Model of Drug-Loaded Fluorocarbon-Based Micelles Studied by

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Model of Drug-Loaded Fluorocarbon-Based Micelles Studied by Electron-Spin Induced 19F Relaxation NMR and Molecular Dynamics Simulation Errol V. Mathias,† Xiangli Liu,† Osmundo Franco,† Imran Khan,† Yong Ba,*,† and Julia A. Kornfield‡ Department of Chemistry and Biochemistry, California State UniVersity Los Angeles, Los Angeles, California 90032, and DiVision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 ReceiVed June 21, 2007. In Final Form: October 4, 2007 Rf-IPDU-PEGs belong to a class of fluoroalkyl-ended poly(ethylene glycol) polymers (Rf-PEGs), where the IPDU (isophorone diurethane) functions as a linker to connect each end of the PEG chain to a fluoroalkyl group. The Rf-IPDU-PEGs form hydrogels in water with favorable sol-gel coexistence properties. Thus, they are promising for use as drug delivery agents. In this study, we introduce an electron-spin induced 19F relaxation NMR technique to probe the location and drug-loading capacity for an electron-spin labeled hydrophobic drug, CT (chlorambucil-tempol adduct), enclosed in the Rf-IPDU-PEG micelle. With the assistance of molecular dynamics simulations, a clear idea regarding the structures of the Rf-IPDU-PEG micelle and its CT-loaded micelle was revealed. The significance of this research lies in the finding that the hydrophobic drug molecules were loaded within the intermediate IPDU shells of the Rf-IPDU-PEG micelles. The molecular structures of IPDU and that of CT are favorably comparable. Consequently, it appears that this study opens a window to modify the linker between the Rf group and the PEG chain for achieving customized structure-based drug-loading capabilities for these hydrogels, while the advantage of the strong affinity among the Rf groups to hold individual micelles together and to interconnect the micellar network is still retained in hopes of maintaining the sol-gel coexistence of the Rf-PEGs.

Introduction The role of polymeric carriers as nanobased drug delivery agents has gained importance in recent years.1-6 The majority of the applied polymeric molecules is amphiphilic in nature and possesses a unique capability to assemble into micelles in aqueous media.4-9 The assembly consists of a hydrophobic core surrounded by a hydrophilic outer sphere. The design of drug delivery systems using polymeric micelles depends on their ability to incorporate drugs, which is an area of great interest.10,11 Polymeric micelles possess good drug-loading capacities for hydrophobic drugs,4,12,13 including anticancer agents.10,11,14-17 Many of the core-sphere polymeric drug carriers are based on PEG (poly* Corresponding author. Tel.: (323) 343-2360; fax: (323) 343-6490; e-mail: [email protected]. † California State University Los Angeles. ‡ California Institute of Technology. (1) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113-131. (2) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature (London, U.K.) 1997, 388, 860-862. (3) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science (Washington, DC, U.S.) 2003, 300, 615-618. (4) Ge, H.; Hu, Y.; Jiang, X.; Cheng, D.; Yuan, Y.; Bi, H.; Yang, C. J. Pharm. Sci. 2002, 91, 1463-1473. (5) Nishiyama, N.; Kataoka, K. Pharmacol. Ther. 2006, 112, 630-648. (6) Otsuka, H.; Nagasaki, Y.; Kataoka, K. AdV. Drug DeliVery ReV. 2003, 55, 403-419. (7) Kwon, G. S.; Naito, M.; Kataoka, K.; Yokoyama, M.; Sakurai, Y.; Okano, T. Colloids Surf., B 1994, 2, 429-434. (8) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3-27. (9) Zhang, J. X.; Qiu, L. Y.; Jin, Y.; Zhu, K. J. Colloids Surf., B 2005, 43, 123-130. (10) Opanasopit, P.; Yokoyama, M.; Watanabe, M.; Kawano, K.; Maitani, Y.; Okano, T. Pharm. Res. 2004, 21, 2001-2008. (11) Nakayama, M.; Okano, T.; Miyazaki, T.; Kohori, F.; Sakai, K.; Yokoyama, M. J. Controlled Release 2006, 115, 46-56. (12) Trimaille, T.; Mondon, K.; Gurny, R.; Moller, M. Int. J. Pharm. 2006, 319, 147-154. (13) Francis, M. F.; Lavoie, L.; Winnik, F. M.; Leroux, J.-C. Eur. J. Pharm. Biopharm. 2003, 56, 337-346.

(ethylene glycol))-type polymers due to their favorable properties of biocompatibility, nontoxicity, and nonimmunogenicity.6 PEG modified with hydrophobes at both ends has been investigated widely as a model associative polymer.18 The majority of the systems studied so far forms a single phase over the entire concentration range. Some hydrogels formed by polymeric micelles possess the unique ability of possessing a sol-gel coexistence, and this can provide added benefits for drug delivery.19,20 Recently, phase separation has been reported for PEG modified with long alkyl groups,21 and a sol-gel coexistent system of hydrogels made from the self-assembly of PEG molecules modified at both ends with fluoroalkyl groups (RfPEGs) was also found.22,23 The synthesis of these Rf-PEGs involves the use of isophorone diisocyanate (IPDI), which functions as a linker between the Rf group and the PEG group. Upon reacting, the diisocyanate converts to a diurethane (IPDU), which is the final structure of the linker. Both PEG and fluorocarbons separately have been applied as drug delivery agents.24 In addition to fluorocarbon’s hydrophobic nature, they (14) Shuai, X.; Merdan, T.; Schaper, A. K.; Xi, F.; Kissel, T. Bioconjugate Chem. 2004, 15, 441-448. (15) Opanasopit, P.; Ngawhirunpat, T.; Chaidedgumjorn, A.; Rojanarata, T.; Apirakaramwong, A.; Phongying, S.; Choochottiros, C.; Chirachanchai, S. Eur. J. Pharm. Biopharm. 2006, 64, 269-276. (16) Wang, J.; Mongayt, D.; Torchilin, V. P. J. Drug Target. 2005, 13, 73-80. (17) Brigger, I.; Chaminade, P.; Marsaud, V.; Appel, M.; Besnard, M.; Gurny, R.; Renoir, M.; Couvreur, P. Int. J. Pharm. 2001, 214, 37-42. (18) Rubinstein, M.; Dobrynin, A. V. Curr. Opin. Colloid Int. 1999, 4, 83-87. (19) Shim, W. S.; Yoo, J. S.; Bae, Y. H.; Lee, D. S. Biomacromolecules 2005, 6, 2930-2934. (20) Lee, J. W.; Hua, F.; Lee, D. S. J. Controlled Release 2001, 73, 315-327. (21) Francois, J.; Maitre, S.; Rawiso, M.; Sarazin, D.; Beinert, G. Colloids Surf., A 1996, 112, 251-265. (22) Xu, B.; Li, L.; Yekta, A.; Masoumi, M.; Kanagalingam, S.; Winnik, M. A.; Zhang, K.; Macdonald, P. M.; Menchen, S. Langmuir 1997, 13, 2447-2456. (23) Giyoong, T.; Kornfield, J. A.; Hubbell, J. A.; Johannsmann, D.; HogenEsch, T. E. Macromolecules 2001, 34, 6409-6419. (24) Krafft, M. P. AdV. Drug DeliVery ReV. 2001, 47, 209-228.

10.1021/la701833w CCC: $40.75 © 2008 American Chemical Society Published on Web 12/01/2007

Model of Drug-Loaded Fluorocarbon-Based Micelles

are also lipophobic25 (i.e., do not dissolve in lipids). Fluorocarbons tend to strongly aggregate together, even in the presence of hydrocarbon chains.26 In an aqueous medium, Rf-PEGs assemble into micelles with the Rf groups forming the core and the PEG chains forming the outer sphere. Some of the Rf-PEG chains also cross-link between the micelles to physically hold the micellar network together to form a hydrogel system.23 It is also known that Rf-PEGs can dissociate in biocompatible organic solvents to produce a low viscosity and injectable formulation.23 An interesting feature of the Rf-PEG system is that the gel phase erodes in a predictable manner through the desorption of micelles from the surface followed by their diffusion into solution, while the sol-gel equilibrium composition is maintained by the gel phase concentration during the erosion.23 Many methods have been used to determine micelle formation and size including scattering experiments,9,13,27 microscope techniques such as TEM,4,9,14 19F NMR,28 SANS,29-31 and SAXS.30,31 Specifically, the SANS method has been used to determine the Rf-PEG micelle size.32 The micelle sizes for various fluorinated surfactants have also been calculated theoretically.30 The determination of encapsulation of drugs into micelles is of utmost importance. Methods for this study include spectroscopic methods such as UV,4,14 NIR,17 fluorescence,4,15,27 and chromatography.13-15,17,27 NMR has played an important role in the study of macromolecules. For example, relaxation NMR techniques have been applied to study molecular tumbling and the local motion of polymers,33 as well as binding of biological macromolecules.34,35 19F NMR has been used in the study of polymeric micelles and surfactants.36,37 The use of paramagnetic probes to study micellar properties by NMR have been well-documented.38-45 Changes in 19F relaxation times of fluorinated polymers have also been monitored using paramagnetic probes.40-42 In this paper, we introduce a method using electron-spin induced 19F and 1H spin lattice relaxation (T1) NMR to study the encapsulation of an electronspin labeled hydrophobic drug into the Rf-PEG micelles. We (25) Krafft, M. P.; Riess, J. G. Biochimie 1998, 80, 489-514. (26) Barthe´le´my, P.; Tomao, V.; Selb, J.; Chaudier, Y.; Pucci, B. Langmuir 2002, 18, 2557-2563. (27) Jette, K. K.; Law, D.; Schmitt, E. A.; Kwon, G. S. Pharm. Res. 2004, 21, 1184-1191. (28) Hoang, K. C.; Mecozzi, S. Langmuir 2004, 20, 7347-7350. (29) Downer, A.; Eastoe, J.; Pitt, A. R.; Penfold, J.; Heenan, R. K. Colloids Surf., A 1999, 156, 33-48. (30) Buhler, E.; Oelschlaeger, C.; Waton, G.; Rawiso, M.; Schmidt, J.; Talmon, Y.; Candau, S. J. Langmuir 2006, 22, 2534-2542. (31) Matsumoto, K.; Ishizuka, T.; Harada, T.; Matsuoka, H. Langmuir 2004, 20, 7270-7282. (32) Giyoong, T.; Kornfield, J. A.; Hubbell, J. A.; Lal, J. Macromolecules 2002, 35, 4448-4457. (33) Sillerud, L. O.; Larson, R. S. Methods Mol. Biol. (Totowa, NJ, U.S.) 2006, 316, 227-289. (34) Igumenova, T. I.; Frederick, K. K.; Wand, A. J. Chem. ReV. 2006, 106, 1672-1699. (35) Kay, L. E. J. Magn. Reson. 2005, 173, 193-207. (36) Asakawa, T.; Miyagishi, S.; Nishida, M. J. Colloid Interface Sci. 1985, 104, 279-281. (37) Clapperton, R. M.; Ottewill, R. H.; Ingram, B. T. Langmuir 1994, 10, 51-56. (38) Closs, G. L.; Forbes, M. D. E.; Norris, J. R. J. J. Phys. Chem. 1987, 91, 3592-3599. (39) Al-Abdul-Wahid, M. S.; Yu, C. H.; Batruch, I.; Evanics, F.; Pome`s, R.; Prosser, R. S. Biochemistry 2006, 45, 10719-10728. (40) Prosser, R. S.; Luchette, P. A. J. Magn. Reson. 2004, 171, 225-232. (41) Prosser, R. S.; Luchette, P. A.; Westerman, P. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9967-9971. (42) Kameo, Y.; Takahashi, S.; Krieg-Kowald, M.; Ohmachi, T.; Takagi, S.; Inoue, H. J. Phys. Chem. B 1999, 103, 9562-9568. (43) Gao, Z.; Wasylishen, R. E.; Kwak, J. C. T. J. Phys. Chem. 1991, 95, 462-467. (44) Hilty, C.; Wider, G.; Fernandez, C.; Wuthrich, K. ChemBioChem 2004, 5, 467-473. (45) Waggoner, A. S.; Griffith, O. H.; Christensen, C. R. Proc. Natl. Acad. Sci. U.S.A. 1967, 57, 1198-1205.

Langmuir, Vol. 24, No. 3, 2008 693 Scheme 1. Synthesis of CT Adduct

used an earlier synthesized spin-labeled anticancer drug, the chlorambucil-tempol (CT) adduct,46 as a model compound for this purpose. When an electron spin is in close proximity to a nuclear magnetic moment (or spin), the local magnetic field generated by the electron spin will be modulated as a function of time due to local molecular motion, which in turn induces a strong relaxation mechanism for that nucleus. We used this property to probe the location of CT in the Rf-PEG micelle and to monitor the molecular-loading capacity of the drug (CT), which could be encapsulated by the micelles. We further applied NMR relaxation theory to determine the average interspin distance of the free electron radical in CT and the -CF3 19F nucleus in the Rf group. Using the interspin distance information as a restraint for molecular dynamics simulations, a model for the CT-loaded Rf-PEG micelle was then fabricated. Experimental Procedures Materials. Chlorambucil (99%, 4-[bis(2-chlorethyl)amino]benzenebutanoic acid) was supplied by MP Biomedicals Inc., tempol (98%, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was supplied by Sigma-Aldrich, 6000 PEG (100%, poly(ethylene glycol) with a molecular mass of 6 KDa) was supplied by Alfa Aesar, 1H,1H,2H,2Hperfluorooctanol (97%) was supplied by Lancaster, isophorone diisocyanate (IPDI, 99.3%) was supplied by MP Biomedicals, and dibutyltin diacetate (Catalyst, Sn content: 33.0%) was supplied by Fisher Scientific. All organic solvents including dichloromethane, hexane, glyme (ethylene glycol dimethyl ether), diethyl ether, and methanol were purchased from Fisher Scientific as reagent or ACS grades. Synthesis of CT Adduct. Our method for the synthesis of the CT adduct was previously published.46 The product was purified by column chromatography and characterized by NMR and MALDITOF. Scheme 1 shows the synthesis route. Synthesis of 6KC6 (PEG MW 6000, Rf C6F13-CH2-CH2OH) Rf-PEG. The synthesis of the polymer was carried out by methods previously reported in the literature.22 The product was synthesized with a yield of ∼80%. Purity as determined by HPLC was found to be greater than 98%. The typical symbolic notation for the material is Rf-IPDU-[PEG]n-IPDU-Rf. To specify the IPDU linker and its specific role in hydrophobic drug loading (as will be shown shortly), in the rest of the paper we use the notation RfIPDU-PEG to denote the polymer. Its structural diagram is given in Scheme 2. Preparation of Drug-Loaded Samples. A total of 100 mg of 6KC6 Rf-IPDU-PEG and 1 mg of CT were first dissolved in 1 mL of methylene chloride. The homogeneous mixture was allowed to (46) Prabhutendolkar, A.; Liu, X.; Mathias, E. V.; Ba, Y.; Kornfield, J. A. Drug DeliV. 2006, 13, 433-440.

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Scheme 2. Schematic Representation of the Rf-IPDU-PEG Structure

completely air-dry. Then, 0.9 mL of D2O was added. The mixture was then sonicated using a Branson sonicator to yield 2.4 mM CT in the hydrogel. This gel sample was allowed to stabilize for 24 h before NMR studies. For comparison purposes, we also prepared a similar concentration (2.4 mM) of tempol in the hydrogel. Different concentrations of CT relative to Rf-IPDU-PEG were also prepared by the same method. They were prepared by mixing 0.4-8 mg of CT in 100 mg of 6KC6 Rf-IPDU-PEG. NMR Experiments. All the NMR experiments were carried out on a Bruker Avance DRX 400 MHz NMR instrument equipped with a 5 mm QNP probe tuned to proton and fluorine frequencies. T1 relaxation times were measured using the inversion recovery pulse sequence.47 A recycling delay time of 8 s and typically 256 scans were used. T2 relaxation times were measured using the CarrPurcell-Meiboom-Gill (CPMG) spin echo pulse sequence.48,49 The 1H chemical shifts were referenced with a proton signal at 4.8 ppm remaining in the D2O solvent, and the 19F chemical shifts were referenced with TFA (trifluoroacetic acid) as an external standard at -78.5 ppm. Molecular Dynamics (MD) Simulations. MD simulations using molecular mechanic (MM) methods were run on the discover module of the Insight II suite by Accelrys. A simulated annealing protocol was used, and potentials were set using the consistent valence force field (CVFF). Previously, the CVFF has been employed for perfluoroalkyl containing polymeric assemblies.50,51 After minimizing the assembly with the steepest descents and conjugate gradients, the dynamics was initialized at 300 K. Then, the assembly was heated to 1000 K in 2000 iterations and finally cooled to 300 K with 2000 iterations. Trajectories of 25 structures were collected after a final minimization using the steepest descents and conjugate gradients to achieve the rms derivatives of less than 0.001. Previous SANS studies for these micelles have suggested that the aggregation number for 6KC6 Rf-IPDU-PEG is close to but not larger than 32 Rf groups.32 We, therefore, used the micelle script in the Insight II program to prepare an initial structure consisting of 32 Rf groups. Throughout this study, two types of micelles were prepared. One was with only the Rf and IPDU groups called the Rf-IPDU structure, and the other type also included a short PEG chain for each Rf-IPDU unit, called the Rf-IPDU-PEG structure. Two such structures including one with 10 PEG units and the other with 20 PEG units were used. For the Rf-IPDU structure, each of the 32 Rf chains was attached to an IPDU unit that was endcapped with -CH3, and for the Rf-IPDU-PEG structure, endcapped -CH3 was replaced by PEG units. The Rf-IPDU micelle was prepared by using a script provided by Accelrys, and a restraint range within 10 Å was applied for each -CF3 group to the remaining 31 -CF3 groups using strong upper bound force restraints of 10 kcal/Å. In the case where CT needed to be added into the micelle with restraints, a distance restraint between the oxygen in the tempol group and a carbon in the IPDU ring was set to be about 4-5 Å with loose restraints of upper bound force constants at 4 kcal/Å. Our calculations did not include explicit water, as it would be time-consuming and beyond the affordability of the computation time. Because of the hydrophilic nature of the PEG chains and the hydrophobic nature of the IPDU and Rf groups, we do not expect that the exclusion of water would qualitatively mislead the micellar structure and the location of CT in the micelle. (47) Vold, R. L.; Waugh, J. S.; Klein, M. P.; Phelps, D. E. J. Chem. Phys. 1968, 48, 3831-3832. (48) Carr, H. Y.; Purcell, E. M. Phys. ReV. 1954, 94, 630-638. (49) Meiboom, S.; Gill, D. ReV. Sci. Instrum. 1958, 29, 688-691. (50) Koike, A. J. Phys. Chem. B 1999, 103, 4578-4589. (51) Senapati, S.; Berkowitz, M. L. J. Phys. Chem. B 2003, 107, 1290612916.

Relaxation Theory for Probing Electron-Nucleus Interspin Proximity One of the issues of this research was to find the location of CT enclosed in the Rf-IPDU-PEG micelle. The motional averaged distance between the electron spin in CT and the nuclear spin in 19F in the Rf -CF3 group provides such information. It is known that the relaxation rate caused by dipole-dipole interaction is inversely proportional to the sixth power of the interspin distance. Thus, we will use electron-nuclear relaxation to find the location of CT in the micelle. The spin lattice relaxation rate (R p1) of a nuclear spin (here 19F) due to the time dependent perturbation of a local magnetic field generated by electron spins can be written as52,53

Rp1 )

[]

1 2 µ0 2 2 2 2 ) N γf ge µB S(S + 1) × e Tp1 15r6 4π 3τc 7τc + (1) 2 2 1 + ωe τc 1 + ωf2τc2

[

]

where the superscript p indicates that the relaxation is caused by a paramagnetic species, Ne is the number of electron spins (in this case, the number of CT molecules), r is the motionally averaged value of the electron-nuclear interspin distance, γf ) 25.1815 × 107 rad s-1 T-1 is the gyromagnetic ratio of 19F, µ0 ) 1.2558 × 10-6 J s2 C-2 m-1 is the permeability of free space for 19F, ge ) 2.00232 is the average electronic ge factor, µB ) 9.2740154 × 10-24 J T-1 is the value of the Bohr magneton, S ) 1/2 is the electron-spin quantum number, τc is the correlation time for the related motion, ωf ) 376.4517587 MHz is the resonance frequency of 19F at a magnetic field corresponding to 400 MHz for a proton’s resonance frequency, and ωe ) 1.6548 × 1012 s-1 is the corresponding resonance frequency of an electron in the same field. The gyromagnetic ratio of an electron (1.7609 × 1011 rad s-1 T-1) is much larger than that of 19F or 1H (2.6751965 × 108 rad s-1 T-1). Thus, when unpaired electrons exist, the electron-nuclear dipole-dipole contribution dominates the spin lattice relaxation mechanism, and the nuclear dipole-dipole contribution to T1 becomes insignificant. Therefore, in calculating the electron-19F internuclear distance as seen shortly in this paper, we neglected 1H-19F and 19F-19F dipole-dipole relaxation mechanisms. Eq 1 shows that to estimate the electron-19F interspin proximity, the correlation time, τc, needs to be known in the first place. Assuming that the primary motional mode that causes relaxation is the C-CF3 chemical bond rotation, and perturbation to the motion due to the inclusion of CT in the micelle is negligible, the correlation time can be found from the following spin lattice relaxation rate due to the 19F-19F dipole-dipole mechanism53,54 (52) Bertini, I.; Luchinat, C. NMR of Paramagnetic Molecules in Biological Systems; Benjamin/Cummings: Menlo Park, CA, 1986. (53) Sudmeier, J. L.; Anderson, S. E.; Frye, J. S. Concepts Magn. Reson. 1990, 2, 197-212. (54) Farrar, T. C.; Becker, E. D. Pulse and Fourier Transform NMR; Academic Press: New York, 1971.

Model of Drug-Loaded Fluorocarbon-Based Micelles

R1 )

[]

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µ0 2 2τc 1 ) h2γf4 S(S + 1) × T1 4π 5r6 4 1 + (2) 1 + (ωfτc)2 1 + (2ωfτc)2

[

]

where r is the F-F distance in the -CF3 group. In a nonideal case, where the rotational motion of the Rf group and the micelle’s tumbling also contribute to the relaxation, the strategy using the ratio of R2/R1 can be employed,55 where R2 represents the spinspin relaxation rate as given in the following equation:53,54

R2 )

[]

µ0 2 τc 1 ) h2γf4 S(S + 1) × T2 4π 5r6 5 2 3+ + (3) 2 1 + (ωfτc) 1 + (2ωfτc)2

[

]

The internuclear distance r will be cancelled in the ratio. Thus, τc can be extracted provided that R1 and R2 have been obtained experimentally. τc obtained from this method reflects the effective correlation time due to the combination of many motional modes but may not solely represent that of the single C-CF3 chemical bond rotation. In the presence of CT in a micelle, the correlation time is made up of contributions from rotational motion and electron spinspin relaxation times, T2e, which is assumed to be replaced by an electron correlation time, τe.53 To find the effect of τe on the correlation time, we can include the electron-19F dipole-dipole contribution to the ratio R2/R1. If the electron-19F dipole-dipole mechanism dominates the relaxation, the R p2/R p1 ratio can be used to find the correlation time, where R p2 represents the electron19F dipole-dipole contribution to the spin-spin relaxation time as given by52,53

Rp2 )

[]

1 1 µ0 2 2 2 2 ) Ne 6 γf ge µB S(S + 1) × p T2 15r 4π 3τc 13τc + + 4τc (4) 2 2 1 + ωe τ c 1 + ωf2τc2

[

]

Results and Discussion Encapsulation of CT in the Rf-IPDU-PEG Micelles. To assess the property of the Rf-IPDU-PEG micelles to encapsulate CT molecules, we carried out 19F and 1H T1 relaxation experiments for three hydrogel samples. The first was the blank 6KC6 RfIPDU-PEG hydrogel, the second was the CT-loaded 6KC6 RfIPDU-PEG hydrogel (2.4 mM corresponding to 1% CT in 6KC6 Rf-IPDU-PEG), and the third was the tempol-loaded 6KC6 RfIPDU-PEG hydrogel (2.4 mM). It is known that the Rf groups form the core, the IPDU units form the intermediate shell, and the PEG chains form the outer sphere of the Rf-IPDU-PEG micelle. CT is quite hydrophobic, and tempol is hydrophilic due to its hydroxyl group. Thus, comparison of the 1H and 19F T1 values for these samples gives us an understanding of the locations of the free radical groups of CT and tempol in the micelle. The experimental results are summarized in Table 1. It can be seen that the 1H relaxation times changed to some extent from 0.61 s for the blank hydrogel to 0.52 s for the CT-loaded hydrogel, while for the tempol-loaded hydrogel, the corresponding T1 times changed significantly from 0.61 to 0.31 s. The larger T1 time (55) Kay, L. E.; Torchia, D. A.; Bax, A. Biochemistry 1989, 28, 8972-8979.

Figure 1. 19F T1 values for the Rf terminal -CF3 groups of the 6KC6 Rf-IPDU-PEG micelles vs CT-loading level. Table 1. Summary of 1H and 19F T1 Relaxation Times sample hydrogel blank (6KC6) CT-loaded hydrogel tempol-loaded hydrogel

1H

T1 value (s)

0.61( 0.02 0.52 ( 0.03 0.31 ( 0.02

19F

T1 value (s)

0.56 ( 0.02 0.24 ( 0.02 0.44 ( 0.01

change for the tempol-loaded sample shows the closer proximity of tempol molecules to the PEG chains. The opposite trend of changes was observed for the -CF3 19F T1 times. The CT-loaded hydrogel caused the 19F T1 time to decrease from 0.56 s for the blank hydrogel to 0.24 s, while the tempol-loaded sample only caused a decrease to 0.44 s. Thus, the 19F T1 results show that CT molecules are in molecular proximity to the Rf groups. This is a logical conclusion because CT is much more hydrophobic than tempol and the Rf-IPDU-PEG micelle is constituted by the hydrophobic Rf core and the hydrophobic intermediate IPDU shell. CT-Loading Capacity. Figure 1 shows the 19F T1 values of the Rf terminal -CF3 groups for the CT-loaded 6KC6 Rf-IPDUPEG hydrogels as a function of CT-loading levels (mg of CT/ 100 mg of Rf-IPDU-PEG). Figure 1 shows that the T1 values decrease as the CT-loading level increases. The decrease leveled off after more than 2 mg of CT was introduced to 100 mg of Rf-IPDU-PEG. The relaxation mechanism is primarily attributed to the nucleus-electron dipole-dipole interaction. The 19F relaxation rate is approximately proportional to the number of nitroxide radicals in molecular proximity to the 19F nucleus. Thus, the plateau in the T1 curve illustrates that the CT-loading level has reached a maximum. The excess amount of CT stayed in the hydrogel as solid particles. In theory, the -CF3 19F and electron interspin distance is determined by the micelle size and location of the CT molecules in the micelle. Previous studies by SANS have shown that the hydrophobic Rf cores order into a body-centered-cubic structure in the hydrogel and that the Rf aggregation number of the hydrophobic core is related to the length of the hydrophobic end groups but insensitive to polymer concentration and temperature.32 The SANS results also demonstrate a trend that the aggregation number decreases with a decrease in the length of the Rf hydrophobe. According to their data, the Rf aggregation number for 6KC6 Rf-IPDU-PEG is close to but not larger than 32. Using the number 32, we can estimate from Figure 1 that the maximum number of CT molecules that can be encapsulated in one micelle is about 4. Electron-19F Interspin Proximity. Eq 1 shows that the electron-19F interspin distance can be determined as long as T1 and the correlation time are known. To determine the correlation

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Figure 2. (a) Stacked plot of spectra vs recovery time and (b) exponential decay of -CF3 19F intensity vs recovery time for the blank Rf-IPDU-PEG hydrogel.

Figure 3. (a) Stacked plot of spectra at different delay times and (b) -CF3 19F T2 decay curve vs spin echo time for the blank RfIPDU-PEG hydrogel.

time using the ratio of R2/R1, we carried out the 19F T1 and T2 NMR experiments for the blank Rf-IPDU-PEG hydrogel. The stacked plot of the 19F NMR spectra versus the recovery time as labeled with the spectra is shown in Figure 2 a. The peak at around -112 ppm corresponds to the 19F nuclei of the -CF2groups adjacent to the methylene carbons of the Rf chain., followed by a broad peak from -115 to -125 ppm corresponding to the 19F nuclei of the -CF - backbone, and last the broad peak at 2 -125 to -127 ppm is assigned to the -CF2- groups adjacent to the terminal -CF3.56 The exponential decay curve for -CF3 19F versus recovery time is shown in Figure 2b, which generates T1 ) 0.56 ( 0.02 s. The -CF3 19F T2 experimental result is shown in Figure 3 a (stacked plot) and Figure 3b (exponential decay curve). The resulting T2 value is 0.00032 ( 0.00003 s. By applying R2/R1 ) 0.56/0.00032 ) 1750 to R2/R1 determined by eqs 3 and 2, τc ) 128 ( 20 ns was obtained. τc could represent the effective correlation time of multiple motions, including the major C-CF3 bond rotation, and the Rf group’s motion and the micelle’s tumbling.

It was reported that fluoroalcohols in a water solution have correlation times in the picosecond scale.57 However, our τc value was found to have the same order in other systems where the -CF3 groups were linked to bulky groups and their rotations were hindered.58,59 We presume that our correlation time was due to the crowded intergroup interactions in the Rf core of the hydrogel and that the motional mode of the -CF3 group is in the slow motion regime. To confirm the slow motion, we carried out a variable temperature (VT) dependent T1 NMR experiment for the blank hydrogel sample. A plot of T1 versus temperature as displayed in Figure 4 shows that T1 decreases with an increase of temperature, which in turn demonstrates that T1 increases with an increase in correlation time. Therefore, the VT dependent T1 experiment reveals that the rotation of the C-CF3 chemical bond is in the slow motion regime. By applying the correlation time τc ) 128 ( 20 ns and T1 ) 0.22 ( 0.02 s for the sample with a maximum CT-loading level

(56) Zhang, H.; Pan, J.; Hogen-Esch, T. E. Macromolecules 1998, 31, 28152821.

(57) Mizutani, Y.; Kamogawa, K.; Kitagawa, T.; Shimizu, A.; Taniguchi, Y.; Nakanishi, K. J. Phys. Chem. 1991, 95, 1790-1794. (58) Phillips, L.; Separovic, E.; Cornell, B. A.; Barden, J. A.; dos Remedios, C. G. Eur. Biophys. J. 1991, 19, 147-155. (59) Kay, L. E.; Pascone, J. M.; Sykest, B. D.; Shriver, J. W. J. Biol. Chem. 1987, 262, 1984-1988.

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Figure 4. -CF3 19F T1 vs temperature for the blank Rf-IPDU-PEG hydrogel.

(Ne ) 4) to eq 1, the interspin distance was estimated to be 12.3 ( 1 Å. The T1 values for other loading levels could also be used to determine the interspin proximity. However, the distribution of CT for the unsaturated CT-loaded samples ranging from 1 CT to 4 CT per micelle could not be determined in this study. We thus consider that the most reasonable result can be attained by using the T1 value of the maximally loaded sample. To study the effect of τe on the correlation time of the CTloaded Rf-IPDU-PEG micelle, we also carried out the T1 and T2 NMR experiment for a sample constituting 1% CT. The T1 value for this sample was found to be 0.24 s ( 0.02 s, but it failed to measure T2 as it was too short. Thus, we were not able to use the R p2/R p1 expression to find the corresponding correlation time. A previous study on tempone, a nitroxide spin label similar to tempol, found that the electron spin-spin relaxation time in the slow motion regime is in the same order (T2e ) 300 ns ≈ τe)60 as that of our measured τc for the blank Rf-IPDU-PEG hydrogel sample. Using τe ) 300 ns and τc ) 128 ns as an example, we see that the effective correlation time (τef) is 89.7 ns. (Here, we have used 1/τef ) 1/τc + 1/τe.)52,53 By substituting this τef value instead of τc in eq 1, we found that the electron-19F interspin distance was 12.8 ( 1 Å. This distance has no significant difference from the interspin distance estimated by using τc alone. Therefore, although T2 for the CT-loaded hydrogel sample was not obtained, the distance estimated using τc derived from the blank hydrogel sample could be within experimental error. We used the -CF3 group’s relaxation times to determine the electron-19F interspin distance. The accuracy of the relaxation times is crucial for the distance estimation. Possible spin diffusion between the -CF3 19F and the neighboring -CF2- 19F could exist. This might cause severe errors for the intrinsic -CF3 19F relaxation time. For this reason, we compared the T1 values for the backbone -CF2- groups with the terminal -CF3 group for three samples, the blank Rf-IPDU-PEG hydrogel, 0.6% CT (0.6 mg of CT/100 mg of Rf-IPDU-PEG) in the hydrogel, and 1.6% CT in the hydrogel. The results are tabulated in Table 2. In all three samples, it was found that the T1 values for the -CF2 groups were much larger than those of the -CF3 groups. This means that the effects of spin diffusion to the T1 measurement are negligible for the micelles. To confirm this conclusion further, we also carried out (60) Millhauser, G. L.; Freed, J. H. J. Chem. Phys. 1984, 81, 37-48.

Figure 5. (a) Structure of Rf-IPDU aggregation, (b) Rf-IPDU individual unit, and (c) structure of CT. Table 2. Comparison of 19F T1 Relaxation Times between -CF2 and -CF3 Groups samples

-CF3 19F T1 value (s)

-CF2-19F T1 value (s)

blank hydrogel 0.6% CT in hydrogel 1.6% CT in hydrogel

0.55 ( 0.02 0.28 ( 0.02 0.22 ( 0.02

0.83 ( 0.06 0.56 ( 0.1 0.49 ( 0.1

two-dimensional 19F-19F diffusion experiments to check for spin diffusion between the two kinds of groups. With mixing times ranging from 100 ms to 2 s, we could not observe any crosspeaks between the -CF3 group and the -CF2- groups, which further confirms that effects of spin diffusion to determine the interspin distance could be neglected. Molecular Modeling for the Rf-IPDU Structure. Many models for the shape of micelles formed by fluorinated surfactants have been hypothesized, including spherical, tubular, and ellipsoidal shapes.30,61,62 Figure 5 a shows the aggregation model of 32 Rf-IPDU units after MD simulation. (In all the succeeding figures, the fluorine atoms in the Rf chains are represented by turquoise, and the other atoms are in standard notations, i.e., carbon, green; hydrogen, white; nitrogen, blue; and oxygen, red.) Figure 5 displays a sandwich structure with Rf groups forming the core (the hypothetical meat) and IPDU groups forming the double shells (the hypothetical bread). The average thickness of the sandwich was 24 Å. This length was measured between the furthermost carbons of IPDU on the two opposite sides of the sandwich. It is shown in Figure 5b that the length of the naturally curved Rf-IPDU is about 16.6 Å. Thus, the thickness (24 Å) indicates that a section of the Rf chains on one side overlaps with (61) Sun, H.; Xu, G.; Li, Y.; Chen, Y. J. Fluorine Chem. 2006, 127, 187-192. (62) Ito, H.; Imae, T.; Nakamura, T.; Sugiura, M.; Oshibe, Y. J. Colloid Interface Sci. 2004, 276, 290-298.

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Figure 6. Models of encapsulated CT in the Rf-IPDU structures after molecular dynamics simulations with these starting structures: (a) CT in the IPDU shell with restraint, (b) CT within the Rf core with restraint to the IPDU shell, and (c) CT within the Rf core without restraints.

Figure 7. (a) CT was initially placed outside the micelle core and (b) was mixed with the IPDU shell after the dynamics simulation.

that on the other side. This overlap enhances the strength of the Rf cores to hold the individual micelle together and to interconnect the micellar network to form the gel system. Figure 5c shows the structure of the CT molecule. It has a naturally curved length of 15.3 Å. (The naturally curved structure resulted from an energy minimization process.) Because the 19F-electron interspin distance was estimated to be 12.3 ( 1 Å, which is about half of the thickness of the Rf-IPDU sandwich, the tempol groups in CT appear to be mixed with the IPDU shells. We used this information as a restraint to model the CT-loaded Rf-IPDU structure and the CT-loaded Rf-IPDU-PEG structure. Molecular Modeling for the CT-Loaded Rf-IPDU Structure. To study the encapsulation of CT into the micelle core, many models of CT with Rf-IPDU were prepared. In some cases, we included distance restraints between CT and IPDU groups. The following is a summary of our results. Figure 6a shows the final CT Rf-IPDU structure after the dynamics simulation starting with CT in the IPDU shell by reinforcing a restraint for the tempol group with the IPDU shell. It displays a structure in which CT (yellow) is still within the IPDU shell. Figure 6b shows the final CT Rf-IPDU structure after the dynamics simulation starting with CT inside the Rf core with restraint for the tempol group in the IPDU shell. The result shows that CT’s position was not changed much after the dynamic heating and cooling process. Figure 6c shows the structure starting with CT inside the Rf core without any restraints. After the dynamics simulation, the CT molecule moved out to the IPDU shell. Panels a and c indicate that the CT molecule has a higher tendency to mix with the IPDU groups instead of the Rf groups. Panel b indicates that when the motional freedom of CT is restricted, the kinetic energy given for the simulation is not high enough to allow CT to come out to the IPDU shell. It has been known that fluorocarbons do not mix well with hydrocarbons.26 In addition, as shown in Figure 5b,c, the IPDU unit and CT have comparable

structural features so that CT possesses a stronger affinity to the IPDU units than to the Rf groups. We think that panel b in Figure 6 is less likely to happen during the drug-loading process. To show the spontaneous trend that CT could enter the RfIPDU unit during the drug-loading period, we started a structure with CT outside the Rf-IPDU unit. After the dynamics simulation, as displayed in Figure 7a,b, we found that CT came into the IPDU shell. As a summary, the previous dynamics simulations show that CT molecules were most likely to be loaded in the IPDU shell of the Rf-IPDU-PEG micelle. As shown next, owing to the hydrophilic property of the PEG outer sphere, we did not expect that the Rf-IPDU structure and the CT drug-loading position would be changed significantly for a full Rf-IPDU-PEG micelle. Molecular Modeling for the CT-Loaded Rf-IPDU-PEG Structure. To assess the validity of the dynamics simulation for using the Rf-IPDU units without PEG chains to find the micelle core structure and the CT drug-loading position, we carried out another set of simulations by attaching 10 and 20 PEG chains to each Rf-IPDU unit, respectively. This result could show a closer scenario where up to possibly 137 PEG units are present. Figure 8a depicts the model of 32 Rf-IPDU-PEG units, each containing 10 PEG units after the dynamics simulation. As compared to Figure 6, this demonstrates that the Rf-IPDU units are almost covered by the PEG chains. The slight gap that allows the Rf core to be noticeable is due to the shortened length of the PEG chains. Figure 8b shows the corresponding model for CT being restrained to the IPDU shell. Just as in the case of Figure 6b, CT is still in the IPDU shell after the dynamics simulation. (For clearly indicating the location of CT, we used a ball-andstick structure to represent the Rf-IPDU-PEG units.) Simulation with CT in the IPDU shell without restraints also resulted in CT being trapped within the IPDU shell as shown in Figure 8c. The same results were actually obtained as those without using the PEG units.

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Figure 8. (a) Model for the Rf-IPDU-PEG micelle with 10 PEG units after dynamics simulation, (b) with CT in the IPDU shell with restraints, and (c) with CT in the IPDU shell without restraints.

In the absence of commercial software for modeling highly fluorinated assemblies, we chose to use the CVFF force field. As mentioned earlier, this force field has been successfully used by other groups to model perfluoroalkyl containing polymeric aggregates.50,51 After coupling our results with the SANS data for the aggregation number of the micelle,32 we believe that this calculation gives a general idea about the aggregation of the Rf-IPDU-PEG micelle and the location of the CT drug in the micelle. The dynamics run with 20 PEG units took 23 days to complete using our computational facility. Running simulations is an expensive task, especially if we want to run macromolecular assemblies. Using our facilities, to run our dynamic simulations using explicit water solvation models for our micelle and to model the entire PEG backbone for our dynamic studies is too time-consuming. Using a solvation model would be a more realistic approach for modeling our micelles, but the micellar core is composed of highly hydrophobic rigid fluorocarbons, and the IPDU shell is also quite hydrophobic, and the chance for water molecules being in the core/shell is minimal. Water will mix with the hydrophilic PEG chains. Thus, we believe that including water in the simulation may not change the micellar structure and the CT location to any significant extent. Nevertheless, explicit solvation will provide a differently sized micelle due to the different folding that PEG will exhibit in the presence of water molecules. A different folding of the PEG moieties will also necessarily have an effect on the corresponding exposure of the micellar inner core to the solvent as well as on the diffusion rate and tumbling of the encapsulated drugs. Therefore, MD simulations should only be considered to give a plausible model calculation but without any real insight on the system dynamics. Running a simulation with 4 CT molecules in our Rf-IPDU-20 PEG micelle model is also time-consuming and, thus, was not done. Figure 9. Structure of a CT-loaded Rf-IPDU-PEG micelle containing 20 PEG units. Panel a represents a space filling model, and panel b shows the location of CT (space-filled yellow molecule) in the IPDU shell in ball-and-stick notation for the Rf-IPDU-PEG units.

As a further more time-consuming trial, we carried out a simulation using 20 PEG units for a model of CT mixed in the IPDU shell without restraints. The result is shown in Figure 9a,b. The Rf-IPDU units and CT were completely covered by the PEG outer structure. To illustrate the location of CT in the micelle, we re-plotted the figure using the ball-and-stick structure for the Rf-IPDU-PEG units as shown in Figure 9b.

Conclusion We have shown that the Rf-IPDU-PEG micelle is an interesting carrier for enclosing hydrophobic drugs. The encapsulation of CT in the micelles was experimentally demonstrated by 1H and 19F NMR relaxation techniques. Using the CT concentration dependent 19F T1 relaxation times, we could calculate the loading capacity of the hydrogel. The method for calculating the 19Felectron interspin distance proved to be useful for locating the position of the drug molecule in the Rf-IPDU-PEG micelle. This interspin distance was used as a restraint for molecular dynamics simulations to find the location of CT in the Rf-IPDU-PEG micelles. MD simulations displayed the Rf-IPDU-PEG micelle

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structure and its CT-loaded structure starting the simulation with and without the restraint. The significance of this research is the finding that the hydrophobic drugs were loaded in the intermediate IPDU shells of the Rf-IPDU-PEG micelles. This result shows that we can customize specific structure-based drug-loading hydrogels by selecting the right intermediate units. As the parent PEG backbone

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and the terminal Rf groups remain intact, the physical properties of the parent hydrogel are expected to be retained. Acknowledgment. This research was supported by NSF Grant 0351848 and NSF Grant 0619147 for a NMR facility upgrade at CSULA. LA701833W