitman: Boston, 1984. (23) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 229-242. (24) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213-227. (25) Huggins, M. L. J. Phys. Chem. 1938, 42, 911. (26) Cowman, M. K.; Balazs, E. A.; Bergmann, C. W.; Meyer, K. Biochemistry 1981, 20, 1379-1385. (27) Knudsen, K. D.; Lauten, R. A.; Kjoniksen, A. L.; Nystrom, B. Eur. Polym. J. 2004, 40, 721-733. (28) Scott, J. E.; Heatley, F. Biomacromolecules 2002, 3, 547-553. (29) Heatley, F.; Scott, J. E. Biochem. J. 1988, 254, 489-493. (30) Valentini, M.; Vaccaro, A.; Rehor, A.; Napoli, A.; Hubbell, J. A.; Tirelli, N. J. Am. Chem. Soc. 2004, 126, 2142-2147.
BM049221D
