Characterization of Controlled Release Starch-Nimodipine Implant for Antispasmodic and Neuroprotective Therapies in the Brain.

Parenteral depot systems can provide a constant release of drugs over a few days to months. Most of the parenteral depot products on the market are based on poly(lactic acid) and poly(lactide-co-glycolide) (PLGA). Studies have shown that acidic monomers of these polymers can lead to nonlinear release profiles or even drug inactivation before release. Therefore, finding alternatives for these polymers is of great importance. Our previous study showed the potential of starch as a natural and biodegradable polymer to form a controlled release system. Subarachnoid hemorrhage (SAH) is a life-threatening type of stroke and a major cause of death and disability in patients. Nimotop® (nimodipine (NMD)) is an FDA-approved drug for treating SAH-induced vasospasms. In addition, NMD has, in contrast to other Ca antagonists, unique neuroprotective effects. The oral administration of NMD is linked to variable absorption and systemic side effects. Therefore, the development of a local parenteral depot formulation is desirable. To avoid the formation of an acidic microenvironment and autocatalytic polymer degradation, we avoided PLGA as a matrix and investigated starch as an alternative. Implants with drug loads of 20 and 40% NMD were prepared by hot melt extrusion (HME) and sterilized with an electron beam. The effects of HME and electron beam on NMD and starch were evaluated with NMR, IR, and Raman spectroscopy. The release profile of NMD from the systems was assessed by high-performance liquid chromatography. Different spectroscopy methods confirmed the stability of NMD during the sterilization process. The homogeneity of the produced system was proven by Raman spectroscopy and scanning electron microscopy images. In vitro release studies demonstrated the sustained release of NMD over more than 3 months from both NMD systems. In summary, homogeneous nimodipine-starch implants were produced and characterized, which can be used for therapeutic purposes in the brain.

Reproduction of a conventional anthropomorphic female chest phantom by 3D-printing: Comparison of image contrasts and absorbed doses in CT.

BACKGROUND: The production of individualized anthropomorphic phantoms via three-dimensional (3D) printing methods offers promising possibilities to assess and optimize radiation exposures for specifically relevant patient groups (i.e., overweighed or pregnant persons) that are not adequately represented by standardized anthropomorphic phantoms. However, the equivalence of printed phantoms must be demonstrated exemplarily with respect to the resulting image contrasts and dose distributions. PURPOSE: To reproduce a conventionally produced anthropomorphic phantom of a female chest and breasts and to evaluate their equivalence with respect to image contrasts and absorbed doses at the example of a computed tomography (CT) examination of the chest. METHODS: In a first step, the effect of different print settings on the CT values of printed samples was systematically investigated. Subsequently, a transversal slice and breast add-ons of a conventionally produced female body phantom were reproduced using a multi-material extrusion-based printer, considering six different types of tissues (muscle, lung, adipose, and glandular breast tissue, as well as bone and cartilage). CT images of the printed and conventionally produced phantom parts were evaluated with respect to their geometric correspondence, image contrasts, and absorbed doses measured using thermoluminescent dosimeters. RESULTS: CT values of printed objects are highly sensitive to the selected print settings. The soft tissues of the conventionally produced phantom could be reproduced with a good agreement. Minor differences in CT values were observed for bone and lung tissue, whereas absorbed doses to the relevant tissues were identical within the measurement uncertainties. CONCLUSION: 3D-printed phantoms are with exception of minor contrast differences equivalent to their conventionally manufactured counterparts. When comparing the two production techniques, it is important to note that conventionally manufactured phantoms should not be considered as absolute benchmarks, as they also only approximate the human body in terms of its absorption, and attenuation of x-rays as well as its geometry.

Graphene Oxide-Grafted Hybrid Diblock Copolymer Brush (GO-graft-PEG6k-block-P(MA-POSS)) as Nanofillers for Enhanced Lithium Ion Conductivity of PEO-Based Nanocomposite Solid Polymer Electrolytes.

Nanocomposite solid polymer electrolytes (NSPEs) with PEO as the matrix and (i) GO or (ii) GO-graft-PEG6k or (iii) GO-graft-PEG6k-block-P(MA-POSS) as nanofillers have been fabricated to elucidate the impact of the filler morphology on the lithium ion conductivity. GO-graft-PEG6k was obtained by grafting PEG6k onto GO via esterification. GO-graft-PEG6k-block-P(MA-POSS) was prepared via surface-initiated atom transfer radical polymerization. Fourier-transform infrared spectroscopy revealed enhanced salt dissociation and complexation between the filler and PEO host that could be attributed to Lewis acid-base interactions. Electrochemical impedance spectroscopy revealed the improved ion conductivity of the fabricated NSPEs as compared with the pristine PEO-LiClO4. As an example, at 50 °C, the ion conductivity increased to 4.01 × 10-5 and 6.31 × 10-5 S cm-1 with 0.3% GO and 0.3% GO-graft-PEG6k, respectively, from 2.36 × 10-5 S cm-1 of PEO-LiClO4, suggesting that the filler with brush-like architecture (GO-graft-PEG6k) is more efficient in enhancing the ion conductivity. Further increase in filler content resulted in lowering of the ion conductivity that could be ascribed to aggregation of the filler. The most dramatic impact on conductivity was observed with the incorporation of brush-like GO-graft-PEG6k-block-P(MA-POSS) as a nanofiller (3.0 × 10-4 S cm-1 at 50 °C with 1.0 wt % filler content). The increase in ion conductivity in the current systems, as opposed to the conventional view, could not be correlated with the content of the amorphous phase of the matrix. The conduction mechanism is still unclear; nevertheless, it could be assumed that in addition to the ion conduction through the PEO matrix, the filler forms additional low-energy ion conducting channels at its interface with the matrix. The pendent POSS nanocages of GO-graft-PEG6k-block-P(MAPOSS) might probably increase the free volume at the interface with the matrix that is associated with higher chain and ion mobility, thus further enhancing the ion conductivity as compared with GO and GO-graft-PEG6k. The faster ion dynamics in 1.0 wt % GO-graft-PEG6k-block-P(MAPOSS) NSPEs has also been verified by the dielectric relaxation studies. Thus, integration of both the PEG and POSS nanocages into GO-grafted brush-like architecture offers a new tool for tuning the lithium ion conductivity for potential Li ion battery applications.

Tissue equivalence of 3D printing materials with respect to attenuation and absorption of X-rays used for diagnostic and interventional imaging.

INTRODUCTION: Three-dimensional printing is a promising technology to produce phantoms for quality assurance and dosimetry in X-ray imaging. Crucial to this, however, is the use of tissue equivalent printing materials. It was thus the aim of this study to evaluate the properties of a larger number of commercially available printing filaments with respect to their attenuation and absorption of X-rays. MATERIALS AND METHODS: Apparent kerma attenuation coefficients (AKACs) and absorbed doses for different X-ray spectra (tube voltages, 70-140 kV) were measured and simulated by Monte-Carlo computations for a larger number of fused-deposition-modeling (FDM) materials. The results were compared with the respective values simulated for reference body tissues. In addition, the properties of polylactide acid samples printed with reduced infill densities were investigated. RESULTS: Measured and simulated AKACs and absorbed doses agreed well with each other and in case of AKACs also with attenuation coefficients derived from the reference database of the National Institute of Standards and Technology (NIST). For lung, adipose, muscle, and bulk soft tissue as well as for spongiosa (cancellous bone), printed materials with equivalent attenuation as well as absorption properties could be identified. In contrast, none of the considered printed materials was equivalent to cortical bone. CONCLUSION: Several FDM materials have been identified as well-suited substitutes for body tissues in terms of the investigated material characteristics. They can therefore be used for in-house production of individualized and task-specific phantoms for image quality assessment and dose measurements in X-ray imaging.

Polymer Networks Synthesized from Poly(Sorbitol Adipate) and Functionalized Poly(Ethylene Glycol).

Polymer networks were prepared by Steglich esterification using poly(sorbitol adipate) (PSA) and poly(sorbitol adipate)-graft-poly(ethylene glycol) mono methyl ether (PSA-g-mPEG12) copolymer. Utilizing multi-hydroxyl functionalities of PSA, poly(ethylene glycol) (PEG) was first grafted onto a PSA backbone. Then the cross-linking of PSA or PSA-g-mPEG12 was carried out with disuccinyl PEG of different molar masses (Suc-PEGn-Suc). Polymers were characterized through nuclear magnetic resonance (NMR) spectroscopy, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). The degree of swelling of networks was investigated through water (D2O) uptake studies, while for detailed examination of their structural dynamics, networks were studied using 13C magic angle spinning NMR (13C MAS NMR) spectroscopy, 1H double quantum NMR (1H DQ NMR) spectroscopy, and 1H pulsed field gradient NMR (1H PFG NMR) spectroscopy. These solid state NMR results revealed that the networks were composed of a two component structure, having different dipolar coupling constants. The diffusion of solvent molecules depended on the degree of swelling that was imparted to the network by the varying chain length of the PEG based cross-linking agent.

Miniaturized Measurement of Drug-Polymer Interactions via Viscosity Increase for Polymer Selection in Amorphous Solid Dispersions.

Drug-polymer interactions have a substantial impact on stability and performance of amorphous solid dispersions (ASD) but are difficult to analyze. Whereas there are many screening methods described for polymer selection based for example on glass forming ability, drug-polymer miscibility, supersaturation, or inhibition of recrystallization, the distinct detection of physico-chemical interactions mostly lacks miniaturized techniques. This work presents an interaction screening assessing the relative viscosity increase between highly concentrated polymer solutions with and without the model drug ketoconazole (KTZ). The fluorescent molecular rotor 9-(2-carboxy-2-cyanovinyl)julolidine was added to the solutions in a miniaturized setup in µL-scale. Due to its environment-sensitive emission behavior, the integrated fluorescence intensity can be used as a viscosity dye within this screening approach (FluViSc). Differences in relative viscosity increases through addition of KTZ were proposed to rank polymers regarding KTZ-polymer interactions. Absolute viscosities were measured with a cone-plate rheometer as a complimentary method and supported the results acquired by the FluViSc. Solid-state nuclear magnetic resonance (ss-NMR) relaxation time measurements and Raman spectroscopy were utilized to investigate drug-polymer interactions at a molecular level. Whereas Raman spectroscopy was not suited to reveal KTZ-polymer interactions, ss-NMR relaxation time measurements differentiated between the selected polymeric carriers hydroxypropylmethylcellulose acetate succinate (HPMCAS) and polyvinylpyrrolidone vinyl acetate 60:40 (PVP-VA64). Interactions were detected for HPMCAS/KTZ ASD while there was no hint for interactions between KTZ and PVP-VA64. These results were in correlation with the FluViSc. The findings were correlated with the dissolution performance of ASD and found to be predictive for supersaturation and inhibition of precipitation during dissolution.

Ion Transport Properties and Ionicity of 1,3-Dimethyl-1,2,3-Triazolium Salts with Fluorinated Anions.

1,2,3-Triazolium salts are an important class of materials with a plethora of sophisticated applications. A series of three novel 1,3-dimethyl-1,2,3-triazolium salts with fluorine, containing anions of various size, is synthesized by methylation of 1,2,3-triazole. Their ion conductivity is measured by impedance spectroscopy, and the corresponding ionicities are determined by diffusion coefficients obtained from ¹H and 19F pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy data, revealing that the anion strongly influences their ion conductive properties. Since the molar ion conductivities and ionicities of the 1,3-dimethyl-1,2,3-triazolium salts are enhanced in comparison to other 1,2,3-triazolium salts with longer alkyl substituents, they are promising candidates for applications as electrolytes in electrochemical devices.

Molecular Dynamics in the Crystalline Regions of Poly(ethylene oxide) Containing a Well-Defined Point Defect in the Middle of the Polymer Chain.

The chain mobility in crystals of a homopolymer of poly(ethylene oxide) (PEO) with 22 monomer units (PEO22) is compared with that of a PEO having the identical number of monomer units but additionally a 1,4-disubstituted 1,2,3-triazole (TR) point defect in the middle of the chain (PEO11-TR-PEO11). In crystals of PEO22, the characteristic αc-relaxation (helix jumps) is detected and the activation energy of this process is calculated from the pure crystalline 1H FIDs to 67 kJ/mol. PEO11-TR-PEO11 exhibits a more complex behavior, i.e. a transition into the high temperature phase HTPh is noticed during heating in the temperature range between -5 and 10 °C which is attributed to the incorporation of the TR ring into the crystalline lamellae. The crystal mobility of the low temperature phase LTPh of PEO11-TR-PEO11 is in good agreement with PEO22 since helical jump motions could also be detected by analysis of the 1H FIDs and the corresponding values of their second moments M2. In contrast, the high temperature phase of PEO11-TR-PEO11 shows a completely different behavior of the crystal mobility. The crystalline PEO chains are rigid in this HTPh on the time scale of both, the 1H time-domain technique and in 13C MAS CODEX NMR spectroscopy, i.e. the αc-mobility of PEO in the HTPh of PEO11-TR-PEO11 is completely suppressed and the PEO11 chains are converted into a crystal-fixed polymer due to the incorporation of the TR rings into the crystal structure. However, the TR defect of PEO11-TR-PEO11 shows in the HTPh characteristic π-flip motions with an Arrhenius type activation energy of 223 kJ/mol measured by dielectric relaxation spectroscopy. This motion cannot be observed by corresponding 13C MAS CODEX NMR measurements due to an interfering spin-dynamic effect.

Chain Tilt and Crystallization of Ethylene Oxide Oligomers with Midchain Defects.

Many text books and publications do not focus on the necessity of chain tilting in crystalline lamellae of oligomers and polymers, a fundamental aspect of their crystallization already discussed by Flory. Herein we investigate the chain tilt of ethylene oxide oligomers (EOs) containing various midchain defects by WAXS, SAXS and solid state 13C MAS NMR spectroscopy. At low temperatures, one out of the two EO chains of EO9-meta-EO9 and EO11-TR-EO11 containing a 1,3-disubstituted benzene or a 1,4-disubstituted 1,2,3-triazole defect in central position of the oligomer chain forms crystals and the other EO chain as well as the defect remain in the amorphous phase. The aromatic midchain defect of these two oligomers can be incorporated into the crystalline lamella upon heating below Tm. Then, the adjoining amorphous EO chain crosses from the lamellae to the amorphous regions at an angle ξ, which is preordained by the substitution pattern of the aromatic defect, revealing that the chain tilt angle ranges between 36° ≤ ϕ ≤ 60°.

New insights into the interaction of proteins and disaccharides-The effect of pH and concentration.

To gain new insights into the interaction of proteins and disaccharides, we investigated the hydrodynamic radii, RhProt, of lysozyme molecules in solution and in a ternary protein-sugar-water system by PFG-NMR. Our approach is based on the assumption that the anhydrobiotic properties of disaccharides like trehalose are based on aggregation of sugar molecules to the proteins, i.e., accumulation of sugar molecules close to the protein, and that this process can be investigated by the experimentally detectable RhProt value of the protein. The Rh values are calculated from the experimentally determined diffusion coefficients and the application of a viscosity correction using the inert molecule dioxane as an internal viscosity reference. The experiments were performed as a function of sugar concentration, the overall particle concentration and the pH value. We investigated the disaccharides trehalose and sucrose, mainly for the reason that trehalose has well know cryptobiotic properties while sucrose, which is similar in size and structure, lacks these properties. The results show the formation of a protective sugar shell around the proteins over a wider range of concentrations and pH values in the case of trehalose.

Proton conductivity and phase transitions in 1,2,3-triazole.

1,2,3-Triazole (TR) is a good proton conductor which is tidely related to formation of a hydrogen bond network along the N-HN trajectory and its self-dissociation into diH-1,2,3-triazolium and 1,2,3-triazolate. To gain a deeper understanding, the proton conductivity of TR is measured by impedance spectroscopy (IS) across its melting temperature and an additionally discovered solid-solid phase transition. The orthorhombic high temperature phase and the monoclinic low temperature modification are investigated by polarized optical microscopy, DSC- and WAXS measurements. Furthermore, the diffusion coefficients of TR are determined from IS data and measured by (1)H PFG NMR spectroscopy in the melt which allows for separate evaluation of contributions of proton hopping across the hydrogen bond network and the vehicle mechanism to the proton conductivity where the vehicles are defined as charged species generated by TR self-dissociation. Finally, the degree of dissociation of TR is calculated and the influence of the self-dissociation of TR on the proton conductivity is discussed in the context of the dielectric constant.

Helical jump motions of poly(L-lactic acid) chains in the α phase as revealed by solid-state NMR.

The molecular dynamics of Poly(L-lactic Acid) (PLLA) chains in the α phase was investigated by Solid-State NMR spectroscopy. (13)C high-resolution NMR clearly indicates that the crystalline signals split into 2, 3, and 4 signals for the CH3, CH and CO groups, respectively at 25 °C, while the amorphous signals give a broad component at the bottom of the crystalline signals. (13)C NMR spectra show that the crystalline line shape changes with increasing temperatures well above the glass transition temperature (Tg) and imply the presence of the molecular dynamics in the crystalline region. Comparisons of the evolution-time dependence of CODEX data and simulation results based on reorientation of chemical shift anisotropy (CSA) indicate that the chains in the α phase perform helical jump motions in the slow dynamic range at temperatures above 115 °C. The mixing-time dependence of the CODEX data yields an activation energy of Ea of (95 ± 8) kJ/mol for the helical jump motions. Moreover, two-dimensional exchange NMR with highly resolved signals for the CO group provides cross peaks among four well resolved signals due to the helical jumps. Comparison of 2D buildup curves of the cross peaks and calculated data determines that helical jump motions prefer largely uncorrelated random back-and-forth motions between the neighboring sites, possibly enabling large-scale chain diffusion in the crystalline regions.

A double-component Anderson-Weiss approach for describing NMR signals of mobile SIn units: application to constant-time DIPSHIFT experiments.

A composed Gaussian local field is proposed to describe the effect of molecular motions on NMR signals of SIn units (e.g., CHn or NHn), based upon the well-know Anderson-Weiss (AW) approximation. The approach is exemplified on constant-time recoupled dipolar chemical-shift correlation (tC-recDIPSHIFT) experiments, providing an analytical formula that can be used as a fitting function in studies of intermediate-regime motions. By comparison of analytical tC-recDIPSHIFT curves and dynamic spin dynamics simulations, we show that for heteronuclear spin pairs (SI system), the AW treatment assuming the usual Gaussian local field is accurate. However, the approximation fails for the case of SIn spin systems for motional rates higher than a few kHz. Based on earlier work of Terao et al., who proposed a decomposition of CHn dipolar powder patterns into to 2(n) spin-pair-type patterns, we propose an AW approach based upon a double-Gaussian local field. We derive an analytical formula for tC-recDIPSHIFT signals, and demonstrate its accuracy by comparison with simulations of several motional geometries and rates, and with experimental results for a model sample. The approach is not limited to the tC-recDIPSHIFT experiment and should be of general use in dipolar-coupling based experiments probing (partially) mobile SIn molecular moieties.

Solid-state NMR approaches to internal dynamics of proteins: from picoseconds to microseconds and seconds.

Solid-state nuclear magnetic resonance (NMR) spectroscopy has matured to the point that it is possible to determine the structure of proteins in immobilized states, such as within microcrystals or embedded in membranes. Currently, researchers continue to develop and apply NMR techniques that can deliver site-resolved dynamic information toward the goal of understanding protein function at the atomic scale. As a widely-used, natural approach, researchers have mostly measured longitudinal (T1) relaxation times, which, like in solution-state NMR, are sensitive to picosecond and nanosecond motions, and motionally averaged dipolar couplings, which provide an integral amplitude of all motions with a correlation time of up to a few microseconds. While overall Brownian tumbling in solution mostly precludes access to slower internal dynamics, dedicated solid-state NMR approaches are now emerging as powerful new options. In this Account, we give an overview of the classes of solid-state NMR experiments that have expanded the accessible range correlation times from microseconds to many milliseconds. The measurement of relaxation times in the rotating frame, T1ρ, now allows researchers to access the microsecond range. Using our recent theoretical work, researchers can now quantitatively analyze this data to distinguish relaxation due to chemical-shift anisotropy (CSA) from that due to dipole-dipole couplings. Off-resonance irradiation allows researchers to extend the frequency range of such experiments. We have built multidimensional analogues of T2-type or line shape experiments using variants of the dipolar-chemical shift correlation (DIPSHIFT) experiment that are particularly suited to extract intermediate time scale motions in the millisecond range. In addition, we have continuously improved variants of exchange experiments, mostly relying on the recoupling of anisotropic interactions to address ultraslow motions in the ms to s ranges. The NH dipolar coupling offers a useful probe of local dynamics, especially with proton-depleted samples that suppress the adverse effect of strong proton dipolar couplings. We demonstrate how these techniques have provided a concise picture of the internal dynamics in a popular model system, the SH3 domain of α-spectrin. T1-based methods have shown that large-amplitude bond orientation fluctuations in the picosecond range and slower 10 ns low-amplitude motions coexist in these structures. When we include T1ρ data, we observe that many residues undergo low amplitude motions slower than 100 ns. On the millisecond to second scale, mostly localized but potentially cooperative motions occur. Comparing different exchange experiments, we found that terminal NH2 groups in side chains can even undergo a combination of ultraslow large-angle two-site jumps accompanied by small-angle fluctuations that occur 10 times more quickly.

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data.

In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state nuclear magnetic resonance experiments reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.

Recoupled separated-local-field experiments and applications to study intermediate-regime molecular motions.

A specific separated-local-field NMR experiment, dubbed Dipolar-Chemical-Shift Correlation (DIPSHIFT) is frequently used to study molecular motions by probing reorientations through the changes in XH dipolar coupling and T2. In systems where the coupling is weak or the reorientation angle is small, a recoupled variant of the DIPSHIFT experiment is applied, where the effective dipolar coupling is amplified by a REDOR-like π-pulse train. However, a previously described constant-time variant of this experiment is not sensitive to the motion-induced T2 effect, which precludes the observation of motions over a large range of rates ranging from hundreds of Hz to around a MHz. We present a DIPSHIFT implementation which amplifies the dipolar couplings and is still sensitive to T2 effects. Spin dynamics simulations, analytical calculations and experiments demonstrate the sensitivity of the technique to molecular motions, and suggest the best experimental conditions to avoid imperfections. Furthermore, an in-depth theoretical analysis of the interplay of REDOR-like recoupling and proton decoupling based on Average-Hamiltonian Theory was performed, which allowed explaining the origin of many artifacts found in literature data.

The trehalose coating effect on the internal protein dynamics.

(15)N and (13)C NMR experiments were applied to conduct a comparative study of a cold shock protein (Csp) in two states-lyophilized powder and a protein embedded in a glassy trehalose matrix. Both samples were studied at various levels of rehydration. The experiments used (measuring relaxation rates R(1) and R(1ρ), motionally averaged dipolar couplings and solid state exchange method detecting reorientation of the chemical shift anisotropy tensor) allow obtaining abundant information on the protein structural features and internal motions in a range of correlation times from nanoseconds to seconds. The main results are: (a) the trehalose coating makes the protein structure more native in comparison with the dehydrated lyophilized powder, however, trehalose still cannot remove all non-native hydrogen bonds which are present in a dehydrated protein; (b) trehalose has an appreciable effect on the internal dynamics: the motion of the backbone N-H groups in the nanosecond and microsecond time scales becomes slower while the motional amplitude remains constant; (c) upon adding water to the Csp-trehalose mixture, water molecules accumulate around proteins forming a layer between the protein surface and the trehalose matrix. The protein dynamics become faster, however, not as fast as in the fully hydrated state; (d) the hydration response of dynamics of the NH and CH(CH(2)) groups in a protein is qualitatively different: upon increasing protein hydration, the correlation times of the N-H motions become shorter and the amplitude remains stable, and for CH(CH(2)) groups the motional amplitude increases and the correlation times do not change. This can be explained by a different ability of the NH and CH(CH(2)) groups to form hydrogen bonds.

Understanding the molecular dynamics associated with polymorphic transitions of DL-norvaline with solid-state NMR methods.

DL-Norvaline (NVA) is an important pharmaceutical intermediate and undergoes two polymorphic transitions between 140 and 300 K. To understand molecular dynamics of NVA accompanied with these transitions, we conducted a comprehensive study on its molecular motions at multiple time scales (10(-9)-1 s) with various solid-state NMR methods. (13)C CPMAS NMR spectra revealed four sets of clearly resolved signals for NVA carbons corresponding to at least three crystal modifications with two polymorphic transitions. Proton and (13)C relaxation results showed that, apart from the reorientations of methyl and amino groups, NVA had another relaxation process following the second transition with the activation energy of 16-21 kJ/mol corresponding to the side-chain motions. This was further confirmed with the data from dipolar and chemical shift experiments. No motions were detected at CODEX time scale (ms-s). These results provide essential information for understanding the mechanistic aspects of the polymorphic transitions in aliphatic α-amino acids with linear side-chains.

Microsecond time scale mobility in a solid protein as studied by the 15N R(1rho) site-specific NMR relaxation rates.

For the first time, we have demonstrated the site-resolved measurement of reliable (i.e., free of interfering effects) (15)N R(1rho) relaxation rates from a solid protein to extract dynamic information on the microsecond time scale. (15)N R(1rho) NMR relaxation rates were measured as a function of the residue number in a (15)N,(2)H-enriched (with 10-20% back-exchanged protons at labile sites) microcrystalline SH3 domain of chicken alpha-spectrin. The experiments were performed at different temperatures and at different spin-lock frequencies, which were realized by on- and off-resonance spin-lock irradiation. The results obtained indicate that the interfering spin-spin contribution to the R(1rho) rate in a perdeuterated protein is negligible even at low spin-lock fields, in contrast to the case for normal protonated samples. Through correlation plots, the R(1rho) rates were compared with previous data for the same protein characterizing different kinds of internal mobility.

The mobility of chondroitin sulfate in articular and artificial cartilage characterized by 13C magic-angle spinning NMR spectroscopy.

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in natural and artificial cartilage are different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue.