Synthesis of Folate Conjugated Fluorescent Nanoparticle Probe and Its Application in Cervical Cancer Cell Imaging

Abstract

Cervical cancer is the second most common cancer in women worldwide with 80% of cases arising in the developing world, following cancer of the breast. The mortality associated with cervical cancer can be reduced if this disease is detected in a timely fashion. In this study, a folate conjugated fluorescent nanoparticle (FCFN) probe was used to detect cervical cancer cells. Fluorescent nanoparticles (FNs), with excellent characteristics such as nontoxicity and photostability, were first synthesized with a simple, cost-effective, and environmentally friendly modified St?ber synthesis method and then successfully modified with folate. This kind of fluorescence probe based on FCFNs has been used to detect cervical cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the FCFNs can effectively recognize cervical cancer cells and exhibit good sensitivity and exceptional photostability; they would provide a novel way for the diagnosis and curative effect observation of cervical cancer cells and offer a new method in detecting folate receptors (FR).     

Anti-Her-2 monoclonal antibody conjugated polymer fluorescent nanoparticles probe for ovarian cancer imaging

Fluorescent nanoparticles (FNPs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. A novel kind of polymer fluorescent nanoparticles (PFNPs) was synthesized and its application for ovarian cancer imaging with fluorescence microscopy imaging technology was presented in this study. The PFNPs were synthesized with precipitation polymerization by using methacrylic acid (MAA) as monomer, trimethylolpropane trimethacrylate (Trim) as cross-linker, azobisisobutyronitrile (AIBN) as radical initiator and butyl rhodamine B (BTRB) as fluorescent dye. And the fluorescent dye was embedded into the three-dimensional network of the polymer when the polymer was produced. With this method the PFNPs can be prepared easily. And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody. The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-Her-2 monoclonal antibody conjugated PFNPs can effectively recognize ovarian cancer cells and exhibit good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of ovarian cancer cells.     

A Comparative Study of Receptor-Targeted Magnetosome and HSA-Coated Iron Oxide Nanoparticles as MRI Contrast-Enhancing Agent in Animal Cancer Model

Magnetosomes are specialized organelles arranged in intracellular chains in magnetotactic bacteria. The superparamagnetic property of these magnetite crystals provides potential applications as contrast-enhancing agents for magnetic resonance imaging. In this study, we compared two different nanoparticles that are bacterial magnetosome and HSA-coated iron oxide nanoparticles for targeting breast cancer. Both magnetosomes and HSA-coated iron oxide nanoparticles were chemically conjugated to fluorescent-labeled anti-EGFR antibodies. Antibody-conjugated nanoparticles were able to bind the MDA-MB-231 cell line, as assessed by flow cytometry. To compare the cytotoxic effect of nanoparticles, MTT assay was used, and according to the results, HSA-coated iron oxide nanoparticles were less cytotoxic to breast cancer cells than magnetosomes. Magnetosomes were bound with higher rate to breast cancer cells than HSA-coated iron oxide nanoparticles. While 250 μg/ml of magnetosomes was bound 92 ± 0.2%, 250 μg/ml of HSA-coated iron oxide nanoparticles was bound with a rate of 65 ± 5%. In vivo efficiencies of these nanoparticles on breast cancer generated in nude mice were assessed by MRI imaging. Anti-EGFR-modified nanoparticles provide higher resolution images than unmodified nanoparticles. Also, magnetosome with anti-EGFR produced darker image of the tumor tissue in T2-weighted MRI than HSA-coated iron oxide nanoparticles with anti-EGFR. In vivo MR imaging in a mouse breast cancer model shows effective intratumoral distribution of both nanoparticles in the tumor tissue. However, magnetosome demonstrated higher distribution than HSA-coated iron oxide nanoparticles according to fluorescence microscopy evaluation. According to the results of in vitro and in vivo study results, magnetosomes are promising for targeting and therapy applications of the breast cancer cells.     

水溶性量子点荧光探针用于胃癌细胞相关抗原CA242的检测

基于量子点荧光探针对胃癌细胞相关抗原CA242进行了检测。首先在水溶液中直接合成性能优良的量子点荧光纳米颗粒,并在其表面成功修饰了羊抗小鼠IgG和聚乙二醇,制得功能化的水溶性量子点荧光探针,并利用探针对胃癌细胞相关抗原CA242进行检测,进一步与传统的基于荧光染料标记的免疫荧光分析方法进行了比较。实验结果表明:该功能化的探针能够有效地识别胃癌细胞相关抗原CA242,并且在光稳定性和灵敏度方面都较传统的基于荧光染料标记的免疫荧光分析方法有明显的改善,从而为CA242的相关检测以及胃癌的诊断与愈后判断提供了新的方法。     

FSiNPs mediated improved double immunofluorescence staining for gastric cancer cells imaging

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen-antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.     

A Novel Magneto-fluorescent Nano-bioprobe for Cancer Cell Targeting,Imaging and Collection

Silica-coated magnetic polystyrene nanospheres (MPN) containing CdTe/CdS quantum dots (QDs) and Fe₃O₄ nanoparticles were prepared, and novel anti-EGFR antibodies were conjugated onto these magneto-fluorescent nanocomposites (MPN–QDs–SiO₂) for cancer cell targeting, imaging and collection. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) images and energy-dispersive x-ray spectrometry (EDS) data showed that the MPN had been successfully coated with QDs and a silica shell, and the nanocomposites obtained with negative charged surfaces were well dispersed. The bioconjugates could be used for specifically labeling and separating cancer cells (MDA-MB-435S, SMMC-7721), but did not recognize and separate the K562 cells because the human epidermal growth factor receptor (EGFR) was not expressed on the surface. Because the anti-EGFR antibody, which we have developed, could specifically recognize certain cancer cells that highly expressed EGFR on their surface, these nanoscale bioconjugates, synchronously exhibiting fluorescence and magnetism, may be used in novel bioprobes for labeling and collecting rare cancer cells, which may be beneficial for early cancer diagnosis.     

Biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging of pancreas cancer

We report on the fabrication and characterization of biofunctional magnetic nanoparticles as contrast agents for magnetic resonance imaging. The anti-cancer antigen 19-9 monoclonal antibody (a cancer-targeting antibody) was conjugated onto the magnetic contrast agents in an effort to detect pancreatic tumor. The structure, size, morphology and magnetic property of the biofunctional magnetic nanoparticles are characterized systematically by means of transmission electron microscopy and X-ray diffractometry. Furthermore, the interaction between the nanoparticles and pancreas cancers cells are investigated by atomic force microscope and transmission electron microscopy. Magnetic resonance imaging demonstrates that the conjugated nanoparticles can effectively target cancer cells both in vitro and in vivo, suggesting that they potentially can be used as contrast agents for magnetic resonance imaging of pancreas cancer.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

The synthesis of highly water-dispersible and targeted CdS quantum dots and it is used for bioimaging by confocal microscopy

Synthesis of a highly dispersed hydrophilic CdS nanocrystals and their use as fluorescence labeling for live cell imaging is reported here. By carefully manipulating the surface of CdS nanocrystals, the dispersions of CdS-MAA-PEI-FA nanocrystals with high photostability is prepared. The receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as CBRH7919 liver cancer cells was demonstrated by confocal microscopy. In the future, the further modified CdS nanoparticles can be used for the tissue imaging in vivo studies.     

水溶性CdTe量子点荧光探针检测肠癌相关T抗原

直接合成性能优良的水溶性CdTe量子点,然后在其表面成功修饰花生凝集素,经过凝胶柱的分离纯化获得功能化的量子点荧光探针.基于T抗原选择性与花生凝集素(PNA)结合的特性,利用该探针对肠癌中高表达的T抗原进行检测,且与传统的荧光染料标记的免疫荧光分析进行了比较.实验结果表明:该功能化的荧光探针能够有效地识别肠癌的相关T抗原,从而为T抗原的检测以及肠癌的临床诊断与愈后判断提供了一种新的方法.     

Blue-emissive upconversion nanoparticles for low-power-excited bioimaging in vivo

Water-soluble upconversion luminescent (UCL) nanoparticles based on triplet-triplet annihilation (TTA) were successfully prepared by coloading sensitizer (octaethylporphyrin Pd complex) and annihilator (9,10-diphenylanthracene) into silica nanoparticles. The upconversion luminescence quantum yield of the nanoparticles can be as high as 4.5% in aqueous solution. As determined by continuous kinetic scan, the nanoparticles have excellent photostability. Such TTA-based upconversion nanoparticles show low cytotoxicity and were successfully used to label living cells with very high signal-to-noise ratio. UCL imaging with the nanoparticles as probe is capable of completely eliminating background fluorescence from either endogenous fluorophores of biological sample or the colabeled fluorescent probe. In particular, such blue-emissive upconversion nanoparticles were successfully applied in lymph node imaging in vivo of living mouse with excellent signal-to-noise ratio (>25), upon low-power density excitation of continuous-wave 532 laser (8.5 mW cm(-2)). Such high-contrast and low-power excited bioimaging in vivo with a blue-emissive upconversion nanoparticle as probe may extend the arsenal of currently available luminescent bioimaging in vitro and in vivo.     

Photodynamic action of Rose Bengal silica nanoparticle complex on breast and oral cancer cell lines

Rose Bengal, an anionic photosensitizer was conjugated to organically modified silica nanoparticles having 3-amino propyl groups by electrostatic or covalent interaction. The drug-nanoparticle complexes were characterized by FTIR, light scattering and zeta potential measurements. Significant changes were observed in the spectroscopic properties of the drug when it is conjugated with nanoparticles. The toxicity of the free drug and drug-nanoparticle complex was studied against oral (4451) and breast (MCF-7) cancer cell lines. Both complexes with nanoparticles were more phototoxic than free Rose Bengal, with the covalent complex being the more effective. Studies carried out on cellular uptake, photostability and singlet oxygen generation suggest that enhanced phototoxicity is primarily due to the enhanced uptake of the drug-nanoparticle complex.     

Synthesis and characterization of new fluorescent nanoparticles

A novel kind of fluorescent nanoparticles（FNPs）has been prepared using a precipitation polymerization method.Methacrylic acid,trimethylolpropane trimethacrylate and azobisisobutyronitrile were used as functional-monomer,cross-linker and initiator, respectively.Compared with other fluorescent nanoparticles,the FNPs have the characteristics including low dye leakage and good photostability.The fluorescence microscopy imaging indicates that the FNPs can be used as fluorescent labels in bioanalysis.     

Targeting cancer cells through iron(III) complexes of di(picolyl)amine modified silica core-shell nanospheres

In this report, we aim at optimizing the approach of delivering and imaging cancer cell targeting using anti-proliferative nanoparticle complex. Rhodamine B isothiocyanate doped silica-coated (RBITC-SiO?) were prepared by microemulsion method. Fe(III) complex of di(picolyl)amine was conjugated on to the surface RBITC-SiO? to produce final nanosphere (RBITC-SiO? @dpa-Fe) with an average hydrodynamic diameter of 74 nm. The Fe(III)-di(picolyl)amine complex modified nanospheres displayed enhanced HeLa cells uptake in vitro suggesting selective cancer cell payload delivery. RBITC-SiO? @dpa-Fe also showed reduced off-target cytotoxicity. The conjugate of dpa-Fe(III) complex and fluorescence core-shell nanoparticles RBITC-SiO? represents a class of novel multi-functional nanoparticles that combines the advantages of active cancer-targeting through Fe(III) complex mediated intracellular drug delivery and compatibility with fluorescence imaging.     

A cancer cell turn-on protein-CuSMn nanoparticle as the sensor of breast cancer cell and CH3O-PEG-phosphatide

A cancer activated protein-inorganic nanoparticle was used as breast cancer cell turn-on fluorescence sensor and NIR activated attenuator.     

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation-Induced NIR-I/II Fluorescence Imaging

An activatable nanoprobe for imaging breast cancer metastases through near infrared-I (NIR-I)/NIR-II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D-π-A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation-induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.     

pH值响应的共轭聚合物纳米粒子的制备、表征与细胞成像研究

共轭聚合物纳米粒子（CPNs）因其高荧光亮度、低毒性、表面易修饰的特性,近年来在生物材料和生物医药领域备受关注。本论文中我们设计、合成了一种新的pH 值响应共轭聚合物（PFPA）,并通过纳米沉淀方法制备了其纳米粒子。动态光散射实验表明PFPA纳米粒子在水中分散性较好,其粒径约为8 nm。 PFPA纳米粒子的最大吸收峰为379 nm,其摩尔吸光系数为2.1×10⁶ L·mol -1·cm -1;另外该纳米粒子的荧光最大发射峰为422 nm,其荧光量子产率为35%。PFPA纳米粒子在汞灯（100瓦）照射下表现出较好的光稳定性,另外MTT实验表明其具有较低的细胞毒性。该纳米粒子具有pH响应的光学特性,并可以用于活细胞成像。PFPA纳米粒子在癌症诊断、药物与基因传递等方面具有潜在的应用价值。     

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation‐Induced NIR‐I/II Fluorescence Imaging

An activatable nanoprobe for imaging breast cancer metastases through near infrared‐I (NIR‐I)/NIR‐II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D‐π‐A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation‐induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.     

Differentiation Between Anti-Epidermal Growth Factor Receptors Antibody Conjugated and Unconjugated Gold Nanoparticles Using Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) along with UV-Vis was used to distinguish between structural differences between a normal human breast cell line (MCF-12 F) and a cancerous breast cell line (MCF-7) after both being treated with conjugated gold nanoparticles and unconjugated gold nanoparticles. The MCF-7 cell line possessed a small amount of epidermal growth factor receptors (EGFR) on its cell surface whereas the MCF-12 F cell line did not. The anti-EGFR antibodies blocked the binding of epidermal growth factors to this receptor which plays an important role in the regulation of cell growth, proliferation, and differentiation. The infrared (IR) spectra for both cell lines yield several differences between normal biological samples and cancerous samples. As the malignancy of the sample increases, the peak intensity and wavenumber positions decrease. The same conclusion was drawn from thin-layers of conjugated nanoparticles and nonconjugated nanoparticles. The presence of conjugated nanoparticles increased the peak intensity of the MCF-7 cell line whereas it decreased the peak intensity for the MCF-12 F cell line. UV-Vis was used to show the presence of the anti-EGFR antibodies on the surface of the gold nanoparticles which was 5 nm red shifted compared to the gold nanoparticles solution.     

Cell-permeable and biocompatible polymeric nanoparticles for apoptosis imaging

Here, we report for the first time cell-permeable and biocompatible polymeric nanoparticles consisting of a polymer conjugated to a near-infrared (NIR) fluorescence (Cy5.5)-linked effector caspase-specific peptide. The close spatial proximity of the NIR fluorochromes in polymeric nanoparticles results in an autoquenched state, but polymer nanoparticles give rise to strong NIR fluorescence signal under apoptotic cells. Thus, the smart polymeric nanoparticle developed here is an attractive probe for real-time imaging of apoptosis in single cells.