4852
Langmuir 2003, 19, 4852-4855
Precise Determination of the Hydrophobic/ Hydrophilic Junction in Polymeric Vesicles M. Valentini,† A. Napoli,† N. Tirelli,*,†,‡ and J. A. Hubbell† Institute for Biomedical Engineering and Department of Materials, Swiss Federal Institute of Technology (ETH) and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland, and School of Pharmacy, University of Manchester, M13 9PL Manchester, United Kingdom Received November 23, 2002. In Final Form: January 30, 2003
Introduction Polymeric vesicles are materials increasingly regarded with interest because of their potentialities in targeted and controlled drug delivery.1-7 Our research has recently focused on a new class of amphiphilic polysulfide-blockpolyethers [poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG)],8 which showed high facility in the formation of vesicular aggregates;9 several features contribute to render their aggregates attractive for delivery purposes: the easy and versatile synthesis of the PPSPEG polymers;8 the PEG-ylated and, thus, proteinrepellant surface;10-12 and finally, the possibility of inserting bioactive functional groups and incorporating cleavable sites. Furthermore, PPS-PEG vesicles showed very promising behavior in preliminary biocompatibility assessments (human foreskin fibroblasts) and exhibit oxidation-sensitive release possibilities (destabilization of the vesicular structures upon sulfide oxidation to sulfones), which may be used for the targeting of inflammatory states. A key issue in the characterization of such selfassembled structures is the level of water penetration, that is, the precise localization of the border between the hydrophobic and hydrophilic domains. This parameter is of high significance for a correct and rational design of the macromolecular structure; according to our synthetic procedure (Figure 1), functional groups can be inserted in the PPS-block-PEG structures in both symmetric and asymmetric fashions. The presence of * To whom correspondence should be addressed. Telephone: +41 1 6326348. Fax: +41 1 6321214. E-mail:
[email protected];
[email protected]. † Swiss Federal Institute of Technology and University of Zurich. ‡ University of Manchester. (1) Nardin, C.; Winterhalter, M.; Meier, W. Langmuir 2000, 16, 7708. (2) Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Chem. Commun. 2000, 1433. (3) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035. (4) Lee, J. C. M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y. Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 73, 135. (5) Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y. Y.; Bates, F. S. J. Phys. Chem. B 2002, 106, 2848. (6) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. J. Am. Chem. Soc. 2002, 124, 1658. (7) Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C. M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (8) Napoli, A.; Tirelli, N.; Kilcher, G.; Hubbell, J. A. Macromolecules 2001, 34, 8913. (9) Napoli, A.; Tirelli, N.; Wehrli, E.; Hubbell, J. A. Langmuir, accepted for publication. (10) Szleifer, I. Curr. Opin. Solid State Mater. Sci. 1997, 2, 337. (11) Stolnik, S.; Illum, L.; Davis, S. S. Adv. Drug Delivery Rev. 1995, 16, 195. (12) Li, J. T.; Carlsson, J.; Huang, S. C.; Caldwell, K. D. Adsorption of poly(ethylene oxide)-containing block copolymerssA route to protein resistance. Hydrophilic Polymers; Advances in Chemistry Series 248; American Chemical Society: Washington, DC, 1996; p 61.
esters or other hydrolytically or enzymatically cleavable groups can allow the destabilization of the vesicular structure through chain scission and a consequent change in the hydrophilic/lipophilic balance (HLB) of the amphiphilic macromolecule; even the cleavage of only one PEG chain per molecule (the one exposed to the external water environment) is likely enough to cause a reorganization of the aggregate. A viable release mechanism of any material encapsulated in the vesicular cavity can, therefore, make use of reactions with species in the water phase. The highest destabilization efficiency should be obtained through a very large HLB change, ideally having the reaction occur as close as possible to the hydrophobic domain. In the present paper, we present a complete characterization of the PPS-PEG block copolymers first at a molecular level, through a complete assignment of 1H and 13C NMR spectraa of the triblock copolymers P1 and P2 (the assignments here reported for the PPS main chain correct a partially erroneous assignment reported in ref 8, where the presence of diastereotopic protons was not taken into account), then as lyotropic aggregates, with the precise localization of the hydrophilic/hydrophobic border of their vesicles. In this study, we used multidimensional NMR measurements13 for the 1H and 13C assignments (two-dimensional NMR studies of the conformation and molecular dynamics in colloidal aggregates are relatively common)14 and 1H CPMG (Carr-PurcellMeiboom-Gill) experiments15,16 for the determination of the transverse relaxation times [T2’s, inversely proportional to the full width at half-height (fwhh) of the peaks], which are well-known for relating molecular mobility in an inversely proportional fashion.17 We have used this feature for discriminating a hydrophobic domain (bulk PPS, viscous phase with short T2’s) from a water-swollen, solution-like one (highly mobile PEG with longer T2’s). Furthermore, the signals characterized by short transverse relaxation times, T2’s, can be filtered off from the 1H NMR spectra, which will result in only peaks having short spin-lattice relaxation times (few milliseconds) and long T2’s (tens of milliseconds or more), allowing an easy individuation of the water-exposed groups. The same approach has been very recently used for investigating the central solidlike core of poly(lactic acid)poly(ethylene oxide) nanoparticles;18 excluding the polystyrene signals from the NMR spectra of solid-phase synthesis substrates;19 analyzing the composition of human aqueous humor, which contains several proteins, short T2’s, many metabolites, and longer relaxation times;20 and determining quantitatively the amount of bound water in wheat starch.21 (13) Claridge, T. D. W. High-Resolution NMR Techniques in Organic Chemistry; Elsevier Science: Oxford, U.K., 1999. (14) Lindblom, G. Curr. Opin. Colloid Interface Sci. 1996, 1, 287. (15) Carr, H. Y.; Purcell, E. M. Phys. Rev. 1954, 94, 630. (16) Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958, 29, 688. (17) Lu¨sse, S.; Arnold, K. Macromolecules 1996, 29, 4251. (18) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2002, 18, 3669. (19) Grøtli, M.; Gotfredsen, C. H.; Rademann, J.; Buchardt, J.; Clark, A. J.; Duus, J. Ø.; Meldal, M. J. Comb. Chem. 2000, 2, 108. (20) Tkadlecova`, M.; Havlicek, J.; Volka, K.; Soucek, P.; Karel, I. J. Mol. Struct. 1999, 480-481, 601. (21) Le Botlan, D.; Rugraff, Y.; Martin, C.; Colonna, P. Carbohydr. Res. 1998, 308, 29.
10.1021/la026896y CCC: $25.00 © 2003 American Chemical Society Published on Web 05/02/2003
Notes
Langmuir, Vol. 19, No. 11, 2003 4853
Figure 1. Synthetic procedure of P1 and P2 and corresponding lettering used for the 1H and 13C NMR assignments; n ) 16, m ) 25, and p ) 8 (values estimated by 1H NMR).
Experimental Section The vesicles were prepared from polymer films by soaking and then sonicating them in D2O (1% w/w), according to a procedure described in forthcoming publications. All the vesicular samples were extruded with a mini-extruder by Avanti Lipids by using a 200-nm-pore-size polycarbonate membrane, and their average size and size distribution were measured on a Polymer Laboratories hydrodynamic chromatograph (PL-PSDA). All the samples prepared gave a width at half-height of about 70 nm. The NMR measurements were performed on a Bruker AVANCE 500-MHz spectrometer equipped with a multinuclear probe head. The polymers were measured in CD2Cl2 and the vesicles in D2O. Deuterated solvents were purchased from Cambridge Isotope Laboratories and used without further purification. The two-dimensional NMR experiments were conducted according to a standard literature procedure.13 The 1H CPMG spectra and T2 measurements were performed using the standard π/2-[τ-π-τ]n CPMG sequence. For the spin-spin relaxationtime experiments, τ was equal to 400 µs and the number of echoes collected was chosen depending on the proton observed. The 1H NMR data may likely be confirmed by studying the 13C relaxation times; however, our attempts to measure the T1’s22 and T2’s of the carbon atoms were inconclusive as a result of the low sensibility and broadness of the peaks (v1/2 ≈ 10-50 Hz). (PEG)16(PPS)25(PEG)8 (P1). 1H NMR (CD2Cl2, 298 K, δ): 4.27 [m, k]; 3.71 [m, l]; 3.65 [s, b]; 3.56 [m, c]; 3.39 [s, a]; 3.08 [m, i]; 2.95 [br, g]; 2.93 [br, f or f′]; 2.67 [br, f or f′]; 2.63 [m, e]; 1.88 [m, d]; 1.41 [br, h]. 13C NMR (δ): 172.5 [s, COO]; 70.9 [s, b]; 69.9 [s, c]; 64.2 [s, k]; 59.0 [s, a]; 52.0 [s, l]; 41.6 [s, g]; 38.7 [br, f]; 37.7 [s, i]; 35.2 [s, e]; 30.4 [s, d]; 21.0 [s, h]. (PEG)16(PPS)50(PEG)16 (P2). 1H NMR (CD2Cl2, 298 K, δ): 3.65 [s, b]; 3.56 [m, c]; 3.39 [s, a]; 2.95 [br, g]; 2.93 [br, f or f′]; 2.67 [br, f or f′]; 2.63 [m, e]; 1.88 [m, d]; 1.41 [br, h]. 13C NMR (δ): 70.9 [s, b]; 69.9 [s, c]; 59.0 [s, a]; 41.6 [s, g]; 38.7 [br, f]; 35.2 [s, e]; 30.4 [s, d]; 21.0 [s, h]. The vesicles formed by P1 and P2 in D2O were also characterized by 1H and 13C NMR, with chemical shifts almost identical with those observed for P1 and P2; the above values can also be used for the corresponding colloidal aggregates. (22) Nirmala, N. R.; Wagner, G. J. Am. Chem. Soc. 1988, 110, 7557.
Figure 2. Sections of the COSY spectrum for P1 showing the cross peaks between the protons of the hydrophobic region.
Results and Discussion In the present study, we first provide complete 1H and C spectral assignments of the two triblock copolymers of ethylene glycol and propylene sulfide (Figure 1) with asymmetric (P1) or symmetric (P2) structure, recording the spectra in a homogeneous organic solution (CD2Cl2). The resonances arising from PEG, PPS, and the three methylene groups (c-e) at the junction are present in both P1 and P2. In the 1H NMR spectrum, the PEG b protons exhibit a strong signal at 3.65 ppm, while those of PPS show intense resonances at 1.41 and 2.95 ppm, 13
4854
Langmuir, Vol. 19, No. 11, 2003
Notes
Figure 3. Left: 1H NMR spectrum of the vesicles formed by using the asymmetric triblock copolymer P1, showing very larger peaks for the protons of the PPS units. Right: same spectrum recorded by using the CPMG sequence (T2 filter); the large bands are removed while the sharp signals are unchanged. Table 1. T1 and T2 Values for the PPS and PEG Protons in Vesicular Aggregates protona
T1 (ms)
T2 (ms)
a b f and g
902.8 778.6 752.3
45.9 53.7 2.7
a
protona
T1 (ms)
T2 (ms)
f′ h
610.1 730.6
3.1 3.6
See Figure 1.
which correspond respectively to the methyl h and methine g protons. Protons f and f ′, at 2.93 and 2.67 ppm, present different chemical shifts because they are diastereotopic. The assignments for g, h, f, and f ′ are properly done via a 1H-1H correlation spectroscopy (COSY) measurement, which clearly shows (Figure 2) cross peaks between the methyl resonance at 1.41 ppm and the left part of the signal at about 2.95 ppm, indicating that the latter is the proton on the chiral carbon. Furthermore, the absence of cross peaks between h and the signals at 2.67 and 2.93 ppm supports this observation. The last set of common signals for P1 and P2 is that for protons c, d, and e at 3.56, 1.88, and 2.63 ppm, respectively. Finally, P1 shows multiplets at 3.08, 4.27, and 3.71 ppm, which correspond to i, k, and l, respectively. Both polymers P1 and P2 formed lamellar phases in water,9 which can be easily converted to vesicular aggregates, showing, after extrusion with a 200-nm-poresize membrane, a diameter in the range of 15-250 nm. The 1H NMR spectrum of the P1 vesicular aggregates (Figure 3, left) shows very large featureless signals (fwhh ca. 150-200 Hz) for the PPS protons, while the other resonances are only slightly broadened compared to the CD2Cl2 spectrum. The sound increase of the PPS band’s fwhh derives from the shortening of the T2 values, characteristic of protons in a bulk phase. PPS protons show T2 values of a few milliseconds, while those of the PEG protons are 1 order of magnitude bigger (Table 1). This clear difference can be conveniently used for determining the level of penetration of water defined as the limit of “long T2’s”. However, as a result of the small
signals of the protons around the hydrophilic/hydrophobic junction, a precise determination of their T2’s is difficult; we have chosen an alternative method for gathering the same information, removing the short T2 signals from the spectrum using the standard CPMG sequence,15,16 and the resonances characterized by relatively long transverse relaxation times (i.e., in contact with water) are left unchanged. As expected, the P1 vesicles (Figure 3, right) show the PEG intense resonance at 3.65 ppm and a sharp singlet at 3.39 ppm arising from a, a result of the water environment surrounding the PEG chain. More important features are (a) the presence of the multiplet at 4.27 ppm (proton k) and a very weak signal at 3.55 ppm (proton c), detectable only via a 1H-13C correlation, and (b) the absence of protons i and j and the resonance in the range 1.8-2.0 ppm (proton d). These observations allow us to conclude that polymer chains are accessible to water up to the ester group on one side and the first methylene group of the PEG-PPS junction on the other side; in the last case, the very low intensity of the proton c signal likely arises from its “borderline” nature (Figure 4). The same measurements were also performed on the symmetric triblock copolymer P2 vesicles, obtaining similar results. As for P1, the 1H CPMG NMR spectrum displays only the methyl group a, the ethylene glycol units b, and a very weak signal for proton c, again detectable only by a two-dimensional heteronuclear correlation, while d is completely absent. This indicates that the high mobility (solution-like) of the chains, due to solvation with water, is relevant only up to the first methylene group of the bridge between the PEG chain and the hydrophobic PPS part. The presented method is applicable to any polymeric amphiphile and, in a general case, can give valuable feedback for the synthetic procedure. For example, in our specific case, we were able to show that the ester group on one side of P1 is not hydrophobically shielded; if hydrolysis is not desired, the polymer structure should display hydrophobic groups on both sides of the ester. Furthermore, if sensitive groups have to be
Notes
Langmuir, Vol. 19, No. 11, 2003 4855
Figure 4. Pictorial view of a vesicular aggregate and the level of water exposure of the polymer chains in it.
present in the other part of P1 or P2, they must be inserted before the three-methylene spacer, that is, before the stage of end functionalization of PEG with allyl ethers (first synthetic step in Figure 1). Polymeric vesicles are systems of potential high significance for targeted and controlled drug release; the importance of the characterization method presented here
is in the possibility of precisely locating some key points of the vesicular structure. A rational macromolecular design based on this knowledge can largely improve the efficacy of environmentally sensitive groups and the controlled-release strategies based on them. LA026896Y
