Super blinking and biocompatible nanoprobes based on dye doped BSA nanoparticles for super resolution imaging.

As one of the super-resolved optical imaging techniques, single molecule localization microscopy (SMLM) received considerable attention due to its impressive spatial resolution. Compared with other fluorescence imaging techniques, SMLM has one particular request for the fluorophores, that is, continuous 'on' and 'off' behaviors of their signals (referred to as 'blinking'). Hence, we present here a kind of super blinking and biocompatible nanoprobes (denoted as SBNs) for SMLM. The SBNs have two main advantages, first, they possess an outstanding fluorescence blinking. Second, they are biocompatible since they are based on bovine serum albumin (BSA). The SBNs are fabricated by doping organic dyes into BSA nanoparticles. We fabricated two kinds of SBNs, one was doped with Alexa Fluor 647 (A647) and the other was doped with Alexa Fluor 594 (A594). Especially for A594 doped SBNs, the improved blinking of A594 doped SBNs induced a better localization precision as compared with A594 alone. Moreover, SMLM imaging of breast cancer cells and exosomes using the SBNs was successfully realized with high spatial resolutions. The work demonstrated here provides a new strategy to prepare novel kinds of super blinking fluorescent agents for SMLM, which broadens the selection of suitable fluorophores for SMLM.

Investigating the Intracellular Behaviors of Liposomal Nanohybrids via SERS: Insights into the Influence of Metal Nanoparticles.

The recent proposition to combine liposomes with nanoparticles presents great opportunities to develop multifunctional drug delivery platforms. Although impressive progress has been made, attempts to elucidate the role nanoparticles play in the integral nanohybrids are still rather limited. Here, using surface-enhanced Raman scattering (SERS) technique, we investigate the influence of metal nanoparticles on the liposomal properties, ranging from drug release to intracellular movement. Specifically, we prepared SERS-active nanohybrids by attaching metal nanoparticles to liposomes and employed SERS signals to explore the intracellular behavior of the nanohybrids. Once deposited on the cell membrane, the nanohybrids entered tumor cells via clathrin-mediated endocytosis and then moved to lysosomes. In comparison with pure liposomes, metal nanoparticles in the nanohybrids had little influence on the properties of liposomes. This study fills the gap of the function of nanoparticles in the overall nanohybrids, which provides a significant prerequisite for efficient drug delivery in therapeutic applications.

Dual-mode tracking of tumor-cell-specific drug delivery using fluorescence and label-free SERS techniques.

We developed a dual-mode detection method for tumor cell specific targeting and intracellular delivery of the chemotherapeutic agent Doxorubicin (DOX) using folic acid functionalized mesoporous silica nanoparticles (FA-MSNs) as carrier systems. In this method, label free surface enhanced Raman scattering (SERS) spectra were utilized to monitor the dynamic release of DOX inside tumor cells in combination with fluorescence images. To investigate the targeting delivery performance of the carrier system, both normal cells (MRC-5) and tumor cells (HeLa) were used as the model cells. The real-time release of DOX from FA-MSNs inside MRC-5 and HeLa cells was monitored. As demonstrated by both fluorescence and SERS results, the DOX loaded FA-MSNs can actively target FA receptor overexpressed tumor cells. Moreover, the releasing behavior of DOX from FA-MSNs in tumor and in normal cells was quantitatively analyzed. Compared with the traditional sole fluorescence or SERS method, this dual-mode detection is more powerful and more accurate, which should have a potential application in drug tracking in living cells.

Distinguishing breast cancer cells using surface-enhanced Raman scattering.

The detection and identification of epidermal growth factor receptor 2 (HER2)-positive breast cancer cells is crucial for the clinic therapy of breast cancer. For the aim of the detection, a novel surface-enhanced Raman scattering (SERS) probe for distinguishing breast cancers at different HER2 statuses is reported in this paper. In such a probe, anti-HER2 antibody-conjugated silver nanoparticles have been synthesized for specific targeting of HER2-positive breast cancer cells. More importantly, different from the previously reported SERS probe for targeting cancer cells, p-mercaptobenzoic acid is utilized as both the Raman reporter and the conjugation agent for attaching antibody molecules, which leads to a much simplified structure. For investigating the ability of such a probe to distinguish breast cancer cells, SKBR3 and MCF7 cells were chosen as two model systems, which are HER2-positive- and HER2-negative-expressing cells, respectively. The experimental results reveal that SKBR3 cells exhibit much stronger SERS signals than MCF7 cells, indicating that the probe could be utilized to distinguish breast cancer cells at different HER2 statuses. This kind of SERS probe holds a potential for a direct detection of living breast cancer cells with the advantages of easy fabrication, high SERS sensitivity, and biocompatibility.

A straightforward route to the synthesis of a surface-enhanced Raman scattering probe for targeting transferrin receptor-overexpressed cells.

A tumor cell targeting surface-enhanced Raman scattering (SERS) probe has been successfully synthesized by using p-mercaptobenzoic acid (pMBA) as both the SERS reporter and the conjugation agent for attaching transferrin molecules, which shows experimentally the targeting ability for transferrin receptor-overexpressed HeLa cells and exhibits strong SERS signals when being incubated inside cells. To prove that the uptake of such a SERS probe is through a Tf-receptor-mediated endocytosis process, two control experiments: (1) HeLa cells being incubated with the probe at 4 degrees C and (2) HeLa cells being pre-blocked with free transferrin at 37 degrees C, were employed. The difference of SERS intensity between the transferrin-overexpressed HeLa cells and transferrin-pre-blocked HeLa cells indicates that the probe has the potential to selectively target tumor cells.

Polyvinylpyrrolidone- (PVP-) coated silver aggregates for high performance surface-enhanced Raman scattering in living cells.

A biocompatible and stable surface-enhanced Raman scattering (SERS) probe has been successfully synthesized through a simple route with silver aggregates. Polyvinylpyrrolidone (PVP), a biocompatible polymer, was utilized to control the aggregation process and improve the chemical stability of the aggregates. Extinction spectroscopy and TEM results show the aggregation degree and core-shell structure of the probe. It is found that when we employ 4-mercaptobenzoic acid (4MBA), crystal violet (CV), Rhodamine 6G (R6G) or 4,4'-bipyridine molecules as Raman reporters, the SERS signal from the proposed probe can remain at a high level under aggressive chemical environments, even after being incorporated into living cells. In comparison with the traditional probes without the PVP shell, the new ones exhibit strong surface-enhanced effects and low toxicity towards living cells. We demonstrate that the PVP-coated silver aggregates are highly SERS effective, for which the fabrication protocol is advantageous in its simplicity and reproducibility.