Biophysical model for high-throughput tumor and epithelial cell co-culture in complex biochemical microenvironments

The in vivo tumor microenvironment is a complex niche that includes heterogeneous physical structures,unique biochemical gradients and multiple cell interactions.Its high-fidelity in vitro reconstruction is of fundamental importance to improve current understandings of cell behavior,efficacy predictions and drug safety.In this study,we have developed a high-throughput biochip with hundreds of composite extracellular matrix(ECM)microchambers to co-culture invasive breast cancer cells(MDA-MB-231-RFP)and normal breast epithelial cells(MCF-10 A-GFP).The composite ECM is composed of type I collagen and Matrigel which provides a heterogeneous microenvironment that is similar to that of in vivo cell growth.Additionally,the growth factors and drug gradients that involve human epidermal growth factor(EGF),discoidin domain receptor 1(DDR1)inhibitor 7 rh and matrix metalloproteinase inhibitor batimastat allow for the mimicking of the complex in vivo biochemical microenvironment to investigate their effect on the spatial-temporal dynamics of cell growth.Our results demonstrate that the MDA-MB-231-RFP cells and MCF-10 A-GFP cells exhibit different spatial proliferation behaviors under the combination of growth factors and drugs.Basing on the experimental data,we have also developed a cellular automata(CA)model that incorporated drug diffusion to describe the experimental phenomenon,as well as employed Shannon entropy(SE)to explore the effect of the drug diffusion coefficient on the spatial-temporal dynamics of cell growth.The results indicate that the uniform cell growth is related to the drug diffusion coefficient,which reveals that the pore size of the ECM plays a key role in the formation of complex biochemical gradients.Therefore,our integrated,biomimetic and high-throughput co-culture platforms,as well as the computational model can be used as an effective tool for investigating cancer pathogenesis and drug development.     

三维微纳米制造技术在癌症生物物理研究中的应用

癌症致命的主要原因是癌细胞在临床上的转移性. 癌细胞的侵袭和转移是一个非常复杂的三维过程, 但现有的癌症研究在活体上有诸多观测和操作上的困难. 而体外实验又通常在培养皿中进行, 其二维的生长环境已完全不能满足对癌细胞空间转移性的深入研究, 故在活体外构建出癌细胞侵袭和转移的三维物理模型具有十分重要的意义. 然而如何在体外尽可能真实地模拟体内癌细胞的生长微环境一直是困扰科学家的难题. 本文系统介绍了三维微纳米制造的几种主流技术, 探讨了它们在癌症生物物理研究中的应用和发展. 在此基础上为了在未来实现对体外三维模型的制造、观测和精确操作, 文章还创新性地提出了一种结合紫外线固化生物型水凝胶的三维成型技术、光片三维成像技术以及微纳米探针控制技术的一体化研究平台. 这些先进的技术和理念, 势必会逐步升级现有传统的癌症研究手段, 为未来理解和治疗癌症揭开全新的篇章.     

胶原纤维网络和癌细胞的力学微环境

文章以第一类胶原纤维网络为例, 着重分析了癌细胞三维微环境的多尺度结构及力学特征. 对于细胞与细胞外介质结合的蛋白集团、单个细胞以及细胞群体, 分别由单个纤维(或亚纤维)、纤维集束以及纤维网络整体来决定相应的力学环境. 文章同时也讨论了胶原纤维(及其类似材料) 的局限性.     

鼠S180肉瘤的体内31P MRS研究

利用表面线圈³¹P NMR研究了小鼠S180肉瘤生长过程中能量代谢和磷脂类变化的特点.结果发现:随着肿瘤体积的增大,(1)Pi和PME升高;(2)PCr降低,在肿瘤体积较大时常检测不到;(3)β-NTP(通常用来表示ATP的量)变化较小;(4)PDE波动性较大;(5)PCr/Pi和β-NTP/Pi比值均下降,且PCr/Pi比β-NTP/Pi下降得快;(6)PME/β-NTP比值升高;(7)肿瘤pH下降,且与PCr/Pi、β-NTP/Pi或(PCr+β-NTP)/Pi比值有相关性.讨论了与这些参数变化相关联的生物学意义.     

Confirmation of the protective effect of cysteamine in in vitro ultrasound exposures

Armour and Corry (Radiat. Res. (1982) 89 369–380) reported that ultrasound-induced damage to in vitro Chinese hamster ovary cells was significantly reduced in the presence of cysteamine. The objective of this study was to attempt verification of this result. Four series of experiments were undertaken using in vitro cell suspensions, namely: (1) determination of the effect of cysteamine concentration on cell growth; (2) determination of the temperature dependence of ultrasonically induced cell damage; (3) determination of a dose-response relationship for the cytotoxicity of cysteamine; and (4) assessment of cell integrity and reproductive capacity in the presence or absence of cysteamine during ultrasonic exposure. Ultrasound parameters included a resonance frequency of 1 MHz, a continuous wave exposure duration of 5 min, and intensities from 0 to 21.6 W cm⁻². The results indicated a dependence of ultrasound's efficacy on the medium's temperature during insonation and a significant reduction of ultrasound efficacy in compromising cellular integrity in the presence of cysteamine.     

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quantification of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell's Franz device with receptor medium container with a PBS/EtOH 20% solution (10 mM, pH 7.4) at 37 °C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall.     

Angiostatin抑制转移性肿瘤血液灌注和间质液输运的数值模拟

数值研究抗血管生成因子Angiostatin对转移性肿瘤微循环内血液灌注和间质液输运的抑制效应.肿瘤微血管网生成数学模型包括angiostatin的抑制效应、促血管生成因子的趋化效应和内皮细胞自身的扩散效应,血液灌注、间质液输运和跨壁流体交换分别采用Poiseuille、Darcy和Starling定律来描述.结果表明:angiostatin能抑制转移性肿瘤内外微血管网的生长速率、毛细血管芽的分叉、融合与增殖;进一步angiostatin可改善转移性肿瘤微环境内血液灌注和间质液输运的特性,从而增加微血管内外流体对流和物质交换的能力,降低肿瘤内药物输运困难,结果与实验相符,可为临床抗血管生成治疗肿瘤提供有益信息.     

PEG化氘代单磷酸三苯甲基自由基的高效合成及性能研究

电子顺磁共振（EPR）波谱联用外源性自旋探针技术是测量体内外pH的有效手段.有研究表明,单磷酸取代三苯甲基（p₁TAM）自由基是目前用于检测pH的最理想的EPR探针.然而,这类探针的合成产率低、易受蛋白影响,极大限制了其生物应用.针对上述问题,本文提出了高效合成p₁TAM自由基的方法,使其总产率从文献报道的1.6%提高到了25%;同时采用PEG修饰的方法避免了白蛋白（BSA）对其产生的干扰.进一步氘代实验结果证实：非氘代PEG化（p₁TAM-H）衍生物（POP）虽与氘代PEG化（p₁TAM-D）衍生物（dPOP）具有相似的pH敏感性（其pKa分别为6.80和6.79）,但POP的EPR谱图较为复杂,而dPOP无论在低pH还是在高pH条件下均具有简单的EPR双峰信号;而且,dPOP具有比POP和p₁TAM-H更好的检测灵敏性;此外,dPOP还具有较好的生物稳定性和氧气敏感性.因此,dPOP能够用来同时检测pH和氧气,有望在生物医学方面得到更好的应用.     

Molecular beam epitaxy growth of HgCdTe for high performance infrared photon detectors

Significant progresses have been made in the molecular beam epitaxy (MBE) growth of HgCdTe for high performance infrared photon detectors with the aid of in situ and ex situ characterization techniques. Superlattice interfacial layers compensate in part for the influence of non-ideal CdZnTe substrates and hence improved the material quality as well as yield. They result in photoconductive carrier recombination lifetimes approaching theoretical limits set by the intrinsic radiative and Auger recombination mechanisms for 8–14 μm long-wavelength infrared HgCdTe. Very high composition and thickness uniformities have also been achieved. However, the Urbach tail energy, which is associated with structural disorder, was found to be non-uniform for both large wafer (up to 20 × 20 mm²) and very small area (down to 200 × 200 μm²). After several years of improvements in MBE HgCdTe growth techniques, substrates once again have become a bottleneck to further improvements.     

Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This technique replaces the traditional sectioning of cartilage or culturing of chondrocytes. Whole ‘chunks’ of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other stains have been used, the stains presented include phalloidin and propidium iodide which stain filamentous actin (F-actin) and the DNA and RNA of cells, respectively. Collagen secreting endoplasmic reticulum (ER) was localized with a primary antibody to chick prolyl hydroxylase (CPH) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed using in situ hybridization. The cDNA probe specific for the C-propeptide region of the 1 type (II) collagen mRNA was nick translated and labeled with biotin-16-dUTP. Biotin labeled probes were visualized with avidin-FITC. Depending on the intensity of the stain, we were able to analyze approximately 3–10 layers of chondrocytes. Stains penetrated into the cartilage better than antibodies and biotin-avidin labeled cDNA probes. The F-actin was located as bands of filaments in the superficial layers of the cartilage and was associated with the membranes that marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinuclear regions of the cells, but the antibody was only able to penetrate 4–5 cell layers. Single label in situ hybridization studies show that chondrocytes are positive for type II collagen mRNA. Similar to the immunohistochemistry, in situ hybridization probes were only able to penetrate 4–5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.     

基于CEST机制的pH成像方法、原理和应用

化学交换饱和转移（Chemical Exchange Saturation Transfer,CEST）技术作为一种新型的磁共振成像（Magnetic Resonance Imaging,MRI）技术.它的原理为溶质池中被激发饱和的质子与游离水中未被饱和的质子间的化学交换,能够引起水质子磁共振信号的下降,从而获得组织内生物分子的相关信息.由于质子间的交换速率k_ex与组织微环境的pH值之间存在直接联系,因而可以通过溶质质子的CEST信号对活体组织进行pH成像.目前用于pH成像的溶质分子既包括内源性游离的蛋白质、多肽分子,还包括一系列的外源性小分子和金属螯合物.通过不同类型的比率法、内源性胺和酰胺浓度-独立检测（Amine and Amide Concentration-independent Detection,AACID）等成像方法,能够获得肾脏、中风脑组织以及肿瘤组织的pH图谱.本文详细总结了2000年以来利用CEST技术进行pH成像方面的研究进展,包括对比剂、成像方法和相关应用,展望了活体组织pH成像的发展趋势和应用前景.     

Improved performance of MoS2 FET by in situ NH3 doping in ALD Al2O3 dielectric

Since defects such as traps and oxygen vacancies exist in dielectrics, it is difficult to fabricate a high-performance MoS$_{2}$ field-effect transistor (FET) using atomic layer deposition (ALD) Al$_{2}$O$_{3}$ as the gate dielectric layer. In this paper, NH$_{3}$ in situ doping, a process treatment approach during ALD growth of Al$_{2}$O$_{3}$, is used to decrease these defects for better device characteristics. MoS$_{2}$ FET has been well fabricated with this technique and the effect of different NH$_{3}$ in situ doping sequences in the growth cycle has been investigated in detail. Compared with counterparts, those devices with NH$_{3}$ in situ doping demonstrate obvious performance enhancements: $I_{\rm on}/I_{\rm off}$ is improved by one order of magnitude, from $1.33\times 10^{5}$ to $3.56\times 10^{6}$, the threshold voltage shifts from $-0.74 $ V to $-0.12$ V and a small subthreshold swing of 105 mV/dec is achieved. The improved MoS$_{2}$ FET performance is attributed to nitrogen doping by the introduction of NH$_{3}$ during the Al$_{2}$O$_{3}$ ALD growth process, which leads to a reduction in the surface roughness of the dielectric layer and the repair of oxygen vacancies in the Al$_{2}$O$_{3}$ layer. Furthermore, the MoS$_{2}$ FET processed by in situ NH$_{3}$ doping after the Al and O precursor filling cycles demonstrates the best performance; this may be because the final NH$_{3}$ doping after film growth restores more oxygen vacancies to screen more charge scattering in the MoS$_{2}$ channel. The reported method provides a promising way to reduce charge scattering in carrier transport for high-performance MoS$_{2 }$ devices.     

High-speed photography of transient excitation

Transient excitation can cause the unstable growth and collapse of bubble nuclei, as well as enhancing the stable cavitation of larger bubbles. These unstable collapses have been photographed at 8000 f.p.s., the resulting pictures agreeing well with predicted numerical solutions. These cavities, unlike those normally studied with high-speed photography, are generated by purely acoustic methods, and so the events are as would occur during in situ acoustic cavitation.     

Multiscale models for the growth of avascular tumors

In the past 30 years we have witnessed an extraordinary progress on the research in the molecular biology of cancer, but its medical treatment, widely based on empirically established protocols, still has many limitations. One of the reasons for that is the limited quantitative understanding of the dynamics of tumor growth and drug response in the organism. In this review we shall discuss in general terms the use of mathematical modeling and computer simulations related to cancer growth and its applications to improve tumor therapy. Particular emphasis is devoted to multiscale models which permit integration of the rapidly expanding knowledge concerning the molecular basis of cancer and the complex, nonlinear interactions among tumor cells and their microenvironment that will determine the neoplastic growth at the tissue level.     

原位液体核磁共振在高分子材料表征领域的应用

随着原位系统研究的进步以及灵敏度的提高，原位核磁共振在高分子材料领域的应用越来越广泛.原位液体核磁共振具有分析速度快、适用范围宽、无损检测、无需标样等特点，多用于表征聚合反应动力学及反应机理.除此之外，它还可以通过与其他仪器进行联用、匹配适用的探头等手段分离并解析未知物的化学结构.因此，原位液体核磁共振波谱技术将成为高分子科学研究领域不可或缺的通用工具.     

Projection structure of photosystem II In vivo determined by cryo-electron crystallography

Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4 nm, b = 23.1 nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.     

Nonlinear inversion of ultrasonic guided waves for in vivo evaluation of cortical bone properties

Ultrasonic guided waves (UGWs), which propagate throughout the entire thickness of cortical bone, are attractive for the early diagnosis of osteoporosis. However, this is challenging due to the impact of soft tissue and the inherent difficulties related to multiparametric inversion of cortical bone quality factors, such as cortical thickness and bulk wave velocity. Therefore, in this research, a UGW-based multi-parameter inversion algorithm is developed to predict strength-related factors. In simulation, a free plate (cortical bone) and a bilayer plate (soft tissue and cortical bone) are used to validate the proposed method. The inversed cortical thickness (CTh), longitudinal velocity (V_L) and transverse velocity (V_T) are in accordance with the true values. Then four bovine cortical bone plates were used in in vitro experiments. Compared with the reference values, the relative errors for cortical thickness were 3.96%, 0.83%, 2.87%, and 4.25%, respectively. In the in vivo measurements, UGWs are collected from the tibias of 10 volunteers. The theoretical dispersion curves depicted by the estimated parameters (V_T, V_L, CTh) match well with the extracted experimental ones. In comparison with dual-energy x-ray absorptiometry, our results show that the estimated transverse velocity and cortical thickness are highly sensitive to osteoporosis. Therefore, these two parameters (CTh and V_T) of long bones have potential to be used for diagnosis of bone status in clinical applications.     

Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound

In order to quantify the effects of exposure parameters under therapeutic conditions such as sonodynamic therapy, it is necessary initially to evaluate the inertial cavitation activity in vitro. In this study, the dependence of cavitation activity induced by the low-level dual-frequency ultrasound irradiation on exposure parameters has been studied. Experiments were performed in the near 150 kHz and 1 MHz fields in the progressive wave mode. It has been shown that at constant ultrasound energy the fluorescence intensity for continuous sonication is higher than for pulsed mode. With increasing the duty cycle of pulsed field, the inertial cavitation activity is increased. The activity of cavitation produced by simultaneous combined sonication by two ultrasound fields is remarkably higher than the algebraic sum of effects produced by fields separately (p-value < 0.05). This study shows that simultaneous combined dual-frequency ultrasound sonication in continuous mode is more effective in producing inertial cavitation activity at low-level intensity. Therefore, it is concluded that investigations in this combined ultrasound sonication can be useful in sonodynamic therapy for superficial tumors.     

The 3-D electrostatic potential surrounding a square array of surface charges

Simple scaling laws have been derived which relate the 3-D potential to the inter-surfacestate spacing, the field-plate spacing, the dielectric constants of both insulators bordering the interface, and the dimensions x,y,z. This 3-D potential, obtained by using a pair of images and Neumann boundary conditions, contains the usual 1-D potential as the zeroth harmonic in a Fourier series. The 3-D and 1-D potentials give equivalent densities of conduction electrons in an accumulation layer only for a surface state density σ < 3 × 10¹¹/cm². For higher σ, the 1-D potential seriously underestimates the amount of space charge possible. There is a low saddle point in the 3-D potential configuration which allows surface conduction electrons to transport laterally in the presence of a transverse field.