Langmuir 2004, 20, 8839-8845
8839
Application of NMR Spectroscopy to the Characterization of PEG-Stabilized Lipid Nanoparticles Marcos Garcia-Fuentes,† Dolores Torres,† Manuel Martı´n-Pastor,‡ and Maria J. Alonso*,† Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, and Laboratorio Integral de Dina´ mica e Estructura de Biomole´ culas Jose´ R. Carracido, Unidade de Resonancia Magne´ tica, RIAIDT, Edif. Cactus, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Received February 25, 2004. In Final Form: June 16, 2004 The main purpose of the present work was to apply NMR techniques to characterize the nanostructural organization of a new drug nanocarrier composed of tripalmitin, lecithin, and poly(ethylene glycol) (PEG)stearate. These nanocarriers were prepared by an emulsification-solvent evaporation technique and were characterized for their composition and nanostructural architecture. The results showed that tripalmitin, present in the core of the nanoparticles, is the main component of these systems, whereas PEG-stearate is firmly attached to the surface of the nanoparticles, forming a hydrated polymeric layer. Furthermore, the results indicate that, by selecting appropriately the composition of the lipid mixtures used for nanoparticle preparation, it was possible to modulate the PEG-coating density. This rigorous characterization by NMR provided very useful information about the architectural organization of this new colloidal drug carrier and showed the potential of modern NMR techniques for the characterization of core-coated nanostructures intended for drug delivery.
Introduction In past years, poly(ethylene glycol) (PEG)-grafted biomaterials have been extensively studied because of their capacity to control the adhesion of proteins to biomaterial surfaces.1 A specific application of this technology with great impact in drug delivery has been the design of long-circulating (“stealth”) liposomes and nanoparticles (NPs).2 Surprisingly, despite the recognized biological advantages of these so-called PEGylated systems, their design continues to be mostly performed on an empirical basis. Therefore, to advance toward a rational design of these carriers, it appears critical to develop analytical tools that enable one to characterize qualitatively and quantitatively this colloidal surface modification. Currently, there are several methods for the characterization of the composition and surface morphology of the NP. However, most of them require sample preparations that alter their structure and/or do not give information about the structural composition. For example, techniques such as transmission electron microscopy (TEM), electron spectroscopy for chemical analysis (ESCA), and secondary ion mass spectroscopy (SIMS)3-5 can only be applied to dried samples in which the PEGlayer is collapsed and, hence, in a different state from that presented in biological fluids. Alternatively, using * Corresponding author. Telephone: +34 981584627. Fax: +34 981547148. E-mail:
[email protected]. † School of Pharmacy. ‡ Unidade de Resonancia Magne ´ tica. (1) Hong, D.; Chandoroy, P.; Hui, S. W. Biochim. Biophys. Acta. 1997, 1326, 236-248. (2) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (3) Garcia-Fuentes, M.; Torres, D.; Alonso, M. J. Colloids Surf., B 2002, 27, 159-168. (4) Reddy, B. M.; Chowdhury, B.; Reddy, E. P.; Ferna´ndez, A. Langmuir 2001, 17, 1132-1137. (5) Gref, R.; Quellec, P.; Sanchez, A.; Calvo, P.; Dellacherie, E.; Alonso, M. J. Eur. J. Pharm. Biopharm. 2001, 51, 111-118.
atomic force microscopy (ATM), cryo-transmission electron microscopy, and freeze fracture electron microscopy6 allows the imaging of the NP in less destructive conditions. Unfortunately, despite the suitability of these methods for morphological characterization, they do not provide information of the architecture of the systems at a molecular level. Other techniques such as micropipet manipulation7 or titration of the amount of proteins adsorbed8 based on the inhibition of adsorption processes have been applied to characterize the effectiveness of the steric layer of PEG. Nevertheless, these are indirect determinations that give evidence of the PEG-coating but do not inform about its architectural organization and density. More recently, the use of NMR techniques, classically applied to characterize micellar systems,9-11 has attracted attention for the characterization of colloids of pharmaceutical interest such as NPs12,13 and nanocapsules.14 Most of the reported work has focused on the qualitative analysis of the structural features of these carriers, while a few exceptions have additionally quantified the degree of PEGylation of liposomes15 and poly(lactic-co-glycolic acid) NPs.16 (6) Coldren, B.; Van Zanten, R.; Mackel, M. J.; Zasadzinski, J. A.; Jung, H.-T. Langmuir 2003, 19, 5632-5639. (7) Needham, D.; Kim, D. H. Colloids Surf., B 2000, 18, 183-195. (8) Ostumi, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343. (9) Strubenrauch, C.; Frank, C.; Strey, R. Langmuir 2002, 18, 50275030. (10) Bilia, A. R.; Bergonzi, M. C.; Vincieri, F. F.; Lo Nostro, P.; Morris, G. A. J. Pharm. Sci. 2002, 91, 2265-2270. (11) Alami, E.; Abrahmse´n-Alami, S.; Eastroe, J. Langmuir 2003, 19, 18-23. (12) Stolnik, S.; Dunn, S. E.; Garnett, M. C.; Davies, M. C.; Coombes, A. G. A.; Taylor, D. C.; Irving, M. P.; Purkiss, S. C.; Tadros, T. F.; Davis, S. S.; Illum, L. Pharm. Res. 1994, 11, 1800-1808. (13) Hrkach, J. S.; Peracchia, M. T.; Domb, A.; Noah, L.; Langer, R. Biomaterials 1997, 18, 27-30. (14) Mayer, C.; Hoffmann, D.; Wohlgemuth, M. Int. J. Pharm. 2002, 242, 37-46.
10.1021/la049505j CCC: $27.50 © 2004 American Chemical Society Published on Web 08/24/2004
8840
Langmuir, Vol. 20, No. 20, 2004
In a preceding paper, we have disclosed the preparation of NPs composed of tripalmitin, lecithin, and PEGstearate. These NPs were able to associate and release bioactive molecules such as insulin.3 In addition, we showed that the association of PEG-stearate to the lipid NPs was critical to prevent their enzymatic degradation and aggregation in gastrointestinal fluids. These results led us to hypothesize that PEG-stearate formed a stabilizing/enzyme repellent coating onto the lipid NPs.3 Taking into account the promising features of these drug nanocarriers, the present work was aimed at analyzing the structural composition and architectural organization of these new NPs theoretically coated by PEG. With this purpose in mind, we have first analyzed the mixture of colloidal systems resulting from the preparation method. Once the different colloidal structures present in the final preparation were identified, we have determined the composition and structural features of the larger fraction of those, which are the lipid NPs. Finally, we analyzed some factors that affect the PEG-coating density of the NPs. Experimental Section Materials. Tripalmitin [Dynasan 116, Hu¨lls, Germany]; L-Rlecithin [R-phosphatidylcholine, Sigma, Spain]; PEG-stearate of two chain lengths: 40 ethylene oxide units (1760 Mw, PEG40stearate) [Simulsol M 52, Seppic, France] and 100 ethylene oxide units (4400 Mw, PEG100-stearate) [Simulsol M 59, Seppic, France]. Benzenosulfonic acid (Merck, Spain) was dissolved, stoichiometrically neutralized with sodium hydroxide, and dried at 60 °C. Other reagents used were analytical grade or better. The deuteration of the NMR solvents used was always at least 99.9%. NMR Signal Assignment. Tripalmitin, lecithin, and PEGstearate were dissolved in deuterated chloroform, and their 1H and 13C chemical shifts were assigned by standard NMR methods: 1H, 13C, 1H-13C heteronuclear correlation (HMQC), multiple bonds 1H-13C correlation (HMBC), and 1H-1H homonuclear correlation (COSY) spectra. Unidimensional spectra were acquired in a Bruker AMX operating at 300 MHz, and twodimensional experiments were carried out in a Bruker Advance DRX operating at 500 MHz. Preparation of the Lipid NPs. Nanoparticles were prepared using the double emulsion technique as was previously reported.3 Briefly, 100 µL of ultrapure water was emulsified in a 1 mL solution of tripalmitin (123.9 µmol/100 mg), lecithin (63.76 µmol/ 50 mg), and PEG40-stearate (28.4 µmol/50 mg) in methylene chloride, using sonication (15 s, 20 W, Branson Sonifier 250). Next, 2 mL of ultrapure water was poured over this primary emulsion and sonicated again for 1 min (20 W, Branson Sonifier 250), thus leading to the formation of a w/o/w emulsion. The double emulsion was diluted to 10 mL, and the solvent was allowed to evaporate for 30 min at room conditions and for another 30 min under reduced pressure at 30 °C. The NP suspensions intended for direct NMR analysis were prepared using deuterated water (d-water) instead of ultrapure water. To investigate the influence of the PEG40-stearate added during nanoparticle preparation in the PEG-coating density of the resulting NPs, three different amounts of PEG40-stearate were incorporated in the lipid mixture: 2.84, 28.4, and 56.80 µmol. The rest of the ingredients and the procedure were as described above. Similarly, to study the effect of the PEG chain length, NPs were prepared as described, but by substituting the PEG40-stearate by 28.4 µmol of PEG100-stearate, a fatty acid derivative with a 100 monomer-long PEG chain. NPs were isolated by ultracentrifugation at 85 000g, washed twice with ultrapure water, and resuspended in d-water. (15) Vernooij, E. A. A. M.; Gentry, C. A.; Herron, J. N.; Crommelin, D. J. A.; Kettenes-van den Bosh, J. J. Pharm. Res. 1999, 16, 16581661. (16) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Ilum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2002, 18, 3669-3675.
Garcia-Fuentes et al. Characterization of the Colloidal System Resulting from NPs Preparation (Nonisolated NPs). These experiments were intended to identify the physical state (true solutions, micellar suspension, or NP) of each specific compound used for the formation of the NP in the final preparation. For this purpose, the diffusion and the relaxation times of the components involved in the preparation procedure were analyzed via NMR before and after the NP isolation step. As a starting point, these NMR parameters were measured in a solution of the pure compounds. Tripalmitin and PEG40-stearate were dissolved in d-chloroform, while lecithin, because it forms reverse-micelles in d-chloroform, was analyzed in d4-methanol. Next, lecithin and PEG40-stearate were dispersed in d-water using the same procedure as was described for the formation of NPs. The NMR diffusion and relaxation parameters of these control samples were considered to be representative of their typical self-aggregation in water. Using the information obtained from these controls, it was possible to compare the diffusion and relaxation times of the colloidal mixture formed after the NP preparation procedure and also after the NP isolation step. NMR diffusion measurements were performed using a stimulated echo DOSY experiment modified to select a certain slice of the NMR sample (slice selection). This modification has been previously described as a way to improve the precision in the determination of diffusion values, eliminating some of the potential pitfalls of the original DOSY method.17 For this analysis, the signal intensity was fitted to a single parameter exponential equation to determine the diffusion coefficient. Spin-lattice (T1) relaxation times were analyzed using an inversion-recovery experiment and were fitted with a three-parameter nonlinear equation. Spin-spin (T2) relaxation times were analyzed using a CPMG (Carr Purcell Meibom Gill) sequence, and the relative integrals were fitted with a two-parameter nonlinear equation.18 Relaxation measurements were performed in a 500 MHz Bruker Avance spectrometer, and DOSY studies were acquired in a 750 MHz Varian Inova NMR instrument. Analysis of the Composition of Isolated Lipid NPs. This study was intended to show the real composition of the NPs once isolated by ultracentrifugation. Isolated NPs were freeze-dried, and the dry powder was dissolved in d-chloroform. This solvent was selected because it can dissolve all of the components of the NPs. Three different NMR methods were used to quantify the final composition of the lipid NPs: (I) integration of the 1H NMR spectrum of the NP and calculation of the different mole fractions and weight fractions of each component; (II) integration of the 1H NMR spectra of various physical mixtures of tripalmitin/ lecithin/PEG40-stearate and calculation of response factors (mass/integral) for the signal representative of each one compound; the calculated response factors were then applied to the determination of the composition of the NPs; and (III) adaptation of the preceding method to the 2D-HMQC by measuring the peak volumes. For all methods, the relaxation delay was set to 5 × T1, and the number of accumulated transients was set to 16. In the case of method III, the delays for the heteronuclear transference of magnetization were adjusted to a nominal 1H-13C one-bond coupling of 145 Hz. In all cases, the composition of the NP was calculated by taking into account the most relevant signal of each compound: δ ) 3.2 ppm of the trimethylamonium protons of lecithin, δ ) 3.6 ppm of the ethylene glycol protons of PEG40-stearate, and δ ) 4.14.3 ppm of the 1,3 glycerol protons of tripalmitin. Overlapping signals were corrected in the calculations. Analysis of the Structural Composition of the NPs. These studies were aimed at identifying the architectural disposition of the different components of the isolated NPs. The 1H NMR spectrum of the isolated NPs suspended in D2O was obtained at three different temperatures (room temperature, 55 °C, and 66 °C). In addition, to detect the chemical moieties that were exposed to the external water phase, a 1D sel-water LOGSY experiment was performed in isolated NPs samples resuspended in 90% H2O/ 10% D2O. This experiment was designed to show nuclear Overhauser effect contacts between the bulk external water and (17) Antalek, B. Concepts Magn. Reson. 2002, 4, 225-258. (18) Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958, 29, 688-691.
NMR Characterization of PEG-Lipid Nanoparticles
Langmuir, Vol. 20, No. 20, 2004 8841
The integral values of the compounds identified in the H NMR spectra agree with the molecular structures of tripalmitin and PEG40-stearate. On the other hand, from the NMR characterization and also from the GPC analysis (unpublished data), lecithin, a natural compound, was found to be a mixture of 84% diacyl-phosphatidylcholine and 16% diacyl-phosphatidylglycerol esterified with different fatty acids. The structure depicted in Figure 1 is that of a molecule of phosphatidylcholine with aliphatic chains of 18.6 carbon-long chains and 1.48 double bonds per chain. On the other hand, the integral values of PEG100-stearate 1H NMR spectra were not in perfect agreement with those described by the commercial source. According to our data, PEG100-stearate has 127 ethylene glycol units per chain instead of 100. Consequently, we have considered the calculated value as the real polymerization degree for further calculations. Characterization of the Colloidal System Resulting from the NP Preparation Method (Nonisolated NPs). Lipids with surface-active properties show considerable complexity in their behavior in water, due to their ability to structure into different liquid crystalline phases.22 We have previously prepared PEG-coated NPs consisting of tripalmitin/lecithin/PEG40-stearate using a multiple emulsion technique. The size and morphology of the NPs were confirmed by photon correlation spectroscopy (PCS) and transmission electron microscopy.3 However, this preliminary characterization did not allow us to determine the coexistence of micelles or true solutions of the lipid compounds with the NPs. Therefore, prior to studying the structural composition of the isolated NPs, we found it important to identify the physical state in which each individual component used in the NP preparation was present in the resulting suspension. This resulting suspension was called a colloidal system mixture or nonisolated NPs. It was assumed that these experiments would lead us to a better understanding of the dynamics of the components used for the NP formation. As a first step, we have studied the dynamic behavior of our systems by diffusion-ordered NMR spectroscopy (DOSY). DOSY experiments measure the diffusion coefficient of the molecules whose peaks are observed in a 1H NMR experiment. The diffusion coefficient is related to the loss of magnetization of each NMR signal, which depends on the random walk of the molecule during a period of time which is given by the so-called diffusion delay period inserted in the DOSY NMR sequence. This technique is based on the principle that signals of different molecules evolve differently in a DOSY sequence due to their dissimilar diffusion. Consequently, this technique offers interesting information in the case of mixtures of molecules or aggregation structures.23 Unfortunately, there are limitations for the case of very slow diffusing species, as is the case of NPs. Indeed, the detection of small diffusion values requires the use of a special NMR probe and a very high-field gradient in the fringe field of the NMR magnet,24,25 which is not the case of the conventional NMR probes. However, by a careful setting of the diffusion delay period incorporated in the pulse sequence, the DOSY sensitivity can be maximized and detect slow diffusion species with conventional NMR probes. Using the theory involved in the DOSY experiment, 1
Figure 1. Molecular structure of the components used for the preparation of lipid NPs. Lecithin substituted by top “x” radical becomes phosphatidylcholine; if substituted by the bottom one, it becomes phosphatidylglycerol. For PEG40-stearate, n in the written formula is 40, and for PEG100-stearate, n is 100. TP2*, P2*, and L2* correspond to bulk aliphatic chains unless otherwise noted. the NPs chemical groups exposed to it.19 T1 and T2 relaxation measurements were performed as was indicated before. Quantification of the PEG-Coating Density. NPs with different amounts of PEG40-stearate or different PEG chain lengths were isolated by standard methods and were resuspended in d-water with a known concentration of benzylsulfonic sodium salt. The quantity of PEG was calculated from the 1H integrals of the benzylsulfonic peak (standard) and the P6 (δ ) 3.6 ppm, ethylene oxide) peak. The average PEG chain density (chain/ nm2) on the surface of the NPs was calculated by taking into account the total quantity of PEG detected on the surface of the NPs and their surface area. The surface area was calculated considering that the sample is made of individual particles of a diameter equal to that measured by photon correlation spectroscopy (PCS) (Zetasizer 3000HS, Malvern, UK) and a density of 1 g/mL.20 The zeta potential (ζ) of the NPs was also measured by laser doppler anemometry (Zetasizer 3000HS, Malvern, UK). Spectra Analysis. NMR spectra processing and analysis were performed with MestRe C v3.0.21
Results and Discussion NMR Signal Assignment. The molecular structures of tripalmitin, phosphatidylcholine, phosphatidylglycerol, and PEG-stearate are depicted in Figure 1. 1H NMR signals were assigned by standard procedures as follows (functional group labels are the same as those in Figure 1). δ ) 0.9 ppm: TP1, P1, L1. δ ) 1.1-1.3 ppm: TP2 *, P2*, L2* (bulk aliphatic chains). δ ) 1.6 ppm: TP3, P3, L5. δ ) 2.0: L4. δ ) 2.3 ppm: TP4, P4, L6. δ ) 2.8: L7. δ ) 3.2 ppm: PC3 and P6-13C satellites. δ ) 3.6 ppm: P6, PG3, and PC3-13C satellites. δ ) 3.7-4.0 ppm: L10, PC2, PG1, PG2. δ ) 4.0-4.3 ppm: TP5, P5, L8, PC1. δ ) 5.15.4 ppm: TP6, L3, L9. (19) Dalvit, C.; Pavarello, P.; Tato, M.; Veronesi, M.; Vulpetti, A.; Sundstrom, M. J. Biomol. NMR 2000, 18, 65-68. (20) Gref, R.; Lu¨ck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Mu¨ller, R. H. Colloids Surf., B 2000, 18, 301313. (21) Cobas, J. C.; Sardina, F. J. Concepts Magn. Reson. 2003, 19A, 80-96.
(22) Larsson, K.; Quinn, P. J. The Lipid Handbook, 2nd ed.; Chapman & Hall Chemical Database: New York, 1994; Chapter 8. (23) Griffiths, P. C.; Cheung, A. Y. F.; Davies, J. A.; Paul, A.; Tipples, C. N.; Winnington, A. L. Magn. Reson. Chem. 2002, 40, S40-S50. (24) Kimmich, R.; Unrath, W.; Schnur, G.; Rommel, E. J. Magn. Reson. 1991, 91, 136-140. (25) Wu, D.; Johnson, C. S., Jr. J. Magn. Reson., Ser. A 1995, 116, 270-272.
8842
Langmuir, Vol. 20, No. 20, 2004
Garcia-Fuentes et al.
Table 1. Diffusion Coefficients (D) of the Isolated NPs, the Mixed Colloidal System, and the Compounds Used for Nanoparticle Preparation Both in Solution in an Organic Solvent or Suspended in d-Watera diffusion (×10-10 m2/s) sample controls
mixed colloidal system (nonisolated NPs)
isolated NPs
tripalmitin
molecule/δ
organic solvent
d-water
tripalmitin lecithin PEG40-stearate
12.4 17.9 5.72
3.47 0.647
1.2 ppm 3.2 ppm 3.6 ppm
0.819 0.807 0.957
3.6 ppm
0.0412
a
Values of controls are the D average of whole signals of the molecules. For the mixed colloidal system and isolated NPs, the D of each peak (d) is indicated separately.
we have deduced an equation that allowed us to identify the DOSY optimized experimental conditions (see Supporting Information). Using this equation, we were able to calculate accurately their diffusion coefficient values. The diffusion coefficients are related to the effective radii of the colloid structures through the well-known Stokes-Einstein equation:
D)
KT 6πηR
Table 2. Spin-Lattice (T1) and Spin-Spin (T2) Relaxation Times for Different Signals of Tripalmitin, Lecithin, and PEG40-Stearate in Solution in an Organic Solvent: Tripalmitin and PEG40-Stearate in d-Chloroform and Lecithin in d4-Methanol
(1)
In this equation, K is the Boltzmann constant, T is the temperature, η is the viscosity, and R is the particle radius. On the other hand, the relaxation times of the molecules identified in the NMR spectra can also be used to obtain complementary information about the structure of the colloids. Indeed, the spin-lattice (T1) and spin-spin (T2) are parameters that are very sensitive to the molecular motions in the environment of the chemical groups. The similarity of the values of both relaxation parameters is an indication of the high mobility of the corresponding chemical group. On the other hand, when T2 is much smaller than T1, the conclusion is that these chemical groups have limited mobility (e.g., forming entities of higher molecular weight due to strong interaction with other groups).26,27 This is likely to be the case of adsorbed or complexed molecules. The diffusion coefficient and relaxation times of the pure components in solution were characterized for further comparison with those of the materials forming colloidal structures. For this analysis, the various components were dissolved in organic solvents (i.e., d-chloroform and d-methanol). We understand that the constants measured in organic solvents are not comparable to those of a hypothetical solution in d-water. However, these values, especially the diffusion coefficient, provide useful information if we take into consideration the viscosity of the solvents. The diffusion values of the pure compounds are displayed in Table 1, and their relaxation times are given in Table 2. Typical values for the diffusion of tripalmitin and PEG40-stearate were found in d-chloroform: the diffusion coefficient of PEG40-stearate was one-half of that of tripalmitin because of its higher molecular weight (26) Song, Y.-Q.; Venkataramanan, L.; Hu¨rlimann, M. D.; Flaum, M.; Frulla, P.; Straley, C. J. Magn. Reson. 2002, 154, 261-268. (27) Rossi, C.; Bonechi, C.; Martini, S.; Ricci, M.; Corbini, G.; Corti, P.; Donati, A. Magn. Reson. Chem. 2001, 39, 457-462.
lecithin
PEG40-stearate
δ (ppm)
T1 (s)
T2 (s)
T1 (s)
T2 (s)
T1 (s)
T2 (s)
0.9 1.1-1.3 2.3 3.2 3.6-4.0 4.0-4.3 5.1-5.4
1.5 0.98 0.81
1.0 0.87 0.49
1.1 0.98 0.74
1.0
0.87
0.19
0.60 1.3 0.78 1.1 0.84 0.90 0.80
1.6 1.1 1.1
0.57
4.7 2.7 2.2 2.0 1.56 1.6 3.6
and thus larger effective radius. The diffusion coefficient and relaxation times of lecithin were measured in d4methanol instead of d-chloroform because it was found that this compound self-aggregates in chloroform.28,29 In a second step, we examined, when possible, the configuration of the pure compounds dispersed in water. To obtain colloidal dispersions of PEG40-stearate and lecithin in water, we processed them according to the double emulsion method used for the nanoparticle formation (only the compound under investigation was included in the preparation). Unfortunately, in the case of tripalmitin, we could not obtain a colloidal dispersion, due to its high hydrophobicity and the absence of surfactants. The diffusion coefficients and relaxation times of the PEG40-stearate and lecithin structures are shown in Tables 1 and 3, respectively. The lecithin diffusion coefficient indicates that it forms aggregates of 1.1 nm of diameter. In addition, in d-water, the aliphatic chains (δ ) 0.9-2.8 ppm) of lecithin showed T2 values that were much smaller than their T1 value. This indicates that these groups are tightly packed, forming a micelle-type structure. The same pattern for the relaxation times was observed for PEG40-stearate in d-water. However, because of its larger molecular volume, PEG40-stearate presents also larger aggregation structures. Indeed, the difference in size between those structures can be clearly appreciated from the differences in diffusion in d-water: lecithin micelles are 5-fold quicker than those of PEG40-stearate. According to the Stokes-Einstein equation, this diffusion coefficient value is expected for particles with radii of 3.1 nm, this value being approximately equal to the Flory radius of this polymer in water (3.2 nm).30 Finally, from the diffusion coefficients obtained for the mixed colloidal system (before NP isolation), we could deduce the presence of aggregation structures with a size similar to that of PEG40-stearate micelles (Table 1). In contrast to what happened with the pure-component micelles, the diffusion of lecithin (δ ) 3.2 ppm) and PEG40stearate (δ ) 3.6 ppm) was very similar. This similarity may indicate that lecithin and PEG40-stearate are forming blended micelles coexisting with the NPs. The fact that most of the bulk aliphatic groups (δ ) 1.2 ppm) in the mixed colloidal system are not observed by liquid-state NMR is consistent with the idea that tripalmitin is forming solid structures. Consequently, the peak at 1.2 ppm observed for the mixed colloidal system could be most probably associated with the aliphatic protons of PEG40-stearate and lecithin forming micellar (28) Walde, P.; Giuliani, A. M.; Boicelli, C. A.; Luisi, P. L. Chem. Phys. Lipids 1990, 53, 265-288. (29) Angelico, R.; Ambrosone, L.; Ceglie, A.; Palazzo, G.; Mortensen, K.; Olsson, U. Prog. Colloid Polym. Sci. 2000, 116, 37-41. (30) Needham, D.; Zhelev, D. V.; McIntosh, T. J. Surface Chemistry of the Sterically Stabilized PEG-liposomes. General Principles; MarcelDekker: New York, 1999; Chapter 2.
NMR Characterization of PEG-Lipid Nanoparticles
Langmuir, Vol. 20, No. 20, 2004 8843
Table 3. Spin-Lattice (T1) and Spin-Spin (T2) Relaxation Times for Different Signals of Lecithin, PEG40-Stearate, the Mixed Colloidal System (Nonisolated NPs), and Isolated NPs Dispersed in d-Water lecithin
PEG40-stearate
mixed colloidal system
isolated NPs
δ (ppm)
T1 (s)
T2 (s)
T1 (s)
T2 (s)
T1 (s)
T2 (s)
T1 (s)
T2 (s)
0.9 1.1-1.3 2.3 3.2 3.6-4.0 4.0-4.3 5.1-5.4
0.85 0.77 N/A 0.48 0.45 0.54 0.99
0.021 0.044 N/A 0.039 0.30 0.41 0.14
1.00 0.65 0.57
0.14 0.082 0.053
0.72
0.44
1.00 0.67 N/A 0.53 0.74 N/A N/A
0.14 0.072 N/A 0.16 0.54 N/A N/A
N/Aa 0.64 N/A N/A 0.66 N/A N/A
N/A 0.022 N/A N/A 0.21 N/A N/A
a
N/A: not available. Table 4. Composition of Lipid NPs Obtained by Integration of 1H NMR and HMQC Experimentsa
method I II III a
tripalmitin (% mol)
lecithin (% mol)
PEG40-stearate (% mol)
tripalmitin (% w/w)
lecithin (% w/w)
PEG40-stearate (% w/w)
88.3 ( 1.3
5.3 ( 0.1
6.4 ( 1.2
80.4 ( 2.5 82.3 ( 1.7 83.8 ( 1.1
4.9 ( 0.0 6.1 ( 0.1 7.1 ( 0.2
14.8 ( 2.5 11.6 ( 1.6 9.0 ( 0.9
The values represent the percentage of each component with respect to the total composition of the NPs (mean ( SD, n ) 3).
structures. This hypothesis was also experimentally supported by the similar relaxation time values calculated from this signal in the spectrum of the pure component micelles and that of the mixed colloidal system. In addition, the hypothesis of a population of blended micelles was corroborated by the similarity of the diffusion coefficients associated with this signal, in both spectra. In the case of the mixed colloidal system, the effect of the quick moving micelles in the dislocation of the NMR signal during a DOSY experiment was prominent, making it impossible to precisely isolate the contribution of the NPs to the diffusion value. In contrast, the suspension of isolated NPs presented a clear change in their 1H NMR spectrum: a distinct signal could only be detected at δ ) 1.2 ppm and at δ ) 3.6 ppm, while very weak peaks, in the limit of the resolution, were seen at 0.8 and 3.2 ppm. The peak assigned to the bulk aliphatic groups (1.2 ppm) was also weak and considerably broadened as compared to that of the mixed colloidal system. Consequently, the DOSY-NMR studies performed on the isolated NPs were restricted to the analysis of the δ ) 3.6 peak because this was the only high-resolution signal. Under optimum conditions, the experiment permitted the detection of the very slow diffusion coefficients typical of NPs (D ) 4.12 × 10-12 m2 s-1, χ2 ) 305493.2) (Table 1). This calculated diffusion coefficient corresponds to a NP of 90 nm diameter. The discrepancy in the size values obtained from this experiment and from PCS (226 nm) could be justified by the different fundamentals of both techniques. In conclusion, the mixed colloidal system composed of tripalmitin, lecithin, and PEG40-stearate forms two populations of colloidal systems: NPs and mixed micelles of lecithin and PEG40-stearate. Analytical Composition of the Isolated Lipid NPs. NMR analysis has been applied to quantify the composition of lipid mixtures in solution.15,31 In the present work, we have used 1H NMR and HMQC spectra for the estimation of the composition of the NPs after their isolation from the mixed colloidal system. As was indicated in the Experimental Section, for the determination of the actual composition of the NPs, we have used three different approximations. The results corresponding to these approximations are shown in Table 4. Method I involves the calculation, from the integrals of the signals, of the relative (31) Sparling, M. L. Comput. Appl. Biosci. 1990, 6, 29-42.
mole fractions of the different compounds. The multiplication of the resulting values by the respective molecular weight of each compound leads to the relative weight fractions. On the other hand, methods II and III make use of external standards to define the response factor between the integral and the mass of each compound. Therefore, these latter methods permit the calculation of the composition in the form of weight fraction, without the necessity of assuming a specific molecular weight for each compound. The results show good agreement between the compositions calculated by the different methods. A clear conclusion from these results is that tripalmitin is the major component of the particles, accounting for more than 80% of the final weight. On the other hand, lecithin and PEG40-stearate are only present in minor fractions of final composition (∼10%). The calculated composition can be understood as a logical consequence of the formation of the mixed colloidal system, as was discussed in the previous section. After the preparation method, PEG40-stearate and lecithin are mostly present in the form of micelles, which do not sediment during the ultracentrifugation step and, consequently, are discarded in the isolation process. Analysis of the Structural Composition of the NPs. Important differences can be appreciated between the 1H spectrum of isolated NPs dissolved in d-chloroform and that of the same particles suspended in d-water. The NPs dissolved in d-chloroform display most of their functional groups in the aliphatic region (below 2.8 ppm), especially in a second order peak at 1.2 ppm. In contrast, the NPs suspended in d-water showed no appreciable aliphatic functional group signals except for a broad and small peak at 1.2 ppm (Figure 2, bottom). In this solvent, the only well defined signal, at 3.6 ppm, was mainly associated with the ethylene glycol (P6) functional groups of PEG. Logically, tripalmitin, which accounts for most of the aliphatic functional groups in the NPs, cannot be detected by liquid-state NMR because it is in the form of solid nanomatrixes. The fact that PEG showed a narrow peak and long relaxation times (T1 ≈ T2) is indicative of high molecular mobility. On the other hand, the extremely slow diffusion of PEG chains (Table 1) suggests that these chains are attached to the NPs. Consequently, a conclusion from these observations is that PEG-stearate is in the external water phase, forming a loose coating around the NPs. Very similar conclusions were obtained by other authors
8844
Langmuir, Vol. 20, No. 20, 2004
Garcia-Fuentes et al.
Figure 3. NOE spectrum between H2O and the nanoparticle functional groups.
Figure 2. 1H NMR spectra of isolated lipid NPs suspended in d-water obtained at different temperatures. From top to bottom, the spectra correspond to room temperature, 55 °C, and 66 °C.
following analysis of the 1H NMR spectra of poly(lactic acid)-PEG NPs previously dissolved in organic solvents or suspended in d-water.13,16 More detailed information about the nanostructural organization of the NP components was obtained when 1H NMR spectra were obtained at different temperatures. At 55 °C, the 1H spectrum of the suspended NPs exhibits an increment in the signal at 1.2 ppm (Figure 2), which should be associated with the lipids that melt below 55 °C, as it is the case of stearate linked to PEG. At higher temperatures (66 °C), tripalmitin melts, and, consequently, the presence of this material as well as that of lecithin can be noted in the 1H spectrum of the NPs (Figure 2). At this temperature, all of the lipids are melted and the observed spectrum is almost equal to that of the NPs dissolved in d-chloroform (difference in peaks’ integrals
