荧光光谱对自组装多肽作为药物载体的初步研究

为解决疏水性药物普遍存在的因水中溶解度低而给药困难、生物利用度低的问题,采用了新型两亲性自组装多肽RGA16(Ac-RADAGAGARADAGAGS-NH₂)作为载体包裹和释放疏水性模型药物。以芘为模型疏水性药物,以鸡蛋卵磷脂脂质体模拟细胞膜,通过稳态荧光光谱表征和测定芘的存在形式和浓度。两亲性自组装多肽RGA16能够在水溶液中稳定模型疏水性药物芘的晶体。扫描电镜图像显示多肽RGA16与芘晶体相互吸引,两者形成10 μm以上大小的复合体。在机械搅拌下多肽RGA16与水溶液中的芘相互作用5 d左右形成稳定的胶体混悬液(多肽-芘复合体)。被多肽包裹时,芘以晶体的形式存在。而当与EPC脂质体溶液混合时,芘可从多肽的包裹中以分子形式释放到EPC的双层膜中。芘从自组装多肽所稳定的胶态晶体向EPC脂质体释放的过程采用连续时间扫描稳态荧光光谱加以观察。通过将释放过程中芘单体的荧光强度与标准曲线相比较,确定了特定时间点EPC脂质体中芘的转移量。以上结果表明：该两亲性自组装多肽RGA16具有作为小分子量疏水性药物载体的潜力。     

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at ?20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.     

Fluorescence lifetime imaging of molecular rotors to map microviscosity in cells

Fluorescence liftime imaging （FLIM） of modified hydrophobic bodipy dyes that act as fluorescent molecular rotors shows that the fluorescence lifetime of these probes is a function of the microviscosity of their environment. Incubating cells with these dyes, we find a punctate and continuous distribution of the dye in cells. The viscosity value obtained in what appears to be endocytotic vesicles in living cells is around 100 times higher than that of water and of cellular cytoplasm.Time-resolved fluorescence anisotropy measurements also yield rotational correlation times consistent with large microviscosity values. In this way, we successfully develop a practical and versatile approach to map the microviscosity in cells based on imaging fluorescent molecular rotors.     

Styryl Dyes as Two-Photon Excited Fluorescent Probes for DNA Detection and Two-Photon Laser Scanning Fluorescence Microscopy of Living Cells

Spectral-fluorescent properties of benzothiazole styryl monomer (Bos-3) and homodimer (DBos-21) dyes in presence of DNA were studied. The dyes enhance their fluorescence intensity in 2–3 orders of magnitude upon interaction with DNA. Studied styrylcyanines in DNA presence demonstrate rather high values of two-photon absorption (TPA) cross-section, which are comparable with the values of TPA cross section of the rhodamine dyes. An applicability of the styrylcyanines as probes for the fluorescence microscopy of living cells was studied. It was shown that both dyes are cell-permeable but homodimer dye DBos-21 produces noticeably brighter staining of HeLa cells comparing with monomer dye Bos-3. Molecules of DBos-21 initially bind to the nucleic acids- containing cell organelles (presumable mitochondria) and are able to penetrate into the cell nucleus. Thus, homodimer styryl DBos-21 dye is viewed as efficient stain for single-photon and two-photon excitation fluorescence imaging of living cells.     

Quiescent cells: A natural way to resist chemotherapy

Most chemotherapeutic treatments use drugs that target proliferating cancer cells. Therefore, they do not affect quiescent cells which are naturally resistant. Surviving cancer cells can reactivate their cell cycles in the intervals between doses, becoming proliferative again and thus restarting tumor growth. In this work, we present a mathematical model to study the impact of quiescent cells on chemotherapy effectiveness. Our simulations show that, although tumor growth is delayed after the beginning of each dose, the resistance of quiescent cells is enough to reactivate it due to accelerated repopulation, eventually causing therapy failure even in the absence of acquired resistance.     

Novel,Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity

The dimeric cyanine dyes, YOYO-1 and TOTO-1, are widely used as DNA probes because of their excellent fluorescent properties. They have a higher fluorescence quantum yield than ethidium homodimer, DAPI and Hoechst dyes and bind to double-stranded DNA with high affinity. However, these dyes are limited by heterogeneous staining at high dye loading, photocleavage of DNA under extended illumination, nicking of DNA, and inhibition of the activity of DNA binding enzymes. To overcome these limitations, seven novel cyanine dyes (Cyan-2, DC-21, DM, DM-1, DMB-2OH, SH-0367, SH1015-OH) were synthesized and tested for fluorescence emission, resistance to displacement by Mg²⁺, and the ability to function as reporters for DNA unwinding. Results show that Cyan-2, DM-1, SH-0367 and SH1015-OH formed highly fluorescent complexes with dsDNA. Of these, only Cyan-2 and DM-1 exhibited a large fluorescence enhancement in buffers, and were resistant to displacement by Mg²⁺. The potential of these two dyes to function as reporter molecules was evaluated using continuous fluorescence, DNA helicase assays. The rate of DNA unwinding was not significantly affected by either of these two dyes. Therefore, Cyan-2 and DM-1 form the basis for the synthesis of novel cyanine dyes with the potential to overcome the limitations of YOYO-1 and TOTO-1.     

The DNA content of mouse two-cell embryos can be measured by microfluorimetric image analysis under conditions of cell viability

Video-enhanced fluorescence imaging was used to quantify the DNA content in live two-cell mouse embryos. DNA was stained with the vital fluorophore Hoechst 33342. Conditions of dye concentration and irradiation were such that two-cell embryos could be kept in the constant presence of the dye for about 24 h without a major effect on their furtherin vitro viability. Total nuclear fluorescence intensity of stained two-cell embryos was measured twice under these conditions, i.e., in G1 (1 h after cleavage) and in G2 (15–18 h after cleavage), by image analysis. After correcting for the fluctuations in excitation intensity and for the spatial nonhomogeneities of the optical system (lenses and sensor), the mean total nuclear fluorescence intensity was about twofold higher in G2 than in G1 (R=1.99 to 2.25), and this increase was abolished by the addition of aphidicolin, an inhibitor of replication. The fluorescence increase did not depend on the Hoechst concentration in the range of 10–40 ng/ml, i.e., in the range of embryo viability. The coefficient of variation of the total nuclear fluorescence intensity measured under these conditions was rather large (10 to 20%). Nevertheless, the mean value of fluorescence intensity in G1 of nuclei of a given pool represents an appropriate reference to measure the increase in fluorescence intensity between G1 and G2.     

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.     

双光子成像观测衰老对小鼠卵母细胞内DNA甲基转移酶表达的影响

量子点(QD)具有抗漂白能力强、量子产率高、激发谱宽和发射谱窄等优点,在生物荧光标记领域有较广泛的应用。利用QD 585和Hoechst 33342 (H342)双标记小鼠卵母细胞中DNA甲基转移酶(Dnmt)和染色体,通过双光子成像同时双通道探测它们的荧光图像,获得了Dnmt蛋白表达的三维空间分布。发现老龄小鼠卵母细胞不适合体外培养成熟：该成熟方式不仅引起老龄小鼠卵母细胞Dnmt蛋白含量的变化,而且还改变了它们在胞质中的空间分布。这些改变可能与异常的甲基化修饰有关。     

Core–shell-structured nanothermites synthesized by atomic layer deposition

Chitosan was conjugated with folic acid (FA) and the resulting chitosan derivatives with a FA-substitution degree of around 6 % was used to synthesize FA-conjugated chitosan–polylactide (FA–CH–PLA) copolymers to build a drug carrier with active targeting characteristics for the anticancer drug of paclitaxel (PTX). Selected FA–CH–PLAs with various polylactide percentages of about 40 wt% or lower were employed to fabricate nanoparticles using sodium tripolyphosphate as a crosslinker, and different types of nanoparticles were endued with similar average particle-sizes located in a range between 100 and 200 nm. Certain types of PTX-loaded FA–CH–PLA nanoparticles having encapsulation efficiency of around 90 % and initial load of about 12 % were able to release PTX in a controlled manner with significant regulation by polylactide content in FA–CH–PLAs. Targeting characteristic of achieved nanoparticles was confirmed using FA-receptor-expressed MCF-7 breast cancer cells. The uptake of PTX revealed that optimized FA–CH–PLA nanoparticles with an equivalent PTX-dose of around 1 μg/mL could have more than sixfold increasing abilities to facilitate intracellular paclitaxel accumulation in MCF-7 cells after 24 h treatment as compared to free PTX. At a relatively safe equivalent PTX-dose for normal MCF-10A mammary epithelial cells, the obtained results from Hoechst 33342 staining indicated that optimized PTX-loaded FA–CH–PLA nanoparticles had more than threefold increasing abilities to induce MCF-7 cell apoptosis in comparison to free PTX.     

Enhancing the performance of poly(3-hexylthiophene)/ZnO nanorod arrays based hybrid solar cells through incorporation of a third component

Sparse ZnO nanorod arrays(NRAs)are fabricated on transparent conducting oxide coated glass substrates by using a modified liquid phase epitaxial growth method.By adjusting the polymer concentrations and the spin-coating parameters,full infiltration of poly(3-hexylthiophene)(P3HT)into the as-prepared ZnO NRAs is achieved at 130°C in vacuum.A third component is incorporated into the P3HT/ZnO NRAs ordered bulk heterojunctions(BHJs)either through ZnO surface modification with N719dye or CdS shell layer or by inclusion of a fullerene derivative into the P3HT matrix.Experimental results indicate that performances of the hybrid solar cells are improved greatly with the incorporation of a third component.However,the working principles of these third components differ from one another,according to morphology,structure,optical property,charge transfer and interfacial properties of the composite structures.An ideal device architecture for hybrid solar cells based on P3HT/ZnO NRAs ordered BHJs is proposed,which can be used as a guidance to further increase the power conversion efficiency of such solar cells.     

Triple-combination therapy assisted with ultrasound-active gold nanoparticles and ultrasound therapy against 3D cisplatin-resistant ovarian cancer model

Cancer chemotherapy suffers from drug resistance and side effects of the drugs. Combination therapies have been attracted attention to overcome these limitations of traditional cancer treatments. Recently, increasing in intracellular chemotherapeutic concentration in the presence of ultrasonic waves (US) has been shown in the preclinical stage. In addition, some recent studies have shown that nanoparticles increase the effectiveness of ultrasound therapy. In this study, the US-active property of gold nanocones (AuNCs) was utilized for combinational US and cisplatin (Cis) to overcome drug resistance. The effect of the triple combination therapy US + AuNCs + Cis with low-dose Cis on 2/3D models of cisplatin-resistant ovarian cancer cell line (A2780cis) were investigated. In the 2D cell culture, 60% of the A2780cis cell population was suppressed with triple combination therapy; and the long-term therapeutic efficacy of the US + AuNCs + Cis with the low-dose drug was demonstrated by suppressing 83% of colony formation. According to the results in the 3D cell model, 60% of the spheroid formation was suppressed by the triple combination therapy with low-dose Cis. These results not only demonstrate the success of the US + AuNCs + Cis triple combination therapy for its long-term therapeutic effect on resistant cancer cells but also verified that it might enable effective cancer therapy in vivo and clinical stages based on the 3D tumor models. In addition, enhanced anti-cancer activity was demonstrated at the low-dose Cis on drug-resistant cancer cells indicating the triple-combination therapy successfully overcame drug resistance and this is a promising strategy to reduce the side effects of chemotherapy. This work exhibits a novel US and AuNCs-mediated combination cancer therapy, which demonstrates the role of ultrasound-active AuNCs to combat drug resistance with low-dose chemotherapy.     

碲镉汞焦平面光伏器件的实时γ辐照效应研究

对碲镉汞长波和中波焦平面光伏器件进行了实时γ射线辐照效应研究,通过辐照过程中实时测试器件的电流-电压特性,发现随着辐照剂量的增加,中波器件比长波器件表现出更好的抗辐照能力.对于长波器件,随着辐照剂量的增大,能够反映器件性能的零偏电阻逐渐降低;对于中波器件,零偏电阻随着辐照剂量的增加无固定变化趋势,辐照效应主要表现在电阻-电压曲线随着辐照剂量增加出现越来越明显的扰动.根据光伏器件的暗电流机理,对长波器件的电阻-电压曲线进行数值拟合,发现辐照引起少子产生-复合寿命逐渐降低,缺陷密度逐渐增大,主要影响的电流机理 关键词： γ辐照 辐照效应 光伏器件 碲镉汞     

Reversal of adriamycin resistance in ovarian carcinoma cell line by combination of verapamil and low-level ultrasound

In order to determine whether ultrasound, alone or combined with verapamil, could reverse resistance in adriamycin resistant human ovarian carcinoma cell line SKOV(3)/ADR in vitro, cells were subjected to a variable concentration of adriamycin. Verapamil, ultrasound exposure and both of the two were used concurrently or sequentially. Survival rates were decreased in groups in which acoustic irradiation was exerted, or verapamil pretreated and both of which applied. Intracellular adriamycin levels were high where cytotoxicity was enhanced. These results revealed that ultrasound reverse drug resistance in ovarian carcinoma cells, and synergism also existed between verapamil and acoustic exposure if administrated sequentially. These effects were ascribed to increase of intracellular adriamycin accumulation.     

Quantification of optison bubble size and lifetime during sonication dominant role of secondary cavitation bubbles causing acoustic bioeffects

Acoustic cavitation has been shown to deliver molecules into viable cells, which is of interest for drug and gene delivery applications. To address mechanisms of these acoustic bioeffects, this work measured the lifetime of albumin-stabilized cavitation bubbles (Optison) and correlated it with desirable (intracellular uptake of molecules) and undesirable (loss of cell viability) bioeffects. Optison was exposed to 500 kHz ultrasound (acoustic pressures of 0.6-3.0 MPa and energy exposures of 0.2-200 J/cm2) either with or without the presence of DU145 prostate cancer cells (10(6) cells/ml) bathed in calcein, a cell-impermeant tracer molecule. Bubble lifetime was determined using a Coulter counter and flow cytometer, while bioeffects were evaluated by flow cytometry. The lifetime of Optison cavitation nuclei was found to decrease and bioeffects (molecular uptake and loss of cell viability) were found to increase with increasing acoustic energy exposure. These bioeffects correlated well with the disappearance of bubbles, suggesting that contrast agent destruction either directly or indirectly affected cells, probably involving unstabilized cavitation nuclei created upon the destruction of Optison. Because Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects, the indirect mechanism involving secondary cavitation bubbles is more likely.     

A scanning and transmission electron microscopic study of the development of the surface structure of neuroepithelial bodies in the mouse lung

Neuroepithelial bodies (NEBs) are groups of neuroepithelial (NE) cells that are localized on mounds on the bronchiolar epithelium of the lung. The present study examined NEBs in mice ranging in age from 2 days before birth to 80 days after birth. The position and surface architecture of NEBs was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In foetal mice, 2 days before birth, NEBs were distinguished from the rest of the bronchiolar epithelium by a slight elevation of non-ciliated Clara-like cells arranged in a cobbestone-like pattern. The exposed surface of the NEB was identified by small protrusions with regular microvilli intermittenly located at the base of deep clefts between the Clara-like cells. The surface of the Clara-like cells had fewer and smaller microvilli and could be easily distinguished from the apical surface of the NEB. Before birth, the surface of all of the apical cells was covered by regularly placed microville, however after birth some of the more prominently positional apical cells revealed a bare patch at the centre of the portion of apical cell exposed to the lumen of the lung. As the mice aged there was an increase in the number of apical cell protusions observed with centrally positioned bare patches. These two morphologically distinct surfaces of apical cells may have separate specialized functions. The exposed surfaces of apical cells were often observed in pairs and this feature has been observed in various sensory organs providing support for chemoreceptive function. However small bright spheres resembling vesicles were frequently observed on the lumenal surface of apical cells of the centrally placed bare patch. Transmission electron microscopy confirmed the presence of vesicles on the surface of apical cells and due to their location these vesicles were thought to contain a substance secreted into the lumen of the lung by apical cells. The significance of the bare region on the apical cells is not clear in terms of the proposed chemoreceptive function usually attributed to NEBs. It may be possible that the morphological changes observed in apical cells after birth are more appropriate for secretion of than for chemoreception. This is supported by the observation in the present study of vesicles lying on the lumenal surface of the bare region of the apical cell, however the mechanism for secretion of whoel vesicles is not clear and requires further investigation.     

Enhanced Protein Adsorption,Cell Attachment,and Neural Differentiation with the Help of Amine Functionalized Polycaprolactone Scaffolds

Today, neurodegenerative diseases are very common among people. As a result, researchers are investigating methods for treatment of these diseases. One therapeutic approach is differentiating stem cells into neural cells to replace damaged areas of the brain. Cell attachment is the first, necessary step for the process of differentiation. Hence, we tried to enhance cell adhesion and proliferation of bone marrow stem cells on poly(?-caprolactone) (PCL) scaffolds through modifying this substrate with amine functional groups. The presence of amine groups was confirmed by Fourier transform infrared spectrometry (FTIR). Protein adsorption was measured at 280 nm via UV-spectrometry. The proliferation of differentiated neurons was assessed by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (a dye) and cresyl violet staining. Finally, the morphology of differentiated neurons was shown by scanning electron microscopy (SEM). Results showed that amine modification of PCL scaffolds enhanced protein absorption and, consequently, cell adhesion and proliferation.     

Rutacridone as a Fluorescent Dye for the Study of Pollen

Rutacridone, alkaloid from plant Ruta graveolens was used for the histochemical staining of Hippeastrum hybridum pollen. The rutacridone fluorescence (excited by UV light at 360–380 nm) has been studied with Karnaukhov-constructed microspectrofluorimeters. Nucleus and DNA were stained by this dye in cells and fluoresced by green light with maxima of 530 and 555 nm, respectively. Development of the pollen was analyzed with this dye.     

Morphological characterization via light and electron microscopy of Atlantic jackknife clam (Ensis directus) hemocytes

The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim’s stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96 × 10⁶ cells mL⁻¹ while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function.     

Modeling of the surface nucleation and growth on active centers from the vapor phase

The formation of nuclei of a new phase and their growth on the inhomogeneous substrate from a vapor phase are studied. Basic kinetic equations describing such a process are solved numerically. The effect of the depletion of active sites during the growth of nuclei is taken into account. Basic characteristics of the nucleation process, such as size distribution function, nucleation rate and number of nuclei formed on the unit surface are determined. It is shown that the size distribution of nuclei evolves by a nontrivial way as a function of time. This process is fully nonstationary from the viewpoint of nucleation rate. The total number of nuclei reaches the number of active centers for a sufficiently long time. Presented at the VIII-th Symposium on Surface Physics, Třešt’ Castle, Czech Republic, June 28 – July 2, 1999. This work was supported by Grant No. 202/99/0403 of the Grant Agency of the Czech Republic.