1H T1ρ Research Articles

Predicting the Stability of Formulations Containing Lyophilized Human Serum Albumin and Sucrose/Trehalose Using Solid-State NMR Spectroscopy.

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the β-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.

Miscibility of Amorphous Solid Dispersions: A Rheological and Solid-State NMR Spectroscopy Study

Miscibility is critical in the prediction of stability against crystallization of amorphous solid dispersions (ASDs) in the solid state. However, currently available approaches for its determination are limited by both theoretical and practical considerations. Recently, a rheological approach guided by the polymer overlap concentration (c*) has been proposed for miscibility quantification of ASDs [J. Pharm. Sci., 112 (2023) 204−212] and shown to be useful in predicting both accelerated and long term physical stability in the absence of moisture. However, this approach can only be performed at high temperatures (slightly above the melting temperature, Tm, of drugs), and little is known about the difference in miscibility between high and low temperatures (e.g., below the glass transition temperature, Tg). Here we compare the miscibility of nifedipine (NIF)/polyvinylpyrrolidone (PVP) ASDs as determined by the rheological approach at 175°C (∼3°C above Tm of NIF) and solid state NMR (ssNMR) 1H T1 and T1ρ relaxation times at -20°C (∼66°C below Tg of NIF). Our results indicate agreement between the two methods. For low molecular weight (Mw) PVP, T1ρ measurements are more consistent with the rheological approach, while T1 measurements are closer for relatively high Mw PVP. Our findings support the use of the c* based rheological approach for inferring miscibility of deeply cooled ASDs.

Kinetics of 1H →31P NMR cross-polarization and dynamics in a layered crystalline α-Sn(IV) phosphate

The proton-phosphorus (H–P) cross-polarization (CP) is effective in Sn(HPO4)2·H2O despite of the presence of paramagnetic ion impurities. Polarization constants TH-P and 1H T1ρ times are measured in static Sn(HPO4)2·H2O by the kinetic variable-temperature H–P CP experiments. The temperature dependence of the 1H T1ρ times is interpreted in terms of proton movements in the interlayer space occurring between the phosphate groups without participation of the water molecules. The process requires an activation energy of 8.7 ± 0.7 kcal/mol. The MAS effect on the 1H T1ρ times is shown and discussed.

Analytical descriptions of (multiple-contact) cross-polarization dynamics and spin-lattice relaxation in solid alanine

In this work, several exact and approximate analytical solutions to the quantum master equation are derived using both classical and non-classical coupling models to describe the kinetics of Hartmann-Hahn cross-polarization (HHCP) and multiple-contact CP (MCCP). Moreover, the analytical solution originally obtained by Naito and McDowell [J. Chem. Phys. 84 (1986) 4181.] is shown to be incorrect and the different regimes of spin diffusion and T1ρ relaxation are characterized by the amplitude of the second stage of the HHCP dynamics and the HHCP/MCCP crossing time. The analysis of the 1H–13C HHCP and MCCP dynamics together with (Lee-Goldburg) 1H T1ρ relaxation experimental data provides a consistent picture of spin dynamics in solid alanine and explains the apparent discrepancies previously observed between T1ρ and T1 relaxation measurements. The CH and CH3 protons relax as expected via spin diffusion towards the NH3 protons but the assumption of common proton spin temperature, in which the bottleneck of relaxation is at the NH3 sites, generally valid for T1 relaxation breaks down for T1ρ relaxation. A diffusion-limited situation in which nuclear Zeeman energy is transferred to the lattice faster than can be supplied by spin diffusion is observed instead.

SPICY: a method for single scan rotating frame relaxometry.

T 1ρ is an NMR relaxation mode that is sensitive to low frequency molecular motions, making it an especially valuable tool in biomolecular research. Here, we introduce a new method, SPICY, for measuring T1ρ relaxation times. In contrast to conventional T1ρ experiments, in which the sequence is repeated many times to determine the T1ρ time, the SPICY sequence allows determination of T1ρ within a single scan, shortening the experiment time remarkably. We demonstrate the method using 1H T1ρ relaxation dispersion experiments. Additionally, we combine the sequence with spatial encoding to produce 1D images in a single scan. We show that T1ρ relaxation times obtained using the single scan approach are in good agreement with those obtained using the traditional experiments.

1H T1ρ based ROSY: NMR spectral fingerprints for nanoscale phase separated structure of block copolymers

1H T1ρ based ROSY: NMR spectral fingerprints for nanoscale phase separated structure of block copolymers

Molecular dynamics between amorphous and crystalline phases of e-beam irradiated piezoelectric PVDF thin films employing solid-state NMR spectroscopy

Piezoelectric bi-axially and uni-axially stretched PVDF films subjected to e-beam irradiation doses of 5 kGy, 500 kGy and 1 MGy and films not irradiated have been studied. It was found that the piezoelectric responses of both untreated and e-beam irradiated PVDF films under strain strongly depends on their initial manufacturing processing. The initial structural differences were mainly based on crystalline phase composition, notably the α to β polymorph ratio. 19F MAS Solid State NMR measurements performed with fast sample spinning (31.25 kHz) allow us to accurately determine the proportion of crystal and amorphous phases. Considering only the crystalline phases, it was concluded that the uni-axial ((β)-PVDF) film was almost composed of 100% of β-phase while the biaxially-oriented ((α-β)-PVDF) was composed of 81% β-phase and 19% of α-phase. It was also shown that the changes of local dynamic processes of PVDF chains in kHz timescale enable to reflect the degradation of both the crystalline and amorphous phases. Presence of small molecular-weight chemical compounds was evidenced by Carr–Purcell–Meiboom–Gill (CPMG) 1H MAS NMR experiment. The degradation process was in agreement with melting peak decrease registered by DSC. 19F/1H T1ρ relaxation times and analysis of cross-polarization (CP) buildup curves are discussed. Significant scatter of relaxation parameters (19F T1ρ and 1H T1ρ) suggested an important degradation of polymer structure. The 1H T1ρ values for PVDF samples irradiated at 1 MGy mainly imply chain scission mechanisms of degradation rather than cross-linking processes. SS NMR spectroscopy data supported assumption that e-beam irradiation brings flexibility to piezoelectric PVDF films which improved their piezoelectric voltage output generated by deformation up to doses of 500kGy. Above this dose range, degradations were very important and affected severely PVDF films mechanical properties.

Thermal stability, cation dynamics, and ferroelastic domain walls in the α→β→γ phase transitions of perovskite (C2H5NH3)2MnCl4 crystals

Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) confirmed that the (C2H5NH3)2MnCl4 crystal undergoes α-β-γ phase transitions at approximately TC2 = 225 K and TC1 = 424 K, remains stable until 470 K, and displays a weight loss of 25.24% at around 579 K due to partial thermal decomposition. Cation dynamics near these temperatures was investigated by MAS 1H NMR and MAS 13C NMR experiments. The small changes in the 1H chemical shift for C2H5 near TC2 are consistent with the transition from the orthorhombic γ-phase to the orthorhombic β-phase. Also, the abrupt changes in NMR signals near TC1 correspond to structural change from the orthorhombic β-phase to the tetragonal α-phase. The 13C spin-lattice relaxation time in the rotating frame (T1ρ) below TC2 is shorter for CH3 than that for CH2, due to the greater mobility at the free end of the alkyl chain. In addition, the temperature-dependent ferroelastic domain structure was studied near the α-β phase transition. There was no noticeable temperature dependence in 1H and 13C T1ρ values at TC1 and TC2, whereas the change in the domain wall was prominent near TC1.

Determining effect of partial substitution of paramagnetic Mn2+ ions in perovskite (MA)2Zn1-xMnxCl4 mixed crystals through MAS NMR relaxation times

This study discusses the effects of the partial replacement of Zn2+ ions with paramagnetic Mn2+ ions (x = 0.1, 0.3, 0.5, and 0.7) in ecofriendly (MA)2Zn1-xMnxCl4 lead-free perovskite crystals through magic angle spinning nuclear magnetic resonance relaxation times. These crystals are grown and their structures are measured through X-ray diffraction experiments. Further, the spin-lattice relaxation time, T1ρ, in the rotating frame is measured for 1H and 13C nuclei of (MA)+ cations to understand the local environments with respect to the Mn2+ ion content. The structures and chemical shifts for CH3 and NH3 with x = 0.1 and 0.3 were highly similar to those with x = 0, whereas those for CH3 and NH3 with x = 0.5 and 0.7 were highly similar to those with x = 1. Nonetheless, the 1H T1ρ depending on the paramagnetic ion content became shorter because (MA)+ cations were bound to the paramagnetic Mn2+ layer through N–H⋯Cl bonds. The main effects were the differences in the metal Zn2+ and Mn2+ ions.

Thermal decomposition and structural dynamics in perovskite (C2H5NH3)2CdCl4 crystals

In this study, (C2H5NH3)2CdCl4 single crystals were grown, and their thermal properties were studied by the thermogravimetric analysis and differential scanning calorimetry. Phase transition temperature above room temperature was observed at 470 K. In addition, the cation dynamics in this crystal were investigated by 1H magic-angle spinning nuclear magnetic resonance (MAS NMR) and 13C cross-polarization (CP)/MAS NMR experiments. The Bloembergen–Purcell–Pound curves for 1H T1ρ and 13C T1ρ in C2H5NH3 cation exhibited minimum values, and these curves represent C2H5NH3 and C2H5 rotational motions. The activation energy for 1H in the C2H5NH3 cation is Ea = 22.63 kJ mol−1, whereas those for 13C in CH3 and CH2 are Ea = 18.05 and 19.14 kJ mol−1, respectively. Furthermore, the proton dynamics of C2H5NH3 undergo faster rotation than carbons. This implies that the molecular motion for 1H is enhanced at the C-end and N-end of an organic cation, whereas the molecular motion is not free at the main chain carbons of the organic cation.

Phase Behavior of Amorphous Solid Dispersions of Felodipine: Homogeneity and Drug-Polymer Interactions.

In the current investigation, the role of drug-polymer hydrogen bonding (H-bonding) with respect to the phase behavior of amorphous solid dispersions (ASDs) is studied in depth on a nanometer level. Melt-quenched dispersions of felodipine (FEL) with poly(vinylpyrrolidone), or PVP, poly(vinylpyrrolidone-co-vinylacetate), or PVP/VA, and poly(vinylacetate), or PVAc, were prepared at drug loadings of 50-90% w/w. Modulated differential scanning calorimetry (MDSC) was used to detect microscopic homogeneity for each set of ASDs. A single composition dependent glass transition temperature (Tg) was observed over the entire composition range in MDSC data for each set of ASDs; however some samples within each set of ASDs showed a crystallization exotherm and corresponding melting endotherm in the first heating scan. Solid-state nuclear magnetic resonance spectroscopy (SSNMR) was further employed to understand phase homogeneity in these systems. The proton spin-lattice relaxation times in the laboratory and rotating frame (1H T1 and T1ρ) for the drug and individual polymer for each set of ASDs were measured to evaluate phase homogeneity. On the basis of proton relaxation measurements, it was revealed that FEL:PVP and FEL:PVP/VA ASDs exhibited better compositional homogeneity than FEL:PVAc ASDs. The strength and the extent of H-bonding were studied by using 13C SSNMR spectra. In addition, deconvolution of the carbonyl region of amorphous FEL revealed that 40% of amorphous FEL molecules were hydrogen bonded (H-bonded) through dimers and the remaining 60% were free/non H-bonded. The dimer fraction decreased as the polymer content increased for each set of ASDs, while the free fraction increased. This indicated that the polymers containing hydrogen bond acceptor groups disrupted dimers and formed intermolecular H-bonding interactions with FEL. The strength and extent of FEL:polymer H-bonding was rank ordered as PVP > PVP/VA > PVAc. These findings were also confirmed through DFT calculations on these systems. Our results suggest that drug-polymer H-bonding interaction may impact the phase homogeneity in ASDs formulated by a specific method. The data from the current study further demonstrate that SSNMR is a powerful tool for characterizing phase homogeneity in ASDs with sub-50 nm resolution. In addition, SSNMR can provide insights into drug-polymer interactions and speciation in ASDs.

Study on Paramagnetic Interactions of (CH3NH3)2CoBr4 Hybrid Perovskites Based on Nuclear Magnetic Resonance (NMR) Relaxation Time.

The thermal properties of organic–inorganic (CH3NH3)2CoBr4 crystals were investigated using differential scanning calorimetry and thermogravimetric analysis. The phase transition and partial decomposition temperatures were observed at 460 K and 572 K. Nuclear magnetic resonance (NMR) chemical shifts depend on the local field at the site of the resonating nucleus. In addition, temperature-dependent spin–lattice relaxation times (T1ρ) were measured using 1H and 13C magic angle spinning NMR to elucidate the paramagnetic interactions of the (CH3NH3)+ cations. The shortening of 1H and 13C T1ρ of the (CH3NH3)2CoBr4 crystals are due to the paramagnetic Co2+ effect. Moreover, the physical properties of (CH3NH3)2CoBr4 with paramagnetic ions and those of (CH3NH3)2CdBr4 without paramagnetic ions are reported and compared.

Polymer Infiltration into Metal-Organic Frameworks in Mixed-Matrix Membranes Detected in Situ by NMR.

Solid-state NMR has been used to study mixed-matrix membranes (MMMs) prepared with a metal-organic framework (MOF, UiO-66) and two different high molecular weight polymers (PEO and PVDF). 13C and 1H NMR data provide overwhelming evidence that most UiO-66 organic linkers are within 1 nm of PEO, which indicates that PEO is homogeneously distributed throughout the MOF. Systematic changes in MOF 13C NMR peak positions and 1H NMR line widths, as well as dramatic reductions in the MOF 1H T1ρ relaxation times, are observed as the PEO content increases, and when the pores have been filled, a further increase in PEO results in the formation of semicrystalline PEO outside the UiO-66 particles. In contrast, similar studies on PVDF MMMs show that the polymer contacts only a small fraction (<20%) of the MOF linkers. Simulations confirm that PEO penetrates into UiO-66 more easily than does PVDF. These studies are among the first to provide experimental insights into MOF-polymer interactions in an MMM.

Rubber-Filler Interactions in Polyisoprene Filled with In Situ Generated Silica: A Solid State NMR Study.

In this paper we used high- and low-resolution solid state Nuclear Magnetic Resonance (NMR) techniques to investigate a series of polyisoprene samples filled with silica generated in situ from tetraethoxysilane by sol-gel process. In particular, 1H spin-lattice and spin-spin relaxation times allowed us to get insights into the dynamic properties of both the polymer bulk and the bound rubber, and to obtain a comparative estimate of the amount of bound rubber in samples prepared with different compositions and sol-gel reaction times. In all samples, three fractions with different mobility could be distinguished by 1H T2 and ascribed to loosely bound rubber, polymer bulk, and free chain ends. The amount of bound rubber was found to be dependent on sample preparation, and it resulted maximum in the sample showing the best dispersion of silica domains in the rubber matrix. The interpretation of the loosely bound rubber in terms of “glassy” behaviour was discussed, also on the basis of 1H T1 and T1ρ data.

Solid-State NMR Investigation of Drug-Excipient Interactions and Phase Behavior in Indomethacin-Eudragit E Amorphous Solid Dispersions.

To investigate the nature of drug-excipient interactions between indomethacin (IMC) and methacrylate copolymer Eudragit® E (EE) in the amorphous state, and evaluate the effects on formulation and stability of these amorphous systems. Amorphous solid dispersions containing IMC and EE were spray dried with drug loadings from 20% to 90%. PXRD was used to confirm the amorphous nature of the dispersions, and DSC was used to measure glass transition temperatures (Tg). 13C and 15N solid-state NMR was utilized to investigate changes in local structure and protonation state, while 1H T1 and T1ρ relaxation measurements were used to probe miscibility and phase behavior of the dispersions. Tg values for IMC-EE solid dispersions showed significant positive deviations from predicted values in the drug loading range of 40-90%, indicating a relatively strong drug-excipient interaction. 15N solid-state NMR exhibited a change in protonation state of the EE basic amine, with two distinct populations for the EE amine at -360.7ppm (unprotonated) and -344.4ppm (protonated). Additionally, 1H relaxation measurements showed phase separation at high drug load, indicating an amorphous ionic complex and free IMC-rich phase. PXRD data showed all ASDs up to 90% drug load remained physically stable after 2years. 15N solid-state NMR experiments show a change in protonation state of EE, indicating that an ionic complex indeed forms between IMC and EE in amorphous solid dispersions. Phase behavior was determined to exhibit nanoscale phase separation at high drug load between the amorphous ionic complex and excess free IMC.

Structural Phase Transition of Perovskite-Type N(CH3)4CdBr3 Studied by MAS NMR and Static NMR

The structural geometry change in the perovskite-type N(CH3)4CdBr3 single crystal near the phase transition temperature of TC = 390 K was investigated using magic angle spinning nuclear magnetic resonance techniques. For 1H and 13C nuclei, the temperature dependences of their chemical shift, spectral intensity, and spin–lattice relaxation time (T1ρ) in the rotating frame were obtained and analyzed. While the chemical shift and T1ρ of 1H showed change near TC, those of 13C did not. In addition, the 113Cd spin–lattice relaxation time T1 in the laboratory frame near TC show no evidence of anomalous change near the phase transition temperature, which coincides with the measured changes in the 1H T1ρ. The driving force for this phase transition was connected to the 1H in the CH3 groups.

Investigating the two inequivalent NH2(CH3)2 ions in [NH2(CH3)2]2CuCl4 using magic angle spinning nuclear magnetic resonance

The structural change near the phase transition temperatures of [NH2(CH3)2]2CuCl4 is discussed in terms of the chemical shifts and the spin-lattice relaxation times T1ρ in the rotating frame for 1H MAS NMR and 13C CP/MAS NMR. The 1H T1ρ undergoes molecular motion near the phase-transition temperature (TC2 = 253 K). In addition, the two inequivalent [NH2(CH3)2] (1) and [NH2(CH3)2] (2) sites were distinguishable by the 13C chemical shift. And, the most significant change was observed at TC2 for the 13C CP/MAS NMR spectrum; this temperature corresponds to a ferroelastic phase transition with different orientations.

31P, 1H NMR Relaxation and Molecular Mobility in Layered α-Zirconium Phosphate: Variable-Temperature NMR Experiments

Internal rotations in HPO4– groups and proton transfer between them have been characterized in static α-zirconium phosphate by variable-temperature measurements of 31P T1 and 1H T1ρ times. The 1H T1ρ times have been obtained by the kinetic of phosphorus–proton cross-polarization treated with the I-I*-S model. The calculations of the temperature T1 relaxation dependences have resulted in the rotational activation energy of 2.6 kcal/mol (or 3.9 kcal/mol in account for the τC distribution) and the proton transfer activation energy of 5.5 kcal/mol. Thus, two types of motions have been found in α-zirconium phosphate: the low-frequency proton transfer with the correlation time τc of 1.5 × 10–5 s at 295 K and the high-frequency rotation in P–OH groups with the correlation time τc of 2.2 × 10–8 s at 295 K.

Investigation of drug-excipient interactions in lapatinib amorphous solid dispersions using solid-state NMR spectroscopy.

This study investigated the presence of specific drug-excipient interactions in amorphous solid dispersions of lapatinib (LB) and four commonly used pharmaceutical polymers, including Soluplus, polyvinylpyrrolidone vinyl acetate (PVPVA), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and hydroxypropylmethylcellulose phthalate (HPMCP). Based on predicted pKa differences, LB was hypothesized to exhibit a specific ionic interaction with HPMCP, and possibly with HPMCAS, while Soluplus and PVPVA were studied as controls without ionizable functionality. Thermal studies showed a single glass transition (Tg) for each dispersion, in close agreement with predicted values for Soluplus, PVPVA, and HPMCAS systems. However, the Tg values of LB-HPMCP solid dispersions were markedly higher than predicted values, indicating a strong intermolecular interaction between LB and HPMCP. (15)N solid-state NMR provided direct spectroscopic evidence for protonation of LB (i.e., salt formation) within the HPMCP solid dispersions. (1)H T1 and (1)H T1ρ relaxation studies of the dispersions supported the ionic interaction hypothesis, and indicated multiple phases in the cases of excess drug or polymer. In addition, the dissolution and stability behavior of each system was examined. Both acidic polymers, HPMCAS and HPMCP, effectively inhibited the crystallization of LB on accelerated stability, likely owing to beneficial strong intermolecular hydrogen and/or specific ionic bonds with the acidic polymers. Soluplus and PVPVA showed poor physical properties on stability and subsequently poor crystallization inhibition.

Structure and Dynamics of Brachypodium Primary Cell Wall Polysaccharides from Two-Dimensional 13C Solid-State Nuclear Magnetic Resonance Spectroscopy

The polysaccharide structure and dynamics in the primary cell wall of the model grass Brachypodium distachyon are investigated for the first time using solid-state nuclear magnetic resonance (NMR). While both grass and non-grass cell walls contain cellulose as the main structural scaffold, the former contains xylan with arabinose and glucuronic acid substitutions as the main hemicellulose, with a small amount of xyloglucan (XyG) and pectins, while the latter contains XyG as the main hemicellulose and significant amounts of pectins. We labeled the Brachypodium cell wall with (13)C to allow two-dimensional (2D) (13)C correlation NMR experiments under magic-angle spinning. Well-resolved 2D spectra are obtained in which the (13)C signals of cellulose, glucuronoarabinoxylan (GAX), and other matrix polysaccharides can be assigned. The assigned (13)C chemical shifts indicate that there are a large number of arabinose and xylose linkages in the wall, and GAX is significantly branched at the developmental stage of 2 weeks. 2D (13)C-(13)C correlation spectra measured with long spin diffusion mixing times indicate that the branched GAX approaches cellulose microfibrils on the nanometer scale, contrary to the conventional model in which only unbranched GAX can bind cellulose. The GAX chains are highly dynamic, with average order parameters of ~0.4. Biexponential (13)C T1 and (1)H T1ρ relaxation indicates that there are two dynamically distinct domains in GAX: the more rigid domain may be responsible for cross-linking cellulose microfibrils, while the more mobile domain may fill the interfibrillar space. This dynamic heterogeneity is more pronounced than that of the non-grass hemicellulose, XyG, suggesting that GAX adopts the mixed characteristics of XyG and pectins. Moderate differences in cellulose rigidity are observed between the Brachypodium and Arabidopsis cell walls, suggesting different effects of the matrix polysaccharides on cellulose. These data provide the first molecular-level structural information about the three-dimensional organization of the polysaccharides in the grass primary wall.