Characterization of Variant Soft Nanoparticle Structure and

Article pubs.acs.org/JPCC

Characterization of Variant Soft Nanoparticle Structure and Morphology in Solution by NMR Spectroscopy Muzaddid Sarker,† Robin E. Fraser,§ Michael D. Lumsden,‡ Darren J. Anderson,§ and Jan K. Rainey*,†,‡ †

Department of Biochemistry & Molecular Biology and ‡Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada § Vive Crop Protection Inc., Toronto, Ontario M5G 1Z6, Canada S Supporting Information *

ABSTRACT: The physicochemical properties of soft nanoparticles, most notably size, morphology, and ligand interactions, typically depend upon the solution environment in which they are suspended. Comprehensive characterization in a given environment is therefore essential. We have employed high-resolution solution nuclear magnetic resonance (NMR) spectroscopy, nuclear spin relaxation measurements, and diffusion ordered NMR spectroscopy (DOSY) techniques to thoroughly characterize polymeric nanoparticles formed by salt-induced collapse of a methacrylic acid−ethyl acrylate copolymer stabilized by ultraviolet (UV) irradiation. UV-dosedependent production of new chemical species is apparent from 1H and 13C chemical shift patterns. 1H−13C correlation spectroscopy reveals that cross-linking is likely responsible for nanoparticle structural integrity. Paramagnetic relaxation enhancement (PRE) unambiguously shows protection of photochemically derived moieties from solvent, with a UV-dosedependent decrease in particle size. Temperature-dependent swelling and solvent-induced contraction demonstrate that increased UV dose leads to an increase in the proportion of compact, solvent protected particle core relative to more dynamic, solvent accessible shell. Correlation of disparate solution-state NMR observables allowed for these conclusions and is readily generalizable to the in situ characterization of the exact state of a wide variety of soft nanoparticles as a function of environment.

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INTRODUCTION The robust physical and chemical characterization of nanoparticles is key for their optimization. Nanoparticles can be generally classified as rigid or soft, with soft nanoparticles susceptible to change in shape and/or component distribution.1 Soft nanoparticles are typically polymeric in composition. Since these particles are often biocompatible and biodegradable, they are a popular vehicle for delivering active ingredients such as drugs.2,3 However, their malleability makes characterization by techniques such as electron microscopy or atomic force microscopy difficult, since the conformation observed upon substrate deposition for microscopy may not be representative of that found in solvent. Nuclear magnetic resonance (NMR) spectroscopy provides the unique capability to characterize particle structure, dynamics, hydrodynamic properties, and interactions from the atomic to supramolecular levels in solution. While the physicochemical description of a nanoparticle system usually entails a correlative analysis obtained by comparative measurement and characterization from an array of techniques, NMR possesses the potential to provide an all-inclusive, general toolkit for biofunctionalized soft nanoparticle characterization.4 Although the scope for NMR characterization of metallic nanoparticles may be inherently limited, NMR can comprehensively probe the surface chemistry of colloidal nanocrystals and their ligand interactions.5 © 2015 American Chemical Society

Nanoparticles may be likened to macromolecules in terms of their physicochemical behavior.6 There is a rich history of studying biomolecular systems of similar nanoscale dimensions (e.g., proteins, nucleic acids, carbohydrates, and lipid complexes) using high-resolution solution-state NMR spectroscopy, and many of the techniques employed are increasingly being applied in nanoparticle characterization. Historically, characterization of nanoparticles has mainly relied upon application of solid-state NMR methodology,7 which is not limited by molecular mass (or supramolecular assembly size) or on use of a nonexhaustive subset of solution-state NMR experiments. Soft nanoparticle solution-state NMR studies have included 1H NMR testing of functional group deprotection during nanoparticle synthesis8 and differentiation between immobile core vs mobile shell moieties.9−11 1H based, 1 H−13C correlation, and nuclear spin relaxation NMR experiments have been used to examine density of shell moieties on a lipid core.12 Investigations of nanoparticle structure, size, and cargo loading mechanism have been performed using nuclear spin relaxation and/or pulsed field gradient (PFG) diffusion NMR experiments.13,14 PFG diffusion NMR has also been validated as an alternative to electron microscopy for highly Received: January 5, 2015 Revised: March 10, 2015 Published: March 11, 2015 7461

DOI: 10.1021/acs.jpcc.5b00097 J. Phys. Chem. C 2015, 119, 7461−7471

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The Journal of Physical Chemistry C accurate rigid metal nanoparticle sizing.15 Although some recent reviews on nanoparticle characterization either ignore NMR altogether16,17 or even go so far as to state that it is inapplicable to nanoparticles formed following polymer collapse,18 NMR spectroscopy has certainly been widely applied for the characterization of nanoparticle systems, at least in the past decade.4 Natural abundance studies of nuclei with spin-1/2 in soft polymeric nanoparticle systems, most notably 1H (near 100% natural abundance) and 13C (∼1.1% natural abundance), make them amenable to high-resolution studies by NMR. NMR can be employed to accurately characterize the most investigated physicochemical properties, including size, shape, composition, morphology, accessibility, and surface behavior. Here, we demonstrate the application of solution-state, relaxation, and diffusion NMR techniques to comprehensively differentiate and characterize two nanoparticle species photochemically produced from the same starting copolymer with different doses of ultraviolet (UV) irradiation. Through examination of temperature- and solvent-dependent behavior, the hydrodynamics and core−shell morphology of each of these porous nanoparticle species are demonstrated to differ dramatically. The experimental approach we have used is readily applicable and generalizable to other traditionally challenging to characterize soft nanoparticles.

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EXPERIMENTAL METHODS Nanoparticle Production. Allosperse nanoparticles (NPs; Vive Crop Protection, Toronto, ON, Canada) are being explored as a potential delivery system for crop-protection agents. In this study, two types of Allosperse NPs produced from a copolymer (the “starting polymer”, or SP) of methacrylic acid (MAA) and ethyl acrylate (EA) (90.9:9.1 molar ratio of MAA to EA; Apic Laboratories, Suzhou, Jiangsu, P.R. China; Figure 1A) were employed. These NPs were produced by collapsing SP species into a coil configuration by addition of NaCl (0.5 M), followed by exposure to UV irradiation (254 nm) at pH > 6, and subsequent removal of salt by diafiltration (NF270-4040 Cross-flow Membrane, Dow, Midland, MI). As UV treatment is both critical for retention of NP properties following the dialysis step and affects NP properties, NPs produced with two different UV doses were characterized in this study. These are referred to as NP(1xUV) and NP(4xUV), which indicate NPs produced using a dose of 4.98 kJ/g standard to Allosperse preparation and a roughly quadruple dose of 18.52 kJ/g during synthesis. Titration, Gel Permeation Chromatography, and Viscometry. Apparent carboxylic acid (COOH) contents of each NP type were determined by titration against NaOH. The apparent molecular weight (Mw) and polydispersity index (PdI) of each sample were determined by gel permeation chromatography (GPC; Viscotek gel permeation chromatograph, Malvern, Westborough, MA; G4000PWXL and G2500PWXL columns, Tosoh Bioscience, King of Prussia, PA; PMAA standards, Sigma-Aldrich, Oakville, ON, for calibration curve). Calibration curves for all PMAA-EA materials were constructed employing a pure PMAA standard as the closest approximation in the absence of any copolymer standard. Viscosities of SP and NP(1xUV) samples were measured using an SV-10 Series Sine-wave Vibro viscometer (A&D, Tokyo, Japan). NMR Sample Preparation. NMR samples of the SP and both NP types were prepared in D2O at three different

Figure 1. (A) Chemical structures of SP constituents MAA and EA (without stereochemical consideration, ∼9:1 MAA:EA molar ratio). (B) 1D 1H (bottom) and 1D 1H TOCSY (top; irradiation frequency indicated by an asterisk) spectra. (C) Overlaid 2D 1H−13C HSQC spectra of indicated SP (20 mg/mL) at 22 °C. 7462


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The Journal of Physical Chemistry C Table 1. NMR Experimental Parameters Used for the SP and NP Samples experimenta

spectrometer (1H strength, MHz)

pulse programb

no. of scans

recycle delay (s)

no. of increments/points

500 500 500 500 700 700 500 300 500 300 500

zgesgp selmlgp udeft hsqcetgpsisp2 hsqcetgpsp.2 hmbcgplpndqf t1ir t1ir cpmgh cpmg dstebpgp3sj

64−256 32 4000 256 64 64 64 32−48 64 16−32 32−256

2 2 4 2 1 1 5−10 2 5 2 2

64 256 256 16 16 16 16 16−32

1

1D H 1D 1H TOCSYc 13 C UDEFTd 2D 1H−13C HSQCd 2D 1H−13C HMBCd,e T1 relaxationf T2 relaxationg 2D 1H DOSYi a

AbbreviationsTOCSY: total correlation spectroscopy; UDEFT: uniform driven equilibrium Fourier transform; HSQC: heteronuclear single quantum coherence; HMBC: heteronuclear multiple bond correlation; DOSY: diffusion ordered spectroscopy. bStandard pulse program included in Bruker catalogue. cTOCSY mixing time: 50 ms. dPerformed at natural 13C abundance. eLong-range coupling evolution delays: 60 and 90 ms. f Inversion−recovery delays: 10, 20, 40, 70, 110, 150, 200, 250, 300, 400, 500, 600, 800, 1000, 1500, and 2000 ms. gCPMG delays: 2, 4, 6, 10, 14, 20, 28, 36, 46, 58, 72, 88, 106, 130, 160, and 200 ms. hIn-house modification performed to incorporate presaturation solvent suppression. iDetails of diffusion time, gradient pulse length, and signal attenuation are in Supporting Information Table S1. jDOSY pulse program with convection compensation.

constants R1 and R2 for dipolar relaxation of a pair of identical spin-1/2 nuclei are given by21,22

concentrations: 1, 4, and 20 mg/mL. For chemical shift assignment purposes, a pure MAA polymer sample was also prepared. Upon reconstitution in D2O, the SP material remained undissolved in the resulting acidic solution (pH ∼3). Addition of 0.225 mg of NaOH per 1 mg of SP allowed for dissolution. The same gravimetric ratio of NaOH was added to each NP sample for consistency. Each sample was sonicated (5−10 min) and stirred until the material was fully dispersed, and a clear solution was observed. For paramagnetic relaxation enhancement (PRE) experiments, MnCl2 was added at 1 mM to samples containing 20 mg/mL SP or NP. Samples of SP and NP(1xUV) at 20 mg/mL concentration were also prepared in CD3OD, in which all materials dissolved instantly. NMR Data Acquisition. NMR experiments (listed with detailed parameters in Table 1) were performed at 37 °C using an Avance 300 MHz spectrometer (Bruker Canada, Milton, ON) equipped with a 5 mm BBFO probe and a z-axis gradient, at 22 and 37 °C using an Avance 500 MHz spectrometer equipped with a 5 mm TXI probe and a z-axis gradient and at 47 °C using a Avance III 700 MHz spectrometer equipped with a 5 mm TCI probe and a z-axis gradient. The NMR data were processed and analyzed using Bruker Topspin 3.1. Nuclear Spin Relaxation Measurement and Analysis. T1 (longitudinal) and T2 (transverse) 1H spin relaxation time constants were extracted through fits to series of inversion− recovery and Carr−Purcell−Meiboom−Gill (CPMG) experiments with varying delays, respectively. The time constants were determined using Bruker Dynamics Center from the exponential fits of the observed signal intensities as a function of delay using the relationships I(t ) = I(0)(1 − 2A e−t / T1)

(1)

I(t ) = P e−t / T2

(2)

R1 =

1 3 γ 4ℏ2 = f (τC) T1 20 r 6 1

(3)

R2 =

1 3 γ 4ℏ2 = f (τC) T2 20 r 6 2

(4)

where γ is the gyromagnetic ratio, ℏ the reduced Planck constant, r is the distance between the interacting spin-1/2 nuclei, and f1 (τC) =

2τC 2

1 + ωO τC

f2 (τC) = 3τC +

2

+

8τC 1 + 4ωO2τC 2

5τC 2

1 + ωO τC

2

+

(5)

2τC 1 + 4ωO2τC 2

(6)

−1

where ωO is the Larmor frequency (in rad s ) of the nucleus in question. A measured ratio of T1/T2 can be matched by inspection to the ratio of eq 4/eq 3 to determine the corresponding value of τC;19 alternatively, Carper and Keller provide convenient polynomial approximations for τC as a function of T1/T2.20 Diffusion-Ordered Spectroscopy (DOSY). The translational diffusion coefficient (DC) of each species was determined using pulsed field gradient (PFG) NMR diffusion-ordered spectroscopy (DOSY). In DOSY experiments, the envelope of 1 H signals was attenuated to ∼3% of its initial amplitude or to the maximum achievable under instrumental limitations (attenuation levels detailed in Supporting Information Table S1) by linearly ramping up the gradient strength from 2% to 95% of the maximum (53.5 G/cm) in 16 or 32 steps. DC was determined using Bruker Dynamics Center from the exponential fit of the observed signal intensity as a function of gradient strength for each series of experiments using the Stejskal−Tanner formula:23

where I(t) is the observed signal intensity at delay time t, I(0) is the intensity of the full signal, A is a normalization factor, and P is the amplitude of transverse magnetization at zero delay. The ratio of T1/T2 can be used to estimate the rotational correlation time (τC) for the tumbling of a spin undergoing relaxation.19,20 Assuming isotropic random tumbling, the rate

2

4

I = I(0)e(−DC(2πγgδ) (Δ− δ /3)10 )

(7)

where I is the observed signal intensity, I(0) is the unattenuated signal intensity, γ is the gyromagnetic ratio of the observed 7463


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The Journal of Physical Chemistry C nucleus, g is the gradient strength, δ is the gradient pulse length (optimized for a given sample between 4 and 8 ms), and Δ is the diffusion time (optimized for a given sample over 100−500 ms). The values used for Δ and δ (Supporting Information Table S1) were automatically adjusted by Topspin or Dynamics Centre during both data acquisition and processing according to the pulse program used; a double-stimulated echo24 with bipolar gradients and convection compensation25 in this case. Particle Size Determination. Three methods of particle size determination were compared. Apparent molecular weight (Mw) from gel permeation chromatography was employed to predict the hydrodynamic diameter (dH[Mw]) for an ideal spherical conformation from the volume−diameter relationship

dH[MW ] =

3

6VMW πNA

According to GPC, a drastic, UV-dose-dependent reduction of apparent mass was observed for each NP relative to the SP (Table 2). This would be consistent with compaction of the polymeric material due to UV-induced cross-linking but also with scission.30,31 Prior to this study, no direct experimental data are available to confirm cross-linking, although microscopy and dialysis (results not shown) provided indirect support for this. Also consistent with formation of a compact cross-linked species, viscosities of D2O solutions containing NP species were identical to the D2O blank, whereas SP solutions were found to be highly viscous (Table 2; details in Supporting Information Tables S2 and S3). As detailed below, NMR spectroscopy allowed for distinguishing between these possibilities for UV-induced photochemistry. NMR Chemical Shift Assignment of the SP. Chemical shifts of SP 1H and 13C nuclei (labeled A−G; Figure 1A) were assigned in part by comparison of 1D 1H32 and 2D 1H−13C heteronuclear single quantum coherence (HSQC)33 spectra of a pure MAA polymer and the MAA+EA copolymer (Figure 1). 1D 1H total correlation spectroscopy (TOCSY)34 experiments (Figure 1B) facilitated unambiguous assignment of the EA moiety spin system (C−D and E−F, Figure 1) and the EA− MAA polymer backbone linkage derived spin system (D−A, Figure 1). The presence of multiple signals generated by TOCSY transfer between sites D and A is consistent with the subunits of MAA and EA being randomly distributed in the SP, as opposed to in a uniform block copolymer arrangement. 2D 1 H−13C HSQC experiments allowed site-specific assignment of all covalently bonded 13C−1H spin pairs (Figure 1C). Both the 1D 1H and 2D 1H−13C HSQC spectra of the SP show more than one chemical shift for almost all moieties, indicative of a random sequence distribution and/or variable tacticity of the monomer units in the polymer chains of MAA−EA copolymer. Additionally, slow conformational exchange on the NMR time scale is likely, potentially increasing the number of observed chemical shifts attributable to a given site.35 The combination of 1D 1H, 1D 1H TOCSY, and 2D 1H−13C HSQC experiments thus allowed full resonance assignment for all nonexchangeable 1 H nuclei and 13C−1H spin pairs of the SP (labeled A−F, Figure 1). Spectral Changes for NP Samples. 1D 1H and 2D 1 H−13C HSQC spectra demonstrate characteristic changes in chemical shift patterns for NPs vs SP (Figure 2). Specifically, a number of new 1H−13C correlations are observed near the cross-peaks corresponding to sites B, C, and D, while few spectral changes are seen near those of sites A, E, and F. Many of these peaks appear quite narrow in comparison to the SP spectrum, and quadrupling of the UV intensity causes an increase in these new peaks compared to the single dose treatment (Figure 2). Unfortunately, the extensive degree of 1H overlap for the SP (as is very evident in the 2D 1H−13C HSQC) is only exacerbated with the formation of new moieties upon UV-induced NP formation. Notably, comparison of relative intensities of peaks arising specifically from either MAA or EA (i.e., those corresponding to methyl groups B and F, respectively) indicates that the ratio of MAA to EA subunits in each class of NP is not modified relative to SP (Figure 2 and Supporting Information Table S2). Photochemical Modifications in NPs. The fact that the MAA:EA stoichiometry of the NP species relative to the SP is unchanged implies that UV-treatment is not causing specific decarboxylation or scission reactions. 2D 1H−13C heteronuclear

(8) 3

−1

where V is the specific volume (0.992 cm g ; calculated for 90.9:9.1 molar ratio of MAA (density 1.015 g/cm3) and EA (density 0.9405 g/cm3) of the SP), MW is the molecular weight (g mol−1), and NA is Avogadro’s number. Based upon τc, as may be estimated on the basis of the ratio of the T1 to T2 relaxation time constants (or, eq 4/eq 3), Stokes’ law for an isotropic sphere was employed for obtaining the second estimate of the hydrodynamic diameter (dH[τC]):26,27

dH[τC] =

3

6kBTτC πη

(9)

where η is the solution viscosity (Supporting Information Tables S2 and S3), kB is the Boltzmann constant, and T is the absolute temperature. Finally, based upon the observed translational diffusion coefficient (DC) from DOSY, the Stokes−Einstein equation was employed for calculating the experimental hydrodynamic diameter (dH[DC]):27 dH[DC] =

■

kBT 3πηDC

(10)

RESULTS AND DISCUSSION Physicochemical Comparison of SP to NP. Clear differences in SP and NP solutions were evident. First, the apparent COOH content of NP samples vs the SP, as determined by titration against NaOH, showed a UV dosedependent decrease for the NPs (Table 2). Without additional experimental characterization, this could be taken as evidence of UV-induced decarboxylation. While photochemical decarboxylation is perfectly plausible,28,29 an alternative explanation is the rendering of some population of COOH groups inaccessible to reaction. Table 2. Comparison of Apparent COOH Content, Polydispersity Index (PdI), Apparent Molecular Weight (MW), and Measured Viscosity (η; at 20 mg/mL and 22 °C) of SP to Allosperse NPs sample SP NP(1xUV) NP(4xUV)

UV dose (kJ/g)

COOH content (mmol/g)

PdI

MW (Da)

η (cP)

4.98 18.52

9.9 9.7 8.5

1.74 2.73 6.15

86 588 21 383 12 055

64.70 1.26 1.26 7464


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The Journal of Physical Chemistry C

expected in the SP. Further long-range 1H correlations are observed for both the alkene and N1 13C species at 1.86 and 2.23 ppm. The three other major UV-induced CH species, N2− N4, with 1H chemical shifts between 1.37 and 1.56 ppm, all correlate to a new 13C species (13C chemical shift 84.5 ppm; Figure 3B) in the HMBC of the NPs. According to chemical shift, this species is consistent with an alkyne or R3C−O functionality but cannot be better defined at present due to lack of further correlations. Taken together, these 2D 1H−13C spectra allow development of an NMR-based model for photochemical modification of the SP chemical structure leading to the formation of NP species. This NMR-based NP structural model relies on the following three sets of observations. First, comparison of HSQC spectra of NP formed from pure MAA SP vs from MAA+EA copolymer SP demonstrates that the UV-induced spin-systems detailed above arise only when EA is present (Supporting Information Figure S1). Therefore, the origin of these new moieties is likely arising from photochemical modification to EA subunits. Second, sites C and D of the EA subunits are substantially affected by the UV irradiation while sites E and F are not affected to any noticeable extent (Figure 2B). E and F also do not show any correlations to the HMBC propagation patterns of the new N1−N4 signals. Thus, it is clear that sites C and D are photochemically modified while sites E and F are not. Third, the carbonyl C at site Q3 is modified. This is demonstrated by the fact that correlation to site E in the HMBC, observed weakly for the SP, is drastically reduced in the NP species (Figure 3A,B). In theory, the C(Q3)/H(E) three-bond correlation should be observed in the HMBC for all SP/NP species. However, this signal is weak even for the SP, possibly due to inefficient three-bond magnetization transfer at two tested long-range coupling evolution delays of 60 and 90 ms. The signal becomes much weaker for the NPs, indicating a change in the local chemical environment surrounding a Q3 subpopulation with further decrease of magnetization transfer efficiency. We therefore conclude that the chemical structure of the UV-modified EA subpopulation is such that site C is the origin of the new signal N1 and site D lacks an attached H and is double-bonded to carbon Q3, with conversion to a singly bonded C−O species (Figure 3C). This modified EA would then cross-link to another subunit through ether formation, inducing the collapsed NP structure. Signals N2−N4 correspond to sites A/C of the previous/next subunits. It is clear that not all EA units are photochemically modified in this way, since site D is still observed at reduced intensity (correlated to UV exposure) in the HSQC (Figures 2 and 3). Since the observed stoichiometries and chemical shifts of other moieties are generally unchanged, we believe that the primary mechanism for UV-induced nanoparticle stabilization is cross-linking through the EA carbonyl moiety in a preferential manner. Variability in Solvent Accessibility. We employed PREbased attenuation of NMR signals38 to probe the solvent accessibility of each class of moiety within SP/NPs. The Mn2+ ion was employed as a paramagnetic agent due to its five unpaired 3d electrons. Mn2+ partitions to the aqueous phase, thus attenuating NMR signals through enhanced transverse relaxation at sites that are readily solvent accessible while sites protected from solvent are correspondingly protected from signal attenuation.39 All SP NMR signals disappear upon addition of Mn2+, indicating that all SP sites are fully solvent accessible (Figure 4A). In contrast, both NPs retain significant NMR signal in the presence of Mn2+ in a site-specific manner.

Figure 2. 1D 1H (A) and 2D 1H−13C HSQC (B; overlaid) spectra of indicated species (20 mg/mL) at 22 °C.

multiple bond correlation (HMBC)36 experiments provide significant insight into the nature of photochemical modifications to the SP structure. It should be noted that, to reduce effects of transverse relaxation,37 the HMBC experiments, alongside HSQC experiments to control for temperaturedependent chemical shift variation, were performed at an elevated temperature of 47 °C. Four distinct UV-induced 1 H−13C HSQC cross-peaks indicative of new CH bonds, labeled N 1 −N 4 , have 1 H moieties with clear HMBC correlations to nonprotonated 13C nuclei that are not present in the SP (Figure 3, A vs B). N1 (1H chemical shift 1.68 ppm, 13 C shift 23.5 ppm) is consistent with a CH2 moiety. In the HMBC, N1 correlates to a pair of 13C nuclei (13C chemical shifts of 113.5 and 144.3 ppm; Figure 3, A vs B) consistent with a fully substituted double bond that is neither observed nor 7465


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Figure 3. (A, B) Overlaid 2D 1H−13C HSQC and HMBC spectra of indicated species (20 mg/mL) at 47 °C. Long-range HMBC correlations to neighboring A or C moieties are indicated by A′/C′. (C) Proposed photochemical modifications to the chemical structure of a subpopulation of EA implicating cross-linking in the production of NPs from the SP.

Figure 4. Overlaid 2D 1H−13C HSQC spectra, with and without 1 mM MnCl2, of indicated species (20 mg/mL) at 22 °C.

the nanoparticles to the interior. The lack of solvent exposure for a significant proportion of the NP species also helps to rationalize the apparent decrease of COOH content in the NPs vs the SP (Table 2), likely arising from the lower accessibility of these moieties that are inside the NPs rather than from a UVinduced decarboxylation process. 1 H Spin Relaxation Analysis. Despite clear differences in solvent accessibility between the SP and the NPs, the 1D 1H and 2D 1H−13C HSQC spectra demonstrate unambiguously, and somewhat counterintuitively, that chemical shifts and peak intensities are not dramatically different between the SP and the NPs. This implies strong similarity in local chemical environment between the SP and bulk NP materials, despite sequestration of some species away from solvent and production of new chemical moieties. Features other than

Strikingly, sites E and F from the ethyl moiety of the EA component are largely protected and the photochemically derived species are almost fully protected from Mn2+ (Figure 4B,C). Corresponding to the increased presence of photochemically derived moieties, NP(4xUV) shows greater protection from PRE than NP(1xUV). The observed differences in PRE imply a compact solventexcluded core in both NP species, made up preferentially of the most hydrophobic moiety of the SP and of the photochemically derived species. This is logical based upon the fact that chemical modification appears to preferentially take place in EA (Figure 3). Despite this compactness, however, solvent can still access the interior of the NPs to a degree; hence, it is plausible that the NPs are porous core−shell structures or, more likely, are a network structure with varying density from the surface of 7466


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Table 3. 1H T1 and T2 Relaxation Time Constants for Sites A−F (Figure 1; 1H Spin Type Indicated in Parentheses) at 500 and 300 MHz, Their Ratio, and the Corresponding Estimated Rotational Correlation Time (τC) of the SP and the NPs (20 mg/mL Samples) at 22 °C 500 MHz sample SP

NP (1xUV)

NP (4xUV)

site A, C (−H2) B (−H3) D (−H) E (−H2) F (−H3) A, C (−H2) B (−H3) D (−H) E (−H2) F (−H3) A, C (−H2) B (−H3) D (−H) E (−H2) F (−H3)

T1 (ms) 596 411 530 1200 958 548 367 403 882 1000 328 230 315 908 704

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17 9 34 74 19 20 11 62 151 10 10 8 17 42 21

300 MHz

T2 (ms)

T1/T2

τC (ns)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

45.5 15.6 15.4 40.8 5.9 29.9 11.3 9.5 12.9 6.3 11.3 5.9 6.1 11.3 4.3

2.43 1.36 1.35 2.29 0.75 1.95 1.13 1.02 1.22 0.78 1.13 0.75 0.77 1.13 0.59

13.1 26.3 34.4 29.4 162 18.3 32.6 42.6 68.2 159 29.1 38.9 52.0 80.6 164

0.8 1.5 12.2 7.1 42 1.4 1.5 13.2 15.6 46 3.5 2.6 14.6 23.4 46

a

T1 (ms) 284 183 310 783 614 261 159 254 1020 570 234 113 153 1040 433

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

18 15 20 48 21 17 12 36 25 24 18 9 43 41 26

T2 (ms)

T1/T2

τCa (ns)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

30.2 8.0 17.1 15.5 4.0 13.2 4.5 5.0 10.4 2.8 2.8 3.0 2.0 8.1 2.4

3.26 1.53 2.40 2.27 0.93 2.07 1.02 1.11 1.80 0.67 0.67 0.72 0.45 1.54 0.57

9.4 23.0 18.1 50.5 152 19.8 35.6 50.4 98.4 205 84.2 38.1 76.9 128 184

0.6 1.5 1.4 10.3 39 1.6 1.9 5.1 34.0 39 24.3 2.6 9.2 40 35

τC values were determined by iteratively matching the experimental value to the ratio of eq 4/eq 3;19 τC estimates using the polynomial approximation of Carper and Keller20 were approximately identical (not shown).

a

therefore, the ratio T1/T2 can be used to accurately estimate τc through eqs 3−6.19,20 However, these expressions assume random isotropic tumbling. It should be noted that internal motion can, in turn, affect relaxation. For a rotating methyl group,42 for example, perturbation to relaxation leading to an underestimate of τC is observed if the internal motion is on a similar time scale to the isotropic tumbling.19 In order to test the utility of this τC estimation method for soft nanoparticle characterization, τC was determined for each species according to the T1/T2 ratio observed at each site (Table 3). Notably, the τC estimates for a given species are widely variable from site to site, without a clear relationship to the conjugation state (and hence local spin network) at a given site. In general, τC depends upon the translational frictional coefficient of the rotating species, a parameter which, in turn, depends upon the shape and size of the species as well as the solution viscosity, η.27 In the case of the SP sample, the observed higher values of τC may therefore be directly attributable to the dramatically elevated solution η (Table 3 and Supporting Information Table S3). In direct contrast to this, the η of the NP(1xUV) solution was unchanged from that of D2O even at the highest concentration (20 mg/mL) employed (Supporting Information Table S3). Following from this, we have assumed that NP(4xUV) solutions would also have unperturbed η. The invariant η observed in the NP samples implies that it is change in shape and/or size that is giving rise to τC variation. Assuming spherical collapse trapped by UV-induced cross-linking, the qualitative trend of decreased particle size with increased UV dose implied by the T1 and T2 values observed is reflected with estimation of a decrease of τC. While qualitatively useful, however, determination of τC from T1/T2 is of limited value for quantitative characterization of NP (or SP) hydrodynamic behavior as reflected in the wide variability of τC estimates. This will become even more apparent in the subsequent discussion. Hydrodynamic Measurements. For direct experimental characterization of the hydrodynamics of the SP and NPs in D2O, we performed 1H PFG DOSY.43 The translational diffusion coefficient, DC, for which DOSY is one of the most

chemical shifts are therefore needed to characterize the inherent disparity in structural conformation between SP and NP. Nuclear spin longitudinal (T1) and transverse (T2) relaxation time constants are highly sensitive to motion, both within a molecule or supramolecular structure and for its tumbling as a whole.37,40 To test for differences in nuclear spin relaxation arising from differences in conjugation and to minimize overlap of multiple chemical species, the 1H T1 and T2 time constants based upon the intensity corresponding to the strongest signal for each of sites A−F (with A and C considered together due to overlap) were examined for each material (Table 3). In progression from SP to NP(1xUV) to NP(4xUV), T1 decreases while T2 increases. Since all species are in the macromolecular tumbling regime, as demonstrated by decrease of T1 values at 300 MHz strength compared to 500 MHz strength, a spin relaxation trend of this nature at constant static magnetic field strength is consistent with a more rapid molecular reorientation and smaller effective size as T1 decreases and T2 increases. Relaxation of spin-1/2 nuclei is dominated by contributions from two sources: a dipolar mechanism and a chemical shift anisotropy mechanism.37,40 The effectiveness of a given relaxation mechanism at a given frequency depends upon the magnitude of motion sampled at that frequency (the spectral density function), which is directly related to the τC for the molecule/supramolecular assembly containing the spin in question. The high gyromagnetic ratio and small chemical shift range of the 1H nucleus lead to domination of the dipolar relaxation mechanism over the chemical shift anisotropy mechanism for 1H−1H relaxation, simplifying treatment to some degree.21,41 Assuming domination of the dipolar relaxation mechanism, with a known number of interacting spin-1/2 nuclei, the ratio of T1 to T2 depends simply upon field strength and τC (eqs 3−6). It should be noted that the inverse sixth-power distance dependence for the dipolar mechanism (eqs 3 and 4) means that only 1H nuclei within ∼5 Å contribute significantly to relaxation,37 meaning that spinsystem topology for relaxation is often predictable strictly on the basis of covalent structure. Under ideal circumstances, 7467


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component exponential fits reduces significantly as a function of decreased NP concentration, a relative degree of crowding at higher concentrations is apparent and likely the major contributor to anomalous diffusion behavior. Furthermore, the smooth exponential decay of the NP signals in all cases, unlike the SP, implies that the NPs are mostly uniformly structured and are diffusing as rigid-body particles. Particle Size Characterization. Size is a key factor in nanoparticle description. Since the DOSY data indicate that the NPs are mostly uniformly structured rigid bodies, and polymer collapse theory suggests formation of spherical particles,46 they can be modeled as spherical hydrated particles diffusing freely in solution for the purpose of size estimation. While a spherical hydrodynamic configuration for the SP is not reasonable, the corresponding extrapolated SP dimensions are included for the purpose of comparison. Based upon the data detailed above, three different size estimations may be performed. First, using GPC elution time relative to GPC molecular weight standards, the apparent molecular weight of a given species (Table 2) may be employed to calculate a hydrodynamic diameter (dH[MW]) on the basis of spherical shape and an estimated specific volume (eq 8). Second, based upon nuclear spin relaxation-derived τC values (Table 3), dH may be estimated (dH[τC]) using Stokes’ law (eq 9). Both GPC and nuclear spin relaxation measurements imply that increased UV irradiation dose following NP collapse decreases the resulting cross-linked NP diameter (Table 5). However, comparison of dH[τC] and dH[MW] for

accurate measurement techniques,44,45 is inversely proportional to particle size.27 DOSY experiments were performed for each material at three different concentrations (1, 4, and 20 mg/mL) and at two different temperatures (22 and 37 °C). In each case, the DC (Table 4) was determined with a single component exponential fit to eq 7 (Supporting Information Figure S2). Table 4. Translational Diffusion Coefficients of the SP and the NPs Measured Using PFG-Based 1H DOSY NMRa diffusion coefficient, DC (× 10−12 m2/s) temp (°C) SPb NP(1xUV) NP(4xUV)

22 37 22 37 22 37

20 mg/mL 1.20 1.35 47.3 58.7 145 185

± ± ± ± ± ±

0.14 0.09 1.6 2.0 6 8

4 mg/mL 2.58 2.97 53.8 66.8 145 190

± ± ± ± ± ±

0.37 0.38 3.0 3.6 6 11

1 mg/mL 4.38 5.10 54.7 68.3 150 196

± ± ± ± ± ±

1.29 1.70 2.2 3.3 7 10

a

The diffusion coefficient represents the average of three values obtained from the methyl protons (two peaks for site B and a single peak for site F; Figure 2A). The error corresponds to the average of the fit errors. bDC values for SP are inherently inaccurate due to singlecomponent fits of very scattered data (Supporting Information Figure S2).

The trends for DC are the same in the SP and the NPs, demonstrating increased translational diffusion with increased temperature and with decreased concentration (Table 4). However, for the SP, significant deviation from ideal exponential decay as a function of applied gradient strength was observed for all conditions (Supporting Information Figure S2). Hypothetically, this may be due to non-rigid-body translational motion resulting from large-scale conformational exchange due to winding/unwinding of polymer chains. This also implies that the SP structure is inhomogeneous and transient. Diffusion of particles in a solution is inversely related to the shear η.27 Interestingly, the η of SP solutions was far higher than any of the NP solutions and showed a dramatic increase as a function of concentration over the 1−20 mg/mL range examined (Supporting Information Table S3). This is indicative of the SP sampling an extended conformation with significant interaction between neighboring polymer chains, increasing solution η. The extended nature of the conformation arises from the fact that the SP is a nonuniform random copolymer with high hydrophilic content; therefore, there are not enough nearby hydrophobic domains to self-associate and exclude water. This agrees with the PRE observations where the entire SP is exposed to aqueous solution (Figure 4A). The cross-linked collapsed configuration of the NP reduces the interaction between NPs (compared to interaction observed between extended polymer chains of SP); therefore, the η remained unchanged over the concentration range studied here. In contrast to the SP, both NPs exhibited uniform signal decay, although in some cases a single component exponential fit did not include all data points, especially at the highest concentration employed of 20 mg/mL (Supporting Information Figure S2). The observed deviation from single component exponential fits in high concentration NP samples may result from two possible scenarios. In the first, at high concentration, the DOSY data could be affected by restricted diffusion due to crowding; in the second, particle size heterogeneity within the population would lead to convolution due to multiple DC values. Because the deviation for the single

Table 5. Predicted Diameters (in nm) from Apparent Molecular Weight Determined by Gel Filtration Chromatography (dH[MW]) and from Rotational Correlation Time Calculated Using Relaxation Time Constants (dH[τC]) Compared to Experimental Hydrodynamic Diameters Determined by DOSY NMR (dH[DC]) for the SP and Each NP (20 mg/mL Samples) in D2O at 22 °C dH[τC] sample

dH[MW]

500 MHz

300 MHz

dH[DC]

SP NP(1xUV) NP(4xUV)

6.5 4.1 3.4

0.45−0.66 1.69−2.29 1.54−1.91

0.48−0.66 1.61−2.34 1.52−2.12

5.6 ± 0.6a 7.3 ± 0.2 2.4 ± 0.1

a None of the DC values are physically meaningful as the SP is not spherically structured, and it is determined from an inherently inaccurate DC (included here only for the purpose of comparison).

each species demonstrates highly inconsistent values (Table 5). Since both methods correspond to indirect probing, the discrepancy observed is indicative that the sizes of these species need to be compared using a direct measurement. The DOSY NMR experiment provides a direct measurement of DC (eq 7). In the context of a known viscosity, the Stokes− Einstein equation (eq 10) in turn provides an accurate dH value for a spherical particle. A striking NP contraction is apparent for NP(4xUV) vs NP(1xUV) according to dH on the basis of DC (dH[DC]; Table 5). Although this contraction was also implied by both dH[MW] and dH[τC], the values of dH[DC] are dramatically different from either dH[MW] or dH[τC]. In the case of the GPC-derived MW values, the deviation in size determination is likely due to the fact that the set of polymeric standard samples employed is not suitable for size determination of a compact species such as a nanoparticle. Unless a set of more appropriate standards becomes available, GPC is probably inherently limited in value for accurate soft nano7468


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Figure 5. Apparent hydrodynamic diameters (dH’s) of the NPs as calculated from the observed translational diffusion coefficients (DC’s; Table 4 for D2O, Supporting Information Table S4 for CD3OD) using the Stokes−Einstein equation (eq 10).

diffusion at 22 and 37 °C were calculated identically to those in D2O. As in D2O, the size of NP(1xUV) was increased at 37 °C relative to 22 °C. Hence, the malleability, i.e., the softness attributable to the core−shell NP structuring, is maintained in both solvents. Interestingly, in contrast to the relaxation data, the dH values obtained from the corresponding DOSY-derived DC values demonstrate the NP(1xUV) particles are significantly larger in D2O than in CD3OD. This is most likely due to a methanol-induced contraction of the shell layer surrounding the NP core, directly attributable to the nonpolar nature of the solvent. Diffusion NMR measurements are therefore clearly capable of capturing solvent-dependent size variation in soft nanoparticles. Core−Shell Soft Nanoparticle Structuring. Several pieces of data are indicative of spherical NP formation comprising a malleable polymeric shell surrounding a mostly solvent-excluded core, with an increased proportion of core relative to shell as a function of UV-induced cross-linking. The DOSY and PRE observations of increased compactness and solvent protection for NP(4xUV) vs NP(1xUV) are fully consistent with the type of behavior. This also fits well with the improved estimate of spin relaxation-based dH measurement on the basis of τC, where decreased internal motion would be expected on the basis of a decreased shell region. Decreased discrepancy between the τC-based dH and DC-based dH values in methanol also follow this trend; a solvent-induced compaction of the shell is consistent with a decrease in shell diameter and, correspondingly, decrease in polymer mobility within the shell. It thus seems reasonable that the less compact NP(1xUV) species has a thicker shell region, allowing greater local mobility of the polymer, while increased UV-induced photochemistry leads to formation of a more compact core, as in the NP(4xUV) species.

particle size determination. The 1H relaxation-based τC values consistently underestimate particle size. Because the methodology in question specifically relies upon uniform reorientation of the relaxing spin with the entire particle,19 any segmental motion on a time scale similar to isotropic tumbling would decrease the calculated value of dH. The estimated dH values are thus likely indicative of significant internal motion within the NP species. The observation that the maximum of the NP(4xUV) dH[τC] range is much closer to dH[DC] than for NP(1xUV) may imply a lesser contribution of internal motion. For the purpose of comparison between the SP and NP species, we also calculated and report the apparent dH of the SP on the basis of DC using the Stokes−Einstein hydrodynamic model (Supporting Information Figure S3) and on the basis of τC (Table 5). The very low apparent dH[τC] values of the SP (Table 5) can likely be directly attributed to extensive internal motion on the same time scale as the overall tumbling. As with the NPs, the apparent size of the SP increases with temperature. However, implausibly, the apparent dH increases with decreased concentration. This indicates that the Stokes−Einstein spherical model is not appropriate for the SP. Instead, this may imply that the SP is able to occupy a greater volume when not restricted by crowding. Environmental Effects upon Nanoparticle Morphology. The concentration and temperature dependence of dH[DC] for both types of NP is highly informative (Figure 5). The larger NP(1xUV) exhibits a degree of concentrationdependent dH reduction in going from 20 to 4 mg/mL but is unchanged between 4 and 1 mg/mL. Conversely, dH is practically insensitive to concentration for the far more compact NP(4xUV). This implies that the larger NP(1xUV) is affected to a degree by crowding at the higher concentration (albeit still only ∼2% by weight, assuming ∼1 g/mL density) and is illustrative of a reliable dH determination over 1−4 mg/ mL range. Soft nanoparticles typically exhibit solvent- and temperaturedependent conformational change.1 Nuclear spin relaxation and DOSY-based translational diffusion measurements on NP(1xUV) samples in CD3OD allowed direct quantitation of the first of these effects. The measured T1/T2 ratios of 3.7−14.9 correspond to predicted dH of 1.85−2.53 nm, an apparent increase relative to 1.69−2.29 nm in D2O. Temperaturedependent swelling is also clear for both NP forms in D2O, with dH at all concentrations increased at 37 °C relative to 22 °C (Figure 5). This swelling is less pronounced for NP(4xUV) (∼7%) than for NP(1xUV) (∼12%). The apparent dH values of a 20 mg/mL sample in CD3OD based upon translational

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CONCLUSIONS This work makes combined use of high-resolution NMR, determination of nuclear spin relaxation properties, and translational diffusion measurements by PFG-based DOSY NMR to comprehensively characterize soft, polymeric Allosperse nanoparticles in the solution state. 1H and 13C NMR experiments demonstrate the production of photochemically induced moieties in these nanoparticles, with Mn2+ PRE showing clear evidence of sequestration of those species away from the aqueous environment. Combination of single-bond and multiple-bond 2D 1H−13C correlation spectra, even when 13 C nuclei are available only at a natural abundance, reveals UVinduced cross-linking within the EA subunits, allowing proposal 7469


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(3) Faraji, A. H.; Wipf, P. Nanoparticles in cellular drug delivery. Bioorg. Med. Chem. 2009, 17, 2950−2962. (4) Lopez-Cebral, R.; Martin-Pastor, M.; Seijo, B.; Sanchez, A. Progress in the characterization of bio-functionalized nanoparticles using NMR methods and their applications as MRI contrast agents. Prog. Nucl. Magn. Reson. Spectrosc. 2014, 79, 1−13. (5) Hens, Z.; Martins, J. C. A solution NMR toolbox for characterizing the surface chemistry of colloidal nanocrystals. Chem. Mater. 2013, 25, 1211−1221. (6) Miller, J. B.; Hobbie, E. K. Nanoparticles as macromolecules. J. Polym. Sci., Polym. Phys. 2013, 51, 1195−1208. (7) Mayer, C. NMR studies of nanoparticles. Annu. Rep. NMR Spectrosc. 2005, 55, 205−258. (8) Jiang, J.; Thayumanavan, S. Synthesis and characterization of amine-functionalized polystyrene nanoparticles. Macromolecules 2005, 38, 5886−5891. (9) Hrkach, J. S.; Peracchia, M. T.; Domb, A.; Lotan, N.; Langer, R. Nanotechnology for biomaterials engineering: Structural characterization of amphiphilic polymeric nanoparticles by H-1 NMR spectroscopy. Biomaterials 1997, 18, 27−30. (10) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Poly(lactic acid)-poly(ethylene oxide) (PLA-PEG) nanoparticles: NMR studies of the central solidlike PLA core and the liquid PEG corona. Langmuir 2002, 18, 3669−3675. (11) Jang, M. K.; Jeong, Y. I.; Nah, J. W. Characterization and preparation of core-shell type nanoparticle for encapsulation of anticancer drug. Colloids Surf., B 2010, 81, 530−536. (12) Garcia-Fuentes, M.; Torres, D.; Martin-Pastor, M.; Alonso, M. J. Application of NMR spectroscopy to the characterization of PEGstabilized lipid nanoparticles. Langmuir 2004, 20, 8839−8845. (13) Simeonova, M.; Rangel, M.; Ivanova, G. NMR study of the supramolecular structure of dual drug-loaded poly(butylcyanoacrylate) nanoparticles. Phys. Chem. Chem. Phys. 2013, 15, 16657−16664. (14) Ivanova, G.; Simeonova, M.; Cabrita, E. J.; Rangel, M. NMR Insight into the supramolecular structure of daunorubicin loaded polymer nanoparticles. J. Phys. Chem. B 2011, 115, 902−909. (15) Canzi, G.; Mrse, A. A.; Kubiak, C. P. Diffusion-ordered NMR spectroscopy as a reliable alternative to TEM for determining the size of gold nanoparticles in organic solutions. J. Phys. Chem. C 2011, 115, 7972−7978. (16) Chaudhuri, R. G.; Paria, S. Core/shell nanoparticles: classes, properties, synthesis mechanisms, characterization, and applications. Chem. Rev. 2012, 112, 2373−2433. (17) Cho, E. J.; Holback, H.; Liu, K. C.; Abouelmagd, S. A.; Park, J.; Yeo, Y. Nanoparticle characterization: State of the art, challenges, and emerging technologies. Mol. Pharmaceutics 2013, 10, 2093−2110. (18) Aiertza, M. K.; Odriozola, I.; Cabanero, G.; Grande, H. J.; Loinaz, I. Single-chain polymer nanoparticles. Cell. Mol. Life Sci. 2012, 69, 337−346. (19) Navon, G.; Lanir, A. NMR relaxation by intermolecular and intramolecular dipolar interactions in small molecules bound to an enzyme. J. Magn. Reson. 1972, 8, 144−151. (20) Carper, W. R.; Keller, C. E. Direct determination of NMR correlation times from spin-lattice and spin-spin relaxation times. J. Phys. Chem. A 1997, 101, 3246−3250. (21) Solomon, I. Relaxation processes in a system of 2 spins. Phys. Rev. 1955, 99, 559−565. (22) Kubo, R.; Tomita, K. A general theory of magnetic resonance absorption. J. Phys. Soc. Jpn. 1954, 9, 888−919. (23) Stejskal, E. O.; Tanner, J. E. Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J. Chem. Phys. 1965, 42, 288−292. (24) Sinnaeve, D. The Stejskal-Tanner equation generalized for any gradient shape-an overview of most pulse sequences measuring free diffusion. Concepts Magn. Reson., Part A 2012, 40A, 39−65. (25) Jerschow, A.; Muller, N. Suppression of convection artifacts in stimulated-echo diffusion experiments. Double-stimulated-echo experiments. J. Magn. Reson. 1997, 125, 372−375.

of a structural model for NP stabilization by UV irradiation. Extremely accurate solution-state nanoparticle sizing is obtained with DOSY NMR experiments, and we demonstrate that rotational diffusion characterization by nuclear spin relaxation provides a qualitative measure of relative particle dimensions and, more importantly, direct demonstration of deviation from ideal “hard sphere” behavior. This latter point is particularly useful in characterization of core (mostly inaccessible to solvent) vs shell (solvent accessible) regions of soft nanoparticles. Effects of environmental variation upon Allosperse nanoparticle conformation were also readily determinable, as observed both with temperature-dependent particle swelling and with solvent-induced contraction of solvent-accessible shell regions. Solution-state NMR spectroscopy therefore provides a broad and comprehensive ability to both physically and chemically characterize soft nanoparticles in the environment directly relevant to their application.

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ASSOCIATED CONTENT

* Supporting Information S

Diffusion time and gradient pulse length used in the DOSY experiments, viscosity data of D2O solutions containing the SP and the NPs, translational diffusion coefficients of NP(1xUV) in CD3OD, 2D 1H−13C HSQC spectra of pure MAA NP and MAA+EA SP/NP, pseudo-2D 1H DOSY spectra and single component exponential fits for attenuation of the most intense peak, and apparent hydrodynamic diameters of the SP. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone (902) 494-4632; Fax (902) 494-1355; e-mail jan. [email protected] (J.K.R.). Notes

The authors declare the following competing financial interest(s): Allosperse nanoparticle formation and non-NMR spectroscopy based characterization was funded directly by Vive Crop Protection. This had no bearing upon the presented experimental interpretation.

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ACKNOWLEDGMENTS Funding was provided initially by a Natural Sciences and Engineering Research Council of Canada (NSERC) Engage Grant (EGP/419106-2011 to J.K.R.) with follow-up analysis and data elaboration funded by an NSERC Discovery Grant (RGPIN/342034-2012 to J.K.R.). The TCI probe on the 700 MHz instrument at the National Research Council Biological Magnetic Resonance Facility (NRC-BMRF) was provided by Dalhousie University through an Atlantic Canada Opportunities Agency grant. We are grateful to Bruce Stewart for expert technical assistance and to Dr. Peng Zhang (Chemistry, Dalhousie University) for helpful comments on the manuscript. J.K.R. is grateful for support in the form of a Canadian Institutes of Health Research New Investigator Award.

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REFERENCES

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Characterization of Variant Soft Nanoparticle Structure and

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