Furong Tian

Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging

Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimers, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered.

Gold nanoprisms as optoacoustic signal nanoamplifiers for in vivo bioimaging of gastrointestinal cancers.

Early detection of cancer greatly increases the chances of a simpler and more effective treatment. Traditional imaging techniques are often limited by shallow penetration, low sensitivity, low specificity, poor spatial resolution or the use of ionizing radiation. Hybrid modalities, like optoacoustic imaging, an emerging molecular imaging modality, contribute to improving most of these limitations. However, this imaging method is hindered by relatively low signal contrast. Here, gold nanoprisms (AuNPrs) are used as signal amplifiers in multispectral optoacoustic tomography (MSOT) to visualize gastrointestinal cancer. PEGylated AuNPrs are successfully internalized by HT-29 gastrointestinal cancer cells in vitro. Moreover, the particles show good biocompatibility and exhibit a surface plasmon band centered at 830 nm, a suitable wavelength for optoacoustic imaging purposes. These findings extend well to an in vivo setting, in which mice are injected with PEGylated AuNPrs in order to visualize tumor angiogenesis in gastrointestinal cancer cells. Overall, both our in vitro and in vivo results show that PEGylated AuNPrs have the capacity to penetrate tumors and provide a high-resolution signal amplifier for optoacoustic imaging. The combination of PEGylated AuNPrs and MSOT represents a significant advance for the in vivo imaging of cancers.

Effects of single-walled carbon nanotubes on the polymerase chain reaction

The effects of single-walled carbon nanotubes on the polymerase chain reaction (PCR) were investigated via quantitative PCR product measurements, scanning electron microscopy (SEM), high-resolution transmission electron microscopy (TEM) and x-ray photoelectron spectroscopy (XPS). The results showed that adding single-walled carbon nanotubes (SWCNTs) into the reaction liquid increases the amount of PCR product at SWCNT concentrations below 3 µg µl−1, but this effect is reversed at higher SWCNT concentrations. Similar effects were observed in PCR reactions with or without Mg2+. Both SEM and HRTEM results showed that the DNA templates and Taq enzymes are attached to bundles of SWCNTs in PCR products. XPS results showed that the C 1s binding energy in PCR products increased after reaction, with the emergence of two new peaks beside the main peak compared with carbon nanotubes before reaction, suggesting that there might be a chemical reaction between SWCNTs and PCR components. In conclusion, SWCNTs may increase the PCR efficiency at a concentration range of less than 3 µg µl−1 in the reaction liquid and have the potential to act as catalysts in a variety of biochemical reactions.

In vivo tumor targeting via nanoparticle-mediated therapeutic siRNA coupled to inflammatory response in lung cancer mouse models

Up to now, functionalized gold nanoparticles have been optimized as an effective intracellular in vitro delivery vehicle for siRNAs to interfere with the expression of specific genes by selective targeting, and provide protection against nucleases. Few examples however of suchlike in vivo applications have been described so far. In this study, we report the use of siRNA/RGD gold nanoparticles capable of targeting tumor cells in a lung cancer syngeneic orthotopic murine model. Therapeutic RGD-nanoparticle treatment resulted in successful targeting evident from significant c-myc oncogene down-regulation followed by tumor growth inhibition and prolonged survival of lung tumor bearing mice, possibly via αvβ3 integrin interaction. Our results suggest that RGD gold nanoparticles-mediated delivery of siRNA by intratracheal instillation in mice leads to successful suppression of tumor cell proliferation and respective tumor size reduction. These results reiterate the capability of functionalized gold nanoparticles for targeted delivery of siRNA to cancer cells towards effective silencing of the specific target oncogene. What is more, we demonstrate that the gold-nanoconjugates trigger a complex inflammatory and immune response that might promote the therapeutic effect of the RNAi to reduce tumor size with low doses of siRNA.

Surface enhanced Raman scattering with gold nanoparticles: effect of particle shape

The dependence of the Surface Enhanced Raman Scattering (SERS) by gold nanoparticles on their shape is examined using the organic dye, rhodamine 6G (R6G) as probe molecule. SERS has been explored extensively for applications in sensing and imaging, but the design and optimisation of efficient substrates is still challenging. In order to understand and optimise the SERS process in nanoparticles, gold nanospheres and their aggregates, nanotriangles, and nanostars of similar dimensions were synthesised and characterised according to their average size, zeta potential and UV/visible absorption. SERS from R6G was negligible for unaggregated nanospheres at 532 nm, close to the maximum of the surface plasmon resonance (SPR) at 560 nm. Upon aggregation of the nanospheres, the SPR shifts to ∼660 nm, attributable to local surface plasmon “hotspots” between the spheres, and the SERS signal of R6G is significantly increased, at 785 nm. In monodisperse gold nanotriangles, the SPR is located at ∼800 nm, and significant SERS of R6G is observed using 785 nm as source, as is the case for gold nanostars, which exhibit a double SPR with maxima at ∼600 nm and ∼785 nm, attributable to the core sphere and vertices of the structures, respectively. In suspensions of equal nanoparticle and dye concentration, the SERS effect increases as nanospheres < nanosphere aggregates < nanotriangles < nanostars, clearly indicating that control over the number of local field hotspots can optimise the SERS efficiency. Notably, it is demonstrated that the SERS intensity per nanoparticle scales with the magnitude of the SPR absorbance at the excitation wavelength (785 nm), providing a clear guide to optimisation of the process experimentally.

Antibody–drug gold nanoantennas with Raman spectroscopic fingerprints for in vivo tumour theranostics

Inspired by the ability of SERS nanoantennas to provide an integrated platform to enhance disease targeting in vivo, we developed a highly sensitive probe for in vivo tumour recognition with the capacity to target specific cancer biomarkers such as epidermal growth factor receptors (EGFR) on human cancer cells and xenograft tumour models. Here, we used ~90nm gold nanoparticles capped by a Raman reporter, encapsulated and entrapped by larger polymers and a FDA antibody-drug conjugate - Cetuximab (Erbitux®) - that specifically targets EGFR and turns off a main signalling cascade for cancer cells to proliferate and survive. These drug/SERS gold nanoantennas present a high Raman signal both in cancer cells and in mice bearing xenograft tumours. Moreover, the Raman detection signal is accomplished simultaneously by extensive tumour growth inhibition in mice, making these gold nanoantennas ideal for cancer nanotheranostics, i.e. tumour detection and tumour cell inhibition at the same time.

Exposure to 2.45 GHz electromagnetic fields induces hsp70 at a high SAR of more than 20 W/kg but not at 5W/kg in human glioma MO54 cells.

Purpose : To determine potential hazards from exposure to a high-frequency electromagnetic field (HFEMF) at 2.45 GHz by studies of the expression of heat-shock protein 70 (hsp70) in MO54 cells. Method : MO54 cells were exposed to a HFEMF at average specific absorption rates (SAR) of 5, 20, 50 and 100 W/kg, using input powers of 0.8, 3.2, 7.8 and 13 W, at a temperature of up to 39°C. An annular culture dish provided three levels of exposure for a given input power, designated inner, middle and outer rings. Two control groups were used: the first was subjected to sham exposure and the second was a temperature control, used to determine the effect of high temperature using incubation in a conventional incubator at 39°C. Cell survival was determined in intervals up to 24 h. Protein was extracted from MO54 cells in both groups after 2, 4, 8 and 16 h exposure times. Changes in the hsp70 protein levels were analysed by Western blots. Results : Little or no cell death was observed in the sham-exposed cells, nor for incubation at 39°C for up to 16 h. Cell survival decreased to about 30% after exposure to HFEMF for 24 h at an average SAR of 100 W/kg. A slight increase in hsp70 was observed in cells in both the inner and outer rings of the plate after exposure at SAR levels of 25 and 78 W/kg, respectively, for 2 h. With increasing exposure time, hsp70 expression increased except for an SAR of 5 W/kg. In the raised temperature control at 39°C, hsp70 expression also increased as the incubation time increased. However, the expression level of hsp70 for the HFEMF exposure was greater than that for the raised temperature control. Conclusion : HFEMF can produce an increased level of hsp70 expression in MO54 cells at SAR levels above 20 W/kg, even when the effect of raised temperature is taken into account.

Systematic selection of housekeeping genes for gene expression normalization in chicken embryo fibroblasts infected with Newcastle disease virus

Gene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize gene expression. Here we describe the first systematic selection and evaluation of suitable HKGs for gene expression analysis in chicken embryo fibroblasts (CEF) infected with NDV adapted to the guidelines from Gorzelniak and Ferguson. Our results indicate that ACTB, HPRT1 and HMBS were valuable and stable HKGs, while 18S RNA, GAPDH and SHDA are considerably regulated during the course of infection and thus precluded for normalization. Normalizing the infection dependent gene IFN-a and the infection independent gene B2M to inappropriate HKGs consequently misleads to significant errors in estimating their regulations. Our study emphasizes that even the most popular HKGs like 18S RNA and GAPDH can lead to divergent and inaccurate data interpretation of significant magnitude if not carefully analyzed for stability before.

Surface modification and size dependence in particle translocation during early embryonic development

Since the mid-1990s, the number of studies linking air pollutants to preterm and low birth weight, as well as to cardiac birth defects, has grown steadily each year. The critical period in the development of mouse embryos begins with the commencement of gastrulation at day 7.5 of gestation. Our aim is to examine the role of particles size and surface modification in particle translocation during this early embryonic development. Fluorescent polystyrene particles (PS) were employed because they offer an efficient and safe tracking method. Pregnant female mice were sacrificed at 7.5 days of gestation. After cutting open the deciduas, the parietal endoderm was carefully separated and removed. Different sizes of amine- and carboxyl-modified PS beads were injected via the extraembryonic tissue. The embryos were incubated for 12 h, and were investigated under fluorescent microscopy, confocal microscopy, and mesoscopic fluorescence tomography. The results show that 20-nm carboxylic PS distribute in the embryonic and extraembryonic germ layers of ectoderm, mesoderm, and endoderm. Moreover, when the particles are bigger than 100 nm, PS accumulate in extraembryonic tissue, but nevertheless 200-nm amine-modified particles can pass into the embryos. Interestingly, a growth inhibition was observed in the embryos containing nanoparticles. Finally, the stronger translocation effect is associated with amine- modified PS beads (200 nm) instead of the smaller (20 nm, 100 nm) carboxyl ones.

Gold nanoprisms as a hybrid in vivo cancer theranostic platform for in situ photoacoustic imaging, angiography, and localized hyperthermia

The development of high-resolution nanosized photoacoustic contrast agents is an exciting yet challenging technological advance. Herein, antibody (breast cancer-associated antigen 1 (Brcaa1) monoclonal antibody)- and peptide (RGD)-functionalized gold nanoprisms (AuNprs) were used as a combinatorial methodology for in situ photoacoustic imaging, angiography, and localized hyperthermia using orthotopic and subcutaneous murine gastric carcinoma models. RGD-conjugated PEGylated AuNprs are available for tumor angiography, and Brcaa1 monoclonal antibody-conjugated PEGylated AuNprs are used for targeting and for in situ imaging of gastric carcinoma in orthotopic tumor models. In situ photoacoustic imaging allowed for anatomical and functional imaging at the tumor site. In vivo tumor angiography imaging showed enhancement of the photoacoustic signal in a time-dependent manner. Furthermore, photoacoustic imaging demonstrated that tumor vessels were clearly damaged after localized hyperthermia. This is the first proof-of-concept using two AuNprs probes as highly sensitive contrasts and therapeutic agents for in situ tumor detection and inhibition. These smart antibody/peptide AuNprs can be used as an efficient nanotheranostic platform for in vivo tumor detection with high sensitivity, as well as for tumor targeting therapy, which, with a single-dose injection, results in tumor size reduction and increases mice survival after localized hyperthermia treatment.