Core−Shell Structure of PLA−PEG Nanoparticles Used for Drug

The splayed PEG chains of the PLA(d)−PEG 3:5 assemblies were characteristic ..... The core polydispersities of all three systems and, in particular,...
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Langmuir 2003, 19, 8428-8435

Core-Shell Structure of PLA-PEG Nanoparticles Used for Drug Delivery T. Riley, C. R. Heald, S. Stolnik, M. C. Garnett, L. Illum, and S. S. Davis* School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.

S. M. King and R. K. Heenan ISIS Facility, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 OQX, U.K.

S. C. Purkiss, R. J. Barlow, P. R. Gellert, and C. Washington AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K. Received November 15, 2002. In Final Form: April 2, 2003 Small-angle neutron scattering (SANS) has been used to study the internal structure of poly(lactic acid)-poly(ethylene glycol) (PLA(d)-PEG) block copolymer assemblies, which are being investigated as particulate drug carriers. Three PLA(d)-PEG copolymers with a fixed PEG of 5 kDa and a fully deuterated PLA(d) block of either 3, 15, or 45 kDa were synthesized by the ring opening polymerization of d8-D,Llactide, using stannous octoate as a catalyst. These copolymers assembled to form nanoparticles in aqueous media, following precipitation from a water miscible organic solvent. The hydrodynamic radius of the PLA(d)-PEG nanoparticles increased with the molecular weight of the PLA(d) block. SANS data obtained at three different solvent contrasts were analyzed simultaneously using core-shell models, which assumed a homogeneous core of uniform scattering length density and a simple functional form for the scattering length density profile of the shell. The thickness and structure of the stabilizing PEG layer were found to depend on the molecular weight of the PLA(d) block. The splayed PEG chains of the PLA(d)-PEG 3:5 assemblies were characteristic of those found in polymeric micelles. However, as the molecular weight of the PLA(d) block was increased, the PEG brush became more radially homogeneous, in accord with recent Scheutjens-Fleer mean-field theory predictions.

Introduction The inherent core-shell structure of micelles and nanoparticles assembled from amphiphilic block copolymers makes them interesting candidates as drug carriers for targeted delivery.1,2 Drugs may be incorporated into the core of the assemblies by either covalent or noncovalent bonding, such as hydrophobic or ionic interactions.2-4 Poly(ethylene glycol) (PEG) is usually chosen as the hydrophilic buoy block whose role is to provide a hydrated steric barrier. This steric barrier is generally accepted to minimize the adsorption of circulating blood components, which ordinarily facilitate the recognition and subsequent engulfment of colloidal carriers by the macrophages of the reticuloendothelial system. In some cases where adsorbed block copolymers have been used to provide a steric layer, the coating has been found to desorb from the particle surface in the blood circulation.1 However, in this work, the stabilizing PEG chains of block copolymer assemblies are effectively grafted to the surface of the core, and the possibility of displacement by serum components is eliminated. * To whom correspondence should be addressed. Phone: +44 (0) 115 9515 121. Fax: +44 (0) 115 9515 122. E-mail address: [email protected]. (1) Stolnik, S.; Illum, L.; Davis, S. S. Adv. Drug Delivery Rev. 1995, 16, 195-214. (2) Kwon, G. S. Crit. Rev. Ther. Drug Carrier Syst. 1998, 15, 481512. (3) Yokoyama, M.; Okano, T.; Sakurai, Y.; Suwa, S.; Kataoka, K. J. Controlled Release 1996, 39, 351-356. (4) Zhang, X.; Jackson, J. K.; Burt, H. Int. J. Pharm. 1996, 132, 195-206.

AB block copolymers of PEG with either poly(lactic acid) (PLA-PEG) or poly(lactic acid-co-glycolic acid) (PLGAPEG) have been extensively investigated as drug delivery vehicles, since the core-forming block is fully biodegradable.1,5-10 By varying the molecular weight ratio of the hydrophobic and hydrophilic blocks, it is possible to control the aggregation behavior of the copolymer. Water soluble PLA-PEG copolymers with relatively low molecular weight PLA blocks self-disperse in water to form polymeric micelles11,12 which have been used to solubilize strongly hydrophobic anticancer drugs by micellar incorporation.4 PLA-PEG copolymers with higher PLA to PEG weight ratios are water insoluble but can be assembled to form micellar-like nanoparticles in aqueous media, either using an emulsification procedure or by precipitation from water miscible organic solvents.1,5-8 Reports on the biological performance of such systems are encouraging from a drug delivery perspective. For (5) Piskin, E.; Kaitian, X.; Denkbas, E. B.; Ku¨c¸ u¨kyavuz, Z. J. Biomater. Sci. Polym. Ed. 1995, 7, 359-373. (6) Bazile, D.; Prud’homme, C.; Bassoullet, M.-T.; Marlard, M.; Spenlehauer, G.; Veillard, M. J. Pharm. Sci. 1995, 84, 493-498. (7) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (8) Govender, T.; Riley, T.; Ehtezazi, T.; Garnett, M. C.; Stolnik, S.; Illum, L.; Davis, S. S. Int. J. Pharm. 2000, 199, 95-110. (9) Yasugi, K.; Nakamura, T.; Nagasaki, Y.; Kato, K. M.; Kataoka, K. Macromolecules 1999, 32, 8024-8032. (10) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113. (11) Hagan, S.; Coombes, A. G. A.; Garnett, M. C.; Dunn, S. E.; Davies, M. C.; Illum, L.; Davis, S. S. Langmuir 1996, 12, 2153-2161. (12) Tanodekaew, S.; Pannu, R.; Heatley, F.; Attwood, D.; Booth, C. Macromol. Chem. Phys. 1997, 198, 927-944.

10.1021/la020911h CCC: $25.00 © 2003 American Chemical Society Published on Web 08/21/2003

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analyzed the structure of polymeric micelles using an adaption of the Scheutjens-Fleer mean-field lattice theory. Figure 1. Chemical structure of the selectively deuterated PLA(d)-PEG block copolymers.

example, the blood circulation time of nanoparticles assembled from a PLA-PEG block copolymer with a PLA block of 30 kDa and a PEG block of 2 kDa was dramatically increased relative to that of albumin coated PLA nanoparticles of a similar size.6 Nevertheless, little is known about how the physicochemical characteristics of the PLAPEG assemblies influence their biological fate, although their size and the thickness of the stabilizing PEG layer are thought to be important.1 To aid in this investigation, we have recently described the preparation of a series of PLA-PEG nanoparticles, in which the particle size of the assembly was strictly controlled by the molecular weight of the PLA block.13,14 A variety of spectroscopic, light scattering, magnetic resonance, and rheological techniques have been successfully used to study these systems.13-15 However, many of these techniques only provide information about the structure of the assembly as a whole, such as the total hydrodynamic radius of the core plus the stabilizing PEG layer. In this study, small angle neutron scattering (SANS) has been used to provide direct structural information about the internal structure of the PLA-PEG assemblies. Although SANS studies of polymers at a contrast-matched interface are abundant,16-20 this work is, as far as we are aware, unique due to the small size of the core particle and the fact that it is the off-match data which provide the more detailed structural information via solvent contrast variation. Selectively deuterated PLA(d)-PEG copolymers with fully deuterated PLA(d) blocks (Figure 1) were synthesized specifically for use in these experiments. The high neutron scattering length density of deuterated PLA(d) enabled the cores of the PLA(d)-PEG nanoparticles to be contrastmatched to a deuterium-rich dispersion medium. This enables diffraction to be seen from a “hollow shell” of PEG, which is highly sensitive to the composition and structure of the PEG layer. Structural parameters were extracted by fitting core-shell models to the scattering data, obtained at three different contrasts. In this way it was possible to construct scattering length density profiles for the PLA(d)-PEG nanoparticles, which help to describe how the volume fractions of PLA(d), PEG, and solvent vary with radial distance R from the center of the assembly. These experimental volume fraction profiles were compared to those obtained by Leermakers et al.,21 who (13) Riley, T.; Govender, T.; Stolnik, S.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S. Colloids Surf., B: Biointerfaces 1999, 16, 147159. (14) 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, 3168-3174. (15) Hagan, S. A.; Davis, S. S.; Illum, L.; Davies, M. C.; Garnett, M. C.; Taylor, D. C.; Irving, M. P.; Tadros, Th. F. Langmuir 1995, 11, 1482-1485. (16) King, S. M.; Griffiths, P. C.; Cosgrove, T. Using SANS to study adsorbed layers in colloidal dipsersions. In Applications of neutrons in soft condensed matter; Gabrys, B. J., Ed.; Gordon and Breach: New York, 1998. (17) Washington, C.; King, S. M.; Heenan, R. J. Phys. Chem. 1996, 100, 7603-7609. (18) Washington, C.; King, S. M. Langmuir 1997, 13, 4545-4550. (19) Cosgrove, T.; Ryan, K. Langmuir 1990, 6, 136-142. (20) Cosgrove, T.; Vincent, B.; Crowley, T. L.; Cohen Stuart, M. A. ACS Symp. Ser. 1984, 240, 147-159. (21) Leermakers, F. A. M.; Wijmans, C. M.; Fleer, G. J. Macromolecules 1995, 28, 3434-3443.

Experimental Section Materials. Poly(ethylene glycol) monomethyl ether (MePEG, MW 5000 Da) obtained from Fluka, Gillingham, U.K., was purified by extraction from aqueous solution by dichloromethane, followed by precipitation into an excess of diethyl ether. Fully deuterated d8-D,L-lactide purchased from Goss Scientific Instruments Ltd., Great Baddow, U.K., was purified by recrystalization from dried ethyl acetate. The polymerization catalyst, tin(II) octoate (Sigma, Poole, U.K.), was prepared as a 0.3% w/v solution in dried toluene. All organic solvents used were of HPLC grade and supplied by Fisher Scientific, Loughborough, U.K. Deuterium oxide (D2O), 99.9% D, was purchased from Goss Scientific Instruments Ltd., while deionized water (Elgastat Ltd., U.K.) was used in all experiments. Deuterated chloroform and sodium 3-trimethylsilyl d4-propionate (TSP) were also obtained from Goss Scientific Instruments Ltd., while all other materials were obtained from the Sigma Chemical Company. Synthesis of PLA(d)-PEG Copolymers. Three selectively deuterated PLA(d)-PEG copolymers were synthesized by the ring opening polymerization of d8-D,L-lactide in the presence of MePEG (5 kDa), using stannous octoate as a catalyst.22 This synthesis procedure has been described in detail elsewhere.13,14 Briefly, appropriate amounts of d8-D,L-lactide and MePEG were placed in a dried polymerization tube, and the stannous octoate catalyst (0.016% w/w) was added as a solution in toluene. The reactants were dried under reduced pressure, at 70 °C for 2 h. The tube was then sealed under vacuum, and the copolymerization was carried out at 170 °C for 5 h. The product was dissolved in dichloromethane and precipitated into an excess of petroleum ether at 40-60 °C, cooling if necessary with liquid nitrogen. The purified copolymers were dried in a vacuum oven at 70 °C for approximately 24 h and stored in a vacuum desiccator at +4 °C. 1H NMR spectra of each of the selectively deuterated PLA(d)-PEG copolymers dissolved in deuterated chloroform (CDCl3) were obtained using a Bruker AC250 spectrophotometer operating at 250 MHz. Since the deuterated PLA(d) block is not observed by 1H NMR, the PEG signal was calibrated using a known mass of each copolymer (2.0 mg) together with benzoic acid (2.0 mg) as an external reference, contained within a coaxial NMR tube. The 1H NMR spectra revealed strong signals corresponding to the PEG methyene groups (CH2CH2, δ 3.63 pm, singlet). The integration ratio of the peaks corresponding to the benzoic acid allylic protons (δ 7.25-8.15 ppm) and PEG methylene groups enabled the number of moles of PEG protons observed to be determined. Since the average number of protons in each PEG chain is known (455), it is possible to estimate the number average molecular weight of the PLA block, M h n,PLA, as follows:

M h n,PLA )

[

]

mass of copolymer in the sample moles of 1H(PEG) observed/no. of 1H in PEG 5kDa 5000 (1)

The values of M h n,PLA obtained (Table 1) were similar to those expected from the nominal PLA(d)-PEG copolymerization feed ratios, which have therefore been used as nomenclature in the text. Preparation of Dispersions. Aqueous dispersions of PLA(d)-PEG nanoparticles were prepared by a precipitation/solvent evaporation technique.23 A solution of the copolymer in acetone (20 mg mL-1, 10 mL) was added dropwise with stirring to 30 mL of D2O. The preparation vessel was sealed under a constant stream of nitrogen gas, to facilitate the removal of the acetone with minimal exposure to water vapor. Once the acetone had been removed, the dispersions were concentrated to approximately half their initial volume by ultrafiltration under nitrogen (22) Deng, X. M.; Xiong, C. D.; Cheng, L. M.; Huang, H. H.; Xu, R. P. J. Appl. Polym. Sci. 1995, 55, 1193-1196. (23) Fessi, H.; Devissaguet, J. P.; Puisieux, F.; Thies, C. French Patent 2,608,988, 1986.

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Table 1. Characterization of the PLA(d)-PEG Copolymers and the Nanoparticles They Form in Aqueous Mediaa PLA(d)-PEG M h n,PLA/Da NPLA hydrodynamic radius (Rhyd)/nm CFPT/(mol dm-3 Na2SO4)

3:5 3 200 42 16.0 0.55

15:5 15 600 205 24.2 0.50

Table 2. Coherent Neutron Scattering Length Densities

45:5 42 400 558 55.4 0.35

a Description of the parameters: M h n,PLA ) number average molecular weight of the PLA(d) block as determined by 1H NMR of the copolymer dissolved in CDCl3; NPLA ) average number of chemical repeat units in the PLA(d) block; Rhyd ) hydrodynamic radius of the PLA(d)-PEG assemblies as determined by PCS; CFPT ) critical flocculation point (concentration of electrolyte required to induce flocculation).

pressure (Amicon ultrafiltration cell with Diaflo PM30 ultrafilters, Amicon Inc., U.S.A.). The concentrated PLA(d)-PEG dispersions were filtered (1.2 µm pore size filters, Sartorius AG, Go¨ttingen, Germany) and stored at +4 °C in nitrogen purged glass vials, sealed with rubber Suba-seal septa. A known volume (5 mL) of each of the dispersions was removed and freeze-dried (Edwards Modulyo freeze-drier, York, U.K.), to determine the exact final nanosphere concentration (w/w). The dispersion media of these samples were taken to be 100% D2O. Physicochemical Properties of PLA(d)-PEG Nanoparticles. The size distributions of the nanoparticles were measured by dynamic light scattering at the temperature 25.0 ( 0.1 °C, using a Malvern 4700 dynamic light scattering instrument (Malvern Instruments, U.K.). The cumulants method was used to determine the average particle radius. In addition, the colloidal stability of the nanoparticles to added electrolyte was studied by adding 0.5 mL of each dispersion to 2.5 mL of Na2SO4 solutions of varying concentration (0-0.8 M) and measuring the turbidity at a wavelength of 400 nm after 15 min (Beckman BD/640 spectrophotometer, High Wycombe, U.K.). The critical flocculation point (CFPT) was taken as the electrolyte concentration at which a dramatic increase in the turbidity was first detected. The geometries of the PLA(d)-PEG copolymers used in this study are such that they are water insoluble and consequently do not spontaneously form micelles in equilibrium with dissolved copolymer units. Nevertheless, when precipitated from a water miscible organic solvent, they reproducibly assemble to form micellar-like nanoparticles of a defined size, which do not dissociate due to the insolubility of the individual copolymer units. The hydrodynamic diameter, polydispersity index (determined by PCS), and critical flocculation point (CFPT) of the nanoparticles assembled from the PLA(d)-PEG block copolymers used here are presented in Table 1. As we have recently shown for a comprehensive series of fully hydrogenated PLA-PEG nanoparticles,13,14 the particle size of the assemblies is dependent on the molecular weight of the PLA(d) block. This increase in particle size is accompanied by a decrease in the colloidal stability of the dispersions, as indicated by a reduction in the critical flocculation point (CFPT). Flocculation in solvents better than Θ-solvents at stabilizing PEG chains is attributed to a reduction in the PEG surface coverage. In addition, 1H NMR spectra of the PLA(d)-PEG nanoparticles dispersed in D2O have been used to confirm that the PEG chains are all in a solvated state, with negligible penetration into the solid PLA(d) core.14 This is consistent with the core-shell structure of the PLA(d)-PEG assemblies. Small-Angle Neutron Scattering. (a) Collection and Reduction of SANS Data. SANS data were obtained on the LOQ small-angle diffractometer at the ISIS spallation neutron source (Rutherford Appleton Laboratory, Didcot, U.K.). This is a fixedgeometry “white beam” time-of-flight instrument which utilizes neutrons with wavelengths of between 2.2 and 10 Å to provide a simultaneous Q-range of 0.008-0.24 Å-1. Q is the modulus of the scattering vector:

Q)

(4πλ) sin(θ/2)

(2)

where λ is the neutron wavelength and θ is the scattering angle.

solvent/polymer

bulk density, Fbulk/(g cm-3)

scattering length density, F/(×1010 cm-2)

100% D2O 80% D2O/20% H2O 75% D2O/25% H2O 65% D2O/35% H2O 100% H2O PEG (hydrogenated) PLA(d) (perdeuterated)

1.11 1.09 1.08 1.07 1.00 1.13 (as solid18) 1.29 (as solid24)

+6.36 +5.00 +4.65 +3.95 -0.56 +0.64 +5.95

The coherent neutron scattering from each component of a composite system is dependent on the square of the difference (or contrast) between the neutron scattering length densities (F) of that component and the dispersion medium. If the cores of core-shell type assemblies are selectively deuterated, then it is possible to make them “invisible” to neutrons by adjusting the scattering length density of the aqueous dispersion medium through its H2O to D2O ratio. The scattering length densities of the hydrogen- and deuterium-containing materials used in this study are tabulated in Table 2. SANS samples with different contrasts were prepared by accurately diluting the dispersions prepared as described above, with H2O (the data analysis allowed for the resultant changes in concentration). The scattering and transmission of each sample and an appropriate background were measured at the temperature 25 ( 0.2 °C. The backgrounds used consisted of H2O/D2O mixtures in the same ratios as those for the continuous phases of the dispersions. Each raw scattering data set was corrected for the sample transmission and background scattering and converted to the scattering cross section data, (∂Σ/∂Ω)(Q), using instrumentspecific software.25,26 The reduced data were placed on an absolute scale using a well characterized blend of hydrogenous and perdeuterated polystyrene as a calibration standard.27 (b) Data Analysis Using Core-Shell Models. The core-shell models used in the analysis of the SANS data from the PLA(d)-PEG assemblies assumed a homogeneous core with a fixed scattering length density, Fc, and a simple functional form for the scattering length density profile of the shell, Fs(R). The overall scattering cross section from such composite structures consists of a contrast weighted summation of the scattering from the core and shell components, together with a contribution from interference terms, and it may be expressed as

∂Σ (Q) ) nP(Q) S(Q) + B ∂Ω

(3)

where n is the number density of particles, P(Q) is the particle form factor, S(Q) is the interparticle structure factor, and B is the background signal. At low particle concentrations, S(Q) tends to unity and can be disregarded. In the calculation of P(Q), the particles were assumed to have polydisperse cores with radii Rc distributed according to the Schultz distribution, characterized by a mean radius R h c and polydispersity index (σ/R h c). The scattering law, P(Q), for spherical particles composed of a core plus multiple shells, with sharp interfaces, was given by Ottewill et al.28 In general, the particle form factor for a core-shell assembly may be expressed as

P(Q) ) ((F1 - F2)F(Q,R1) + (F3 - F2)F(Q,R2) + ...)2

(4)

where the form factor for each step or feature, F(Q,R), depends on the scattering length density profile, F(R) (see Appendix). The scattering length density profile of the core-shell model used to fit the SANS data obtained from the PLA(d)-PEG (24) Eling, B.; Gogolewski, S.; Pennings, A. J. Polymer 1982, 23, 1587-1593. (25) King, S. M.; Heenan, R. K. Using COLETTE; Rutherford Appleton Laboratory Report RAL-95-005; 1995. (26) Heenan, R. K.; Penfold, J.; King, S. M. J. Appl. Crystallogr. 1997, 30, 1140-1147. (27) Wignall, G. D.; Bates, F. S. J. Appl. Crystallogr. 1987, 20, 2840. (28) Markovic, I.; Ottewill, R.; Cebula, D. J.; Field, I.; Marsh, J. Colloid Polym. Sci. 1984, 262, 648.

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Figure 2. Scattering length density profile of the core-shell model used to fit the SANS data from micellar-like structures, incorporating a diffuse shell. assemblies is illustrated in Figure 2. The core was assumed to be homogeneous with a uniform scattering length density, while the scattering length density profile of the PEG shell was allowed to be diffuse. The principle adjustable parameters in this diffuseshell model determine how the scattering length of the shell increases (or decreases) linearly from F2 at R h c to F3 at Rm before reaching the scattering length density of the medium F4 at Rt (Figure 2). The scattering length density of the shell at radius R > Rc depends on the volume fraction of PEG (φPEG) and solvent (φwater) in the layer at that radius (φPEG + φwater ) 1). Additive and multiplicative versions of this diffuse-shell model were compared. In the additive diffuse-shell model, the shell thickness is assumed to be constant, and at each core radius (Rc) in the polydispersity, Rm and Rt are given by

Rm ) Rc + δ1

(5a)

Rt ) Rc + δ1 + δ2

(5b)

It should be noted that in this model δ1 is not necessarily equal to δ2. In the multiplicative diffuse-shell model, Rm and Rt are multiples (defined by the dimensionless parameter A) of the core radius in the integration over polydispersity, which is closer to keeping the shell/core volume fixed:

Rm ) 0.5Rc(1 + A)

(6a)

Rt ) RcA

(6b)

Hence, in this model δ1 is constrained to be equal to δ2. The average values of the total micellar radius, Rt, are approximately given by eqs 5b and 6b with Rc ) R h c. For each PLA(d)-PEG system, the SANS data sets obtained at three different contrasts were fitted simultaneously to the core-shell models. In this approach, any change in the parameters for a particular data set must be reflected in the other two. Hence, this is expected to yield more representative structural parameters than would be the case if the data sets were fitted individually. The scattering length densities of the core, shell, and solvents were initially set at their calculated values (Table 2), although all were then allowed to float during the data-fitting process. The parameters of the model were adjusted to achieve the “best-fit” to the scattering data using a least-squares fitting program.29 For any given set of structural parameters, the core volume fraction defines the absolute scattering intensity. The model which best represents the internal structure of the PLA(d)-PEG assemblies must (a) be physically realistic and (b) minimize the residuals in the fit.

Figure 3. SANS (∂Σ/∂Ω versus Q) from selectively deuterated PLA(d)-PEG 3:5 nanoparticles at different solvent contrasts: (a) 100% D2O/0% H2O; (b) 80% D2O/20% H2O; (c) 65% D2O/35% H2O (vertically displaced). The continuous lines are a simultaneous fit of all three data sets to the additive diffuse-shell model (parameters in Table 3).

Figure 4. SANS (∂Σ/∂Ω versus Q) from selectively deuterated PLA(d)-PEG 15:5 nanoparticles at different solvent contrasts: (a) 100% D2O/0% H2O; (b) 80% D2O/20% H2O; (c) 65% D2O/35% H2O (vertically displaced). The continuous lines are a simultaneous fit of all three data sets to the multiplicative diffuse-shell model (parameters in Table 3).

Figure 5. SANS (∂Σ/∂Ω versus Q) from selectively deuterated PLA(d)-PEG 45:5 nanoparticles at different solvent contrasts: (a) 100% D2O/0% H2O; (b) 80% D2O/20% H2O; (c) 75% D2O/25% H2O (vertically displaced). The continuous lines are a simultaneous fit of all three data sets to the multiplicative diffuse-shell model (parameters in Table 3).

Results and Discussion SANS Scattering Cross Sections. Figures 3-5 show the fully reduced neutron scattering data (∂Σ/∂Ω versus Q) for each of the PLA(d)-PEG nanoparticle dispersions, (29) Heenan, R. K. FISH Data Analysis Program; Rutherford Appleton Laboratory Report RAL-89-129; 1989.

at three different solvent contrasts (H2O/D2O ratios). The form and magnitude of the scattering cross sections of the two smaller PLA(d)-PEG 3:5 (Figure 3) and 15:5 (Figure 4) assemblies are clearly dependent on the contrast. With an aqueous medium of 100% D2O, the scattering length densities of the PLA(d) core (6.0 × 1010 cm-2) and medium

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(6.4 × 1010 cm-2) are such that the core is close to being at contrast match. Therefore, there is minimal scattering from the PLA(d) core, and the scattering is predominantly from the PEG corona (Figure 3a). The observed scattering data are thus similar to those displayed by terminally grafted or adsorbed polymer chains: I(Q) ≈ Q-2 exp(-σ2Q2).30 When diluted with H2O in order to vary the relative amount of core and shell scattering included in eq 4, two of the samples studied fortuitously gave peaks at low Q in the SANS data. Though the presence of a peak in scattering cross sections often indicates an interparticle structure factor, S(Q),31 this is not the case here, as an interparticle S(Q) peak would be present at all scattering contrasts and not only appear as the contrast is varied. Furthermore, the peaks appear at Q values that are lower than would be consistent with an S(Q) for close packed particles of this size (i.e. 2π/[particle diameter]). Instead, peaks are also known to occur when the volumes of the core and shell are comparable and the scattering length differences in eq 4 are of opposite sign.32 When F4 in Figure 2 equals the volume weighted mean of F1, F2, and F3, then on a long distance scale (small Q) the SANS signal tends to zero, while at shorter distances (higher Q) the shell structure is still visible. This intramolecular interference or cancellation between the terms in eq 4 is very sensitive to the composition, structure, and polydispersity of the particles. More usually, for large radius cores and/or thin shells, the point at which the solvent scattering density matches the volume average scattering density of the whole particle becomes close to the match point for the core alone. In such cases, as demonstrated by the PLA-PEG 45:5 sample here, the core term in eq 4 dominates off-contrast, and the shell structure may only produce characteristic oscillations in the SANS signal at higher Q. The continuous lines in Figures 3-5 represent the best fits obtained in the simultaneous analysis of the multiplecontrast SANS data sets, using the core-shell models described above. Successful simultaneous fitting of the data sets exhibiting interference peaks (Figures 3 and 4) is difficult to achieve and is therefore sensitive to the details of the core-shell model in a quite remarkable way. In all cases, the residuals for the fits to these diffuse-shell models were substantially lower than could be achieved if the PEG shell was constrained to be homogeneous. The values of the fitted parameters are given in Table 3. The core and shell volume fractions obtained from the absolute fit scale factors of the fits (φfit core, φshell) were in good agreeexpected expected ) on the basis ment with those expected (φcore , φshell of the solids content of the dispersions. Core-Shell Dimensions. As with the overall dimensions of the PLA-PEG nanoparticles, the mean core radius (R h c) increased with the molecular weight of the PLA(d) block (Table 3). However, the values obtained were considerably lower than the extended (all trans) lengths of the PLA(d) 3, 15, and 45 kDa chains, calculated to be 17, 82, and 223 nm, respectively, assuming that the length of a lactic acid unit ( three bonds) is approximately 4 Å.12 This suggests that the PLA(d) chains adopt a fairly collapsed configuration within the core, which is typical of diblock copolymer micelles.33-36 However, the PLA(d) chains of the PLA(d)-PEG 3:5 micellar-like assemblies (30) Cosgrove, T.; Heath, T. G.; Ryan, K.; Crowley, T. L. Macromolecules 1987, 20, 2879-2883. (31) Wu, G.; Chu, B.; Schneider, D. K. J. Phys. Chem. 1995, 99, 50945101. (32) Markovic, I.; Ottewill, R. H. Colloid Polym. Sci. 1986, 264, 6576.

Riley et al. Table 3. Values of Parameters Obtained from a Simultaneous Analysis of the Multiple-Contrast SANS Data Obtained from PLA(d)-PEG Nanoparticles, Using a Core-Shell Model Incorporating a Diffuse Shella PLA(d)-PEG shell type Fc/1010 cm-2 R h c/nm σ/R hc ≈R h m/nm ≈R h t/nm δ1/nm δt/nm φPEG at R hc φPEG at Rm Nagg/nm2 (Sc/Nagg)/nm2 (St/Nagg)/nm2 φexpected core φfit core φexpected shell φfit shell

3:5 additive 6.16 5.8 0.28 9.3 15.8 3.6 10.1 0.43 0.075 242 1.9 13 0.0057 0.0058 0.0102 0.0061

15:5 multiplicative 5.98 11.0 0.27 14.4 17.7 3.35 6.7 0.18 0.11 341 4.8 12 0.0125 0.0165 0.0046 0.0044

45:5 multiplicative 6.05 13.8 0.52 23.6 9.7 0.18 393 7.8 23 0.0119 0.0143 0.0016 0.0018

a The actual fits are shown graphically in Figures 3-5 and 7. Description of the parameters: Fc ) scattering length density of the PLA(d) core; R h c ) mean core radius; σ/R h c ) polydispersity of the core radius; Rm ) shell midradius; Rt ) total micellar radius; δt ) δ1 + δ2 ) thickness of the PEG shell; φPEG ) volume fraction of PEG in the shell; Nagg ) micellar aggregation number; Sc/Naggand St/Nagg ) surface area per PEG chain at the core/shell interface expected and the periphery of the shell, respectively; φcore/shell ) expected core/shell volume fraction (calculated from the dispersion concenfit tration); φcore/shell ) core/shell volume fraction obtained from the absolute scale factor of the fit.

appeared to be more linearly extended (34% of extended length) than those of the 15:5 (13% of extended length) or 45:5 (11% of extended length) systems. The free energy of a block copolymer micelle is essentially a balance between the elastic deformation of the core chains, the osmotic energy of the shell chains, and the interfacial tension between the core chains and the solvent.33-36 The relative contribution of these three components to the total free energy of the assembly is dependent on the number of chemical repeat units in the core (Ncore) and shell (Nshell) blocks. Micellar assemblies formed by association of block copolymers with Ncore , Nshell15/11 have been described by star models, where the external radius of the shell is much larger than the core size.33,35 On the basis of the copolymer geometry, the PLA(d)-PEG 3:5 assemblies should fall into this category, with the osmotic contribution of the shell dominating. In this case, the linear extension of the relatively short PLA(d) chains is determined by how close PEG stabilizing moieties can approach one another. On increasing the molecular weight of the PLA(d) block to 15 or 45 kDa, the increased number of hydrophobic interactions between the PLA chains draws the PEG chains closer together and the PLA(d) blocks are in a more collapsed state relative to that of the PLA(d)-PEG 3:5 system. The core polydispersities of all three systems and, in particular, the PLA-PEG 45:5 assemblies were high (σ/ R h c > 0.2), corresponding to broad Schultz core-size distributions.37 The polydispersity of the nanoparticles smears some of the features in the diffraction patterns. However, by including polydispersity in the models used, (33) Izzo, D.; Marques, C. M. Macromolecules 1993, 26, 7189-7194. (34) Tuzar, Z.; Kratochvil, P. In Surface and Colloid Science; Matijevic, E., Ed.; Plenum: New York, 1993; Vol. 15. (35) Halperin, A. Macromolecules 1987, 20, 2943-2946. (36) Noolandi, J.; Hong, M. H. Macromolecules 1983, 16, 1443-1456. (37) Eastoe, J.; Dong, J. F.; Hetherington, K. J.; Steytler, D.; Heenan, R. K. J. Chem. Soc., Faraday Trans. 1996, 92, 65-72.

Core-Shell Structure of Nanoparticles

Figure 6. Schematic representation of the internal structure of the PLA(d)-PEG assemblies.

this was taken into account, such that the average shell structure was determined across the distribution of core sizes. The mean core sizes of the systems studied were sufficiently different that the dependency of shell structure on particle size could be elucidated. The additive version of the diffuse-shell model with a core-size independent PEG/solvent layer thickness fixed at δt ) 10.1 nm was best suited to fitting the PLA(d)-PEG 3:5 scattering data (eqs 5). This again implies that for the micellar-like PLA(d)-PEG 3:5 assemblies, where NPLA < NPEG, the molecular weight of the shell-forming block independently determines the structure of the shell. In contrast, analysis of the PLA(d)-PEG 15:5 and 45:5 scattering data required the multiplicative version of the diffuse-shell model (eqs 6), suggesting that when NPLA > NPEG, the core-forming block has a stronger influence on the structure of the shell, with larger cores in the population having correspondingly thicker PEG layers. The average thickness of the multiplicative PEG shell for the PLA(d)-PEG 45:5 assemblies was found to be slightly greater than that of the PLA(d)-PEG 15:5 assemblies (Table 3). The average total radius of the PLA(d)-PEG 3:5 assemblies obtained by SANS (R h t, Table 3) was in good agreement with the PCS-determined hydrodynamic radius (Rhyd, Table 2). However, as the molecular weight of the PLA(d) block was increased, so R h t decreased relative to Rhyd. It is suggested that dispersions of the larger PLA(d)-PEG assemblies, in particular the highly polydisperse PLA(d)-PEG 45:5 system, contain small subpopulations of larger aggregates that the PCS technique is sensitive to. The average aggregation number, Nagg, of the PLA(d)PEG assemblies was obtained using the molar volume of the PLA(d)-PEG chains and numerically integrating over the fitted polydispersity. These values were then used in conjunction with the surface areas of the core (Sc ) 4πR h cc2) and the total particle (St ) 4πR h t2), again summed over the polydispersity, to obtain an estimate of the surface area available per PEG chain at the core/shell interface (Sc/ Nagg) and at the periphery of the shell (St/Nagg), as illustrated in Figure 6. As shown in Table 3, the surface area available per PEG chain at the periphery of the shell of the PLA(d)-PEG 45:5 system was considerably higher than that for the PLA(d)-PEG 3:5 and 15:5 assemblies. This is likely to be the root cause of this system’s comparatively poor colloidal stability (Table 2). Similarly, the area available per PEG chain at the core/shell interface increases with the molecular weight of the PLA block. A reported study of the adsorption behavior of PLA(d)-PEG 2:5 copolymers onto the surface of 170 nm polystyrene particles found that each PEG chain occupied an average surface area of 4.5 nm2 at the surface of the particle.38 This is approximately the same as the area per PEG chain at the core surface of PLA(d)-PEG 15:5 assemblies, while

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the 3:5 and 45:5 structures would appear to have higher and lower grafting densities, respectively. In all cases, however, the surface area available to each PEG chain is considerably lower than that required for the 5 kDa chains to be considered isolated from one another; that is, Sc > πRg2 ≈ 30 nm2 (radius of gyration of the PEG 5 kDa chains, Rg ) 3.1 nm, calculated according to ref 39). This implies that the PEG chains in the PLA(d)-PEG assemblies are likely to interact and adopt a brushlike configuration.21,40 De Gennes scaling theory predicts that the layer thickness of a grafted polymer brush should exhibit an inverse dependence on the distance between grafting points.40 This is indeed the case here, with the PLA(d)PEG 3:5 assemblies having the highest PEG chain grafting density and correspondingly thickest PEG layer. The span of the solvated PEG chains is also the highest for the assembly with the smallest core The surface curvature at the PLA/PEG interface is higher for the PLA(d)-PEG 3:5 nanoparticles than for either of the other two systems studied, and therefore, the conical region to which the PEG chains are confined expands rapidly with radial distance from the core surface. Volume Fraction Profiles. For a particle of large radius, presenting an effectively flat interface, the volume fraction profiles, φ(R), of adsorbed or grafted polymer layers may be determined by a mathematical transformation (“inversion”) of the SANS data obtained from the shell only, that is, with the particle core at contrast match.16-18,41 Such methods are not appropriate for the systems studied here due to the small particle radius compared to the layer thickness and the lack of an experimental method to obtain the neutron scattering length density of the PLA(d) core. In an alternative approach, structural information obtained from the core-diffuse shell analysis enabled the construction of volume fraction profiles for the PLA(d)PEG nanoparticles which are shown in Figure 7. Eastoe et al.33,42,43 have also used this method of analyzing multiple-contrast SANS data, in a study of the internal structure of water-in-oil microemulsions containing dichained surfactants. The main intent of the two-linearshell model used here was to detect any possible maxima in the PEG volume fraction profiles (i.e. a “mushroom” profile40) using a model with a minimal number of adjustable parameters. The accuracy of the structural information obtained was ensured by fitting to multiplecontrast SANS data sets, in that for each system only a unique set of parameter values generated the contrastdependent interference peaks observed. The use of models incorporating a different power law for the profile did not improve the fit. For all three PLA(d)-PEG systems studied, the scattering length density of the cores (Table 3) suggested that their bulk density approached that of solid PLA(d). In contrast, the PEG shells were found to be relatively solvent rich, as has also been reported for PEO13-PPO30-PEO13 block copolymer micelles.44,45 (38) Stolnik, S.; Felumb, N. C.; Heald, C. R.; Garnett, M. C.; Illum, L.; Davis, S. S. Colloids Surf., A: Physicochem. Eng. Aspects 1997, 122, 151-159. (39) Devanand, K.; Selser, J. C. Macromolecules 1991, 24, 59435947. (40) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman & Hall: U.K., 1993. (41) Cosgrove, T. J. Chem. Soc., Faraday Trans. 1990, 86, 13231332. (42) Eastoe, J.; Hetherington, K. J.; Sharpe, D.; Dong, J. F.; Hetherington, G. J.; Heenan, R. K.; Steytler, D. Langmuir 1996, 12, 3876-3880. (43) Eastoe, J.; Hetherington, K. J.; Sharpe, D.; Dong, J. F.; Hetherington, G. J.; Heenan, R. K.; Steytler, D. Colloids Surf., A: Physicochem. Eng. Aspects 1997, 128, 209-215.

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Riley et al.

grafted to the surface of polystyrene nanoparticles has a distinct maximum away from the surface. Although no such maxima were observed here (the two PEG shells in the model would allow a maximum to develop in the fitting process), it is possible that the core-shell analysis would be insensitive to the presence of a relatively thin depleted layer near the core surface. The general form of the PLA(d)-PEG 3:5 volume fraction profile, although constrained by the model, is similar to that predicted for AB block copolymer micelles. Using Scheutjens-Fleer mean-field theory in a spherical lattice geometry, Leermakers et al.21 found that if the curvature of the core is significant, then the volume fraction profile of the buoy block consists of two distinct regions: (i) a power-law part which decays steeply due to the high curvature near the core/shell interface and (ii) a parabolic profile at the periphery of the brush. The two regions identified in the PEG volume fraction of the PLA(d)-PEG 3:5 assembly appear to mimic these theoretical profiles. The PEG volume fraction diminishes rapidly away from the core surface, as the space available to each chain increases dramatically with radial distance. In contrast, at the periphery of the brush, where the curvature is reduced, the volume fraction profile decays less abruptly. The decrease in PEG chain grafting density as the molecular weight of the PLA(d) block was increased to 15 kDa (Table 3) manifested itself as a reduction in the PEG volume fraction near the core, as shown in Figure 7b. Furthermore, with a reduced curvature in the brush, the PEG volume fraction profile decayed less steeply away from the core surface than that for the PLA(d)-PEG 3:5 assemblies, and consequently the shell appeared to be more radially homogeneous. This is again in accord with the Scheutjens-Fleer mean-field theory predictions, which demonstrated that on increasing the core size of a polymeric micelle the parabolic part of the profile becomes larger at the expense of the steeply decaying power-law region.21 Only one region could be identified in the PEG volume fraction profile of the PLA(d)-PEG 45:5 nanoparticles shown in Figure 7c, and the shell appeared to be even less splayed. Although the PEG chain grafting density of this system is less than that of the PLA(d)-PEG 15:5 assemblies, the surface of the core is relatively flat, and so the volume accessible to each PEG chain does not increase significantly away from the core surface. Consequently, the brushlike PEG chains are constrained to extend further away from the core surface to avoid unfavorable overlap, with an increase in the average shell thickness (Table 3). Figure 7. Volume fraction profiles of PLA(d)-PEG nanoparticles, at the mean core size of the core Rc ) R h c: (a) PLA(d)PEG 3:5; (b) PLA(d)-PEG 15:5; (c) PLA(d)-PEG 45:5.

In the case of the PLA(d)-PEG 3:5 nanoparticles, the PEG layer was found to be fairly diffuse, with a high PEG volume fraction at the core/shell interface (φPEG ) 0.4 at R h c) which decayed steeply, reaching 0.08 at a distance 3.6 nm away from the surface of the core (Figure 7a). In addition, there were some highly extended PEG tails stretched out to approximately 10 nm. By direct inversion of SANS data, Cosgrove and Ryan17 have shown that the volume fraction profile of PEG 5 kDa chains terminally (44) Wu, G.; Chu, B.; Schneider, D. K. J. Phys. Chem. 1995, 99, 50945101. (45) Chu, B. Structure and dynamics of block copolymer colloids. Langmuir 1995, 11, 414-421.

Conclusions Using SANS, it was possible to probe the internal structure of nanoparticles assembled from PLA(d)-PEG copolymers, with a fully deuterated core-forming PLA(d) block. Scattering cross sections were obtained at different contrasts, with prominent form factor interference peaks observable when the PLA(d) cores of the PLA(d)-PEG 3:5 and 15:5 nanoparticles were not contrast matched to the dispersion medium. Simultaneous analysis of the multiple-contrast SANS data sets using core-shell models enabled the construction of volume fraction profiles for the PLA(d)-PEG assemblies. As anticipated, the mean core radius increased with the molecular weight of the PLA(d) block. The shells of all the PLA(d)-PEG assemblies were very solvent (water) rich. However, differences in the volume fraction profiles suggested that the conformation of the stabilizing chains

Core-Shell Structure of Nanoparticles

was dependent on the length of the PLA(d) block. The highly splayed PEG chains of the PLA(d)-PEG 3:5 assemblies were characteristic of polymeric micelles with a small core size. On increasing the molecular weight of the PLA(d) block, the PEG chain grafting density decreased but the shell became more radially homogeneous, a consequence of the reduced curvature in the brush. In summary, SANS has provided rich structural information about the PLA(d)-PEG assemblies, not previously accessible by other complimentary techniques. The differences in particle size and the structure of the PEG layer are likely to have a profound influence on the

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biological behavior of these systems. This is to be investigated. Acknowledgment. The authors are grateful to the EPSRC and CLRC for the provision of the neutron scattering facilities. Funds to purchase the deuterated monomers for these experiments were provided by AstraZeneca Pharmaceuticals as part of a DTI Link Scheme (GR/J57889). T.R. is grateful to the BBSRC for provision of a postgraduate studentship and would like to thank Prof. Th.F. Tadros for many helpful discussions. LA020911H