Structure Pharmaceutics Based on Synchrotron Radiation X-Ray Micro- Computed Tomography: From Characterization to Evaluation and Innovation of Pharmaceutical Structures

Drug delivery systems (DDS) are essentially pharmaceutical products for human therapy, typically involving a mixture of active ingredients and excipients. Based upon quantitative characterization of structure, the thesis introduces the concept of classifying the architecture of DDS into four levels by their spatial scale and the life time period. The primary level is recognised as the static structure of the whole dosage form with a size from μm to cm with the final structure generated by formulation design. The secondary level categorises the structures of particles or sub-units to form a DDS with sizes from nm to mm as key units in processing such as mixing, grinding, granulation and packing; The tertiary level represents the dynamic structures of DDS during the drug release phase in vitro or in vivo incorporating the structure size range from nm to mm, which undergo changes during dissolution, swelling, erosion or diffusion. The spatial scale for the quaternary level is defined as the meso or micro scale architecture of active and non-active molecules within a DDS with sizes from Å to μm for the molecular structure of drug and excipients. Methods combining X-ray tomography, image processing, and 3D reconstructions have been devised and evaluated to study systematically pharmaceutical structures and correlate them with drug release kinetics of DDS. Based on the quantitative structural information of pharmaceutical intermediates and dosage forms, it is possible now to correlate structures with production processing, behaviour and function, and the static and dynamic structures of DDS with the release kinetics. Thus, a structure-guided methodology has been established for the research of DDS.Chinese Academy of Science

Simultaneously expressed miR-424 and miR-381 synergistically suppress the proliferation and survival of renal cancer cells---Cdc2 activity is up-regulated by targeting WEE1

OBJECTIVES: MiRNAs are intrinsic RNAs that interfere with protein translation. Few studies on the synergistic effects of miRNAs have been reported. Both miR-424 and miR-381 have been individually reported to be involved in carcinogenesis. They share a common putative target, WEE1, which is described as an inhibitor of G2/M progression. Here, we studied the synergistic effects of miR-424 and miR-381 on renal cancer cells. METHODS: The viability of 786-O cells was analyzed after transfection with either a combination of miR-424 and miR-381 or each miRNA alone. We investigated cell cycle progression and apoptosis with flow cytometry. To confirm apoptosis and the abrogation of G2/M arrest, we determined the level of pHH3, which is an indicator of mitosis, and caspase-3/7 activity. The expression levels of WEE1, Cdc25, γH2AX, and Cdc2 were manipulated to investigate the roles of these proteins in the miRNA-induced anti-tumor effects. To verify that WEE1 was a direct target of both miR-424 and miR-381, we performed a dual luciferase reporter assay. RESULTS: We showed that the combination of these miRNAs synergistically inhibited proliferation, abrogated G2/M arrest, and induced apoptosis. This combination led to Cdc2 activation through WEE1 inhibition. This regulation was more effective when cells were treated with both miRNAs than with either miRNA alone, indicating synergy between these miRNAs. WEE1 was verified to be a direct target of each miRNA according to the luciferase reporter assay. CONCLUSIONS: These data clearly demonstrate that these two miRNAs might synergistically act as novel modulators of tumorigenesis by down-regulating WEE1 expression in renal cell cancer cells

Non-destructive 3D Microtomography of Cerebral Angioarchitecture Changes Following Ischemic Stroke in Rats Using Synchrotron Radiation

A better understanding of functional changes in the cerebral microvasculature following ischemic injury is essential to elucidate the pathogenesis of stroke. Up to now, the simultaneous depiction and stereological analysis of 3D micro-architectural changes of brain vasculature with network disorders remains a technical challenge. We aimed to explore the three dimensional (3D) microstructural changes of microvasculature in the rat brain on 4, 6 hours, 3 and 18 days post-ischemia using synchrotron radiation micro-computed tomography (SRμCT) with a per pixel size of 5.2 μm. The plasticity of angioarchitecture was distinctly visualized. Quantitative assessments of time-related trends after focal ischemia, including number of branches, number of nodes, and frequency distribution of vessel diameter, reached a peak at 6 h and significantly decreased at 3 days and initiated to form cavities. The detected pathological changes were also proven by histological tests. We depicted a novel methodology for the 3D analysis of vascular repair in ischemic injury, both qualitatively and quantitatively. Cerebral angioarchitecture sustained 3D remodeling and modification during the healing process. The results might provide a deeper insight into the compensatory mechanisms of microvasculature after injury, suggesting that SRμCT is able to provide a potential new platform for deepening imaging pathological changes in complicated angioarchitecture and evaluating potential therapeutic targets for stroke

Optimization of taste-masking on ibuprofen microspheres with selected structure features

The microsphere was a primary particulate system for taste-masking with unique structural features defined by production process. In this article, ibuprofen lipid microspheres of octadecanol and glycerin monostearate were prepared to mask the undesirable taste of ibuprofen via three kinds of spray congealing processes, namely, air-cooling, water-cooling and citric acid solution-cooling. The stereoscopic and internal structures of ibuprofen microspheres were quantitatively analyzed by synchrotron radiation X-ray micro-computed tomography (SR-µCT) to establish the relationship between the preparation process and microsphere architectures. It was found that the microstructure and morphology of the microspheres were significantly influenced by preparation processes as the primary factors to determine the release profiles and taste-masking effects. The sphericity of ibuprofen microspheres congealed in citric acid solution was higher than that of other two and its morphology was more regular than that being congealed in air or distilled water, and the contact angles between congealing media and melted ibuprofen in octadecanol and glycerin monostearate well demonstrated the structure differences among microspheres of three processes which controlled the release characteristics of the microspheres. The structure parameters like porosity, sphericity, and radius ratio from quantitative analysis were correlated well with drug release behaviors. The results demonstrated that the exterior morphology and internal structure of microspheres had considerable influences on the drug release behaviors as well as taste-masking effects. Keywords: Ibuprofen microsphere, Spray congealing, Internal structure, Synchrotron radiation X-ray micro-computed tomograph

The Angiogenic Effect of microRNA-21 Targeting TIMP3 through the Regulation of MMP2 and MMP9

<div><p>microRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. <i>In vivo</i>, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.</p></div

Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden.

Alterations of mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs) and the risk of prostate cancer (PCa) has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001). Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio= 1.85, 95% confidence interval: 1.21-2.83). A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (Ptrend = 0.011). Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively). These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden

Effect of miR-21 on HUVEC migration.

<p>(A) Scratch wound migration assay <i>in vitro</i>. After transfection with miR-21 mimics, miR-21 mimic negative control, antagomir-21 or antagomir-21 negative control, confluent HUVECs monolayers were exposed to OGD and then scratched to induce horizontal migration of HUVECs (magnification, x100). (C) Comparison of the distance of HUVECs horizontal migration at 0 h and 24 h between treatment groups and negative control groups. Values represent means±S.D; n = 3; *p<0.05. (B) Transwell chamber migration assay <i>in vitro</i>. HUVECs subjected to transfection and OGD mentioned above were trypsinized and seeded into the upper chamber and then allowed to vertically migrate for 24 h. Cells on the lower surface of the membrane were stained with 0.1% crystal violet (lavender) (magnification, x100). (D) Comparison of the rate of cell migration in the treatment groups relative to their respective negative control groups at 24 h. Values represent means±S.D; n = 3; *p<0.05, ^#p<0.05.</p

Effect of miR-21 on TIMP3, MMP2 and MMP9 expression.

<p>HUVECs were transfected with miR-21 mimics, miR-21 mimic negative control, antagomir-21 or antagomir-21 negative control and exposed to OGD for 4 h and then recovered for 24h. (A-C) qRT-PCR reveals the different mRNA expression levels of TIMP3, MMP2 and MMP9 between the treatment groups and respective negative groups. Values represent means±S.D; n = 3; *p<0.05. (D) Western blot bands show the protein expression of TIMP3, MMP2 and MMP9. (E-G) Comparison of TIMP3, MMP2 and MMP9 protein expression between the treatment groups and respective negative groups. Values represent means±S.D; n = 3; *p<0.05.</p

3D vascular morphologic changes and quantitative analysis after injury <i>in vivo</i>.

<p>(A) 3D vascular morphology changes after SCI in rats treated with antagomir-21 or antagomir-21 negative control. (B) Effect of antagomir-21 on the density of bifurcation points (DBP). (C) Effect of antagomir-21 on the sum of vascular volume (SVV). These results revealed that the down-regulation of miR-21 was adverse to remodeling of the vasculature 3,7 or 14 days after SCI in rats (Bar = 500 μm, Values represent means±S.D; n = 3; *p<0.05).</p