# 1H-19F cross-polarization magic angle spinning dynamic nuclear polarization NMR investigation of advanced pharmaceutical formulations

Mária Šoltésová, Arthur C Pinon, Fabien Aussenac, Judith Schlagnitweit, Christian Reiter, Armin Purea, Roberto Melzi, Frank Engelke, Dave Martin, Stefanie Krambeck, et al.

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Mária Šoltésová, Arthur C Pinon, Fabien Aussenac, Judith Schlagnitweit, Christian Reiter, et al. 1H-19F cross-polarization magic angle spinning dynamic nuclear polarization NMR investigation of advanced pharmaceutical formulations. Journal of Magnetic Resonance, 2024, 371, pp.107827. 10.1016/j.jmr.2024.107827. hal-05374311

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# ¹H–¹⁹F cross-polarization magic angle spinning dynamic nuclear polarization NMR investigation of advanced pharmaceutical formulations

Mária Šoltésová<sup>a</sup>, Arthur C. Pinon<sup>a, *</sup>, Fabien Aussenac<sup>b</sup>, Judith Schlagnitweit<sup>c</sup>, Christian Reiter<sup>d</sup>, Armin Purea<sup>d</sup>, Roberto Melzi<sup>e</sup>, Frank Engelke<sup>d</sup>, Dave Martin<sup>f</sup>, Stefanie Krambeck<sup>g</sup>, Annabelle Biscans<sup>g</sup>, Emma Kay<sup>h</sup>, Lyndon Emsley<sup>i</sup>, Staffan Schantz<sup>j</sup>

## ARTICLE INFO

Keywords:

Nuclear magnetic resonance
Dynamic nuclear polarization
Fluorine
NMR probe
Pharmaceutical formulation

## ABSTRACT

A new 3.2 mm ¹H–¹⁹F–X magic angle spinning dynamic nuclear polarization NMR (MAS DNP-NMR) probe was developed with a unique coil design with separate radiofrequency channels for ¹H excitation and ¹³C or ¹⁹F detection to enable acquisition of ¹H–¹⁹F cross-polarization (CP) MAS experiments, direct-detected ¹⁹F spectra with proton decoupling, and acquisition on ¹³C with simultaneous double decoupling on the ¹H and ¹⁹F channels as well as ¹H–¹⁹F–¹³C double-CP experiments under low temperature MAS DNP conditions. We use these sequences to study AZD2811, which is an active pharmaceutical ingredient (API), in its pure dry state as well as in its corresponding drug delivery formulation consisting of drug-loaded polymeric nanoparticles (PNPs). Included in this study are also small interfering RNAs (siRNAs) for therapeutic targeting of peptidyl-prolyl cis–trans isomerase B (Ppib) mRNA. We demonstrate that ¹H–¹⁹F CP MAS experiments performed on the new HFX probe represent a notable advantage over usually acquired direct-detected ¹⁹F experiments. The indirect ¹⁹F DNP enhancement $ε_{on/off}(¹⁹F) = 26$ was obtained via ¹H–¹⁹F CP for the pure API impregnated with DNP solution, with an overall 30-fold sensitivity gain compared to the direct-detected ¹⁹F experiment under similar conditions. DNP enhancement value of $ε_{on/off}(¹⁹F) = 42$ was obtained via ¹H–¹⁹F CP for the polymeric nanoparticle suspension and $ε_{on/off}(¹⁹F) \approx 150$ were obtained for two different siRNAs in frozen DNP solution.

## 1. Introduction

Solid-state nuclear magnetic resonance (ssNMR) is an indispensable method for the characterization of the structure and dynamics of various solid-state compounds, including pharmaceutical formulations. It has been successfully used, for example, to investigate and differentiate structural polymorphism, which is an issue of great importance for pharmaceutically relevant compounds [1–7]. Related areas of recent interest include studying pharmaceuticals in-vivo or in-cell, thus in biologically realistic environments and at relevant concentrations, i.e.

low concentrations of the nuclei of interest [8,9].

Solid-state MAS spectroscopy is not utilizable for abundant high-gamma nuclei such as ¹H, unless spinning frequencies over 100 kHz are used, due to the strong homonuclear interactions causing substantially broadened lines [10,11]. Moreover, some background signal from ¹H-containing components of the NMR probe is often present in the spectra. Besides, detection of low-gamma nuclei (¹³C, ¹⁵N...) at natural abundance is impractical due to the inherently low sensitivity of NMR unless isotopic labeling is used. A possible way to overcome these sensitivity limitations is dynamic nuclear polarization (DNP), a method

<footnote><sup>a</sup> Swedish NMR Centre, University of Gothenburg, 413 90 Gothenburg, Sweden</footnote>

<footnote><sup>b</sup> Bruker BioSpin France, Wissembourg, France</footnote>

<footnote><sup>c</sup> Centre de RMN à Très Hauts Champs de Lyon, UMR5082 CNRS/ENS-Lyon/Université Claude Bernard Lyon 1, Villeurbanne, France</footnote>

<footnote><sup>d</sup> Bruker BioSpin Germany, Ettlingen, Germany</footnote>

<footnote><sup>e</sup> Bruker BioSpin Italy, Milan, Italy</footnote>

<footnote><sup>f</sup> Oral Product Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Macclesfield, UK</footnote>

<footnote><sup>g</sup> Cell, Gene and RNA Therapy, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Sweden</footnote>

<footnote><sup>h</sup> Assays, Profiling and Cell Sciences, Discovery Sciences, AstraZeneca, Gothenburg, Sweden</footnote>

<footnote><sup>i</sup> Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland</footnote>

<footnote><sup>j</sup> Oral Product Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Gothenburg, Sweden</footnote>

<footnote>* Corresponding author.
E-mail address: arthur.pinon@gu.se (A.C. Pinon).</footnote>
that can enhance NMR signals by transferring the high magnetization of unpaired electrons from a radical molecule to coupled nuclei using microwave irradiation. Although the concept of DNP is known since the 1950s [12,13], it has not been successfully integrated into high-field MAS NMR until recently, after the introduction of high-power microwave source and low temperature MAS (LT-MAS) [14–16].

In recent years, DNP-enhanced ¹⁹F solid-state NMR has gained popularity as a promising approach for studying fluorine-containing molecules, such as pharmaceuticals [17], fluorinated proteins in cells [18,19] and small molecules [20]. The advantage of ¹⁹F experiments is the high natural abundance of fluorine and a large chemical shift range. Besides that, fluorine is present in many pharmaceuticals, while hardly any excipients contain it. It can also easily be incorporated in biomolecules such as nucleic acids or proteins, while it does not occur naturally in the cell. This facilitates the study of the fluorinated molecule of interest directly in pharmaceutical formulations or in cells without the need to de-crowd spectra of signals arising from excipients or the cell background. Moreover, ¹⁹F has sometimes long magnetization buildup times in solids compared to ¹H, which implies that direct-detected experiments are very time-consuming.

When performing a DNP experiment of an organic solid sample, the unpaired electron magnetization is transferred to ¹H nuclei surrounding the radical molecule. Over the years, DNP solution compositions have mainly been optimized for maximizing the proton enhancement for MAS DNP. Despite the fact that the ¹⁹F gyromagnetic ratio is close to the one of ¹H, direct ¹⁹F DNP remains less effective and yields a more modest signal enhancement than the one of protons [17]. This is partially because finding proton-free, fluorinated solvents and radicals that fulfill the criteria for efficient direct ¹⁹F DNP remains challenging. This is however an active and promising area of research [21]. Consequently, a DNP solution is either optimized for direct DNP to the nucleus of interest (other than ¹H) or optimized for ¹H nuclei, in which case a CP step is performed, where the hyperpolarized ¹H magnetization is transferred to the nucleus of interest.

¹H-¹⁹F CP MAS DNP experiments would represent a great advantage in terms of sensitivity improvement and shortening of the experimental time. Indeed, such experiments would gain from the transfer of hyperpolarization of ¹H nuclei to ¹⁹F nuclei with CP, while benefiting from typically much faster build-up times of DNP-enhanced ¹H polarization allowing the use of shorter recycle delay. ¹H-¹⁹F CP MAS experiments were successfully utilized previously [22–24] however, up to our best knowledge, no such experiments using DNP have been reported. Up until now, the limitations for the employment of ¹H-¹⁹F CP MAS under DNP conditions have been the available instrumentation.

Currently, there are two types of HFX probes for room temperature measurements [25–27]: the ones with separate channels for ¹H and ¹⁹F, and the ones with a single channel with the use of external splitters. While the probe requirements for utilizing external splitting of the proton channel are rather low, tuning the two frequencies is little user-friendly and it comes at the expense of RF sensitivity. This is because every additional RF component in the line comes with some insertion loss, and it is therefore preferable to reduce their number to a minimum. Implementing two independently tunable channels for ¹H and ¹⁹F on the other hand is more challenging regarding the probe's RF circuit but yields an improved NMR performance and it is easy to use. There are two main approaches for such HFX probes: Either connecting the three RF channels to one coil and isolate them by using transmission lines or having two orthogonal coils for the nearby frequencies of ¹H and ¹⁹F.

In the existing DNP probe platform, the implementation of the latter was easier and Bruker, using the design developed by Peter Gor'kov [28], decided to develop a 400 MHz widebore 3.2 mm MAS HFX DNP probe with ¹⁹F and ¹H resonating on two separate coils – the outer coil tunable to ¹H, the inner one tuning to ¹⁹F and X nuclei (see Fig. 1). A probe with the RF circuit and two-coil design as proposed is generally suitable for ¹H/¹⁹F/X triple resonance experiments.

In this study, we present the first experiments testing the

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Radiofrequency coil setup of the new HXF solid-state MAS DNP probe. The setup employs two separate coils: solenoid doubly tuned to ¹⁹F and X-nuclei, and a capacitively tuned cage coil for ¹H. The unique low-E field-type design of the cage coil is shown, which reduces sample heating during the high-power cross-polarization and decoupling pulses. The crosstalk between the close resonance frequencies of ¹H and ¹⁹F nuclei is avoided by RF fields of the solenoid and cage coil being perpendicular to each other.</figcaption></figure>

performance of this probe, and we apply ¹H-¹⁹F cross polarization with ¹H decoupling during ¹⁹F acquisition to study advanced pharmaceutical formulations.

We demonstrate the performance of the new HFX probe on the AZD2811 API in its pure powder form as well as its corresponding drug delivery formulation consisting of drug-loaded polymeric nanoparticles (PNPs). Included in this study were also two therapeutical siRNAs compounds, for which we present DNP-enhanced ¹H-¹⁹F CP MAS spectra and ¹H-¹⁹F-¹³C double-CP spectra.

## 2. Experimental section

### 2.1. Compound preparation

#### 2.1.1. AZD2811 nanosuspension preparation

The active pharmaceutical ingredient AZD2811 and the polymeric nanoparticles were obtained from AstraZeneca Ltd. AZD2811 has the molecular formula C₂₆H₃₀FN₇O₃, containing about 3.7 % fluorine in its molar mass. Polymeric nanoparticle suspensions containing polylactic acid-polyethylene glycol (PLA-PEG) block copolymer (a number average molecular weight of approximately 16 kDa for PLA and approximately 5 kDa for PEG), pamoic acid and AZD2811 were prepared following the method of Song et al. [29] at a concentration of about 48 mg/mL, equivalent to about 10 mg/mL of API.

#### 2.1.2. siRNAs preparation

For details about the oligonucleotide synthesis, see the Supporting Information.

### 2.2. DNP sample preparation

#### 2.2.1. AZD2811

Three samples of API were prepared: dry powder sample containing 20 mg of pure crystalline AZD2811; impregnated powder sample where
34 mg of crystalline AZD2811 was impregnated with 40 µL of DNP solution containing glycerol-d₈/H₂O (6/4 vol ratio) with 12 mM AMUPol radical by incipient wetness impregnation [30,31]; The PNP sample was prepared by making a DNP solution of glycerol-d₈/D₂O/PNP suspension (in a 6/3/1 vol ratio) and containing 12 mM of AMUPol, resulting in a final concentration of 1.2 mg of nanoparticles per 1 mL of the DNP solution, or about 0.25 mg/mL of the API. The total volume of the sample was 25 µL. All samples were transferred into a 3.2 mm sapphire rotor, sealed with a silicone insert and closed with a Vespel cap, transferred into a precooled LT-MAS DNP probe.

### 2.2.2. siRNAs

Lyophilized siRNA was dissolved at a concentration of 9 mM in a DNP solution containing 6/3/1/ volume ratio of glycerol-d₈/D₂O/H₂O with 12 mM of AMUPol. 25 µL of the solution were transferred into a 3.2 mm sapphire rotor, sealed with a silicone insert, closed with a zirconia cap, and transferred into a precooled LT-MAS DNP probe.

## 2.3. NMR spectroscopy

All NMR experiments were performed on a 400 MHz Avance Neo Bruker DNP solid-state NMR spectrometer. The spectrometer was equipped with an LT-MAS DNP 3.2 mm ¹H/¹⁹F/X triple resonance probe and a 263 GHz gyrotron capable of outputting approximately 25 W of CW microwaves. The X channel of the probe was tuned to the frequency of ¹³C during the experiments. The main magnetic field was adjusted to match the maximum positive ¹H DNP enhancement of the biradical agent AMUPol.

Isotropic chemical shifts in ¹H spectra were referenced according to the signal of the silicon insert at 0.1 ppm. In the case of ¹⁹F spectra, chemical shifts were referenced according to the ¹⁹F signal of ammonium trifluoroacetate (ATFA), used as an external standard, at –76 ppm. In the case of dry crystalline AZD2811, the direct-detected ¹⁹F and ¹H–¹⁹F CP MAS experiments were recorded at 100 K and 10 kHz MAS frequency, with 4 scans and recycle delay ($d_1$) of 270 s and 16 scans and $d_1$ of 70 s, respectively, resulting in overall experimental time of approximately 18 min in both cases. The amplitude of ¹H pulse during the contact time was ramped from 30 to 60 kHz.

Sensitivity enhancements and DNP enhancements were determined by comparing the peak areas of the spectra acquired with and without microwave irradiation. Peak areas were obtained by spectral integration. In the case of ¹H, only the central peak (excluding silicone insert signal) was integrated. In the case of ¹⁹F spectra, the whole sideband manifold was considered. The same integral ranges were used for determination of ¹H and ¹⁹F longitudinal relaxation times, $T_1$, and buildup times of DNP-enhanced magnetization, $T_B$. Variable amplitude cross-polarization was used to transfer polarization from ¹H to ¹³C. SPINAL-64 heteronuclear ¹H decoupling [32] with an RF field of 60 kHz was applied during the acquisition in all cases. Radiofrequency fields at 100 K for ¹H, ¹⁹F and ¹³C were 60 kHz at 120 W, 60 kHz at 50 W and 55 kHz at 50 W, respectively.

## 2.4. HFX probe

The new 3.2 mm HFX solid-state MAS DNP probe (400 MHz/263 GHz) has three independent radiofrequency channels. Two of them are operated at Larmor frequencies of ¹H and ¹⁹F nuclei, the third channel is an X channel tunable to frequencies from 40 (¹⁵N) to 100 MHz (¹³C), with an additional range coil, frequencies up to 162 MHz (³¹P) can be achieved (Fig. 1). Two separate coils are employed – a solenoid doubly tuned to ¹⁹F and X-nuclei, and a capacitively tuned cage coil for ¹H. The cage coil for the ¹H channel has a unique low-E field-type design which reduces sample heating during the high-power cross-polarization and decoupling pulses. Moreover, the ¹H cage coil can deliver an RF field perpendicular to the one of the second solenoid coil which presents an advantage of avoiding the crosstalk between the close resonance

frequencies of ¹H and ¹⁹F nuclei.

This design using a solenoid for ¹⁹F and ¹³C leads to an optimized sensitivity on ¹⁹F and ¹³C, as there are only these two channels connected to the solenoid: from a ¹⁹F and X point of view, the probe is a double resonance probe. This comes at the expense of lower filling factor and lower sensitivity of the ¹H channel, which is not problematic in this experimental scenario as the ¹H channel is rarely observed in a 3.2 mm probe under DNP conditions. A decoupling field of 60 kHz can still be achieved through the outer cage. We note that the maximum RF field amplitude on ¹H channel is limited compared to the usually reported ¹H decoupling power levels (order of 100 kHz) for moderate MAS frequencies at room temperature [24,33]. Nevertheless, substantial improvement in signal intensity and linewidth is achieved even with the ¹H decoupling at the reduced power level (60 kHz) during the acquisition of the ¹H–¹⁹F CP MAS experiment, as opposed to the non-decoupled spectra, as demonstrated in Fig. S2c.

This unique setup of the probe opens a possibility for experiments that were not available before under LT-MAS DNP conditions. It is possible to acquire, among others, ¹H–¹⁹F CP MAS experiment with ¹H decoupling during the acquisition, direct detected ¹⁹F spectra with ¹H decoupling, direct detected ¹H spectra with ¹⁹F decoupling, direct ¹³C observation with simultaneous ¹H and ¹⁹F decoupling, and ¹H–¹⁹F–¹³C double-CP MAS experiment. Common experiments, such as ¹H–¹³C and ¹⁹F–¹³C CP MAS are as well available.

In the current probe prototype, the stator and the coil block are made of the glass–ceramic material Macor®, which contains 4 % of ¹⁹F. Consequently, direct-detected ¹⁹F experiments on the empty probe without the sample at 295 K and 100 K reveal a broad peak of a probe background at approximately –180 ppm. The material of the stator was later exchanged for boron nitride, and the coil block material was replaced by quartz, which resulted in a significant reduction of the ¹⁹F probe background.

## 3. Results and discussion

### 3.1. AZD2811 dry powder

Direct-detected ¹⁹F and ¹H–¹⁹F CP MAS experiments were first recorded for the dry crystalline AZD2811 powder at 100 K and 10 kHz MAS frequency (Fig. S1a). The isotropic fluorine chemical shift of the only fluorine site of the AZD2811 molecule is approximately –110 ppm. Due to the large CSA of ¹⁹F, a number of spinning sidebands are observed, the isotropic peak is the most intense one.

To ensure the correct setting of the recycle delay $d_1$ for the experiments above, low temperature (100 K) ¹H and ¹⁹F spin–lattice relaxation times ($T_1$) were measured by saturation-recovery echo experiments on the dry crystalline AZD2811 drug. The $T_1$ relaxation time for ¹H was 56 s, with the $T_1$ for ¹⁹F found to be at least 4 orders of magnitude longer than ¹H $T_1$ (Fig. S1b–d).

The sensitivity (expressed as signal-to-noise ratio (SNR) per mg of sample per square root of experimental time) is 0.25 $mg^{-1}H^{-1/2}$ for the direct detected ¹⁹F experiment as opposed to 11.5 $mg^{-1}H^{-1/2}$ for the ¹H–¹⁹F CP MAS measurement. This gain in sensitivity is predominantly given by the choice of the CP MAS experiment over the direct-detected ¹⁹F one (higher gyromagnetic ratio of protons also contributes to 15 % increase of the ¹H–¹⁹F CP MAS signal). Shorter $T_1$ of ¹H compared to a very long $T_1$ of ¹⁹F results in significant shortening of the recycle delay $d_1$ (70 s for the ¹H–¹⁹F CP MAS as opposed to 270 s for the direct-detected ¹⁹F experiment) in the ¹H–¹⁹F CP MAS experiment, thus of the overall experimental time. Furthermore, suppression of the probe background signal is much more efficient in the CP MAS experiment due to the phase cycling excluding any ¹⁹F signal not arising from nearby ¹H spins.
## 3.2. AZD2811 impregnated powder

On the AZD2811 powder impregnated with the DNP solution, ¹H spin echo, ¹⁹F spin echo, and ¹H-¹⁹F CP MAS experiments were acquired both under microwave irradiation (MW on) and without microwave irradiation (MW off) to assess the DNP enhancements (Fig. S2a). Also in this case, magnetization build-up times of ¹H and ¹⁹F were measured under MW on conditions by saturation-recovery echo experiments, confirming much longer fluorine build-up time ($T_b(^{19F}) = 184$ s) as opposed to the proton build-up time ($T_b(^{1H}) = 2.5$ s). The magnetization build-up times $T_b$ of proton and fluorine in the impregnated AZD2811 powder are much shorter than the $T_1$ relaxation times measured for the dry AZD2811 powder. This is due to the presence of paramagnetic radical, causing paramagnetic relaxation enhancement, as well as spin diffusion causing the enhanced magnetization to build-up faster. It was also shown that the possibility of ¹H decoupling during the acquisition of the ¹H-¹⁹F CP MAS experiment presents notable improvement in signal intensity and linewidth as opposed to the non-decoupled spectra (Fig. S2b).

The ¹H DNP enhancement is $\epsilon_{on/off}(^{1H}) = 59 \pm 5$. In the case of microcrystalline organic solids, proton-proton spin diffusion relays the polarization from the surface of the crystallites inside [34,35], and reasonably high DNP enhancement can be achieved depending on the build-up time of the DNP-enhanced ¹H polarization.

For direct-detected ¹⁹F experiment on the AZD2811 impregnated powder, rather large values of direct fluorine DNP enhancement $\epsilon_{on/off}(^{19F}) = 28 \pm 1$ were obtained. Even though the DNP solution used for sample impregnation here is not optimized for fluorine hyperpolarization, the very long magnetization build-up time of ¹⁹F ensures

sufficient time for spin diffusion to relay the enhanced magnetization from the surface of the particle into its core, ensuring it reaches a large amount of fluorine nuclei. The drawback of this experiment is the ¹⁹F probe background appearing in the spectra, although it is significantly weaker compared to the DNP enhanced API signal. Another downside is the necessary long recycle delay, resulting in extended experimental time.

¹H-¹⁹F CP experiment on the impregnated AZD2811 powder was recorded with and without microwaves and resulting ¹⁹F enhancement was $\epsilon_{on/off}(^{19F}) = 26 \pm 1$. This is a demonstration of indirect DNP – electrons are polarizing ¹H nuclei, the magnetization then undergoes spin diffusion and gets distributed from the surface of the particles into the core. The spin diffusion is very effective given the high amount of ¹H in the organic solid. Subsequently, ¹H magnetization is transferred to ¹⁹F by CP and detected. Despite a slightly lower enhancement than direct-detected spectra, the overall sensitivity is $425 \text{ mg}^{-1} \sqrt{\text{h}^{-1}}$ as opposed to $14 \text{ mg}^{-1} \sqrt{\text{h}^{-1}}$ for the direct-detected ¹⁹F experiment, benefiting from much shorter $d_1$ due to shorter proton $T_1$. The probe background signal is virtually not present in DNP enhanced CP spectra. These benefits highlight the usefulness of the ¹H-¹⁹F CP MAS experiment and make this experiment the method of choice, especially for organic solids such as pharmaceuticals containing relatively low amount of fluorine compared to protons and where the fluorine relaxation time is much longer than the one of proton.

### 3.2.1. AZD2811 nanoparticles suspension

The ability of the new hardware to transfer ¹H enhancements onto ¹⁹F nuclei of APIs was further evaluated using a novel PNP formulation. Encapsulation of an API within a functionalized polymeric

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Solid-state CP MAS DNP experiment on impregnated AZD2811 nanoparticle formulation. (a) Layered structure of the polymeric nanoparticle formulation of the AZD2811 drug. (b) Molecular formula of the AZD2811 API. Solid-state spectra of AZD2811 nanoparticle suspension with (blue) and without (orange) microwaves and a direct-detected ¹⁹F spectrum (yellow) measured at 100 K, 9.4 T, and 10 kHz MAS. The ¹H spectra (c) show the bulk proton DNP enhancement of $\epsilon_{on/off}(^{1H}) = 256 \pm 1$. ¹H-¹⁹F CP MAS spectra (d) show the indirect fluorine DNP enhancement of $\epsilon_{on/off}(^{19F}) = 42 \pm 15$. The DNP enhanced spectrum (MW on) was recorded with 1410 scans, resulting in total experimental time of 1 h 30 min. The spectrum without enhancement (MW off) was recorded over 15 h 24 min with 15,512 scans. The direct-detected fluorine spectrum was detected over 1 h 12 min and shows no distinguishable signal. We note that the noise level is smaller for the direct detected experiment since much less scans were recorded.</figcaption></figure>
nanoparticle has shown potential as an alternative drug delivery system compared to classical oral tablets. In this case, the polymeric nano-particles are based upon poly(d,l-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer containing a hydrophobic ion-pair (salt) between pamoic acid and the API (AZD2811). Recent studies (Song, [29,36]) confirmed that the nanoparticles have a multilayer structure, consisting of a drug-rich core (≈ 21 nm radius), surrounded by a PLA-rich polymer layer (≈ 10 nm) and a hydrated PEG-rich layer on the surface (≈ 10 nm) (Fig. 2a,b).

While the polymeric layer as well as the drug contain a rather high density of protons, fluorine is only present in the drug-rich core. Direct DNP enhancement of fluorine is thus not only hampered by the ineffective direct transfer of polarization to fluorine nuclei, but also by the

spatial separation from the radical, as shown later. This is a situation that can commonly occur in pharmaceutical formulations, multicomponent materials, or other type of heterogeneous solids.

A diluted suspension of AZD2811 nanoparticles in DNP solution with the solid content of 1.2 mg of nanoparticles per 1 mL of the DNP solution was prepared and investigated. This is equivalent to about 0.25 mg/mL of API. Furthermore, the API contains 1 fluorine atom per 30 hydrogen atoms, which is less than 4 % of the API's molecular weight. ¹H solid state MAS DNP spectra of the frozen nanosuspension at 100 K were recorded with and without microwave irradiation to obtain the ¹H DNP enhancement of ε<sub>on/off</sub>(¹H) = 256 ± 1, which mostly comes from the DNP solution, and is in line with the DNP solution being optimized for ¹H enhancement. Both spectra show a peak centered at 5.81 ppm

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Solid-state CP MAS DNP spectra of siRNA compounds. Spectra were measured with (blue) and without (orange) microwave irradiation measured at 100 K, 9.4 T, and 8 kHz MAS. The measurement was performed on (a) 2'-OMethyl-rich and (b) 2'-Fluoro-rich siRNAs dissolved in DNP solution. ¹H Hahn echo spectra under microwave (MW) on/off conditions demonstrate the bulk proton DNP enhancement of ε<sub>on/off</sub>(¹H) = 161 ± 8 for (c) 2'-OMethyl-rich compound and ε<sub>on/off</sub>(¹H) = 156 ± 8 for (e) 2'-Fluoro-rich compound. ¹H-¹⁹F CP MAS spectra of siRNA (d) 2'-OMethyl-rich and (f) 2'-Fluoro-rich duplexes comprise of one fluorine signal centred at -200 ppm and demonstrate indirect ¹⁹F DNP enhancement of ε<sub>on/off</sub>(¹⁹F) = 141 ± 18 and 149 ± 9 for 2'-OMethyl-rich and 2'-Fluoro-rich compounds, respectively. ¹H-¹⁹F CP MAS are compared to the direct-detected ¹⁹F spectra with ¹H decoupling acquired over similarly long experimental time (d and f, yellow). Lower DNP enhancement and longer recycle delay of the direct-detected experiments resulted in approximately an order of magnitude lower sensitivity.</figcaption></figure>
(Fig. 2c), assigned jointly to all ¹H contained within the sample, the major contribution to this signal is thus H₂O from the DNP solution.

The ¹H buildup times T<sub>b</sub> of the AZD2811 nanosuspension were measured under both MW on and off conditions by ¹H saturation-recovery experiment (see Fig. S3 in SI). Under MW off conditions, the magnetization buildup was found to comprise of two components, the slower component with the buildup time T<sub>b,OFF</sub>(¹H) = 2.8 s was ascribed to the sample (DNP solution), while the faster component (340 ms) belongs to the silicone insert and is not detectable under the MW on conditions since it is not enhanced. Only one component (i.e. the one of the DNP solution), with a buildup time similar to that of MW off, T<sub>b, ON</sub>(¹H) = 3.0 s, was measured under the MW on conditions.

¹H–¹⁹F CP DNP enhanced spectrum of the AZD2811 nanosuspension was acquired, using 1410 scans, with a recycle delay of 3.6 s, resulting in a total acquisition time of approximately 1 h 30 min. The spectrum is presented in Fig. 2. The MW off counterpart was acquired over 15 h 24 min with 15,512 scans, recycle delay of 3.6 s to determine the DNP enhancement of ε<sub>on/off</sub> (¹⁹F) = 42 ± 15. A direct detected fluorine spectrum was recorded as well using a recycle delay of 270 s and 16 scans, and no signal was observed after 1 h 12 min, which underlines the fact that the ¹H–¹⁹F CP experiment is crucial for fluorine detection in present diluted nanoparticle suspension (Fig. 2d). We note that, although the DNP enhancement in this experiment is 42, a much higher gain in sensitivity is expected in comparison with the direct-detected fluorine experiment. This is due to the much shorter ¹H buildup times which are in order of seconds in the presence of radical, in contrast with the ¹⁹F buildup times, which are often hundreds of seconds.

### 3.2.2. siRNAs

Therapeutic siRNAs commonly contain chemical modifications at the 2' position of the ribose, such as 2'-O-Methyl and 2-deoxy-2-fluoro (2'-Fluoro) to improve their potency and stability *in vivo* [37,38]. Solutions of two siRNA duplexes targeting mRNA peptidyl-prolyl cis-trans isomerase B (Ppib) mRNA in DNP solution were prepared at a concentration of 9 mM and measured under LT-MAS DNP conditions. The first of the duplexes, referred to as 2'-OMethyl-rich, contains an alternating pattern of 2'-Fluoro and 2'-OMethyl in the antisense strand, whereas the sense strand is fully 2'-OMethyl-substituted, resulting in a total of 10 fluorine atoms per duplex (Fig. 3a). This design was chosen to produce a fluorine signal only from the antisense strand, while still potentially retaining the silencing activity of the duplex [39]. The second compound, denoted as 2'-Fluoro-rich, has an alternating 2'-OMethyl/2'-Fluoro pattern in both the antisense and sense strand, resulting in a total of 19 fluorines per duplex (Fig. 3b). Confirmation of the fluorine signal chemical shift and enhancement for both siRNAs will allow us to exploit the different chemical patterns *in vitro* for antisense and sense detection and quantification.

¹H direct detected experiment shows high DNP enhancement of the DNP matrix ε<sub>on/off</sub>(¹H) = 161 ± 8 (2'-OMethyl-rich) and ε<sub>on/off</sub>(¹H) = 156 ± 8 (2'-Fluoro-rich) typical for frozen radical solution optimized for proton polarization (Fig. 3c, e).

¹H–¹⁹F CP MAS spectra were recorded for both siRNA duplexes, one signal from all fluorine positions present in the molecule is observed at around –200 ppm. The indirect DNP enhancement was estimated as ε<sub>on/off</sub>(¹⁹F) = 141 ± 18 for 2'-OMethyl-rich compound (Fig. 3d) and as 149 ± 9 for the 2'-Fluoro-rich compound (Fig. 3f) from the MW on and off spectra. Direct-detected ¹⁹F spectra with proton decoupling were recorded over similar experimental time as ¹H–¹⁹F CP MAS experiments (1 scan and 300 s recycle delay) and are displayed on Fig. 3d and f. The direct DNP enhancement was estimated as ε<sub>on/off</sub>(¹⁹F direct) = 7 for 2'-OMethyl-rich compound and as 17 for the 2'-Fluoro-rich compound from the MW on spectra and MW off spectra (not shown). The sensitivity of the direct-detected experiments is thus roughly an order of magnitude lower as compared to the ¹H–¹⁹F CP MAS experiments. This is in line with the fact that in these experiments, the DNP solution (the radical and the matrix) was optimized for proton DNP [40]. Direct fluorine

enhancement could be improved using fluorinated matrices [17]; however, these are not bio-compatible, i.e. cannot be used under relevant conditions to study these siRNAs in cells.

The new HFX probe also enables recording of experiments such as ¹H–¹⁹F–¹³C double CP with ¹⁹F decoupling (Fig. 4a). During this experiment, the polarization from hyperpolarized protons is first transferred to fluorine spins and subsequently to the carbons that are in the vicinity of fluorine. This type of experiment, together with 2D ¹⁹F–¹³C correlation experiments, was used previously for resonance assignment of fluorinated proteins [41]. Spectra from both 2'-OMethyl-rich and 2'-Fluoro-rich compounds are presented in Fig. 4b and display ¹³C signals centered at around 84 ppm arising from the carbons in the vicinity of fluorine nuclei, in our case most probably from the C₁', C₂' and C₃' carbons of the ribose ring.

## 4. Conclusions

We demonstrate ¹H–¹⁹F CP MAS experiments that are possible with the new 3.2 mm solid-state DNP HFX probe equipped with two separate radiofrequency channels for ¹H and ¹⁹F nuclei, representing a notable advantage over commonly acquired direct-detected ¹⁹F experiments. Moreover, the possibility of ¹H decoupling during the acquisition further improves the spectral resolution. The indirect ¹⁹F DNP enhancement ε<sub>on/off</sub>(¹⁹F) = 26 ± 1 was obtained for impregnated crystalline AZD2811 powder, and the overall sensitivity gain was 30-fold (expressed as signal-to-noise ratio per mg of sample per square root of experimental time) compared to the direct-detected ¹⁹F experiment under similar conditions. An enhancement of ε<sub>on/off</sub>(¹⁹F) = 42 ± 15 was obtained for the AZD2811 nanoparticle suspension and ε<sub>on/off</sub>(¹⁹F) = 141 ± 18 and 149 ± 9 were obtained for two different (2'-OMethyl-rich and 2'-Fluoro-rich) siRNA designs in frozen DNP solution. We anticipate that this kind of experiment will find a broad range of applications especially for materials where fluorine nuclei are present in low amount, but protons are abundant, including but not limited to pharmaceutical formulations in low concentrations. Finally, we hope that the development of this new probe will significantly contribute to broader availability of fluorine solid-state NMR.

### CRediT authorship contribution statement

**Mária Šoltésová:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Arthur C. Pinon:** Writing – review & editing, Visualization, Validation, Software, Resources, Methodology, Formal analysis, Data curation, Conceptualization. **Fabien Aussenac:** Writing – review & editing, Validation, Project administration, Methodology, Data curation, Conceptualization. **Judith Schlagnitweit:** Writing – review & editing, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. **Christian Reiter:** Writing – review & editing, Software, Methodology. **Armin Purea:** Writing – review & editing, Software, Methodology. **Roberto Melzi:** Writing – review & editing, Software, Methodology. **Frank Engelke:** Writing – review & editing, Software, Methodology. **Dave Martin:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Stefanie Krambeck:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Annabelle Biscans:** Writing – review & editing, Methodology, Investigation. **Emma Kay:** Writing – review & editing, Validation, Methodology. **Lyndon Emsley:** Validation, Supervision, Project administration, Methodology, Conceptualization. **Staffan Schantz:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
<figure><img src="image_7.png" /><figcaption>Fig. 4. (a) Double-CP experiment on siRNAs. Double-CP pulse sequence comprised of two CP transfer blocks. During acquisition, only fluorine decoupling was used due to the issues with probe arcing. (b) Double-CP spectra of 2'-OMethyl-rich (upper) and 2'-Fluoro-rich (lower) siRNA compounds showing carbon peaks, most likely of the C₁', C₂' and C₃' carbons of ribose.</figcaption></figure>

the work reported in this paper.

## Acknowledgements

The authors acknowledge the Horizon Europe project PANACEA Grant (H2020, contract no.101008500). Judith Schlagnitweit acknowledges funding from the Agence Nationale de la Recherche (ANR-23-CE29-0027).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmr.2024.107827.

## Data availability

Data will be made available on request.

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