Diffusion 19F-NMR of Nanofluorides: In Situ Quantification of Colloidal Diameters and Protein Corona Formation in Solution

The NMR-detectability of elements of organic ligands that stabilize colloidal inorganic nanocrystals (NCs) allow the study of their diffusion characteristics in solutions. Nevertheless, these measurements are sensitive to dynamic ligand exchange and often lead to overestimation of diffusion coefficients of dispersed colloids. Here, we present an approach for the quantitative assessment of the diffusion properties of colloidal NCs based on the NMR signals of the elements of their inorganic cores. Benefiting from the robust 19F-NMR signals of the fluorides in the core of colloidal CaF2 and SrF2, we show the immunity of 19F-diffusion NMR to dynamic ligand exchange and, thus, the ability to quantify, with high accuracy, the colloidal diameters of different types of nanofluorides in situ. With the demonstrated ability to characterize the formation of protein corona at the surface of nanofluorides, we envision that this study can be extended to additional formulations and applications.

The hydrodynamic diameter and size distribution of the obtained nanoparticles were evaluated by dynamic light scattering, Malvern Nano-ZS. Measurements were taken in a 12.5 mm diameter cylindrical quartz cuvette (for cyclohexane dispersed NCs) or plastic (for water dispersed NCs) according to the appropriate solvent viscosity.  Cit-CaF2 1mmol of NaF was added to 820mg citric acid in 50ml of ddH2O at pH=7, and neutralized with ammonium hydroxide. The solution was heated to 75°C while stirring. Then, 1/2mmol CaCl2 was dissolved in 2ml of ddH2O and was rapidly injected to the hot solution. The solution was removed from the heating plate immediately after the injection and cooled to room temperature. Then, the solution was centrifuged and washed with ethanol/ddH2O.     the studied colloid. Colored spheres represent the diffusion NMR signal decay obtained from the 1 H-NMR peaks of the AEP ligand. Dashed line represents the bi-exponential fitting of the experimental data resulted in two equal populations of AEP, one that is represented by a slow diffusion coefficient (D= 0.72´10 10 m 2 sec -1 ) and one by a fast diffusion coefficient (D= 4.6´10 10 m 2 sec -1 ). For comparison, the diffusion coefficient of the free AEP ligand in water was found to be D= 5.2´10 10 m 2 sec -1 and the diffusion coefficient of the AEP-CaF2 colloids as extracted from 19 F-NMR experiment was evaluated to be D= 0.78´10 10 m 2 sec -1 .

Sequence optimization for 19 F diffusion of NCs
To examine the feasibility of performing 19 F-diffusion NMR experiments, a solution containing OA-CaF2 in cyclohexane was first studied using a PGSTE-sequence.
Interestingly, even when a very weak pulse gradient was applied (G= 0.681 gauss/cm) for a duration of only 1.2 msec in the 19 F-NMR PGSTE experiment, the characteristic highresolution 19 F-NMR signal of the studied OA-CaF2 was totally eliminated (Fig. S3,

Equation S1
The normalized 19 F-NMR signal decay of the nanofluorides as a function of the experimental b-values is plotted in Fig. 2c-f and were calculated according to Equation S1: where is the gyromagnetic ratio of the studied nuclei ( 2 / = 40.04 $% for 19 F) 1 , G is the applied gradient, is the duration time of the applied G, and ∆ is the diffusion time.

Equation S2
Importantly, using Equation 2, the diffusion coefficient, D, can be extracted from the linear slope of the plot in Fig. 2c-f or Fig.3a: where I is the 19 F-NMR signal at a given gradient strength (G) and I0 is the signal without the pulsed-gradient application; D is the apparent diffusion coefficient.

Equation S3
In order to consider both the viscosity of the NCs solution (compared to the pure solvent viscosity η) and the gradient calibration constants 7 we calculated a calibration factor ( 1 ) in Eq S3; (Eq. S3) Where Dsolvent measured is the diffusion coefficient measured from the diffusion NMR experiment of the solvent in the studied solution and Dsolvent known is the diffusion coefficients from literature (values in Table S2).