How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

Targeted drug delivery in cancer typically focuses on maximising the endocytosis of drugs into the diseased cells. However, there has been less focus on exploiting the differences in the endocytosis pathways of cancer cells versus non-cancer cells. An understanding of the endocytosis pathways in both cancer and non-cancer cells allows for the design of nanoparticles to deliver drugs to cancer cells whilst restricting healthy cells from taking up anticancer drugs, thus efficiently killing the cancer cells. Herein we compare the differences in the endocytosis pathways of cancer and healthy cells. Second, we highlight the importance of the physicochemical properties of nanoparticles (size, shape, stiffness, and surface chemistry) on cellular uptake and how they can be adjusted to selectively target the dominated endocytosis pathway of cancer cells over healthy cells and to deliver anticancer drug to the target cells. The review generates new thought in the design of cancer-selective nanoparticles based on the endocytosis pathways.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

How to exploit different endocytosis pathways to allow selective delivery of anticancer drugs to cancer cells over healthy cells.

It was recently shown that it is possible to exploit the nanoparticle shape to selectively target endocytosis pathways found in cancer and not healthy cells. It is important to understand and compare the endocytosis pathways of nanoparticles in both cancer and healthy cells to restrict the healthy cells from taking up anticancer drugs to help reduce the side effects for patients. Here, the clathrin-mediated endocytosis inhibitor, hydroxychloroquine, and the anticancer drug, doxorubicin, are loaded into the same mesoporous silica nanorods. The use of nanorods was found to restrict the uptake by healthy cells but allowed cancer cells to take up the nanorods via the macropinocytosis pathway. Furthermore, it is shown that the nanorods can selectively deliver doxorubicin to the nucleus of breast cancer cells and to the cytoplasm of pancreatic cancer cells. The dual-drug-loaded nanorods were able to selectively kill the breast cancer cells in the presence of healthy breast cells. This study opens exciting possibilities of targeting cancer cells based on the material shape rather than targeting antibodies.

Rod-shaped mesoporous silica nanoparticles for nanomedicine: recent progress and perspectives.

INTRODUCTION: Interest in mesoporous silica nanoparticles for drug delivery has resulted in a good understanding of the impact of size and surface chemistry of these nanoparticles on their performance as drug carriers. Shape has emerged as an additional factor that can have a significant effect on delivery efficacy. Rod-shaped mesoporous silica nanoparticles show improvements in drug delivery relative to spherical mesoporous silica nanoparticles. AREAS COVERED: This review summarises the synthesis methods for producing rod-shaped mesoporous silica nanoparticles for use in nanomedicine. The second part covers recent progress of mesoporous silica nanorods by comparing the impact of sphere and rod-shape on drug delivery efficiency. EXPERT OPINION: As hollow mesoporous silica nanorods are capable of higher drug loads than most other drug delivery vehicles, such particles will reduce the amount of mesoporous silica in the body for efficient therapy. However, the importance of nanoparticle shape on drug delivery efficiency is not well understood for mesoporous silica. Studies that visualize and quantify the uptake pathway of mesoporous silica nanorods in specific cell types and compare the cellular uptake to the well-studied nanospheres should be the focus of research to better understand the role of shape in uptake.

Silica-encapsulated gold nanoparticle dimers for organelle-targeted cellular delivery.

Silica-encapsulated gold nanoparticle dimers were self-assembled through a single-insertion process using capillary force and can be utilized as an advanced drug-delivery and sensing platform for organelle-targeting in cancer cells.

Fabrication of peptide stabilized fluorescent gold nanocluster/graphene oxide nanocomplex and its application in turn-on detection of metalloproteinase-9.

This study introduces the double-ligands stabilizing gold nanoclusters and the fabrication of gold nanocluster/graphene nanocomplex as a "turn-on" fluorescent probe for the detection of cancer-related enzyme matrix metalloproteinase-9. A facile, one-step approach was developed for the synthesis of fluorescent gold nanoclusters using peptides and mercaptoundecanoic acid as co-templating ligands. The peptide was designed to possess a metalloproteinase-9 cleavage site and to act not only as a stabilizer but also as a targeting ligand for the enzyme detection. The prepared gold nanoclusters show an intense red fluorescence with a broad adsorption spectrum. In the presence of the enzyme, due to the excellent quenching properties and the negligible background of graphene oxide, the developed peptide-gold nanocluster/graphene nanocomplex yielded an intense "turn-on" fluorescent response, which strongly correlated with the enzyme concentration. The limit of detection of the nanocomplex was 0.15nM. The sensor was successfully applied for "turn-on" detection of metalloproteinase-9 secreted from human breast adenocarcinoma MCF-7 cells with high sensitivity, selectivity, significant improvement in terms of detection time and simplicity.

Self-assembly of an upconverting nanocomplex and its application to turn-on detection of metalloproteinase-9 in living cells.

Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml(-1). Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.

Fabrication of Magnetic Upconversion Nanohybrid for Luminescent Resonance Energy Transfer-Based Detection of Glutathione.

This study introduces the facile fabrication of a bimodal nanohybrid for the luminescent ON/OFF detection of glutathione. The proposed nanosensor consists of magnetic (Fe3O4) and upconversion nanoparticles (UCP) co-encapsulated in a silica matrix, and decorated with gold nanoparticle (AuNP) as a luminescent quencher. The detection mechanism is based on the Luminescent Resonance Energy Transfer (LRET) between the donor (UCP) and the acceptor (AuNP) with the help of a disulfide bond as a bridging element. In the presence of glutathione, the disulfide bridges between AuNPs and Fe3O4/UCP@SiO2 was cleaved and the amount of glutathione could be traced by the restored luminescence (ON state) of the nanohybrids after magnetic separation.

Gold nanoparticle silica nanopeapods.

A subnanometer gap-separated linear chain gold nanoparticle (AuNP) silica nanotube peapod (SNTP) was fabricated by self-assembly. The geometrical configurations of the AuNPs inside the SNTPs were managed in order to pose either a single-line or a double-line nanostructure by controlling the diameters of the AuNPs and the orifice in the silica nanotubes (SNTs). The AuNPs were internalized and self-assembled linearly inside the SNTs by capillary force using a repeated wet-dry process on a rocking plate. Transmission electron microscopy (TEM) images clearly indicated that numerous nanogap junctions with sub-1-nm distances were formed among AuNPs inside SNTs. Finite-dimension time domain (FDTD) calculations were performed to estimate the electric field enhancements. Polarization-dependent surface-enhanced Raman scattering (SERS) spectra of bifunctional aromatic linker p-mercaptobenzoic acid (p-MBA)-coated AuNP-embedded SNTs supported the linearly aligned nanogaps. We could demonstrate a silica wall-protected nanopeapod sensor with single nanotube sensitivity. SNTPs have potential application to intracellular pH sensors after endocytosis in mammalian cells for practical purposes. The TEM images indicated that the nanogaps were preserved inside the cellular constituents. SNTPs exhibited superior quality SERS spectra in vivo due to well-sustained nanogap junctions inside the SNTs, when compared to simply using AuNPs without any silica encapsulation. By using these SNTPs, a robust intracellular optical pH sensor could be developed with the advantage of the sustained nanogaps, due to silica wall-protection.

Upconversion nanoparticle-based Förster resonance energy transfer for detecting the IS6110 sequence of Mycobacterium tuberculosis complex in sputum.

Upconversion nanoparticles (UCNPs), which are excited at near-infrared wavelength (980 nm), emit high-energy photons. Since UCNPs display a high signal-to-noise ratio and no photobleaching, they are extremely useful for diagnostic application. In this study, we applied UCNPs for detecting the IS6110 sequence of the Mycobacterium tuberculosis complex (MTBC) and evaluated the feasibility of the system for use in molecular diagnostics. Using biotinylated primers, IS6110 DNA PCR was performed and the PCR amplicon was then mixed with streptavidin-conjugated UCNPs, followed by intercalation with SYTOX Orange dye. Fluorescence detection for the Förster resonance energy transfer (FRET) of the UCNPs (UCNP-FRET) was then performed. The estimated lowest detection by UCNP-FRET was 10(2) copies/µL of IS6110 DNA (157 bp). The kappa agreement of the UCNP-FRET assay with conventional PCR was 0.8464 (95% confidence interval, 0.7442-0.9486) and false-negative results were reduced. Our results demonstrated the successful implementation of the UCNP-FRET system in detecting the IS6110 sequence of the MTBC and its potential application for molecular diagnostics.

Effects of upconversion nanoparticles on polymerase chain reaction.

Nanoparticles (NPs) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR) specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix) and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit). Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C-45°C), addition of the 40 nm UCNP (1 µg/µL) to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.