ABSTRACT
The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3-10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.
Subject(s)
Gold , Metal Nanoparticles , Cricetinae , Animals , Humans , HeLa Cells , Cricetulus , Transfection , Coloring Agents , Microscopy, FluorescenceABSTRACT
Fluorescence spectroscopy is a sensitive, fast and non-invasive tool for a diagnostics of cancerous gastrointestinal lesions. It could be applied for in situ detection of tumours during primary endoscopic observations or as add-on measurement modality during microscopic observations of tissue histology slides for their initial or retrospective diagnosis. Therefore, we are looking for diagnostically important features of normal and cancerous tissue areas in a broad spectral range for gastrointestinal tissues ex vivo using two steady-state macroscopic fluorescent spectroscopic modalities and by confocal fluorescent microscopic detection. Results obtained from autofluorescence spectroscopy of benign and malignant lower part gastrointestinal tract (GIT) lesions from freshly excised tissues during surgical removal of the lesions in 18 patients (22 lesions), were compared with the spectral measurements obtained during confocal fluorescent microscopy observations of unstained tissue slides using 405 nm excitation. Excitation-emission matrices (EEMs) were used for ex vivo measurements with applied excitation in 280-440 nm spectral region and emission observed between 300 and 700 nm. Synchronous fluorescence spectroscopy (SFS) approach was also applied to improve the spectral resolution of the observed complex emission spectra. Specific fluorescent features observed, related to presence of structural proteins, co-enzymes and endogenous porphyrins in the tissues investigated, allow discriminating normal mucosa from benign polyps and malignant carcinoma lesions with diagnostic accuracy up to 94.4%.
Subject(s)
Deficiency Diseases/etiology , Metabolic Diseases/etiology , Parenteral Nutrition/adverse effects , Adult , Aged , Avitaminosis/etiology , Clinical Trials as Topic , Deficiency Diseases/diagnosis , Humans , Hyperglycemia/etiology , Hypertriglyceridemia/etiology , Hypoglycemia/etiology , Hypokalemia/etiology , Metabolic Diseases/diagnosis , Middle Aged , Monitoring, Physiologic , Time Factors , Trace Elements/deficiencyABSTRACT
The correlation between biological purification of sewage and concentrations of carcinogens in it is studied. The balance between the income and outcome of benza(a)pyrene (BP) with water and surplus active mud of paper in aero-tanks, performed under both industrial and laboratory conditions is examined. It is demonstrated that BP is not destroyed by biological purification but is accumulated by the active sludge cells. Thus, the biologic purification of sewage prevents from pollution by carcinogenic polycyclic hydrocarbons, but does not remove these agents from the environment.
