Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity.

Objective: Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa. Methods: Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention. Results: Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen. Disccusion: Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

Synchrotron Radiation-Based Three-Dimensional Visualization of Angioarchitectural Remodeling in Hippocampus of Epileptic Rats.

Characterizing the three-dimensional (3D) morphological alterations of microvessels under both normal and seizure conditions is crucial for a better understanding of epilepsy. However, conventional imaging techniques cannot detect microvessels on micron/sub-micron scales without angiography. In this study, synchrotron radiation (SR)-based X-ray in-line phase-contrast imaging (ILPCI) and quantitative 3D characterization were used to acquire high-resolution, high-contrast images of rat brain tissue under both normal and seizure conditions. The number of blood microvessels was markedly increased on days 1 and 14, but decreased on day 60 after seizures. The surface area, diameter distribution, mean tortuosity, and number of bifurcations and network segments also showed similar trends. These pathological changes were confirmed by histological tests. Thus, SR-based ILPCI provides systematic and detailed views of cerebrovascular anatomy at the micron level without using contrast-enhancing agents. This holds considerable promise for better diagnosis and understanding of the pathogenesis and development of epilepsy.

Comparison of high-resolution synchrotron-radiation-based phase-contrast imaging and absorption-contrast imaging for evaluating microstructure of vascular networks in rat brain: from 2D to 3D views.

Conventional imaging methods such as magnetic resonance imaging, computed tomography and digital subtraction angiography have limited temporospatial resolutions and shortcomings like invasive angiography, potential allergy to contrast agents, and image deformation, that restrict their application in high-resolution visualization of the structure of microvessels. In this study, through comparing synchrotron radiation (SR) absorption-contrast imaging to absorption phase-contrast imaging, it was found that SR-based phase-contrast imaging could provide more detailed ultra-high-pixel images of microvascular networks than absorption phase-contrast imaging. Simultaneously, SR-based phase-contrast imaging was used to perform high-quality, multi-dimensional and multi-scale imaging of rat brain angioarchitecture. With the aid of image post-processing, high-pixel-size two-dimensional virtual slices can be obtained without sectioning. The distribution of blood supply is in accordance with the results of traditional tissue staining. Three-dimensional anatomical maps of cerebral angioarchitecture can also be acquired. Functional partitions of regions of interest are reproduced in the reconstructed rat cerebral vascular networks. Imaging analysis of the same sample can also be displayed simultaneously in two- and three-dimensional views, which provides abundant anatomical information together with parenchyma and vessels. In conclusion, SR-based phase-contrast imaging holds great promise for visualizing microstructure of microvascular networks in two- and three-dimensional perspectives during the development of neurovascular diseases.

Peptide-Tethered Hydrogel Scaffold Promotes Recovery from Spinal Cord Transection via Synergism with Mesenchymal Stem Cells.

Spinal cord injury (SCI) is one of the most devastating injuries. Treatment strategies for SCI are required to overcome comprehensive issues. Implantation of biomaterial scaffolds and stem cells has been demonstrated to be a promising strategy. However, a comprehensive recovery effect is difficult to achieve. In the comprehensive treatment process, the specific roles of the implanted scaffolds and of stem cells in combined strategy are usually neglected. In this study, a peptide-modified scaffold is developed based on hyaluronic acid and an adhesive peptide PPFLMLLKGSTR. Synchrotron radiation micro computed tomography measurement provides insights to the three-dimensional inner topographical property and perspective porous structure of the scaffold. The modified scaffold significantly improves cellular survival and adhesive growth of mesenchymal stem cells during 3D culture in vitro. After implantation in transected spinal cord, the modified scaffold and mesenchymal stems are found to function in synergy to restore injured spinal cord tissue, with respective strengths. Hindlimb motor function scores exhibit the most significant impact of the composite implant at 2 weeks post injury, which is the time secondary injury factors begin to take hold. Investigation on the secondary injury factors including inflammatory response and astrocyte overactivity at 10 days post injury reveals the possible underlying reason. Implants of the scaffold, cells, and especially the combination of both elicit inhibitory effects on these adverse factors. The study develops a promising implant for spinal cord tissue engineering and reveals the roles of the scaffold and stem cells. More importantly, the results provide the first understanding of the bioactive peptide PPFLMLLKGSTR concerning its functions on mesenchymal stem cells and spinal cord tissue restoration.

In situ 3D topographic and shape analysis by synchrotron radiation X-ray microtomography for crystal form identification in polymorphic mixtures.

Polymorphism denotes the existence of more than one crystal structure of a substance, and great practical and theoretical interest for the chemical and pharmaceutical industries. In many cases, it is challenging to produce a pure crystal form and establish a sensitive detection method for the identification of crystal form in a mixture of polymorphs. In this study, an accurate and sensitive method based on synchrotron radiation X-ray computed microtomography (SR-µCT) was devised to identify the polymorphs of clopidogrel bisulphate (CLP). After 3D reconstruction, crystal particles were extracted and dozens of structural parameters were calculated. Whilst, the particle shapes of the two crystal forms were all irregular, the surface of CLP II was found to be rougher than CLP I. In order to classify the crystal form based on the quantitative morphological property of particles, Volume Bias Percentage based on Surface Smoothing (VBP) was defined and a new method based on VBP was successfully developed, with a total matching rate of 99.91% for 4544 particles and a lowest detectable limit of 1%. More important for the mixtures in solid pharmaceutical formulations, the interference of excipients can be avoided, a feature cannot achieved by other available analytical methods.

Visualization and quantification of deformation behavior of clopidogrel bisulfate polymorphs during tableting.

The deformation behavior of particles under pressure dominates the mechanical properties of solid dosage forms. In this study, the in situ 3D deformation of two polymorphs (I and II) of clopidogrel bisulfate (CLP) was determined to illustrate pressure distribution profiles within the tablet by the deformation of the crystalline particles for the first time. Synchrotron radiation X-ray computed microtomography (SR-µCT) was utilized to visualize and quantify the morphology of thousands crystalline particles of CLP I and CLP II before and after compression. As a result, the deformation was examined across scale dimensions from microns to the size of the final dosage form. Three dimensional parameters such as volume, sphericity, oblate and prolate of individual particle and distributions were computed and analyzed for quantitative comparison to CLP I and CLP II. The different degrees of deformation under the same compression conditions of CLP I and CLP II were observed and characterized quantitatively. The map of deformation degrees within the tablet illustrated the heterogeneous pressure distribution in various regions of the compacted tablet. In conclusion, the polymorph deformation behaviors demonstrated by SR-µCT quantitative structure analysis deepen understanding of tableting across dimensions from microns to millimeters for the macrostrcuture of tablet.

[Formation mechanism of Pickering emulsions induced by self-assembly of medium chain triglycerides and α-cyclodextrin].

In this study, the Pickering emulsions were prepared using medium chain triglycerides(MCT) and α-cyclodextrin(α-CD) and the formation mechanism was studied by means of several physicochemical techniques. The MCT/α-CD microparticles, which stabilized the emulsions, were characterized by the measurement of interfacial tension and the contact angle(θ(ow)), powder X ray diffraction(XRD), scanning electron microscope(SEM), high performance liquid chromatography(HPLC), differential interference microscope(DIM), Cryo-scanning electron microscopy(Cryo-SEM). The physical stability of emulsions with different α-CD content in the continuous aqueous phase was investigated by determination of the droplet size and sedimentation rate, combined with the observation of droplet morphologies by the inverted phase contrast microscope. As a result, it was observed that the amphiphilic supramolecule of MCT and α-CD were indeed formed. Furthermore, MCT/α-CD microparticles formed by the aggregation of MCT/α-CD supramolecule absorbed at the oil/water interface, and then forming a membrane structure to stabilize emulsion. In addition, the average θ(ow) for the MCT/α-CD microparticles was(46.1 ± 3.4)° which stabilized O/W emulsion. When the content of α-CD was increased in the continuous phase, there were more microparticles formed at the oil/water interface and in the continuous aqueous phase, which resulted in smaller particle size of droplet and higher viscosity of the continuous phase. In summary, the study suggest that α-CD/MCT/water emulsions were of O/W Pickering emulsions and the physical stability was better for emulsions with higher content of α-CD in the continuous phase.

[Taste-masking mechanism of layer-by-layer self-assembly coating investigated by synchrotron radiation-based Fourier-transform infrared spectromicroscopy].

Ibuprofen lipid pellets prepared by melting method could mask the bitter taste of the drug to some extent. The pellets were further coated with chitosan (cationic) and gelatin (anionic) by ionic interaction layer- by-layer self-assembly (LBL) coating to improve masking effects. In this paper, the release percentage of drugs in short time (1 min) was utilized as an indicator for the taste-masking, and it had confirmed the LBL coating inhibited the release of model drug of ibuprofen. Synchrotron radiation-based Fourier-transform infrared spectromicroscopy (SR-FTIR) has been applied to investigate the material distributions on the cross section of pellets and film. Characteristic absorptions of the compositions were obtained by SR-FTIR single spectrum scanning. The distributions of the drug and materials in coated films were determined by SR-FTIR mapping. The FTIR absorptions of chitosan and gelatin on the surface of lipid pellets was examined to verify the existence of chitosan and gelatin on the surface and a film formed using SR-FTIR ratio analysis. Whilst pellets coated only by chitosan or gelatin did not show the typical absorption of chitosan or gelatin, which confirmed the effects of ionic interaction on the film forming process. In conclusion, the method of SR-FTIR established for the study of the existence and distribution of materials in coated film offers a new choice for researches on membranes/films in drug delivery systems and pharmaceutical preparations.

[Quantitative structure characteristics and fractal dimension of Chinese medicine granules measured by synchrotron radiation X-ray computed micro tomography].

The shape and structure of granules are controlled by the granulation process, which is one of the main factors to determine the nature of the solid dosage forms. In this article, three kinds of granules of a traditional Chinese medicine for improving appetite and promoting digestion, namely, Jianwei Granules, were prepared using granulation technologies as pendular granulation, high speed stirring granulation, and fluidized bed granulation and the powder properties of them were investigated. Meanwhile, synchrotron radiation X-ray computed micro tomography (SR-µCT) was applied to quantitatively determine the irregular internal structures of the granules. The three-dimensional (3D) structure models were obtained by 3D reconstruction, which were more accurately to characterize the three-dimensional structures of the particles through the quantitative data. The models were also used to quantitatively compare the structural differences of granules prepared by different granulation processes with the same formula, so as to characterize how the production process plays a role in the pharmaceutical behaviors of the granules. To focus on the irregularity of the particle structure, the box counting method was used to calculate the fractal dimensions of the granules. The results showed that the fractal dimension is more sensitive to reflect the minor differences in the structure features than the conventional parameters, and capable to specifically distinct granules in structure. It is proved that the fractal dimension could quantitatively characterize the structural information of irregular granules. It is the first time suggested by our research that the fractal dimension difference (Df,c) between two fractal dimension parameters, namely, the volume matrix fractal dimension and the surface matrix fractal dimension, is a new index to characterize granules with irregular structures and evaluate the effects of production processes on the structures of granules as a new indicator for the granulating process control and optimization.

Ultra-high-resolution 3D digitalized imaging of the cerebral angioarchitecture in rats using synchrotron radiation.

The angioarchitecture is a fundamental aspect of brain development and physiology. However, available imaging tools are unsuited for non-destructive cerebral mapping of the functionally important three-dimensional (3D) vascular microstructures. To address this issue, we developed an ultra-high resolution 3D digitalized angioarchitectural map for rat brain, based on synchrotron radiation phase contrast imaging (SR-PCI) with pixel size of 5.92 µm. This approach provides a systematic and detailed view of the cerebrovascular anatomy at the micrometer level without any need for contrast agents. From qualitative and quantitative perspectives, the present 3D data provide a considerable insight into the spatial vascular network for whole rodent brain, particularly for functionally important regions of interest, such as the hippocampus, pre-frontal cerebral cortex and the corpus striatum. We extended these results to synchrotron-based virtual micro-endoscopy, thus revealing the trajectory of targeted vessels in 3D. The SR-PCI method for systematic visualization of cerebral microvasculature holds considerable promise for wider application in life sciences, including 3D micro-imaging in experimental models of neurodevelopmental and vascular disorders.

[Release kinetics of single pellets and the multi-pellet system of tamsulosin hydrochloride sustained release pellets].

The release behavior of single pellet was investigated by LC/MS/MS method with tamsulosin hydrochloride (TSH) as the model drug of the research and then the pellets were divided into four groups according to the drug loading. Comparison of dissolution profiles of each group and capsule were performed using f1 and f2 factor methods to study the difference and similarity. The release profiles of single pellet, each group and capsule were analyzed using principle component analysis (PCA). The particle system was built through Matlab to get the target release profile. The result of this research demonstrated the release behavior of single pellet correlated well with the drug loading. While the dissolution profile of capsule as a reference, the similarity factor of dissolution profiles of the lower drug loading groups were 62.2, 67.1, 53.9, respectively and, 43.3 for highest drug loading group. The particle systems with different pellet distribution and same release profiles were built through release behavior of single pellet. It is of significance to investigate the release behavior of single pellets for studying the release regularity of multiple-unit drug delivery system.

[Identification of the polymorphs of clopidogrel bisulfate based on the steric morphology parameters of crystals].

The crystal form of solid substance had intrinsic correlation with its three dimensional crystal morphology. Based on the characterization of the three dimensional crystal morphology of clopidogrel bisulfate, this research is to establish a model based on the three dimensional morphological parameters. The granular samples composed of polymorphs of clopidogrel bisulfate and microcrystalline cellulose (MCC) were scanned by synchrotron radiation X-ray microscopic CT technology (SR-microCT) and the three dimensional structural models for which were constructed. Seven groups of three dimensional morphological parameters were calculated. Finally, the mathematical model was established with the multi-layer perception (MLP) artificial neutral network methods to identify and predict the polymorphs of clopidogrel bisulfate. The success rate of the model prediction for the polymorphs of clopidogrel bisulfate was 92.7% and the area under the ROC curve was 96.2%. The polymorphs of drugs could be identified and predicted through the numerical description of the three dimensional morphology. The volume, number of the vertices and the surface area were the major determinants for the identification of the polymorphs of clopidogrel bisulfate.

[Applicability of a natural swelling matrix as the propellant of osmotic pump tablets].

The purpose of this study is to investigate the applicability of a natural swelling matrix derived from boat-fruited sterculia seed (SMS) as the propellant of osmotic pump tablets. The sugar components, static swelling, water uptake and viscosity of SMS were determined and compared with that of polythylene oxide (WSR-N10 and WSR-303). Both ribavirin and glipizide were used as water-soluble and water-insoluble model drugs. Then, the monolayer osmotic pump tablets of ribavirin and the bilayer osmotic pump tablets of glipizide were prepared using SMS as the osmotically active substance and propellant. SMS was mainly composed of rhamnose, arabinose, xylose and galactose and exhibited relatively high swelling ability. The area of the disintegrated matrix tablet was 20.1 times as that at initial after swelling for 600 s. SMS swelled rapidly and was fully swelled (0.5%) in aqueous solution with relative low viscosity (3.66 +/- 0.03) mPa x s at 25 degrees C. The monolayer osmotic pump tablets of ribavirin and the bilayer osmotic pump tablets of glipizide using SMS as propellant exhibited typical drug release features of osmotic pumps. In conclusion, the swelling matrix derived from boat-fruited sterculia seed, with low viscosity and high swelling, is a potential propellant in the application of osmotic pump tablets.