Biocompatible and photostable AIE dots with red emission for in vivo two-photon bioimaging.

Materials and instruments The TTF fluorogen was prepared following our previously published protocol 22 . DSPE-mPEG 5000 was purchased from Creative PEGWorks, Inc. Other chemical reagents, which were not specially pointed out, were obtained from Sigma Inc. Deioned (DI) water was used in all the experimental procedures. A Shimadzu 2550 UV-vis scanning spectrophotometer and a HITACHI F-2500 fluorescence spectrophotometer were used to measure the absorption and photoluminescence (PL) spectra of samples. Dynamic light scattering (DLS) analysis was conducted using a Dawn® Heleos light scatter from Wyatt Technology Corp at room temperature. TEM images were taken by a JEOL JEM-1230 transmission electron microscope operating at 160 kV in bright-field mode.

Preparation and characterization of AIE-dots

AIE dots were fabricated through a modified nanoprecipitation procedure 10 . Briefly, 0.25 mL of TTF solutions in chloroform (1 mg/mL) and a certain amount of DSPE-mPEG 5000 solutions in chloroform (1 mg/mL) were mixed in a flask (25 mL). The mixture was sonicated for several minutes to form a homogeneous red solution and dried under vacuum in a rotary evaporator at 70°C. After the chloroform was completely removed, 2 mL of deionized water was added into the flask and the solution was sonicated for 2 min. Subsequently, an optically clear suspension containing AIE-dots was prepared. By varying the amount of DSPE-mPEG 5000 added into the mixture, AIE-dots with different TTF feeding ratios were prepared. For the preparation of TTF feeding ratio of 5 wt%, 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50, and 60 wt%, the volume of DSPE-mPEG 5000 solutions added was 4.75 mL, 2.25 mL, 1 mL, 0.58 mL, 0.375 mL, 0.25 mL and 0.17 mL, respectively. To determine the loading ratio of TTF in AIE-dots, solutions of AIE-dots were measured by a Shimadzu 2550 UV-vis scanning spectrophotometer. The TTF concentration was determined by comparing its absorption intensity with that of a sample with known concentration at 500 nm. The feeding ratio was calculated by dividing the weight of TTF by the total weight of TTF and DSPE-mPEG 5000 in the feed mixture. The loading ratio was calculated by dividing the weight of the loaded TTF by the weight of the AIE-dots. The encapsulation efficiency was calculated by dividing the loading ratio by the feeding ratio. The average sizes of AIE-dots were measured by Dynamic Light Scattering (DLS) analysis. For release kinetics studies, solutions of AIE-dots were incubated with 1 mL Tween-20 solutions (1% in DI water) at 40°C. After a certain time, the sample was spin-filtered using microfuge membrane-filter (NANOSEP 100 K OMEGA, Pall Corporation, USA) at 12,000 rpm for 15 min (spin filtration). The filtrated solution, which passed through the membrane, was collected. Its extinction spectrum, as well as that of the original aqueous solution of AIE-dots, was measured. The ratio of the two extinction peak intensities could be used to characterize the release percentage of TTF from AIE-dots. One- and two-photon fluorescence of AIE-dots To compare the microcosmic one-photon luminescence (1 PL) and two-photon luminescence (2 PL) signals quantitatively from AIE-dots with different TTF loading ratios, aqueous suspensions of AIE-dots with different TTF loading ratios were dropped on glass slides and imaged with a laser scanning confocal microscope (Olympus, BX61W1-FV1000). The 1 PL and 2 PL of samples were performed by a CW laser at 543 nm and a femtosecond laser at 800 nm, respectively. A water-immersion objective lens (20×, NA = 0.75) was used to focus the laser beam onto the samples, and an external detector was used to collect emission signals. Through statistics and analysis of a mass of spots from the fluorescence images, we obtained an average intensity for each picture which can be used to reflect the fluorescence intensity for single dot.

Animals Male black mice

(C57 line) were obtained from the Laboratory Animal Center of Zhejiang University (Hangzhou, China). Mice were housed in cages in groups (5 mice per cage) and fed with standard mouse chow and water. The cages were maintained in a room with controlled temperature (25 ± 1°C) and a 12 h light/dark cycle. The protocol of animal experiments was approved by the Institutional Ethical Committee of Animal Experimentation of Zhejiang University in China, and the experiments were performed strictly according to governmental and international guidelines on animal experimentation. According to requirements for Biosafety and Animal Ethics, all efforts were made to minimize the number of animals used and their suffering.

Biodistribution of AIE-dots in mice

Mice were intravenously injected with suspensions of AIE-dots (with 20 wt% TTF loading, 1 mg/mL in 1× phosphate buffered saline (PBS), 100 μL solutions per mouse). The mice were put down various hours after injection. Three mice were used at each time point after injection. Their major organs were taken out and imaged by utilizing a Maestro in vivo optical imaging system (CRI, Inc. Woburn, MA). The organs of mice were excited with laser of 595 nm peak wavelength. The fluorescence images were captured with a constant exposure time of 2000 ms for all the samples. In vivo imaging of ear blood vessels Six mice were randomly divided into two groups. Three mice in one group were intravenously injected with suspensions of AIE-dots (with 20 wt% TTF loading, 1 mg/mL in 1× PBS, 100 μL solutions per mouse) as experimental group, and the mice in the other group was intravenously injected with PBS solution (100 μL solutions per mouse) as control group. For ear blood vessels imaging, the mice were anaesthetized with an intraperitoneal injection of 0.5% pentobarbital sodium and placed on a Petri dish with one ear attached to the coverslip. An upright two-photon scanning microscope (Olympus, BX61W1-FV1000) was used for 2 PL imaging. A long work distance (objective length = 2 mm) water-immersion objective lens (25×, NA = 1.05) was used to focus the laser beam onto the water-immersed earlobe, and an external photomultiplier tube (PMT) was used to collect two-photon induced emission signals via non-descanned detection (NDD). In vivo imaging of brain blood vessels Six mice were random divided into two groups. Three mice in one group were intravenously injected with suspensions of AIE-dots (with 20 wt% TTF loading, 1 mg/mL in 1× PBS, 100 μL solutions per mouse) as experimental group, and the mice in the other group was intravenously injected with PBS solution (100 μL solutions per mouse) as control group. For in vivo brain imaging of mice, the mice were anaesthetized with an intraperitoneal injection of 0.5% pentobarbital sodium and their skulls were opened up through microsurgery. An upright two-photon scanning microscope was used to image the blood vessels of the mice. For the description of the immobilization of mice's heads and how the objective of the upright two-photon scanning microscope was arranged to contact the brain, we refer to our previous work [ref. 7 ].

Materials and instruments The TTF fluorogen was prepared following our previously published protocol 22 . DSPE-mPEG 5000 was purchased from Creative PEGWorks, Inc. Other chemical reagents, which were not specially pointed out, were obtained from Sigma Inc. Deioned (DI) water was used in all the experimental procedures. A Shimadzu 2550 UV-vis scanning spectrophotometer and a HITACHI F-2500 fluorescence spectrophotometer were used to measure the absorption and photoluminescence (PL) spectra of samples. Dynamic light scattering (DLS) analysis was conducted using a Dawn® Heleos light scatter from Wyatt Technology Corp at room temperature. TEM images were taken by a JEOL JEM-1230 transmission electron microscope operating at 160 kV in bright-field mode.