Poly(lactic acid)−Poly(ethylene oxide) (PLA−PEG) Nanoparticles

Mar 23, 2002 - Hrkach et al.5 used 1H NMR to study the structure of PLA−PEG nanoparticles. ..... not used in the study as PEG loses its solubility a...
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Langmuir 2002, 18, 3669-3675

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Poly(lactic acid)-Poly(ethylene oxide) (PLA-PEG) Nanoparticles: NMR Studies of the Central Solidlike PLA Core and the Liquid PEG Corona C. R. Heald,† S. Stolnik,† K. S. Kujawinski,† C. De Matteis,† M. C. Garnett,† L. Illum,† S. S. Davis,*,† S. C. Purkiss,‡ R. J. Barlow,‡ and P. R. Gellert‡ Institute of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom, and Pharmaceutical & Analytical R & D, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TD, United Kingdom Received September 4, 2001. In Final Form: February 6, 2002 NMR studies on a series of poly(lactic acid)-poly(ethylene oxide) (PLA-PEG) diblock copolymers have been carried out in d6-acetone and in D2O. Nanoparticles of the PLA-PEG copolymers were obtained using a modified interfacial polymer deposition-solvent evaporation technique (Fessi, H.; Devissaguet, J. P.; Puisieux, F.; Thies, C. French Patent 2 608 988, 1986). In D2O, the PLA block forms a central hydrophobic core, while the PEG block forms an hydrophilic corona layer. In D2O, the hydrophobic core of the nanoparticle is generally not seen, while the PEG corona is observed. Only the PLA methyl protons at the interface between the two regions are observed, and these are seen as a double doublet structure. For nanoparticles with a low molecular weight PLA block length, an additional methyl multiplet signal is seen suggesting that PLA methyl groups are in more than one chemical environment. This is not seen for nanoparticles with a high molecular weight PLA block length indicating more uniform structure in the core interfacial region. As temperature is increased, the core of the latter becomes more liquidlike. Quantitative calibration studies of the PEG corona layer show that most of the PEG layer is seen indicating that it is in the liquid phase and on the surface of the nanoparticle. 13C solid-state NMR spectroscopy studies indicate the presence of a central solidlike core and a more mobile interfacial region at the PLA-PEG interface, while the relaxation rate of the nanoparticle obtained from T1 studies indicates that the PEG corona is a much more mobile environment than the interfacial methyl protons.

Introduction Poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles have shown potential as drug delivery systems due to their prolonged circulation in the bloodstream after intravenous injection.2,3 The nanoparticles can be produced from PLA-PEG diblock copolymers with a PLA portion of varying molecular weight (between 2 and 110 kDa)4 and a PEG portion which can be varied between 750 Da and 20 kDa. It has been shown that the PLA blocks form an hydrophobic central core, while the PEG blocks form a solvated, hydrophilic corona2,4 which energetically stabilizes the nanoparticle. Drug molecules may be incorporated into the hydrophobic region of the nanoparticle and released by a diffusion mechanism.5-7 Characterization of these nanoparticles in terms of size, * To whom correspondence should be addressed. Phone: +44 (115) 9515121. Fax: +44 (115) 9515122. E-mail: stanley.davis@ nottingham.ac.uk. † University of Nottingham. ‡ AstraZeneca Pharmaceuticals. (1) Fessi, H.; Devissaguet, J. P.; Puisieux, F.; Thies, C. French Patent 2 608 988, 1986. (2) Stolnik, S.; Heald, C. R.; Neal, J.; Garnett, M. C.; Davis, S. S.; Illum, L.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. J. Drug Targeting 2001, 9, 361. (3) Peracchia, M. T.; Gref, R.; Minamitake, Y.; Domb, A.; Lotan, N.; Langer, R. J. Controlled Release 1997, 46, 223. (4) Riley, T.; Stolnik, S.; Heald, C. R.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2001, 17, 3168. (5) Hrkach, J. S.; Peracchia, M. T.; Domb, A.; Lotan, N.; Langer, R. Biomaterials 1997, 18, 27. (6) Govender, T.; Riley, T.; Ehtezazi, T.; Garnett, M. C.; Stolnik, S.; Illum, L.; Davis, S. S. Int. J. Pharm. 2000, 199, 95. (7) Riley, T.; Govender, T.; Stolnik, S.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S. Colloids Surf., B 1999, 16, 147.

shape, and amount of PEG on the surface has been performed by various techniques.8,9 However, a detailed structural characterization of these nanoparticles is difficult since few methods allow a direct analysis of the structure and conformation of such nanoparticles in the solvated state. Transmission electron microscopy (TEM) experiments which are performed in the dried state give information on the size and the spherical nature of these nanoparticles4 but little information on the conformation of the individual polymer units within the nanoparticles. Static light scattering (SLS) can be used to identify the molecular aggregation number of the PLA-PEG nanoparticles.4,10 It is evident from photon correlation spectrometry (PCS) experiments that the hydrodynamic diameter of the nanoparticles in the solvated state is larger than that obtained from TEM experiments.4 The difference in nanoparticle radii measured by TEM and PCS indicates that the nanoparticles are present in a different conformational state in the two types of experiments. Secondary ion mass spectroscopy (SIMS) has frequently been used to evaluate the presence and density of the PEG layer but again cannot provide a true picture of the conformation of the PEG layer as the method is performed on nanoparticles in the dried state where the PEG layer is collapsed and consequently in a different conformation.11 (8) McGurk, S.; Sanders, G. H. W.; Davies, M. C.; Davis, S. S.; Illum, L.; Roberts, C. J.; Stolnik, S.; Tendler, S. J. B.; Williams, P. M. Polym. J. (Tokyo) 2000, 32, 444. (9) Kwon, G. S. Crit. Rev. Ther. Drug Carrier Syst. 1998, 15, 481. (10) Tanodekaew, S.; Pannu, R.; Heatley, F.; Attwood, D.; Booth, C. Macromol. Chem. Phys. 1997, 198, 927. (11) Dunn, S. E.; Brindley, A.; Davis, S. S.; Davies, M. C.; Illum, L. Pharm. Res. 1994, 11, 1016.

10.1021/la011393y CCC: $22.00 © 2002 American Chemical Society Published on Web 03/23/2002

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Self-consistent field (SCF) modeling has also been used to describe the PLA-PEG nanoparticles.12,13 Self-consistent field modeling is one of the few techniques, apart from small-angle neutron scattering,14 which can be used to assess conformation of the core and corona regions of such PLA-PEG nanoparticles. The SCF model used was based on the mean field theory developed in the 1980s for homopolymer adsorption to interfaces and later developed further for particle formation from various diblock copolymers. Results from the modeling include information on the conformation of the central hydrophobic core and the corona regions, together with the nanoparticle radii, the aggregation number, and the stability to dilution of the PLA-PEG nanoparticles. In the present work, NMR studies have been employed to obtain structural and conformational data about the hydrophobic core and hydrophilic regions of these PLAPEG nanoparticles in the solvated state. This is possible since the nanoparticle system is in the same conformational state whether the PLA-PEG copolymer is dissolved in water or in D2O, that is, the PEG corona is in an extended solvated state due to H-bonding between the PEG and the water molecules.15 Consequently, NMR is an easily accessible technique which can be used to study the PLAPEG nanoparticles. NMR spectroscopy has been used in the past to study a wide range of cationic,16,17 anionic,18,19 and amphiphilic4,5,20,21 type micellar and nanoparticle type structures. Podo et al.20 performed NMR studies on Triton X-100 in different solvents. They found that the molecule dissolved in NMR solvents (benzene, dioxane), where the molecules existed as an homogeneous solution of unimer molecules, whereas the core and the corona of the micelle were seen when Triton was dissolved in D2O. These results suggest that the hydrophobic protons in the micelle structure are mobile and therefore liquidlike. 13C NMR performed on micellar nanoparticles of Triton X-100 in D2O22 again suggested that the core protons were seen, showing the “liquidlike” structure of the core, that is, the dynamic nature of these micelles. Hrkach et al.5 used 1H NMR to study the structure of PLA-PEG nanoparticles. They showed that the signal obtained corresponded to the PEG coating with no signal detected from the PLA core. The aim of the NMR experiments performed in the present work was to determine the composition of the PLA-PEG diblock copolymer when dissolved in a deuterated organic solvent and to assess the structure of the copolymer when formed into a PLA-PEG nanoparticle using temperature studies, solid-state NMR, and T1 relaxation experiments. The Mw of the PLA block varied (12) Heald, C. R.; Stolnik, S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Leermakers, F. A. M. Colloids Surf., A 2001, 179, 79. (13) Heald, C. R.; Stolnik, S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Leermakers, F. A. M. Colloids Surf., A, submitted. (14) Washington, C.; King, S. M. Langmuir 1997, 13, 4545. (15) Poly(ethyleneglycol) Chemistry - Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992. (16) El Seoud, O. A.; Blasko´, A.; Bunton, C. A. Langmuir 1994, 10, 653. (17) El Seoud, O. A.; Farah, J. P. S.; El Seoud, O. A. Ber. Bunsen-Ges. Phys. Chem. 1989, 93, 180. (18) El Seoud, O. A.; Farah, J. P. S.; Vieira, P. C.; El Seoud, M. I. J. Phys. Chem. 1987, 91, 2950. (19) Nagy, J. B.; Bodart-Ravet, I.; Derouane, E. G.; Gourgue, A.; Verfaillie, J. P. Colloids Surf. 1989, 36, 229. (20) Podo, F.; Ray, A.; Nemethy, G. J. Am. Chem. Soc. 1973, 95, 6164. (21) Godward, J.; Heatley, F.; Booth, C. J. Chem. Soc., Faraday Trans. 1993, 89, 3471. (22) Ribeiro, A. A.; Dennis, E. A. J. Colloid Interface Sci. 1976, 55, 94.

Heald et al.

Figure 1. Diagram of the PLA-PEG structure where A can vary from 28 to 1500 lactic acid units (Mw ) 2-110 kDa) and B is 114 ethylene oxide units (Mw ) 5 kDa).

from 2 kDa (28 repeat units) to 25 kDa (417 repeat units). The PEG block remained constant throughout the series at 5 kDa. Materials and Methods A series of biodegradable PLA-PEG diblock copolymers derived from poly(D,L-lactic acid) and monomethoxy poly(ethylene glycol) (PEG 5kDa) were synthesized by AstraZeneca23 using the ring-opening polymerization of D,L-lactide using stannous octanoate as a catalyst to form diblock copolymers. The basic structure of the PLA-PEG diblock copolymer is shown in Figure 1. A series of these diblock polymers was used to perform various NMR experiments. Poly(lactide-co-glycolide) (PLGA) was synthesized by Zeneca Pharmaceuticals with a Mw of 28 kDa at a ratio of 50:50 for the lactide and glycolide groups. The polydispersity of the sample was 1.7 as measured by size exclusion chromatography. The polymers were designated by the relative molecular weight of the PLA and PEG units in thousands; for example, PLA-PEG 2:5 contains a PLA segment of 2 kDa and a PEG segment of 5 kDa. Preparation of PLA-PEG Nanoparticles. A series of PLA-PEG nanoparticles, with constant size of the PEG chain (5 kDa) but varying PLA chain length (2:5, 3:5, 4:5, 6:5, 10:5, 13:5, 25:5), were obtained using a modified interfacial polymer deposition-solvent evaporation technique.1 To facilitate 1H NMR studies, deuterated water was used as the solvent. PLA-PEG (33.3 mg), D2O (10 mL, Goss Scientific Instruments Ltd., U.K.), and acetone (3.33 mL, HPLC grade) were mixed. The PLA-PEG samples were dissolved in acetone and added dropwise into the aqueous solution. The acetone solvent was removed from the reaction vessel by blowing nitrogen gas over the sample until all the acetone had evaporated. The reaction vessel was enclosed with sodium sulfate water-vapor traps so that water vapor could not reach the deuterated solvent and be exchanged. PLA-PEG 2:5, which is water soluble, forms micellar-type nanoparticles when added directly to D2O and consequently was produced by simply dissolving the PLA-PEG copolymer in D2O. Approximately 0.5 mL samples of the prepared solutions were then used for NMR analysis. In addition, for each PLA-PEG polymer a solution in d6-acetone (Cambridge Isotope Laboratories, U.S.) was prepared. Approximately 2 mg of the PLA-PEG polymers was dissolved in d6-acetone (0.5 mL) which was then used for NMR analysis. NMR Analysis. For each of the PLA-PEG polymers, 1H NMR spectra were obtained at 250 MHz using a Bruker AC250 spectrometer in both aqueous (D2O) and d6-acetone environments. For the NMR temperature studies (at 20, 30, 40, 50, 60, and 70 °C), the sample was equilibrated for 5 min before shimming, locking, and scanning the PLA-PEG. A calibration study was performed for the PEG signal and used to calculate how much PEG was seen on the surface of the nanoparticles. A known amount of sodium 3-trimethylsilylpropionate-d4 (TSP, 2 mg, Goss Scientific Instruments Ltd., U.K.) dissolved in D2O (0.2 mL) was employed as an external reference using a narrow bore tube placed coaxially within an NMR tube to hold the reference sample. PLA-PEG nanoparticles (0.5 mL) were also added to the coaxial NMR tube in which the total weight of the polymer was found by freeze-drying the sample after the NMR experiment. A known weight of PEG 5000 (Fluka, U.K.) homopolymer in D2O (5 mg in 0.5 mL) was used to check and compare the TSP signal (2 mg in 0.2 mL). By determining the number of moles of each sample present and relating this to the number of protons present in each sample, one can obtain a calibration between the two samples. Excellent agreement (99%) was observed indicating (23) Churchill, J. R.; Hutchinson, F. G. European Patent Application 0 092 918, 1983; European Patent Application 0 166 596, 1986.

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Table 1. Table of the Chemical Shifts for the Different Series of PLA-PEG Type Diblock Copolymers in d6-Acetone (Unimer Form) and D2O (Particle Form) at Room Temperature, Where s ) Singlet, m ) Multiplet, and dd ) Double Doubleta d6-acetone

D2O

PLA-PEG

δ CH

δ CH2CH2

δ CH3

δ CH

δ CH2CH2

δ CH3

percentage CH3

2:5 3:5 4:5 6:5 10:5 13:5 25:5

5.23 m 5.23 m 5.22 m 5.25 m 5.26 m 5.23 m 5.24 m

3.62 s 3.62 s 3.65 s 3.63 s 3.64 s 3.64 s 3.66 s

1.59 m 1.53 m 1.58 m 1.61 m 1.60 m 1.63 m 1.61 m

5.21 m

3.67 s 3.68 s 3.68 s 3.67 s 3.68 s 3.67 s 3.67 s

1.56 m, 1.41 dd 1.55 m, 1.45 dd 1.54 m, 1.47 dd 1.42 dd 1.44 dd 1.45 dd 1.45 dd

33 18 35 13 17 14 21

a The CH double doublet is seen from the splitting of the D- and L- forms of poly(lactic acid). The percentage CH observed is obtained 3 3 from the ratios of the CH3 signal seen between the D2O and d6-acetone spectra assuming that 100% CH3 is seen in the d6-acetone spectra.

that the method used, employing TSP as an external reference, is a satisfactory way of determining the amount of PEG present. Consequently, TSP was used as the calibration reference sample because it dissolves easily in D2O and gives a singlet at 0.0 ppm which can easily be integrated; the singlet does not affect any of the peaks of the PLA-PEG nanoparticle structure, and it had excellent agreement when calibrating using PEG 5000. For the solid-state NMR experiments (13C and 1H), a depolarization magic angle spinning (DPMAS) experiment was used in which the PLA-PEG nanoparticles (10:5 and 25:5) remained in the D2O liquid phase. Although the nanoparticles were in an aqueous environment, the solid-state experiments were able to pick up any solidlike regions in the nanoparticles. Consequently, the molecular structure of the nanoparticles remained in the same conformational state and aqueous environment as in the liquid-state 1H NMR experiments. The only difference was that more concentrated samples were needed in these experiments, so the samples were prepared as above but concentrated up to 10 times using an ultrafiltration cell (Amicon, U.S.). PCS experiments (Malvern 4700 PCS using the cumulants method and CONTIN for the distribution analysis) indicated that the mean size (Z average) of the nanoparticles did not alter after being concentrated. T1 relaxation rate 1H NMR experiments were also performed at 300 MHz on the 2:5 and 25:5 nanoparticles using Bruker NMR machines at a temperature of 293 K and variable delays of 10, 5, 4, 3, 2, 1, 0.5, 0.25, 0.10, and 0.01 s. Comparisons between the T1 values obtained from the two different types of nanoparticles and the different regions of the nanoparticles, namely, the core and the corona regions, were made. Standard Bruker software (xwinnmr 1.2) was used for the analysis of all of the results. Control 1H NMR experiments were also performed on PLGA nanoparticles. The nanoparticles were prepared using the Fessi method1 (33.3 mg of PLGA, 10 mL of D2O) to ascertain the influence of the central, hydrophobic core of the nanoparticle on the NMR signal resonances. PLA particles were also produced, but they did not form very stable structures due to aggregation of the particles and consequently were not analyzed by NMR experiments.

Results and Discussion 1H

NMR Liquid-State Studies. Initial studies on (1) the PLA-PEG nanoparticles were conducted at 293 K. The position of resonances in d6-acetone and in D2O are described in Table 1, together with the percentages of methyl protons in the PLA core that were observed. Examples of 250 MHz 1H NMR spectra of PLA-PEG 10:5 obtained in d6-acetone and in D2O at 293 K are shown in Figure 2. These results show that in d6-acetone, a nonselective solvent for the PLA-PEG polymer, complete structural resolution of the whole PLA-PEG diblock copolymer was observed. The PLA methyl (CH3) and methine (CH) groups together with the PEG (OCH2CH2) groups were completely resolved; that is, the diblock copolymer was dissolved as an homogeneous solution of unimer molecules, and the chemical shift of the PEG groups was upfield to approximately 3.6 ppm. However,

Figure 2. 1H NMR spectra of PLA-PEG 10:5 in D2O and d6acetone at room temperature indicating the different structures of the PLA-PEG molecules in different solvents.

in D2O, only the PEG signal and a very small methyl signal were seen. The methine protons and most of the methyl protons have disappeared from the spectrum. This indicates that these latter groups are in a different environment to those in d6-acetone. The NMR spectrum suggests that the PLA groups form a central solidlike hydrophobic core to minimize their interaction with the solvent. The PEG blocks interact favorably with the water molecules through the formation of H-bonds to create an exterior hydrophilic corona which extends into the aqueous media and stabilizes the nanoparticle structure.2,5 In the case of all the PLA-PEG nanoparticles, similar signals were seen (Table 1) with the strength of the CH3 signals weaker in the D2O spectra than in the d6-acetone spectra. For all the systems studied, small but well-defined double doublet resonances were seen at a chemical shift value appropriate for the CH3 group indicating splitting by the methine proton in the D- and L- forms of poly(lactic acid). This indicates that there is a region of intermediary phase between the two different phases in the nanoparticle. It is likely that these visible protons represent a more mobile region of PLA, probably located at the PLAPEG interface. Furthermore, for the low molecular weight PLA-PEG nanoparticle series (2:5, 3:5, and 4:5) an additional multiplet is seen at approximately δ ) 1.55 ppm. A 1H NMR spectrum of PLA-PEG 2:5 in D2O is shown in Figure 3 as an example of this behavior. This spectrum shows the singlet corresponding to the PEG protons and two sets of signals corresponding to the methyl protons in the interfacial region of the core, that is, the double doublet and the multiplet which is not seen with the high molecular weight particles (Figure 2). The solidlike region of the PLA core, that is, the immobile methine and methyl groups in the core, is still not observed. The methyl protons of the PLA, which are sufficiently mobile to be observed in the 1H NMR spectrum, appear to have a different structure in the low molecular weight particles as compared to those of higher molecular weight.

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Heald et al.

Table 2. Table of the Chemical Shifts and Integral Values for the PLA-PEG Nanoparticle Series for the Three Signals (CH, CH2CH2, and CH3) Together with the Percentage of the Methyl Signal Observed at the Various Temperaturesa PLA-PEG

temp (°C)

δ(CH)

integral

δ(CH2CH2)

integral

δ(CH3)

integral

percentage CH3

2:5

20 30 40 50 60 70 20 50 70 20 50 70 20 50 70 20 50 70 20 50 70 20 50 70

5.21 5.14 4.99 5.49 5.58 5.69

0.01 0.07 0.06 0.04 0.06 0.08

5.25 5.86

0.05 0.08

5.18 5.58

0.07 0.13

5.20 5.71

0.07 0.14

5.25 5.49

0.08 0.26

5.29 5.94

0.04 0.35

5.23 5.46

0.10 0.77

3.67 3.83 3.66 4.00 4.11 4.21 3.68 3.80 4.39 3.68 3.77 4.11 3.67 3.75 4.23 3.68 3.82 4.01 3.67 3.91 4.45 3.67 3.81 4.04

1.0 1.0 1.0 1.0 1.0 1.0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

1.54 1.69 1.53 1.80 1.91 1.99 1.50 1.60 2.19 1.50 1.58 1.93 1.42 1.52 2.02 1.44 1.61 1.79 1.45 1.68 2.27 1.45 1.55 2.52

0.06 0.07 0.10 0.14 0.16 0.18 0.04 0.12 0.26 0.13 0.24 0.39 0.04 0.17 0.39 0.06 0.20 0.69 0.16 0.17 1.11 0.48 0.46 1.86

33 39 56 78 89 100 18 43 95 35 65 105 13 31 71 17 22 75 14 15 96 21 20 80

3:5 4:5 6:5 10:5 13:5 25:5

a The percentage CH observed at each temperature is obtained as the ratio of the CH integral seen compared to that in d -acetone 3 3 6 assuming that 100% CH3 is seen in the room-temperature d6-acetone spectra.

Figure 3. 1H NMR spectra of PLA-PEG 2:5 in D2O at room temperature. Inserted is a more detailed figure of the methyl double doublet between 1.3 and 1.7 ppm.

The multiplet at approximately δ ) 1.55 ppm, which is observed only in the particles with the low molecular weight PLA blocks in the PLA-PEG series, has a different splitting pattern and is shifted downfield when compared to the double doublet signal which is observed in the complete PLA-PEG series. The existence of these two sets of resonances at differing chemical shifts, together with the broad nature of the multiplet signal, suggests that the PLA methyl protons exist in a number of different chemical environments within this relatively mobile interfacial region. This may arise from differences in the proximity to the PEG groups or differences in the packing of adjacent poly(lactic acid) chains. In the high molecular weight particles, only the double doublet signal is obtained, suggesting that the structure of the methyl protons in the interfacial regions of these larger particles is more uniform. The methine signal was always absent from the 1H NMR spectra (except for PLA-PEG 2:5) even though there was always an equivalent small double doublet methyl signal. This could suggest that the methine proton group was in a state which could not be resolved by the NMR experiment and was therefore in a solidlike environment. However, since some of the methyl groups were seen as double doublets, it would be expected that a small methine signal would also be present. The lack of methine signal may

simply be due to the weakness of this signal, since the corresponding methyl resonance is also weak. When the relaxation decay time of the NMR experiment was increased from 0.1 to 10 s, the methine proton was still not observed, again supporting the suggestion of the methine and methyl protons being embedded in a solidlike core. The PEG signal on the other hand was very similar throughout the PLA-PEG nanoparticle series and appeared as a single peak at a chemical shift of about 3.68 ppm. This signal was in the form of a singlet structure with a broad base indicating the enhanced mobility of the PEG protons as they become further away from the central PLA core. (2) Variable Temperature Studies. Variable temperature NMR studies were performed to observe the effects of temperature on the nanoparticle structures and in particular how the PLA core was affected. Experiments were conducted at temperature intervals of 10 K from 208 to 343 K. Higher temperatures were not used in the study as PEG loses its solubility at temperatures higher than 343 K (cloud point) due to breakage of a H-bonding water structure15 which stabilizes the PEG corona. The chemical shifts and the integral results for the PLA-PEG 2:5 nanoparticle at varying temperature are shown in Table 2. The data show that the chemical shifts for the constituent groups moved further downfield as the temperature was raised and that the amount of CH3 observed (in terms of its integral and percentage amount) increased as the temperature was raised until all the core methyl protons were seen at 343 K. The percentage of methyl signal observed refers to a comparison with the methyl signal obtained when the diblock copolymer is dissolved as an homogeneous solution in d6-acetone. These results indicate that changes have taken place in the nanoparticle conformation and structure as the temperature was increased, that is, the higher temperature was able to make the PLA core more mobile until all the core protons were observed at 343 K. Furthermore, the glass transition temperature (Tg) of PLA-PEG diblock copolymers is approximately 333 K suggesting that it would be

NMR Studies of PLA-PEG Nanoparticles

easier to observe the solidlike PLA core above this temperature since the mobility of the PLA groups would be greater. The chemical shifts, integral values, and percentage of methyl groups seen in the 1H NMR spectra at various temperatures for the complete PLA-PEG nanoparticle series are given in Table 2. These data show how the spectra change; namely, the methyl (δ ) 1.5 ppm) and methine (δ ) 5 ppm) signals increase in size and shift downfield as the temperature increases. Some changes in the shape of these signals are also observed with an increase in temperature. Furthermore, the PEG signal shifts downfield also as the temperature is increased, although the size and shape of the signal remains fairly constant. The shift downfield of all the proton resonances as the temperature is increased, which is observed with all the PLA-PEG nanoparticle systems, suggests a clear change in the structure of the particles with heating which affects all the protons in the diblock copolymer. However, this observation is not easily explained. The chemical shifts of carbon-bonded protons are not normally changed greatly by temperature. Perhaps the changes in chemical shifts reflect an alteration in the tumbling of the molecules as the system becomes more liquidlike at higher temperatures, which would improve the relaxation of the protons. Certainly the methine and methyl protons appear initially to form broader singlets as the temperature is increased, which then become sharper singlet peaks at a higher temperature. This observation is in keeping with enhanced relaxation effects. Alternatively, the increase in temperature may result in such a radical change in diblock copolymer structure that each of the protons is now located in a very different chemical environment and large changes in chemical shift are observed. For the low molecular weight PLA-PEG nanoparticle systems (2:5, 3:5, and 4:5), an increase in temperature to 323 K had a significant effect on the structures. An increased amount of the PLA core of these low molecular weight systems, as expressed by the amount of methyl and methine protons detectable, was observed at 323 K, whereas for the high molecular weight PLA-PEG nanoparticle systems (6:5 and above) this increase in temperature had little effect. The presence of an additional multiplet in the spectra of the smaller PLA-PEG nanoparticles at room temperature corresponding to methyl protons (Figure 3) suggests, as described previously, the presence of PLA methyl protons in a number of different chemical environments. As the temperature is raised to 323 K, the mobility of this region is enhanced and these different resonances are no longer distinguishable. At this temperature, the percentage of the PLA core observed was increased. For the higher molecular weight PLAPEG nanoparticles, little change in the amount of the PLA core protons was observed at 323 K. A small methine signal was seen, only at 323 K, while the small methyl double doublet and the PEG singlet remained constant in size and shape in this lower temperature range. Above 323 K, a greater part of the PLA core (expressed in terms of the methyl and methine protons observed) for all the PLA-PEG nanoparticles was seen as the groups became more mobile and liquidlike. As the core becomes more mobile, the double doublet signal from the interfacial protons becomes smaller and the CH3 signal turns gradually into a broad multiplet. This suggests that the PLA protons in the interfacial region and those within the hydrophobic core are now located in more similar chemical environments, thus resonating at similar chemical shifts. This would be expected as the polymer becomes more mobile when approaching its Tg value.

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At 343 K, most of the methyl protons were seen for both the low molecular weight and high molecular weight nanoparticles indicating an essentially liquid core in these PLA-PEG nanoparticles (Table 2). Most of the methine protons were also visible indicating that when the core is in a liquid state, these resonances can be observed. Furthermore, the PEG methylene signals, seen at approximately δ ) 3.68 ppm at room temperature, shifted downfield to δ ) 4.04 ppm as the temperature was increased to 343 K, maintaining fairly constant size and shape. By observing the methine signal with increasing temperature, it could be seen that the methine protons could relax under the time scale of the NMR experiment. Consequently, the methine peak could be measured and would be visible at room temperature if the group was in the liquid state. The ratio of the methyl/methine groups was approximately 3:1 at 343 K (as expected) indicating that the phase of the core was in the liquid state. This 3:1 ratio of the methyl/methine signal is not observed at lower temperatures. This could be due to either the relaxation rate of the methine group and/or the lack of enough methine protons being in the liquid state to be observed. These results confirm that at room temperature the core demonstrated a solidlike environment in which most of the PLA signals (both CH3 and CH) were not seen and that a more liquidlike, mobile core was observed at elevated temperatures. Equivalent experiments performed on PLGA nanoparticles also show this phenomenon, where the nanoparticle core was not observed at room temperature by liquidstate 1H NMR experiments. However, as the temperature was raised to above 323 K the PLGA nanoparticle core was observed as indicated by a series of resonances. Again, this temperature (323 K) is close to the Tg of the PLGA copolymer (data not shown). 1 H NMR studies on varying length nonionic alkyl poly(oxyethylene) (CnEOm) ether micelles have shown similar temperature dependence on the core protons resonances.24,25 Ribeiro and Dennis26 used 13C and 1H NMR to study C12EO8 micelles in D2O where the alkyl hydrophobic core and the hydrophilic corona were seen. Again, due to the small size of the hydrophobic core, the core is in a liquid state and is detected by the NMR experiment. 1H NMR studies of the micellar structure of polystyreneβ-poly(ethylene/propylene) block copolymer in octane21,27,28 at room temperature demonstrated that the polystyrene peaks were absent in the spectra, indicating that the polymer core was in a glassy state. As the temperature was increased, the polystyrene peaks gradually appeared as the core chains become more mobile and liquidlike. (3) Quantification of the Amount of PEG Observed Using TSP as an External Reference. Quantification studies of the PEG corona of the PLA-PEG nanoparticle series using TSP as an external reference sample indicate that virtually all of the PEG protons were seen in the liquid-state 1H NMR experiments (Table 3). For all members of the PLA-PEG nanoparticle series, more than 90% of the PEG protons were seen in 1H NMR experiments using a known amount as an external reference. The only exception to this was the PLA-PEG 25:5 and 10:5 nanoparticles. For reference, the particle sizes of the (24) Elworthy, P. H.; Patel, M. S. J. Pharm. Pharmacol. 1984, 36, 565. (25) Nilsson, P. G.; Wennerstroem, H.; Lindman, B. J. Phys. Chem. 1983, 87, 1377. (26) Ribeiro, A. A.; Dennis, E. A. J. Phys. Chem. 1977, 81, 957. (27) Cabdau, F.; Heatley, F.; Price, C.; Stubbersfield, R. B. Eur. Polym. J. 1984, 20, 685. (28) Godward, J.; Heatley, F.; Booth, C. J. Chem. Soc., Faraday Trans. 1995, 91, 1491.

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Heald et al.

Figure 4. 13C solid-state NMR spectrum of PLA-PEG 25:5 showing the solid central PLA core as compared to Figure 2 where the central core is not seen in the liquid-state 1H NMR experiment. Table 3. Table of the Percentage of PEG Detected by 1H NMR for the PLA-PEG Nanoparticle Systems as Compared to a Standard TSP (2 mg) Reference Samplea PLA-PEG

% PEG detected

diameter ( std dev (nm)

2:5 3:5 4:5 6:5 10:5 13:5 25:5

94 92 99 90 88 97 85

24.8 ( 0.5 26.6 ( 0.4 27.4 ( 0.3 30.3 ( 0.7 42.7 ( 1.9 76.4 ( 0.2 71.2 ( 0.3

a Particle size data obtained by photon correlation spectroscopy from ref 2.

nanoparticles2 are included in the table showing a significant increase in particle diameter with increasing molecular weight of the PLA block. Despite the increasing particle size, the percentage of PEG on the surface remains remarkably constant. The results from these quantification experiments again lead to the conclusion that PEG is in a liquid corona on the outside of the nanoparticle being solvated by water (D2O) molecules and not trapped in the central more rigid hydrophobic core of the nanoparticle. (4) Solid-State NMR Studies. The solid-state 13C NMR spectrum of PLA-PEG 25:5 in D2O is shown in Figure 4. The spectrum shows the resonances of the methyl group

at 17 ppm and the OCH2/OCH groups at 70.3 ppm in which the two different groups were not resolved. This lack of resolution of the OCH2/OCH groups has also been seen in the 13C NMR spectra obtained for the PLA-PEG copolymers dissolved in d6-acetone. The carbonyl carbon was not seen for the copolymer in nanoparticle form (dissolved in D2O) due to the short relaxation delay used in the experiment. When the polymer was dissolved in d6acetone, the carbonyl signal was observed since the group can relax more easily under the time scale of the liquidstate experiment. In the methyl region (∼17 ppm), a number of signals can be seen, that is, one broad and two sharp signals. The sharp signals were from relatively mobile species (the CH3 groups near the interface), and the broad signal was from methyl carbon atoms which were in a more rigid, less mobile region of the nanoparticle. From these results, it can be concluded that the PLA blocks exist in a two-phase system. The 13C solid-state spectra resolved the two different central core environments for the CH3 carbon atoms: a solidlike core and a more mobile interfacial region. The OCH2/OCH signal was seen at 70.3 ppm. The OCH2 signal would be expected to represent highly mobile PEG carbon atoms and indicates therefore that equivalent to the PEG signal at 3.68 ppm in the 1H liquid-state spectra, 13C solid-state NMR provides resonances for groups within the liquid phase.

NMR Studies of PLA-PEG Nanoparticles

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Table 4. Table of the T1 Relaxation Data of the PEG Group and the Interfacial Methyl Groups for PLA-PEG 2:5 and 25:5 Nanoparticles Performed at 300 MHz PLA-PEG

peak (ppm)

T1

PLA-PEG

peak (ppm)

T1

2:5

3.63 (PEG) 1.43 (CH3)

0.62 1.17

25:5

3.63 (PEG) 1.41 (CH3)

0.63 1.17

The 1H solid-state NMR spectra showed similar characteristics to the liquid-state NMR spectra. A small double doublet signal was seen at 1.35 ppm for the methyl group in the 1H solid-state spectra which corresponds to that observed in the 13C spectra. The PEG group was seen at 3.6 ppm, in agreement with the liquid-state spectra. The methine signal was not resolved in this 1H solid-state experiment. The 1H solid-state experiment indicated the presence of interfacial methyl protons as described above but could not pick up the rigid core methyl protons. In the case of PLA-PEG 10:5, the 13C solid-state spectrum showed peaks at 70.3 ppm (CH2O/CHO) and a broad signal at 17.0 ppm (CH3). A small signal was seen at 170 ppm, which probably indicates the presence of the carbonyl group. Complete resolution of the methyl protons for the 10:5 nanoparticle at 17 ppm was not observed, since the singlet signals for the interfacial methyl carbons were not seen. It is not clear why this difference is observed, but it suggests either that the interfacial methyl proton signals are weaker as compared to the methyl proton signals in the solid region of these particles or that subtle differences in the structure of the cores of these particles renders the mobility or chemical environment of all the methyl protons to be very similar. The 13C solid-state experiments provide evidence for the existence of two different regions within the PLA core of the nanoparticles: a solidlike central core and a more mobile interfacial region. The spectra showed that the more immobile methyl carbon atoms gave rise to a broad resonance, while the more mobile methyl carbon atoms around the PLA-PEG interface appeared as two sharp singlets. This indicates a two-phase hydrophobic central core. The 1H NMR solid-state experiments were not able to detect the protons in the solid central core, although the interfacial methyl protons were detected. (5) 1H NMR T1 Relaxation Measurements. The results for the 1H NMR T1 relaxation experiments on PLAPEG 2:5 and 25:5 nanoparticles at room temperature are shown in Table 4. The T1 relaxation rates were obtained for the PEG protons (singlet at approximately 3.63 ppm) and the interfacial CH3 protons of the PLA group (double doublet signal at approximately 1.43 ppm). T1 relaxation rates for the central PLA core could not be obtained as the CH3 core protons cannot be detected by the 1H NMR experiment. The results show that the T1 relaxation rates for the mobile protons at the PLA/PEG interface were larger than those of the PEG protons at approximately 1.1 and 0.68 s, respectively. This indicates that although the interfacial methyl protons were observed and were clearly in a mobile state, the PEG protons were in a much more mobile environment since the protons could relax much more quickly. T1 values have also been obtained for some alkyl and butylene oxide poly(oxyethylene) ether micelles.29 These micelles have their hydrophobic corona region in a mobile state, and as a consequence, the two

T1 values from each micelle system are more comparable. The T1 relaxation value for C16EO27 was 0.56 and 0.47 s for the ethylene oxide and alkyl regions,29 while for C22EO27 the relaxation rates were 0.53 and 0.40 s, respectively.24 For a butylene oxide core, the T1 values were 0.6 and 0.5 s for the corona and core regions,30 respectively. These results show that if the core was much more mobile, that is, in a liquidlike state, the relaxation time of the core protons would be quicker than that of the corona protons since it is easier for the liquidlike core groups to relax. The T1 relaxation measurements of the PLA-PEG nanoparticles indicate an increased rigidity of the interfacial PLA methyl protons as compared to the more fluid central core region of the alkyl poly(oxyethylene) ether micelles. Comparing the T1 results from the literature and those for the PLA-PEG nanoparticles of the corona PEG protons, similar values were seen indicating the mobility and freedom of movement of these PEG protons in aqueous media. In comparing the two different PLA-PEG nanoparticles, very little difference was seen in the T1 relaxation rates between the two PEG groups and the two methyl groups. This indicates that both groups are in similar chemical and structural environments in the two different nanoparticles. However, 1H NMR experiments at room temperature have indicated that the interfacial methyl groups experience different chemical environments in the low molecular weight particles, since two distinct methyl resonances are obtained in these systems. This is not the case for the high molecular weight PLA-PEG system where the size and the packing density of the PLA core result in the core being in a more solidlike environment. Conclusions These studies have shown that a combination of 1H NMR liquid-state and 13C solid-state NMR spectroscopy studies performed on the PLA-PEG diblock copolymer series can provide information on the structure of the nanoparticles formed from these copolymers. It was shown that as the size of the PLA block increases, the central hydrophobic core of the nanoparticles becomes more solidlike. Acknowledgment. We thank Dr. D. Apperley at Durham University, Solid State NMR service, for the solidstate NMR spectra of PLA-PEG 10:5 and 25:5 and Mr. J. Fleming at the NMR service, Department of Chemistry, Nottingham University, for the T1 measurements of the PLA-PEG 2:5 and 25:5 samples. Finally, thanks are given to the DTI (Link Scheme Grant Number GR/J57889) and our industrial partners (AstraZeneca, Oxford Materials, CSMA, and DanBioSyst (UK) Ltd.) for financial assistance. Supporting Information Available: The variable temperature 1H NMR spectra for PLA-PEG 13:5 from 20 to 70 °C. 1H NMR spectra for the T1 relaxation for the PLA-PEG 2.5 nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. LA011393Y (29) Heatley, F.; Teo, H. H.; Booth, C. J. Chem. Soc., Faraday Trans. 1984, 80, 981. (30) Godward, J.; Heatley, F.; Smith, S.; Tanodekaew, S.; Yang, Y.W.; Booth, C. J. Chem. Soc., Faraday Trans. 1995, 91, 3461.