M. H. J. Koch

Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential

Abstract Drug carrier systems based on lipid nanosuspensions prepared by melt emulsification present a number of severe stability problems such as a high gelation tendency, considerable particle growth and drug expulsion. Destabilization of the emulsified lipidic carriers is related to recrystallization of the lipids. The choice of stabilizers for colloidal lipid suspensions is, therefore, restricted. Systematic surface modifications are thus limited. In addition, the drug payload of crystalline nanosuspension particles is generally low. Improved stability and loading capacities were found for amorphous lipid nanoparticles which present the characteristic signals of supercooled melts in high resolution 1 H-NMR. The NMR data indicate that such liquid but viscous carriers can, however, not immobilize the incorporated drug molecules to the same extent as a solid matrix. Sustained release over days or weeks as in slowly biodegraded solid matrices thus seems difficult to achieve with a supercooled melt. Attempts to combine the advantages of the solid crystalline lipids and the amorphous nature of the supercooled melts by generating solid but amorphous lipid suspension particles with a satisfactory long-term stability by a variation of the lipid matrix material have hitherto not been successful. Even a satisfactory stabilization of the α-modification using complex lipid mixtures to improve the loading capacity or to slow down the drug expulsion process could not be achieved. The rates of the polymorphic transitions were much higher in the colloidal lipid dispersions than in the bulk for the hard fats under investigation. Despite the fact that the properties of the lipids are superimposed with colloidal properties, significant differences between monoacid triglycerides and complex lipids were, however, found.

Use of SAXS and linear correlation functions for the determination of the crystallinity and morphology of semi-crystalline polymers. Application to linear polyethylene

The use of correlation functions to obtain the morphological parameters of crystalline-amorphous two-phase lamellar systems is critically reviewed and extended. It is shown that processing of the experimental SAXS-patterns only significantly affects the curvature of the autocorrelation triangle and that the parameters of the corresponding ideal two-phase structure can be determined independently of the data processing procedure. The methods to be used depend on the normalization of the correlation function. The validity of the formulation is illustrated for a sample of linear polyethylene, cooled and heated at 10°C per min. Crystallite thickening during crystallization and surface melting during heating are observed. The overall crystallinity and the fraction of semi-crystalline stacks during crystallization and melting are determined quantitatively as a function of temperature using the total scattering power of the corresponding ideal two-phase structure, correlation functions, and a scaling procedure. Absolute intensities are not required. The SAXS results are confirmed by independent techniques (DSC, WAXD, and SALLS). During crystallization, amorphous regions are present outside the semi-crystalline regions because growing spherulites do not fill space completely. During melting, larger amorphous regions develop in the spherulites because of the complete melting of stacks.

Miscibility, crystallization kinetics and real-time small-angle X-ray scattering investigation of the semicrystalline morphology in thermosetting polymer blends of epoxy resin and poly(ethylene oxide)

Thermosetting polymer blends of poly(ethylene oxide) (PEO) and bisphenol-A-type epoxy resin (ER) were prepared using 4,4′-methylenebis(3-chloro-2,6-diethylaniline) (MCDEA) as curing agent. The miscibility and crystallization behavior of MCDEA-cured ER/PEO blends were investigated by differential scanning calorimetry (DSC). The existence of a single composition-dependent glass transition temperature (Tg) indicates that PEO is completely miscible with MCDEA-cured ER in the melt and in the amorphous state over the entire composition range. Fourier-transform infrared (FTIR) investigations indicated hydrogen-bonding interaction between the hydroxyl groups of MCDEA-cured ER and the ether oxygens of PEO in the blends, which is an important driving force for the miscibility of the blends. The average strength of the hydrogen bond in the cured ER/PEO blends is higher than in the pure MCDEA-cured ER. Crystallization kinetics of PEO from the melt is strongly influenced by the blend composition and the crystallization temperature. At high conversion, the time dependence of the relative degree of crystallinity deviated from the Avrami equation. The addition of a non-crystallizable ER component into PEO causes a depression of both the overall crystallization rate and the melting temperature. The surface free energy of folding σe displays a minimum with variation of composition. The spherulitic morphology of PEO in the ER/PEO blends exhibits typical characteristics of miscible crystalline/amorphous blends, and the PEO spherulites in the blends are always completely volume-filling. Real-time small-angle X-ray scattering (SAXS) experiments reveal that the long period L increases drastically with increasing ER content at the same temperatures. The amorphous cured ER component segregates interlamellarly during the crystallization process of PEO because of the low chain mobility of the cured ER. A model describing the semicrystalline morphology of MCDEA-cured ER/PEO blends is proposed based on the SAXS results. The semicrystalline morphology is a stack of crystalline lamellae; the amorphous fraction of PEO, the branched ER chains and imperfect ER network are located between PEO lamellae.

Miscibility, crystallization and real-time small-angle X-ray scattering investigation of the semicrystalline morphology in thermosetting polymer blends

The results of the study of a completely miscible thermosetting polymer blend containing a crystallizable component are reported. Blends of poly(ϵ-caprolactone) (PCL) and bisphenol-A-type epoxy resin (ER) cured with 4,4′-methylenebis(3-chloro-2,6-diethylaniline) (MCDEA) were prepared and compared with blends of PCL with uncured bisphenol A-type epoxy resin, i.e. diglycidylether of bisphenol A (DGEBA). The miscibility, crystallization behavior and spherulitic morphology of both uncured DGEBA/PCL blends and MCDEA-cured ER/PCL blends were investigated by differential scanning calorimetry (DSC), optical microscopy, and Fourier-transform infrared (FTIR) spectroscopy. It was found that PCL is completely miscible with both DGEBA and MCDEA-cured ER in the melt and in the amorphous state over the entire composition range, as shown by the existence of a single composition-dependent glass transition temperature (Tg). The overall crystallization rate and crystallinity of PCL in the MCDEA-cured ER/PCL blends decrease much more rapidly with increasing amorphous content than those of the DGEBA/PCL blends. The spherulitic morphology of PCL in both the uncured and the cured blends is characteristic of miscible crystalline/amorphous blends, and the PCL spherulites in these blends are always completely volume-filling. The miscibility of the uncured DGEBA/PCL blends is considered to be due predominately to the entropic contribution, whereas that of the cured ER/PCL blends is due to the enthalpic contribution. FTIR investigations indicated hydrogen bonding interaction between the hydroxyl groups of MCDEA-cured ER and the carbonyl groups of PCL in the cured system, which is an important driving force for the miscibility of the cured ER/PCL blends. Real-time small-angle X-ray scattering (SAXS) experiments revealed that the amorphous cured ER segregated interlamellarly during the crystallization process of PCL, which is considered to result from the low chain mobility of the cured ER in the ER/PCL blends. On the basis of the SAXS results, a model describing the semicrystalline morphology of MCDEA-cured ER/PCL blends is proposed. The amorphous fraction of PCL, the branched ER chains and imperfect ER network are located between PCL lamellae in the crystallized blend.

Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation

Abstract Differential scanning calorimetry, high and small angle X-ray diffraction analyses have been carried out on air-dried and rehydrated rat tail tendon collagen in order to test the reversibility of collagen thermal denaturation. The mean enthalpy values calculated for the denaturation process of air-dried and rehydrated samples are ΔH D = 9.0 ± 0.8 cal / g and ΔH D = 11.9 ±0.7 cal / g respectively, while the denaturation temperatures are T D = 112 ± 1° C and T D = 51 ± 1° C . Partial reversibility of the coiled coil—random coil process can be obtained by storing the samples in air or more rapidly by equilibration in water. After denaturation air-dried collagen fibres recover not only their molecular structure but also their characteristic fibrillar structure. The latter does not greatly influence the mean experimental enthalpy values.

Shape Determination from Solution Scattering of Biopolymers

Practical aspects of low-resolution shape determination in small-angle scattering studies of biological macromolecules in solution are considered. The shape restoration method using spherical harmonics [Svergun, Volkov, Kozin & Stuhrmann (1996). Acta Cryst. A52, 419–426] is extended to account for deviations from the idealized model and to work directly on raw experimental data sets. An algorithm to restore the structure of homodimeric particles in terms of the shape of the monomer and the separation between the monomers is implemented. Applications of the program to the shape restoration of several proteins, with known and unknown crystal structures, from X-ray solution-scattering data are presented.

Quantitative analysis of the molecular sliding mechanisms in native tendon collagen — time-resolved dynamic studies using synchrotron radiation

Abstract The stretching of native fibres from rat tail tendons (RTT) was monitored in time-resolved X-ray measurements using synchotron radiation, by registering one meridional small angle diffraction pattern every second. The time course of this dynamic molecular process was analyzed quantitatively with the help of model calculations based on the amino acid sequence. The results show that two mechanisms contribute to the elongation of fibrils, namely the stretching of the collagen triple helices and their sliding relative to each other (increase of the D stagger). The results further show that these two processes do not take place simultaneously. The first increase of the D period from 67.0 nm to about 67.6 nm is correlated with a stretching of the triple helices. The further increase of the D period is due to a continuous increase of the D stagger. This succession is independent of the age of the animals and also independent of the stretching velocity. The stretching process is shown to be reversible at the molecular level up to a D period of about 68.4 nm.

Structural analysis of turkey tendon collagen upon removal of the inorganic phase.

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.

The superstructure of chromatin and its condensation mechanism

Solutions of rat liver and chicken erythrocyte chromatin at different ionic strngths were characterized by synchrotron X-ray solution scattering, ultracentrifugation, density and viscosity measurements. Previous observations on nuclei were extended to rat liver, calf thymus and yeast nuclei.It is shown that with monovalent cations condensation is independent of the nature of the cation whereas with divalent cations there are significant differences related to the preference of base binding over phosphate binding. The consistency of hydrodynamic and scattering results confirm the view that chromatin in solution at low ionic strength has a helix-like superstructure. A survey of X-ray and neutron scattering results in the literature shows that previous interpretations, e.g. in terms of a 10 nm filament, are incompatible with the experimental data at low resolution.

Structural dynamic of native tendon collagen

The dynamic behaviour of collagen fibrils is revealed by time-resolved X-ray investigations of native rat tail tendon fibres in tensile tests.