量子点体外对小鼠腹腔巨噬细胞细胞化学的影响

目的 探讨量子点(QDs)体外对巨噬细胞细胞化学及酶活性的影响.方法 用倒置相差显微镜、荧光显微镜、细胞化学方法,在细胞水平观察QDs对巨噬细胞的生物相容性及对PAS反应、Feulgen反应、ATP酶、酸性磷酸酶(AcP)、碱性磷酸酶(ALP)、α-醋酸萘酚酯酶(ANAE)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)活性的影响.结果 3.125mg/L剂量的QDs对巨噬细胞的结构没有影响,但细胞内的细胞化学及酶活性发生了不同的变化,即PAS反应、Feulgen反应、AcP、ALP、ANAE和Mg2 -ATP酶表现为阳性,而SDH、LDH则为阴性.阳性结果中,Feulgen反应、ANAE、ALP和Mg2 -ATP组中QDs组与空白组比较,有统计学意义(P＜0.05),而PAS反应、AcP组中QDs组与空白组比较,无统计学意义(P＞0.05).结论 在细胞学水平上,QDs虽然可以使巨噬细胞内的某些酶发生变化,但不影响其结构及吞噬功能.3.125mg/L剂量的QDs可以很好地应用于生物医学领域标记活细胞,对细胞没有明显的影响.     

仿生量子点脂质体纳米探针的合成

目的:合成仿生量子点脂质体纳米探针（PEG-LP-QDs）,优化合成条件并初步探究PEG-LP-QDs的性质,为PEG-LP-QDs在体内示踪、肿瘤成像、多重检测等生物医学领域的应用奠定基础。 方法:采用薄膜分散法,以磷脂酰胆碱、聚乙二醇（PEG）-磷脂酰乙醇胺、胆固醇为膜材,包裹一定量的油溶性ZnCdSe/ZnS量子点（QDs）合成PEG-LP-QDs;通过透射电子显微镜(TEM)、纳米颗粒追踪技术（NTA）、荧光分光光度计鉴定并分析PEG-LP-QDs的形貌、粒径和荧光强度,进而优化合成温度及QDs数量。 结果:根据合成的LPs粒径与QDs的荧光强度变化,选取40 ℃为最佳合成温度;TEM和NTA结果证实PEG-LP-QDs合成成功,粒径集中分布于128 nm左右;且当加入10 μL浓度为3 mg/mL的QDs时,PEG-LP-QDs的荧光强度达到最大值。 结论:成功合成新型PEG-LP-QDs,具有良好的生物相容性和独特的发光特性,其在生物医学领域中的应用价值有待进一步研究。     

量子点探针对人肝癌裸鼠模型的体内靶向成像研究

目的研究量子点标记靶向探针对人肝癌裸鼠模型的体内成像技术。方法将巯基乙酸修饰的水溶性量子点结合鼠抗人甲胎蛋白（AFP）单克隆抗体制备成水溶性量子点-AFP-Ab复合物探针。荧光、紫外光光谱分析及透射电镜研究其特性。通过直接免疫荧光法,用该复合物探针特异性识别肝癌细胞株HCCLM6 AFP抗原。将体外培养的肝癌细胞株HCCLM6通过皮下接种和尾静脉注射裸鼠分别建立人肝癌裸鼠模型和肺转移模型。尾静脉注射量子点-AFP-Ab探针,用蓝光二极管照射获得活体荧光成像;用掺Ti蓝宝石激光器照射,对肿瘤部位和正常部位进行光谱分析。取血清检测丙氨酸转氨酶、天冬氨酸转氨酶、尿素氮和肌苷水平。取裸鼠肝、脾、肾、肺、心和脑6种主要实质性器官行振荡切片,共聚焦显微镜观察,研究量子点-AFP-Ab探针在裸鼠体内的非特异性摄取。结果量子点-AFP-Ab复合物探针具有激发光谱宽、荧光强度高的特点,能特异性与肝癌细胞AFP抗原高亲和力结合,在体内能特异性靶向肿瘤组织进行活体成像,无明显急性毒性。光谱分析显示量子点-AFP-Ab复合物探针主要分布于肿瘤的外周部位,少数该探针被肝、脾和肺非特异性摄取。结论量子点-AFP-Ab复合物探针具有优良的光学特性和生物相容性,能够进行肝癌体内靶向成像,将有助于肝癌的分子靶向研究。     

生长抑素和血管活性肠肽对腹腔巨噬细胞吞噬能力的调节和细胞内钙离子浓度的调节

生长抑素(SS)和血管活性肠肽(VIP)是两种具有多种生物活性的神经肽,在胃肠道内含量丰富,近年来研究发现,在淋巴细胞和巨噬细胞的表面有SS和VIP的特异性受体,它们对免疫的调节作用日益受到重视。本实验的目的是观察SS和VIP对体外培养的腹腔巨噬细胞吞噬能力的影响和对细胞内钙离子浓度的调节作用。方法:1巨噬细胞吞噬中性红后,溶解细胞,用分光光度计测定光密度,反映巨噬细胞的吞噬能力;2巨噬细胞与45Ca共同培养后,用液体闪烁计数测定其放射性,来反映巨噬细胞内Ca2+的浓度;3Fluo-3/AM作为荧光探针,应用激光共聚焦扫描显微镜观察巨噬细…     

固定剂、封片剂、温度及除菌方式对量子点标记细胞的影响

目的:观察不同固定剂、封片剂、温度及除菌方式对3种量子点标记小鼠腹腔巨噬细胞和正常皮肤的影响.方法:利用发射波长为610、 523和576 nm的3种量子点,以具有吞噬能力的小鼠腹腔巨噬细胞及正常皮肤为载体,观察不同的固定剂、封片剂、温度及除菌方式对量子点标记细胞及组织的影响.结果:3种量子点对小鼠腹腔巨噬细胞和皮肤组织没有明显的毒性,并且在6～72 h内保持荧光不衰减.高于56℃的温度会使量子点失去发射荧光的特性.较好的除菌方式为 60Co照射和微孔滤膜过滤.最稳定的封片剂为水溶性的ClearmountTM.吞噬了量子点523的巨噬细胞,可使用多种固定液固定.结论:不同波长量子点标记小鼠腹腔巨噬细胞和皮肤组织的效能受固定剂、封片剂、温度和除灭菌方式的影响.     

人参皂甙对小鼠腹腔巨噬细胞活性的影响及其作用机制初步探讨

目的:观察人参皂甙(GS)对巨噬细胞的免疫激活作用,探讨GS活化巨噬细胞及免疫调节机制。方法:在体外培养的小鼠腹腔巨噬细胞中加入不同浓度的GS后,观察巨噬细胞一氧化氮(NO)合成及MTT比色法检测活化后的巨噬细胞对肿瘤细胞杀伤活性的影响;扫描电子显微镜(SEM)观察巨噬细胞的超微结构改变;激光扫描共聚焦显微镜(LSCM)观察巨噬细胞表面组织相容性复合体Ⅱ(MHCⅡ)的变化;以特异性荧光探针Fluo-3/AM负载细胞,应用LSCM检测巨噬细胞内Ca2+浓度。结果:小鼠腹腔巨噬细胞经GS作用后,细胞形态发生活化性改变,杀瘤活性增强,细胞表面MHCⅡ表达增加并增强对H22细胞杀伤活性;GS作用细胞4小时后,25~200 mg/L GS细胞内Ca2+浓度升高,并与药物浓度呈正相关。结论:GS在体外能激活小鼠巨噬细胞,促进其发挥免疫防御功能,其免疫调节机制可能与细胞内Ca2+浓度升高有关。     

核酸适配体W3介导靶向乳腺癌的量子点成像

目的 利用核酸适配体W3对乳腺癌细胞的结合特异性，对乳腺癌细胞以及临床乳腺癌组织石蜡切片标本进行量子点(QDs)成像。方法 利用流式细胞测量术检测核酸适配体W3对乳腺癌细胞系的结合特异性，基于生物素与链霉亲和素的结合反应原理，通过荧光显微镜观察核酸适配体W3对乳腺癌细胞系以及临床乳腺癌患者组织标本的量子点靶向成像，并进一步分析其临床意义。结果 核酸适配体W3能特异性结合恶性程度较高的乳腺癌细胞，偶联量子点QD605形成量子点探针(W3-QDs),能够对恶性程度较高的乳腺癌细胞系以及乳腺癌组织实现靶向成像，并且具有高恶性程度的乳腺癌组织的荧光强度明显高于恶性程度低的乳腺癌组织。结论 核酸适配体W3偶联量子点后能够实现乳腺癌的靶向成像，可用于乳腺癌的早期诊断。     

量子点与多肽LyP-1的连接、表征及对肿瘤细胞的识别

利用水相合成的量子点(Quantum dots,QDs)纳米粒子,通过交联剂[N-succinimidyl3-(2-pyridyldithio)propionate,SPDP]将其与多肽LyP-1(CGNKRTRGC)进行连接以形成一种纳米荧光探针。毛细管电泳、吸收光谱以及荧光光谱测试结果表明,LyP-1已成功地连接到了QDs表面,所得荧光探针能较好地识别SPCA-1肺腺癌细胞,但不识别HL-60原髓细胞白血病成淋巴细胞。     

EMB基因330密码子分子DNA探针设计及荧光检测

目的 针对结核杆菌耐乙胺丁醇embB330密码子位点设计分子DNA探针,尝试运用荧光分光光度计直接观测液相中分子DNA探针与embB330密码子扩增产物杂交后的荧光信号,从而检出该位点突变.方法 运用软件Beacon designer设计embB基因包含330密码子的分子DNA探针,应用荧光分光光度计检测embB306密码子扩增片段与探针杂交后荧光信号,比较扩增产物测序结果.结果 通过荧光分光光度计观测到结核标准株及embB330密码子突变株PCR产物与探针杂交后荧光信号存在显著差异;33株耐乙胺丁醇组与10株H37RV标准株对照组荧光信号强度比较,耐乙胺丁醇组embB330密码子突变检出率为3%,测序法突变检出率为3%.结论 分子DNA探针技术可以有效检测embB330密码子单碱基靶点突变;应用荧光分光光度计直接观测液相荧光杂交信号简单、灵敏.     

人树突状细胞摄取量子点后的表面标志与超微结构

目的:观察人树突状细胞体外摄取量子点后,其形态、表面标志、超微结构的变化.方法:两种量子点与人未成熟树突状细胞体外共孵育,荧光显微镜下观察量子点在树突状细胞内的分布;免疫细胞化学方法检测树突状细胞表面标志;扫描电镜及透射电镜观察树突状细胞超微结构的改变.结果:与量子点共培养的树突状细胞的细胞质内充满了较强荧光颗粒,共培养前后的树突状细胞表面标志均呈阳性反应.扫描电镜下可见树突状细胞表面有丰富的典型树枝样的突起.透射电镜下可见树突状细胞以胞饮的方式摄入量子点,形成有膜包被的囊泡分布在细胞质内.结论:树突状细胞是通过胞饮作用摄取量子点,且量子点对树突状细胞的形态、表面标志及超微结构没有影响,生物相容性好.     

The cytotoxicity of cadmium based, aqueous phase - synthesized, quantum dots and its modulation by surface coating

In this report, we evaluated the cytotoxicity of a series of quantum dots (QDs) directly synthesized in aqueous phase, i.e., thiols-stabilized CdTe, CdTe/CdS core-shell structured and CdTe/CdS/ZnS core-shell-shell structured QDs, with a variety of cell lines including K562 and HEK293T. We have demonstrated that the CdTe QDs are highly toxic for cells due to the release of cadmium ions. Epitaxial growth of a CdS layer reduces the cytotoxicity of QDs to a small extent. However, the presence of a ZnS outlayer greatly improves the biocompatibility of QDs, with no observed cytotoxicity even at very high concentration and long-time exposure in cells. Our systematic investigation clearly shows that the cytotoxicity of QDs can be modulated through elaborate surface coatings and that the CdTe/CdS/ZnS core-shell-shell structured QDs directly synthesized in aqueous phase are highly promising biological fluorescent probes for cellular imaging.     

Fluorescent cadmium telluride quantum dots embedded chitosan nanoparticles: a stable,biocompatible preparation for bio-imaging

Fluorescent cadmium telluride quantum dots (CdTe QDs) are an optically attractive option for bioimaging, but are known to display high cytotoxicity. Nanoparticles synthesized from chitosan, a natural biopolymer of β 1-4 linked glucosamine, display good biocompatibility and cellular uptake. A facile, green synthetic strategy has been developed to embed green fluorescent cadmium telluride quantum dots (CdTe QDs) in biocompatible CNPs to obtain a safer preparation than ‘as is’ QDs. High-resolution transmission electron microscopy showed the crystal lattice corresponding to CdTe QDs embedded in CNPs while thermogravimetry confirmed their polymeric composition. Electrostatic interactions between thiol-capped QDs (4 nm, ?57 mV) and CNPs (~300 nm, +38 mV) generated CdTe QDs-embedded CNPs that were stable up to three months. Further, viability of NIH3T3 mouse fibroblast cells in vitro increased in presence of QDs-embedded CNPs as compared to bare QDs. At the highest concentration (10 μg/ml), the former shows 34 and 39% increase in viability at 24 and 48 h, respectively, as compared to the latter. This shows that chitosan nanoparticles do not release the QDs up to 48 h and do not cause extended toxicity. Furthermore, hydrolytic enzymes such as lysozyme and chitinase did not degrade chitosan nanoparticles. Moreover, QDs-embedded CNPs show enhanced internalization in NIH3T3 cells as compared to bare QDs. This method offers ease of synthesis and handling of stable, luminescent, biocompatible CdTe QDs-embedded CNPs with a favorable toxicity profile and better cellular uptake with potential for bioimaging and targeted detection of cellular components.     

Photostable water-dispersible NIR-emitting CdTe/CdS/ZnS core–shell–shell quantum dots for high-resolution tumor targeting

Near-infrared (NIR, 700–900 nm) fluorescent quantum dots are highly promising as NIR bioprobes for high-resolution and high-sensitivity bioimaging applications. In this article, we present a class of NIR-emitting CdTe/CdS/ZnS core–shell–shell quantum dots (QDs), which are directly prepared in aqueous phase via a facile microwave synthesis. Significantly, the prepared NIR-emitting QDs possess excellent aqueous dispersibility, strong photoluminescence, favorable biocompatibility, robust storage-, chemical-, and photo-stability, and finely tunable emission in the NIR range (700–800 nm). The QDs are readily functionalized with antibodies for use in immunofluorescent bioimaging, yielding highly spectrally and spatially resolved emission for in vitro and in vivo imaging. In comparison to the large size of 15–30 nm of the conventional NIR QDs, the extremely small size (∼4.2 nm or 7.5 nm measured by TEM or DLS, respectively) of our QDs offers great opportunities for high-efficiency and high-sensitivity targeted imaging in cells and animals.     

Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots

Quantum dots (QDs) are emerging as alternative or complementary tools to the organic fluorescent dyes currently used in bioimaging. QDs hold several advantages over conventional fluorescent dyes including greater photostability and a wider range of excitation/emission wavelengths. However, recent work suggests that QDs exert deleterious effects on cellular processes. This study examined the subcellular localization and toxicity of cadmium telluride (CdTe) QDs and pharmacological means of preventing QD-induced cell death. The localization of CdTe QDs was found to depend upon QD size. CdTe QDs exhibited marked cytotoxicity in PC12 and N9 cells at concentrations as low as 10 µg/ml in chronic treatment paradigms. QD-induced cell death was characterized by chromatin condensation and membrane blebbing and was more pronounced with small (2r=2.2±0.1 nm), green emitting positively charged QDs than large (2r=5.2±0.1 nm), equally charged red emitting QDs. Pretreatment of cells with the antioxidant N-acetylcysteine and with bovine serum albumin, but not Trolox, significantly reduced the QD-induced cell death. These findings suggest that the size of QDs contributes to their subcellular distribution and that drugs can alter QD-induced cytotoxicity.     

Dual-labelled immunoassay with goldmag nanoparticles and quantum dots for quantification of casein in milk

A dual-labelled immunoassay using goldmag nanoparticles (GMNPs) and CdTe quantum dots (QDs) was applied for the quantification of casein in milk. In this method, anti-casein monoclonal antibody bound to GMNPs was used as capture probe, and the anti-casein polyclonal antibody, labelled with CdTe QDs, was employed as detection probe. This dual-labelled immunoassay was optimized and applied in the testing of casein content of three brands of commercial milks. The results showed a good linear tendency between casein concentrations and fluorescent intensities in the range of 4.617–289.9?ng/mL, and the half inhibition concentration (IC₅₀) was 36.59?ng/mL. Besides, the recovery rates of this developed immunoassay were in accordance with those in traditional enzyme-linked immunosorbent assay for rapid detection of casein content in commercial milks. It suggested that this dual-labelled immunoassay was reliable, and could provide important theoretical value and practical significance in the identification and quantification of milk allergens.     

Folic acid-conjugated core/shell ZnS:Mn/ZnS quantum dots as targeted probes for two photon fluorescence imaging of cancer cells

This work presents a novel approach to producing water soluble manganese-doped core/shell ZnS/ZnS quantum dots (ZnS:Mn/ZnS). The Mn-doped ZnS core was prepared through a nucleation doping strategy and a ZnS shell was grown on ZnS:Mn d-dots by decomposition of Zn(2+)-3-mercaptopropionic acid (MPA) complexes at 100 °C. It was found that the Mn2+(4)T1→6A1 fluorescence emission at ～590 nm significantly increased after growth of the shell when the Mn2+ doping content was 4.0 at.%. A photoluminescence quantum yield of ～22% was obtained for core/shell nanocrystals. The nanoparticles were structurally and compositionally characterized by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and dynamic light scattering. The surface MPA molecules favor the dispersion of ZnS:Mn/ZnS QDs in aqueous media and make possible conjugation with targeting folic acid molecules. The folate receptor-mediated delivery of folic acid-conjugated ZnS:Mn/ZnS QDs was demonstrated using confocal microscopy with biphotonic excitation. Bare and folate-conjugated QDs exhibit only weak cytotoxicity towards folate receptor-positive T47D cancer cells and MCF-7 cells, used as a reference, at high concentrations (mmolar range) after 72h incubation.     

Ultra-photostable, non-cytotoxic, and highly fluorescent quantum nanospheres for long-term, high-specificity cell imaging

A new class of fluorescent quantum nanospheres (QNs) is directly achieved in aqueous phase through a facile one-pot microwave irradiation (MWI) strategy. Multi-color QNs with maximum emission wavelengths ranging from 525 to 610 nm and PLQY of 30-60% are facilely prepared through this new MWI strategy. In addition to strong fluorescence, these QNs possess excellent photostability, preserving ～90% of the original intensity after 70 min high-power UV irradiation (100 W Xeon lamp). In sharp contrast, the fluorescence of CdTe/CdS/ZnS core-shell-shell quantum dots (QDs), recognized as established fluorescent probes with robust photostability, decrease to ～50% under the same conditions. Besides, cytotoxicity assessment demonstrates that the prepared QNs exhibit favorable cytocompatibility to K562 cells with high concentration (3 μmol) and long-time incubation (24 h). Furthermore, cellular imaging results demonstrate that the as-prepared QNs are remarkably efficacious for long-term and high-specificity immunofluorescent cellular labeling, and multi-color cell imaging. Our systematical investigation clearly shows that these high-performance QNs may serve as practical and powerful tools for various biological researches, such as in vivo and in vitro imaging.     

Extracellular microbial synthesis of biocompatible CdTe quantum dots

An efficient bacterial synthesis method to harvest cadmium telluride (CdTe) quantum dots (QDs) with tunable fluorescence emission using Escherichia coli is demonstrated. Ultraviolet–visible, photoluminescence, X-ray diffraction and transmission electron microscopy analysis confirmed the superior size-tunable optical properties, with fluorescence emission from 488 to 551 nm, and the good crystallinity of the as synthesized QDs. A surface protein capping layer was confirmed by hydrodynamic size, ζ potential and Fourier transform infrared spectroscopy measurements, which could maintain the viability (92.9%) of cells in an environment with a QD concentration as high as 2 μM. After functionalization with folic acid the QDs were used to image cultured cervical cancer cells in vitro. Investigations of bacterial growth and morphology and the biosynthesis of CdTe QDs in Luria–Bertani medium containing E. coli-secreted proteins showed that extracellular synthesis directly relied on the E. coli-secreted proteins, and a mechanism for protein-assisted biosynthesis of QDs is proposed. This work provides an economical approach to fabricate highly fluorescent biocompatible CdTe QDs via an environmentally friendly production process. The biosynthesized QDs may have great potential in broad bio-imaging and bio-labeling applications.     

The cytotoxicity of cadmium-based quantum dots

Semiconductor Quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. More recently, there have been intense concerns on cytotoxicity assessment of QDs. Most QDs are made of heavy metal ions (e.g., Cd²⁺), which may result in potential in vitro toxicity that hampers their practical applications. In this article, we aim to summarize recent progress on mechanistic studies of cytotoxicity of II-IV QDs. We have studied the cytotoxicity of a series of aqueous synthesized QDs (aqQDs), i.e. CdTe, CdTe/CdS core-shell structured and CdTe/CdS/ZnS core-shell-shell structured aqQDs. Our results suggested that released cadmium ions are responsible for the observed cytotoxicity of cadmium-based QDs. The fact that CdTe/CdS/ZnS core-shell-shell structured QDs are nearly nontoxic to cells further confirmed the role of released cadmium ions on cytotoxicity, and the effective protection of the ZnS shell. However, intracellular level of Cd²⁺ ions cannot be the only reason since the comparison with CdCl₂-treated cells suggests there are other factors contributed to the cytotoxicity of aqQDs. Our studies on genome-wide gene expression profiling and subcellular localization of aqQDs with synchrotron-based scanning transmission X-ray microscopy (STXM) further suggest that the cytotoxicity of CdTe QDs not only comes from the release of Cd²⁺ ions but also intracellular distribution of QD nanoparticles in cells and the associated nanoscale effects.