Direct cytoplasm delivery of gold nanoparticles for real-time apoptosis detection

Gold nanoparticles(AuNPs)assembled with fluorescent peptides through Au-S bonds(pep-AuNPs)have been widely used in biomolecular detection.However,due to the endo/lysosomal trapping after the nanoprobes enter cells,the direct delivery of AuNP probes into the cytoplasm for real-time imaging remains a difficult barrier for many cytoplasm-targeting agents.Here,we prepare AuNP@gel by wrapping a multi-functional nanogel structure on the surface of a single AuNP probe by in-situ polymerization in order to directly deliver AuNP probes into the cell cytoplasm.Compared with the pep-AuNP probes,which are trapped inside lysosomes for long periods,the AuNP@gel probes use the proton-sponge effect to effectively disrupt endo/lysosomal membranes and remain in the cytoplasm.In addition,the AuNP@gel probes rapidly escape from endo/lysosomes to avoid the complex environment that interferes with the stability of the AuNP probes and the lysosomal-storage trigger the upregulation of oxidative stress into the cells.The nanogel structure enables the AuNP probes to avoid some detrimental effects and to achieve high-fidelity fluorescence signals in the cells.Compared to traditional strategies for lysosomal escape,this one-step in-situ polymerization procedure avoids the complicated modification of additional ligands and is generally applicable to peptide-,DNA-,and polymerlinked AuNP probes.

Detecting the apoptosis of dopamine neurons with immunohistochemical staining and double-staining technique

BACKGROUND： It is proved that the onset of Parkinson disease companies with neuronal apoptosis of dopamine in substantia nigra of midbrain. Previous researches on neuronal apoptosis of dopamine were analyzed on their consecutive tissue sections with immunohistochemical single-labeling method, immunofluorescence and electron microscope, and there are significant differences.OBJECTIVE ： To observe the feasibility of neuronal apoptosis of dopamine with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique.DESIGN ： Controlled study.SETTING： College of Pharmacology of Taishan Medical College; College of Management of Taishan Medical College. MATERIALS ： Wistar rats with 2 weeks old and of clean grade were provided by the Animal Center of Taishan Medical College. In situ end labeling kit （terminal deoxynucleotidyl transferase, mixed reactive solution of nucleotide, transfusion-POD）, monoclonal antibody of rat antibody against tyrosine hydroxylase （Boehriuser）. METHODS： The experiment was completed at the Pharmacological Laboratory of Taishan Medical College from February to December 2005. Tissue from midbrain of rats was taken out to make paraffin sections to observe the neuronal apoptosis of dopamine under microscope with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique.MAIN OUTCOME MEASURES ： Neuronal apoptosis of dopamine with in situ end labeling and tyrosine-hydroxylase antibody immunohistochemical double-labeling staining technique. RESULTS： ① After double-labeling staining, two kinks of positive products were observed in neurons of dopamine which were suffered from apoptosis. One stained with tyrosine hydroxylase was hyacinthine, and the other stained with in situ end labeling was buffy. Cells of positive products stained with in situ end labeling shaped as strap and bend and was distributed in clustering. Cytoplasm was hyacinthine, staining was symmetrical, and cellular ecphyma was observed. Nucleus was stained vacantly which was coincidence with form of neurons of dopamine. ②Apoptosis showed strictly in cytoplasm and nucleus at the aspect of morphology. Cytoplasm stained with in situ end labeling was hardly to recognize because of the usage of double-labeling staining technique, but nucleus was still characterized by apoptosis. The behavior of positive products stained with in situ end labeling was described as following： nucleus was buffy; karyopycnosis was round and irregular; caryotin was integrated into clump which was distributed at the border of nucleus and shaped as demilune and anular; positive signals were limited in nucleus and coincidence with morphological changes of apoptosis. However, blue and positive products were observed in cytoplasm of neurons of dopamine which did not occur apoptosis, and the nucleus was not labeled. Therefore, processing apoptosis of neurons of dopamine could be recognized. CONCULSION： Double-labeling staining technique can be used to correctly reveal histological and morphological changes of neuronal apoptosis of dopamine during its onset and development.